PHYTOCHEMICAL ANTIOXIDENTAL AND ETHNOMEDICINAL ACTIVITIES OF HIBISCUS HISPIDISSIMUS (MALVACEAE) Dissertation submitted to University of Kerala In partial fulfillment of the requirements for the award of B.Sc. Botany degree 2019 -20. 1.Gayathri B P-245171240 2. .Kavyamohan M S-24517124005 3.Anjali A-245171240011 4.Indu Nair B A-24517124011 5.Jincy Lukose-24517124019 6.Shefeena R S-24517124028 7.Sangeetha.G S-24517124026 DEPARTMENT OF BOTANY N.S.S.COLLEGE, NILAMEL KOLLAM

CERTIFICATE Certified that the Project entitled “PHYTOCHEMICAL ANTIOXIDENTAL AND ETHNOMEDICINAL ACTIVITIES OF HIBISCUS HISPIDISSIMUS (MALVACEAE)” is a bonafide record of the project work carried out by the candidates Gayathri B P-245171240, Kavyamohan M S- 24517124005, Anjali A-245171240011, Indu Nair B A-24517124011, Jincy Lukose-24517124019, Shefeena R S-24517124028, Sangeetha.G S-24517124026 under the guidance of Geetha. R. Nair, Assistant Professor , Department of Botany,NSS College,Nilamel during the year 2019-2020. 1.Gayathri B P-245171240 2. .Kavyamohan M S-24517124005 3.Anjali A-245171240011 4.Indu Nair B A-24517124011 5.Jincy Lukose-24517124019 6.Shefeena R S-24517124028 7.Sangeetha.G S-24517124026 Geetha R Nair, Geetha. R. Nair, HOD, Department of Botany, Assistant Professor, NSS College Nilamel, Department of Botany, Kollam. NSS College Nilamel, Kollam.

DECLARATION We hereby declare that the project work entitled “Phytochemical ,Antioxidental and Ethnomedicinal activities of Hibiscus hispidissimus(Malvaceae)” is a bonafide record of the original work carried out under the supervision of Geetha.R.Nair, Assistant Professor, Department of Botany, NSS College, Nilamel, Kollam in partial fulfilment of the requirements for the award of Degree of Bachelor of Science in Botany of the University of Kerala. No part of this work has been previously formed the basis of any other Degree or Diploma. 1.Gayathri B P-245171240 Kollam 25-05-2020 2..Kavyamohan 24517124005 3.Anjali A-245171240011 4.Indu Nair B A-24517124011 5.Jincy Lukose-24517124019 6.Shefeena R S-24517124028 7.Sangeetha.G S-24517124026

ACKNOWLEDGEMENT The present work was carried out under the guidance and constant supervision of Geetha. R. Nair, Assistant Professor, Department of Botany, NSS College, Nilamel, Kollam. We wish to place on record our sincere gratitude and deep indebtedness to our esteemed guide, for suggesting the topic and for valuable guidance, advice and help throughout the course of this investigation. We are very grateful to Dr.Sindhu Rani, Head of the Department of Biochemistry , NSS College Nilamel, Kollam for providing the necessary facilities for carrying out this work. Our sincere thanks to all other teaching and non-teaching staff of the Department of Botany, NSS College, Nilamel, Kollam for their help and for providing laboratory facilities, for completing the study. Lastly, but not least, our gratitude to all classmates for their valuable support and help. .Gayathri B P Kavyamohan Anjali A Indu Nair B A Jincy Lukose Shefeena R S Sangeetha.G

CONTENTS Page No. 1. INTRODUCTION 1-3 2. REVIEW OF LITERATURE 4-7 3. MATERIALS AND METHODS 8-11 4. RESULTS 12-15 5. DISCUSSION 16-19 6. S U M M A R Y & CONCLUSION 20 7. REFERENCES 21-23

INTRODUCTION The plants provide foodstuff, attire, shelter and medicine. Most of the herbal benefits seem to have been developed through observation of wild animals and by trial and error methods. As time goes on, people started to find and to utilize more herbs having medicinal power. They systematically brought together information on herbs and developed to well-defined herbal pharmacopoeias i.e. traditional medicinal system (2016, Nov21) . Traditional use of medicine is identified as a way to learn about potential future medicines. Because of wide biological and medicinal values, high safety margins and lesser cost of herbal medicine, it has great demand and used as source of basic health care in both developed and developing countries (2016, Nov.21,Kamboj, 2000) . WHO notes that around 200 pharmaceutical medicines are derived from the plant, in modern medicinal system around 74% of which are used in ways that can directly correlated to their ancient medicinal uses (Akerele,1993). Medicinal plants or botanical medicines have been used traditionally by mankind for the prevention and treatment of various ailments. The plant kingdom plays a vital role in the life of human beings and animals. The plants are the major source of various compounds that are widely used in pharma industries and other nutraceutical sectors. The rise in population, inadequate supply of drugs, side effects of allopathic medicines, resistance to drugs and high cost treatments have made human beings to use plant as a source of medicine for a variety of diseases. Green plants which are usually the reservoir of many biochemical products can be extracted and used for various scientific experiments thus leading to the development of plant based non-toxic, non-reactive product (Kumar et al;2014). The method of using medicinal plants can be a mixture of many active components or a single active component. The plant Hibiscus hispidissimus belongs to the family Malvaceae (Mallow family). The common name of this plant is Wild Hibiscus, Comfort root, Big thicket Hibiscus. The plant is a large climber having reddish stems that are covered with hooked prickles (https,KFRI, 2011, Warrier et al., 2003, Bindu et al., 1997, Barve et al., 2010). The leaves are alternately arranged, 6-8cm, palmately 3-5 lobed, hairy and heart shaped at the base. Leaf margins are toothed, lobes are long pointed, leaf stalks 5-10cm long and prickly. Stipules are lane shaped. Yellow flowers arise singly from leaf axils which are carried on 3-5cm long prickly stalks. 8-12 bracts below the flowers with leafy appendages. Seed capsules are 1cm long, ovoid, pointed, enclosed in enlarged sepal cup. This plant is commonly found in the evergreen forests of Western Ghats. The flowering period is November-January. It is distributed throughout India. The method of propagation of the plant is by seeds (html). Ethnomedicinal uses of Hibiscus hispidissimus The sour leaves are used as food ingredient and used in the preparation of South Indian cuisine. The leaves are the source of an ayurvedic drug Sathambasthi. This drug is one of 1

the five acid drugs (pancamla). It is a major constituent of pancamlatailam, an oil preparation for body-anointing. The leaves are also an ingredient in Ayurvedic drug Annabhedi sinduram and Abhram. The leaves are anti inflammatory and anthelmintic (Sivarajan et al., 1996) . Tribal healers of Kerala region use this plant to treat Liver diseases. It is said to improve digestion and have anthelmintic action(Gauthaman et al., 2006). The Leaves of the plant are acidic and eaten after cooking. The juice of the leaves are mixed with honey and used in treatment of eye diseases (Salem MZM, 2014). In summer the roots of the plant is fused in water is used as a cooling drink. Decoction of the root bark is used as remedy for poisons, swellings and cleansing the kidneys (Senthil Kumar, 2014) . Considering the ethno medicinal value of Hibiscus hispidissimus, the present work has been taken up to document the phytochemial profile and antioxidant activity of the plant. The leaf of Hibiscus hispidissimus shows the presence of Hibiscus acid, the chemical name being Tetrahydro-3- hydroxy-5-oxo-2, 3 -furandicarboxylic acid. The optical isomer of hydroxycitric acid, Garcinia acid is also an important organic acid present in the plant giving it the medicinal properties (Masao et al., 1999, Singh et al., 2005). The plant is also a rich source of triterpene and flavanoid compounds like hibiscatin and gossyptrin which is giving the plant its antioxidant properties ( Nair et al., 1981 ). The plant also contains various acids like Citric acid, Malic acid, Tartaric acid and allo-hydroxycitric acid lactone (Hibiscus acid). The Flowers of the plant reported the presence of gossypin, gossypitrin and hibiscatin. The plant also found to contain alkaloids, anthocyanins and quercetin. All these phytochemicals present in Hibiscus hispidissimus contribute to the medicinal properties (Nwaiwu et al., 2012, Obi et al., 1998, Harborne 2015). Systematic position Kingdom : Plantae Subkingdom : Trcheobionta Super Division : Spermatophyta Division : Magnoliophyta Class : Magnoliopsida Sub class : Dilleniidae Order : Malvales Family : Malvaceae. 2

Genus : Hibiscus Species : hispidissimus Objective of the study 1. Providing local name, botanical name and family of collected plants for easy identification of plants without any confusion 2. To study the morphological characters of collected plant. 3. Providing information about morphologically useful parts of the plant. 4. Contribution of ethnomedicinal importance of the plant . 5. To study the phytochemical analysis of the plant 6. To understand the antioxidental activities of the plant. 3

REVIEW OF LITERATURE In the last few decades there has been an exponential growth in the field of herbal medicine.More than 70 % of Indians use non- allopathic systems of medicines. Medicinal plants have a long-standing history in many indigenous communities and continue to provide useful tools for treating various diseases. The practices of traditional medicine are based on hundreds of years of belief and observations, which predate the development and spread of modern medicine. (Jayaprakash et al; 2011) Today, there is widespread interest in herbal drugs. This interest primarily stems from the belief that herbal medicines are safe, inexpensive and have no adverse effects. (Kaur et al; 2011) Medicinal plants are moving from fringe to main stream use with a greater number of people seeking remedies and health approach. (Saha et al; 2010) It is no wonder that the world’s one-fourth population i.e. 1.42 billion people, are dependent on traditional medicines for the treatment of various ailments(Jena et al., 2011). However a key obstacle, which has hindered the acceptance of the alternative medicines in the developed countries, is the lack of documentation and stringent quality control. There is a need for documentation of research work carried out on traditional medicines. With this backdrop, it becomes extremely important to make an effort towards standardization of the plant material to be used as a medicine. The process of standardization can be achieved by stepwise pharmacognostic and phytochemical studies. These studies help in identification and standardization of the plant material. Correct identification and quality assurance of the starting materials is an essential prerequisite to ensure reproducible quality of herbal medicine which will contribute to its safety and efficacy. Medicinal plants have traditionally served as man’s novel weapons against different ailments. Besides the modern medical practices existing today, about 65% of the Indian population depends on the traditional medical systems for their primary healthcare. Standardization can be achieved by stepwise pharmacognostic studies. Simple pharmacognostic techniques used in standardization of plant material include its morphological, anatomical and biochemical characteristics(Gowda et al; 2009). The standards are utmost importance in not only finding out genuity but also in detection of adulterants in marketed drugs(Hariprasad et al; 2011). Medicinal plants are usually playing a significant part in traditional medicines intended for therapy of various health issues. However a crucial hurdle, which has impeded the promotion in the usage of alternative medications in the developed countries, is lack of evidence of documentation and absence of stringent quality control measures. Additionally, there is a dependence on the data of all study meted out on traditional medicines by way of documentation. Keeping this issue, it is now quite necessary to generate assurance about the standardization of the plant as well as its parts to be used like a medication. During the process of standardization, we are able to take advantage of various techniques and methodology to achieve our goal in a phase wise approach e.g. pharmacognostic and phytochemical studies. These techniques and 4

methods are helpful in recognition and standardization of the plant material. Appropriate characterization and quality assurance of starting material is a crucial step to ensure reproducible quality of herbal medicine to assist people in order to justify its safety and effectiveness. Phytochemical Screening is the search for the major families of secondary metabolites was made in the dried plant powder and in the aqueous and organic extracts of the aerial part of Anabasis aretioïdes Coss. & Moq. using a qualitative analysis based on coloring reactions and/or precipitation.Flavonoids were searched by the cyanidin reaction( Guessan et al., 2009). For the characterization of catechic and gallic tannins, we used ferric chloride(Karumi et al., 2004)]. Identificatio n of saponins was based on their ability to form a mousse (Beckro et al., 2008), the sterols were searched BioMed Research International by the reaction of Liebermann (Guessan et al., 2009) , and characterized the alkaloids by Dragendorff reagent (potassium iodobismuthate) and Valser-Mayer reagent (potassium tetra-iodomercurate) (Reifer and S. Niziolek,1957). The presence of anthracenosides was searched by the Borntraeger reaction (Houta ., 2012),the KOH reagent was used for the detection of anthraquinones ( Suleiman, et al., 2015) and the presence of free quinones is confirmed by adding a few drops of NaOH (Dohou, 2003) .The dosage of secondary metabolites of Anabasis aretioïdes Coss. & Moq. was conducted according to the results of phytochemical screening tests. Plants are nature's \"Chemical factories\" providing the richest source of organic chemicals on earth. But, considering the species richness of the plant world, the knowledge of plants acquired by humans is still insufficient. Therefore, systematic exploration of plant wealth, along with investigations of phytochemicals in plant and their biological activity is needed for scientific support of its traditional claim. Alotaibi et al., 2018 evaluated the presence of different phytochemical in ethanolic extract and successive fractions of Ruta chalepensis L. and four phytoconstituents namely, scopletin, kaempferol, quercetin, quercetin 3-O-α-L-rhamno glucopyranosyl (Rutin) w ere isolated, which were evaluated for their and biological activities. The crude ethanolic extract and successive fractions like chloroform, ethyl acetate and butanol showed excellent antimicrobial activities against gram negative bacteria, gram positive bacteria and fungi. From the result ethyl acetate extract was found to be the best for inhibition of the microorganism’s growth. All extracts and fractions showed DPPH radical scavenging activity in a concentration-dependent manner. Most promising activity was obtained for ethyl acetate and butanol extract. These extracts also showed anticoagulant activity at higher concentration with prolonged clotting time 6:30 and 4:30 s at 10 mg/mL concentrations. Thus, the study indicated that the bioactivity of R. chalepensis L. is mainly due to the presence of active compounds like scopletin, kaempferol and quercetin. Fomogne-Fodjo, 2017 isolated two novel secondary metabolites named tetraceranoate and N-hydroxy imidate-tetracerane, together with five known 5

compounds β-stigmasterol, stigmast-5-en-3β-yl acetate, betulinic acid, betulin and lupeol were isolated from the stem bark of Tetracera potatoria. Tetraceranoate exhibited the best activity against Mycobacterium smegmatis with a minimum inhibitory concentration of 7.8 µg/mL, while β-stigmasterol, betulinic acid and betulin showed appreciable anti-mycobacterial activity against both strains. Only tetraceranoate one of the isolated compounds showed antibacterial activity against M. smegmatis having efficacy as high as standard rifampicin. Thus, the result of the study concluded that tetraceranoate might be an interesting target for systematic testing of anti-TB treatment and management. Das et al., 2017 evaluated in vivo hepatoprotective effects of isolated compounds from Neanotis wightiana aerial parts in CCl4 induced hepatotoxicity to validate its traditional use. During the study, two known triterpenoids, ursolic acid and oleanolic acid along with a new compound named Neanoside B was isolated from the polar n-butanol fraction of methanolic extract of N. wightiana aerial parts by repeated column chromatography. The new compound structure was established as naphthalene diglucoside 1 on the basis of extensive spectroscopic (including 2D NMR) analysis. The compound exhibited significant in vivo hepatoprotective effect in a dose-dependent manner by normalizing the elevated levels of hepatic injury markers and amelioration of the damage of liver tissue by reducing the necrosis and vacuoles indicating its use as a potential drug in liver disorders. Mbaveng et al., 2017 isolated five naturally occuring compounds: Betulin, Mundulea lactone, Seputhecarpan A, Seputheisoflavone and Epunctanone from medicinal plants Garcinia epunctata and Ptycholobium contortum and evaluated their bioactivity towards 9 multi-factorial drug resistant cancer cell lines. Compounds 1-5 displayed cytotoxic effects in cancer cell lines with IC50 values below 70 µM. The IC50 values varied from 8.20 µM (in HCT116 (p53−/−) colon cancer cells) to 35.10 µM (against HepG2 hepatocarcinoma cells) for Betulin. Epunctanone induced apoptosis in CCRF-CEM cells through alteration of MMP and increase in ROS production. In addition to apoptosis, ferroptosis was also identified as another mode of cell death induced by Epunctanone. Fernandes et al., 2017 extracted and characterised natural products from the trunk bark of Helietta apiculata Benth (Rutaceae). They isolated nine alkaloids, eight furoquinoline and one quinolone, limonine, three cinnamic acid derivatives, three neolignans, tetracosanoic acid, six coumarins. This include Apiculin A, Apiculin B (neolignans), and Tanizin (coumarin), which are previously undescribed compounds. Spectroscopic methods were used to determine the structures of all compounds and the crystal structures of two of the newly undescribed compounds, Apiculin A and Apiculin B, were determined by X-ray analysis. The extracts and pure compounds isolated from H. apiculata showed promising antimicrobial and anti- inflammatory activities against the strain tested. . Xia et al., 2016 isolated pyranocoumarins from the stems of Clausena emarginata and evaluated its hepatoprotective effect on D-galactosamine- induced damage in WBF344 cells. Seven pyranocoumarins, clauemarmarins A-G, along with 6 6

known analogues were isolated from the stems of Clausena emarginata. Their structures were elucidated by extensive spectroscopic analyses and the absolute stereochemistries at C-6″ of Clauemarmarin B, C & D and the absolute configurations of Clauemarmarin E, F & G were determined by ECD experiments. The compounds Clauemarmarin C, D and two known analogues exhibited potent hepatoprotective activities against D-galactosamine-induced damage in WB-F344 cells. El Sohafy et al., 2016 fractionated the the ethanol extract of Cynara cornigera L. and the fractions were subjected to in vivo hepatoprotective assays. The liver injury was induced using CCl4 which caused elevation of biochemical parameters such as AST, ALT, ALP and total bilirubin, reflecting liver injury, when compared with standard Silymarin control. Phytochemical investigation and chromatographic separation of the hepatoprotective fractions led to the isolation of a new sesqui-lignan namely Cornigerin, along with eight known compounds like apigenin, luteolin, β-sitosterol glycoside, apigenin 7-O-β-D-glucopyranoside, luteolin-7-O-β-D-glucopyranoside, apigenin-7-O-rutinoside, cynarin 1,5-di-O-caffeoylquinic acid and apigenin-7-O-β-D-glucuronide possessing various bioactivites. Dahiy et al., 2016 isolated and characterised an anxiolytic constituent named Mahanimbine from the leaves of from Murraya koenigii L. Hydroalcoholic extract of M. koenigii leaves was evaluated for antianxiety activity using Elevated plus-maze (EPM) and further partitioned successively. All the fractions were evaluated for antianxiety activity and ethyl acetate fraction was column chromatographed to get 5 fractions, which showed maximum potential. A pure compound, separated out from the F-2 sub fraction of ethyl acetate fraction, was characterized using standard spectroscopic techniques, and its anxiolytic activity was evaluated using EPM. Antianxiety activity of isolated compound Mahanimbine was further evaluated and TLC-densitometric method was developed to quantify Mahanimbine in the plant. The result of the study validates the ethnopharmacological use of M. koenigii and indicated that Mahanimbine is responsible for the antianxiety effect. Ethnomedical Information Hibiscus hispidissimus is well known for its medicinal values. Traditionally it is used as an antiasthmatic and the leaves have good hypoglycemic and hypolipidemic effect. The tribal people use the leaves and flowers with honey for giddiness and liver disorders. The literature revealed that this plant has not been scientifically validated for their hepatoprotective and antioxidant activity. Therefore this plant is selected for the present study. 7

MATERIALS AND METHODS The plant specimen for the present study was taxonomically identified with the help of Flora Of Presidency Of Madras and collected from different localities of our campus NSS College, Nilamel, Kollam.Care was taken to select healthy plants and for normal organs. The whole plant were collected and thoroughly washed with running water to remove the adherent impurities. The collected plants were dried in shade, powdered using mechanical grinder and stored in air-tight glass containers. The powder was then passed through Sohxlet apparatus and extracted with ethanol .The extract was filtered and evaporated to get crude extract (HHCRD). This was then used for phytochemical investigation. 1.1.Phytochemical Investigations 1.1. 1 Preliminary phytochemical analysis of crude extract of leaf of H.hispidimus were carried out according to the standard procedures ( Harborne 1998; Raman ,2008). Tests for proteins Millon’s test:2 ml of Millon’s reagent mixed with the entire plant crude extract, appeared white precipitate, which upon gentle heating turned into red color which indicated the presence of protein in the plant. Ninhydrin test:Boil 2 ml of 0.2% Ninhydrin solution with the entire plant Crude extract, appeared violet color indicate the presence of proteins and amino acids. Tests for carbohydrates Fehling’s solutions test:Boil a mixture of Fehling solutions A and B with equal volumes were added to crude plant extract. A red color precipitate indicated the presence of reducing sugars. Benedict’s reagent test:Boil 2 ml of Benedict’s reagent with a crude extract, a reddish brown color indicated the presence of the carbohydrates. Molisch’s solution test: Shake 2 ml of Molisch’s solution with crude plant extract then add 2 ml of H2SO4 concentrated and poured carefully along the side of the test tube. a violet ring appeared at the inter phase of the test tube indicated the presence of carbohydrate. Iodine test:2 ml of iodine solution mixed with crude plant extract. Purple or dark blue colors prove the presence of the carbohydrate. Test for phenols and tannins Two milliliter of 2% solution of FeCl3 mixed with crude extract. Black or blue-green color 9

indicated the presence of tannins and phenols. Phenol test: 400ml of the crude acetone extract was treated with 5ml of dis.H2O and 5% ferric chloride solution and observed for formation of dark greenish colour Tannin test: 2ml of water extract was treated with 0.1% of FeCl3 solution and observed for blue black colouration for the presence of tannins. Tests for flavonoids Shinoda test:pieces of magnesium ribbon and HCl concentrated were mixed with crude plant extract after few minutes pink colored scarlet appeared that indicated the presence of flavonoids. Alkaline reagent test: 2 ml of 2% NaOH solution was mixed with plant crude extract, intensive yellow color was formed, which turned into colorless when added 2 drops of diluted acid(may HCL) to solution, this result indicated the presence of flavonoids. Test for saponins Five milliliter of distilled water was added to crude plant extract in a test tube and it was shaken vigorously. The foam formation indicated the presence of saponins. Tests for glycosides Liebermann’s test: 2 ml of acetic acid and 2 ml of chloroform mixed with entire plant crude extract. The mixture was then cooled and added H2SO4 concentrated, green color indicated the entity of aglycone steroidal part of glycosides. Salkowski’s test:H2SO4 concentrated (about 2 ml) was added to the entire plant crude extract. A reddish brown color produced indicated the entity of steroidal aglycone part of the glycoside. Alkaloids test: 2ml of methanol extract was taken with 1%HCl, 1ml filtrate with 6 drops of Wagner s reagent (1.27gm of iodine, 2gm of potassium iodide and 100ml of water). Formation of brownish red precipitation confirms the presence of alkaloids. 1.1.2 .Evalutaion of invitro antioxidant activity Antioxidant activity of ethanolic extract of H.hispidimus were evaluated using standard procedures as mentioned below. 1.1.2.1. Estimation of DPPH radical scavenging activist The effect of extracts on DPPH radicals were assayed 10

using the standard method (Blois , 1958).A methanolic solution of 2ml of DPPH (0.025g/l) was added to 200ml of the different concentrations (25mg/ml to 200mg/ml) of plant extract or serial fractions allowed to react at room temperature for 30m in dark and the absorbance was measured at 517 nm. Methanol served as the blank 200ml of methanol was added to DPPH in positive control tubes , instead of plant extract . Ascorbic acid was used as standard . % of inhibition= Ascorbance of control- Ascorbance of sample/Ascorbance of controlX100 From the calculated DPPH radical scavenging activity the EC50 was calculated , which represents the concentration of the scavenging compound that caused 50% neutralization. 11

RESULT AND DISCUSSION 2.1. Phytochemical analysis. 2.1.1. Qualitative phytochemical analysis. Qualitative phytochemical investigations revealed the presence of alkaloids, flavonoids, carbohydrates, glycosides, phenolic compounds and fixed fat and oils in HH CRD as shown in Table. 1. All other constituents like coumarins, saponins, proteins & amino acids, phytosterols and terpenoids etc. were absent. Table:1. Qualitative phytochemical analysis of H.hispidissimus Phytochemicals Test conducted Results 1 Alkaloids Mayer's test Wagner’s test + 2 Flavonoids Hanger’s test ++ 3 Carbohydrates ++ 4 Coumarins Dragendorff’s test - 5 Glycosides Alkaline reagent test ++ ++ Shinoda test - Iodine test ++ Benedict test + Molisch’s test - Alcohol-Sodium hydroxide test ++ Keller-killani test ++ Borntrager’s test

Legal’s test ++ - 6 Saponins Foam test - - Proteins Biuret test - ++ 7& Millon’s test ++ ++ Amino Acids Ninhydrin test - Phenols Lead acetate test - 8& Gelatin test + - Tannins Ferric chloride test Phytosterols Salkowski test 9& Libermann-Burchard test Terpenoids 10 Fixed oils & Fat Spot test 11 Gum & Mucilage Stirring test ‘+’ Present, ‘++’ strongly present and ‘–’ Absent. 2.1.In vitro Antioxidant Studies In- vitro antioxidant studies of Hibiscus hispidissimus were carried out using standard procedures and the results are as shown below. 2.1.1. DPPH radical scavenging activity The extracts of HH CRD showed potent antioxidant activity by inhibiting DPPH radical in a concentration dependent manner (Fig. 1). HH CRD showed most potent DPPH radical scavenging of 81 % at 320µg/mL and the EC50 was found to be 104.51 ± 2.64 µg/mL which is comparable to the ascorbic acid standard with EC50 value of 11.69 ±0.93 µg/mL.

Fig.1. DPPH radical scavenging activity of standard ascorbic acid . DPPH-STD Graph 100 y = 3.26x + 11.9 90 R² = 0.9978 80 %Inhibition 70 AA 60 50 5 10 15 20 25 30 40 Concentration 30 20 10 0 0 12 Fig.2. DPPH radical scavenging activity of H.hispidissimus crude extrcat

% Inhibition 100 DPPH Radical Scavenging Activity 90 80 y = 0.4034x + 7.8415 70 R² = 0.9874 60 50 HH CRD 40 30 50 100 150 200 250 20 Concentration 10 0 0 15

DISCUSSION In vitro antioxidant activity Herbaceous plants have a long history of use as medicine, food and variety of daily needs. Many epidemiological studies suggest that an increased consumption of several medicinal plants containing antioxidants can protect against DNA damage and carcinogenesis, and often exhibit a wide range of pharmacological activities such as anti-inflammatory, anti- bacterial and anti- fungal properties (Rasineni et al .,2008). Flavonoids have good antioxidant efficiencies and are common in leafy vegetables. A number of phytochemicals commonly used in research have antioxidant activity that can protect cells from reactive oxygen species (ROS)- mediated DNA damage that results in mutation and subsequent carcinogenesis. (Szeto et al.,2002), (Lazze et al .,2003). (Cao et al.,1996) indicated that increased consumption of vegetables and fruits increases the plasma antioxidant capacity in humans. Free radicals released during oxidative stress pose the major endogenous damage in the biological system. This type of damage is often associated with various degenerative diseases and disorders such as cancer, cardiovascular diseases and immunofunction decline and aging. Free radicals are highly reactive molecules having unpaired electrons and produced by radiation or as by-products of the metabolic processes (Cheung et al.,2003), (Kaur et al., 2001). To gain stability, free radicals capture the electrons quickly from other compounds and the attacked compound becomes a free radical itself, which continues to attack other compounds and leads to a chain reaction. These results in the disintegration of cell membranes and cell compounds, including lipid, protein and nucleic acids. Besides damage to living cells, free radicals are the major cause of food deterioration through lipid oxidation, which ultimately affects the organoleptic properties and edibility of foods (Kaur et al., 2001). Recent research suggested that synthetic antioxidants could promote tumor formation as well as anticarcinogenic properties. Due to these contradictory properties, the application and exploration of natural antioxidants has received more attention.(Halliwell,1989). Ascorbic acid , the standard antioxidant used in the present study , acts as a chain breaking scavenging agent that impaires the formation of free radicals in the process of intracellular substances formation throughout the body, including collagen bone matrix and tooth.(Aquil et al., 2006).Several methods have been developed to estimate the antioxidant capacity of different plant materials(Guo et al .,2003). A single assay is not sufficient to evaluate the total antioxidant activity (Frankel and Meyer, 2000: Silva et al .,2006). Hence in the present study,the ethanolic extract of H.hispidissimus were investigated for their antioxidant activity 16

using DPPH radical scavenging activity. The DPPH radical scavenging assay is used for evaluation of antioxidant potential of natural products because of its stability in the radical form, accuracy and simplicity of the assay (Bozin et al., 2007).The radical scavenging activities of ethnolic extract of H.hispidissimus were tested using the ‘stable’ free radical , DPPH. Unlike laboratory generated free radicals such as the hydroxyl radical and superoxide anion, DPPH has the advantage of being unaffected by certain side reactions, such as metal ion chelating and enzyme inhibition (Yen and Hung, 2000; Mau et al .,2002; Cheung et al .,2003; Amarowicz et al .,2004).DPPH is very popular for the study of natural antioxidants (Bhagya et al .,2013). DPPH radical scavenging is considered to be a good in vitro model widely used to asses antioxidant efficacy within a very short time. In its radical form, DPPH disappears, on reduction by an antioxidant compound or a radical species, to become a stable diamagnetic molecule resulting in the colour change from purple to yellow, due to the formation of diphenyl picryl hydrazine (DPPH), which could be taken as an indication of the hydrogen donating ability of the tested samples (Oktay et al .,2003; Shon et al .,2003 ;Lee et al., 2007). The relatively stable organic radical , DPPH, has been widely used in the determination of antioxidant activity of a single compound, as well as of different plant extracts(Katalinic et al.,2006). .In the present study , among the solvent tested , the HH CRD extract exhibited the highest DPPH radical scavenging activities. The result indicate that the plant extracts with their proton donating ability, could serve as free radical inhibitors or scavengers, acting possibly as primary antioxidants (Marxen et al.,2007). DPPH containing an odd electron, gives maximum absorption at 517 nm. As the odd electron of the radical becomes paired off in the presence of a hydrogen donor, the absorption strength is decreased and the resulting decolorisation (Violet to yellow) is stoichiometric with respect to the number of electrons quenched (Blios, 1958). During this study, the stabilized DPPH radical produces an intense violet colour in ethanol solution and the antioxidants present in H.hispidissimus reacted with the DPPH free radicals and convert them into reduced form either by donating a hydrogen atom or transferring an electron followed by proton. The decrease in absorbance is taken as a measure of the extent of radical scavenging.These IC50 values obtained for HH CRD is (104.51 ± 2.64 µg/mL) and is almost comparable to the standard Ascorbic acid (11.69 ±0.93 µg/mL) used. 5.8. Phytochemical analysis . Phytochemicals are chemicals that occurs naturally in plants .Medicinal plants are rich source of bioactive phytochemicals or bionutrients. Studies shows that these phytochemicals have an important role in preventing many diseases. It describes the structure of large number of secondary metabolic compounds found in plants. Traditional herbal medicines have received much attention as a source of novel therapeutic drugs since they are carefully chosen and utilized by humans from generations to generations and besides being therapeutic agents, they are a big source of information for a wide variety of chemical constituents which could be developed as drugs with precise selectivity (Vijyalakshmi and Ravindran, 2012). This

finding has raised hopes of obtaining novel bioactive phytoconstituents from plants with multitherapeutic and least side effects. 5.8.1. Qualitative phytochemical analysis. Medicinal plants contain an array of therapeutically active ingredients. Knowledge of these chemical constituents play an important role in evaluating its pharmacological activity. The therapeutic efficacy of traditionally used medicinal plants is mainly due to the phytochemicals present in them and these phytochemicals is mostly secondary metabolites such as alkaloids, tannins, total phenolics etc. (Ashokkumar et al. 2010). Phytochemical screening of plants is the need of the hour in order to discover and develop novel therapeutic agents with improved efficacy.Among all the phytoconstituents, phenolic compounds are one of the largest and most umbiquitous groups of plant metabolites (Singh et al., 2007). Comparative preliminary phytochemical analysis of various fractions and crude extract of HH CRD leaf showed rich in bioactive phytoconstituents like alkaloids, flavonoids, phenolic compounds , steroid and saponins.They are immunomodulatory (Chiang and Lin, 2003), antioxidant (Kahkonen et al., 1999), anti-apoptosis, anticarcinogen, antiinflammation, anti-atherosclerosis, cardiovascular protection and exhibit improvement of endothelial function, as well as inhibition of angiogenesis and cell proliferation activities. Phenolic structures often have the potential to strongly interact with proteins which gives them the ability to act as antioxidants and capacity to inhibit some enzyme involved in radical generation, such as various cytochrome P450 isoforms, lipoxygenases, cyclooxygenase and xanthine oxidase (Parr and Bolwell., 2002). Flavanoids have been reported to exhibit their actions through effects on membrane permeability and by inhibition of membrane bound enzymes such as the ATPase and phospholipase A2 (Li et al. 2003). Tannins are antioxidants possessing antiviral, antibacterial and antitumor activity. They are also reported to selectively inhibit HIV replication (Khanbabaee et al., 2001; Kashiwada et al., 1992). Alkaloids of plant origin also showed hepatoprotective, antioxidant, ant-viral, anti- microbial and anti-cancerous activities (Rajangama and Christina, 2012). Cardiac glycoside has been used in the treatment of congestive heart failure due to its direct action which increases the force of myocardial contraction (Braunwald et al., 1961). Phytosterols are able to interact with metabolic process and help in regulating the immune response (Shah et al., 2008; Fraile et al., 2012).Among the detected phytoconstituents , phenolics, alkaloids and phytosterols may contribute towards its pharmacological activities.

19

SUMMARY AND CONCLUSION The treatment of diseases in man using plants, plant extracts and pure compounds is increasing day by day. Medicinal plants and the active principles isolated from them are an important discovery in human beings to fight against diseases and disorders. Plants contain various phytochemicals like alkaloids, terpenoids, glycosides, phenols, tannins, and saponnins which have an important role in the defense mechanism of the body. The plant Hibiscus hispidissimus is being used by Traditional healers and tribal communities for various ailments. The phytochemical, antioxidant and ethnomedicinal studies have showed significant results and this may be due to the presence of phytochemical compounds like triterpenes and flavanoids. As there are very limited researches have been carried out on this plant, there is a need of extensive research on this plant focusing on its pharmacological and biochemical aspects of studies . 20

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