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B Journal of Medicinal 

Plants Research 

Volume 6 Number 27 18 July, 2012 

ISSN 1996-0875

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Elazıg Directorate of National Education 

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Information Center, 

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100029, 

China. 

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Postgraduate Institute of Medical Education and 

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AIMST University 

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India. 

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Lagos State University, Ojo, Lagos, 

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Omdurman Islamic University, Botany Department, 

Sudan. 

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Abdul Wali Khan University Mardan, 

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Department of Biology and Environment Engineering, 

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Division of plant Biology, 

Bose Institute 

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Indian Vetreinary Research Institute, 

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Veterinary Medicine, 

India. 

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Lorestan University, 

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Prof Dr Cemşit Karakurt 

Pediatrics and Pediatric Cardiology 

Inonu University Faculty of Medicine, 

Turkey. 

Samuel Adelani Babarinde 

Department of Crop and Environmental Protection, 

Ladoke Akintola University of Technology, 

Ogbomoso 

Nigeria. 

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Cairo 

Egypt.

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International Journal of Medicine and Medical Sciences 

Journal of Medicinal Plants Research 

Table of Contents: Volume 6 Number 27 18 July, 2012 

ences 

ARTICLES 

Review 

Medicinal properties of Moringa oleifera: An overview of promising 

healer 4368 

Fozia Farooq, Meenu Rai, Avinash Tiwari, Abdul Arif Khan and Shaila Farooq 

Research Articles 

Protein tyrosine kinase (PTK) as a novel target for some natural anti- 

cancer molecules extracted from plants 4375 

Mehrdad Hashemi, Newshan Behrangi, Hojat Borna and Maliheh Entezari 

In vitro antibacterial activity of seven plants used traditionally to treat 

wound myiasis in animals in Southern Africa 4379 

Lillian Mukandiwa, Vinasan Naidoo and Jacobus N. Eloff 

Comparative study on different methods for Lonicera japonica Thunb. 

micropropagation and acclimatization 4389 

Jiang Xiang Hui, She Chao Wen, Zhu Yong Hua and Liu Xuan Ming 

Effect of water stress on essential oil yield and storage capability of 

Matricaria chamomilla L. 4394 

Alireza Pirzad 

Enhanced callus induction and high-efficiency plant regeneration in 

Tribulus terrestris L., an important medicinal plant 4401 

Sara Sharifi, Taher Nejad Sattari, Alireza Zebarjadi, Ahmad Majd, and 

Hamid Reza Ghasempour

ences 

Table of Contents: Volume 6 Number 27 18 July, 2012 

ARTICLES 

Cytotoxic saikosaponins from Bupleurum yinchowense 4409 

LI Zong yang, Cao Li, Liu Xin min, Chang Qi and Pan Rui le 

Chemical composition and antioxidant activity of Lippia species 4416 

Junya de Lacorte Singulani, Pâmela Souza Silva, Nádia Rezende Barbosa 

Raposo, Ezequias Pessoa de Siqueira, Carlos Leomar Zani, Tânia Maria 

Almeida Alves and Lyderson Facio Viccini 

Influencing factors of consumers’ willingness to pay for Crocus sativus: 

An analysis of survey data from China 4423 

Lin Hong, Guangtong Gu, Wenchuan Li, Dan Fan, Jun Wu, Yanying Duan, 

Haijun Peng, and Qingsong Shao 

Evaluation of biochemical, hematological and histopathological 

parameters of albino rats treated with Stemona aphylla Craib. extract 4429 

Wararut Buncharoen, Supap Saenphet, Siriwadee Chomdej and 

Kanokporn Saenphet 

Assessment of fluoride, chloride and sulfate contamination of herbal teas, 

and possible interference with the medicinal properties 4436 

Mohamed Yehia Abouleish and Naser Abdo 

Effects of Moringa oleifera methanolic leaf extract on the morbidity and 

mortality of chickens experimentally infected with Newcastle disease 

virus (Kudu 113) strain 4443 

Didacus Chukwuemeka Eze, Emmanuel Chukwudi Okwor, Okoye John 

Osita A., Onah Denis Nnabuike and Shoyinka S. Vincent Olu 

Antioxidant and antimicrobial activities of Chowlai (Amaranthus viridisL.) 

leaf and seed extracts 4450 

Muhammad Javid Iqbal, Sumaira Hanif, Zahed Mahmood, Farooq Anwar 

and Amer Jamil

Journal of Medicinal Plants Research Vol. 6(27), pp. 4368-4374, 18 July, 2012 

Available online at http://www.academicjournals.org/JMPR 

DOI: 10.5897/JMPR12.279 

ISSN 1996-0875 ©2012 Academic Journals 

Review 

Medicinal properties of Moringa oleifera: An overview 

of promising healer 

Fozia Farooq 1 *, Meenu Rai 2 , Avinash Tiwari 1 , Abdul Arif Khan 3 and Shaila Farooq 4 

1 School of Studies in Botany, Jiwaji University, Gwalior-474001 (MP), India. 

2 Life Science Department, Vijayaraje Institute of Science and Management, Turari, NH 75, Gwalior (MP), India. 

3 Department of Pharmaceutics, College of Pharmacy, P. O. Box 2457, King Saud University, Riyadh 11451, 

Saudi Arabia. 

4 School of Studies in Biotechnology, Jiwaji University, Gwalior-474001 (MP), India. 

Accepted 3 May, 2012 

Moringa oleifera Lam. (MO) is a small size tree with approximately 5 to 10 m height. It is cultivated all 

over the world due to its multiple utilities. Every part of Moringa is used for certain nutritional and/or 

medicinal propose. Besides being a good source of protein, vitamins, oils, fatty acids, micro-macro 

minerals elements and various phenolics, it is also reported as anti-inflammatory, antimicrobial, 

antioxidant, anticancer, cardiovascular, hepatoprotective, anti-ulcer, diuretic, antiurolithiatic, and 

antihelmintic. Its multiple pharmaceutical effects are capitalized as therapeutic remedy for various 

diseases in traditional medicinal system. Further research on this charismatic healer may lead to the 

development of novel agents for various diseases. This study provides a brief overview about medicinal 

potential of Moringa and its future as a component of modern medicinal system. This study concludes 

that Moringa needs legitimate appraisal to establish its pharmaceutical knack in modern medicine. 

Key word: Moringa oleifera, medicinal plant, anti-inflammatory, anti-microbial, antioxidant, antiulcer, diuretic. 

INTRODUCTION 

Moringa oleifera (MO) is an aboriginal of Indian 

subcontinent and has become naturalized in the tropical 

and subtropical areas around the world. Nearly thirteen 

species of Moringa are included in the family 

Moringaceae (Nadkarni, 1976). Indians have been using 

it as a regular component of conventional eatables for 

nearly 5000 years (Anwar et al., 2005; Anwar and 

Bhanger, 2003; D'Souza and Kulkarni, 1993). Moringa 

tree can grow well in the humid tropic or hot dry land with 

average height that ranges from 5 to 10 m. It can survive 

in harsh climatic condition including destitute soil without 

being much affected by drought (Morton, 1991). It can 

tolerate wide range of rainfall requirements estimated at 

250 mm and maximum at over 3000 mm and a pH of 5.0 

to 9.0 (Palada and Chang, 2003). Its trunk is soft, white 

*Corresponding author. E-mail: foziarifkhan@gmail.com. 

Abbreviations: MO, Moringa oleifera; GK, Goto-Kakizaki; ISP, 

isoproterenol. 

corky and branches bearing a gummy bark. Each 

tripinnately compound leaves bear several small leaf 

legs. The flowers are white and the three wings seeds 

are scattered by the winds. The flowers, tenders leaves 

and pods are eaten as vegetables. The leaves are rich in 

iron and therefore highly recommended for expected 

mothers. In some part of the world, MO is referred to as 

the ‘drum stick tree’ or the ‘horse radish tree’, whereas in 

others, it is known as the kelor, marango, mlonge, 

moonga, mulangay, nebeday, saijhan, sajna or Ben oil 

tree (Anwar and Bhanger, 2003; Prabhu et al., 2011). In 

India and Pakistan, MO is locally known as Sohanjna and 

is grown and cultivated all over the country (Anwar et al., 

2005; Qaisar, 1973). It has been reported by Bureau of 

plant industry that Moringa is an outstanding source 

nutritional components. Its leaves (weight per weight) 

have the calcium equivalent of four times that of milk, the 

vitamin C content is seven times that of oranges, while its 

potassium is three times that of bananas, three times the 

iron of spinach, four times the amount of vitamin A in 

carrots, and two times the protein in milk (Kamal, 2008). 

Besides, Moringa is also suggested as a viable

supplement of dietary minerals. The pods and leaves of 

Moringa contains high amount of Ca, Mg, K, Mn, P, Zn, 

Na, Cu, and Fe (Aslam et al., 2005). Although, minerals 

content of Moringa shows variation in composition with 

changes in location (Anjorin et al., 2010). 

Ancient medicinal system relies on several plant 

products used by traditionally human communities in 

many parts of the world for different diseases. Among 

these plants, MO has its great contribution from ancient 

time. It is a plant with exceptional medicinal properties 

which can resolves the health care needs in several 

situations. Easy cultivation of Moringa within adverse 

environmental condition and wide availability attract 

attention for economic and health related potential in 

resource limited developing countries. This study 

discusses medicinal potential of this exceptional plant 

and its potential as a commercial medicinal and 

nutritional supplement. 

MEDICINAL PROPERTIES OF MORINGA 

MO has enormous medicinal potential, which has long 

been recognized in the Ayurvedic and Unani system 

(Mughal et al., 1999). Nearly every part of this plant, 

including root, bark, gum, leaf, fruit (pods), flowers, seed, 

and seed oil have been used for various ailments in the 

indigenous medicine (Odebiyi and Sofowora, 1999), but 

recent research is also indicating about several active 

constituents for accepting its applicability in modern 

medicine (Table 1). Few representatives of these are 

discussed in this article. 

Antimicrobial and antihelmintic effects 

Antimicrobial components of MO have been validated 

after the discovery of inhibitory activity against several 

microorganisms. In a recent study, aqueous extracts of 

MO was found to be inhibitory against many pathogenic 

bacteria, including Staphylococcus aureus, Bacillus 

subtilis, Escherichia coli, and Pseudomonas aeruginosa 

in dose dependent manner (Saadabi and Abu Zaid, 

2011). MO extracts was also found to be inhibitory 

against Mycobacterium phlei and B. subtilis (Eilert et al., 

1981). Leaf extract of MO was found to be effective in 

checking growth of fungi Basidiobolus haptosporus and 

Basidiobolus ranarums (Nwosu and Okafor, 1995). 

Another study involving aqueous methanolic extract and 

fixed oil against microorganisms was performed using 

Scenedesmus obliquus (green algae), E. coli ATCC 

13706, P. aeruginosa ATCC10145, S. aureus NAMRU 3 

25923, Bacillus stearothermophilus (bacterial strains) and 

Herpes Simplex virus type 1 (HSV 1) and Polio virus type 

1 (sabin vaccine). Varying degree of antimicrobial activity 

was observed ranging from sensitive for B. 

stearothermophilus to resistant for P. aeruginosa (Ali et 

Farooq et al. 4369 

al., 2004). Beside antibacterial activity of MO oils, it also 

posses anti-fungal activity (Chuang et al., 2007). Study 

comparing relative antimicrobial activity of seed extracts 

against bacteria (Pasturella multocida, E. coli, B. subtilis 

and S. aureus) and fungi (Fusarium solani and Rhizopus 

solani) revealed that P. multocida and B. subtilis were the 

most sensitive strains, and their activity was influenced 

by cations (Na + , K + , Mg 2+ and Ca 2+ ) (Jabeen et al., 2008). 

Another relative comparison of antibacterial and 

antifungal efficacy of MO steam distillate observed more 

inhibition for E. coli followed by S. aureus, Klebsiella 

pneumoniae, P. aeruginosa and B. subtilis. In case of 

fungi, Aspergillus niger was strongly inhibited followed by 

Aspergillus oryzae, Aspergillus terreus and Aspergillus 

nidulans (Prashith Kekuda et al., 2010). Contrary to 

resistance against P. aeruginosa and Candida albicans 

for MO in other studies, one study using ethanolic extract 

of leaves, seeds and flowers showed the antimicrobial 

activity against E. coli, K. pneumoniae, Enterobacter 

species, Proteus mirabilis, P. aeruginosa, Salmonella 

typhi A, S. aureus, Streptococcus and Candida albicans 

(Nepolean et al., 2009). Moringa contains pterygospermin 

(originally found in Moringa pterygosperma) which has 

powerful antibacterial and fungicidal effects (Rao et al., 

1946). Several other specific components of Moringa 

have been reported with antibacterial activity, including 4- 

(4'-O-acetyl-a-L-rhamnopyranosyloxy) benzyl 

isothiocyanate, 4-(a-L-rhamnopyranosyloxy) benzyl 

isothiocyanate, niazimicin, benzyl isothiocyanate, and 4- 

(a-L-rhamnopyranosyloxy) benzyl glucosinolate (Fahey, 

2005). Other bioactive compounds, such as Spirochin 

and Anthonine are found in root and are active against 

several bacteria. Anthonine has potent inhibitory activity 

against Vibrio cholerae (Nwosu and Okafor, 1995). MO 

flower and leaves are also capable of controlling parasitic 

worms, their antihelmintic activity has been demonstrated 

during several studies (Bhattacharya et al., 1982). 

Moreover, it has also been reported to inhibit Indian 

earthworm Pheritima posthuma with MO leaves ethanolic 

extracts (Rastogi et al., 2009). 

Anti-inflammatory activity 

Moringa plant parts have substantial anti-inflammatory 

activity. For instance, the root extract exhibits significant 

anti-inflammatory activity in carrageenan induced rat paw 

oedema (Ezeamuzie et al., 1996; Khare et al., 1997). The 

crude methanol extract of the root inhibits carrageenaninduced 

rat paw oedema in a dose dependent manner 

after oral administration (Anonymous, 2005). Moreover, 

n-butanol extract of the seeds of MO shows antiinflammatory 

activity against ovalbumin-induced airway 

inflammation in guinea pigs (Mahajan et al., 2009). 

Amelioration of inflammation associated chronic diseases 

can be possible with the potent anti-inflammatory activity 

of MO bioactive compounds (Muangnoi et al., 2011).

4370 J. Med. Plants Res. 

Considering potent anti-inflammatory activity of Moringa 

plant, it can be surmised that this plant shows profound 

influence on inflammation associated diseases and 

resultant symptoms. As a consequence, this plant shows 

beneficial effects on asthma, pain, and other resultant 

symptoms. 

Anti-asthmatic activity 

It has been reported a long time ago that Moringa plant 

alkaloid closely resembles ephedrine in action and can 

be used for the treatment of asthma. Alkaloid moringine 

relaxes bronchioles (Kirtikar and Basu, 1975). The seed 

kernels of MO also showed promising effect in the 

treatment of bronchial asthma, during a study to analyze 

efficacy and safety of seed kernels for the management 

of asthmatic patients. The study showed significant 

decrease in the severity of asthma symptoms and also 

concurrent respiratory functions improvement (Agrawal 

and Mehta, 2008). 

Analgesic activity 

The analgesic activity of Moringa has been reported in 

several Moringa species. In a study using ethanolic 

extracts of Moringa concanensis tender pod-like fruits in 

experimental animals, a significant analgesic activity was 

observed (Rao et al., 2008). Furthermore, alcoholic 

extract of the leaves and seeds of MO also possess 

marked analgesic activity as evidenced through hot plate 

and tail immersion method (Sutar et al., 2008). 

Antipyretic activity 

As a result of anti-inflammatory action of Moringa 

bioactive constituents, the antipyretic activity can be 

hypothesized. A study was designed to assess antipyretic 

effect of ethanol, petroleum ether, solvent ether and ethyl 

acetate extracts of MO seeds using yeast induced 

hyperpyrexia method. Paracetamol was used as control 

during the study. Not surprisingly, ethanol and ethyl 

acetate extracts of seeds showed significant antipyretic 

activity in rats (Hukkeri et al., 2006). 

Antihypertensive, diuretic and cholesterol lowering 

activities 

Moringa leaves contain several bio active compounds, 

they exert direct effect on blood pressure, and thus these 

can be used for stabilizing blood pressure. MO compounds 

leading to blood pressure lowering effect includes 

nitrile, mustard oil glycosides and thiocarbamate 

glycosides present in Moringa leaves (Anwar et al., 

2007). In addition, diuretic activity of Moringa exists in its 

roots, leaves, flowers, gum and the aqueous infusion of 

seeds (Morton, 1991). Moreover, Moringa leaves also 

contain bioactive phytoconstituent, (that is, b-sitosterol) 

with cholesterol lowering effect. This compound is 

capable to reduce cholesterol level from the serum of 

high fat diet fed rats (Ghasi et al., 2000). 

Antidiabetic activity 

Several medicinal plants have been evaluated for their 

potential as therapeutic agent for diabetes. MO is also an 

important component in this category. MO leaves 

significantly decrease blood glucose concentration in 

Wistar rats and Goto-Kakizaki (GK) rats, modeled type 2 

diabetes (Ndong et al., 2007). Another study indicated 

that the extract from Moringa leaf is effective in lowering 

blood sugar levels within 3 h after ingestion (Mittal et al., 

2007). As a mechanistic model for antidiabetic activity of 

MO, it has been indicated that dark chocolate 

polyphenols (Grassi et al., 2005) and other polyphenols 

(Al-Awwadi et al., 2004; Moharram et al., 2003) are 

responsible for hypoglycemic activity. Moringa leaves are 

potent source of polyphenols, including quercetin-3glycoside, 

rutin, kaempferol glycosides, and other 

polyphenols (Ndong et al., 2007). Thus, potential antidiabetic 

activity of MO can be commercialized through 

the development of suitable technology with achieving 

anti-diabetic activity up to conventional drugs. 

Antioxidant activity 

MO is a rich source of antioxidant (Chumark et al., 2008). 

It has been reported that aqueous extracts of leaf, fruit 

and seed of MO act as an antioxidant (Singh et al., 

2009). During a study reporting antioxidant property of 

freeze dried Moringa leaves from different extraction 

procedures, it was found that methanol and ethanol 

extracts of Indian origin MO have the highest antioxidant 

activity with 65.1 and 66.8%, respectively (Lalas and 

Tsaknis, 2002; Siddhuraju and Becker, 2003). It was also 

reported that the major bioactive compounds of 

phenolics, such as quercetin and kaempferol are 

responsible for antioxidant activity (Bajpai et al., 2005; 

Siddhuraju and Becker, 2003). During another study, 

quercetin and kaempferol have shown good antioxidant 

activity on hepatocyte growth factor (HGF) induced Met 

phosphorylation with IC50 value for 12 and ~6 µM/L, 

respectively (Labbe et al., 2009). Another recent study 

comparing palm oil with MO seeds for their antioxidant 

potential found out that MO seed are superiors for radical 

scavenging (Ogbunugafor et al., 2011). 

Hepatoprotective activity 

MO has shown significant hepatoprotective activity in

several studies. MO leaves ethanolic extracts showed 

significant protection against liver damage induced by 

antitubercular drugs [isoniazid (INH), rifampicin (RMP), 

and pyrazinamide (PZA)] in rats. It was found that 

hepatoprotective activity of MO is medicated by its effect 

on the levels of glutamic oxaloacetic transaminase 

(aspartate aminotransferase), glutamic pyruvic 

transaminase (alanine aminotransferase), alkaline 

phosphatase, and bilirubin in the serum; lipids, and lipid 

peroxidation levels in liver (Pari and Kumar, 2002). 

Moreover, methanolic and chloroform extracts of MO 

leaves also showed significant protection against CCl4 

induced liver damage in albino rats. Besides 

hepatoprotective activity of MO leaves, its root and 

flowers also possess strong hepatoprotective activity. 

Moringa flowers contain a well recognized flavonoid 

(Quercetin), which may be responsible for its potent 

hepatoprotective activity (Ruckmani et al., 1998; 

Selvakumar and Natarajan, 2008). In a recent study 

evaluating the effect of MO seed extract on liver fibrosis, 

it was found that MO seed extract has the ability to 

subside liver fibrosis. This study involved CCl4 induced 

liver fibrosis and concurrent administration of MO seed 

extract. MO seed extract control the elevation of serum 

aminotransferase activities and globulin level induced by 

CCl4. Moreover, immunohistochemical studies also 

showed that MO reduces liver fibrosis (Hamza, 2010). 

Antitumor activity 

MO has been found as a potent anticancer plant and 

several bioactive compounds with significant antitumor 

activity have been discovered from MO. Among bioactive 

compounds from MO, niazimicin, a MO leaves 

thiocarbamate was found to have potent anticancer 

activity (Guevaraa et al., 1999). Furthermore, niazimicin 

also shows the inhibition of tumor promoter teleocidin B- 

4-induced Epstein-Barr virus (EBV) activation (Murakami 

et al., 1998). Another study involving 11 plants used in 

Bangladeshi folk medicine, MO was considered as 

potential source of anticancer compounds. During this 

study, the plant extract were analyzed for cytotoxicity 

through brine shrimp lethality assay, sea urchin eggs 

assay, hemolysis assay and MTT assay using tumor cell 

lines. The study also indicated the potential cytotoxic 

effects of MO leaf extract on human multiple myeloma 

cell lines (Costa-Lotufo et al., 2005; Parvathy and 

Umamaheshwari, 2007). Beside leaves, MO seed 

extracts also have anticancer activity through its effects 

on hepatic carcinogen metabolizing enzymes, and 

antioxidant property (Bharali et al., 2003). 

Antifertility activity 

MO plant also has pertinent antifertility activity. The 

aqueous extract obtained from root and bark of MO 


showed post-coital antifertility effect in rat and also 

induced foetal resorption at late pregnancy (Prakash et 

al., 1987). Moreover, aqueous extract of MO roots was 

also evaluated for estrogenic, anti-estrogenic, progestational 

and antiprogestational activities. This extract 

induces several consequences for affecting its antifertility 

property (Shukla et al., 1988). During another study 

analyzing anti reproductive potential of folk medicine 

plants, MO leaf extracts were found to be 100% abortive 

with doses equivalent to 175 mg/kg of starting dry 

material (Nath et al., 1992). 

Antispasmodic and antiulcer effects 

Moringa root and leaves contain several compounds with 

spasmolytic activity. These compounds include 4- (alpha- 

L-rhamnosyloxybenzyl)-o-methyl thiocarbamate which is 

possibly affected through calcium channel blockade, 

niazinin A, niazinin B, niazimicin, etc., with hypotensive 

and bradycardiac effect. The spasmolytic activity of 

different constituents support for traditional uses of this 

plant in gastrointestinal motility disorder (Gilani et al., 

1994). MO methanolic extract is also capable in 

protecting experimental rats from gastric lesions induced 

by acetylsalicylic acid, serotonin and indomethacin. In 

addition, it also enhances healing process of chronic 

gastric lesions induced by acetic acid in experimental 

animals (Pal et al., 1995). Another study have reported 

the antiulcer effect of MO leaves aqueous extract on 

adult Holtzman albino rats (Debnath and Guha, 2007). 

Cardiac and circulatory stimulant 

In addition to earlier mentioned bradycardiac effect of MO 

leaves, all parts of MO are reported with somewhat 

cardiac and circulatory stimulant activity. Root bark of 

Moringa contains alkaloid moringinine which acts as 

cardiac stimulant through its effect on sympathetic 

nervous system (Duke, 2001). The aforementioned 

effects can also result due to the prevention of 

hyperlipidemia. It has been demonstrated that MO 

prevent hyperlipidemia in male Wister rat due to iron 

deficiency (Ndong et al., 2007). During a study 

performing comparison of MO leaf extract with antenolol 

(a selective β1 receptor antagonist drug, used for 

cardiovascular diseases) on serum cholesterol level, 

serum triglyceride level, blood glucose level, heart weight 

and body weight of adrenaline induced rats, it was found 

that MO leaf extract cause significant changes in 

cardiovascular parameters. This study reported MO leaf 

extract as hypolipidimic, lowering body weight, heart 

weight, serum triglyceride level and serum cholesterol 

level in experimental animals (Ara et al., 2008). In 

addition to the aforementioned studies, antiatherosclerotic 

and hypolipidaemic effect of MO leaves were also


Table 1. Major pharmaceutical components present in Moringa and their importance. 

S/N Compound Method used for detection 

1 Pterygospermin 

2 4-(4'-O-acetyl-a-L-rhamnopyranosyloxy) 

benzyl isothiocyanate, 4-(a-Lrhamnopyranosyloxy) 

benzyl 

isothiocyanate, niazimicin, benzyl 

isothiocyanate, and 4-(a-Lrhamnopyranosyloxy) 

benzyl 

glucosinolate, Anthonine and Spirochin 

3 Alkaloid Moringine 

4 

Nitrile, mustard oil glycosides and 

thiocarbamate glycosides 

5 b-sitosterol 

6 

Dark chocolate polyphenols and other 

polyphenols 

7 Quecertin and kaempferol 

8 Niazimicin, 

9 

4- (alpha- L-rhamnosyloxybenzyl)-omethyl 

thiocarbamate, niazinin A, 

niazinin B, niazimicin etc. 

analyzed in another study using simvastatin as control 

(Chumark et al., 2008). MO also causes cardio protective 

effects in isoproterenol (ISP)-induced myocardial 

infarction in male Wistar albino rats. It was reported that 

MO treatment plays favorable modulation on biochemical 

Solvent extraction followed by MIC 

analysis 

Solvent extraction followed by MIC 

analysis (Busani et al., 2012) 

Clinical study involving 

consumption of Moringa followed 

by antiasthmatic activity evaluation 

using spirometer 

Potential 

application 

Antibacterial and 

fungicidal effects 

Antibacterial 

Antiasthmatic 

Bioassay directed isolation Hypotensive 

Study involved consumption of 

Moringa leaves with cholesterol 

and subsequent measurement of 

cholesterol lowering activity (Ghasi 

et al., 2000). 

Administration of MO leaves in 

diabetic and control rats and 

hypoglycemic activity evaluation 

and characterization of 

polyphenols using HPLC (Ndong 

et al., 2007). 

Solvent extraction followed by 

antioxidant activity analysis 

Solvent extraction followed by in 

vitro anticancer activity 

Solvent extraction for purification 

of compounds followed by 

intravenous administration of each 

compound in anaesthetized rats 

and subsequent evaluation of their 

activity in experimental animals 

Cholesterol 

lowering effects 

Hypoglycemic 

effects 

Antioxidant, 

hepatoprotective 

Reference 

Rao et al. (1946) 

Fahey (2005) and 

Nwosu and Okafor 

(1995) 

Agrawal and Mehta 

(2008) and Kirtikar and 

Basu (1975) 

Anwar et al. (2007) and 

Faizi et al. (1995) 

Ghasi et al. (2000) 

Grassi et al. (2005), Al- 

Awwadi et al. (2004) 

and Moharram et al. 

(2003) 

Bajpai et al. (2005), 

Siddhuraju and Becker 

(2003), Ruckmani et al. 

(1998) and Selvakumar 

and Natarajan (2008) 

Anticancer Guevaraa et al. (1999) 

Spasmolytic, 

hypotensive and 

bradycardiac 

Gilani et al. (1994) 

enzymatic parameters including, superoxide dismutase, 

catalase, glutathione peroxidase, lactate dehydrogenase, 

and creatine kinase-MB. Moreover, it also prevents 

histopathological damage and ultra-structure perturbation 

caused due to ISP induced myocardial infarction

(Nandave et al., 2009). 

In ocular diseases 

Vitamin A deficiency is a major cause of blindness, which 

ranges from impaired dark adaptation to night blindness. 

Consumption of MO leaves, and pods and leaf powder 

which contain high proportion of vitamin A can help to 

prevent night blindness and eye problems in children. 

Ingesting drumstick leaves with oils can improve vitamin 

A nutrition and can delay the development of cataract 

(Pullakhandam and Failla, 2007). In fact the use of MO 

as a supplementary food was highly accepted for 

integrated child development scheme supplementary 

food (ICDS-SFP) for its potential as vitamin A source 

(Nambiar et al., 2003). 

Conclusion 

Medicinal potential of MO is enormous and difficult to 

cover in a single article, despite this current article 

provided glimpses of MO applications for performing 

appraisal of this promising nutrition and medicinal plant. 

Although, many bioactive compounds have been 

discovered from Moringa, still the knowledge is in infancy, 

in term of its total reserve. Perhaps, future rigorous 

studies directed towards the detection, and 

commercialization of MO bioactive compounds can lead 

to the development of remedies for several ailments. 

Thus, it can also prove the validity of traditional utility of 

MO in various folklores. 

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DOI: 10.5897/JMPR11.1005 


Full Length Research Paper 

Protein tyrosine kinase (PTK) as a novel target for some 

natural anti-cancer molecules extracted from plants 

Mehrdad Hashemi 1 *, Newshan Behrangi 2 , Hojat Borna 1 and Maliheh Entezari 1 

1 Department of Genetics, Islamic Azad University, Science and Research Branch, Tehran, Iran. 

2 Department of Genetics, Islamic Azad University, Tehran Medical Branch, Tehran, Iran. 

Accepted 6 January, 2012 

For the fact that protein tyrosine kinases (PTKs) are important components of signal transduction 

pathway, they also are involved in regulating cell growth, differentiation and oncogenesis. Therefore, 

protein tyrosine kinases represent a potential target for cancer treatment. Drugs known as protein 

tyrosine kinase (PTK) inhibitors play a key role in developing therapeutic strategies for cancer therapy 

and plants are critical sources of natural PTK inhibitors. In the present study, we evaluated some 

natural anticancer molecules with PASS software in order to predict their possible targets involved in 

cancer. About 14 molecules (Afrormosin, Iriflogenin, Irigenin, Irisolidone, Irilone, biochanin A, 

Pseudobaptigenin, Pinosylvin, Galangin, Luteolin, Apigenin, Formononetin, Piceatannol and Daidzein) 

exhibited PTK inhibitor activity more than 0.6 theresholds. The results obtained reveal that Daidzein 

shows the highest PTK inhibitor activity with 0.780 score. However, Galangin has the lowest PTK 

inhibitory with 0.605 score. Furthermore, all of these compounds have CYP1A1 human substrate 

activity with score more than 0.6; therefore, Biochanin A and Daidzein with score 0.815 are the most 

potent CYP1A1 human substrate. In addition, all molecules had high druglikeness score and it means 

that they are been applied as drugs. Consequently, Biochanin A and Diadzein with 0.79 mean score are 

the most potent anticancer agents in our study. 

Key words: Protein tyrosine kinase, plants, bioinformatics tools. 

INTRODUCTION 

In multicellular organisms, all aspects of cell behavior 

such as metabolism, movement, proliferation and 

differentiation are regulated by cell signaling. Signal 

transduction pathway transmits a signal into cell by a 

series of consecutive events that regulate the activity of a 

gene. Protein tyrosin kinases mediate the transduction 

and process of many extra-and intracellular signals. They 

are involved in regulating cell growth, differentiation as 

well as oncogenesis. 

There are two general classes of protein tyrosin 

kinases: The receptor tyrosine kinase and receptorassociated 

tyrosine kinase. The receptor tyrosine kinase 

consists of an extracellular ligand binding domain and 

and intracellular catalytic domain which has tyrosine 

kinase activity. When a ligand is bound to the receptor, 

the various downstream effects including stimulation of 

*Corresponding author. E-mail: mhashemi@iautmu.ac.ir. 

other tyrosine kinases, elevation of intracellular calcium 

levels, activation of serine/threonine kinases, 

phospholipase C and phosphatidylinositol-3’-kinase, and 

ultimately changes in gene expression are going to occur. 

The receptor-associated tyrosine kinase interacts with the 

cytoplasmic domain of membrane protein in order to 

transmits signal from the membrane (Strachan and Read, 

2011; Hyde et al., 2009). 

Protein tyrosine kinases have enormous roles in cancer 

molecular pathogenesis, so currently they are as a 

potential target for anticancer drugs. There are two 

classes of protein tyrosine kinase inhibitors. One is bound 

to the ATPbinding site and the other is bound to the 

substrate binding site of the enzyme (Fabbro et al., 

2002). Most of the anticancer drugs that target protein 

tyrosine kinase are extracted from plants, fruits or 

microorganisms (Brunton et al., 2011). 

Bioinformatics is the mathematical, statistical and 

computing methods that aim to solve biological problems 

(Vaidya and Dawkha, 2010). Bioinformatics can be


Figure 1. 3D structure of Apigenin with ChemAxon within MDL SD file. 

applied in the field of medical sciences to know the 

molecular pathways of diseases. By developing 

sophisticated bioinformatics software such as prediction 

of activity spectra for substances (PASS), it is now 

possible to predict some targets of anticancer molecules 

on basis of the structure formula of a substance with high 

accuracy (Poroikov et al., 2003; Ali et al., 2011). 

This study is focused on PASS score of some natural 

anticancer molecules and selected molecules based on 

specific target throughout cancer pathway. As a result, 

molecules which exhibited protein tyrosine kinase 

inhibitory with PASS score more than 0.6 were screened, 

and they are includes: Afrormosin, Iriflogenin, Irigenin, 

Irisolidone, Irilone, biochanin A, Pseudobaptigenin, 

Pinosylvin, Galangin, Luteolin, Apigenin, Formononetin, 

Piceatannol and Daidzein. Although Iriflogenin, Irigenin, 

Irisolidone, Irilone are isoflavones isolated from rhizome 

of Iris germanica, other compounds are extracted from 

different plants around the world (Ludwiczuk et al., 2011; 

Entezari et al., 2009). 

MATERIALS AND METHODS 

Data 

A paractical database is the main step in bioinformatics projects. 

Collections of data from Pubmed database were accomplished with 

general keyword “anticancer”. Most data were gathered from 2010 

papers; therefore, anticancer molecules were extracted from these 

papers, and their targets in apoptotic pathway defined. In this case, 

molecules were classified based on their origins, as a result we had 

7 groups of anticancer molecule such as molecules in Drug Bank, 

plants, fruits, microorganisms, semi-synthetic agents, synthetic 

agents and finally ungrouped anticancer agents which their origins 

were unknown (Behrangi et al., 2011). 

Structure 

Structural formula of these molecules were investigated from 

Chemspider, Pubcheme and Wikipedia, respectively, and the 

original molecular structure of all compounds were found; their 

skeletal structures drawn with Chemschetch, Chemaxon, version 

5.4 software in order to reach 3D structures of molecules within 

MDL SD file, the same software is used with molecular mechanics 

algorithm for structural optimization. ChemAxon is a leader in 

providing Java based chemical software development platform for 

biotechnology and pharmaceutical industries. Protein Data Bank 

(PDB), Tripos MOL2, MDL MOL and SD file formats were saved as 

well (Figure 1). 

Software 

Predicition of activity spectra for substance (PASS) is a simple 

computational tool that can predict more than 1500 

pharmacological effects, molecular mechanisms of action, and 

toxicities on basis of structural descriptors of compounds with over 

80% accuracy and has capability to predict many types of activity

Table 1. Pa and Pi of PTK inhibitory, CYP1A1 human substrate and drug likeness of molecules. 

Label Molecule 

PASS activity 

(PTK Inhibitory) 

PASS inactivity 

(PTK inhibitory) 

PASS activity 

(CYP1A1 human substrate ) 

Hashemi et al. 4377 

PASS inactivity 

(CYP1A1 human substrate) 

Drug 

likeness 

A Afrormosin 0.699 0.004 0.738 0.005 0.865 

B Apigenin 0.638 0.005 0.716 0.005 0.940 

C Biochanin A 0.758 0.003 0.815 0.004 0.887 

D Daidzein 0.780 0.002 0.814 0.004 0.857 

E Formononetin 0.724 0.003 0.759 0.005 0.817 

F Galangin 0.605 0.006 0.678 0.006 0.957 

G Iriflogenin 0.654 0.005 0.641 0.007 0.968 

H Irigenin 0.706 0.004 0.710 0.005 0.928 

I Irilone 0.701 0.004 0.683 0.006 0.978 

J Irisolidone 0.657 0.005 0.696 0.006 0.983 

K Luteolin 0.635 0.005 0.707 0.006 0.959 

L Piceatannol 0.657 0.005 0.679 0.006 0.795 

M Pinosylvin 0.638 0.005 0.686 0.006 0.683 

N Pseudobaptigenin 0.702 0.004 0.689 0.006 0.916 

for a new substance. 

PASS utilizes input data with molecular structure Protein Data 

Bank (PDB), Tripos MOL2, MDL MOL and SD file formats for 

representing the structural information about molecules under 

study. PASS prediction can be interpreted by Pa and Pi values. Pa 

and Pi values are as measures that determine activity and inactivity 

of compounds. Pa is the probabilities of being active and close to 

1.000, and Pi is the probabilities of being inactive close to 0.000; 

therefore, the Pa and Pi values vary from 0.000 to 1.000 and in 

general Pa + Pi0.6); therefore, it is predicted that 

indicated agents can inhibit protein tyrosine kinase with 

high strength and by increasing the Pa score of them 

,their strength will be improved simultaneously. Thus, 

according to Table 1, Daidzein with Pa 0.780 is the most 

potent PTK inhibitor in our research. In our research, we 

have confronted interesting issue that all of our PTK 

inhibitor agents are as CYP1A1 human substrate and 

they exhibit these properties with Pa score more than 0.6. 

Consequently, Daidzein exhibited the highest P activity 

(0.814) compare to other molecules. As can be seen from 

Table 1, Irisolidone with score 0.983 have the highest 

drug-likeness score and it means that this agent might 

posses functional groups or have physical properties 

which is consistent with most of known drugs (Walters et 

al., 1998). 

DISCUSSION 

Predication of activity spectra for substances (PASS) 

software capable to anticipate more than 1500 

pharmacological effect can be efficiently applied to find 

new targets for some ligands to reveal new biological 

activity of various substances (Lagunin et al., 2000; Jin et 

al., 2010). As this study has focused on anticancer 

molecules, we tried to find out efficient target in cancer 

pathway and screen molecules on basis of their strength 

in specified activity. 

Tyrosine Kinase is an enzyme which transports 

phosphates from ATP to a proteins tyrosine residue. 

Therefore, a tyrosene kinase inhibitor prevents the 

phosphate groups from being transferred. In many cases 

of human malignancies, some mutations activate proten 

tyrosine kinase constitutively in order to implicate 

malignant transformation. Thus, protein tyrosine kinase is 

an appropriate target for cancer treatment. In this study, 

we screened molecules which have Pa>0.6 and 

Pi


in ATP-competitive manner, in order to inhibits 

PKC(epsilon) and Src kinase activity (Gyémánt et al., 

2005). In addition, as Yin et al. (2001) demonstrated, 

Apigenin decreases growth factor receptor (EGF-R) 

tyrosine phosphorylation and phosphorylation of ERK 

mitogen-activated protein (MAP) kinase. Likewise, PASS 

results (Table 1) were so consistent with previous 

research and demonstrated molecules showed high Pa 

too. For instance, Luteolin and Apigenin with Pa 0.635 

and 0.638 respectively are as promising PTK inhibitor 

agents. Moreover, Daidzein has the most potent PTK 

inhibitor activity with 0.780 score, in contrast Galangin 

exhibits the lowest PTK inhibitory with score 0.605 (Heo 

et al., 2001). Biochanin A with 0.758 score is the second 

strong protein tyrosine kinase inhibitor agent. 

Interestingly, all of the 14 molecules with PTK inhibitor 

activity show CYP1A1 human substrate activity too. As 

Cytochrome P450 1A1 isoenzyme involving in metabolic 

conversion of paracarcinogens into carcinogens, thus 

Cyp1A1 is as a target for cancer chemoprevention. 

Previous research also shows that some of indicated 

molecules are CYP1A1 Human substaret. For example, 

Wollenweber et al. (2003) exhibited Iriflogenin, Irigenin, 

Irisolidone and Irilone are as potent inhibitors of 

cytochrome P450 1A enzyme. Our study demonstrated 

that Biochanin A and Daidzein with score 0.815 are the 

most potent CYP1A1 human substrate and they show 

this property with high strength (Moon et al., 2008; 

Sehdev et al., 2009; Peterson and Barnes, 1993). 

In conclusion, the Table 1 clearly shows that all of the 

14 natural anticancer molecules are as a potent protein 

tyrosine kinase inhibitor and CYP1A1 human substrate. 

In addition, their high drug likeness candidate them as 

functional anticancer drugs which can have therapeutic 

effects. Because these molecules are extracted from 

nature, we can reduce side effect of chemotherapeutic 

drugs efficiently, but application of these drugs in clinic 

demands in vitro and in vivo experiments and we hope 

that future experimental research can candidate them as 

known anticancer drugs. 

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DOI: 10.5897/JMPR11.1130 



In vitro antibacterial activity of seven plants used 

traditionally to treat wound myiasis in animals in 

Southern Africa 

Lillian Mukandiwa 1 *, Vinasan Naidoo 2 and Jacobus N. Eloff 1 

1 Department of Paraclinical Sciences, Faculty of Veterinary Science, University of Pretoria, P. Bag X04, Onderstepoort 

0110, South Africa. 

2 Biomedical Research Centre, Faculty of Veterinary Science, University of Pretoria, Onderstepoort 0110, South Africa. 

Accepted 20 October, 2011 

In the extreme situation of subsistence farming where insecticides and other veterinary medicines are 

either unavailable or unaffordable, the use of plants in the treatment of wound myiasis in livestock has 

been reported worldwide. However, the exact effect of these plants on myiatic wounds has not been 

established. This study was therefore undertaken to establish the biological activity of seven species of 

plants which are used traditionally and are claimed to be effective in the treatment of wound myiasis. 

Plants that have a wide distribution in southern Africa were selected. This paper focuses on the 

antibacterial activity of these plants on bacteria known to be among the common contaminants of 

wounds. It has been shown that bacterial action on wounds produce compounds which have an odour 

that serve as an attractant of myiasis-causing flies. The antibacterial activity of the plants was 

investigated using a microdilution assay and bioautography methods. All the tested plants had 

inhibitory activity against the test bacteria. Inhibiting bacterial activity reduces the attractants of 

myiasis-causing flies to the wound. Thus, inhibiting bacteria action on wounds will interfere with the 

development of wound myiasis. This could be one of the mechanism through which the plants that are 

used traditionally in the treatment of wound myiasis work. 

Key words: Wound myiasis, ethnoveterinary medicine, antibacterial activity. 

INTRODUCTION 

Wound myiasis (infestation of wounds by dipterous 

larvae) in livestock can be devastating due to production 

losses, veterinary costs and sometimes death (OIE, 

2008). The role of bacteria in the attraction of myiasis- 

causing flies and oviposition has been established in 

*Corresponding author. E-mail: lmukandiwa@yahoo.com or 

kobus.eloff@up.ac.za. Tel: +27 12 529 8525. Fax: +27 12 529 

8525. 

Abbreviations: INT, p-iodonitrotetrazolium violet; MIC, minimal 

inhibitory concentration; MH, Müller-Hinton; TLC, thin layer 

chromatography; EMW, ethyl acetate/methanol/water; CEF, 

chloroform/ethyl acetate/formic acid; BEA, 

benzene/ethanol/ammonia hydroxide; LPS, 

lipopolysaccharides. 

a number of studies (Chaudhury et al., 2010). Bacteria 

such as Streptococcus pyogenes, Enterococcus faecalis, 

Staphylococcus aureus, Pseudomonas aeruginosa, 

Escherichia coli, Proteus mirabilis and Klebsiella spp. 

found on wounds produce volatile organic, sulphurcontaining 

compounds with an odour that attracts the 

myiasis-causing flies (Khoga et al., 2002). These 

compounds can also act as ovipository stimuli to the 

myiasis-causing flies (Emmens and Murray, 1982). 

Extracts from unsterile sheep fleeces seeded with P. 

aeruginosa, P. mirabilis, Enterobacter cloacae and 

Bacillus subtilis stimulate oviposition by females of Lucilia 

cuprina (Wied.) (Eisemann and Rice, 1987). Wounds 

already infested with larvae are also more attractive to 

the gravid females (Hammack and Holt, 1983). The 

presence of larvae in wounds by themselves is not 

enough to attract gravid females, but their activity in


Table 1. Plants used traditionally to treat wound myiasis in South Africa and Zimbabwe. 

Scientific name Family Plant part used Distribution Preparation and administration 

Aloe marlothii Berger (Van der 

Merwe et al., 2001) 

Aloe zebrina Baker (Luseba and 

Van der Merwe, 2006) 

Calpurnia aurea (Ait.) Benth. 

(Hutchings et al., 1996) 

Psydrax livida (Canthium huillense) 

(Chavunduka, 1976) 

Clausena anisata (Chavunduka, 

1976) 

Erythrina lysistemon Hutch (Van 

Wyk et al., 1997) 

Spirostachys africana Sond 

(Hutchings et al., 1996) 

Asphodelaceae leaves 

Asphodelaceae leaves 

Botswana, Mozambique, South Africa (North-West, Gauteng, Limpopo, 

Mpumulanga, KwaZulu-Natal north of Durban), Swaziland, Zimbabwe. 

Angola, Botswana, Malawi, Mozambique, Namibia, South Africa (Gauteng, 

Mpumalanga, Limpopo), Zambia, Zimbabwe. 

The leaves are crushed and the juice is 

applied onto the wounds 

Succulent fresh leaves are crushed 

and applied onto the wound 

Fabaceae leaves Angola, Mozambique, South Africa, Swaziland, Zimbabwe Leaf sap is squeezed onto the wound 

Rubiaceae leaves 

Rutaceae leaves 

Fabaceae leaves 

media contaminated with bacteria increases 

attractiveness of the wound (Eisemann and Rice, 

1987). As such it is clear that bacterial 

contamination of wounds is important in the 

pathogenesis of wound myiasis. 

In orthodox veterinary medicine, 

organophosphate insecticides in conjunction with 

antibiotics are recommended for the treatment of 

wound myiasis. The insecticides serve to expel 

and kill the larvae from the wound (OIE, 2008). 

The antibiotics deal with the microbial infection on 

the wound, which promotes wound healing and 

prevents secondary re-infestation by flies. In the 

difficult situation of subsistence farming where 

insecticides and other veterinary medicines are 

Botswana, Malawi, Mozambique, Zambia, Zimbabwe, Angola, Kenya, Namibia, 

South Africa( North-West, Limpopo, Mpumalanga) 

Angola, Malawi, Mozambique, Zambia, Zimbabwe, South Africa(Limpopo, 

Mpumalanga, Eastern Cape, KwaZulu-Natal) 

South Africa (North West, Limpopo, Gauteng, Mpumalanga, KwaZulu-Natal, Eastern 

Cape), Swaziland, Zimbabwe, Botswana, Angola 

Euphorbiaceae Zimbabwe, Mozambique, Swaziland, South Africa (Mpumalanga, KwaZulu-Natal) 

either unavailable or unaffordable, plants have 

been used in the treatment of wound myiasis in 

Africa and Asia (Chavunduka, 1976; Van der 

Merwe et al., 2001; Luseba and Van der Merwe, 

2006). However, the exact effect of most of these 

plants on myiatic wounds has not been 

established. We therefore, undertook a study to 

establish the biological activity of 7 species of 

plants which are used traditionally and are 

claimed to be effective in the treatment of wound 

myiasis in South Africa and Zimbabwe (Table 1). 

The study was conducted in an endeavour to 

validate the traditional use of the plants and 

determine those that are highly active. This paper 

focuses on the antibacterial activity of extracts of 

Leaves crushed and packed into the 

wound 

Leaves crushed and packed into the 

wound 

Leaves crushed and placed on a 

maggot-infested wound 

The sap is applied onto the maggot 

infested wound 

these plants on bacteria that are common 

contaminants of wounds. 


Plant materials 

After a study of the literature, seven plant species 

traditionally used in the treatment of cutaneous myiasis: 

Aloe marlothii A. Berger (Van der Merwe et al., 2001), Aloe 

zebrina Baker (Luseba and Van der Merwe, 2006), 

Calpurnia aurea (Aiton) Benth (Hutchings et al., 1996), 

Psydrax livida (Hiern) Bridson (Canthium huillense), 

Clausena anisata (Willd) Hook (Chavunduka, 1976), 

Erythrina lysistemon Hutch (Van Wyk et al., 1997), and 

Spirostachys africana Sond (Hutchings et al., 1996), were 

selected for further study. More information is provided in

Table 1. 

Plant collection and storage 

The plant material was collected from the Pretoria National 

Botanical Garden, South Africa. Voucher specimens and origins of 

the trees are kept in the garden herbarium. It was dried at room 

temperature in a well-ventilated room. Collection, drying and 

storage of plant material guidelines outlined elsewhere were 

followed (McGaw and Eloff, 2010). 

Preparation of plant extracts 

Dried leaf material was ground to fine powder using a KIKA- 

WERKE M20 mill (GMBH and Co., Germany). To obtain the 

acetone, methanol, dichloromethane and hexane extracts, four 

separate aliquots of 4 g of the leaf material of each plant were 

shaken vigorously for 30 min in 40 ml of the respective solvents on 

an orbital shaker (Labotec ® , model 20.2, South Africa). The extracts 

were allowed to settle, centrifuged at 2000 x g for 10 min and the 

supernatant filtered through Whatman No. 1 filter paper into preweighed 

glass vials. The extraction process was repeated 3 times 

for each aliquot of plant material. The extracts were dried in a 

stream of cold air at room temperature and the mass extracted with 

each solvent was determined. The dried extracts were reconstituted 

in acetone to make 10 mg/ml stock extracts which were used for the 

antibacterial assays. Acetone was used for the reconstitution 

because of its efficacy in dissolving extracts with a range of 

polarities (Eloff, 1998a) and its low toxicity to microorganisms (Eloff 

et al., 2007). Twenty-eight extracts were prepared in total. 

Antibacterial assay 

A serial microplate dilution method (Eloff, 1998b) was used to 

screen the plant extracts for antibacterial activity. This method 

allows for the determination of the minimal inhibitory concentration 

(MIC) of each plant extract against each bacterial species by 

measuring the reduction of tetrazolium violet. The test organisms in 

this study included two Gram-positive bacteria, S. aureus (ATCC 

29213), and E. faecalis (ATCC 29212), and two Gram-negative 

ones, P. aeruginosa (ATCC 27853) and E. coli (ATCC 25922). 

These are some of the most common bacteria known for infecting 

wounds. The specific strains used are recommended for use in 

research (NCCLS, 1990). The bacterial cultures were incubated in 

Müller-Hinton (MH) broth overnight at 37°C and a 1% dilution of 

each culture in fresh MH broth was prepared prior to use in the 

microdilution assay. Two fold serial dilutions of plant extracts (100 

µL) were prepared in 96-well microtitre plates, and 100 µL of 

bacterial culture were added to each well. The plates were 

incubated overnight at 37°C and bacterial growth was detected by 

adding 40 µL p-iodonitrotetrazolium violet (INT) (Sigma) to each 

well. After incubation at 37°C for 1 h, INT is reduced to a red 

formazan by biologically active organisms, in this case, the dividing 

bacteria. The lowest concentration where there was a reduction of 

the colour intensity was taken to be the MIC. The MIC values were 

read at 1 h and 24 h after the addition of INT to differentiate 

between bacteriostatic and bacteriocidal activities. Acetone and the 

standard antibiotic gentamicin (Sigma) were included in each 

experiment as controls. 

Bactericidal or bacteriostatic? 

To confirm the bactericidal activity of the plant extracts the method 

described by Pankey and Sabbath (2004) was used. Only the 

Mukandiwa et al. 4381 

acetone plant extracts were used in this assay because in most 

cases in the antibacterial assay they were more effective and 

potent. Subcultures of samples from clear dilution wells from the 

MIC assay were made on MH agar plates by plating 100 µl and 

subsequently incubating for 24 h at 37°C. The test organisms in this 

assay were one Gram-negative bacterium, P. aeruginosa (ATCC 

27853) and one Gram-positive bacterium, S. aureus (ATCC 29213). 

A reduction of at least 99.9% of the colony forming units, compared 

with the culture of the initial inoculum, was regarded as evidence of 

bactericidal activity. 

Bioautography 

Bioautography was carried out to confirm the presence and 

determine number of antibacterial compounds in the plant extracts 

(Masoko and Eloff, 2005). Thin layer chromatography (TLC) plates 

(10 x 10 cm aluminium-baked, Merck, F254) were loaded with 100 

�g (10 �l of 10 mg/ml) of the extracts and dried before being eluted 

in three different solvent systems, that is, ethyl 

acetate/methanol/water (40:5.4:5): [EMW] (polar/neutral); 

chloroform/ethyl acetate/formic acid (5:4:1): [CEF] (intermediate 

polarity/acidic); benzene/ethanol/ammonia hydroxide (90:10:1): 

[BEA] (non-polar/basic) (Kotze and Eloff, 2002). The test organisms 

included, S. aureus (ATCC 29213), a Gram-positive bacteria and P. 

aeruginosa (ATCC 27853) a Gram-negative bacteria. The bacterial 

cultures, cultured for 14 h in MH broth were centrifuged at 3500 rpm 

for 5 min and the pellet re-suspended in minimal volume (20 ml) of 

MH broth. Developed plates were sprayed until damp with the 

concentrated bacterial cultures in a Bio safety Class 11 cabinet 

(Labotec, S.A) and incubated in a humidified chamber (100% 

relative humidity) overnight at 37°C. The plates were then sprayed 

with a 2 mg/ml solution of INT and incubated at 37°C for a further 

12 h. Clear zone against the purple background indicate inhibition 

of microbial growth by separated plant constituents on the TLC 

plate. 

To detect the separated compounds, a duplicate set of 

chromatograms developed in the 3 different solvent systems were 

sprayed with vanillin-sulphuric acid (0.1 g vanillin (Sigma®): 28 

methanol: 1 ml sulphuric acid) and heated at 110°C to optimal 

colour development. 

The mass of extract required to inhibit bacterial growth on an 

average size animal wound 

Whatman No 1 filter papers were cut into circles of 4 cm diameter to 

mimic an average wound size in an animal. The filter paper circles 

were weighed and then sprayed with the acetone extracts until they 

were saturated. The filter paper circles were allowed to dry and reweighed. 

The mass of extract required to cover the whole circle was 

calculated and recorded. Mean separation was done using the 

PDIFF option of SAS (2006). The volume needed to give 

determined mass values were determined using the concentration 

of the extracts which was 10 mg/ml. The quantity of extract in mg 

required to inhibit bacterial growth on wound of 4 cm diameter was 

calculated as: 

Volume of extract X required to saturate the filter paper circle in ml 

multiplied by MIC value for a particular bacterium obtained from the 

antibacterial assay for extract X in mg/ml. 

RESULTS 

Antibacterial assay 

Overall, E. coli was the least susceptible bacterium to the 

plant extracts (Table 2). We considered an MIC of 0.16


Table 2. Antibacterial activity of 7 plant species used to treat wound myiasis in Southern Africa. 

Plant species Extract Time (h) Antibacterial activity (MIC in mg ml-1 ) 

E. coli E. feacalis P. aeruginosa S. aureus 

Aloe marlothii 

Aloe zebrina 

Calpurnia aurea 

Clausena anisata 

Erythrina lysistemon 

Acetone 

Methanol 

Dichloromethane 

Hexane 

Acetone 

Methanol 


Hexane 

Acetone 

Methanol 


Hexane 

Acetone 

Methanol 


Hexane 

Acetone 

Methanol 

1 h 0.313 0.039 0.313 0.313 

24 h 0.313 0.039 0.313 0.078 

1 h 1.25 2.5 0.078 0.625 

24 h 0.625 2.5 0.313 0.313 

1 h 0.313 0.625 0.313 0.156 

24 h 0.625 0.625 0.625 0.078 

1 h 2.5 0.313 0.313 0.625 

24 h 2.5 2.5 2.5 2.5 

1 h 0.156 0.02 0.156 0.039 

24 h 0.156 0.02 0.156 0.039 

1 h 0.313 0.625 0.156 0.078 

24 h 0.156 0.625 0.313 0.156 

T1 0.156 0.078 0.156 0.078 

T2 0.156 0.156 0.313 0.039 

T1 2.5 0.156 2.5 0.313 

T2 2.5 2.5 2.5 2.5 

T1 0.625 0.156 0.156 0.156 

T2 0.625 0.156 0.156 0.156 

T1 1.25 1.25 >2.5 >2.5 

T2 1.25 1.25 0.313 

T1 0.625 1.25 0.313 >2.5 

T2 0.625 1.25 0.313 

T1 1.25 2.5 >2.5 >2.5 

T2 1.25 2.5 >2.5 

T1 0.625 0.625 0.313 0.313 

T2 0.625 0.625 0.156 0.625 

T1 0.625 1.25 0.313 0.625 

T2 0.625 1.25 0.313 0.625 

T1 0.313 0.313 0.156 0.313 

T2 0.625 0.313 0.156 0.313 

T1 0.625 2.5 0.625 >2.5 

T2 1.25 2.5 1.25 >2.5 

T1 0.313 0.156 0.078 0.313 

T2 0.313 0.156 0.078 0.313 

T1 0.625 0.625 0.313 0.313 

T2 0.625 0.625 0.156 0.313 

Dichloromethane T1 0.625 0.156 0.313 0.313

Table 2. Count’d. 

Psydrax livida 

Spirostachys africana 

Hexane 

Acetone 

Methanol 


Hexane T2 

Acetone 

Methanol 


Hexane 

Gentamycin 1.56 x 10 -3 


T2 0.625 0.625 0.156 0.313 

T1 2.5 1.25 0.625 1.25 

T2 2.5 1.25 0.625 1.25 

T1 0.313 0.078 0.313 0.156 

T2 0.313 0.313 0.156 0.078 

T1 0.313 1.25 0.313 1.25 

T2 0.313 1.25 0.625 0.625 

T1 0.156 0.156 0.313 0.156 

T2 0.313 0.156 0.313 0.078 

2.5 0.313 0.313 1.25 

2.5 0.625 0.313 1.25 

T1 0.156 0.156 0.156 0.156 

T2 0.156 0.156 0.156 0.156 

T1 0.313 0.625 0.313 0.313 

T2 0.313 0.625 1.25 0.313 

T1 0.313 0.313 0.313 0.313 

T2 0.313 0.625 0.313 0.313 

T1 0.625 2.5 0.313 1.25 

T2 0.625 2.5 0.313 2.5 

3.9 x10 -4 

1.56 x 10 -3 

7.8 x 10 -4 

Acetone >2.5 >2.5 >2.5 >2.5 

mg/ml or less to be significant antibacterial activity based 

on the guidelines in the Phytomedicine Journal 

(Instruction to Authors). Only 4 out of 28 extracts had 

MIC values equal to or less than 0.16 mg/ml against E. 

coli. Nine of the 28 extracts, 11/28 and 8/28 of the plant 

extracts had MIC values equal to or less than 0.16 mg/ml 

against E. faecalis, P. aeruginosa, and S. aureus, 

respectively. Most of the plant extracts were active 

against both Gram-negative and Gram-positive bacteria. 

In 25/28 analyses (89%) hexane extracts had relatively 

poor activity (1.25 to 2.5 mg/ml) or no antibacterial 

activity at the highest concentration tested (2.5 mg/ml). In 

total, 13 extracts (46%) had MIC ≤ 0.16 mg/ml, of which 6 

were acetone extracts, 5 were dichloromethane extracts 

and 2 were methanol extracts. The antibacterial activity of 

the plant extracts against both the Gram-positive and 

Gram-negative bacteria varied with the solvent used to 

extract the plant material (Table 2). As expected, the 

negative control, acetone, was devoid of any antibacterial 

activity. 

The MICs for each extract type, that is, methanol, 

acetone, dichloromethane and hexane, were averaged 

for the four test organisms. The average activity volumes 

indicating to what volume 1 mg of extract from different 

extractants can be diluted and it would still kill the 

bacteria were determined by dividing 1 mg by the 

average MIC for each extract type. Figure 1 shows the 

average activity volumes of the different extractants. 

Acetone is clearly the best extractant, followed by 

dichloromethane, methanol and finally hexane. These 

results confirm many observations in our laboratory that 

the most active antimicrobial compounds have an 

intermediate polarity. On average 1 mg of the acetone 

extracts can be diluted in 4.2 ml and still kill bacteria 

whilst those of hexane can only be diluted in 0.8 ml. 

To establish the plant species with the highest activity, 

the total activity of the different plant species was also 

determined. Total activity indicates the largest volume to 

which the biologically active compounds in 1 g of plant 

material can be diluted and still inhibit the growth of 

bacteria. It is calculated by dividing the quantity of 

material extracted from 1 g of dried plant material in 

milligrams by the minimal inhibitory concentration in 

mg/ml. It is useful to compare the potency of different


Figure 1. Average activity volumes indicating to what volume 1 mg of extract from 

different extractants can be diluted and it would still kill the bacteria. 

Figure 2. Total activity of the different plant species indicating the volume to which the 

biologically active compound present in 1 g of the dried plant material can be diluted 

and still kill the bacteria. 

plants and to detect synergism or loss of activity in 

bioassay guided fractionation. Figure 2 shows the total 

activity of the different plant species. Spirostachys 

africana had the highest activity followed by C. anisata, 

P. livida and E. lysistemon, respectively. Extracts of 5 of 

the study plants became less potent with time as shown 

by the reduced activity volumes after 24 h. Psydrax livida 

and C. aurea were an exception as the extracts seem to 

get more potent with time. 

Bactericidal or bacteriostatic 

Average activity (ml/mg) 

4.5 

4.0 

3.5 

3.0 

2.5 

2.0 

1.5 

1.0 

0.5 

0.0 

A. mariothii 

All the extracts were bacteriostatic at the determined 

MICs since growth was observed after plating of the 

contents of clear wells on MH agar. However they were 

bactericidal at higher concentrations. All of the tested 

plant extracts, except A. zebrina, were bactericidal 

against P. aeruginosa, at 1.25 mg/ml, with A. zebrina 

being most potent, at 0.625 mg/ml. S. aureus was most 

methanol acetone dcm hexane 

A. zebrina 

C. aurea 

Extractant 

C. anisasta 

E. lysistemon 

Plant species 

P. livida 

susceptible to the plant extracts, with all the tested plant 

extracts being bactericidal at 0.625 mg/ml. 

Bioautography 

S. africana 

TLC was used to fingerprint the plant extracts. This 

allowed for visualization of the different compounds in the 

plant extracts and identification of biologically active 

bands on the chromatograms. Bioautography, in general, 

showed more than one active band per plant extract 

(Figures 3 and 4). Although the hexane extracts had poor 

antibacterial activity in the microdilution assay 

bioautography showed that they too contained 

antibacterial compounds. 

The mass of extract required to inhibit bacterial 

growth on an average size animal wound 

Table 3 shows the mass of the acetone extracts of the 

1h 

24h


Figure 3. Chromatograms of acetone, methanol, dichloromethane and hexane leaf extracts of A. zebrina, A. marlothii, 

C. aurea eluted with BEA and sprayed with P. aeruginosa and S. aureus respectively. White areas indicate the 

presence of antibacterial compounds. 

Figure 4. Chromatograms of acetone, methanol, dichloromethane and hexane leaf extracts of C. anisata, E. 

lysistemon, P. livida eluted with BEA and sprayed with P. aeruginosa and S. aureus respectively. White areas 

indicate the presence of antibacterial compounds. 

different plant species required to inhibit bacterial growth 

on a wound of 4 cm diameter. On average the lowest 

mass of extracts is required when A. zebrina is used 

whilst the highest mass is required when A. marlothii is 

used. 

DISCUSSION 

A. A. zebrina zebrine A. A. marlothii marlothii C. aurea 

A. zebrine A. marlothii C. aurea 

Notably, all the plants in this study had antibacterial 

activity, albeit some at low and others at high minimum 

inhibitory concentrations. This observed antibacterial 

property could be one of the mechanisms through which 

the plants that are used traditionally in the treatment of 

wound myiasis work. One has to keep in mind that 

traditional healers mainly use water extracts. The active 

plant extracts had broad spectrum antibacterial activity, 

inhibiting both Gram-negative and Gram-positive 

bacteria, although the MICs were relatively higher for 

Gram-negative bacteria. It is known that, in general, the 

Gram-negative bacteria are less susceptible to 

antibacterials compared to the Gram-positive ones. This 

is due to the outer membrane composed of 

lipopolysaccharides (LPS), phospholipids, and 

lipoproteins that they possess which is absent in the 

Gram-positive bacteria. The outer membrane serves as a 

barrier for the bacterium against the destructive effects of 

various antibacterial compounds (Hodges, 2002). 

P. aeruginosa is an opportunistic pathogen and is a 

common contaminant of wounds. Its action on wounds 

has been put forward as one of the attractants of myiasis 

causing flies (Eisemann and Rice, 1987). The fact that 

the study plants had one or more extracts with activity 

against P. aeruginosa might add validity to their 

traditional use in the treatment of wound myiasis. 

The antibacterial activity of the plant extracts varied 

with the solvent used for extraction, as expected (Kotze 

and Eloff, 2002; Eloff et al., 2005). This can be explained 

in terms of the polarity of the compounds being extracted 

by each solvent and the amount of that compound, in 

addition to their intrinsic bioactivity. Notably extracts of 

the same plant had antimicrobial activity against the 

same microorganism although at varying MIC values. 

This means that the compound responsible for the 

antimicrobial activity was present in each extract, as 

shown by the bioautography, only at different 

concentrations. The acetone extracts were more effective 

and potent and this implies that acetone extracted a 

higher concentration of the antibacterial compound(s) or 

less of inactive compounds.


Table 3. Mass of the acetone extract of different plant species required to inhibit bacterial growth on a wound of 4 cm diameter. 

Plant species 

Average amount of extract 

sprayed on filter paper (mg) 

Volume of extract required to 

cover the 4 cm filter paper (ml) 

Extract required to inhibit bacterial growth 

on a wound of 4 cm diameter (mg) Average 

E. c E. f S. a P. a 

A. marlothii 4.60 ± 0.5354 c 0.460 0.144 0.018 0.144 0.144 0.112 

A. zebrina 2.75 ± 07937 b 0.275 0.043 0.006 0.043 0.011 0.026 

C. aurea 1.98 ± 0.1260 a 0.196 0.123 0.031 0.031 0.031 0.054 

C. anisata 2.35 ± 0.3416 a 0.235 0.147 0.147 0.074 0.074 0.110 

E. lysistemon 3.10 ± 0.5715 b 0.310 0.097 0.048 0.024 0.097 0.066 

P. livida 2.25 ± 0.6856 a 0.225 0.070 0.018 0.070 0.035 0.048 

S. Africana 2.70 ± 0.7528 b 0.270 0.042 0.042 0.042 0.042 0.042 

Means with same superscripts are not significantly different (P < 0.05). 

Most of the plant extracts became less potent with 

time. This can be explained if the active 

component were volatile and being lost from the 

extract with time. This is unlikely seeing that the 

hexane extract did not have the highest activity. It 

is more likely that the active antibacterial 

compounds may have been broken down or the 

bacteria were able to overcome the initial 

inhibitory effects of the antibacterial compounds 

by metabolizing it. Psydrax livida extracts were an 

exception to this trend. This could be attributed to 

some plant compounds within the extract breaking 

down with time and releasing compounds that 

have higher antibacterial activity. 

Aloe zebrina had the best antibacterial activity 

against all the bacteria and had the least quantity 

of extract required to inhibit bacterial growth on an 

averaged sized wound. However the quantity of 

extract from 1 g of plant material was relatively 

low hence its total activity was low. Generally, the 

bulk of Aloe leaves are water (Koroch et al., 

2009). The leaves of A. zebrina are relatively thin 

compared to those of other aloes such as A. 

marlothii. As a result, the leaves are easy to dry 

as a whole and this is how they were used in this 

study. To determine which plants can be used for 

further testing and isolation, not only the MIC 

value is important, but also the total activity. This 

value indicates the volume to which the 

biologically active compound present in 1 g of the 

dried plant material can be diluted and still kill the 

bacteria (Eloff, 1999). Extracts with higher values 

are considered the best to work with. Among the 

plants that are used to treat cutaneous myiasis, 

the best plants in inhibiting bacterial growth are S. 

africana, C. anisata, P. livida and E. lysistemon, 

respectively, based on total activity. 

The antibacterial activity of plants observed in 

this study concurs with previous findings by other 

researchers. The acetone extract of A. marlothii 

was reported to be active against E.coli, E. 

faecalis and S. aureus (Naidoo et al., 2006). C. 

aurea was reported to have antibacterial activity 

against both the Gram-negative bacteria (E. coli, 

Salmonella pooni, Serratia marcescens, P. 

aeruginosa, and Klebsiella pneumoniae) and the 

Gram-positive ones (Bacillus cereus, 

Staphylococcus epidermidis, S. aureus, 

Micrococcus kristinae, and S. pyogenes) 

(Adedapo et al., 2008). Two carbazole alkaloids, 

clausenol and clausenine, isolated from C. anisata 

are active against both Gram-positive and Gram- 

negative bacteria with MIC values ranging 

between 1.3 µg ml -1 and 40 µg ml -1 (Chakraborty 

et al., 1995). The volatile oil from the leaves of C. 

anisata also has significant activity against a 

number of bacteria and fungi (Gundidza et al., 

1994). Phytochemically, E. lysistemon is rich in 

flavonoids and alkaloids and over 30 compounds 

have been isolated from this plant. Three of the 

isolated compounds have weak activity against 

the Gram-negative bacteria (E. coli) and moderate 

activity against Gram-positive bacteria (B. subtilis 

and S. aureus) (Juma and Majinda, 2005). 

According to Pillay et al. (2001) the bark of E. 

lysistemon is far more active than the leaves, 

yielding activity with water, ethanol and ethyl 

acetate extracts against S. aureus, Micrococcus 

luteus and Bacillus subtlis. The main anti-bacterial 

compound in the E. Iysistemon bark was isolated 

and was identified as wighteone. Crude extracts 

from the bark of S. africana have antibacterial 

activity against diarrhoea-causative 

microorganisms (Salmonella typhi, Shigella 

sonnei, Shigella dysentery, Shigella flexneri, 

Shigella boydii and E. coli.) with MIC values 

ranging between 0.156 and 0.625 mg/ml 

(Mathabe et al., 2006). Phytochemically, the

Euphorbiaceae family to which S. africana belongs is rich 

in alkaloids and terpenoids (Webster, 1986). The 

inhibitory activity of terpenoids on bacteria has been 

reported (Drewes et al., 2005). One triterpene compound 

and two diterpenes compounds were isolated from S. 

africana (Mathabe et al., 2008) and are active against 

some of the diarrhoea-causative microorganisms with 

MIC values ranging between 50 and 200 µg ml -1 . 

In some cases the observed results differed from 

previous findings by other researchers. For example, in 

this study the hexane extract of A. marlothii had some 

antibacterial activity contrary to McGaw et al. (2000) who 

reported that crude hexanic, ethanolic and aqueous 

extracts of A. marlothii does not have antibacterial 

activity. The possible reason for this difference in results 

could be the difference in plant chemical composition due 

to different times of plant collection and geographical 

differences. Unfortunately the TLC fingerprint of the plant 

from the previous research was not available for us to 

compare with the results from our study to confirm this 

postulation. 

The antibacterial activities of extracts of A. zebrina and 

P. livida are being reported for the first time in this paper. 

Although the antibacterial activity of the other five study 

plants against some of the microorganisms have been 

reported against some of the test organisms in this study, 

in most of the studies the agar diffusion assay methods 

were used in determining the antimicrobial activity and 

high minimal inhibitory concentrations of up to 5 mg/ml 

were reported. In this study the serial microplate dilution 

method (Eloff, 1998b) was used. This method allows for 

the determination of the MICs of each plant extract 

against each bacterial species by measuring the 

reduction of tetrazolium violet. It is more sensitive and we 

were able to show that some of the plant species had 

antibacterial activity at much lower concentrations than 

previously determined. For example C. aurea was 

reported to have a MIC of 5 mg/ml against E. coli, P. 

aeruginosa, S. aureus (Adedapo et al., 2008) however, in 

this study we showed that it could still exhibit antibacterial 

activity against P. aeruginosa, S. aureus at 0.156 mg/ml 

and E. coli at 0.625 mg/ml. In addition, although the 

antibacterial activity of some of the plant species such as 

S. africana have been previously reported against some 

of the test organisms in this study, this is a first report of 

their antibacterial activity against P. aeruginosa, an 

important bacteria in the pathogenesis of wound myiasis. 

In some cases, the findings of this study add to the 

information on the antibacterial activity of some plant 

species. Pillay et al. (2000) reports that the ethyl acetate, 

ethanol and water extracts of E. lysistemon are 

ineffective against E. coli and P. aeruginosa however our 

results show that extracts from other extractants such as 

acetone, methanol and dichloromethane have reasonable 

to good antibacterial activity against these bacteria, with 

MICs ranging from 0.08 to 0.625 mg/ml. 

All the plant extracts in this study were bacteriostatic at 


the determined MICs and bactericidal at higher 

concentrations. This is in line with the known fact that the 

MIC is simply the concentration of the drug that inhibits 

the growth of bacteria and inhibition of bacterial growth 

does not necessarily mean that the bacteria have been 

killed (Finberg et al., 2004). The bactericidal activity of an 

antimicrobial agent against a particular organism tends to 

be related to its mechanism of action. In general, agents 

that disrupt the cell wall or cell membrane, or interfere 

with essential bacterial enzymes, are likely to be 

bactericidal, whereas those agents that inhibit ribosome 

function and protein synthesis tend to be bacteriostatic. 

The tangible benefit of the extracts to be bactericidal 

comes in its use in the management of topical infection. 

While the concentration required to kill the tested microorganisms 

is high at 0.6 and 1.25 mg/ml their ability to 

reach this concentration at the wound site in combination 

with the poor immune response associated with topical 

wounds make them beneficial in the clinical management 

of wounds. If a compound or extract only has 

bacteriostatic activity it does not mean that it will be 

ineffective as it may allow the natural defence system of 

the organism to take control. Many commercial antibiotics 

have bacteriostatic activity. At higher concentrations they 

may kill the bacteria. The advantage of controlling topical 

infections is that much higher concentrations can be 

used. 

Generally small quantities, ranging from 0.006 to 0.147 

mg, of acetone extracts are required to inhibit bacterial 

growth on an average sized wound. This may have to be 

mixed with a grease to apply to the animals. Traditionally 

the leaves of the plants are crushed and packed onto a 

wound. 

Conclusion 

The bacteria used in this study are known pathogens of 

wounds and their inhibition by the plant extracts in this 

study might validate the traditional use of plants in the 

treatment of wound myiasis. It has been shown that 

bacterial action on wounds produce ammonia and volatile 

organic, sulphur containing compounds which have an 

odour that serve as an attractant of myiasis-causing flies. 

Therefore, inhibiting bacterial activity reduces the 

attractants of myiasis-causing flies to the wound and the 

stimuli for oviposition. Thus inhibiting bacteria action on 

wounds will interfere with the development of wound 

myiasis. This could be one of the mechanism through 

which the plants that are used traditionally in the 

treatment of wound myiasis work. The next step to be 

addressed is to determine effect of these extracts on 

larval survival and subsequent development into adult 

stages. 

ACKNOWLEDGEMENTS 

The University of Pretoria and the National Research


Foundation provided the financial support for this 

research. The South African National Biodiversity 

Institute, allowed the collection plant material from the 

Pretoria National Botanical Garden. Dr. P. Masika of Fort 

Hare University gave some valuable input in the initial 

preparation of this manuscript. L. Mukandiwa gratefully 

acknowledges the financial support from German 

Academic Exchange Service, DAAD, during the period of 

this study. 

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DOI: 10.5897/JMPR11.1715 



Comparative study on different methods for 

Lonicera japonica Thunb. micropropagation and 

acclimatization 

Jiang Xiang Hui 1,2 , She Chao Wen 2 , Zhu Yong Hua 1 and Liu Xuan Ming 1 * 

1 Bioenergy and Biomaterial Research Center, College of Biology; State Key Laboratory of Chemo/Biosensing and 

Chemometrics, Hunan University, Changsha 410082, Hunan, China. 

2 Department of Life Science, Huaihua University, Huaihua, Hunan 418008, China. 

Accepted 1 February, 2012 

In this study, we reported the establishment of a simple protocol for the micropropagation and 

acclimatization of Lonicera japonica Thunb. Branches with dormant buds were collected from mature L. 

japonica and sprouted in a greenhouse. Tip and node segments were used as starting material for in 

vitro proliferation in woody plant medium (WPM). In the first assay in which explants from five different 

species of Lonicera were used, 95.0% of the tip segments produced new axillary shoots, thus proving to 

be the best explant type. Afterwards, material from L. japonica Thunb. was used to test for plant growth 

regulator (PGR) combination. Shoots from L. japonica were used to assay in vitro rooting using six 

different WPM media. Rooting percentages were high for all media and varied between 83 and 95%. For 

acclimatization, two approaches were assayed: the use of previously rooted in vitro plants and the 

direct acclimatization of shoots. After five weeks, 95.0% of the in vitro rooted plants were successfully 

acclimatized and 88.7%, was attained by direct acclimatization of shoots with two years shoot immersed 

in GA3 solution. These results proved that there is no need for a previous in vitro rooting step and that 

direct acclimatization can effectively reduce time and costs. Thus, the micropropagation and 

acclimatization of L. japonica can be divided into only two steps: proliferation of shoots in WPM and 

direct acclimatization of these shoots in a sterile soil mixture, or direct acclimatization of two years 

shoot immersed in GA3 solution for 8 h. 

Key words: Lonicera japonica Thunb., node segment culture, micropropagation, direct acclimatization. 

INTRODUCTION 

Lonicera japonica Thunb. is a genus of woody plants 

(family Caprifoliaceae) that grows extensively in Europe, 

Asia and North America. L. japonica Thunb. is recorded 

in China Pharmacopeia (2005) with the Chinese name 

Jinyinhua. The aqueous extract from L. japonica Thunb. 

flower has been used in Chinese traditional medicine for 

treating fever, arthritis and infectious diseases for 

*Corresponding author. E-mail: xmll05@126.com. 

Abbreviations: ANOVA, Analysis of variance; BA, N6benzylaminopurine; 

IAA, indole-3-acetic acid; IBA, butyric acid; 

NAA, α-naphthaleneacetic acid; WPM, woody plant medium; 

PGR, plant growth regulator. 

thousands of years. This plant has been shown to display 

a wide spectrum of biological and pharmacological 

activities such as antibacterial, antiviral (Houghton et al., 

1993), antioxidant and inhibition of the platelet activating 

factor (Kim et al., 1994). L. japonica could act as an antiinflammatory 

agent through regulation of NF-кB activation 

(Lee et al., 2001). Rutin is one of the key compounds of 

L. japonica; rutin has been shown to provide protection 

against ischemia and reperfusion (I/R) in a variety of 

experimental models and via multiple mechanisms 

(Lanteri et al., 2007; Lao et al., 2005). L. japonica 

contains anti-complementary polysaccharides and polyphenolic 

compound. The polyphenolic compounds inhibit 

the platelet aggregation, thromboxane biosynthesis and 

hydrogen peroxide induced endothelial injury (Chang


and Hsu, 1992). This species is rich in iridoid secologanin 

(Son et al., 1994) and is a potentially useful model for the 

study of secologanin biosynthesis. Secologanin is a 

primary terpenoid intermediate in the biosynthesis of 

monoterpenoid indole alkaloids such as reserpine, 

ajmaline, ajmalicine and vinbiastine (Zenk, 1980). 

L. japonica Thunb. seeds have the problem of low 

germination rate, low orderliness and long seedling time. 

The rapidness of tissue culture techniques can be 

advantageous for the continuous provision of a plantlet 

stock for cultivation and may further compliment breeding 

programmes. The strategies for L. japonica production 

involved somatic embryogenesis and direct acclimatization 

of shoots. Georges et al. (1993) proved that shoot 

regeneration from true-callus (without any part of the 

original explant) was achieved for the three different 

source tissues within 12 weeks, which were morphologically 

and genetically similar to the mother plant. 

Palacios et al. (2002) studied the regeneration of 

Lonicera tatarica plants from cultured stem sections, 

which showed that the age of the donor plant had no 

noticeable effect on either process; however, rooting of 

elongated shoots occurred only with shoots derived from 

2-month-old donor plants, the plants regenerated from 

stem explants were morphologically normal and levels of 

loganin and secologanin were comparable to those 

detected in plants grown from seed. Cambecedes et al. 

(1991) investigated the effect of growth regulators on bud 

regeneration from leaf explants of the shrubby 

ornamental honeysuckle genotype Lonicera nitida, which 

showed that caulogenesis cannot be managed only by 

the classical modification of the exogenous cytokinin/ 

auxin balance, and the right hormonal balance to achieve 

such a goal must also involved a control of the 

endogenous auxin level of explants. 

In view of the demand of amplification culture and the 

drastic reduction in the number of excellent genus, 

micropropagation offers many advantages because it 

potentially can facilitate large-scale production of valuable 

genus and allow plant regeneration from genetically 

modified cultures. In addition, micropropagation may be 

an essential step to obtain plants from frozen collections 

of L. japonica Thunb. Unfortunately, despite the considerable 

number of micropropagation reports aforementioned, 

most of the protocols are poorly described, in 

particular concerning the difficult stage of acclimatization. 

Therefore, our goal was to try to develop a reliable and 

comprehensive protocol for L. japonica Thunb. 

micropropagation and acclimatization. 


In previous experiments, in vitro culture of L. japonica Thunb. 

shoots collected in the field during spring resulted in a contamination 

rate of 85 to 90%. Collection of branches with dormant 

buds reduced culture contamination to 0 to 10% and was thereafter 

the chosen method to obtain starting material for cultures. 

Branches with dormant buds were collected from five different 

species of Lonicera in January to March, such as L. japonica 

Thunb., L. confusa (Sweet) DC., L. dasystyla Rehd., Lonicera 

hypoglauca Miq. and Lonicera macranthoides Hand. -Mazz., 

located at Huaihua University medicinal garden. 

The first assay of shoot proliferation was performed with material 

from these five different species of Lonicera to determine which 

explant behaved better in culture. Sprouts formed from the buds in 

1 week were disinfected in ethanol 75% (v/v) (30 s) and then in a 

commercial bleach solution (Neoblanc, chlorine concentration 5% 

(v/v)) 20% (v/v) with Teepol 0.01% (v/v) (15 min). Sprouts were then 

rinsed in sterile water three times and sectioned in 2 - 3 cm 

segments that contained the shoot apex and 1 - 2 nodes. Shoot 

proliferation was tested in woody plant medium (Huetteman and 

Preece, 1993). Data were recorded 5 weeks after in vitro 

introduction. The in vitro rooting assay was performed using 3 - 4 

cm long shoots obtained from 18 month old L. japonica on WPM 

medium. Eight variant formulations of WPM culture medium were 

tested (Table 2). Shoots were cultivated in WPM basal medium 

without growth regulators for 4 weeks (control conditions), this was 

followed by transference to WPM medium with different 

combination of auxin during 4 weeks. One shoot was placed per 

culture tube and cultures were grown under Osram cool white 

fluorescent lamps providing a light intensity of 250 µmol m -2 s -1 with 

a 16 h daylight period at 24°C. After five weeks, the number of 

shoots that regenerated roots, the number of regenerated roots per 

shoot and the size and morphology of root were recorded. 

In order to establish a system which could be utilized for 

continuous microplant production and subculturing, combinations of 

NAA or IAA and BA were tested for their ability to multiply in vitro of 

L. japonica. After 90 days, the calli was transferred to regeneration 

media (NAA and BA combinations, IAA and BA combinations) 

(Table 3). During this study all media were autoclaved at 121°C and 

101 kPa for 20 min after adjustment of the pH to 5.8 with 1 M 

NaOH. After cooling, 25 ml of the medium was poured into 50 ml 

conical flask. The calli were transferred to a 26 - 28°C growth room 

with 16 h light illumination (250 μmol m -2 s -1 photosynthetic photon 

density) and 8 h dark. The light was provided by „cool-white‟ 

fluorescent tubes (40W/220V×6). Observations were recorded on 

mean number of shoot per explant, rooting percent and survival 

percent after 12 weeks. 

The fourth assay was completed by inducing plants direct rooting 

in soil with growth regulator; there 15 cm long shoots from different 

stems were used for direct acclimatization in soil mixture. Given 

their characteristics, shoots were divided in three groups: one year 

shoot, two years shoot and three years shoot. The shoot apex and 

the basal leaves were cut off and the shoots of each group had their 

basal end immersed in different solution during 8h (Table 3). 

Results were recorded after five weeks. The acclimatization assay 

was performed in this work. After rooting, single plant was transferred 

to 500 ml pots, with an autoclaved soil mixture of soil: plant 

ash: peat (10:2:3; wt.) which were placed in a glass chamber 

located in the greenhouse. Plants in pots were fogged with a 1 gl -1 

Benlate solution. In the first week, plants were also fogged with 

sterile water to avoid leaf fade. In the greenhouse, incandescent 

light gave a light intensity of 500 µmol m -2 s -1 with a 16 h daylight 

period. Temperature was 26 ± 2°C and relative humidity about 85%. 

The results were recorded after five weeks. 

Statistical analysis 

All the percentage values were arcsine transformed and counts of 

shoots and roots were subjected to square root transformation. 

Results were statistically tested using a two-sided t-test after 

analysis of variance (ANOVA) performed by Fisher‟s test (Dytham, 

2011). Statistical significance was assumed at P≤0.05. Results were 

processed using the program Microsoft Excel 2003.

Table 1. Number of axillary shoots produced from tip segments of five different species of Lonicera. 

Species Shoots per explant in WPM medium 

Lonicera japonica Thunb. 3.4 ± 0.55 a 

Lonicera dasystyla Rehd. 3.26 ± 0.57 ab 

Lonicera confusa (Sweet) DC. 2.58 ± 0.32 b 

Lonicera hypoglauca Miq. 2.04 ± 0.21 b 

Lonicera macranthoides Hand. -Mazz. 1.65 ± 0.64 c 

Values are the average mean ± standard error of two experiments registered at 5 weeks. Values followed by a 

similar letter are not significantly different(P >0.05). 

Hui et al. 4391 

Table 2. Rooting of Lonicera japonica Thunb. from dormant buds on different WPM medium with different concentrations of IBA 

and IAA for five weeks. 

Combination of auxin 

(µM) 

Percentage of root 

production (%) 

Mean number of roots per 

plantlet 

Root length (cm) 

2.0 IBA + 0.0 IAA 87 3 ± 1.58 2.22 ± 1.26 c 

4.0 IBA + 0.0 IAA 83 3 ± 1.55 2.75 ± 1.44 c 

0.0 IBA + 2.5 IAA 87 3 ± 2.68 3.01 ± 1.52 b 

0.0 IBA + 5.5 IAA 88 2 ± 1.22 2.82 ± 1.32 c 

2.0 IBA + 2.5 IAA 95 4 ± 1.38 2.55 ± 0.47 c 

3.0 IBA + 5.5 IAA 89 3 ± 2.62 3.61 ± 1.56 a 

2.0 IBA + 5.5 IAA 91 3 ± 2.02 3.42 ± 0.87 a 

3.0 IBA + 2.5 IAA 93 3 ± 2.38 2.49 ± 1.04 c 

Values are the standard error and treatments denoted by the same letter in a column were not different (P ≤ 0.05) using the LSD test. Ten 

replicates were used per treatment and experiments were repeated three times. 

Table 3. Results of differentiation and rooting of Lonicera japonica Thunb. from calli using different media with the basal 

composition of WPM. 

Combination of NAA, IAA and BA (µM) Shoots per explant Rooting percent Survival percent 

9.0 NAA +2.0 BA 5.50 ± 0.56 a 84 ± 4.52 a 75 ± 5.26 a 

6.0 NAA +4.5 BA 5.32 ± 0.66 a 85 ± 5.45 a 73 ± 6.53 a 

2.0 NAA +8.5 BA 5.24 ± 0.52 ab 82 ± 7.20 a 74 ± 6.40 a 

9.0 IAA +2.0 BA 5.00 ± 0.65 b 82 ± 5.35 a 71 ± 7.33 a 

6.0 IAA +4.5 BA 2.45 ± 0.36 c 79 ± 3.71 a 65 ± 5.35 b 

2.0 IAA +8.5 BA 3.15 ± 0.26 c 77 ± 4.43 a 65 ± 3.64 b 

Values are the average mean ± standard error of experiments registered at 12 weeks. Values followed by a similar letter are not 

significantly different (P >0.05). 

RESULTS 

From the assay carried out to test the genotype influence 

in culture, the genus L. japonica was the one that showed 

the best in vitro shoot production (Table 1); 3.4± 0.55 

shoots were produced per explant, the difference being 

statistically significant (P ≤ 0.05). Therefore, L. japonica 

Thunb. was chosen for subsequent experiments of in 

vitro rooting and acclimatization. Shoots formed roots in 

all in vitro tested media. There were no significant 

differences between the number of roots per shoot 

produced in media with IBA and IAA (Table 2), however, a 

significantly higher number of roots per shoot (4.00 ± 

1.38) were induced in WPM medium with 2.0 μM IBA and 

2.5 μM IAA. WPM medium with 5.5 μM IAA promoted the 

lowest number among all media. Morphologically, roots 

were very variable, roots produced in media with 2.0 μM 

IBA and 2.5 μM IAA were thicker and shorter (2.0 - 3.0 

cm) than roots formed in media with 3.0 μM IBA and 5.5 

μM IAA (3.0 - 5.0 cm). The mean number of roots per 

plantlet was 2 to 4; there were no significant differences 

in all media. For the plants of L. japonica Thunb. rooted


Table 4. Percentage of Lonicera japonica Thunb. shoots directly acclimatized in soil mixture after immersion their basal end 

in different solutions. 

Type of shoot Water(control) IBA (10 mgL -1 ) IBA (10 mgL -1 ) + IAA 

(10 mgL -1 ) 

GA3 (10 mgL -1 ) + IBA 

(10 mgL -1 ) 

One year shoot 58.2 ± 6.55 bb 64.4 ± 7.44 bb 66.5 ± 6.47 bb 72.7 ± 7.33 ba 

Two years shoot 80.5 ± 8.42 ab 83.4 ± 5.63 ab 84.2 ± 7.55 ab 88.7 ± 5.84 aa 

Three years shoot 78.5 ± 8.42 ab 80.4 ± 5.63 ab 83.5 ± 7.55 ab 83.7 ± 5.84 aa 

Values are the average mean± standard error of experiments registered at 5 weeks. Values followed by a different letter, in the same 

column, are significantly different (P ≤ 0.05). In the same row, the second same letters indicate values not significantly different (P > 

0.05), the second different letters indicate values significantly different (P ≤ 0.05). 

in vitro that were transferred to soil mixture, 85.7% were 

totally established after 4 weeks. No morphological 

differences were found between these plants and the 

mother plant. Moreover, all media tested for microplant 

production and in vitro rooting provided obvious results. 

When 9.0 µM NAA was combined with BA at 2.0 µM, 

shoot induction differed from the other combinations 

tested with an average of 5.50 shoots being produced 

(Table 3), but their effect on rooting was statistically 

similar to the other IAA and BA combinations. This may 

imply that for shoot regeneration, the ratio of NAA or IAA 

to BA is important for successful plantlet regeneration. 

However, the rooting does not appear to be dependent 

on the auxin: cytokinin ratio. There were significant 

differences with respect to the survival percent, but the 

result was in accord with shoot multiplication. Furthermore, 

we compared the rooting percentages of L. 

japonica Thunb. shoots of different ages. There were 

significant differences in different types of shoot (Table 4); 

the two years old shoot rooted better than one year 

shoot, using two years old shoots the rooting percentage 

was above 80%, but it was statistically similar to the three 

years shoot. The rooting percent of shoot was only 

slightly increased with auxin treatments than immersion 

of their basal end in water, but there were significant 

differences when immersing them in GA3 solution. This 

suggested that the addition of auxins is beneficial to 

rooting and the using of GA3 is essential. This may imply 

that for shoot regeneration, the ratio of NAA or IAA to BA 

is important for successful plantlet regeneration. 

However, the rooting does not appear to be dependent 

on the auxin: cytokinin ratio. 

DISCUSSION 

Direct acclimatization of shoots (Table 4) resulted in a 

high establishment percentage particularly for shoots with 

two years shoot, above 80%. This was the case for three 

years shoot as well as two years shoot. However, taking 

three years shoot could damage the mother plant badly. 

In each kind of shoots, there were no significant 

differences between shoots treated with auxin and water 

(control), and GA3 showed an important influence on 

direct rooting. Morphologically, there were no differences 

between the plants produced and the mother plant. After 

three months in the greenhouse, plants from direct 

rooting and in vitro rooting were transferred to the field 

and all the plants survived (Figure 1H). Morphologically, 

there were no differences between the plants produced 

and the mother plant. 

Most reports on L. japonica micropropagation do not 

refer to the acclimatization process or they only mention 

that the acclimatization was tested with success. In our 

study, acclimatization was given particular attention, and 

two approaches were assayed: in vitro rooting prior to 

transfer to soil mixture (Figure 1B and D) and direct 

transfer of shoots to soil mixture (Figure 1E). Our results 

led us to the conclusion that formation of in vitro roots 

prior to acclimatization is not needed. This result may be 

due to mechanical damage of roots during transfer of 

plants to soil (Figure 1F). Furthermore, Bonga and Von 

Aderkas (1992) mention that for many hard woods, roots 

formed in vitro had enlarged cortical cells and underdeveloped 

vascular systems, and thus were of poorer 

quality than roots formed ex vitro. The same conclusion 

was drawn by Cheng and Shi (1995) and Paula et al. 

(2008) who reported that the in vitro rooting stage could 

waste time and cost. 

In this study, we found that shoots with thin stems 

tolerated the stress of acclimatization better than in vitro 

rooted plants. Direct acclimatization is easy and inexpensive 

and allows an efficient and fast establishment 

of plants in soil. A group of plants was planted in the field 

in Hunan, Western China and they all survived (Figure 

1H); they were similar to their progenitors, in relation to 

size and morphology of the leaves and thickness and 

elongation of the stem. 


The authors wish to thank the Key Project of Hunan 

Technological Department (no. 2009FJ2008) and the 

Educational Commission of Hunan Province of China 

(10K049) for their support.

REFERENCES 

Hui et al. 4393 

Figure 1. Comparative of different methods of micropropagation and acclimatization for Lonicera japonica Thunb. (A) 

Formation of sprouts from dormant buds collected in the field from adult plant. (B) Differentiation of callus by medium with 

3.0 μM IAA and 2.0 μM BA. (C) Shoot organogenesis induced by WPM medium with 9.0 μM NAA and 2.0 μM BA. (D) 

Rooting situation of multiplication seedling in WPM medium with 9.0 μM NAA and 2.0 μM BA after 12 weeks. (E) Type of 

shoots used in direct Acclimatization in soil mixture and situation of rooting. (F) The rooting situation of shoots used in 

direct Acclimatization by immersed in GA3 solution. (G) Acclimatized plants observed 6 weeks after their transfer from in 

vitro rooting medium to soil mixture. (H) Lonicera japonica Thunb. photographed 1 year after being planted in the field. 

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DOI: 10.5897/JMPR12.098 



Effect of water stress on essential oil yield and storage 

capability of Matricaria chamomilla L. 

Alireza Pirzad 

Department of Agronomy and Plant Breeding, Faculty of Agriculture, Urmia University, West Azarbayjan, 

Postal Code: 5715944931, Urmia, Iran. 

Department of Medicinal and Industrial Plants, Institute of Biotechnology, Urmia University, West Azarbayjan, Urmia, 

Iran. E-mail: a.pirzad@urmia.ac.ir. Tel: +98(441)2972399. Fax: +98(441)2779558. 

Accepted 15 March, 2012 

To evaluate the effect of water stress on yield and storage capability (essential oil percentage, yield and 

harvest index during five years storage period) of Matricaria chamomilla L., a field experiment was 

conducted in 2005. Water stress treatments of irrigation after 30, 60, 90 and 120 mm evaporation from 

pan class A, were arranged as randomized complete block design with six replications. The harvested 

material was stored for five years and essential oil was extracted yearly. Results of the first year of 

experiment indicated that the highest (1347 kg/ha and 10084 g/ha) and lowest (952.2 kg/ha and 6672 

g/ha) yields of dried flower and essential oil were obtained from irrigation after 60 and 120 mm 

evaporation from pan, respectively. Results from stored plant material (5-year data of storage) showed 

a significant effect during storage on essential oil percentage, yield of essential oil, harvest index of 

essential oil, reduction of essential oil percentage and yield. Means comparisons from the 5-year data 

indicated that the highest percentage and yield of essential oil (0.715% and 8442 g/ha) was observed at 

first year. After that, there was a 27% reduction trend in essential oil percentage to the 5-year storage 

result. So, the lowest essential oil content and yield (0.194% and 2335 g/ha) was obtained from flowers 

at the fifth year of storage. 

Key words: Chamomilla recutita, dried flower yield, excess water, storage quality, water deficit. 

INTRODUCTION 

According to WHO, 80% of the world’s population is 

currently dependent on plants for health care (Akerele et 

al., 2009). Plants provide a rich source of secondary 

metabolites that have medicinal and aromatic properties 

(Gomez-Galera et al., 2007). The world market for herbal 

medicine, including herb based products and raw 

materials, is growing at an annual rate of 5 to 15%. This 

indicates the likelihood of growing demand for plantderived 

drugs in the coming years (Kumar and Gupta, 

2008). Over harvesting and shrinkage of plant habitats 

from increasing urbanization have caused a significant 

decline in the volume of raw materials produced (Franke 

and Schilcher, 2005). Chamomile (Matricaria chamomilla 

L.), Family: Asteraceae, commonly known as German 

chamomile is a native of Europe and is cultivated 

extensively in Hungary, Germany, Russia and Yugoslavia 

(Singh, 1982). 

M. chamomilla is an important medicinal plant that has 

multi-therapeutic, cosmetic, and nutritional values; 

established through years of traditional and scientific 

application (Schilcher, 1987). German chamomile essential 

oil obtained from flower heads, either by steam 

distillation or by solvent extraction, varies from 0.24 to 

1.9% in fresh plant tissue. Distillation using the 4-h 

European pharmacopeia method typically yields 0.33 

ml/100 g of tissue, whereas distillation, using the 2-h 

German Pharmacopeia method (DAB7) typically yields 

0.29 ml/100 g of tissue (Mechler and Ruckdeschel, 

1980). About 120 chemical constituents have been 

identified in chamomile as secondary metabolites, 

including 28 terpenoids, 36 flavonoids, and 52 additional 

compounds with potential pharmacological activity. 

Though (–)-α-bisabolol and chamazulene have proven to 

have the most bioactive inflammatory activity, the

en-indicycloethers have an antispasmolytic effect 

(Salamon, 1992). 

The highest essential oil and prochamazulene 

concentrations are obtained after drying plant material in 

shade at 22 to 25°C (Schmidt et al., 1991). Slight losses 

(up to 7%) of prochamazulene, with no reduction in plant 

essential oil content, occur with drying in a stationary bulk 

dryer with active ventilation at 40 to 45°C. In another 

study, the greatest loss of active compounds in the 

essential oil generally resulted from storage of plant 

material at −6 to 25°C and 55 to 95% humidity 

(Walenciak and Korzeniowski, 1983). It seems that the 

essential oil content and constituents of essential oil had 

been changed by storage time. In this regard, climate 

factors may affect on quality and quantity of essential oil 

of medicinal plants. However, these factors can affect the 

maintenance of oil content of stored herbal raw material 

tissues in during storage period (Franke and Schilcher, 

2005). 

Water, as an important effective factor on plant growth 

and secondary metabolite production, increases levels of 

all of the compounds analyzed, particularly some of the 

phenolic compounds (Rostami-Ahmadvandi et al., 2011; 

Kahrizi et al., 2012). In flooded soil, the microorganisms 

reacted vigorously to a deficiency of lifesaving oxygen, 

but potentially inhibitory concentrations of carbon dioxide, 

hydrogen sulfide, methane, ethylene, manganese, iron, 

sulfate, and many organic substances may accumulate 

abnormally in large concentrations and lead to plant 

damage as a result of a reduction in oxygen (Rowe and 

Beardsell, 1973). Large quantities of medicinal plants are 

wasted during operations such as logging, chipping 

during semi-processing, powdering of leaves and tender 

stems during drying, there can be a loss of small particles 

during bundling and transportation and a certain 

percentage during washing. Collection and semiprocessing 

at an inappropriate season might also lead to 

considerable waste. Documentation of the ideal season 

for harvest and storage is necessary to protect the active 

plant properties and to preserve their optimum 

therapeutic value (Akerele et al., 2009; Franke and 

Schilcher, 2005). 

Due to defective storage, the raw drugs are often prone 

to attack by insects, fungi and bacteria. There is visible 

remarkable deterioration during storage (Krishnamurthy, 

1993; Tajuddin et al., 1996). But there were few studies 

on reduction of essential oil quality and quantity in long 

time storage period in health condition. And there were 

no report on the effect of varying water availability 

(irrigation regimes) on the maintenance potential of 

chamomile essential oil percentage during long storage 

period. Based on current knowledge, information on the 

response of M. chamomilla, quantity and quality of herbal 

raw material tissue, to different irrigation regimes and 

storage periods is scarce. Therefore, the main objective 

of this study was to evaluate the effects of different 

irrigation regimes on yield of essential oil, and however 

Pirzad 4395 

on storage capability of dried flower (changes in 

percentage, yield and harvest index of essential oil) 

during long term (5 year) storage. 


This research was conducted at the experimental field of the 

Department of Agronomy and Plant Breeding, Faculty of 

Agriculture, Urmia University (latitude 37.53° N, 45.08° E, and 1320 

m above sea level), Urmia, Iran in 2005. The soil texture of the site 

was clay-loam (28% silt, 33% clay, 40% sand) with 22.5% field 

capacity, 1.54 g cm -3 soil density, 1.98% organic mater, pH 7.6. 

The research consisted of two experiments; firstly, a field 

experiment was conducted and secondly, tests on the harvested 

material after 1 to 5 years storage. In the first experiment, the 

treatments were irrigation after 30, 60, 90 and 120 mm evaporation 

from pan class A with six replications. M. chamomilla L. c.v. 

Bodegold, a tetraploide variety seeds were planted on 1st May 

2005. Plant growth was continuously monitored for the duration of 

the experiment by the mechanical control of weeds. The harvested 

crop consisted of freshly gathered typical flower heads, with 

approximately 10% of flowers containing fragments of small flower 

stalks, which were up to 30 mm long. Flower heads were picked 

when they were fully developed and dried in a shady place. Flowers 

were hand harvested at the medium stage of development and 

dried at 25°C for 72 h (Bottcher et al., 2001). 

In the second experiment, harvested dried flowers were stored at 

25°C for 5 years. The air-dried parts of chamomile (15 g of the dry 

sample) were hydro-distilled in a Clevenger-type apparatus in 1000 

ml round bottomed flask with 600 ml deionized water for 4 h 

(Salamon, 1992). The essential oil was extracted each year for with 

5 years. 


Statistical evaluation was performed using SAS software package, 

version 9.1 (SAS Institute, 2004). The effects of the irrigation 

regimes were analyzed with an analysis of variance test. The 

results of statistical analysis are expressed by F-values; asterisks 

indicate p-values: p* ≤ 0.05 and p** ≤ 0.01. The comparison of 

means was carried out with Student-Neuman Keul's test. 

RESULTS 

Field experiment (first year) 

Results of data analysis of variance, from the field 

experiment showed significant effect of irrigation on dried 

flower yield (P ≤ 0.05), biological yield (P ≤ 0.01) and 

essential oil yield (P ≤ 0.01) (Table 1). 

Means comparison indicated that the highest yield of 

dried flower (1347 kg/ha) was obtained from irrigation 

after 60 mm evaporation from pan class A. This greatest 

yield had no significant difference with yield of flower 

obtained from irrigation after 30 and 90 mm evaporation. 

But, the lowest yield of dried flower (952.2 kg/ha) was 

obtained from plants irrigated on 120 mm evaporation 

from pan. Regression function of dried flower yield along 

with irrigation was a binomial function with R 2 = 0.9596 

(Figure 1A).


Table 1. Analysis of variance of yield and harvest index of Matricaria recutita L. affected by irrigation. 

Source of 

variation 

df 

Dried flower 

yield 

Biological 

yield 

Harvest index 

of dried flower 

Essential oil 

percentage 

Essential 

oil yield 

Harvest index 

of essential oil 

Replication 5 47141.9 ns 123626.0 ns 0.005 ns 0.002 ns 0.008 ns 0.007 ns 

Irrigation 3 172697.3* 2618468.3** 0.020 ns 0.006 ns 0.035** 0.029 ns 

Error 15 43266.7 394476 0.014 0.005 0.006 0.014 

Coefficient of 

Variation (%) 

17.65 21.78 7.21 9.67 1.98 4.80 

df, degree of freedom; ns, * and ** ; non-significant, significant at P ≤ 5% and P ≤ 1%. 

Dried Flower Yield (kg/ha) 

Biological Yield (kg/ha) 

Essential Oil Yield (g/ha) 

1800 

1600 

1400 

1200 

1000 

800 

600 

400 

200 

0 

4500 

4000 

3500 

3000 

2500 

2000 

1500 

1000 

500 

0 

14000 

12000 

10000 

8000 

6000 

4000 

2000 

0 

A 

y = -73.45x 2 + 256.51x + 1088.2 

R 2 = 0.9596 

ab a ab b 

30 mm 60 mm 90 mm 120 mm 

B 

Irrigation after evaporation from pan class A 

y = -184.75x 2 + 473.05x + 3086.8 

R 2 = 0.8792 

a a a b 

30 mm 60 mm 90 mm 120 mm 

C 


y = -892.75x 2 + 3787.6x + 5668.3 

R 2 = 0.9357 

ab a ab b 

30 mm 60 mm 90 mm 120 mm 


Figure 1. Means comparison of dried flower yield (A), biological yield (B) and 

essential oil yield (C) of German chamomile under irrigation regimes at first year. 

The same letters showed non significant differences.

Table 2. Analysis of variance of yield and harvest index of Matricaria recutita L. affected by irrigation and storage time. 

Source of variation df 

Essential oil 

percentage 

Essential 

oil yield 

Harvest index 

of essential 

oil 

Reduction of 

essential oil 

percentage 

Pirzad 4397 

Reduction 

of essential 

oil yield 

Replication 5 0.0004 ns 0.068* 0.046 ns 0.042 ns 0.04 ns 

Storage 4 0.077** 1.176** 1.155** 13.57** 13.6** 

Irrigation 3 0.002 ns 0.256** 0.083* 0.055 ns 0.06 ns 

Storage × Irrigation 12 0.001 ns 0.019 ns 0.018 ns 0.063 ns 0.06 ns 

Error 95 0.001 0.026 0.030 0.043 0.04 

Coefficient of variation (%) 21.22 4.42 7.93 15.68 15.7 

df, degree of freedom; ns, * and ** ; non-significant, significant at P ≤ 5% and P ≤ 1%. 

The minimum biological yield of German chamomile 

(1934 kg/ha) belonged to the most strength stress, 

irrigation after 120 mm evaporation from pan class A. 

But, the other three irrigation regimes (irrigation after 30, 

60 and 90 mm evaporation) produced the maximum 

biomass (3464 kg/ha). Trends in biomass changes along 

with irrigation showed binomial regression with R 2 = 

0.8792 (Figure 1B). 

Similarity of changes in essential oil yield and dried 

flower yield indicated that the highest yield of essential oil 

(10084 g/ha) was obtained from irrigation after 60 mm 

evaporation that had any significant differences with yield 

of irrigation after 30 and 90 mm evaporation. But, the 

most strength stress, irrigation after 120 mm evaporation 

caused the lowest yield of essential oil (6672 g/ha). A 

binomial function between irrigation and yield of essential 

oil was made apparent by the means comparison (R 2 = 

0.9357) (Figure 1C). The similarity between yield of dried 

flower and essential oil showed that dried flower yield had 

a correlation with oil yield. 

Storage (five years) 

Data analysis of variance of plant material (dried flower 

obtained from field experiment) stored for five years 

showed significant effect of storage on essential oil 

percentage, yield of essential oil, harvest index of 

essential oil, reduction of essential oil percentage and 

yield (Table 2). 

Means comparison of data from 5-year storage 

indicated that the highest percentage of essential oil 

(0.715%) was observed at first year (harvesting year). 

After that, despite non-significant difference for second 

and third year’s essential oil contents, there was a 

reduction trend in essential oil percentage along with that 

of 5 years’ storage. So, the lowest essential oil content 

(0.194%) was obtained from flowers at the fifth year of 

storage. A polynomial equation of degree 3 (y = - 

0.0286x 3 + 0.2685x 2 - 0.855x + 1.3238; R 2 = 0.9809), 

showed the reduction as results of an analysis of 

variance (Figure 2A). 

Changes in essential oil yield, as for the results of oil 

content, showed a decreasing direction from the first to 

the fifth year of storage. We observed the minimum 

essential oil yield (2335 g/ha) in the fifth year of storage 

compared with the control (8442 g/ha). Polynomial 

equation between storage time (year) and oil yield was 

degree 3 (y = -339.92x 3 + 3182x 2 - 10081x + 15596; R 2 = 

0.9746) (Figure 2B). 

Harvest index of essential oil (HI; proportion of 

economic yield as the results for essential oil to total 

aerial dry matter), had a direction like essential oil yield 

because of consistency of biological yield for the duration 

of the experiment. So, the maximum (0.303%) and the 

minimum (0.083%) HI of essential oil from German 

chamomile was obtained at the first and fifth years of the 

experiment, respectively. This 27% reduction of essential 

oil was underlined with polynomial equation of degree 3 

(y = -0.0115x 3 + 0.1079x 2 - 0.3452x + 0.5491; R 2 = 

0.9785) (Figure 2C). 

Reduction in essential oil content and yield compared 

to the control (first year of harvest) was significant and 

the highest reduction was recorded after storage for 5 

years (73.23%). Polynomial equation of degree 3 (y = 

4.0905x 3 – 38.235x 2 + 120.91x – 85.91; R 2 = 0.9818) 

showed this increasing trend (Figure 2D and E). 

In the first year harvest, the correlation between 

biomass and dried flower yield was positive and 

significant (P ≤ 0.05). But correlations of biomass yield 

versus harvest index of dried flower and harvest index of 

essential oil were negative and highly significant (P ≤ 

0.01). These relations indicate an ever-increasing trend in 

biomass, resulting in parallel changes to dried flower 

yields. However, higher biomass led to a decreasing 

harvest index ratio of both dried flower and essential oil 

because of the non-significant correlation between 

biomass and essential oil percentage. The non significant 

correlation of dried flower yield and harvest index of dried 

flower, like harvest index of essential oil, emphasizes the 

greater effect of biomass, because of its greater value in 

comparison with dried flower yield. The yield of essential 

oil showed significantly positive association with dried 

flower yield. The non-significant correlation between dried


Essential Oil Content (%) 

Harvest Index of Essential Oil 

(%) 

0.8 

0.7 

0.6 

0.5 

0.4 

0.3 

0.2 

0.1 

0 

0.35 

0.3 

0.25 

0.2 

0.15 

0.1 

0.05 

0 

y = -0.0286x 3 + 0.2685x 2 - 0.855x + 1.3238 

R 2 = 0.9809 

a b b c 

d 

1 2 3 4 5 

Storage Time (Year) 

y = -0.0115x 3 + 0.1079x 2 - 0.3452x + 0.5491 

R 2 = 0.9785 

a b 

b b 

1 2 3 4 5 


Reduction of Essential Oil 

Yield (%) 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

E 

c 

A 

C 

Essential Oil Yield (g/ha) 

Reduction of Essential Oil 

Content (%) 

10000 

9000 

8000 

7000 

6000 

5000 

4000 

3000 

2000 

1000 

0 

90 

80 

70 

60 

50 

40 

30 

20 

10 

y = 4.0905x 3 - 38.235x 2 + 120.91x - 85.91 

R 2 = 0.9818 

d bc c b a 

1 2 3 4 5 

0 

D 


y = -339.92x 3 + 3182x 2 - 10081x + 15596 

R 2 = 0.9746 

a b b c d 

1 2 3 4 5 


y = 4.0905x 3 - 38.235x 2 + 120.91x - 85.91 

R 2 = 0.9818 

d bc c b a 

1 2 3 4 5 


Figure 2. Means comparison of essential oil percentage (A), essential oil yield (B), harvest index of essential oil (C), reduction 

of essential oil percentage (D) and reduction of essential oil yield (E) of German chamomile during five storage year. The same 

letters showed non significant differences. 

B

Pirzad 4399 

Table 3. Correlation coefficients among biomass yield, dried flower yield, harvest index of dried flower, essential oil percent, 

and essential oil yield at first year of field experiment. 

Variable 

Biomass 

yield 

Dried flower 

yield 

Harvest index of 

dried flower 

Essential oil 

percent 

Essential oil 

yield 

Dried flower yield 0.431* 

Harvest index of dried flower -0.702** 0.308 ns 

Essential oil percent -0.090 ns 0.076 ns 0.058 ns 

Essential oil yield 0.331 ns 0.904** 0.302 ns 0.490* 

Harvest index of essential oil -0.683** 0.315 ns 0.950** 0.327 ns 0.420* 

ns, * and ** ; non-significant, significant at P ≤ 5% and P ≤ 1%. 

Table 4. Correlation coefficients among biomass yield, dried flower yield, essential oil percent, essential oil yield, harvest index of 

essential oil, essential oil percent reduction, and essential oil yield reduction during five-year storage. 

Variable 

Biomass 

yield 

Dried 

flower yield 

Essential 

oil percent 

Essential 

oil yield 

HI of 

essential oil 

Essential oil 

percent reduction 

Dried flower yield 0.431** 

Essential oil percent 0.075 ns 0.082 ns 

Essential oil yield 0.221* 0.448** 0.909** 

HI of essential oil -0.269** 0.214* 0.859** 0.849** 

Essential oil percent reduction -0.094 ns -0.062 ns -0.976** -0.874** -0.832** 

Essential oil yield reduction -0.094 ns -0.062 ns -0.976** -0.874** -0.832** 1.000** 

ns, * and ** ; non-significant, significant at P ≤ 5% and P ≤ 1% . 

flower yield and harvest index (harvest index of flower 

and essential oil) relates to the importance of biomass in 

comparison with flower yield (Table 3). 

In the 5-year storage, the correlation between biomass 

and dried flower yield (P ≤ 0.01), however between 

biomass and essential oil yield (P ≤ 0.05) was observed 

positive and significant. But the correlation of biomass 

yield to harvest index of essential oil was negative and 

highly significant (P ≤ 0.01). These relations indicate that 

the ever-increasing trend in biomass caused parallel 

changes to dried flower and essential oil yields, but led to 

decreasing trend for harvest index of essential oil. The 

correlation between dried flower and essential oil yield 

was also positive and significant (P ≤ 0.01). The 

significant (P ≤ 0.01) correlation of essential oil 

percentage versus essential oil yield and harvest index of 

essential oil was observed during five years of storage. 

The correlation of essential oil percentage, yield and 

harvest index to reduction of essential oil percentage and 

yield was negative and significant (P ≤ 0.01) (Table 4). 

DISCUSSION 

Based on the results of current study, there was a 

reduction in the yield of biomass, dried flower and 

essential oil in irrigation after 120 mm evaporation from 

pan, because of the strongest water deficit condition. In 

the 5-year storage, there was a sharp slope in reduction 

of essential oil content and yield at the second year of 

storage. And a significant reduction was occurred in the 

percentage and yield of essential oil at the forth and fifth 

year because of destruction in herbal raw material 

tissues. Quality of dried flower will be lost after storage 

for 6 months, because of respiration as a very stringent 

process in living cells of freshly harvested chamomile. It 

mediates the release of chemically bound energy through 

the breakdown of carbon components and the formation 

of carbon skeletons necessary for maintenance and 

synthetic reactions after harvest. A secondary problem of 

prolonged storage is the result of respiration, the release 

of energy as heat. Therefore, heat from respiration needs 

to be monitored and heat needs to be abducted from 

stacks of the stored product. This difference, which was 

statistically significant (P ≤ 0.01), was also maintained 

during postharvest storage. The complex relationship 

between storage temperature, postharvest storage time, 

and respiration course was reported by Franke and 

Schilcher (2005). Thus, the quantity of constituents, in 

general, turned out to have been relatively stable during 

the postharvest period, but there were some unfavorable 

reactions (Bottcher et al., 2001). 

Natural samples of chamomile flowers after a 

postharvest storage period of 80 days contained a 

quantity of +46 ml essential oils/100 g dried herbal raw 

material at 10°C and of +40 ml/100 g dried herbal raw 

material at 20°C in comparison to the quantity at harvest 

date. However, after conditions of 30°C there was a


decline by 9.75 ml/100 g dried herbal raw material. 

Chamazulene was marked by small decreases, rising 

with higher temperatures at 10°C –1.95%; at 20°C -8.8%, 

and at 30°C –10.7% (Bottcher et al., 2001). 

It was determined that the quality of the herb 

deteriorated with storage and its quality was affected 

(Singh et al., 1994). The pattern of accumulation of 

monoterpenes and the effect of storage on the herb prior 

to distillation has been reported in other research (Singh 

et al., 1994, 1996). There are several reports that the oil 

content increases with storage prior to distillation (Singh 

et al., 1994). These show that the safe limit of herb 

storage varied according to the species and storage 

conditions. But, there is no exactly report on the changes 

of essential oil content in longtime storage. Essential oil 

from the leaves of Cymbopogon martinii was tested for 

toxicity against Fusarium oxysporum. Fungitoxicity 

remained unchanged in temperature treatment after a 

long storage period (Akhila, 2010). Storage of the herb 

Cymbopogon lexuosus always caused a reduction in oil 

content except during the summer, when it was not 

affected by 3 days of storage under shade. Little variation 

in the geranial and neral contents of essential oils of C. 

lexuosus leaves was observed during storage for 15 days 

(Singh et al., 1994). Hay storage of C. martinii (during 

summer) either in the shade or in the open increased its 

essential oil content. A slight difference was observed in 

geraniol and geranyl acetate contents of the essential oils 

from leaves of C. martinii (Franke and Schilcher, 2005). 

The infrared spectrum of lemongrass oil proved that 

changes in samples during storage could be detected; 

especially changes that occurred in the carbonyl group 

(Foda et al., 1975). Finally, because of no references on 

changes in essential oil yield from long term storage of 

medicinal plants, that of German chamomile, quality traits 

of other medicinal plants is yet to be discussed. 

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postharvest responses of Matricaria (Matricaria recutita L.) Flowers. 

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Foda YH, Abdullah MA, Zaki MS, Mostafa MM (1975). Identification of 

the volatile constituents of the Egyptian lemongrass oil. III. Infrared 

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Verlagsgesselschaft mbH, Stuttgart, Germany. 

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extracts. Matricin and chamazulene determinations—a comparison of 

GC, HPLC, and photometric methods. Deut. Apotheker Zeitung 

131(5):175–181. 

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CK, Kapur BM (eds), Cultivation and utilization of aromatic plants. 

R.R.L. Jammu-Tawi, pp. 653-657. 

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oil yield and quality in three Cymbopogon species (C. winterianus, C. 

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monoterpenes in the essential oil of citronella Java (C. winterianus) 

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98:166747.



DOI: 10.5897/JMPR12.260 



Enhanced callus induction and high-efficiency plant 

regeneration in Tribulus terrestris L., an important 

medicinal plant 

Sara Sharifi 1 *, Taher Nejad Sattari 1 , Alireza Zebarjadi 2,3 , Ahmad Majd 4 , and Hamid Reza 

Ghasempour 5 

1 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran. 

2 Department of Plant Breeding and Agronomy, Faculty of Agriculture, Razi University, Kermanshah, Iran. 

3 Department of Biotechnology for Drought Resistance, Razi University, Kermanshah, Iran. 

4 Department of Biology, Tehran North Branch, Islamic Azad University, Tehran, Iran. 

5 Department of Biology, Razi University, Kermanshah, Iran. 

Accepted 11 April, 2012 

We described culture conditions for direct and indirect regeneration of Iranian Tribulus terrestris L. 

through epicotyl, hypocotyl and leaf explants. The explants were cultured on MS medium supplemented 

with different concentrations and combinations of auxin and cytokinin. The results indicated that the 

mean of callus induction was influenced by explant type and various phytohormones levels. The 

highest percentage of callus production occurred on MS medium containing 0.1 mg/l naphthalene 

acetic acid (NAA) and 1 mg/l 6-benzylaminopurine (BAP) from epicotyl explants (91.6%), 0.4 mg/l (NAA) 

+ 2 mg/l (BAP) from hypocotyl explants (94.3%), and 0.4 mg/l (NAA) + 0.5 mg/l (BAP) for leaf explants 

(100%). Efficient shoot regeneration (22%) was achieved when the epicotyl explants were incubated on 

MS media amended with 0.1 mg/l (NAA) + 2 mg/l (BAP) and 0.4 mg/l (NAA) + 0.5 mg/l (BAP) within 14 

days of culture. Maximum indirect shoot regeneration (28.4%) was achieved from green-yellowish calli 

derived hypocotyl explants on MS medium with 0.4 mg/l (NAA) + 2 mg/l (BAP) within 21 days of culture. 

Also, an in vitro regeneration system from nodal segments was developed on MS medium 

supplemented with different levels of 6-benzyladenine. Maximum number of shoots per nodal explants 

was developed on a medium containing 3 mg/l (BA) at the rate of 2.5 micro-shoots per nodal explants 

after 4 weeks of culture. Proliferated shoots were elongated in hormone-free MS medium and also, 

shoots were rooted on MS medium and MS with 2 mg/l indol-3-butyric acid. 

Key words: Tribulus terrestris, plant regeneration, callus induction, plant growth regulators, MS (Murashige and 

Skoog) medium. 

INTRODUCTION 

Tribulus terrestris L. (Zygophyllaceae) is a plant native of 

Mediterranean region, but now widely distributed in warm 

*Corresponding author. E-mail: sarah.sharifi.59@gmail.com. 

Tel: 00988318331726. 

Abbreviations: BAP, 6-Benzylaminopurine; IBA, indol-3butyric 

acid; NAA, naphthalene acetic acid; BA, 6benzyladenine; 

PGRs, plant growth regulators. 

regions of Europe, Asia, America, Africa and Australia 

(Frohne, 1999). It is known by several common names 

such as puncturevine, caltrop, goat head, bull’s head, 

ground burr nut, and devil’s thorn. Generally, T. terrestris 

has a considerable seed dormancy lasting over fall and 

winter months (Washington State Noxious Weed Control 

Board, 2001) with some seeds staying dormant for longer 

periods of time. One of the constraints of this 

conventional propagation is its very low germination. No 

reliable data are available about seed germination in vitro 

condition. Seed dormancy and low germination rate in T.


terrestris make this plant a suitable specimen for 

developing an in vitro regeneration method. Therefore, 

establishment of in vitro culture systems is an attractive 

approach to large-scale propagation, germplasm 

resource conservation, and possible future genetic 

modification of this plant. The occurrence of saponins, 

flavonoids, alkaloids, lignamides and cinammic acid 

amides has been reported in T. terrestris (Saleh et al., 

1982; Bourke et al., 1992; Ren et al., 1994; Li et al., 

1998). For this purpose, we have selected T. terrestris L., 

a herb of Zygophyllaceae, endowed with various 

medicinal properties whose fruits are used in curing 

urinary discharges, cough, asthma, pain, 

spermatorrhoea, ophthalmia, anemia, dysentery, skin and 

heart diseases; its leaves purify blood and used in 

aphrodisiac and roots are good for stomachic and 

appetizer (Sarwat et al., 2008). Its purported effects 

include increased luteinizing hormone release and thus 

testosterone production, increased sperm production, 

increased ejaculatory volume and increased libido. The 

original use of T. terrestris extract was as a tonic to treat 

sexual dysfunction. It is an important constituent of 

various medicinal preparations worldwide like 

Dashmularishta, Tribestan, etc., (Sarwat et al., 2008). In 

the search for alternatives to production of desirable 

medicinal compounds from plants, biotechnological 

approaches, specifically plant tissue culture, are found to 

have potentials as a supplement to traditional agriculture 

in the industrial production of bioactive plant metabolites 

(Ramachandra and Ravishankar, 2002). The aim of the 

current research was therefore to determine the role of 

different combinations of naphthalene acetic acid (NAA) 

and 6-benzylaminopurine (BAP) on callus induction and 

shoot regeneration, and to find a suitable explant for in 

vitro multiplication of T. terrestris. 


Plant materials 

Mature fruits of T. terrestris were harvested during July, 2010 from 

Kermanshah province of Iran. 

Seed germination 

In the present study, a mixed method was used for seed 

germination and sterilization. Seeds were treated with captan (1%) 

as an antifungal agent for 30 min and then sterilized with 5% 

sodium hypochlorite for 5 min and 0.1% mercuric chloride for 3 min, 

followed by rinsing with sterile distilled water three times. The 

sterilized seeds were maintained in culture bottles each containing 

25 ml of Murashige and Skoog (MS) and half MS basal medium 

with 0.7% (v/v) agar and 3% (g/l) sucrose and maintained at 32°C 

under 16 h light and at 27°C under 8 h dark condition. In another 

part of these experiments, seeds were cultured in a mixture of soil 

peat, clay, and sand in the same condition. 

Explants such as epicotyl, hypocotyl and leaf were used from 2 

weeks old plants grown in a greenhouse. Then, these tissues were 

surface-disinfected with 0.1% mercuric chloride for 5 min and 

thoroughly washed with sterilized distilled water three times. Also, 

stems (approximately 1 cm in length) were harvested from 

greenhouse grown plants. The nodal segments (approximately 1 

cm) were dissected from the stems and were kept under running 

tap water for about 20 min and treated with captan for 30 min, then 

rinsed with sterile distilled water. 

Media and culture conditions for callus and shoot development 

The surface sterilized explants were inoculated on sterile medium. 

The culture media used throughout the experiments for tissue 

culture consisted of MS (Murashige and Skoog, 1962) basal salts 

supplemented with various concentrations and combinations of 

phytohormones like NAA, BAP, and indol-3-butyric acid (IBA) with 

100 mg/l casein hydrolysate. The pH of media in all case was 

adjusted to 5.8 before autoclaving at 121°C for 20 min. The cultures 

were kept at 25°C under cool-white light with a 16-h photoperiod 

(40 to 60 mmol/m/s) and 50% relative humidity and sub-cultured on 

fresh media at 14 days interval. Callus and direct organogenesis 

were achieved by placing the mentioned explants on MS medium 

supplement with 0.1, 0.2, and 0.4 mg/l NAA in combination with 0.5, 

1, and 2 mg/l BAP. The percentage of the explants producing calli 

and regenerated shoots were recorded on the basis of visual 

observation. Regenerated shoots were excised and transferred to 

hormone-free MS medium for rooting. Also for promoting 

development of the roots, the plantlets were transferred to MS 

Medium and MS medium supplemented with 2 mg/l IBA. Finally, 

plantlets were transferred to soil pots. 


The experiment was laid out as a factorial experiment (3×3×3) 

based on completely randomized design with three replications and 

each replication was made by using 3 Petri dishes per medium 

which contains 9 explants. In the first experiment, the rate of callus 

induction and shoot regeneration from epicotyl, hypocotyl and leaf 

explants were measured in front of different concentrations of NAA 

(0.1, 0.2 and 0.4 mg/l) and BAP (0.5, 1 and 2 mg/l) phytohormones. 

In another experiment, the rate of shoot initiation from nodal 

segments was investigated onto MS media supplemented with 3, 4 

and 5 mg/l 6-benzyladenine (BA). An analysis of variance (ANOVA) 

was carried out for all traits and then mean comparisons were 

performed using Duncan’s multiple range test (Duncan, 1955) at 

P=0.05. Traits such as percentage of callus, rate of shoot 

regeneration and root percentage were recorded in the 

experiments. 

RESULTS 

Approximately 28% of collected seeds were germinated 

after 10 to 14 days in vivo condition. Also in another part 

of investigation, seeds were not germinated in MS basal 

medium and 1/2 MS with 0.7% (g/l) agar and 3% sucrose 

was maintained at 32°C under 16 h light and at 27°C 

under 8 h dark condition (in vitro condition). 

No callus initiated from the different explants on 

hormone-free MS medium. The surface of the epicotyls, 

hypocotyls and leaves showed swelling and friablility; 

pale green callus developed from the cut ends within 10 

to 14 days of inoculation on MS medium supplemented 

with plant growth regulators (PGRs); eventually growing

Table 1. Analysis of variance (ANOVA) for callus formation and shoot regeneration 

of Tribulus terrestris. 

SOV df 

MS (mean square) 

Callus induction Shoot regeneration 

NAA 2 2087.57** 362.74** 

BAP 2 38.62 ns 508.09** 

NAA×BAP 4 566.13* 157.52* 

Explant 2 5342.37** 303.23** 

NAA×Explant 4 2072.5** 100.41** 

BAP×Explant 4 1103.89** 169.72** 

NAA ×BAP ×Explant 8 276.09 ns 154.48** 

Error 54 206.12 2.41 

ns, * and**: non–significant, significant at the 0.05 and 0.01 probability levels, 

respectively. S.O.V: stands for source of variation, and df: stands for degree of freedom. 

Table 2. Effect of different concentrations of NAA and BAP on callus induction from different 

explants of T. terrestris. 

Plant regulator concentration Explant producing callus 

NAA BAP Epicotyl ( ) Hypocotyls ( ) Leaf ( ) 

0.5 65 

0.1 

cdefg 44 ghi 47.6 fghi 

1 91.6 abc 71 bcdefg 33 hi 

2 64.3 cdefg 65.3 cdefg 55 efgh 

0.2 

0.4 

0.5 88.65 abcd 80.3 abcde 44 ghi 

1 90.65 abc 77.6 abcde 33.2 hi 

2 82.3 abcde 77.6 abcde 22 i 

0.5 82 abcde 70 bcdefg 100 a 

1 84.8 abcd 60.4 defg 61.6 defg 

2 67.3 bcdefg 94.3 ab 73.3 abcdef 

Value within a column followed by different letters are significantly different at the 0.05 probability level, 

analyzed by Duncan’s multiple range test. 

to cover the whole explants after 4 weeks of culture. 

The rate of callus formation and regeneration were 

determined after 4 weeks. The callus induction and shoot 

regeneration were variable and depended on the 

combination of growth regulators applied and explant 

types. Significant and non-significant differences among 

main levels of NAA, BAP concentrations and their 

interactions have been represented in Table 1. The 

results showed that all interactions between the BAP and 

NAA concentrations were significant at the 0.05 

probability level, expect the interaction between 

explants×NAA×BAP for callus induction (Table 1). 

The percentage of explants producing callus ranged 

from 22 to 100%. The highest percentage of callus 

induction occurred with 0.4 mg/l NAA and 0.5 mg/l for leaf 

explants (100%), with 0.4 mg/l NAA + 2 mg/l BAP for 

hypocotyls segments (94.3%) and for epicotyl explants 

Sharifi et al. 4403 

(91.6%) with 0.1 mg/l NAA + 1 mg/l BAP (Table 2). 

Maximum shoot regeneration (22%) for epicotyl was 

obtained on MS media supplemented with 0.1 mg/l NAA 

+ 2 mg/l BAP and 0.4 mg/l NAA + 0.5 mg/l BAP within 14 

days of culture (Figure 1a; Table 3). Shoot regeneration 

for hypocotyl segments was observed within 21 days of 

culture, and maximum regeneration for this explant 

(28.4%) was measured on medium with 0.4 mg/l NAA + 2 

mg/l BAP (Figure 1b; Table 3). As a result, according to 

Table 3, the leaf explant could not produce any shoots. 

Also, the results of shoot development and shoot per 

explant from nodal explants showed that the maximum 

shoots initiation from nodal (54%) was obtained on MS 

medium containing 3 mg/l BA, and the highest number of 

shoots per nodal was developed on the same medium at 

the rate of 2.53 micro-shoots per nodal after 4 weeks of 

culture (Figure 2; Table 4).


A B 

Figure 1. Callus formation and in vitro shoot regeneration of Tribulus terrestris L., a) direct shoot regeneration for epicotyl on MS 

media supplemented with 0.1 mg/l NAA + 2 mg/l BAP and b) indirect regeneration of shoots from the hypocotyls on medium with 0.4 

mg/l NAA + 2 mg/l BAP. 

Table 3. Effect of different concentration of NAA and BAP on shoot regeneration from 

hypocotyl and epicotyl of Tribulus terrestris after 14 to 21 days of culture. 

Plant regulator concentration Explant producing shoot 

NAA BAP Epicotyl (%) Hypocotyls (%) Leaf (%) 

0.1 

0.2 

0.4 

0.5 0 0 0 

1 0 0 0 

2 22 b 10 d 0 

0.5 0 0 0 

1 0 0 0 

2 0 0 0 

0.5 22 b 0 0 

1 0 0 0 

2 15.3 c 28.4 a 0 

Value within a column followed by different letters are significantly different at the 0.05 

probability level, analyzed by Duncan’s multiple range test. 

Shoots were excised from the explants and subculture 

on MS medium for the development of plantlets. To 

promote the development of roots, the plantlets were 

transferred to MS medium alone or supplemented with 2 

mg/l IBA (Figure 3a). The rooting of regenerated and 

elongated shoots was observed on MS with IBA and 

without IBA after 14 to 30 days of culture and the 

percentage of rooted shoots fluctuated between 70 and 

100%. Rooted plantlets were then transferred to a bed of 

sterile, moist sand and perlite (Figure 3b). 

DISCUSSION 

Due to its high medicinal value and increasing demands, 

in vitro studies have great importance in the propagation 

and genetic improvement programmer in this species. 

In nature, there are many mechanisms producing the 

crack of the tegumentary barrier in legumes, as 

temperature oscillation and the alternance of dry and wet 

periods (Quinlivan, 1971; Roslton, 1978), bacteria and 

other soil microrganism action, and the chemical

A 

Figure 2. Shoot multiplication from node explants on MS medium 

with 3 mg/l BA after 4 weeks. 

Table 4. Effect of BA (cytokinin) on shoot induction from nodal explants of Tribulus terrestris. 

BA (mg/l) Explant initiating shoots (%) Number of shoots/explant 

3.0 54 a 2.53 a 

4.0 46 ab 2.06 a 

5.0 30 b 2.53 a 

Value within a column followed by different letters are significantly different at the 0.05 probability level, 

analyzed by Duncan’s multiple range test. 

B 


Figure 3. a) Transferring of regenerated shoots to hormone-free MS medium for developing and rooting after 4 weeks and 

b) transferring rooted plantlets to soil. 

scarification suffered through the herbivore digestive 

system (Pereiras et al., 1985). In the current study, it 

seems that soil bacteria and other microrganisms 

stimulate germination of T. terrestris but physical 

scarification of seed coat and chemical treatment (with 

H2SO4) did not have any effect on germination. 

Petkov (2010) reported that cyanobacteria and 

microalgae, being photosynthesizing organisms, enrich 

the soil with oxygen, thus increased seed germination of 

T. terrestris.


A review of the T. terrestris literature revealed that in 

vitro plant regeneration from cotyledonary leaves of 

young seedling (Ali et al., 1997) and somatic 

embryogenesis from stem-derived callus of T. terrestris 

(Mohan et al., 2000) required a few changes of media 

fortified with tested exogenous plant growth regulators. 

Another study showed maximum embryo formation 

from leaf explants of T. terrestris on MS medium 

containing 5 µM BA and 2.5 µM NAA with 75 mg/l casein 

hydrolysate. The embryogenic callus culture of this 

species might offer a potential source for production of 

important pharmaceuticals (Nikam et al., 2009). 

In general, the explants type, its orientation in the 

culture medium, and PGRs play a key role in regulating 

the differentiation process (Chawla, 2000). Selection of 

suitable explants at correct development stage plays a 

key role in the successful establishment of culture under 

in vitro conditions. Morphological integrity of explants 

along with the proper choice of plant growth regulators 

strongly influence induction of optimal callus and shoot 

regeneration (Khawar et al., 2005). 

We also suggested that the balance of auxins and 

cytokinins is a decisive morphogenic factor. The present 

results showed that high concentration of BAP and low 

concentration of NAA was efficient for the induction of 

callus and subsequently shoots regeneration. 

Reports of auxin and cytokinin combinations supporting 

organogenesis differentiation in other species have been 

well documented (Lisowska and Wysonkinska, 2000; 

Pereira et al., 2000; Pretto and Santarém, 2000; 

Tokuhara and Mii, 1993; Tisserat and Jones, 1999; Roy 

and Banerjee, 2003; Janarthanam and Seshadri, 2008). 

In this study, leaf segments cultured on growth 

regulator free and MS medium supplemented with BAP 

and NAA failed to show any regeneration 

(organogenesis) response but remained green up to 2 

weeks. 

It seems the epicotyl explant of T. terrestris has a great 

organogenic potential for direct shoot regeneration. An 

important advantage of direct organogenesis is the 

potential for maintaining genomic stability of regenerated 

plants, whereas regeneration via an intermediated callus 

phase increases the possibility of somaclonal variations 

(Reddy et al., 1998; Tang and Guo, 2001). 

In our research, optimum indirect shoot organogenesis 

(28.4%) was achieved from hypocotyl in green-yellowish 

callus with 0.4 mg/l NAA and 2 mg/l BAP within 21 days 

of culture. Indirect organogenesis is defined as the 

formation of calli on explants and subsequently the 

development of shoots (Sharp et al., 1986). Besides, in 

this investigation, the highest percentage of shoot 

regeneration was attained on a medium containing 0.1 

mg/l NAA + 2 mg/l BAP in calli derived hypocotyl and 

epicotyl explants. It seems that, BAP phytohormone 2 

mg/l has a great potential for shoot regeneration of T. 

terrestris. Motamedi et al. (2011) reported that the 

highest percentage of shoot regeneration was achieved 

on a range of media supplemented with 0.1 mg/l NAA + 2 

mg/l BAP from cotyledon explant of Carthamus tinctorius 

Dincer cultivar. 

Cytokinins are very effective in promoting direct or 

indirect shoot initiation. To encourage the growth of 

axillary buds, and reduce apical dominance in shoot 

cultures, one or more cytokinins are usually 

incorporated into the medium at proliferation stage 

(George et al., 2007). 

Safdari and Kazemitabar (2010) results showed that 

the treatment containing 10 µM BAP was found to be the 

best one for shoot regeneration from nodal segments of 

Portulaca grandiflora L. The treatments with NAA in 

combination with BAP were found to be suitable 

treatments for callus production from leaf explants, and 

shoot regeneration. 

Present study showed that in nodal culture, the 

percentage of response and number of shoot formed per 

node explants was highest on medium supplemented 

with 3 mg/l BA and 2.53 adventitious shoots were 

developed per explants. The multiplication rate is lower 

than the earlier reports. Also, Raghu et al. (2010) 

reported optimum shoot regeneration from nodal explant 

of T. terrestris in woody plant medium supplemented with 

4 mg/l BA with six to seven micro-shoots per nodal 

explant after four weeks of culture. Das and Pal (2005) 

used 3 mg/l BAP (equivalent to 13.3 µM) for shoot 

regeneration from lateral buds of Bambusa balcooa. 

BAP had a significant effect on induction of multiple 

shoot bud in Operculina turpethum although callus 

formation was concomitant with shoot induction. 

Optimum shoot multiplication using BAP is reported in a 

number of plants (Hiregoudar et al., 2006; Sharma, 2006; 

Alderete et al., 2006; Alam et al., 2010b). 

Also, MS medium containing 12.5 mM BA alone was 

effective for inducing multiple shoots of Balanites 

aegyptiaca from nodal segments in 67% of cultures (Anis 

et al., 2010). The stimulating effect of BA on bud break 

and multiple shoot formation has been reported earlier for 

several medicinal woody plant species viz., Acacia tortilis 

ssp. raddiana (Sane et al., 2001), Acacia koa (Skolmen 

and Mapes, 1976), Leucaena leucocephala (Dhawan and 

Bhojwani, 1985), Bupleurum kaoi (Chen et al., 2006), 

Syzygium alternifolium (Sha Villi Khan et al., 1997), 

Pterocarpus marsupium (Chand and Singh, 2004a, b; 

Anis et al., 2005) and Celastrus paniculatus Willd. (Rao 

and Purohit, 2006). 

We observed that rooting had the better response on 

MS medium without any PGRs after 30 days. It seems 

that rooting in the absence of auxins may be attributed to 

endogenous auxin hormones in the plant. This results 

was previously reported too, by other researchers in 

detail for other medicinal plants (Sudhersan and Hussan, 

2003; Lu, 2005; Park et al., 2011). 

Finally, the system described here for continuous 

production of T. terrestris via callus induction and 

regenerated plants without loss of morphogenetic

capacity could be a model for micro-propagation 

systems, not only for the large scale of medicine plants 

(especially Zygophllaceae family) but also for genetic 

improvement of T. terrestris through transformation 

studies. The medicinal property of this plant is mainly due 

to the wide spectrum of alkaloids and saponins. 

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Journal of Medicinal Plants Research Vol.6 (27), pp. 4409-4415, 18 July, 2012 


DOI: 10.5897/JMPR12.344 



Cytotoxic saikosaponins from Bupleurum yinchowense 

LI Zong yang, Cao Li, Liu Xin min, Chang Qi and Pan Rui le* 

Institute of Medicinal Plant Development, Chinese Academy of Medical Science, Peking Union Medical College, 

Beijing 100193, China. 

Accepted 15 March, 2012 

Activity-guided fraction of the ethanolic extract of the root from Bupleurum yinchowense resulted in the 

isolation of 13 saikosaponins. Their structures were identified to be saikosaponin a (1), saikosaponin c 

(2), saikosaponin d (3), saikosaponin b2 (4), saikosaponin f (5), saikosaponin b4 (6), 6"-Oacetylsaikosaponin 

a (7), 3β,16α,23,28-tetrahydroxy-olean-11,13(18)-dien-29-oic acid 3-O-β-D- 

glucopyranosyl-(1→3)-β-D-fucopyranoside (8), chikusaikosideⅠ(9), saikogenin F (10), saikosaponin e 

(11), 6"-O-acetylsaikosaponin d (12), saikosaponin 14 (13) on the basis of their spectral data and 

chemical evidences. Compound 8 is a new natural product and the other 12 compounds were separated 

from this plant for the first time. Compounds 1 to 10 were evaluated in vitro for their inhibitory ability 

against the growth of human esophageal cancer cell lines (Eca-109), human colon cancer cell lines (W- 

48), human cervical cancer cell lines (Hela), human ovarian cancer (SKOV3). Compounds 1, 3 and 7 

exhibited significant inhibitory activities against the tested cell lines, with the IC50 values not more than 

15 μg/ml. 

Key words: Bupleurum yinchowense, umbelliferae, saikosaponins, cytotoxic, 3β,16α,23,28-tetrahydroxy-olean- 

11,13(18)-dien-29-oic acid 3-O-β-D-gluco-pyranosyl-(1→3)-β-D-fucopyranoside. 

INTRODUCTION 

The genus Bupleurum is a well-known and very important 

crude drug in traditional oriental medicine, especially in 

Chinese and Japanese traditional medicine. Preparations 

containing the roots of Bupleurum species have been 

prescribed for more than 2000 years in China where the 

first record about their use appeared in Shen-Nong’s 

Herbal (Xie et al., 2009). Many Bupleurum-containing 

herbal drugs have been traditionally used in the treatment 

of tumours and cancers. Much research has shown that 

*Corresponding author. E-mail: rlpan@implad.ac.cn. Tel: +86- 

10-87533286. Fax: +86-10-87533299. 

Abbreviations: ELISA, Enzyme-linked immuno sorbent assay; 

MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium 

bromide; PBS, phosphate-buffered saline; EDTA, 

ethylenediaminetetraacetic acid; DMSO, dimethyl sulfoxide; 

TBS, tris buffered saline. 

the genus Bupleurum mainly accumulates saikosaponins 

(Ding et al., 1986; Luo et al., 1993; Ebata et al., 

1996;Zhao et al., 1996; Tan et al., 1999; Sánchez- 

Contreras et al., 2000). Moreover, several saikosaponins 

of Bupleurum have been evaluated for the anti-tumour 

and anti-proliferative effects. The extracts from 

Bupleurum scorzonerifolium and Bupleurum kaoi were 

assessed against A549 human lung cancer cells (Cheng 

et al., 2003). The saikosaponins from Bupleurum 

rotundifolium and Bupleurum wenchuanense 

demonstrated cytotoxicity against human gastric 

adenocarcinoma (MK-1), human cervical carcinoma 

(HeLa), murine melanoma (B16F10) cell lines, leukaemia 

P-388 cells and nasopharynx carcinoma KB cells (Fujioka 

et al., 2003, 2006). In our search for antitumour agents 

from Chinese herbs, we found out that the crude 

saikosaponins from the root of Bupleurum yinchowense 

specifically inhibited human esophageal cancer cell lines 

(Eca-109), human colon cancer cell lines (W48), human


ovarian cancer cell lines (SKOV3) and human cervical 

cancer cell lines (Hela) in vitro. B. yinchowense is 

abundantly distributed in the Northwest of China and 

widely used in Chinese folk medicine. No evidence is 

available in the literatures concerning its constituents and 

pharmacological activities. To systematically evaluate its 

potential anticancer activity, the constituents were studied 

by activity-guided fraction and result in the isolation of 13 

saikosaponins. Their structures were identified on the 

basis of spectral data. The isolation, structure elucidation 

and evaluation for cytotoxic activities were described in 

this study. 


General experimental procedure 

Nuclear magnetic resonance (NMR) spectra were recorded on a 

Brucker DRX-500 spectrometer (Brucker Biosciences Corporation, 

Billerica, MA) with tetramethylsilane (TMS) as internal standard 

operating at 500 and 125 MHz for 1 H and 13 C, respectively. Fast 

atom bombardment-mass spectra (FAB-MS) and high resolutionfast 

atom bombardment-mass spectra (HR-FABMS) were recorded 

on a Micromass Autospec-Q instrument (Micromass Ltd., 

Manchester, UK). Infrared (IR) spectra were recorded in KBr discs 

using a Perkin-Elmer 983G spectrophotometer (Perkin-Elmer Ltd., 

USA). Gas chromatography (GC) analysis was carried out on an 

Agilent 6890N gas chromatography (Agilent Co., USA) using an 

HP-5 capillary column. Column chromatography was performed 

with silica gel (100 to 200 mesh, Qingdao Marine Chemical Co., 

Qingdao, P. R. China), Sephadex LH-20 (25 to 100 μm, GE 

Healthcare Bioscences AB, Uppsaki, Sweden), octadecyl silica (25 

to 40 μm, Merck, USA), D101 macroporous resins (Tianjin Gujiao 

Factory, Tianjin, P. R. China), MCI Gel CHP20P (75 to 150 µm, 

Mitsubishi Chemical, Japan). Thin layer chromatography (TLC) was 

performed on precoated silica gel GF254 (0.2 mm thick, Qingdao 

Marine Chemical Co., Qingdao, P. R. China) and spots were 

detected by spraying with 10% ethanolic H2SO4 reagent. 3-(4, 5dimethylthiiazol-2yl)-2,5-diphenyl 

tetrazolium bromide (MTT) was 

purchased from Sigma-Aldrich (St. Louis, MO, USA). 

Plant collection 

The roots of B. yinchowense were collected from Dingxi County, 

Gansu province, China, in August, 2009 and identified by the 

author, Professor Ruile Pan of the Institute of Medicinal Plant, 

Chinese Academy of Medical Sciences and Peking Union Medical 

College, Beijing, China, where a voucher specimen (No. 20090815) 

was deposited. 

Extraction 

The dried and powdered roots (800 g) of B. yinchowense were 

extracted with 60% ethanol containing 0.5% ammoniae aqua (three 

times, 1 L each) at room temperature for 12 h. The ethanol extracts 

were combined and evaporated in vacuo, to yield a dark brown 

residue (120 g), which was dissolved in H2O-MeOH (5:95) solution 

(200 ml), and then portioned with n-hexane of 200 ml) to get the nhexane-soluble 

fraction. The H2O-MeOH (5:95) layer was 

evaporated to remove residual MeOH, and then distilled water (200 

ml) was added. This aqueous solution was subjected to a column 

contained 1 kg D101 macroporous resin and was eluted 

successively with water (2 L), 90% ethanol (2 L), respectively. 

Evaporation of the respective solvents gave n-hexane (12 g), water 

(42 g) and 90% ethanol (32 g) fractions. The 90% ethanol fraction is 

saponin-enriched part. Each fraction was evaluated for the cytotoxic 

activity on the tumor cell lines (Table 1). It was shown that the 

activity resided in the saponin-enriched part. 

Isolation 

Saponin-enriched part (32 g) was subjected to MCI column, eluting 

with a gradient of water-methanol (from 100:0 to 5:95), to yield 5 

fractions. Fraction 2 (8 g) was chromatographed repeatedly on 

silica gel using chloroform-methanol (8:2) and octadecylsilane 

(ODS)-C18 with the elution of methanol-water (7:3) to afford 1 (80 

mg),2 (55 mg),3 (75 mg), 11 (5 mg) and 9 (17 mg). Fraction 3 

(10 g) was separated into three sub-fractions by ODS column using 

methanol-water (7:3) as elution and the second sub-fraction was 

subjected to repeated column chromatography, first on silica gel, 

chloroform:methanol (8:2) and purified on pharmadex LH-20 

(methanol) to obtain 4 (51 mg), 5 (38 mg), 6 (20 mg) and 12 (8 mg). 

Compound 7 (35 mg), 8 (37 mg), 13 (6 mg) and compound 10 (22 

mg) were purified from Fraction 4 (5 g) by repeated semipreparative 

high performance liquid chromatography (HPLC) using methanolwater 

(60:40) as fluent. 

Characterization of isolation 

Saikosaponin a (1): White amorphous powder, mp 229 to 230°C, 

1 H-NMR (C5D5N, 500 MHz): � 0.91, 0.92, 0.99, 1.12, 1.31, 1.37 

(each 3H, s, tert-Me×6), 1.47 (3H, d, J=6.6 Hz, Fuc-CH3), 4.94 (1H, 

d, J=7.8 Hz, Fuc-1′-H), 5.35 (1H, d, J=8.4 Hz, Glc-1″-H), 5.66 (1H, 

dd, J=10.2, 2.4 Hz, 11-H), 6.00 (1H, d, J=10.2 Hz, 12-H). 13 C-NMR 

data (Table 1). 

Saikosaponin c (2): White amorphous powder, mp 207 to 209°C, 

1 H-NMR (C5D5N, 500 MHz): � 0.86, 0.91, 0.96, 0.97, 1.14, 1.28, 

1.35 (each 3H, s, tert-Me × 7), 1.66 (3H, d, J=6.0 Hz, Rha-CH3), 

4.94 (1H, d, J=7.8 Hz, Glc-1′-H), 4.52 (1H, d, J=9.0 Hz, Rha-1″-H), 

4.79 (1H, d, J =7.8 Hz, Glc-1′″-H), 5.64 (1H, dd, J=10.2, 2.4 Hz, 11- 

H), 5.90 (1H, d, J=10.2 Hz, 12-H). 13 C-NMR data (Table 1). 

Saikosaponin d (3): White amorphous powder, mp 227 to 228°C, 

1 H-NMR (C5D5N, 500 MHz): � 0.91, 0.92, 0.99, 1.12, 1.31, 1.37 





Saikosaponin b2 (4): White amorphous powder, mp 201 to 203°C, 

1 H-NMR (C5D5N, 500 MHz): � 0.89, 0.92, 0.99, 1.01, 1.04, 1.68 





Saikosaponin f (5): White amorphous powder, mp198 to 200°C 

1 H-NMR (C5D5N, 500 MHz): � 0.82, 0.95, 0.99, 1.00, 1.01, 1.29, 

1.35 (each 3H, s, tert-Me×7), 1.65 (3H, d, J=6.0 Hz, Rha-CH3), 4.94 

(1H, d, J=7.8 Hz, Glc-1′-H), 4.52 (1H, d, J=9.0 Hz, Rha-1″-H), 4.79 

(1H, d, J=7.8 Hz, Glc-1′″-H), 5.86 (1H, br, 12-H). 13 C-NMR data 

(Table 1). 

Saikosaponin b4 (6): White amorphous powder, mp 206 to 

208°C, 1 H-NMR (C5D5N, 500 MHz): � 0.97, 1.01, 1.01, 1.12, 1.14, 

1.88 (each 3H, s, tert-Me×6), 3.26 (3H, s, OCH3), 1.45 (3H, d, J=6.0 

Hz, Fuc-CH3), 4.96 (1H, d, J=7.2 Hz, Fuc-1′-H), 5.33 (1H, d, J=7.8 

Hz, Glc-1″-H), 5.60 (1H, d, J=3.0 Hz, 12-H). 13 C-NMR data (Table 

1). 

6″-O-acetylsaikosaponin a (7): White amorphous powder, mp 

204 to 205°C, 1 H-NMR (C5D5N, 500 MHz): � 0.89, 0.92, 0.92, 0.98,

Table 1. 13 C-NMR data for compounds 1 to 12 (C5D5N, 125 MHz). 

Li et al. 4411 

1 2 3 4 5 6 7 8 9 10 11 12 

1 38.6 38.5 38.6 38.4 38.8 40.1 38.6 38.4 38.6 38.4 39.8 38.6 

2 26.6 26.5 26.2 26.1 26.6 26.4 25.8 26.1 26.6 26.1 26.3 25.7 

3 88.9 89.0 81.6 81.7 89.1 81.9 81.6 81.7 88.6 81.7 88.8 81.6 

4 39.8 39.7 43.7 43.7 39.5 43.8 43.7 43.7 39.8 43.6 39.8 43.7 

5 55.4 55.3 47.3 47.4 55.7 48.4 47.3 47.3 55.4 47.4 55.7 47.4 

6 18.2 18.1 17.5 18.9 18.5 18.3 17.3 18.2 18.2 17.3 18.3 17.6 

7 31.9 31.9 31.5 32.3 33.4 33.4 31.6 32.3 31.9 31.9 33.3 31.6 

8 42.2 42.2 41.9 41.1 40.2 40.7 42.2 41.1 42.2 41.9 41.0 42.2 

9 53.0 52.8 53.0 54.0 47.1 51.7 53.1 54.1 53.0 53.0 52.0 53.2 

10 36.4 36.3 36.8 35.5 36.8 38.2 36.3 36.5 36.4 36.3 38.1 36.3 

11 132.0 132.1 132.0 126.2 24.0 76.0 132.2 126.1 132.0 132.0 76.0 132.2 

12 131.2 131.2 131.9 126.3 122.7 122.5 131.2 126.8 131.2 131.9 122.4 131.2 

13 84.0 84.0 84.8 136.1 144.0 149.8 83.9 137.4 84.0 84.9 148.1 84.0 

14 45.7 45.7 45.3 41.9 43.8 41.9 45.6 42.1 45.7 43.6 43.9 45.7 

15 36.2 36.2 36.3 32.6 36.8 37.2 36.2 31.9 36.2 35.4 36.8 36.1 

16 64.0 64.0 77.2 67.7 66.6 74.2 64.0 67.7 64.0 77.1 66.2 64.1 

17 47.0 47.0 54.4 45.3 41.1 43.4 47.0 45.5 47.0 45.3 43.6 46.9 

18 52.2 52.2 51.4 133.0 44.5 41.9 52.1 131.4 52.2 51.4 43.9 52.1 

19 37.8 38.5 37.8 39.0 47.1 47.8 37.7 34.0 37.8 38.4 47.0 37.7 

20 31.6 31.6 31.9 31.9 31.1 31.3 31.6 44.0 31.6 31.9 31.1 31.6 

21 34.7 34.7 36.8 36.5 34.3 35.0 34.7 31.1 34.7 36.8 33.4 34.7 

22 25.7 25.7 31.3 24.4 26.6 30.9 25.7 24.2 25.7 31.3 26.3 25.8 

23 64.1 28.0 64.1 64.7 28.2 64.3 64.0 64.1 27.8 64.1 28.2 64.2 

24 13.3 16.3 13.1 13.1 17.0 13.6 13.0 13.1 16.3 13.1 17.0 13.0 

25 18.0 18.1 18.8 18.3 15.7 17.9 18.7 18.9 18.0 18.9 17.3 18.7 

26 20.0 19.9 19.5 17.2 17.0 18.4 20.0 17.3 20.0 19.6 18.5 20.1 

27 20.9 20.9 20.6 21.9 27.1 26.4 20.8 21.9 20.9 18.1 26.3 20.8 

28 73.1 72.7 77.6 64.1 69.1 70.0 73.0 65.1 73.0 78.1 69.1 73.0 

29 33.7 33.7 33.7 25.1 33.4 33.4 33.6 181.2 33.7 33.8 33.3 33.6 

30 23.8 23.8 24.4 32.6 24.1 24.6 23.8 21.5 23.8 24.4 24.0 23.8 

OCH3 - - - - - 53.8 - - - - 54.2 - 

1′ 106.8 106.7 106.0 106.0 106.7 106.0 106.1 106.0 106.8 106.1 106.7 105.1 

2′ 71.6 75.2 71.0 71.8 75.2 71.5 71.4 71.6 71.6 71.5 75.1 71.3 

3′ 85.2 76.8 85.3 85.3 76.8 85.5 85.4 85.3 85.2 84.9 76.8 85.9 

4′ 71.8 80.0 72.2 72.2 80.0 72.2 71.4 72.2 71.8 72.1 79.9 71.7 

5′ 71.0 75.2 71.5 71.0 75.6 71.0 71.0 71.1 71.0 71.0 75.5 70.9 

6′ 17.3 69.2 17.3 17.2 69.1 17.2 17.3 17.3 17.3 17.3 68.5 17.2 

1″ 106.8 102.9 106.8 106.7 102.9 106.7 106.4 106.7 106.8 106.5 105.3 104.5 

2″ 75.9 72.6 75.9 75.8 72.7 75.8 75.3 75.8 75.9 75.3 74.8 86.8 

3″ 78.5 72.5 78.8 78.8 72.6 78.4 78.1 78.3 78.5 77.8 78.3 77.6 

4″ 71.8 73.8 71.8 71.6 73.8 72.2 71.6 71.8 71.8 72.1 71.4 70.5 

5″ 78.4 70.5 78.9 78.5 70.5 78.4 75.3 78.7 78.4 75.6 78.3 78.1 

6″ 62.7 18.1 62.7 62.7 18.4 62.7 64.8 62.7 62.7 64.8 62.5 62.2 

1″′ - 105.2 - - 105.1 - - - - - 103.0 107.8 

2″′ - 74.8 - - 74.8 - - - - - 72.5 76.1 

3″′ - 78.4 - - 78.5 - - - - - 72.7 77.9 

4″′ - 71.4 - - 71.4 - - - - - 73.8 71.0 

5″′ - 78.4 - - 78.4 - - - - - 70.5 67.6 

6″′ - 62.6 - - 62.6 - - - - - 18.3 - 

COCH3 - - - - - - 170.8 - - 170.8 - - 

COCH3 - - - - - - 20.8 - - 20.8 - -


1.10, 1.39 (each 3H, s, tert-Me×6), 1.94 (3H, s, COCH3), 1.48 (3H, 

d, J=6.0 Hz, Fuc-CH3), 4.98 (1H, d, J=7.2 Hz, Fuc-1′-H), 5.25 (1H, 

d, J=7.8 Hz, Glc-1″-H), 6.00 (1H, d, J=10.2 Hz, 12-H), 5.66 (1H, dd, 

J=10.2, 2.4 Hz, 11-H). 13 C-NMR data (Table 1). 

- 3β,16α,23,28-tetrahydroxy-olean-11,13 (18)-dien-29-oic acid 3- 

O-β-D-glucopyranosyl-(1→3)-β-D-fucopyranoside(8): White 

powder, mp 243 to 245°C (MeOH). The HR-ESI-MS m/z 811.4832 

[M +H] + (calcd. for C42H66O15, 811.0039), IR (KBr)νmax 1699 cm -1 . 

1 H-NMR (C5D5N, 500 MHz): � 0.88, 0.91, 0.99, 1.60, 1.70 (each 3H, 

s, tert-Me×5), 6.74 (1H, d, J=10.5 Hz, H-11), 5.72 (1H, d, J=10.5 

Hz, H-12), 4.99 (1H, d, J=7.5 Hz, Fuc-1′-H), 5.34 (1H, d, J=7.5 Hz, 

Glc-1″-H). 13 C-NMR data (Table 1). 

Chikusaikoside (9): White amorphous powder, mp 207 to 209C. 

1 H-NMR (500 MHz, C5D5N) � 0.89, 0.92, 0.93, 0.99, 1.10, 1.39 

(each, 3H, s, tert-Me×6), 1.42 (3H, d, J=6.6 Hz, Fuc-CH3), 4.94 (1H, 

d, J=7.8 Hz, Glu-CH3), 5.15 (1H, d, J=7.8 Hz, Xyl-CH3), 6.70 (1H, d, 

J=10.2 Hz, 11-H), 5.99 (1H, d, J=10.2 Hz, 12-H). 13 C-NMR data 

(Table 1). 

Saikogenin F (10): White amorphous powder, mp 215 to 217°C, 

1 H-NMR (500 MHz, CD3OD): δ 0.70, 0.92, 0.94, 0.97, 1.03, 1.09 

(3H, s, tert-Me×6), 1.90 (1H, br s, H-9), 3.04 (1H, d, J=7.2 Hz, H- 

28), 3.90 (1H, d, J=6.6 Hz, H-28), 4.17 (1H, dd, J=10.5, 6.0 Hz, H- 

16), 5.38 (1H, dd, J=10.5, 3.0 Hz, H-12), 5.95 (1H, d, J=10.8 Hz, 

H-11). 13 C-NMR (125 MHz, CD3OD):δ 12.1 (C-24), 18.4 (C-6), 18.8 

(C-25), 20.2 (C-26), 21.2 (C-27), 24.1 (C-30), 26.1 (C-22), 27.3 (C- 

2), 32.1 (C-7), 32.3 (C-20), 33.9 (C-29), 35.2 (C-21), 36.0 (C-15), 

37.2(C-10), 38.5(C-19), 39.2(C-1), 42.9(C-8), 43.5(C-4), 46.5(C-14), 

47.6(C-14), 48.2(C-5), 53.1(C-30), 53.9(C-9), 65.3(C-16), 66.8(C- 

23), 73.4(C-28), 73.5(C-3), 85.7(C-13), 130.6(C-12) and 134.1(C- 

11). 

Saikosaponin e (11): White amorphous powder, mp 227 to 

228°C. 1 H-NMR (600 MHz, C5D5N): � 0.91, 0.91, 0.96, 0.96, 1.12, 

1.31, 1.37 (each, 3H, s, tert-Me×7), 1.47 (3H, d, J=6.6 Hz, Fuc- 

CH3), 4.74 (1H, d, J=7.8 Hz, Fuc-1′-H), 5.41(1H, d, J=8.4 Hz, Fuc- 

1′-H), 5.66 (1H, d, J=10.2 Hz, 11-H), 5.97 (1H, d, J=10.2 Hz, 12-H). 

13 C-NMR data (Table 1). 

6"-O-acetyl saikosaponin d (12): White amorphous powder, mp 

197 to 198°C. 1 H-NMR (600 MHz, C5D5N): � 0.90, 0.93, 0.99, 0.99, 

1.38, 1.61 (each, 3H, s, tert-Me×6), 1.94 (3H, s, COCH3), 1.49 (3H, 

d, J=6.0 Hz, Fuc-CH3), 4.98 (1H, d, J=7.8 Hz, Fuc-1′-H), 5.25 (1H, 

d, J=7.8 Hz, Fuc-1′-H), 6.02 (1H, d, J=10.2 Hz, 11-H), 5.70 (1H, dd, 

J=10.2, 2.4 Hz, 12-H). 13 C-NMR data (Table 1). 

Saikosaponin 14 (13): White amorphous powder, mp 199 to 

200°C. 1 H-NMR (500 MHz, C5D5N): � 0.91, 0.99, 1.00, 1.02, 1.04, 

131, 1.45 (each, 3H, s, tert-Me×7), 3.24 (3H, s, OCH3), 1.65 (3H, d, 

J=6.0 Hz, Rha-CH3), 4.56 (1H, d, J=9.0 Hz, Glc-1′-H), 4.80 (1H, d, 

J=7.8 Hz, Rha-1″-H), 4.92(1H, d, J=7.8 Hz, Glc-1′″-H), 5.64 (1H, dd, 

J=10.2, 2.4 Hz, 11-H), 5.90 (1H, d, J=10.2 Hz, 12-H). 13 C-NMR data 

(Table 1). 

Sugar identification of compound 8 

Compound 8 (10 mg) was refluxed with 5% HCl in MeOH-water (1:1, 5 

ml) for 6 h. The MeOH was then removed and the solution was 

extracted with EtOAc (2 ml × 3). The aqueous fractions were 

evaporated and the residues were prepared to their derivatives for GC 

analysis according to the methods described in the literature (Tang et 

al., 2005). The D-fucose and D-glucose were confirmed by comparison 

of their retention time (tR, 4.6 and 11.5 min, respectively) with those of 

authentic standards. The authentic samples were purchased from the 

Pfanstienl Chemical Corporation, Waukengan, IL (Lot no, 1279). 

Cytotoxicity assay 

The cytotoxicity was measured by MTT assay (Alley et al., 1988; 

Zhou et al., 1993). Briefly, cells were plated in 96-well plates (5 × 

10 4 cells/well) and incubated in a humidified atmosphere, 95% air, 

5% CO2 at 37°C. After 24 h, additional medium (100 μl) containing 

the test compounds (100, 80, 40, 20, 10 and 5 μg/ml) and vehicle 

(DMSO, final concentration of 0.1%) was added to each well. After 

68 h of incubation, the supernatant was replaced by fresh medium 

containing MTT (0.5 mg/ml). 4 h later, the MTT formazan product 

was dissolved in 150 µl DMSO, and the optical density (OD) was 

read on a microplate ELISA reader (BioRad 680, Molecular Devices, 

USA) at 570 nm. The assays were repeated three times. Media and 

DMSO control wells, in which sample was absent, were included in all 

the experiments, in order to eliminate the influence of DMSO. The 

inhibitory rate of cell proliferation was calculated by the following 

formula: 

Growth inhibition (%) = (ODcontrol – ODtreated / ODcontrol) × 100% 

The cytotoxicity of sample on tumor cell lines was expressed as IC50 

values (the drug concentration reducing by 50% the absorbance in 

treated cells, with respected to untreated cells), which were calculated 

by LOGIT method. 

RESULTS AND DISCUSSION 

Phytochemical investigation 

The saponin-enriched part of B. yinchowense was 

subjected to a succession of chromatographic 

procedures, including silica gel chromatography, 

pharmadex LH-20, ODS-C18 and semipreparative HPLC 

to afford 13 compounds. On the basis of spectroscopic 

data analysis (IR, UV, 1 H-NMR, 13 C-NMR, 1 H- 1 H COSY, 

DEPT, HMBC, HMQC, FAB-MS and HR-FABMS) and 

comparison with reports in the literatures, compounds 1 

to 13 were identified to be saikosaponin a (1) (Liang et 

al., 1998), saikosaponin c (2) (Tori et al., 1976), 

saikosaponin d (3) (Liang et al., 1998), saikosaponin b2 

(4) (Ishii et al., 1980), saikosaponin f (5) (Tori et al., 

1976), saikosaponin b4 (6) (Ishii et al., 1980), 6"-Oacetylsaikosaponin 

a (7) (Ding et al., 1986), 3β, 16α, 23, 

28-tetrahydroxy-olean-11, 13 (18)-dien-29-oic acid 3-O-β- 

D-glucopyranosyl-(1→3)-β-D-fucopyranoside (8) 

(Yoshikawa et al., 1997), chikusaikoside I (9) (Ebata N et 

al., 1996), saikogenin F (10) (Wang et al., 1998), 

saikosaponin e (11) (Hiroshi et al., 1997), 6"-O-acetyl 

saikosaponin d (12) (Ding et al., 1986) and saikosaponin 

14 (13) (Ding et al., 1986). Compound 8 is a new natural 

compound which had been previously synthesized and 

now was isolated from natural source for the first time. 

The other 12 compounds were isolated from this plant for 

the first time. All the 13 compounds were oleanane-type 

saikosaponins (Figure 1). 

3β,16α,23,28-tetrahydroxy-olean-11,13 (18)-dien-29-oic 

acid 3-O-β-D-glucopyranosyl-(1→3)-β-D-fucopyranoside 

(8) was obtained as white powder, mp 243 to 245°C 

(MeOH). The high resolution-electrospray ionizationmass 

spectrometry (HR-ESI-MS) displayed a molecule 

ion peak at m/z 811.4832 [M+H] + (calculate 811.0039), 

consistent with the molecular formula C42H66O15, which 

also was confirmed by 13 C-NMR and 1 H-NMR spectral 

data.

Figure 1. Chemical structures of compounds 1 to 13. 

The 1 H-NMR spectra of compound 8 exhibited five typical 

angular methyl proton signals (δ 0.88, 0.91, 0.99, 1.60 

and 1.70), which indicated that compound 8 was a 

triterpenoid saponin. Its UV spectrum also showed 

absorbances at 242, 251 and 261 nm, suggesting a 

heteroannular diene system at C-11, C-12, C-13 and C- 

18. This was further confirmed by its 1 H-NMR signals at δ 

6.74 (1H, d, J=10.5 Hz, H-11), 5.72 (1H, d, J=10.5 Hz, H- 

12) and 13 C-NMR signals at δ 137.4, 131.4, 126.8 and 

126.1, corresponding to C-13, C-18, C-12 and C-11, 

respectively. The 1 H-NMR and 13 C-NMR spectra also 

showed two anomeric proton signals at 4.99 (1H, d, J=7.5 

Hz) and 5.34 (1H, d, J=7.5 Hz) and two sugar amoneric 

carbons at δ 106.0 and 106.7. All aforementioned 

evidences suggested that compound 8 was a diglycoside 

with a heteroannular diene system at C-11, C-12, C-13 

and C-18 of the aglycone. 

The distortionless enhancement by polarization transfer 

(DEPT) spectrum of compound 8 also showed that the 

aglycone moiety possessed four hydroxyl groups at δ 

81.7, 67.7 (CHOH) and at δ 64.1, 65.1 (CH2OH) and 


carbonyl group at δ 181.2. The 13 C-NMR signals of the 

aglycone moiety were in good agreement with those of 

saikosaponin v (Tan et al., 1999) except for the signal at 

C-30. A comparison of the 13 C-NMR data for their 

aglycone moieties showed that the signals for C-30 of 

compound 8 underwent a downfield shift (+2.6) on going 

from saikosaponin v to compound 8. 

The IR spectrum of compound 8 showed a carbonyl 

band at 1699 cm -1 , and 13 C-NMR spectrum exhibited a 

carbonyl signal at δ 181.2. According to the formula of 

compound 8, it should contain a substitute of carbonyl. In 

heteronuclear multiple bond correlation (HMBC) experiments, 

significant correlation of the carbonyl signal (δ 

181.2) with the methyl protons [δ1.6 (29-CH3)], indicating 

that the carbonyl group should be at C-30. Therefore, the 

aglycone was ultimately determined as 3β, 16α, 23, 28tetrahydroxy-olean-11,13 

(18)-dien-30-oic acid. 

On acid hydrolysis of compound 8, D-fucose and Dglucose 

were detected from the aqueous fraction by GC 

analysis comparing with the authentic samples. Through 

analysis of the 1 H- 1 H COSY, HMQC, HMBC spectral data,


Figure 2. Structure of 8 and key correlations observed from HMBC. 

Table 2. Cytotoxicity of different fractions and isolates from B. 

yinchowense. 

Sample 

SKOV3 

IC50 value (μg/ml) 

SW48 Eca-109 Hela 

n-Hexane fraction >100 >100 >100 >100 

Water fraction >100 >100 >100 >100 

95% Ethanol fraction 14.46 12.12 13.73 22.51 

1 5.14 5.81 4.92 7.82 

2 - - - - 

3 5.32 6.12 5.85 9.21 

4 37.2 35.8 42.3 42.7 

5 - - - - 

6 - - - - 

7 4.53 5.32 4.61 8.67 

8 - - - - 

9 35.2 36.9 30.2 45.4 

10 - 87.5 - - 

5-Fu 11.32 9.31 11.23 14.30 

1 H-NMR and 13 C-NMR signals for compound 8 were fully 

assigned. 13 C-NMR signals are as shown (Table 1). The 

HMBC spectrum showed obvious correlations between δ 

4.99 (H-1') and 81.7 (C-3) and δ 5.34 (H-1’’) and 85.3 (C-3’), 

suggesting the sugar moiety was located at C-3 position, 

the glucosyl was connected with C-3' of the D-fucose. 

According to the coupling constants of sugar anomeric 

proton signals δ 4.99 (1H, d, J=7.5 Hz) and 5.34 (1H, d, 

J=7.5 Hz), the fucose and glucose should be assigned as 

β-anomeric configurations. Thus, compound 8 was 

elucidated as 3β, 16α, 23, 28-tetrahydroxy-olean-11,13 

(18)-dien-29-oic acid 3-O-β-D-glucopyranosyl- (1→3)-β- 

D-fucopyranoside (Figure 2). The spectra data of 

compound 8 were reported for the first time. 

Cytotoxic activity 

Compounds 1 to 10 were evaluated in vitro for their 

inhibitory ability against Eca-109, SW48, Hela, and 

SKOV3 by MTT assay, using cisplatin as a positive 

control. The 50% growth inhibition (IC50) values are 

summarized in Table 2. Saikosaponin a (1), saikosaponin 

d (3) and 6"-O-acety-saikosaponin a (7) displayed strong 

cytotoxic activities against the tested cell lines in a dosedependent 

manner, with IC50 values of 4.53 to 14.3 μg/ml. 

The other compounds showed no or weak 

antiproliferative activity. 

Previous studies have shown many oleanane-type 

Bupleurum saponins, especially those that contained the 

epoxy bridge at C-13 and C-28 in their skeleton were 

very potent against many cancer cells (Chiang et al., 

2003; Fujioka et al., 2006). Saikosaponin d exerted very

potent activity against the HepG2 cell line with an IC50 

value of 12.5 μg/ml (Chiang et al., 2003). 3′-O-acetyl 

derivatives of saikosaponins a and d exhibited a potent 

activities against leukaemia P-388 cells and nasopharynx 

carcinoma KB cells with IC50 values of 0.5 and 6.3 μg/ml 

for the former and 1.2 and 6.3 μg/ml for the latter (Luo et 

al., 1993). In the present study, 10 oleanane-type 

Bupleurum saponins from B. yinchowense were tested 

against Eca-109, W-48, Hela and SKOV3. The result 

revealed that saikosaponin a, saikosaponin d and 6"-Oacetylsaikosaponin 

a exhibited significant inhibitory 

activities against the tested cell lines. The present study 

further demonstrated that Bupleurum saponins with an 

epoxy bridge and their acetyl derivatives were very potent 

against many cancer cells. To the best of our knowledge, 

this is the first report for the cytotoxic activity of 

compound 7. 


This work was financially supported by National Science 

and Technology major projects (2011ZX09307-002-01 

and 2012ZX09301002-001) and by the International 

Science and Technology Collaboration Project (0901). 

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DOI: 10.5897/JMPR12.533 



Chemical composition and antioxidant activity of Lippia 

species 

Junya de Lacorte Singulani 1 , Pâmela Souza Silva 1 *, Nádia Rezende Barbosa Raposo 2 , 

Ezequias Pessoa de Siqueira 3 , Carlos Leomar Zani 3 , Tânia Maria Almeida Alves 3 and 

Lyderson Facio Viccini 1 

1 Laboratório de Genética, Departamento de Biologia, Instituto de Ciências Biológicas, 

Universidade Federal de Juiz de Fora, Juiz de Fora, MG, Brazil. 

2 Núcleo de Pesquisa e Inovação em Ciências da Saúde, Faculdade de Farmácia, 

Universidade Federal de Juiz de Fora, Juiz de Fora, MG, Brazil. 

3 Laboratório de Química de Produtos Naturais, Centro de Pesquisas René Rachou, 

Fundação Oswaldo Cruz - FIOCRUZ, Belo Horizonte, MG, Brazil. 


Chemical composition of the hexanic fraction, antioxidant activity (DPPH assay) and total phenolic 

content (Folin-Ciocalteau assay) of aerial parts of sixteen Lippia species were determined. Hexanic 

fraction was analyzed by gas chromatography-mass spectrometry (GC-MS). The compounds βmyrcene, 

limonene, γ-terpinene, linalool, β-caryophyllene, germacrene D, α–copaene, β-bourbunene, βelemene, 

aloaromadendrene, bicyclogermacrene, and δ-cadinene appeared in the chemical analysis in 

most of the Lippia hexanic fraction studied. This fraction showed low radical scavenging activity; 

however, the ethanolic fraction of Lippia species showed higher radical-scavenging activity than the 

commercial antioxidant butylated hydroxy toluene (BHT). Total phenolic content of the polar fraction, as 

gallic acid equivalents, ranged from 105.5 mg/g in Lippia pseudo-thea to 255.4 mg/g in Lippia sericea. 

The presence of phenolic compounds in the ethanolic fraction of Lippia spp. may also be the major 

cause of its high radical-scavenging activities. 

Key words: Verbenaceae, gas chromatography-mass spectrometry (GC-MS), phenolic content, antioxidant 

assay. 

INTRODUCTION 

The genus Lippia (Verbenaceae) is widely distributed in 

tropical and subtropical America and Africa, and it 

consists of approximately 250 species of herbs, shrubs, 

and small trees which often contain a chemical aromatic 

structure (Moldenke, 1980). One of the main diversity 

centers of the genus is located at Espinhaço range, 

Minas Gerais State, Brazil (Viccini et al., 2006). 

There are many economically important Lippia species 

and some of them have been used in traditional medicine 

mainly for gastrointestinal and respiratory diseases 

*Corresponding author. E-mail: pmlsouz@gmail.com. Tel: +55 

32 2102 3220. Fax: +55 32 2102 3206. 

(Pascual et al., 2001). Brazilian species, such as Lippia 

alba (Mill.) N.E. Br and Lippia sidoides Cham., have 

shown antiulcerogenic, antimicrobial, anti-inflammatory, 

antihelmintic, antioxidant, gastroprotective, and cytostatic 

properties (Aguiar et al., 2008; Camurça-Vasconcelos et 

al., 2007; Carvalho et al., 2003; David et al., 2007; 

Fontenelle et al., 2007; Monteiro et al., 2007; Pascual et 

al., 2001). These effects are partially attributed to the 

essential oils and phenolic compounds produced by both 

species. However, the chemical composition and pharmacological 

aspects of many Lippia spp. still have not 

been studied. 

The present study was done in order to determine: (1) 

the chemical composition of hexanic fraction of sixteen

Lippia spp. using gas chromatography-mass spectrometry 

(GC-MS) analysis, (2) the total phenolic content of 

ethanolic fraction using the Folin-Ciocalteau reagent, and 

(3) the antioxidant capacity, through the capture of radical 

2,2-diphenylpicrylhydrazyl (DPPH) of hexanic and 

ethanolic fractions of Lippia spp. 


Plants 

Leaves of Lippia aff. microphylla Cham., Lippia aristata Schauer., 

Lippia corymbosa Cham., Lippia diamantinensis Glaz., Lippia 

filifolia Mart. & Schauer ex Schauer, Lippia filippei Moldenke, Lippia 

hermannioides Cham., Lippia lupulina Cham., Lippia martiana 

Schauer, Lippia microphylla Cham., Lippia pseudo-thea (St. Hil.) 

Schauer, Lippia pohliana Schauer, Lippia rosella Moldenke, Lippia 

rubella Moldenke, Lippia salviifolia Cham., and Lippia sericea 

Cham. were collected at the Espinhaço Range, Minas Gerais State, 

Brazil, in March and April of 2007. The plants were identified by Dr. 

Fátima Regina Gonçalves Salimena from Botany Department 

(Federal University of Juiz de Fora). The voucher of each species 

was deposited at the CESJ Herbarium of the Federal University of 

Juiz de Fora. 

Preparation of hexanic and ethanolic fractions (HF and EF) 

Fresh leaves of each species (3 g) were collected and transferred 

to a conical tube with ethanol P. A. (30 ml). Each preparation was 

macerated during one week at room temperature. After filtration, 

the extracts were taken and mixed to the same quantity of ultrapure 

water and were partitioned twice with hexane. Ethanolic and 

hexanic fractions were conserved at -20°C until use. 

GC-MS analysis 

The hexanic fractions were analyzed using a Shimadzu GC-MS 

model QP5050A equipped with a flame ionization detector (FID) 

and a DB-5 fused silica capillary column (35 × 0.2 mm, film 

thickness 0.10 μm), using helium as the carrier gas (1.0 ml/min). 

The injection temperature was 200°C and the column oven program 

was 50 to 250°C at 4°C min -1 . Mass spectra were obtained by 

electronic impact 70 eV and the range from 50 to 500 m/z was 

scanned. Data acquisition and handling were done via CLASS 5000 

Shimadzu software. Retention index (RI) in the range of 900 to 

3000 was generated from analysis of a standard mixture containing 

C9 to C30 hydrocarbons. The identification of the compounds was 

based on the comparison of their mass spectra with those in a 

Shimadzu spectral database and retention indices (Adams, 1995). 

Assay for total phenolic content in the ethanolic fraction 

Total phenolic constituents were determined using the Folin- 

Ciocalteu reagent and gallic acid as the standard (Slinkard and 

Singleto, 1997). The reaction mixture contained sample (50 µl), 

Folin-Ciocalteau reagent (250 µl), 20% sodium carbonate (500 µl), 

and distilled water (4.2 ml). Calibration curve was prepared with 

gallic acid solutions ranging from 0 to 700 µg/L. Total phenols of the 

extract, as gallic acid equivalent, were determined using the 

absorbance of the extract measured at 765 nm. Results were 

expressed as µg/L. Tests were carried out in triplicate. 

Antioxidant assay 

Singulani et al. 4417 

The radical scavenging activity of plant extracts was determined by 

DPPH method (Sreejayan and Rao, 1997). An aliquot of ethanol 

solution of the HF or EF (50 μl or 0.97 to 250 μg/ml) was added to a 

0.05 mM ethanol DPPH solution (150 μl) in a 96 well microplate and 

was incubated at room temperature for 30 min. A blank (consisting 

of the extract and ethanol) was used to remove the influence of the 

color of the sample. An ethanolic solution of DPPH was used as 

negative control. Ascorbic acid and butylated hydroxy toluene 

(BHT) were used as antioxidant reference compounds, at the same 

concentrations as those used for the sample. Results were 

expressed as mean of inhibiting concentration (IC50) which was 

calculated using Equation 1 as follow: 

IC50 (%) = 100 × (A0 – As)/A0 (1) 

where A0 and As are the values for the absorbance of the negative 

control and the absorbance of the sample, respectively. Tests were 

carried out in triplicate. The antioxidant activity of the L. lupulina 

and L. microphylla were not analyzed, because there was no 

sufficient amount of extract. 

RESULTS 

The hexanic fractions of Lippia spp. analyzed by GC-MS 

showed mainly terpenoid compounds such as hydrocarbons, 

alcohols, aldehydes, ketones, and esters (Table 

1). Sixty-one compounds have been identified, 

representing 74.88 to 98.49% of the total compounds 

detected. 

The monoterpenes hydrocarbons β-myrcene, limonene, 

γ- terpinene and linalool were identified in at least 50% of 

the hexanic fraction. In the present study, β-myrcene 

concentrations ranging from 0.43 to 12.93% as percentage 

of peak area, being one of the main com-pounds of 

L. filippei, L. lupulina, L. pseudo-thea, and L. rosella. 

Limonene concentration ranging from 0.44 to 10.19%, 

and was one of the main components of L. filifolia and L. 

rubella. However, γ-terpinene appeared in lower concentrations 

(between 0.57 and 5.46%). Linalool 

concentration varied from 0.26 to 6.96% of HF from L. 

filippei and L. pohliana, and was one of the major 

components in these species. 

In addition, the HF of Lippia aristata, L. filifolia, and L. 

rosella exhibited high content of δ-3-carene (15.98%), 

camphene (11.98%) and α-phellandrene (14.46%), 

respectively. α-pinene was the most abundant in L. 

aristata (5.94%) and L. martiana (10.64%) extracts. On 

the other hand, sabinene was mainly found in L. aristata 

(16.01%) and L. pseudo-thea (8.69%). 

Oxygenated monoterpenes were the most abundant 

components observed in some species. The neral and 

geranial appeared in L. diamantinensis with 9.38 and 

14.53%, respectively. Camphor (25.27%) was the main 

component of L. filifolia and cis-limonene epoxide was 

the major component in L. rubella while 1.8-cineole 

appeared as a major compound in L. pseudo-thea and in 

L. rosella (25.47 and 34.64%, respectively).


Table 1. Chemical composition of HF (%) of several Lippia spp. 

Relative percentage 

of compound 

RIa LAFb LARc LCOd LDIe LFFf LFPg LHEh LLUi LMAj LMIk LPSl LPOm LROn LRUo LSAp LSEq Monotepene hydrocarbon 

α-pinene - 6.81 5.94 - - - - - - 10.64 - - - - - 2.91 - 

Canfene - 0.42 - - - 11.98 - - - - - - - - - - - 

Sabinene - 0.92 16.01 - - - - 2.00 - - - 8.69 - 7.81 - 1.02 - 

ß-pinene 978 1.54 0.51 0.46 1.37 - - 2.82 - 1.16 - - - - - 1.32 - 

ß-myrcene 993 0.47 0.99 0.98 - 2.15 8.60 - 12.93 2.86 - 8.24 - 10.27 0.43 - - 

α-phellandrene 1007 - - - - - - - - - - - - 14.46 - - - 

δ-3-carene 1014 0.66 15.98 - - - - - - 4.83 - - - - - - - 

p-cymene 1028 - - 0.61 1.54 1.54 - - - - - 1.44 - 0.52 - - - 

Limonene 1032 0.48 - 0.81 2.02 6.52 3.69 0.44 - 2.98 - 0.68 0.62 - 10.19 0.76 0.93 

β-phellandrene 1037 - - - - - - - - - - - - 2.85 - - - 

cis-ß-ocimene 1041 0.43 - - - 2.15 0.95 - - 7.66 - - - - - - - 

trans-ß-ocimene 1052 1.63 - - 4.06 2.61 1.12 - - 3.80 1.12 0.56 - 1.65 - 0.75 - 

γ-terpinene 1062 - - 2.11 - 5.46 - 0.57 - 0.34 - 2.48 - 0.64 3.70 - - 

Terpinolene 1093 0.47 - - - 1.30 - - - 4.66 - 0.23 - 0.43 - - - 

Oxygenated monoterpene 

1.8-Cineole 1035 - - - - 3.06 - 2.84 - - - 25.47 - 34.64 - - - 

Linalool 1107 0.89 - 0.89 - - 6.71 1.54 - - - 2.57 6.96 0.26 - 0.70 - 

cis-limonene epoxide 1148 - - - - - - - - - - - - - 34,38 - - 

Camphor 1154 - - - - 25.27 - - - - - 2.03 - - - - - 

Myrcenone 1158 - - - - - - - - - - 18.70 - - - - - 

trans-limonene epoxide 1176 - - - - - - - - - - - - - 1.23 - - 

α-terpineol 1200 - - - - - - 0.28 - - - 2.88 - - - - - 

Neral 1257 - - - 9.38 - - - - - - - - - - - - 

Geranial 1288 - - - 14.53 - - - - - - - - - - - - 

Sesquiterpen hydrocarbon 

δ-elemene 1346 - - 3.19 - - - 0.48 3.14 - - - - - 0.23 - - 

α-cubebene 1360 - - 0.46 - - - - - - - - 1.07 - 0.51 - - 

α-copaene 1384 4.79 0.63 1.58 1.61 1.56 2.37 0.94 1.46 10.63 6.02 - 1.85 1.93 0.71 1.82 8.74 

ß-bourbonene 1394 0.89 - 1.84 - - 1.23 2.44 1.31 - - - - - 0.21 0.71 - 

ß-cubebene 1399 1.04 0.50 - - - - 0.94 - - 1.13 - - - 1.58 - 

ß-elemene 1400 - - 18.37 2.02 2.60 4.63 - 2,50 - - - 1.79 0.64 - - -

Table 1. Contd. 


ß-caryophillene 1431 5.57 21.11 9.79 6.68 14.37 16.34 23.40 10.87 20.40 17.82 4.68 33.55 5.43 8.55 7.25 10.19 

ß-gurjunene 1440 0.69 - 0.65 - - 0.49 0.58 - - - - 2.13 0.29 - - 2.40 

γ-elemene 1442 - - 1.43 - - - 0.15 - - - - - - 0.39 - - 

trans- α-bergamotene 1444 0.45 - - - - - - - 1.81 - - - 0.39 - - - 

α-guaiene 1449 2.35 - - - - - - - - - - - - - - 6.01 

α-caryophillene 1465 1.88 1.30 3.61 1.59 3.79 1.21 8.67 3.83 1.52 9.21 0.29 4.07 0.34 5.14 5.10 22.21 

Aloaromadendrene 1473 2.54 0.97 3.36 - - 2..23 2.35 2.37 0.62 2.26 - 3.06 0.97 0.57 2.37 7.07 

γ-muurolene 1489 - - 2.17 - - - - - 0.88 1.68 - 2.76 0.59 - 0.98 4.49 

Germacrene D 1494 39.13 27.42 22.15 8.04 1.75 13.73 27.84 41.97 - 20.37 0.65 4.11 11.47 12.91 25.18 3.87 

ß-selinene 1500 0.72 - 1.69 - - - - - - - - 1.38 - - 0.93 1.79 

Bicyclogermacrene 1510 11.45 3.57 6.78 - 3.57 9.33 5.10 11.27 - 11.67 - 3.99 1.54 0.75 3.08 - 

α-muurolene 1512 - - - - - - - - 1.46 - - - - - - 5.75 

Germacrene A 1519 - - 2.13 - - 0.39 - - - - - - - - 2.30 - 

δ-guaiene 1519 1.15 - - - - - - - - - - - - - - 2.36 

β-curcumene 1527 - - - - - - - - - - - - - 1.34 - - 

γ-cadinene 1529 0.48 - 1.34 - - - - - - - - 1.44 0.39 - 0.68 1.92 

δ-cadinene 1536 3.10 - 2.07 1.05 0.96 - 0.68 1.03 6.95 3.60 - 3.70 0.76 - 1.09 10.44 

Cadina-1,4-diene 1547 - - - - - - - - - - - 0.66 - 1.42 - - 

α-cadinene 1552 - - 1.08 - - - - - - - - 0.74 0.22 - - - 

Germacrene B 1574 - - 2.79 - - - 1.06 4.66 - - - 1.56 - 1.32 - - 

Oxygenated sesquiterpene 

Nerolidol 1575 - - - - - - - - - - - - - - 29.65 - 

Spathulenol 1597 0.51 - - - - 1.78 0.21 - - - - - - - 0.75 - 

Cariophyllene oxide 1600 - - - - - 1.06 - - 0.39 - 0.69 1.42 - - - - 

Ledol 1605 - - - - - - - - - - - - - - - 1.49 

Globulol 1609 - - - - - 1.36 - - - - - 0.66 - - - 1.49 

Guaiol 1611 - 0.95 - - - - - - - - - - - - - - 

α-epi-muurolol 1661 1.12 - - - - - - - - - - - - - - 4.64 

α-muurolol 1665 - - - - - - - - - - - 1.79 - - - 1.31 

α-cadinol 1674 1.34 - - - - - - - - - - - - - - 1.22 

Hydrocarbon 

Undecane 1107 - - - 4.18 - - - - - - - - - - - - 

Tridecane 1319 - - - 4.57 - - - - - - - - - - - - 

Pentadecane 1508 - - - 24.88 - - - - - - - - - - - - 

a, Retention index; b, L. aff. mycrophylla; c, L. aristata; d, L. corymbosa; e, L. diamantinensis; f, L. filifolia; g, L. filippei; h, L. hermannioides; I, L. lupulina; j, L. martiana; k, L. mycrophylla; l, L. 

pseudo-thea; m, L. pohliana; n, L. rosella; o, L. rubella; p, L. salviifolia; q, L. sericea; r, not identified or absent.


Table 2. Antioxidant activity and total phenolic content of Lippia spp. 

Species 

Hexanic fraction (HF) 

IC50 (μg/ml) 

Ethanolic fraction (EF) 

IC50 (μg/ml) 

Total phenolic 

content (mg/g) 

L. aff. microphylla 61.90 10.07 * a 

L. aristata 81.70 4.90 * 

L. corymbosa 218.82 19.22 234.1 

L. diamantinensis 334.68 16.43 163.5 

L. filifolia 171.56 13.11 190.6 

L. filippei 162.05 28.27 157.9 

L. hermannioides 437.78 31.30 109.8 

L. martiana 57.00 5.00 * 

L. pohliana 149.14 18.81 209.5 

L. pseudo-thea 195.69 67.89 105.5 

L. rosella 166.43 39.61 185.3 

L. rubella 62.38 26.62 212.6 

L. salviifolia 68.20 4.80 190.6 

L. sericea 203.63 25.99 255.4 

a Not analyzed. 

In our investigation, β-caryophyllene was present within 

the main component of all analyzed species, and also 

appeared at high concentrations (4.68 to 33.55%). The 

isomer α-caryophyllene was also found in all Lippia spp. 

at low concentrations (0.29 to 9.21%), except in L. 

sericea where it appeared as the most abundant 

component (22.21%). 

Germacrene D was also identified as a common 

component in the studied species, except in L. martiana. 

The compound was found as the major constituent in L. 

aff. microphylla (39.13%), L. aristata (27.42%), L. 

corymbosa (22.15%), L. hermannioides (27.84%), L. 

lupulina (41.97%) and L. microphylla (20.37%), while in L. 

filifolia and L. pseudo-thea the concentrations were lower 

than 2%. Moreover, the sesquiterpenes α–copaene 

(ranging from 0.63 to 10.63%), β-bourbunene (0.21 to 

2.44%), β-elemene (0.64 to 18.37%), aloaromadendrene 

(0.57 to 7.07%), bicyclogermacrene (0.75 to 11.67%), 

and δ-cadinene (0.68 to 10.44%) have been identified for 

most of the Lippia studied. Oxygenated sesquiterpenes 

were detected in minor amounts. However, nerolidol 

(29.65%) was the major constituent of L. salviifolia. 

Besides terpenoids, hydrocarbons common on the 

surface of plants such as undecane, tridecane and 

pentadecane were identified in a high percentage in L. 

diamantinensis (4.18, 4.57, and 24.88%, respectively). 

The antioxidant activity of HF, expressed as the 

concentration that inhibited 50% DPPH free radical (IC50), 

ranged from 57.00 to 437.78 μg m/L (Table 2), showing a 

low radical scavenging activity as compared to ascorbic 

acid (2.50 μg m/L) and BHT (11.82 μg m/L). However, the 

ethanolic fraction of the majority of species examined 

showed high antioxidant activity as compared to BHT 

(11.82 μg m/L). The concentrations that led to 50% 

inhibition (IC50) are given in Table 2. When we analyzed 

the total of phenol content these fractions estimated as 

tannic acid equivalent, it ranged from 105.5 mg/g in L. 

pseudo-thea to 255.4 mg/g in L. sericea (Table 2). 

DISCUSSION 

Hexanic fractions obtained from fresh leaves of sixteen 

Lippia spp. showed a great chemical diversity. Among the 

monoterpenes hydrocarbons, it was possible to detect 

limonene and linalool, two components that can be found 

in higher quantities in essential oils of other Lippia spp. 

(Pascual et al., 2001). Myrcene and myrcenone were 

identified as the major components of the essential oils 

from the leaves and flowers of L. lacunosa, while 

limonene and myrtenal was observed in Lippia 

rotundifolia (Leitão et al., 2008). 

The oxygenated monoterpenes, such as the neral and 

geranial were the most abundant components observed 

in some species (e.g. L. diamantinensis). These compounds 

have been described as important constituents of 

others species of Lippia such as L.citriodora 

(Argyropoulou et al., 2007) and L. rugosa (Tatsadjieu et 

al., 2009). The citral (mixture of neral and geranial) 

showed potent antimicrobial activity against Grampositive 

and Gram-negative bacteria as well as fungi 

(Onawunmi, 1989), while anti-inflammatory, analgesic 

and antifungal properties are assigned to the 1.8-cineole, 

the main compound in L. pseudo-thea and in L. rosella 

(Santos and Rao, 2000; Vilela et al., 2009). 

Sesquiterpenes were mainly represented by βcaryophyllene, 

one component commonly found in 

essential oils of the genus Lippia (Pascual et al., 2001).

Previously, β-caryophyllene was identified as the major 

component of volatile oil from leaves and flowers of 

Lippia chevalieri (Mevy et al., 2007). In our investigation, 

this component was observed as one of the main 

components of all analyzed species, and it also appeared 

at high concentrations. The isomer, α-caryophyllene, was 

also found in Lippia spp. These findings constitute 

evidences that both β-caryophyllene and α-caryophyllene 

have anti-inflammatory and anti-allergic effects (Passos 

et al., 2007). 

In general, the present data are in accordance with 

those previously reported (Pascual et al., 2001; 

Terblanché and Kornelius, 1996), which showed high 

variability of the chemical composition of the essential 

oils in Lippia spp. Thus, the HF represents a qualitative 

alternative to analyze the volatile secondary metabolites. 

The possibility of using HF instead of essential oil 

extraction increases the capability of Lippia chemical 

studies since many species possess few and very small 

leaves. 

The literature emphasizes that, within species, a variety 

of geographical and ecological factors, the isolation 

method and the analytical conditions can influence the 

volatile oil composition (Santos-Gomes et al., 2005). In 

spite of these findings, our data were in accordance with 

previous studies for L. microphylla collected in the 

northeast of Brazil (Costa et al., 2005). Only thymol and 

1, 8-cineole were not identified in L. aff. microphylla and 

L. microphylla analyzed in the present study. 

The antioxidant activity of hexanic fraction of Lippia 

spp. showed low radical scavenging activity. Similar 

results were found for other species of the same genus. 

Both the essential oil from Lippia berlandieri (Rocha- 

Guzmán et al., 2007) and L. chevalieri (Mevy et al., 2007) 

also showed a lower antioxidant property. However, the 

limonene-carvone chemotype of L. alba (Stashenko et 

al., 2004) and L. sidoides (Monteiro et al., 2007), which 

were rich in thymol, showed good antioxidant potential. In 

our investigation, carvone and thymol were not identified. 

Thus, it is noted that the DPPH method shows a better 

response to polar samples when compared with the 

nonpolar samples (Sultana et al., 2007). Literature 

reports that the low response to the test for free radical 

scavenging can be attributed to the absence of 

conjugation to form stable resonance structures during 

the formation of radicals in the low polarity molecules (Di 

Majo et al., 2005). 

On the other hand, the ethanolic fraction of the majority 

of species showed high antioxidant activity. Based on 

previous data, it is possible that the powerful antioxidant 

activity of polar extracts can be attributed to phenolic 

compounds (Mensor et al., 2001), although, in the 

present study, the results did not show a conclusive 

relationship between the total phenolic content and 

antioxidant activity. In addition, the presence of these 

compounds in the ethanolic fraction of Lippia spp. may 

also be the major cause of their high radical-scavenging 


activity. Several studies have also reported the absence 

of this kind of relationship. The molecular antioxidant 

response to free radicals varies markedly, depending on 

the chemical structure and the oxidation conditions. 

Phenol constitutes one of the major groups of compounds 

that act as primary antioxidants (Muchuweti et al, 

2006). Antioxidant compounds neutralize chemically 

active products of the metabolism, such as free radicals 

which can damage cells and tissues. Phenolic 

compounds, with their potential to act as antioxidants, 

may play a key role on the prevention of various 

pathological conditions such as cancer, cardiovascular 

and neurodegenerative diseases that some authors 

believe to be associated with oxidative stress (Losso et 

al., 2007). 

Conclusively, this is the first report of chemical 

composition and antioxidant activity of sixteen Lippia spp. 

Chemical analysis revealed the presence of various 

terpenoid compounds which are common in this genus. 

Furthermore, the compounds of the EF from leaves of 

Lippia spp. showed high antioxidant activity suggesting 

the potential of these plants as a natural source of 

strongly antioxidant substances that can be use as a 

natural additive in food and pharmaceutical industries. 


The authors gratefully acknowledge UFJF and FAPEMIG 

for financial support and Dr. Fátima Regina Gonçalves 

Salimena for plant identification. 

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DOI: 10.5897/JMPR12.573 



Influencing factors of consumers’ willingness to pay for 

Crocus sativus: An analysis of survey data from China 

Lin Hong, Guangtong Gu, Wenchuan Li, Dan Fan, Jun Wu, Yanying Duan, Haijun Peng, and 

Qingsong Shao* 

Zhejiang A & F University, Hangzhou 311300, Peoples Republic of China. 

Accepted 17 April, 2012 

The aim of the present study is to identify the factors that affect consumers' willingness to pay for 

Crocus sativus in China. For this purpose, a total of 467 surveys were carried out in ten cities of China. 

From the collected survey data, a logistic regression analysis was used to estimate the variables of the 

consumers' individual characteristic, health functions of the products, consumers' behavior and 

marketing factors. The results show that gender, household monthly income, marital status, attitudes of 

health functions, attitudes of active ingredient content and taste of the products have a significant 

positive impact on willingness to pay for C. sativus, while the price of the products and comparative 

purchasing behavior have a significant negative impact on willingness to pay for C. sativus. 

Key words: Crocus sativus, consumer behaviour, willingness to pay, influencing factors, logistic model. 

INTRODUCTION 

Crocus sativus L., commonly known as saffron is 

principally produced in Iran and Spain, and has been 

successfully cultivated in various places in China, 

especially in Zhejiang and Shanghai (Chen et al., 2003; 

Wang et al., 2010). C. sativus is regarded as “red gold”, 

for its unparalleled medicinal value. The dried stigmas of 

C. sativus have been used as a traditional Chinese 

medicine for a long time for its notable curative effects on 

promoting blood circulation by removing blood stasis, 

cooling blood detoxification and releasing depression 

(Board of Pharmacopoeia of the People’s Republic of 

China, 2010). Modern pharmacological studies have 

demonstrated that C. sativus have anticonvulsant, 

antidepressant, anti-inflammatory and antitumour effects 

(Hosseinzadeh and Khosravan, 2002; Hosseinzadeh and 

Sadeghnia 2005; Yang et al., 2011). However, natural 

sources of C. sativus are scarce. Overexploitation of their 

natural population for medicinal and commercial use and 

their low reproduction rate at cultivation are the main 

*Corresponding author. E-mail: sqszjfc@126.com. Tel/Fax: +86 

571 63740809. 

reasons for consumers' demand and their high prices. 

Many researches have tried to study artificial cultivation 

techniques of C. sativus since the 1970s in China. So far, 

great progress has been made in breeding, tissue culture 

and cultivation (Karaoğlu et al., 2007; Li, 2008). 

Many studies have examined consumer preferences 

and willingness for organic products and their influencing 

factors (Michael et al., 2008; Briz and Ward, 2009; 

Michaelidou and Hassan, 2010; Chen et al., 2011). 

Sepúlveda et al. (2008) used a logistic regression 

analysis to identify the factors that affect the purchase of 

quality-labelled beef in Spain. Hatirli et al. (2004) 

investigated the main factors affecting fluid milk 

purchasing sources of households in Turkey. Dai et al. 

(2006) investigated the impacts of socioeconomic, 

demographic characteristics, concerns over food safety 

and environment of consumers on organic vegetable 

purchasing behavior. Wang and Zhang (2009) studied 

consumer willingness to pay for Panax ginseng and its 

influencing factors by logistic model. However, influencing 

factors of consumers' willingness to pay for C. sativus 

were not reported. Studies about C. sativus industry are 

basically restricted to description of the qualitative phase. 

Li (2008) proposed that the key to sustainable


Table 1. Definition of variables. 

Variable name Variable definition 

Consumers' individual 

characteristic 

Health functions of the 

products 

Consumers' behavior 

Marketing factors 

A1 Male = 0, Female = 1 

A2 

Less than 29 years old = 1, 30-39 years old = 2, 40-49 years old = 3, 50-59 years old = 4, 

Over 60 years old = 5 

A3 High school graduates and below = 1, University graduates = 2, Post graduates = 3 

A4 

Less than 4000 RMB = 1, 4001-7000 RMB = 2, 7001-10000 RMB = 3, 10001-13000 RMB 

= 4, More than 13001 RMB = 5 

A5 Unmarried = 0, Married = 1 

A6 

Civil servant and public institution personnel = 1, Enterprise employee = 2, Service 

industries personnel = 3, Self employed = 4, Freelance = 5, Student =6, Others = 7 

B1 Very unimportant = 1, Unimportant = 2, No opinion = 3, Important = 4, Very important = 5 

B2 Very unimportant = 1, Unimportant = 2, No opinion = 3, Important = 4, Very important = 5 

C1 Very unimportant = 1, Unimportant = 2, No opinion = 3, Important = 4, Very important = 5 





D1 Very unimportant = 1, Unimportant = 2, No opinion = 3, Important = 4, Very important = 5 



Variables of the model are evaluated as: A1 gender, A2 age, A3 education, A4 household monthly income, A5 marital status, A6 profession, B1 

consumer attitudes of health functions, B2 consumer attitudes of active ingredient content, C1 taste of the products, C2 brand image, C3 comparative 

purchasing behavior, C4 gifts, C5 package, D1 price, D2 place, D3 media advertisement. 

development of industry is to establish a technological 

alliance, and speed up the development of common 

techniques and application of integrated innovations, to 

strengthen self-discipline and monitoring of production, 

and to expand sales market. Chen et al. (2001) 

considered that the potential market demand for C. 

sativus is huge, but we should pay attention to breeding, 

tissue culture and cultivation, guiding the consumers 

properly, and implementing the C. sativus industrialization 

project. With the product demand for C. sativus 

increasing rapidly, it is important for enterprises to 

establish the development strategy and provide policy 

advices based on market demand. 

The objective of this study is twofold: first, to estimate 

willingness to pay for C. sativus, and secondly, to 

examine heterogeneity in consumer valuation of 

functionality in C. sativus and to determine the effect of 

individual characteristics on consumers' choice decisions. 

This study contributes to the improvement and 

broadening of current knowledge about C. sativus 

demand in several ways. For this purpose, we analysed 

the variables of the consumers' individual characteristic, 

health functions of the products, consumers' behaviour 

and marketing factors. In addition, the results will also be 

of interest to government agencies and manufacturers 

who could use the information derived from this study in 

determining marketing strategies and setting up new 

policy tools. 

METHODOLOGY 

The data used in this paper was obtained from a survey on 

consumers' willingness to pay for C. sativus in June and July 2011. 

The survey was conducted face-to-face and through the internet. 

Considering the differences in economic development level and 

density of population among different cities and different districts 

within the cities, we used stratified sampling and random sampling 

to draw our subjects. The specific sites for investigation were 

chosen based on the administrative function zoning in Beijing, 

Shanghai, Hangzhou, Ningbo, Wenzhou, Shenyang, Guangzhou, 

Xi'an, Wuhan and Nanjing. 

Logistic model is used for analysis of consumers' willingness to 

pay for C. sativus. Logistic model describes the behaviour of 

consumers with a common consumption objective when they are 

faced with a variety of goods. However, the goods and choices 

must be highly differentiated by their individual attributes. Assuming 

that consumers' willingness to pay for C. sativus is dependent 

variable y, we divided influencing factors into four dimensions 

(Table 1):

consumers' individual characteristic (6 variables), health functions 

of the products (2 variables), consumers' behavior (5 variables) and 

marketing factors (3 variables). The model assumes that y obeys 

the binomial distribution, the probability y = 1 is equal to P, 

consumers' definite answers are represented by 1, negative 

answers are represented by 0. The probability distribution functions 

of y is expressed as follows: 

f 

y ( y) 

= p ( 1− 

p) 

( 1−y) 

, y = 0, 

1 

The probability of consumers' willingness to pay for C. sativus can 

be calculated according to the following equation: 

⎛ m ⎞ ⎡ ⎛ m ⎞⎤ 

p( y) 

= f ⎜α 

β jχ 

⎟ ij ⎢ ⎜ α β jχ 

⎟ 

⎜ 

+ ∑ ⎟ 

= 1/ 

1+ 

exp 

⎜ 

− + ∑ ij ⎟⎥ 

+ μi 

⎝ j= 

1 ⎠ ⎢⎣ 

⎝ j= 

1 ⎠⎥⎦ 

In this equation, P(y) is the probability of consumers' willingness to 

pay for C. sativus, χij is independent variables of No.j influencing 

factors, βj is the regression coefficients of No.j influencing factors, m 

is the number of the influencing factors, α is the regression 

intercept, µi is the random disturbance term of No.i observed 

objects. All the data analyses were carried out using SPSS17.0 

software. 

RESULTS 

A total of 467 respondents from the survey returned the 

completed questionnaires (response rate 46.7%). 138 of 

the respondents would pay for C. sativus, which 

accounted for 29.6%. Statistical description of the survey 

sample is presented in Table 2. In consumers' individual 

characteristic, gender, age, education, family monthly 

income, marital status and profession were investigated. 

The survey results demonstrate that the male 

respondents constitute 51.8% of the respondents while 

female respondents constitute 48.2% of it. 53.1% of the 

respondents are high school graduates and below, 33.6% 

of the respondents are university graduates and 13.3% of 

the respondents are post graduates. Average monthly 

income of sampled households which is less than 4000 

RMB accounts for 30.6%, 4001 to 7000 RMB accounts 

for 19.9%, 7001 to 10000 RMB accounts for 16.7%, 

10001 to 13000 RMB accounts for 13.9%, and more 

than13001 RMB accounts for 18.8%. In health functions 

of the products, consumer attitudes of health functions 

and active ingredient content are surveyed. Survey 

results revealed that 62.9% of the respondents consider 

health functions important or very important and 68.1% of 

it believes active ingredient content is important or very 

important. In consumers' behaviour, taste of the products, 

brand image, comparative purchasing behavior, gifts and 

package are surveyed. Survey results indicate that 82.0% 

of the respondents consider taste of the products 

important or very important and 73.0% of it believes 

brand image is important or very important. In marketing 

factors, price, place and media advertisement are 

investigated. The survey results demonstrate that 37.0% 

of the respondents consider price as unimportant or fairly 

unimportant while 31.9% of it believe price is important or 

(1) 

(2) 

Hong et al. 4425 

very important. 

KMO is a measure of sampling adequacy that indicates 

the proportion of common variance that might be caused 

by underlying factors. High KMO values (close to 1) 

generally indicate that factor analysis may be useful, 

which is the case in this study: KMO = 0.727, if the KMO 

test value is less than 0.5, factor analysis will not be 

useful. Bartlett’s test of sphericity indicates whether the 

correlation matrix is an identity matrix, indicating that 

variables are unrelated. A significance level less than 

0.05 indicates that there are significant relationships 

among variables, which is the case in this study: χ2 

=5206.487 (P=0.000). The Cronbach's alpha coefficient 

represents the degree of internal consistency of items 

within a test, and values can theoretically range from 0 

(zero internal consistency) to 1 (perfect internal 

consistency). In this study, the value of Cronbach's alpha 

is 0.108, when Cronbach's alpha value is up to 0.542 

after deleting A6 and D3. So, A6 and D3 were deleted; 

the remaining 14 items were selected to evaluate 

consumers' willingness to pay for C. sativus. In order to 

get 467 consumers' cross-sectional data, we used the 

logistic regression analysis. In the course of data 

processing, taking all the variables into it, obtained test 

results are shown in Table 3. Nagelkerke R² and Cox and 

Snell R² statistics attempt to quantify the amount of 

variance explained by the logistic regression model. The 

probability of the observed results, given the estimated 

parameters, is known as the “likelihood”. Since likelihood 

is a small number of less than one, it is customary to use 

“-2LL” (−2 log likelihood) as a measure of the model's 

goodness of fit. A good model is one in which there are 

high values of Nagelkerke R² and Cox and Snell R² 

statistics (the closer the values are to the unit, the higher 

is the variability of the endogenous variables ability to be 

explained) as well as high likelihood of the observed 

results (that is, with a low value of -2LL). In this study, 

Nagelkerke R² is 0.373, Cox and Snell R² is 0.262 and - 

2LL is 424.821. Model can better fit the overall sample 

data; independent variables have a good explanation for 

dependent variable. 

DISCUSSION 

Consumer preferences for certain food attributes are 

important for government agencies and manufacturers 

(Gao and Schroeder, 2009). In the past, different 

preference elicitation methods have been used by 

economists and market researchers to obtain the 

willingness to pay for certain product attributes. In this 

study, consumers' gender has a significant positive 

impact on willingness to pay for C. sativus. The survey 

results illustrate that female consumers are more willing 

to pay for C. sativus. They accept more information about 

C. sativus and are prone to purchase impulsively. 

Moreover, they are more sensitive to the advertisement, 

store promotions, packaging and so on. Therefore,


Table 2. Descriptive statistics of the surveyed consumers. 

Variable name Number/percent 

y No purchased: 329/70.4%, Purchased: 138/29.6% 

A1 Male: 242/51.8%, Female: 225/48.2% 

A2 

Less than 29 years old: 78/16.7%, 30-39 years old: 98/21.0%, 40-49 years old: 90/19.3%, 50-59 years old: 

97/20.8%, Over 60 years old: 104/22.3% 

A3 High school graduates and below: 248/53.1%, University graduates: 157/33.6%, Post graduates: 62/13.3% 

A4 

Less than 4000 RMB: 143/30.6%, 4001-7000 RMB: 93/19.9%, 7001-10000 RMB: 78/16.7%, 10001-13000 

RMB: 65/13.9%, More than 13001 RMB: 88/18.8% 

A5 Unmarried: 104/22.3%, Married: 363/77.7% 

A6 

B1 

B2 

C1 

C2 

C3 

C4 

C5 

D1 

D2 

D3 

Civil servant and public institution personnel: 113/24.2%, Enterprise employee: 50/10.7%, Service industries 

personnel: 59/12.6%, Self employed: 88/18.8%, Freelance: 31/6.6%, Student: 41/8.8%, Others: 85/18.2% 

Very unimportant: 0/0%, Unimportant: 67/14.3%, No opinion: 106/22.7%, Important: 145/31.0%, Very 

important: 149/31.9% 

Very unimportant: 0/0%, Unimportant: 27/5.8%, No opinion: 122/26.1%, Important: 212/45.4%, Very important: 

106/22.7% 

Very unimportant: 3/0.6%, Unimportant: 18/3.9%, No opinion: 63/13.5%, Important: 241/51.6%, Very important: 

142/30.4% 

Very unimportant: 11/2.4%, Unimportant: 43/9.2%, No opinion: 72/15.4%, Important:198/42.4%, Very 


Very unimportant: 40/8.6%, Unimportant: 66/14.1%, No opinion: 98/21.0%, Important: 154/33.0%, Very 

important:109/23.3% 











Variables of the model are evaluated as: A1 gender, A2 age, A3 education, A4 household monthly income, A5 marital status, A6 profession, B1 

consumer attitudes of health functions, B2 consumer attitudes of active ingredient content, C1 taste of the products, C2 brand image, C3 

comparative purchasing behavior, C4 gifts, C5 package, D1 price, D2 place, D3 media advertisement. 

Table 3. The test results of consuming model of C. sativus. 

Variable name Coefficient Standard error Wald test df Significance Exp (B) 

A1 0.968 0.253 14.603 1 0.000** 2.634 

A2 0.052 0.129 0.163 1 0.686 1.053 

A3 0.360 0.245 2.150 1 0.143 1.433 

A4 0.611 0.119 26.163 1 0.000** 1.842 

A5 0.772 0.384 4.052 1 0.044* 2.164 

B1 2.523 0.563 20.082 1 0.000** 12.469 

B2 1.351 0.397 11.558 1 0.001** 3.862 

C1 0.621 0.181 11.717 1 0.001** 1.861

Table 3. Contd. 

Hong et al. 4427 

C2 -0.235 0.133 3.134 1 0.077 0.790 

C3 -0.221 0.111 3.971 1 0.046* 0.802 

C4 0.524 0.365 2.058 1 0.151 1.688 

C5 -0.249 0.347 0.516 1 0.473 0.780 

D1 -1.298 0.493 6.937 1 0.008** 0.273 

D2 0.399 0.232 2.953 1 0.086 1.491 

Constant -18.786 3.281 32.788 1 0.000 0.000 

**, * indicate significance of the estimated non-zero coefficients 1 and 5%. 

gender can be an important part of the market 

segmentation in future research. Consumers' household 

monthly income has a significant positive impact on 

willingness to pay for C. sativus. The higher the 

household income is, the stronger the consumers' 

willingness to pay. As household income levels increase, 

the proportion of consumers to pay for C. sativus will rise. 

C. sativus is a non-necessity in daily life. Actually, it is 

currently considered the world's most expensive herbal 

medicine. Only the higher income level family can 

purchase it regularly. Consumers' marital status has a 

significant positive impact on willingness to pay for C. 

sativus. Married people are more willing to pay for C. 

sativus. Unmarried people lack the recognition of health 

functions of C. sativus, so it is not be included in their 

consumption expenditure. Different from unmarried 

people, married people pay more attention to health. 

Health expenditure accounts for a large proportion in their 

consumption expenditure. Married people have more 

willingness to pay for C. sativus. Consumers' attitude to 

health functions has a significant positive impact on 

willingness to pay for C. sativus. The dried stigmas of C. 

sativus have notable curative effects on promoting blood 

circulation by removing blood stasis, cooling blood 

detoxification, removing depression and anchoring mind. 

The more consumers care about health functions of C. 

sativus, the more it can stimulate their desire to pay for it. 

Consumer attitude of active ingredient content has a 

significant positive impact on willingness to pay for C. 

sativus. The main biologically active ingredient of C. 

sativus are crocins analogues (including crocin 1-4), a 

family of red-colored and water-soluble carotenoids. The 

more consumers care about active ingredient content of 

C. sativus, the more it can stimulate their desire to pay for 

it. Taste of C. sativus has a significant positive impact on 

willingness to pay for C. sativus. If consumers pay more 

attention to the taste of C. sativus, they are more willing 

to accept the product with a good taste. The C. sativus 

products including dried stigmas and extracts are mainly 

sold in the market. Their taste is not very good, with a 

slightly bitter taste. Technology can be used to adjust the 

taste to meet the consumers' demand. Consumers' 

comparative purchasing behavior has a significant 

negative impact on willingness to pay for C. sativus. The 

more discerning the consumers are, the more hesitating 

they are to pay for C. sativus. According to marketing 

theory, in the introduction period, advertisement focuses 

on developing consumers' awareness. In growth period, 

especially when the product is growing rapidly and 

transits to the maturity period, we should focus on 

cultivating consumers' understanding of the overall level 

of products. Only when consumers understand and 

accept the health functions of C. sativus, rather than 

purchase it as a novelty, will they have a positive 

response to the health products. Price of C. sativus has a 

significant negative impact on willingness to pay for C. 

sativus. Due to the high price, willingness to pay for C. 

sativus is reduced. Many consumers cannot afford such 

costly product. New agricultural production technologies 

should be adopted to improve the yield per unit area. Thus, 

the price of C. sativus can be cut down, enabling more 

consumers to pay for C. sativus. 

Based on the foregoing results, some policy 

implications are as follows: first, both government 

agencies and manufacturers should strengthen the 

propaganda of health function of C. sativus. The 

government should provide more information about C. 

sativus on the media, releasing relative scientific, 

objective. Thus, the degree of consumers' awareness 

and concern level regarding C. sativus can be improved. 

Secondly, the government should strengthen the 

supervision and regulation of the implementation of the 

project. Because of high price, there are many 

counterfeits in the market. The government should 

strengthen efforts to fight against counterfeiting, ensuring 

the stable development of the market. Thirdly, 

manufacturers should pay attention to the effect of 

consumers' personal characteristics on their purchase 

behavior, locate the target consumer rightly, choose 

appropriate marketing channels and advance marketing 

strategy for C. sativus. Finally, in the early stages of 

introduction period, the government should give more 

policy support such as providing financial credit at lowinterest 

rate, reducing tax, and offering subsidies. 

Conclusion 

In this study, we examined the impact of various factors


affecting on consumers' willingness to pay for C. sativus. 

For estimation technique, logistic model was specified 

and analyzed using survey data from China. The findings 

of the study reveal that gender, household monthly 

income, marital status, attitudes of health functions, 

attitudes of active ingredient content and taste of the 

products have a significant positive impact on willingness 

to pay for C. sativus, while the prices of the products and 

comparative purchasing behavior have a significant 

negative impact on willingness to pay for C. sativus. As a 

result, female consumers, married, with higher household 

monthly income are more willing to pay for C. sativus with 

good taste at lower prices. Results from this study may 

help government agencies and manufacturers in planning 

marketing strategies, targeting health information and 

anticipating future trends in the market. 

ACKNOWLEDGEMENT 

This study was supported financially by Zhejiang 

Province Traditional Chinese medicine modernization 

project (201124515). 

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Ethnopharmacy 13:45-46.



DOI: 10.5897/JMPR12.595 



Evaluation of biochemical, hematological and 

histopathological parameters of albino rats treated with 

Stemona aphylla Craib. extract 

Wararut Buncharoen, Supap Saenphet, Siriwadee Chomdej and Kanokporn Saenphet* 

Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand. 


In order to measure the safety of Stemona aphylla Craib., an effective insecticidal plant, on mammals, 

the effects of the ethanolic extracts from the root of S. aphylla on blood biochemical, hematological and 

histopathological indices of albino rats have been evaluated. Male rats were given extracts orally at the 

doses of 300 and 500 mg/kg body weight/day for 45 consecutive days. These were compared to control 

rats which received only distilled water. The results of this study showed no significant differences in 

aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, blood urea nitrogen and 

creatinine of all treated groups when compared to those of the control groups (P > 0.05). Nevertheless, 

an increase in lymphocytes count was observed in all treated groups. The alterations in liver and kidney 

tissues of all treated groups showed leukocyte infiltration and haemorrhage in hepatic sinusoids. 

Moreover, the contracted glomerulus, dilated renal tubules and leukocyte infiltration were found in 

kidney tissues. The significant injury of tissues observed in this study is a sign of the toxicity of S. 

aphylla to mammalian species. The use of S. aphylla as bioinsecticide active ingredient should be, 

therefore, thoroughly considered. 

Key words: Biochemistry, hematology, histopathology, rats, Stemona aphylla Craib. 

INTRODUCTION 

The use of synthetic insecticides is still the main strategy 

to control insect pests in agriculture, industry, medicine 

and households. The massive use of synthetic insecticides 

in agriculture has created enormous problems 

worldwide. These insecticides have serious drawbacks, 

for example, accumulation in the environment including 

human health and non-target organisms (Groves and 

Chapman, 2011). Humans can be exposed to insecticides 

by food consumption, inhalation, or direct contact 

with the skin. To reduce these problems, recent interest 

has been focused on using bioinsecticides derived from 

plant materials as alternatives to control insect pests. 

Many plants species have been reported to have 

insecticidal properties such as Azadirachta indica A. 

Juss, Acorus calamus L., Eupatorium odoratum L. and 

*Corresponding author. E-mail: k_saenphet@yahoo.com. Tel: 

+66-86-6050743. Fax: +66-53-892259. 

Mammea siamensis Kosterm (Delobel and Malonga, 

1987; Issakul et al., 2007; Nandi et al., 2008; Satti et al., 

2010). Stemona aphylla Craib. is a member of the 

Stemonaceae family and it is known as Non Tai Yak in 

Thailand. The tuberous root of this plant has long been 

used as an insecticide. Chemical constituents of the plant 

extract include stemofoline, (2’S)-hydroxystemofoline, 

stemaphylline, (11Z)-1’,2’-didehydrostemofoline, 

stemaphylline-N-oxide, (Mungkornasawakul et al., 2009) 

stemofurans M-R, stemofuran E, stemofuran F, 

stemofuran J and stilbostemin F (Sastraruji et al., 2011). 

The insecticidal activity of these compounds have been 

well documented (Brem et al., 2002; Mungkornasawakul 

et al., 2009; Sastraruji et al., 2011; Tang et al., 2008). 

Thus, this plant species may be promoted and developed 

as bioinsecticide products on an industrial scale, if it 

causes no toxicity to non-target organisms. Nevertheless, 

quite a bit of research has been reported on the toxicity of 

bioinsecticide derived from plant materials to mammals 

(Barbosa et al., 2008; Katsayal et al., 2008). Nowadays,


the users increasingly express an interest in the safety of 

bioinsecticides. Therefore, information about the safety of 

plants used as insecticides on mammals is still essential. 

This study aimed to evaluate the effects of the ethanolic 

extract from the root of S. aphylla on biochemical, 

hematological and histopathological indices of male 

albino rats. The data obtained from our study may be 

used to assess the safety levels of this plant in mammals 

and any benefit which may be received from the 

development of bioinsecticide products. 


Plant 

Fresh roots of S. aphylla were collected from Lampang province, 

Thailand. The plant material was identified by the botanist from the 

Department of Biology, Faculty of Science, Chiang Mai University. 

A voucher specimen (number 09-111) was deposited at CMU 

herbarium, Department of Biology, Faculty of Science, Chiang Mai 

University, Chiang Mai, Thailand. 

Preparation of the plant extract 

The roots of S. aphylla were washed with tap water, cut and dried in 

an oven (50°C) to a consistent weight. The dried roots were soaked 

in 95% ethanol for 3 days. The obtained extract was then filtered to 

remove any residue, and then, it was evaporated by a vacuum 

rotary evaporator to obtain the crude ethanolic extract. The 

percentage yield of the extract was 7.92% (w/w). The ethanolic 

extract was stored at 4°C until used. The residue was suspended in 

distilled water, and required doses were prepared for future 

experiments. 

Animals 

Male albino rats (Rattus norvegicus), 4 to 5 weeks of age, weighing 

between 100 to 120 g, were purchased from the National 

Laboratory Animal Center, Mahidol University, Salaya Campus, 

Thailand. 

The animals were housed in sanitary cages and had access to 

tap water and a standard diet (C. P. 082). The room temperature 

was controlled at 24 to 26°C in a 12 h light/dark cycle. All procedures 

involving the animals were conducted with strict adherence 

to guidelines and procedures reviewed and approved by the 

Institutional Animal Care and Use Committee of the Biology 

Department, Faculty of Sciences, Chiang Mai University. 

Animal treatments 

The rats were randomly divided into 3 groups (eight rats per group). 

The animals in each group were given the root extracts of S. 

aphylla orally at the doses of 300 and 500 mg/kg body weight daily 

(1 ml/day) for 45 days. Control rats received only distilled water. 

The selected doses in this study were based on the doses used in 

chronic toxicity tests of this plant reported in a previous study 

(Pandee et al., 2003). 

Serum and tissues preparation 

At the end of the treatment period, the animals were sacrificed and 

blood samples were collected into non anti-coagulated and 

ethylenediaminetetraacetic acid (EDTA) anti-coagulated tubes. The 

non anti-coagulated blood was then centrifuged at 3,000 rpm for 10 

min and serum was collected. Finally, the animals were quickly 

dissected. Livers and kidneys were carefully removed. Portions of 

these organs were fixed in Bouin’s solution for histopathological 

examination. 

Blood biochemical determinations 

The level of clinical biochemistry such as aspartate aminotransferase 

(AST), alanine aminotransferase (ALT), alkaline 

phosphatase (ALP), blood urea nitrogen (BUN) and creatinine were 

evaluated to determine the function of the livers and kidneys of the 

control groups and the treated rats. 

All blood biochemical parameters were determined by automated 

photometric systems with the cooperation of the Veterinary 

Diagnostic Laboratory, Faculty of Veterinary Medicine, Chiang Mai 

University. 

Hematological studies 

EDTA anti-coagulated blood samples were used to assay 

hematological parameters. Total red blood cell counts (TRBC) were 

determined by diluting blood samples with Grower’s solution, and 

then red blood cell were counted in 5 red blood cells square of 

hemocytometer. Total white blood cell counts (TWBC) were 

counted in Neubauer’s hemocytometer after the blood samples 

were diluted (1:20) in Turk’s solution. The hematocrit (HCT) was 

measured by filling blood into a heparinized capillary tubes and 

centrifuged at 11,000 rpm for 5 min. The HCT values were 

measured with hematocrit reader. 

Hemoglobin (HGB) concentration was done by adding 20 µl of 

the blood sample into 5 ml of Drabkin’s solution. The optical density 

was measured in a spectrophotometer at 540 nm and was 

calculated from HGB standard curve. Some hematological parameters 

such as mean cell volume (MCV), mean corpuscular 

hemoglobin (MCH) and mean corpuscular hemoglobin concentration 

(MCHC) were also calculated. For determining the 

percentage of leukocytes, the blood smear was made and stained 

with Wright’s dye. Finally, percentage of lymphocyte, monocyte, 

neutrophil, eosinophil and basophil were counted under light 

microscope. 

Histopathological studies 

The fixed tissues of rats were dehydrated with progressively 

increasing concentrations of ethanol. The tissues were passed 

through xylene solution to clear the ethanol and facilitate molten 

paraffin wax infiltration (55°C). 

After that, they were embedded in a wax block. Paraffin sections 

of 6 µm thickness were cut with the rotary microtome and placed on 

cleaned glass slides. Finally, the sections were stained with 

hematoxylin and eosin. The stained slides were examined using a 

light microscope where the photomicrographs of the tissue samples 

were recorded. 


For statistical analysis, data were analyzed by one-way analysis of 

variance (ANOVA) using Statistical Package of Social Sciences 

(SPSS) software version 16 for Windows. The results were 

expressed as mean ± standard error (SE). A level of P value less 

than 0.05 was considered to be significant.

RESULTS 

Buncharoen et al. 4431 

Table 1. Serum biochemistry in rats treated with 300 and 500 mg/kg body weight of S. aphylla root extracts for 45 days 

as compared to the control group. 

Biochemical index 

Control 

Treatment 

300 (mg/kg body weight) 500 (mg/kg body weight) 

AST (IU/L) 136.67 ± 24.49 a 107.25 ± 18.12 a 115.50 ± 16.70 a 

ALT (IU/L) 28.83 ± 2.23 a 25.25 ± 2.63 a 23.00 ± 2.31 a 

ALP (IU/L) 96.83 ± 7.68 a 96.60 ± 16.55 a 92.25 ± 16.77 a 

BUN (mg/dl) 20.08 ± 2.38 a 19.46 ± 3.80 a 19.88 ± 2.68 a 

creatinine (mg/dl) 0.70 ± 0.03 a 0.64 ± 0.04 a 0.69 ± 0.04 a 

All values are expressed as mean ± SE. A same superscript letters within each row are not significantly different (P > 0.05). 

Table 2. Hematological values in rats treated with 300 and 500 mg/kg body weight of S. aphylla root extracts for 45 days as 

compared to the control group. 

Hematological parameter 

Control 

Treatment 

300 mg/kg body weight 500 mg/kg body weight 

TRBC (cells/ml) 5.70 ± 1.90 a 6.95 ± 1.02 a 6.07 ± 1.22 a 

TWBC (cells/ml) 6.53 ± 2.85 a 5.33 ± 2.02 a 5.26 ± 2.10 a 

HGB (g/dl) 17.05 ± 3.66 a 18.57 ± 3.70 a 18.87 ± 4.09 a 

HCT (%) 48.51 ± 2.93 a 50.67 ± 3.01 a 52.25 ± 2.60 a 

MCV (fl) 95.21 ± 38.43 a 73.97 ± 9.45 a 88.94 ± 17.88 a 

MCH (pg) 32.07 ± 9.52 a 27.27 ± 7.31 a 32.21 ± 8.54 a 

MCHC (g/dl) 35.59 ± 9.35 a 37.04 ± 9.20 a 36.19 ± 7.44 a 

Lymphocyte (%) 76.71 ± 1.11 a 80.71 ± 1.38 b 82.43 ± 1.27 b 

Neutrophil (%) 16.38 ± 3.07 a 14.63 ± 2.13 a 13.88 ± 1.55 a 

Eosinophil (%) 1.13 ± 0.64 a 0.75 ± 0.46 a 0.88 ± 0.64 a 

Basophil (%) 1.00 ± 0.00 a 0.71 ± 0.49 a 0.86 ± 0.69 a 

Monocyte (%) 4.71 ± 2.36 a 2.86 ± 0.69 a 2.71 ± 0.76 a 

All values are expressed as mean ± SE. A different superscript letters within each row are significantly different (P < 0.05). 

Blood biochemical determinations 

The values of biochemical parameters of liver and kidney 

functions in this study are shown in Table 1. There were 

no significant differences (P > 0.05) in AST, ALT, ALP, 

BUN and creatinine of rats treated with S. aphylla extract 

at the doses of 300 and 500 mg/kg body weight when 

compared with those of the control group. All serum 

biochemical values were normal and within the reference 

ranges for rats. The reference ranges of AST, ALT, ALP, 

BUN and creatinine were 50 to 150, 10 to 40 and 30 to 

130 IU/L, 12.0 to 25.8 and 0.4 to 2.3 mg/dl, respectively 

(Sharp and La Regina, 1998). 

Hematological studies 

The results of hematological parameters were listed in 

Table 2. There were no significant changes in TRBC, 

TWBC, HGB, HCT, MCV, MCH and MCHC of rats treated 

with S. aphylla extracts at the doses of 300 and 500 

mg/kg body weight when compared with those of controls 

(P > 0.05). However, an increase in lymphocytes count 

was observed in all the treated groups. 

Histopathological studies 

Histopathological alterations were observed in liver 

tissues of rats treated with S. aphylla extracts at the 

doses of 300 and 500 mg/kg body weight. The sections 

of liver tissue in both treated groups showed similar 

damage, such as infiltration of leukocytes (Figure 1C) 

and haemorrhage in hepatic sinusoids (Figure 1D), 

whereas the normal features of liver tissue in the control 

group showed regular cords of hepatocytes with central 

vein and portal triad (Figure 1A and B). Moreover, the 

histological alteration was also observed in the kidneys of 

all treated groups. The renal tissues of the treated rats 

showed similarly significant changes, such as contracted


Figure 1. Photomicrographs of rat liver sections: A and B showing the normal central vein (CV) with radiating 

cords of hepatocytes and portal traid (circle) in control group; C and D showing leukocyte infiltration (thick arrows) 

and haemorrhage (thin arrows) in liver tissues of rats treated with S. aphylla extracts. (H & E; 20x). 

glomerulus, dilation of renal tubules and infiltration of 

leukocytes around blood vessels (Figure 2B to D). The 

kidney sections of the control group showed normal renal 

corpuscle and renal tubules (Figure 2A). 

DISCUSSION 

Although, many people believe that the use of biological 

agents is safe, quite a few scientific papers have reported 

the toxicity of bioinsecticides derived from plant materials 

on mammals and non-target organisms. Moreover, a lack 

of knowledge of the standardized dosage of biological 

substances may also be leading to toxicity. Therefore, 

any information on plants toxicity is still important. Although, 

the scientific evaluations on insecticidal efficiency 

of S. aphylla have been reported (Brem et al., 2002; Tang 

et al., 2008), its effects on livers and kidneys are not yet 

known. The present study was designed to evaluate the 

effects of the ethanolic extract from the roots of S. aphylla 

on male albino rats by focusing on the alterations of 

structures and functions of livers and kidneys and 

hematological indices. The results of our study showed 

no significant differences in AST, ALT, ALP, BUN and 

creatinine values of rats treated with S. aphylla extracts 

at the doses of 300 and 500 mg/kg body weight when 

compared to those of the control group (Table 1). All 

these clinical parameters were normal and within the 

reference ranges of rats (Shape and La Regina, 1998). 

These results indicated that giving S. aphylla extracts 

orally at the doses and duration investigated in this study 

did not affect liver and kidney functions. Some studies 

reported that female mice treated with the extract from 

Stemona curtisii. Hk. f., a member of Stemonaceae 

family, showed no biochemical changes as compared to 

the control group (Pandee et al., 2003). Livers and 

kidneys are internal organs which have several functions. 

One important role of these organs is the elimination of 

waste products and toxic substances. Dysfunctions of 

these organs lead to leaking of biochemical substances 

into the blood circulatory system (Gaw et al., 1999; Moss 

and Henderson, 1996). Therefore, increasing biochemical 

values in blood is a sign of abnormal liver and kidney 

function due to decreased excretion of waste products. 

Our results reveal that the ethanolic extract from the root 

of S.


Figure 2. Photomicrographs of rat kidney sections showing the normal renal corpuscle (RC) and renal tubules 

(RT) in control group (A). Kidney sections of rats treated with S. aphylla extracts showing contracted glomerulus 

(circle), dilated renal tubules (thin arrows) and leukocyte infiltration surrounding blood vessels (thick arrow) (B, C 

and D). (H & E; 20x). 

aphylla at the doses and treatment period in this study 

did not cause any observable blood biochemical changes 

in male rats. Hematological parameters are important 

indices of the physiological and pathological status for 

both animals and humans (Adeneye et al., 2006). 

Administration of the extract from the root of S. aphylla 

for 45 consecutive days could not alter hematological 

parameters, such as TRBC, TWBC, HGB, HCT, MCV, 

MCH and MCHC of all treated groups when compared 

with those of the control groups. 

However, an elevation in lymphocytes count in all 

treated groups was observed. Lymphocytes are involved 

in a variety of immunological function, such as immunoglobulin 

production and modulation of immune defense 

(Campbell, 1996). The alteration in lymphocyte count 

reflects possible leukopoietic and immunomodulatory 

effects of S. aphylla root extract in rats. It is possible that 

the extract composed of bioactive ingredients containing 

hematopoietin-like principle which is responsible for 

hematopoietins synthesis or release from hematopoietic 

organs, such as the kidneys and liver (Adeneye, 2008; 

Palani et al., 2009). Moreover, an elevation in 

lymphocytes count in treated groups might be associated 

with chronic inflammation of liver and kidney of rats after 

administration of S. aphylla extracts. Histopathological 

finding in a recent study revealed mild inflammation in 

liver and kidney tissues of all treated groups, in 

comparison to the control groups that showed no 

histological alteration. Liver tissue of all treated groups 

showed similar alterations, such as leukocyte infiltration 

around hepatic triad and haemorrhage in hepatic 

sinusoids (Figure 1C and D). 

Furthermore, contracted glomerulus, dilated renal 

tubules and leukocyte infiltration in kidney tissues of all 

treated rats were also found (Figure 2B to D). White 

blood cells contribute to inflammatory mechanisms. 

Therefore, leukocyte infiltration is the responses of the 

immune system to toxic substances which enter into the 

body. Contracted glomeruli were observed with a small 

tuft of capillaries, and a large space between glomerulus 

and Bowman’s capsule in renal corpuscle. 

Renal tubules dilation revealed low cuboidal epithelial 

cells when compared with epithelium of normal tubules. 

Plants which have the efficiency to control insect pests 

have been reported to cause hepato- and reno-toxicity in 

mammals. The administration of A. indica extract in rats


at a high dose showed leukocyte infiltration and 

apoptosis of hepatocytes in liver tissue and congestion of 

red blood cells in glomerulus (Katsayal et al., 2008). 

Microscopic evaluation in goats receiving Nerium 

oleander revealed renal necrosis at convoluted tubules 

and collecting ducts (Barbosa et al., 2008). Furthermore, 

the other plant species, such as Galega officinalis, 

Ageratum conyzoides L., Calendula officinalis and 

Cedrus deodara have been reported as toxic in some 

mammalian species (Adebayo et al., 2010; Lagarto et al., 

2011; Parveen et al., 2010). Histological alteration found 

in this study may be due to the bioactive ingredients 

contained in this crude extract which affects the tissues 

through various mechanisms. It has been reported that 

the roots of S. aphylla contained a variety of alkaloids, 

such as stemofoline, stemaphylline, stemofuran and 

stilbostemin (Mungkornasawakul et al., 2009). Previous 

studies have indicated the hepatotoxic effects of nicotine, 

the principal alkaloid contained in tobacco, on vital 

organs of rats. The results appeared in the deposition of 

adipose around the portal vein in the liver; necrosis, 

congestion and fibrosis in brain, liver and kidney tissues 

(Iranloye and Bolarinwa, 2009). In rats receiving 

swainsonine, the alkaloid contained in the leaves of 

Ipomoea carnea revealed a vacuolation in the renal 

convoluted tubular epithelium (Hueza et al., 2005). 

Moreover, swainsoine has been reported to have 

potential to inhibit the lysosomal �-amannosidase which 

causes accumulation of incompletely processed oligosaccharides 

into lysosomes. This results in the loss of 

cellular function and ultimately cell death (Tulsiani et al., 

1998); therefore, it is possible that the tissue alterations 

which occurred in this study were a result of the action of 

the alkaloids compound contained in the roots of this 

plant. 

In this study, it was concluded that the ethanolic extract 

from the root of S. aphylla administered orally does not 

alter liver and kidney functions in rats. However, a 

significant alteration in some hematological and histological 

parameters obviously found in this study is a sign of 

toxic effects of this plant on mammalian species. The use 

of S. aphylla as bioinsecticide active ingredient should 

be, therefore, thoroughly considered. 


The authors wish to thank Mr. Suluk Wutteerapol for his 

assistance through the consultation in laboratory 

techniques. The authors would like to express their 

appreciation to Mr. James F. Maxwell, the botanist of 

Department of Biology, Faculty of Science, Chiang Mai 

University for plant identification. 

Their sincere thanks also extend to Graduate School, 

Chiang Mai University and the National Research 

University Project under Thailand’s Office of the Higher 

Education Commission for financial support. Finally, they 

thank Mr. Jack Hines from Kiatphatthana Language and 

Computer School (CEC), Chiang Mai for his review on 

the manuscript. 

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DOI: 10.5897/JMPR12.778 



Assessment of fluoride, chloride and sulfate 

contamination of herbal teas, and possible interference 

with the medicinal properties 

Mohamed Yehia Abouleish* and Naser Abdo 

Department of Biology, Chemistry and Environmental Sciences, College of Arts and Sciences, 

American University of Sharjah, P. O. Box 26666, Sharjah, United Arab Emirates. 

Accepted 22 June, 2012 

Herbal teas are sold for medicinal purposes, as well as a beverage. Herbal teas may consist of many 

ingredients, mainly botanical ingredients, and therefore are susceptible to contamination from the 

environment and manufacturing processes, which can interfere with their medicinal purposes. This 

research investigated the contamination of herbal teas by fluoride, chloride and sulfate. The results 

demonstrated that none of the samples contained fluoride. As for chloride and sulfate levels, some 

samples exceeded the US Environmental Protection Agency (EPA), US Food and Drug 

Administration (FDA) and European Commission (EC) permissible levels (250 mg/L) for drinking water, 

while others demonstrated high levels but did not exceed the permissible levels. Some samples 

exceeded the 500 mg/L sulfate level suggested by the World Health Organization (WHO) to cause health 

problems. This study demonstrated that the herbal teas contamination is from the herbal teas 

ingredients and not only from the water used for the preparation. Contamination of the herbal teas and 

sometimes exceeding the permissible levels could lead to health problems, interfere with the function, 

and therefore defy the purpose of using it. Therefore, to ensure the effectiveness of the herbal teas as 

an alternative medicine and avoid health problems, herbal teas should fall under stringent regulations 

concerning planting, harvesting, manufacturing, and ensuring quality control. 

Key words: Fluoride, chloride, sulfate, contamination, herbal tea, botanical product, US Environmental 

Protection Agency (US EPA), World Health Organization (WHO). 

INTRODUCTION 

Herbal tea is sometimes referred to as a botanical 

product, as it may contain either a single or a multivegetable 

ingredient (US FDA, 2004). Herbal teas are 

sold under many brands and serve an extensive list of 

uses from a beverage to serving as a supplement for 

medicinal purposes (AHPA-ERB, 2008; Peter, 2004; 

Woolf, 2003). For example, some herbal teas are used 

for digestion, respiratory and gas problems (Zhang et al., 

2011). Herbal teas consist of botanical extracts - that is 

plants that are grown in the environment, and accordingly 

they are susceptible to contamination from environmental 

pollution caused by human activities (Rembialkowska, 

*Corresponding author. E-mail: mabouleish@aus.edu. Tel: 971- 

6-515-2480. Fax: 971-6-515-2450. 

2007; Woolf, 2003). Herbal teas exist on the market in 

the processed and unprocessed forms, and as a result, 

handling and manufacturing processes may act as a 

source of contamination for the products as well (Woolf, 

2003). 

Both chemical and biological contamination of the 

herbal tea products are a serious matter and can arise 

from the drinking water used for the preparation of the 

herbal teas. Chemical contamination of the drinking water 

is regulated by the United States Environmental 

Protection Agency (US EPA) National Drinking Water 

Regulations (NDWR), United States Food and Drug 

Administration (US FDA) and other international organizations, 

depending on the country (US EPA, 2009; US 

FDA, 2009). Some of the contaminants that are 

considered under the NDWR can cause either health 

problems, or cosmetic and aesthetic effects (US EPA,

Table 1. Herbal tea samples. 

Abouleish and Abdo 4437 

Herbal sample 1 2 3 4 5 6 7 8 9 10 

Herbal Ingredient 

(Label information) 

Recommended age 

(month) 

Fennel Chamomile 

Anise, balm, chamomile, 

fennel, peppermint, thyme 

Fennel Herbal extract 

Hibiscus, orange, peach, 

raspberry, rose hip 

Fennel Fennel 

Anise, balm, chamomile, 

fennel, peppermint, thyme 

Birth 2 6 6 4 6 Birth 6 6 2 

Fluoride (mg/L) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 

Chloride a (mg/L) 445.82±0.77 68.42±0.15 91.43±0.48 76.32±0.60 186.52±0.075 101.85±0.15 3349.5±2.52 79.19±0.82 98.12±0.19 70.47±0.35 

Sulfate a (mg/L) 942.01±1.76 108.90±2.88 298.76±2.28 167.64±0.68 404.48±2.54 158.12±0.40 9352.4±10.28 103.52±0.28 300.64±0.34 113.69±2.04 

a Anion concentration ± standard deviation. 

2009). Some of the contaminants that fall under 

the NDWR include fluoride, chloride and sulfate. 

Contamination of the final product, whether from 

the herbal product’s ingredients or the drinking 

water used for preparation, will add to the overall 

contamination and could have side effects. For 

example, according to the World Health Organization 

(WHO), if sulfate that usually exist in the 

drinking water are present at low levels, there are 

no associated health effects. However, if sulfate 

exists in the drinking water at a very high 

concentration (above 500 mg/L), it may lead to 

gastrointestinal problems such as diarrhea and 

intestinal pain in babies (WDHS, 2000; WHO, 

2011). Therefore, there is a concern over the 

toxicity of such products (Woolf, 2003) and if there 

is any interference with the intended purpose for 

using the herbal teas, since if such conditions 

exist, health problems may occur especially for 

children (Bakerink et al., 1996; Ize-Ludlow et al., 

2004; Saper et al., 2004; Tomassoni and Simone, 

2001; Woolf, 2003). 

Therefore, the goal of this research is to study 

the different herbal teas - that are marketed for 

infant and children use, and could be used by 

adults, in the United Arab Emirates (UAE) market 

- for possible contamination with fluoride, chloride, 

and sulfate. Accordingly, this study will shed light 

on the chemical contamination of the herbal 

products and the quality of the herbs used in the 

preparation of the products, and whether the 

contamination in the final herbal tea product is 

originating from the herbs or from the water used 

for the preparation. In addition, it will indicate 

whether the herbal teas can possibly have side 

effects on human health, especially in sensitive 

people, children and babies, and accordingly, 

interfere with their intended medicinal use. 


Herbal tea samples 

Ten herbal tea brands that represented all the brands on 

the UAE market, were purchased from local markets in 

Dubai, Abu Dhabi, and Sharjah in UAE (Table 1). The ten 

herbal tea brands are marketed for infant and children 

intake and could be used by adults (Table 1). Herbal tea 

samples varied in ingredients, form, flavor, smell and 

texture (Table 1). The herbal tea samples were in the 

processed form (pre-mixed with other ingredients and 

cooked) when purchased, except for sample 7 that was in 

the unprocessed form (stems and leaves). Except for 

Chamomile 

sample 7, all of the other herbal samples contained, in 

addition to the herbal ingredient(s), other ingredients that 

were reported on the labels of the product; for example, 

glucose, sucrose, dextrose, maltodextrin, acids (citric and 

ascorbic), and vegetable oil. The herbal tea samples were 

manufactured in Germany. The samples were analyzed 

after purchase for fluoride (F - ), chloride (Cl - ) and sulfate 

(SO4 2- ) anions. 

Each herbal tea sample was prepared for analysis by 

dissolving 1 g of the sample in 10 mL de-ionized water 

(Millipore “Simplicity” Purification System, USA). The 

samples were then filtered through a 0.45 μm membrane 

filter (Schleicher and Schuell, UK), prior to ion chromatography 

(IC) analysis. The preparation of the samples did 

not involve any heating. 

Fluoride, chloride and sulfate analysis 

A Waters IC system, consisting of an IC-Pak Anion HR 

4.6X75 mm column, 616 pump with 600 S controller, 717 

plus autosampler, 432 conductivity detector, and 

Millennium 32 software, was used for the analysis of the 

anions at room temperature. The isocratic mobile phase 

was prepared by mixing 20 mL of n-butanol (HPLC grade, 

Hipersolv, UK), 120 mL of acetonitrile (HPLC grade, 

Hipersolv, UK), 20 mL of sodium borate/gluconate 

concentrate (18 g boric acid, 16 g sodium gluconate, 25 g 

disodium tetraborate decahydrate and 250 mL glycerin 

diluted to 1 L with deionized water), then diluted to 1 L by


Figure 1. Comparison of sulfate and chloride levels in the herbal tea samples. 

Table 2. Fluoride, chloride, and sulfate set standard limits. 

Organization 

Anions (mg/L) 

Fluoride Chloride Sulfate 

US EPA 2.0* and 4.0** 250.0** 250.0** 

US FDA 1.7 250.0 250.0 

WHO 1.5 - - 

EC 1.5 250.0 250.0 

* US EPA permissible level, under the NPDWR; ** US EPA permissible level, under the NSDWR. 

de-ionized water, and then homogenized, filtered through a 0.2 μm 

membrane filter (Schleicher & Schuell, Germany), and degassed by 

sonication. A 50 μL injection volume was used for the standards, 

and depending on the concentration of the anions in the samples, a 

5 to 100 μL injection volume was used for the samples. A 1.0 

mL/min IC flow rate was used. 

A standard stock solution was prepared from a multi-ion 

standard (Seven Anion Standard II Standard, Dionex, USA) and 

was used for preparing the different calibration curves (correlation 

coefficient ≥ 0.99) for all of the measured anions. The calibration 

curves were prepared with a concentration range of 0 - 10 mg/L 

fluoride; 0 - 50 mg/L chloride; and 0 - 50 mg/L sulphate. The limit of 

detection (LOD) and limit of quantitation (LOQ) for the measured 

anions are as follows, for fluoride 0.10 and 0.35 mg/L, for chloride 

0.39 and 1.29 mg/L, and for sulfate 1.16 and 3.88 mg/L, respectively. 

For the measured anions, the recovery of the applied 

method of analysis was accomplished by spiking the prepared 

herbal tea samples with aliquots of the anion standard, and then 

analyzed. Recovery of fluoride was 108.05 – 127.67%, chloride was 

93.75 – 94.46%, and sulfate was 91.92 – 92.77%; with a standard 

deviation ranging from 0.000 - 0.014 for fluoride, 0.004 - 0.010 for 

chloride, and 0.086 - 0.199 for sulfate. 

RESULTS AND DISCUSSION 

SO4 

The fluoride, chloride and sulfate levels were measured 

for all the herbal tea brands (Table 1 and Figure 1). The 

US EPA, US FDA and European Commission (EC) have 

set standard limits or suggested values for fluoride, 

chloride and sulfate in drinking water (Table 2) based on 

the probable effects that they may have (EC, 1998; US 

EPA, 2009; US FDA, 2009). Unlike the other organizations, 

the WHO provides no guideline value for 

chloride and sulfate, but only for fluoride (Table 2) (WHO, 

2011). Some of the contaminants that are in drinking 

water are considered under the National Primary Drinking 

Water Regulations (NPDWR) standards and cause

Figure 2. Chloride levels in herbal tea samples compared to the set standard limit ( 250 

mg/L). 

health problems, while others cause more of cosmetic 

and aesthetic effects and are considered under the 

National Secondary Drinking Water Regulations 

(NSDWR) standards (US EPA, 2009). Under the US EPA 

regulations, some of those contaminants (e.g., fluoride 

only in this research) fall under both the NPDWR and 

NSDWR, as depending on the level of contamination, 

they could have different side effects (US EPA, 2009). 

Fluoride anion 

Fluoride is found in soil, water, and rocks naturally 

(Sendesh Kannan and Ramasubramanian, 2011). 

Fluoride is usually added to drinking water for dental 

care, but if fluoride is present above the permissible 

levels in drinking water (Table 2), it could lead to teeth 

and bone problems (US EPA, 2009; D’Alessandro et al., 

2008). None of the analyzed herbal tea samples (Table 

2) showed fluoride contamination. 

Chloride and sulfate anions 

Chloride is present naturally in the environment (soil and 

water) as a result of weathering of rocks (Chutia and 

Sarma, 2009; WHO, 2003). Human activities increase the 

levels of chloride in the environment through the use of 

inorganic fertilizers and discharge of agricultural waste 

water, runoff from de-iced roads, leachate from landfills, 

industrial and septic tank effluents, animal feed, and 

intrusion of sea water (D’Alessandro et al., 2008; Chutia 

and Sarma, 2009; DNHW, 1978; WHO, 2003). All such 

activities can lead to contamination of water bodies and 


as a result, contaminate our environment, water and 

food. 

Chloride is considered under the NSDWR regulations 

according to the US EPA; therefore, it has an aesthetic 

and cosmetic effect (US EPA, 2009). At concentrations 

higher than 250 mg/L, the taste of the water or product 

will change (WHO, 2011). Chloride is present in nature in 

different forms, e.g., calcium chloride, sodium chloride 

and potassium chloride (WHO, 2003). Therefore, 

depending on the cation (e.g., calcium, sodium or 

potassium), the taste threshold changes (WHO, 2003). In 

this study, samples 1 and 7 demonstrated chloride 

concentration above the permissible levels for the US 

EPA, US FDA, and EC (Tables 1 and 2, and Figure 2). 

The other samples demonstrated high chloride levels 

(68.42 - 186.52 mg/L range), but below the permissible 

levels set by the U.S. and international organizations 

(Tables 1 and 2). The chloride contamination in the 

samples is from the herbal ingredients, as the water used 

in this research for the preparation of the herbal tea 

samples contained no chloride. The presence of such 

high levels may have an effect on the taste of the herbal 

tea product. 

Sulfates are present naturally in the environment as a 

result of decaying animals and plants, yet human 

activities, such as the excessive use of sulfate fertilizers 

and the release of industrial and residential waste into the 

environment, cause the contamination levels to increase 

in rivers, lakes, and groundwater (Chutia and Sarma, 

2009; WDHS, 2000) and eventually contaminate our 

drinking water and food. Standard limits are set for 

sulfates by the US EPA, US FDA, and EC (Table 2) (EC, 

1998; US EPA, 2009; US FDA, 2009). Sulfate is 

considered under the NSDWR regulations, according to


Figure 3. Sulfate levels in the herbal tea samples compared to the set standard limits ( 250 

mg/L; 500 mg/L). (a) 0 - 10,000 mg/L sulfate; and (b) 0 – 1,000 mg/L sulfate. 

the US EPA (US EPA, 2009). In this study, 5 of the 10 

samples experienced high concentrations above the 

permissible levels, 250 mg/L (Table 1 and Figure 3). 

Therefore, those samples would have a different taste 

that is noticeable when consumed by normal people and 

especially by sensitive people, children, and babies who 

may experience a change in taste at 200 mg/L sulfate 

(WDHS, 2000). According to the WHO and other 

agencies, sulfate levels above 500 mg/L may cause the 

(a) 

(b) 

consumer to experience some gastrointestinal problems, 

e.g., laxative effect and intestinal pain, especially in 

children and sensitive people (WHO, 2011; WDHS, 

2000). Out of the 5 samples that demonstrated sulfate 

levels above the permissible levels (250 mg/L), only 

samples 1 and 7 demonstrated sulfate levels above the 

500 mg/L (Table 1 and Figure 3). Therefore, sensitive 

people, children and babies who consume those samples 

may experience, in addition to change in taste, some

Table 3. Herbal tea ingredients and intended medicinal use (Arrowsmith, 2009; Gaby and Lininger, 2006; Peter, 2004). 

Herbal tea major ingredient Intended medicinal use 


Anise Gas and indigestion problems 

Balm Calming nerves and flatulence problems 

Chamomile Colic, diarrhea, gastritis, heart burn, indigestion, and teething problems 

Fennel Colic, heart burn, and indigestion problems 

Peppermint Colic, gas, flatulence, indigestion, and stomachic problems 

Rose hip Diarrhea problem 

Thyme Colic, constipation, gas, flatulence, indigestion, spasms, and stomachic problems 

gastrointestinal problems. The sulfate contamination in 

the herbal tea samples is from the herbal products’ 

ingredients as the water used in this research for the 

preparation of the herbal tea samples contained no 

sulfate. Such high levels of chloride and sulfate in the 

herbal teas’ ingredients could have resulted from different 

sources such as from the plant itself due to environmental 

contamination during harvesting, and/or after 

harvesting (e.g. handling, cutting, dehydration processes, 

and equipment). 

Herbal tea label 

Not all the herbal tea brands reported the same 

information on the labels. Some of the brands reported 

energy content, concentration of carbohydrates, proteins, 

fats, and sodium and calcium on the labels. Results from 

this research and other research (Abouleish and Abdo, 

2012) suggest that the labels of the herbal teas need to 

report information on the contaminants present, such as 

the levels of fluorides, chlorides, sulfates, nitrates and 

nitrites, since they may cause problems. 

In conclusion, this research demonstrated that the 

studied herbal tea brands are contaminated with chloride 

and sulfate anions, which could lead to a change in the 

taste of the herbal tea product. In addition, some samples 

showed sulfate levels that exceeded the permissible 

levels, therefore, suggesting that those samples could 

cause sensitive people, children and babies to experience 

gastrointestinal problems. The herbal tea samples 

tested in this research are used for treating different 

health conditions such as indigestion, gas, cramps, and 

diarrhea, as presented in Table 3 (Arrowsmith, 2009; 

Gaby and Lininger, 2006; Peter, 2004). The presence of 

sulfate in those herbal tea samples decreases the 

effectiveness of those herbal tea products as they may 

cause other problems, therefore, defying their intended 

purpose. As a result, contamination of such products 

should be taken into consideration when they are 

consumed voluntarily or when prescribed by a physician. 

Therefore, this research suggests that the herbal tea 

products must be tested for chemical contamination, as 

this reflects on the quality of the product as well as on the 

manufacturing process. In addition, herbal tea samples 

are used for medicinal purposes and, as a result, must 

fall under stringent regulations in terms of manufacturing 

and quality control. 

ACKNOWLEDGEMENT 

We would like to thank the American University of 

Sharjah for funding this research and providing the 

necessary equipment. 

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DOI: 10.5897/JMPR12.792 



Effects of Moringa oleifera methanolic leaf extract on 

the morbidity and mortality of chickens experimentally 

infected with Newcastle disease virus (Kudu 113) strain 

Didacus Chukwuemeka Eze 1 *, Emmanuel Chukwudi Okwor 1 , Okoye John Osita A. 1 , Onah 

Denis Nnabuike 2 and Shoyinka S. Vincent Olu 1 

1 Department of Veterinary Pathology and Microbiology, University of Nigeria, Nsukka, Enugu State, Nigeria. 

2 Department of Veterinary Parasitology and Entomology, University of Nigeria, Nsukka, Enugu State, Nigeria. 


Newcastle Disease (ND) is an important disease of poultry worldwide. Its economic impact is high 

because mortality may reach 100% in affected poultry farms. This study was aimed at evaluating the 

protective properties of crude methanolic extract of Moringa oleifera in chickens. Forty two-day old 

chicks were randomly divided into four groups: I, II, III and IV. Groups I and II were given daily oral 

treatment of methanolic extract of M. oleifera at 200 mg/kg body weight until day 56 of age. Groups II 

and III at 42 days of age were vaccinated with the La Sota strain of ND vaccine. Group I was not 

vaccinated, while IV was left as untreated/unvaccinated control. All the groups were challenged with the 

velogenic strain of ND virus on day 56 of age. Feed intake and weight gain were evaluated. Following 

challenge, the birds were monitored for clinical signs, morbidity and mortality. Results of feed intake 

and weight gain were analysed using the statistical package for social sciences (SPSS). Survival was 0, 

32, 88 and 100% in groups IV, I, II and III animals, respectively. Therefore, M. oleifera can protect birds 

from ND and can be used prophylactically against ND. 

Key words: Velogenic Newcastle disease, chickens, Moringa oleifera, morbidity, mortality. 

INTRODUCTION 

Newcastle disease (ND) is a serious threat to the 

aviculturists and the commercial poultry industry 

worldwide. According to the Office International des 

Epizooties/World Organization for Animal Health (OIE), 

ND belongs to List A Group of diseases which have the 

following characteristics: “a transmissible disease that 

has the potential for very serious and rapid spread 

irrespective of national borders, that is of serious socioeconomic 

or public health consequence, and that is of 

major importance in the international trade of animals and 

animal products (OIE, 2005)”. The economic impact of 

ND outbreaks is characterized by high mortality in 

commercial flocks, condemnation of other infected flocks 

*Corresponding author. E-mail: didacus.eze@unn.edu.ng. Tel: 

+2348037292020. 

and trade restrictions associated with quarantine and 

surveillance of affected areas within individual states 

where outbreaks have been detected (Pandey, 1992). 

ND virus (NDV) is an Avian Paramyxovirus serotype 1 

(APMV-1) that belongs to the genus Avulavirus in the 

family Paramyxoviridae (Alexander, 2003). The most 

effective means of controlling NDV has been through 

vaccination and biosecurity. The most common routes of 

inoculation of the vaccine are oral, ocular, and intranasal 

(Spradbrow, 1994). 

Moringa oleifera is the most widely cultivated species of 

a monogeneric family, the Moringaceae that is native to 

the sub-Himalayan tracts of India, Pakistan, Bangladesh 

and Afghanistan. This rapidly growing tree (also known 

as the horseradish tree, drumstick tree, benzolive tree, or 

Ben oil tree), was utilized by the ancient Romans, Greeks 

and Egyptians. It is now widely cultivated and has 

become naturalized in many locations in the tropics. It is


a perennial softwood tree with timber of low quality, but 

which for centuries has been advocated for traditional 

medicinal and industrial uses (Morton, 1991). It is already 

an important crop in India, Ethiopia, the Philippines and 

the Sudan, and is being grown in West, East and South 

Africa, tropical Asia, Latin America, the Caribbean, 

Florida and the Pacific Islands. All parts of the M. oleifera 

tree are edible and have long been consumed by 

humans. M. oleifera is a natural anthelmintic, mild 

antibiotic, detoxifier and outstanding immune builder 

(Dahot, 1998). It is used in many countries to treat 

malnutrition and malaria (Dahot, 1998). M. oleifera is 

widely regarded by water purification experts as one of 

the best hopes for reducing the incidence of waterborne 

diseases. 

Recently, there has been an increased interest in the 

utilization of the M. oleifera, as a protein source for 

livestock (Makkar and Becker, 1997; Sarwatt et al., 

2002). It is a multipurpose tree of significant economic 

importance with industrial and medicinal uses (Morton, 

1991). There is little information available on the use of 

the leaves of this tree as an immunomodulator, especially 

in reducing the mortality rate in chickens infected with 

NDV and also as an adjuvant to vaccination. Therefore, 

in this project, the effects of administration of leaf extract 

of M. oleifera on mortality and morbidity with ND was 

investigated. 


The green leaves of M. oleifera were collected during the months of 

March and April 2010, at Ibagwa-Aka, Nsukka, Enugu State, 

Nigeria. The plant was identified by Mr. Ozioko of Bioresources 

Development and Conservation Programme, Nsukka. Extraction of 

the dried leaves was performed by soaking the plant material in 

absolute methanol (98%) for 24 h at room temperature (28°C). The 

resulting extract was concentrated in vacuo and subsequently air 

dried in a shade. The extract was solubilized in 5% Tween 80 and 

tested for protective activity in chickens experimentally infected with 

Velogenic Newcastle disease virus (VNDV). Phytochemical tests 

were carried out on the absolute methanolic extract using standard 

method (Trease and Evans, 1972). 

Experimental animals 

One hundred and fifty day-old White Harco cockerels were 

purchased from Zartec Ltd, a commercial breeder farm based at 

Ibadan, South West Nigeria. The birds were housed in an isolation 

pen at the Poultry Disease Research Unit of the Department of 

Veterinary Pathology and Microbiology University of Nigeria, 

Nsukka. The poultry house was of the open sided type and deep 

litter floor. Brooding was by kerosene stove and electric bulbs for 

the first two weeks. The birds were given feed and water ad-libitum, 

and were not vaccinated against any disease. 

Experimental challenge 

The birds were randomly divided into four groups- I, II, III, and IV, of 

twenty-five chicks each on day 42 of age. The group VI birds were 

kept in isolation from the other groups. Groups I and II were 

drenched orally with 1 ml 200 mg/kg body weight of M. oleifera daily 

between 42 to 56 days of age. Groups II and III were vaccinated 

with La Sota ® vaccine at 42 days of age. At 56 days of age, all the 

groups were inoculated intramuscularly with 0.2 ml challenge dose 

of VNDV strain (Kudu 113) with titre of 10 9.5 EID50 per ml of the 

inoculum. 

Clinical signs 

The birds in all groups were weighed weekly on days 42, 49, 56, 

63, 70 and 77 of age to determine the mean body weights of the 

birds in the groups. The feed intake was also evaluated by weighing 

the left-over feed every morning before feeding the birds to 

determine the mean feed intake per bird in for the groups. The birds 

were observed daily for clinical signs. Morbidity and mortality were 

recorded. Necropsies were carried out on the birds that died during 

the experiment. 

Statistical analyses 

Body weights and feed intake data were subjected to analysis of 

variance (ANOVA) statistics using the Statistical Package for the 

Social Science (SPSS). Significant means were separated using 

the Duncan’s new multiple range test and tests were considered 

significant at a probability of P < 0.05. 

RESULTS 

Phytochemical analysis 

Qualitative phytochemical tests carried out on the extract 

showed the presence of alkaloid, saponin, glycoside, 

flavonoid, steroid, fats and oil, and reducing sugar (Table 

1). The concentrations of saponin, glycoside, steroid and 

reducing sugars were higher in the methanolic extract of 

M. oleifera when compared to other chemical agents. 

However, tannins and terpenes were not detectable in 

the extract (Table 1). 

Feed intake 

On day 49 of age, the mean daily feed intake in groups I, 

II and IV in the study was not significantly (P> 0.05) 

different from one another, but were significantly (P < 

0.05) lower than that of group III, the vaccinated and 

untreated group (Figure 1). On day 56, there was 

significant (P< 0.05) difference in the mean daily feed 

intake in groups I, II and III and the mean daily feed 

intake in group I was significantly (P< 0.05) lower than 

those in groups II and III, but not significantly (P>0.05) 

different from those of groups IV. Moreover, on day 63, 

the mean daily feed intake in group I was significantly (P< 

0.05) lower that those in groups II and III, but there was 

no significant (P> 0.05) difference between groups I and 

IV. There was no significant (P> 0.05) difference among 

the vaccinated groups II and III (Figure 1). On day 70 and 

77, the mean daily feed intake in group I was significantly 

(P

Mean feed intake (g) 

6 

5 

4 

3 

2 

1 

0 

Table 1. Results of phytochemical analyses of M. oleifera. 

Compounds Presence in methanol extract 

Alkaloid + 

Saponin ++ 

Tannins - 

Glycoside ++ 

Flavonoid + 

Steroid +++ 

Terpenes - 

Fats and Oil + 

Reducing Sugar ++ 

- Absent; + present in trace concentration; ++ present in moderately low concentration; +++ present 

in very high concentration. 

I II III IV 

42 49 56 63 70 

Age (Days) 

Eze et al. 4445 

Figure 1. Feed intake (g) of the different groups of the birds treated with M. oleifera and or NDV vaccination. 

Fig. 1. Feed intake (g) of the different groups of the birds treated with 

M. oleifera and or NDV vaccination. 

there was significant (P0.05) different on day 42 (Figure group II was significantly (P< 0.05) higher than that of


Mean Body Weights (g) 

1200 

1000 

800 

600 

400 

200 

0 

I II III IV 

42 49 56 63 70 77 

Age (Days) 

Figure 2. Body weights (g) of the different groups of the birds treated with M. oleifera and/or NDV 

vaccination. Fig. 2. . Body Weights (g) of the different groups of the birds treated 

with M. oleifera and /or NDV vaccination 

group III. However, throughout the study, there was no 

significant (P> 0.05) difference in the mean body weight 

in the treated groups I and II. Meanwhile, significant (P< 

0.05) differences were observed between the treated 

groups I and II, which was higher than those of the 

untreated groups III and IV from days 49 - 77. There were 

equally no significant (P> 0.05) differences among the 

untreated groups III and IV (Figure 2). 

Clinical signs 

The clinical signs started on day 3 post infection (PI) in 

groups I, II, and IV. The clinical signs were characterized 

by a progressive depression, drop in feed and water 

consumption, nasal discharge of muco-fibrinous fluid, 

listlessness and somnolence, dullness, droopy wing and 

tail feathers, ruffled feathers, facial edema, coughing and 

sneezing were observed in groups II and IV. Greenishyellow 

diarrhea, torticollis and paralysis of the legs, sitting 

on the hock, in-coordination, and muscular twitching were 

seen on day 4 PI in the chickens in group IV. Morbidity 

occurred at day 3 PI up to day 6 PI. Morbidity on day 3PI 

was up to 32% in group I, 12% in group II, 0% in group 

III, and 32% in group IV. On day 6 PI, the morbidity was 

32% in group I, 4% in group II and 0% in group III (Figure 

3). Mortality started from day 4 PI and the total mortality 

by day 6 PI was 68% in group I, 12% in group II, 0% in 

group III, and 100 % in group IV (Figure 4). At the end of 

the study, survivability of the chickens 32% in group I, 

88% in group II, 100% in group III and 0 % in group IV 

(Figure 5). 

Lesion 

Grossly, the post mortem examination showed congested 

breast and the thigh muscles which occurred in (100%) in 

group I, (66.67%) in group II, and (100%) in group IV. 

There were haemorrhages on the proventricular mucosa 

in (50%) in group I, (100%) in group II, and (50%) in 

group IV. Severe intestinal haemorrhagic enteritis and 

ulcers were in (20%) in group I, and nil in groups II and 

IV. The cecal tonsils were also enlarged and 

haemorrhagic in (30%) in group IV, and non in groups I 

and II. The bursa was atrophic in 100, 66.67% and (100% 

in groups I, II, and IV, respectively. Atrophy of the thymus 

was 100% in group IV and nil in groups I and II. Splenic 

atrophy was 80%, 30% and non in groups I, IV, and II, 

respectively. The kidneys were haemorrhagic and 

swollen and mottle in 40% of group I, 100% of group IV 

and non in group II, while the liver was equally congested 

in groups I, II, and IV birds (Table 2). 

DISCUSSION 

In this study, the incubation period of experimental ND 

was three days, which is in agreement with the report of 

Okoye et al. (2000). Hamid et al. (1991) reported an 

incubation period of 2 to 16 days. The morbidity rates 

observed were higher among birds in the unvaccinated

Percentage mortality 

Percentage morbidity 

80 

70 

60 

50 

40 

30 

20 

10 

0 

1 2 3 4 5 6 7 8 9 10 

Time (day) of post infection 

Time (Days) post infection 

Figure 3. Morbidity profile following inoculation with NDV. 

Fig. 3. Morbidity profile following inoculation with NDV 

120 

I II III IV 

100 

80 

60 

40 

20 

0 

1 2 3 4 5 6 7 8 9 10 

Time Time (Days) (day) post of post infection infection 

Figure 4. Mortality profile of birds following inoculation with NDV. 

Fig. 4. Mortality profile of birds following inoculation with 

NDV 

and treated groups I and unvaccinated and untreated IV 

and this agrees with the reports that the morbidity and 

mortality of VND outbreak could be up to 100% in nonimmunized 

birds (Okoye et al., 2000). While the mortality 


in this study was 100% in group IV, it was 68% in group I, 

and also, 12 and 0% in the vaccinated and treated group 

II, and vaccinated and untreated group III, respectively. 

The survival rate in group I was 32%, 88% in group II, 

I 

II 

III 

IV


Percentages of survived birds 

120 

100 

80 

60 

40 

20 

0 

I II III IV 

1 2 3 4 5 6 7 8 9 10 

Time (day) of post infection 

Time (Days) post infection 

Figure 5. Survival profile of the birds following inoculation with NDV. 

Fig.4. Survival profile of the birds following inoculation with NDV 

Table 2. Type and frequency of post mortem lesions found in the different groups. 

Group 

Total number of chickens in 

group after challenge 

Number of 

chickens posted 

Enlarged cecal 

tonsils 

Intestinal 

haemorrhagic ulcer 

Proventricular 

haemorrhage 

Atrophic 

thymus 

Atrophic 

spleen 

Congestion of breast 

and thigh muscle 

I 25 10 - 2/10 5/10 10/10 8/10 10/10 10/10 6/10 4/10 

II 25 3 - - 3/3 - - 3/3 2/3 3/3 - 

III - - - - - - - - - - - 

IV 25 10 3/10 - 5/10 10/10 3/10 10/10 10/10 6/10 10/10 

100% IN group III, and nil in group IV. Mortality in 

ND depends on the nature of the virus, 

susceptibility of the host, age and immune status 

of the host (Hamid et al., 1991). The clinical signs 

observed in groups I, II and IV birds in this study 

were similar to those already described for VVND 

by Alders and Spradbrow (2001) who observed 

depression, partial and/or complete inappettence, 

listlessness, and huddling. Following this was 

greenish diarrhea, which is an indication of 

Atrophic 

bursa 

Intestinal 

ulcer 

Hemorrhagic 

kidney 

gastrointestinal lesion. This is also in agreement 

with what has been described by Okoye et al. 

(2000) and Hamid et al. (1991). Severe nervous 

signs were observed prominent of which were 

ataxia, paralysis and torticollis. The nervous

involvement according to Okoye et al. (2000) was due to 

the intramuscular route of inoculation of the virus. The 

post mortem lesion seen in the challenged birds were 

those of the VVND, but were more severe in group I, 

followed by those of the group IV as reported by Okoye et 

al. (2000). 

The post mortem lesion seen in the challenged birds 

were similar to those reported for VVND by Okoye et al. 

(2000). This is due to the tropism and virulence of the 

virus strain, target species, and their immunity 

(Alexander, 2003). These strains are typically associated 

with hemorrhagic intestinal lesions. The lesions were 

more severe in group IV, followed by groups I and then II. 

There was atrophy of the lymphoid organs such as the 

thymus, spleen, and bursa of Fabricius. While there was 

severe atrophy of the thymus in group IV, the lesion was 

moderate in group I. This was due to the 

immunosuppressive nature of the virus strain which the 

extract reduced in group I. Also observed in the birds was 

enlargement of the caecal tonsils and bursa which was 

due to inflammatory exudates. Intestinal haemorrhagic 

ulcers were equally observed. 

Comparing the results of the unvaccinated but 

challenged groups I and IV, the M. oleifera treated group 

I birds had less mortality (68%) than the untreated group 

IV birds (100%), less feed intake than group IV. They 

also had higher mean body weight gain. Also, the treated 

and vaccinated group II birds had higher mortality (12%) 

than the untreated and vaccinated group III (0%). The 

group II birds had less feed intake but higher mean body 

weight gain than the group III birds. M. oleifera contains 

significant amounts of vitamines A, B and C, minerals 

such as calcum ions, iron, potassium, proteins, as well as 

traces of carotenoids, saponins, phytates and phenolic 

constituents (Ferreira et al., 2008; Siddhuraju and 

Becker, 2003), which may be responsible for the 

immunomodulation of the immune systemobserved in this 

study. The mechanism of action of M. oleifera in 

modulating immune responses could be due to an 

enhanced production of growth factors such as cytokines 

that activates both the innate and adaptive immunity 

(Davis and Kuttan, 1998). 

Conclusion 

It can be concluded that M. oleifera extract had 


advantageous effects on the treated birds; hence, it can 

be recommended as a prophylactic treatment against ND 

in non-vaccinated birds because its advantageous effects 

were compromised in the vaccinated birds. 

REFERENCES 

Alders R, Spradbrow P (2001). Controlling Newcastle Disease in Village 

Chickens, a Field Manual-Australian Center for International 

Agricultural Research (ACIAR), Canberra, Australia. 

Alexander DJ (2003). Newcastle disease, other avian paramyxoviruses, 

and pneumovirus infections. Pages 63-99 in Diseases of Poultry. 

Y.M. Saif ed., Iowa State Press, Ames Iowa. 

Dahot MU (1998). Antimicrobial Activity of Small Protein of Moringa 

oleifera leaves. J. Islam. Acad. Sci., 11:1, 27-32. 

Davis L, Kuttan G (1998). Suppressive effect of cyclophosphamideinduced 

toxicity by Withania somnifera extract in mice. J. 

Ethnopharmacol. 62(3):209-214. 

Ferreira PMP, Farias DF, Oliveira JTA, Carvalho AFU (2008). Moringa 

oleifera: bioactive compounds and nutritional potential, Rev. Nutr. 

Campinas. 21:431-437. 

Hamid H, Campbell RS, Parede L (1991). Studies of the pathology of 

velogenic Newcastle disease: Virus infection in non-immune and 

immune birds. Avian. Pathol. 20:561-575. 

Makkar HPS, Becker K (1997). Nutrients and antiquality factors in 

different morphological parts of the Moringa oleifera tree. J. Agric. 

Sci. 128:311-322. 

Morton JF (1991). The Horseradish tree, Moringa pterygosperma 

(Moringaceae): A plant boon to arid lands? Econ. Bot. 45:318-333. 

OIE (2005). (Office International des Epizooties/World Organization for 

Animal Health). Newcastle disease. In: Manual of standards for 

diagnostic tests and vaccines. p. 2,1,15. 

Okoye JOA, Agu AO, Chineme CN, Echeonwu GON (2000). 

Pathological Characterization of a Velogenic Newcastle Disease 

Virus Isolate from Guinea Fowl. Rev. Élev. Méd. Vét. Pays trop. 

53(4):325-330. 

Pandey VS (1992). Epidemiology and economics of village poultry 

production in Africa: An overview, pp. 124-128. 

Sarwatt SV, Kapange SS, Kakengi AMV (2002). Substituting sunflower 

seed-cake with Moringa oleifera leaves as supplemental goat feed in 

Tanzania. Agrofor. Sys. 56:241-247. 

Siddhuraju P, Becker K (2003). Antioxidant properties of various solvent 

extracts of total phenolic constituents from three different agroclimatic 

origins of drumstick tree (Moringa oleifera Lam.). J. Agric. 

Food Chem. 15(8):2144-2155. 

Spradbrow PB (1994). Newcastle disease in village chickens. Poult. Sci. 

Rev. 5:57-96. 

Trease G, Evans W (1972). Pharmacognosy, Univ. Press, Aberdeen, 

Great Britain. pp. 161-163.

Journal of Medicinal Plants Research Vol. 6(27), pp.4450-4455, 18 July, 2012 


DOI: 10.5897/JMPR12.822 



Antioxidant and antimicrobial activities of Chowlai 

(Amaranthus viridis L.) leaf and seed extracts 

Muhammad Javid Iqbal 1 *, Sumaira Hanif 1 , Zahed Mahmood 1 , Farooq Anwar 2 and Amer Jamil 1 

1 Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad, Pakistan. 

2 Department of Chemistry, University of Sargodha, Sargodha-40100, Pakistan. 


The present study was conducted to evaluate the phenolics, antioxidant and antimicrobial activities of 

leaf and seed extracts from an edible herb namely Amaranthus viridis L. The extract yields of active 

components, produced using pure and aqueous methanol, from the leaves and seeds ranged from 5.4 

to 6.0 and 2.4 to 3.7%, respectively. The extracts contained appreciable levels of total phenolic contents 

(1.03 to 3.64 GAE, g/100 g) and total flavonoid contents (18.4 – 5.42 QE, g/100 g) and also exhibited 

good 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity as revealed by IC50 (14.25 - 83.43 

µg/ml). Besides, the tested extracts showed considerable antimicrobial activity against selected 

bacterial and fungal strains with minimum inhibitory concentrations (MIC) ranging from 179-645 µg/ml. 

Of the parts tested, the seed extracts exhibited superior antioxidant and antimicrobial activity. It was 

concluded that A. viridis leaf and seed can be explored as a potential source for isolation of antioxidant 

and antimicrobial agents for uses in functional food and pharmaceuticals. 

Key words: Amaranthus viridis, phytochemical constituents, minimum inhibitory concentrations (MIC), total 

phenolics, total flavonoids, 1,1-diphenyl-2-picrylhydrazyl (DPPH), radical scavengers. 

INTRODUCTION 

Amaranthus viridis L. (Amaranthaceae), commonly 

known as “Chowlai”, is a fast growing herb mainly 

cultivated in Asia, Africa and Latin America (Amin et al., 

2006). Being resistant to drought, hot climate and pests, 

and with little requirements for its cultivation, this pseudocereal 

has attracted much attention as an important food 

commodity (Sexna et al., 2007). In the last decade, the 

use of amaranth has expanded not only in the common 

diet, but also in diet of people with celiac disease or 

allergies to typical cereals (Berti et al., 2005). Reactive 

oxygen species (ROS) and reactive nitrogen species 

produced as a result of oxidation have been shown to be 

linked with different degenerative disorders such as 

aging, inflammation, cancer, cardiovascular complications, 

and osteoporosis (Wilcox, 2004). Interest in 

search for new natural antioxidants has grown over the 

*Corresponding author. E-mail: imjavid@gmail.com or 

amerjamil@yahoo.com. 

past few years because of their preventive role in 

protecting from oxidative-stress related chronic diseases 

(Halovrson et al., 2002). 

Existence of microorganisms causes food spoilage and 

results in deterioration of the quality and quantity of 

processed food products. Some plant-based biologically 

active compounds isolated from herbs have been 

explored for the growth inhibition of pathogenic microbes 

because of their antimicrobial potential (Abubakar et al., 

2008). The medicinal value and multiple biological 

functionalities of several plants are defined by their 

phytochemical constituents (Fallah et al., 2005). Many 

herbal species being a promising source of bioactive 

compounds such as phenolics, anthocyanins, flavonoids, 

and carotenoids, are usually used to impart flavor and 

enhance the shelf-life of dishes and processed food 

products, recently reported work was (Nisar et al., 2010a, 

b, 2011; Qayum et al., 2012; Zia-Ul-Haq et al., 2008, 

2011a, b, 2012). Due to their high antioxidant potency, 

the consumption of many such plants species is 

recommended (Ozsoy et al., 2009). Antioxidant

properties of green leafy vegetables and herbs including 

different amaranth species have been preliminarily 

studied (Ozbucak et al., 2007). 

The main aim of the present study was to evaluate the 

phenolic compounds, antioxidant and antimicrobial 

activities of pure and aqueous methanol extracted 

components from leaves and seeds of locally grown 

Amaranthus viridis plants to explore their potential 

pharmaceutical and/or functional food uses. 


Collection and pretreatment of plant material 

The leaves and seeds of fully matured A. viridis L. were collected 

during June to July 2009, from the local fields of Faisalabad, 

Pakistan, and identified by the Department of Botany, GC 

University Faisalabad, Pakistan. Collected specimens were dried at 

room temperature and stored in polyethylene bags at 4°C. 

Chemical and reagents 

1,1-diphenyl-2-picrylhydrazyl (DPPH), gallic acid, Folin-Ciocalteu 

reagent, sodium nitrite, butylated hydroxytoluene (BHT) were 

purchased from Sigma Chemical Co (St. Louis, USA) and 

anhydrous sodium carbonate, methanol and ethanol used were 

obtained from Merck (Darmstadt, Germany). All culture media 

antibiotic, discs and sterile solution of 10% (v/v) DMSO in water 

were purchased from Oxoid (Hampshire, UK). 

Preparation of A. viridis extracts 

Ground (80 mesh) leaf and seed samples (100 g each) were 

extracted separately with 1000 ml absolute methanol and 80% 

methanol (80:20, methanol: water, v/v) using an orbital shaker 

(Gallenkamp, UK) for 12 h at room temperature. The extracts were 

separated from solids by filtering through Whatman No. 1 filter 

paper. The residues were extracted thrice and the extracts were 

collected. The solvent was removed under vacuum at 45°C, using a 

rotary vacuum evaporator (N-N Series, Eyela, Rikakikai Co. Ltd. 

Tokyo, Japan) and stored at 4°C till further analysis. 

Phytochemical screening 

The methanol extracts of the tested plant material were screened 

for the presence of various phytoconstituents such as 

phlobatannins, tannins, alkaloids, terpenoids, glycosides, 

flavonoids, and phenolic compounds (Abubakar et al., 2008). 

Antioxidant activity 

Determination of total phenolic contents (TPC) 

Total phenolic contents (TPC) were determined using the Folin- 

Ciocalteu reagent method and gallic acid was used as gallic acid 

equivalent (GAE) (Amin et al., 2006). 

Determination of total flavonoid contents (TFC) 

The total flavonoid contents (TFC) in the leaf and seed extracts 

Iqbal et al. 4451 

were determined following the modified procedure of Edeoga et al. 

(2005), and quercetin was used as standard as quercetin 

equivalent (QE). 

DPPH radical scavenging assay 

1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical assay was carried out 

spectrophotometrically (Miliauskas et al., 2004). The percent 

inhibition was calculated as: 

I (%) = 100 × (Ablank __ Asample / Ablank) 

Where Ablank is absorbance of the control reaction (containing all 

reagents except the test sample), and Asample is the absorbance of 

test samples. Extract concentrations providing 50% inhibition (IC50) 

values were calculated from the plot of percentage scavenging 

versus extracts concentration. 

Antimicrobial activity 

Microbial strains 

The A. viridis leaf and seed extracts were individually tested against 

a panel of microorganisms (locally isolated), including two bacteria, 

Staphylococcus aureus and Escherichia coli, and two pathogenic 

fungi, Fusarium solani and Rhizopus oligosporus. The selected 

strains have strong pathogenic activities against plant and animals 

that lead to a significant loss of lives and food. The pure bacterial 

and fungal strains were obtained from the Bioassay section, Protein 

Molecular Biochemistry laboratory, Department of Chemistry and 

Biochemistry, University of Agriculture Faisalabad, Pakistan. The 

bacterial strains were cultured at 37°C overnight, w hile fungal 

strains were cultured overnight at 28°C in an incuba tor (Memmert, 

Germany). 

Disc diffusion method 

The antimicrobial activity of the leaf and seed extracts was 

determined by using disc diffusion method (CLSI, 2007). The discs 

(6 mm in diameter) were impregnated with 20 µg/ml sample 

extracts (20 µg/disc) and placed on inoculated agar. Rifampicin (20 

µg/disc) (Oxoid) and fulconazole (20 µg/disc) (Oxoid) were used as 

positive reference for bacteria and fungi, respectively. Antimicrobial 

activity was evaluated by measuring the inhibition zones (in mm) by 

zone reader. 

Minimum inhibitory concentration (MIC) assay for 

determination of antimicrobial activity 

Incubator at 35 and 37°C; pipettes of various sizes (G ilson); sterile 

tips, 100, 200, 500 and 1000 µL; 5 ml multi-channel pipette; 

centrifuge tubes; vortex mixer; centrifuge (Fisons); Petri-dishes, 

sterile universal bottles; UV-spectrophotometer (Shimadzu) and 

sterile resazurin tablets (BDH Laboratory Supplies) were used. 

Isosensitest medium was used throughout this assay, as it is pH 

buffered. Although the use of Mueller Hinton medium was 

recommended for susceptibility testing (NCCLS, 2000), the 

isosensitest medium had comparable results for most of the tested 

bacterial strains. 

Use of standardized bacterial colony numbers 

The method wherein turbidity is compared to McFarland standards


usually 0.5, is not able to give a standardized number of CFU for 

bacterial strains only because this is operator-driven and is thus 

subjective. It also makes it difficult to compare different bacterial 

species as they have differing optical densities. A final 

concentration of 5 × 10 5 CFU/ml was adopted for this assay. Thus, 

different strains and different bacterial species could be compared. 

Preparation of microbial culture 

Using aseptic techniques, a single colony was transferred into a 

100 ml bottle of isosensitest broth capped and placed in incubator 

overnight at 35°C. After 12 to 18 h of incubation, u sing aseptic 

preparation and the aid of a centrifuge, clean samples of bacteria 

and fungi were prepared. The broth was spun down using a 

centrifuge at 4000 rpm for 5 min with appropriate aseptic 

precautions. The supernatant was discarded into an appropriately 

labeled contaminated waste beaker. The pellet was re-suspended 

using 20 ml of sterile normal saline and centrifuged again at 4000 

rpm for 5 min. This step was repeated until the supernatant was 

clear. The pellet was then suspended in 20 ml of sterile normal 

saline and was labeled as Bs. The optical density of the Bs was 

recorded at 500 nm, and serial dilutions were carried out with 

appropriate aseptic techniques until the optical density was in the 

range of 0.5 to 1.0. The actual number of colony forming units was 

calculated from the viability graph. The dilution factor needed was 

calculated and the dilution was carried out to obtain a concentration 

of 5 × 10 6 CFU/ml (Winn and Koneman, 2006). 

Preparation of resazurin solution 

The resazurin solution was prepared by dissolving a 270 mg tablet 

in 40 ml of sterile distilled water. A vortex mixer was used to 

dissolve tablet and homogenous solution formation. 

Preparation of the plates 

Plates were prepared under aseptic conditions. A sterile 96 well 

plate was labeled. A volume of 100 µL of test material in 10% (v/v) 

dimethylsulphoxide (DMSO)/sterile water (10 mg/ml for crude 

extracts) was pipetted into the first row of the plate. To all other 

wells, 50 µL of nutrient broth or normal saline was added. Serial 

dilutions were performed using a multichannel pipette. Tips were 

discarded after use such that each well had 50 µL of the test 

material in serially descending concentrations. To each well, 10 µL 

of resazurin indicator solution was added. Furthermore, using a 

pipette, 30 µL of 3.3 × strength isosensitised broths was added to 

each well to ensure that the final volume was single strength of the 

nutrient broth. Finally, 10 µL of bacterial suspension (5 × 10 6 CFU/ 

mL) was added to each well to achieve a concentration of 5 × 10 5 

CFU/ml. Each plate was wrapped loosely with cling film to ensure 

that bacteria did not become dehydrated. Each plate had a set of 

controls: a column with a broad-spectrum antibiotic as positive 

control (usually ciprofloxacin in serial dilution), a column with all 

solutions with the exception of the test compound, and a column 

with all solutions with the exception of the bacterial solution adding 

10 µL of nutrient broth instead. The plates were prepared in 

triplicate, and placed in an incubator set at 37°C fo r 18 to 24 h. The 

color change was then assessed visually. Any color change from 

purple to pink or colorless was recorded as positive. The lowest 

concentration at which color change occurred was taken as the MIC 

value. The average of three values was calculated and that was the 

MIC for the test material and microbial strain. 


Values are given as means ± standard deviation (SD) of each 

measurement. Where appropriate, the data were tested by one-way 

ANOVA using Minitab 15. Pearson correlation coefficients and pvalues 

were used to show correlations and their significance. 

Differences of P

Table 1. Percentage yield extracts from leaves and seeds of Amaranthus viridis. 

Plant used Extract Percent yield (g/100 g) 

Leaves 

100% Methanol 5.4 ± 0.13 a 

80% Methanol 6.0 ± 0.14 a 

Seeds 

100% Methanol 3.7 ± 0.11 ab 

80% Methanol 2.4 ± 0.04 b 


Values are mean ± SD of three samples analyzed individually in triplicate. Different letters in superscript indicate significant and 

non-significant differences with solvents. 

Table 2. Phytochemical constituents of leaf and seed extract of Amaranthus viridis. 

Phytochemical constituent 

Amaranthus viridis 

Leave Seed 

Tannins + + 

Phlobatannins + + 

Saponins - - 

Flavonoids + + 

Terpenoids - - 

Cardiac glycosides + + 

+Represents presence of the phytoconstituents; represents absence of the phytoconstituents. 

Table 3. Antioxidant activity of Amaranthus viridis leaf and seed methanolic extracts. 

Plant used Extracts DPPH, IC50 (µg/ml) TP contents* TF contents** 

Leaves 

100% Methanol 14.25 ± 0.712 a 1.03 ± 0.5 a 2.78 ± 0. 20 b 

80% Methanol 83.43 ± 3.84 a 2.89 ± 0.2 a 18.4 ± 0. 30 a 

Seeds 

100% Methanol 46.50 ± 2.97 a 3.64 ± 0.4 a 5.42 ± 0. 20 a 

80% Methanol 75.91 ± 3.03 a 3.20 ± 0.39 a 2.51 ± 0.07 b 

Values are mean ± SD of samples analyzed in triplicate. *, Total phenolic contents in gallic acid equivalent; **, Total flavonoid 

contents in quercetin equivalent. 

extraction by pure and aqueous methanol. The free 

radical scavenging capacity increased with increasing 

extracts concentrations. The leaves and seed extracts 

showed good hydrogen-donating ability in the presence 

of DPPH stable radicals (Table 3), with IC50 (the extract 

concentration providing 50% inhibition) values ranging 

from 14.25 – 83.43 and 46.50 – 75.91 µg/ml, 

respectively. When compared with the synthetic antioxidant 

BHT (15.7 µg/mL), the tested extracts offered 

slightly lower activity except 100% methanol leaf extract 

(14.25 µg/ml). 

These results were consistent with previous 

observation that Amaranthus varieties contained radical 

scavenging agents that could directly react with and 

quench stable DPPH radicals (Oboh, 2005). The ability of 

an Amaranthus paniculatus extracts to act as a free 

radical scavenger or hydrogen donor has also been 

reported previously (Amin et al., 2006). The betalians 

from plants in the family Amaranthaceae exhibit strong 

antiradical activity, with IC50 values ranging from 3.4 to 

8.4 µM, and representing a new class of dietary 

antioxidants (Cai et al., 2005). These results showed that 

the methanol leave extract contained strongest DPPH 

free radical scavenging compounds; the efficacy of those 

was quite comparable with the positive control BHT (15.7 

µg/ml). 

Total phenolic and total flavonoid contents 

The total phenolic content (TPC) and total flavonoid 

content (TFC) of A. viridis leaf and seed extracts are 

presented in Table 3. The differences in the amount of 

TP and TF may be due to varied efficacy of the extracting


Table 4. Antimicrobial activity and minimum inhibitory concentration of Amaranthus viridis leaf and seed methanolic extracts against the selected strains of bacterial and fungal 

strain. 

Part of plant used Extracts 

S. aureus 

Zone size MIC 

E. coli 

Zone size MIC 

F. solani 

Zone size MIC 

R. oligosporus 

Zone size MIC 

Leaves 

100% Methanol 24 ± 1.9 ab 179 ± 1.28 ab 16 ± 1.4 a 398 ± 1.26 a 17 ± 1.8 ab 436 ± 1.46 19.0 c ± 1.4 302 ± 1.36 c 

80% Methanol 23 ± 2.4 ab 182 ± 1.68 ab 12 ± 0.3 b 603 ± 2.36 b 15 ± 2.2 b 491±1.76 b 18.0 b ± 0.9 352 ± 2.42 b 

Seeds 

100% Methanol 16 ± 2.1 c 428 ± 1.36 c 11 ± 1.5 b 639 ±1.26 b 13 ± 1.7 c 602 ± 1.89 c 15.0 bc ± 1.0 482 ±1.27 bc 

80% Methanol 18 ± 1.5 c 403 ± 2.36 c 10 ± 1.2 b 645 ±1.48 b 10 ± 1.5 c 641 ± 2.38 c 13.0 c ± 1.6 547 ± 2.38 c 

Control Methanol 26 ± 3.1 a 141 ± 1.31 a 17 ± 1.7 a 381 ± 2.39 a 18 ± 1.9 a 391 ± 2.48 a 16 a ± 2.1 436 ± 2.17 a 

Values are mean ± SD of three samples analyzed individually in triplicate. Diameter of inhibition zone (mm) including disc diameter of 6 mm and MIC in µg/ml. Controls used are 

rifampicin and fulconazole for bacterial and fungal strains, respectively. Different letters in superscript indicate significant differences within solvents. 

solvents to dissolve endogenous compounds. 

The ability of different solvents to extract TP 

and TF contents was of the order: for leaf 80%> 

100% methanol and for seed 100% > 80% 

methanol. These values were higher than the 

reported values of A. cruentus (0.3 g/100 g) 

(Nisimba et al., 2007). Gorinstien et al. (2007) 

showed from the values obtained in their work, 

less phenolic content compared to the four 

reported Amaranthus verities (107 g/kg). 

Amaranth plants have been reported as one of 

many vegetables rich in antioxidant compounds 

(Obadoni and Ochuko, 2001). The other 

reported total phenolic contents (TPC) for 

Amaranthus species ranged from 2.95 - 3.75 

GAE, mg/100 g (Pasko et al., 2009). 

Antimicrobial activity 

The antimicrobial activity for A. viridis leave and 

seeds extracts against food-borne and 

pathogenic microorganisms is depicted in Table 

4. The extracts showed considerable 

antimicrobial activity against all the strains 

tested, particularly against Gram-positive 

bacterium. The results from MIC indicated that 

E. coli was most sensitive microbe tested, 

showing the largest inhibition zones (24 mm) for 

leaves and minimum (18 mm) for seeds 

extracts. The least activity is inhibited by 80% 

methanol seeds extract against R. oligosporus, 

with the smallest zone (13 mm). 

In general, the antimicrobial activity of the 

tested A. viridis leaves and seeds extracts was 

comparable with the standard drugs, 

streptomycin and mecanozol. In support to our 

present data, in a previous study, isolation of 

the antifungal peptide from the A. viridis seed 

extracts has been done (Lipkin et al., 2004). 

Resazurin is an oxidation-reduction indicator 

used for the evaluation of cell growth. The 

effectiveness of resazurin oxidation compound 

is higher for the leaves extracts of selected 

Amaranth species. The currents results support 

the earlier findings which demonstrate the 

presence of antimicrobial activity in seeds of 

Amaranthaceae (Cia et al., 2005). 

Conclusion 

The results from the current study indicate that 

A. viridis leaves and seeds extracts contained 

varied types of pharmacologically active 

compounds with antioxidant and antimicrobial 

activities which differed between the two parts 

and extraction solvents used. Further research 

work involving more detailed in vitro and in vivo 

investigations to establish which component of 

the extracts offer best antioxidant and 

antimicrobial activity is needed. Detail toxicological 

studies are also recommended to 

explore the uses this plant extracts as natural 

food preservative. The production of bioactive 

components from such indigenous resources 

and their utilization as potential natural food 

preservatives could be of high economic value. 

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UPCOMING CONFERENCES 

15th European Congress on Biotechnology: "Bio-Crossroads", Istanbul, 

Turkey, 23 Sep 2012 

2012 International Conference on Biotechnology and Food 

Engineering 

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Conferences and Advert 

September 2012 

XVIII International Congress for Tropical Medicine and Malaria, Rio de Janeiro, Brazil, 23 

Sep 2012 

November 2012 

31st World Congress on Internal Medicine (WCIM), Santiago, Chile, 11 Nov 2012 

2013 

September 2013 

61st International Congress and Annual Meeting of the Society for Medicinal Plant and 

Natural Product Research, Muenster, Germany, 1 Sep 2013

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