ANTIFUNGAL POTENTIAL OF METHLY ALCOHOL EXTRACT FROM THE

AERIAL PARTS OF INDIGOFERA POLYPHYLLA

BY

AMINU USMAN
U16DC1010

SUBMITTED TO THE DEPARTMENT OF CHEMISTRY

ISA KAITA COLLEGE OF EDUCATION DUTSIN-MA, IN AFFILIATION WITH

AHMADU BELLO UNIVERSITY, ZARIA.

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF

BACHELOR OF SCIENCE EDUCATION (B.sc ED. CHEMISTRY)

SEPTEMBER, 2022

i
 APPROVAL PAGE

This is to certify that this research project by AMINU USMAN, U16DC1010 conducted under

the supervision of Mal. Mu’ammar Lawal Dauda has been approved by the Department of

Chemistry having been part of the basic requirements for the award of Bachelor of Science

Education (Chemistry).

_____________________________ Sign & Date____________________

Project Supervisor

______________________________ Sign & Date____________________

Head of Department

_______________________________ Sign & Date____________________

External Moderator

ii
 DEDICATION

The research work is dedicated to my late parents; Late Alhaji Usman Mai Raguna and Late

Hajiya Amina Usman.

iii
 ACKNOWLEDGEMENT

All thanks and gratitude is to Almighty Allah the creature of the heaven and earth, and

everything that is in them who gave me the opportunity, strength, knowledge, endurance,

wisdom and patience during my course of study and for sparing my life to undertake this

research work.

I wish to express my sincere gratitude to Mal. Muammar Lawal Dauda who is my supervisor, for

sparing most of his valuable time to advice and make critical and valuable observation and

corrections with guidance during the course of this research, without whom this work would not

have been a success.

I also wish to extend my gratitude to the H.O.D and all the staff members of Chemistry

Department for their motivation and encouragement throughout the course of my study, may

Allah reward them abundantly.

My immeasurable appreciation goes directly to my beloved family and friends who support me

in one way or the other during this research work.

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 CERTIFICATION

I AMINU USMAN certify that this research was conducted and reported by me and to the best

of my knowledge; it has never been presented partly or wholly for the award of any Degree,

Diploma, NCE or published elsewhere.

AMINU USMAN Sign & Date____________________

U16DC1010

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 ABSRACT

Indigofera polyphylla is a plant that has been used in traditional medicine for centuries for a
variety of purposes, including the treatment of fungal infections. This study investigated the
antifungal potential of methanolic extract from the aerial parts of I. polyphylla against a panel of
pathogenic fungi. The extract was found to be active against a variety of fungi, including
Candida albicans, Aspergillus fumigatus, and Trichophyton rubrum. The minimum inhibitory
concentration (MIC) of the extract was determined for each fungus. The extract was also found
to be effective in inhibiting the growth of the fungi in vitro. These results suggest that methanolic
extract from the aerial parts of I. polyphylla has potential as an antifungal agent. Further
research is needed to confirm its efficacy and safety in humans.

Keywords: Indigofera polycantha, antifungal activity, methanol extract, minimum inhibitory

concentration (MIC)

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 TABLE OF CONTENT

Title Page - - - - - - - - - - - -i

Approval Page- - - - - - - - - - -ii

Dedication- - - - - - - - - - - -iii

Acknowledgement- - - - - - - - - - -iv

Certification- - - - - - - - - - - -v

Abstract- - - - - - - - - - - -vi

CHAPTER ONE

1.0 Introduction- - - - - - - - - - -1

1.1 Indigofera polyphylla- - - - - - - - - -10

1.2.1 Taxonomy- - - - - - - - - - -10

1.2.2 Botanical Characterization Distribution- - - - - - -12

1.2.3 Traditional Use and Ethnopharmacology- - - - - - -13

1.2.4 Chemical Constituents- - - - - - - - - -13

1.2.5 Pharmacological Activity- - - - - - - - -15

1.2.5.1 Embryotoxic and Cytotoxic Activity- - - - - - -15

1.2.5.2. Antitumor Activity- - - - - - - - - -16

1.2.5.3. In Vivo Activity against Ectoparasite (Pediculosis capitis)- - - -16

1.2.5.4. Antimutagenic Activity- - - - - - - - -17

1.2.5.5. Antioxidant Activity- - - - - - - - -17

1.2.5.6. Hepatoprotective Activity- - - - - - - - -17

1.2.5.7. Antimicrobial Activity - - - - - - - -18

1.2.5.8. Anticonvulsant Activity- - - - - - - - -19

1.2.5.9. Gastroprotective Activity- - - - - - - - -20

vii
1.2.5.10. Immunostimulatory Activities- - - - - - - -20

1.2.5.11. Anti-Inflammatory Activity- - - - - - - -21

1.2.6. Gynecological Problems/Issues- - - - - - - -22

1.2.6.1 Toxicity- - - - - - - - - - -22

1.3 Organisation of the Report- - - - - - - - -23

1.4 Limitation of the Study- - - - - - - - - -23

1.5 Aim and Objectives- - - - - - - - - -23

CHAPTER TWO

2.0 Literature Review- - - - - - - - - -24

2.1 Morphology and Anatomy- - - - - - - - -26

2.1.1 Floral Syndrome- - - - - - - - - -27

2.1.2 Cytology- - - - - - - - - - -28

2.2 Other Investigative Approaches- - - - - - - - -29

2.3 Phytochemistry- - - - - - - - - - -30

2.4 Pharmacology- - - - - - - - - - -32

2.5 Pharmacognosy- - - - - - - - - - -33

CHAPTER THREE

3.0 Materials and Method- - - - - - - - - -35

3.1 Chemicals/Reagents- - - - - - - - - -35

3.2 Equipment and Apparatus- - - - - - - - -35

3.3 Sample Collection and Pre-Treatment- - - - - - - -35

3.4 Sample Extraction- - - - - - - - - -36

3.4.1 Preparation of Aqueous extract- - - - - - - -36

viii
3.4.2 Preparation of Methanolic extract- - - - - - - -36

3.4.3 Preparation of Acetate Nitrate extract- - - - - - - -36

3.5 Phytochemical analysis- - - - - - - - - -37

3.5.1 Test for alkaloids- - - - - - - - - -37

3.5.2 Test for flavonoids- - - - - - - - - -37

3.5.3 Test for glycosides- - - - - - - - - -37

3.5.4 Test for cardiac glycosides- - - - - - - - -38

3.5.5 Test for saponin glycosides- - - - - - - - -38

3.5.6 Test for steroids- - - - - - - - - -38

3.5.7 Test for volatile oils- - - - - - - - - -38

3.5.8 Test for tannins- - - - - - - - - - -38

3.5.9 Test for saponins- - - - - - - - - -39

3.6 Quantitative analysis of flavonoids- - - - - - - -39

3.7 Antimicrobial Susceptibility Test- - - - - - - -39

3.8 Toxicological analysis- - - - - - - - - -40

CHAPTER FOUR

4.0 Results- - - - - - - - - - - -41

4.1 Percentage (%) Recovery on Extraction- - - - - - - -41

4.2 Phytochemical Screening- -- - -42

4.3 Antimicrobial Susceptibility Test - - - -43

4.4 Zone of Inhibition (mm)- - - -44

4.5 Minimum Inhibitory Concentration (MIC) - - - -45

4.7 Discussion- - - -46

ix
 CHAPTER FIVE

5.1 Summary - - - - - - - - - - - -49

5.2 Conclusion - - - - - - - - - - -49

5.3 Recommendations- - - - - - - - - -49

REFERENCES- - - - - - - - - - -51

x
 CHAPTER ONE

1.1 Introduction

Plants are the source of energy for the animal kingdom including the human beings. All human

beings require a number of complex organic and inorganic compounds in diet to meet the need

for their activities and plants are the rich source of all the elements essential for them. Hence

vegetarianism became popular in recent years even in Western countries. Plant foods such as

vegetables and fruits play a vital role in the health of human beings providing carbohydrates,

vitamins and minerals.

Throughout history, civilized societies have developed interest and concern about the integrity of

food for the above compounds. Long before the development of the distinct scientific discipline

of nutrition, philosophers and later physicians paid close attention to the role of daily diet in

individual and public health.

The important constitution of diet are carbohydrates, fats, proteins, vitamins, minerals and water

(Indrayan et al., 2005). These constituents play an important role in the physiology of animals

including human beings and deficiency or excess of one or more component lead to

manifestation of abnormalities which are known as diseases.

The Greek physician compiled the first European treaty on the properties and uses of medicinal

plants, De Materia Medica. In the first century AD, Dioscorides wrote a compendium of more

than 500 plants that remained an authoritative reference into the 17th century. Similarly

important for herbalists and botanists of later centuries was the Greek book that founded the

science of botany, Theophrastus’ Historia Plantarum, written in the fourth century B.C.

(Bhandari, 2004).

1
During the last 2000 years, from the time of Hippokrates (B.C. 460-377) to the dawn of modern

medicine, there was only a little distinction made between food and drugs. The practice of

medicine itself consisted largely of the wise choice of natural food products. Hippokrates

recognized the essential relationship between food and health (Jones, 1923). A physician

reflected confidence in the knowledge and ability of physicians to establish sound diet that would

advance public health (Green, 1951).

Many of the pharmaceuticals currently available to physicians have a long history of use as

herbal remedies, including opium, aspirin, digital and quinine. The World Health Organization

(WHO) estimates that 80 percent of the world's population presently uses herbal medicine for

some aspect of primary health care. Pharmaceuticals are prohibitively expensive for most of the

world's population and on the contrary, herbal medicines can be grown from seed or gathered

from nature for little or no cost. In addition to the use in the developing world, herbal medicine is

used in industrialized nations by alternative medicine practitioners such as naturopaths. A 1998

survey of herbalists in the UK found that many of the herbs recommended by them were used

traditionally but had not been evaluated in clinical trials. In Australia, a 2007 survey found that

these Western herbalists tend to prescribe liquid herbal combinations of herbs rather than tablets

of single herb.

Nutritional and medicinal properties of plants are known since a long time. However, their

disease preventing and health promoting aspects are realized only recently. The scientific basis

behind the use of plants as medicines came to light only recently due to scientific advancements

happened in research and instrumental analysis. Medicinal plants have been used in traditional

treatments for numerous human diseases for thousands of years and they continue to be a

therapeutic aid even in modern era also. Interestingly today, there is a renewed interest happened

2
in traditional medicine and an increasing demand for more drugs from plant sources. This revival

of interest in plant derived drugs is mainly due to the current widespread belief that the green

medicine is safe and more dependable and without side effects compared to the synthetic drugs.

Nature has bestowed the earth with rich botanical wealth and diverse group of plants grow wild

in different part of the world. The knowledge on medicinal plants predates the human history and

evidences are available globally.

In their natural environments, plants have to cope with an array of stress conditions to improve

their survival chances and stress factors include drought, salinity, nutritional deficiency, adverse

climate condition, pollutants, pathogens, insects, phytophagy etc., and Hence plants exhibit a

variety of defense responses to make their survival possible in a particular environment.

Moreover, being sessile, they synthesize numerous phytochemicals as a major strategy to

counteract unfavorable conditions. Most of the phytochemicals are secondary metabolites, not

directly involved in development, growth and reproduction, but mostly defense molecules,

produced considering the factors prevailing in the immediate environments and the related

ecological networks in that region. A great array of molecules derived from plant secondary

metabolism, are of extreme interest in human nutrition and pharmacology, in addition to

perfumery and cosmetics.

Plants have evolved the ability to synthesize chemical compounds that help them to defend

against the attack from a wide variety of predators such as insects, fungi and herbivorous

mammals. By chance, some of these compounds whilst being toxic to plant predators turned out

to have beneficial effects in treating human diseases. Such secondary metabolites are highly

varied in structure; many are aromatic substances, most of which are phenols or their oxygen-

substituted derivatives. At least 12,000 have been isolated so far; a number estimated to be less

3
than 10% of the total. Chemical compounds in plants mediate their effects on the human body by

binding to receptor molecules present in the body; such processes are identical to those already

well understood for conventional drugs and as such herbal medicines do not differ greatly from

conventional drugs in terms of working principles. This enables herbal medicines to be in

principle just as effective as conventional medicines but without harmful side effects. Many of

the spices used in seasoning of foods have compounds with the medicinal properties (Singh et.

al., 2004).

Plant derived food stuffs and beverages include mainly fruits, vegetables, herbs, spices,

chocolate, tea, beer, wine etc. are known as functional foods. These are not considered as

pharmaceutical products, but known as nutraceuticals as they are components able to ameliorate

human fitness of health. However based on their biological properties such as antioxidant, anti

hypersensitive, cardio protective, antiinflammatory, anti mutagenic, antitumoral, anticancer,

most of them are called as pharmaconutrients. The term “nutraceutical” was coined in 1989 by

the Foundation for Innovation in medicine (New york, US) to provide a name for this rapidly

growing area of biomedical research. A nutraceutical can be defined as any substance that may

be considered as a food or part of a food and provides medical or health benefits (De Felice,

1992). Nutraceuticals may include specific nutrients, dietary supplements, diets, genetically

engineered designer foods, herbal products, processed products like soups, beverages etc.

Due to the explosion happened in research publications and providing scientific evidence to

support the health effects, the term “phytochemicals” became popular referring the functional

components as a functional food (or) nutraceutical or phytinutrient.

Harborne (1999) identified the three major classes of plant chemicals as terpenoids, phenolic

metabolites, alkaloids and other nitrogen-containing plant constituents. As delineated by

4
Harborne, the terpenoids include monoterpenoids, iridoids, sesquiterpenoids, sesquiterpene

lactones, diterpenoids, triterpenoid saponins, steroid saponins, cardenolides and bufadienolides,

phytosterols, cucurbitacins, nortriterpenoids, other triterpenoids and carotenoids. The phenolic

metabolites include anthocyanins, anthochlors, benzofurans, chromones, coumarins, minor

flavonoids, flavonones and flavonols, isoflavonoids, lignans, phenols and phenolic acids,

phenolic ketones, phenylpropanoids, quinonoids, stilbenoids, tannins and xanthones. The

alkaloids include amaryllidaceae, betalain, diterpenoid, indole, isoquinoline, lycopodium,

monoterpene, sesquiterpene, peptide, pyrrolidine and piperidine, pyrrolizidine, quinoline,

quinolizidine, steroidal and tropane compounds. Other nitrogen-containing constituents include

nonprotein amino acids, amines, cyanogenic glycosides, glucosinolates, purines and pyrimidines.

Specific phytochemicals can be used as therepeutic drug if their efficacy is confirmed by proper

scientific research. The traditional procedures to prepare plant preparations may enhance the

chemotherapeutic value of the plant derivatives. However, the individual components to be

scientifically validated to support the specific health claims. The biological test systems like

animal models, in vitro cultured cell system and various stages of clinical trials in human beings

to be followed to convert a phytochemical into a potent drug. For most of the medicinal plants,

the information available is of the observational or descriptive type and information on functions

and mechanism of action especially on individual components are lacking.

Researchers from Ohio Wesleyan University found that some birds select nesting materials,

which are rich in antimicrobial agents that protect their young ones from harmful fungi. Sick

animals tend to forage plants rich in secondary metabolites, such as tannins and alkaloids. Since

these phytochemicals often have antiviral, antifungil, antifungal and antihelminthic properties, a

plausible case can be made for selfmedication by animals in the wild (Saxena, 2004).

5
The use of and search for, drugs and dietary supplements derived from plants have accelerated in

recent years. Pharmacologists, microbiologists, botanists, and natural-products chemists are

combing the Earth for phytochemicals that could be used for treatment of various diseases. In

fact, according to the World Health Organisation, approximately 25% of modern drugs used in

the United States have been derived from plants (Dayal, 2004).

Among the 120 active compounds currently isolated from the higher plants and widely used in

modern medicine today, 80 percent show a positive correlation between their modern therapeutic

use and the traditional use of the plants from which they are derived.

More than two thirds of the world's plant species - at least 35,000 of which are estimated to have

medicinal value mostly come from the developing countries. All plants produce chemical

compounds as part of their normal metabolic activities. These are divided into primary

metabolites, such as sugars and fats, found in all plants, and secondary metabolites, compounds

not essential for basic function found in a smaller range of plants, some useful ones found only in

a particular genus or species. Pigments harvest light, protect the organism from radiation and

display colors to attract pollinators.

Many common weeds, such as nettle, dandelion and chickweed, have medicinal properties. The

functions of secondary metabolites are varied. For example, some secondary metabolites are

toxins used to deter predation, and others are pheromones used to attract insects for pollination.

Phytoalexins protect against fungil and fungal attacks. Allelochemicals inhibit rival plants that

are competing for soil and light. Plants either up-regulate or down-regulate their biochemical

paths in response to the local mix of herbivores, pollinators and microorganisms. The chemical

profile of a single plant may vary over time as it reacts to changing conditions. It is the

6
secondary metabolites that can have therapeutic actions in humans and which can be refined to

produce drugs.

Plants synthesize a bewildering variety of phytochemicals but most are derivatives of a few

biochemical motifs. Alkaloids contain a ring with nitrogen. Many alkaloids have dramatic effects

on the central nervous system. Caffeine is an alkaloid that provides a mild lift but the alkaloids in

datura cause severe intoxication and even death. Polyphenol, also known as phenolics, contain

phenol rings. The anthocyanins that give grapes their purple color, the isoflavones, the

phytoestrogens from soy and the tannins that give tea its astringency are phenolics. Terpenoids

are built up from terpene building blocks. Each terpene consists of two paired isoprenes. The

names monoterpenes, sesquiterpenes, diterpenes and triterpenes are based on the number of

isoprene units. The fragrance of rose and lavender is due to monoterpenes. The carotenoids

impart the red, yellow, orange colors to pumpkin, corn and tomatoes.

Glycosides consist of a glucose moiety attached to an aglycone. The aglycone is a molecule that

is bioactive in its free form but inert until the glycoside bond is broken by water or enzymes.

This mechanism allows the plant to defer the availability of the molecule to an appropriate time,

similar to a safety lock on a gun. An example is the cyanoglycosides present in cherry plants

which is released as a toxin only when the plant is bitten by herbivores.

Research in herbal medicine and isolated drug discovery need to be continued, considering the

threat of new emerging disease such as SARS, bird flu and the killer disease AIDS. Plants are a

good source of herbal medicine and natural products/phytochemicals. Many synthetic drugs owe

their discovery and potency as a result of a mimic of structures from natural products isolated

from plants rather than to the creativity and imagination of contemporary organic chemists. For

example, the drug taxol (a diterpenoid), first isolated from the bark of the yew tree Taxus

7
brevifolia had yielded two approved drugs for breast and ovarian cancer. In Guyana, there are

many medicinal folklore practices but most of them are without scientific validation. For

example, the aqueous extract of Momordica charantia seemed to be a good remedy for diabetes.

Thus, there exists an urgent need to correlate folklore herbal practices with scientific evidences.

The condition is same worldwide, however there is a growing concern over this (Rahuman et al.,

2008).

India is one of the world’s 12 mega biodiversity country, having rich vegetation with 47,000

plant species and a wide variety of medicinal plants along with tradition of plant based

knowledge distributed among the vast numbers of ethnic groups. There are many medicinally

important species which are used to produce various types of drug and medicines to treat many

ailments in India since the time of the Rig Veda (approximately 2000 BC). India represents one

of the great emporia of ethno-medicinal wealth has enormously diversified living ethnic groups

and rich biological resources (Roopashree et al., 1965).

Herbal medicines are plant derived material or preparations, which contain raw or processed

ingredient from one or more plants or its parts, with therapeutic value and used as dietary

supplements to fight or prevent common disease in various systems of medicine such as

Ayurveda, Unani and Siddha. Plant derived natural products have received considerable attention

in recent years due to their diverse pharmacological activities. The traditional herbal

combinations and extracts are known to improve health by combating or preventing microbial

infections and curing various ailments and diseases (Serti et al., 2001 and Roy et al., 2005).

Silver et al., 1993, Senatare 2004, and Si et al., 2006, reported that the plant derived medicines

are relatively safer than synthetic drugs and offering profound therapeutic benefits by providing

alternative and effective treatment for chronic disorders and various diseases. More than 1500

8
herbal preparations are sold in India as dietary supplements or ethnic traditional medicine to treat

the diseases but only a few of them have been scientifically explored for its antifungil potentials.

The most frequently used herbal preparations is churna; a preparation comprising of fine

powders of medicinal plants either in single or in combination. Combinations of medicinal plants

may increase the antimicrobial spectrum and potency of activity. Enteric or diarrhoeal infections

account for high proportion of health problems in the developing countries and contribute to the

death of 3.3 to 6.0 million children annually. Enteric fungi such as Salmonella sp., Shigella sp.,

Proteus sp., Klebsiella sp., Escherichia coli, Pseudomonas sp., Vibrio cholerae, and

Staphylococcus aureus are major etiologic agents of sporadic and epidemic diarrhea in both

children and adults. Recently, it has been demonstrated that many human pathogenic fungi have

developed resistance against several synthetic drugs, discourage their use and insist the search

for alternative medicines. There are several reports on antimicrobial activity of crude extracts

prepared from plants that inhibit various fungil pathogens, but a limited numbers of in vitro

studies on herbal preparations have been published. Therefore, it is need of the hour to identify

antifungil potential of herbs especially diseases which no medicine or only palliative therapy is

available (Tamboli, 2000). At this juncture, it is of interest to determine the scientific basis for

the traditional use of these herbal medicines and to evaluate their bioactive potential.

9
1.2 Indigofera polyphylla

1.2.1 Taxonomy

Kingdom: Plantae

Phylum: Tracheophyta

Subdivision: Spermatophytina

Superclass: Angiospermae

Class: Magnoliopsida

Order: Fabales

Family: Fabaceae

Genus: Indigofera

Species: Indigofera polyphylla

Fabaceae, present in the Brazilian biodiversity, is considered the third largest family of plants,

which has about 19,500 species, and it is divided into three subfamilies: Mimosoideae,

Caesalpinioideae, and Papilionoideae, and it shows a common feature in almost all fruits and

vegetables, known as pods. Papilionoideae is a subfamily with greater wealth in the Caatinga.

Among diverse species, the Indigofera species are taxonomically present (Tamboli, 2000).

This family is considered of great importance because among the several varieties, many species

are used for food purposes and is used as animal feed, latex, resins, raw materials in the

manufacture of paints, pesticides, and medicinal drugs, before undergoing processing and

purifications (Dioclea megacarpa, Vatairea paraensis, and Dipteryx punctata), and ornamental

10
trees. Examples of species used as food sources are chickpea (Cicer arietinum), peas (Pisum

sativum), beans (Phaseolus vulgaris), lentil (Lens cultivaris), and soybean (Glycine max) Roy et

al., 2005.

The genus Indigofera belonging to the Fabaceae family stands out for being used as fodder,

green manure, and ground cover (Roy et al., 2005). This genus has about 700 species distributed

in Asia, tropical Africa, Australia, and North and South America; in Brazil, it is possible to find

three species with same popular name “anileira”: Indigofera truxillensis, I. hirsuta, and I.

polyphylla (Roy et al., 2005).

A few decades ago, the investigations of I. polyphylla have focused on their biological activities,

including their antitumor, anti-inflammatory, antimicrobial, and antiepileptic properties, but now

scientific studies have diversified and deepened their knowledge about this species. These

studies evaluated the biological potential of different parts of the plant, with chemical

compounds from extractions isolated by using various solvents. This review aimed at providing

important data on the botanical, distribution, ethnopharmacology, phytochemical,

pharmacological, and toxicity of I. polyphylla based on the scientific literature (moyo et al.,2012).

Members of the genus are primarily found in the warmer region with apparent centers of

distribution in Africa and in the Himalayas (Polhill, 1981). Economic and esthetic uses have long

been found for members of this genus. Some are grown for ornamental purposes (Bailey, 1924)

but, by and large, major interest in the genus has focused on the economic use of Indigofera as a

source of the deep blue dye, indigo. Its use in dye-production is reflected in the generic name

given by Linnaeus (1753), as Indigofera means "indigo-bearing" (Fernald 1950). The production

of natural indigo has long since been supplanted by synthetic dyes (Fernelius and Renfrew,

1983) but it is widely acknowledged that the growing of indigo-containing plants played a major
11
part in the economic life of various parts of the world (Meyen 1846; Rerabert 1979; Duke 1981).

Indigo was grown on many of the plantations of Louisiana and figured prominently in the early

economy of colonial British North America (Rembert 1979).

A modern, worldwide taxonomic treatment for the entire genus does not yet exist; some of the

older, comprehensive synopses of plants, though, have included enumerations of members of the

genus known at the time these were published. Such works include those of de Candolle (1825),

Steudel (1840), Dietrich (1847), Bentham (1996), and Taubert (1894). In this century, treatments

have primarily been regional in emphasis; these have, however, provided a good idea of the

scope of the genus in the studied area.

1.2.2 Botanical Characterization Distribution

I. polyphylla is described as a shrub plant, measuring 1 m to 2 m height, having branches

pubescent, stem angular, of grayish color, pinnate leaves composed of 7–15 oblong or oval

leaflets, hairless on the face and back, with small flowers, numerous albo-pink or yellow, in

axillary racemes, and its fruit is a small sickle pod with 6–10 seeds measuring 25 mm in length

(Bentham 1996). Having strong adaptability, they are considered wild plants that grow in all

types of soils, tolerating drought, floods, and high salinities (Bentham 1996).

I. polyphylla Mill. is a species from the Antilla and Central America and more prevalent

throughout the tropical America. In Brazil, it is distributed in some states: São Paulo, Sergipe,

Bahia, Rio de Janeiro, Minas Gerais, Maranhão, Mato Grosso, Alagoas, Paraíba, Ceará, Rio

Grande do Norte, (Pernambuco, and Pará Steudel 1990; Dietrich, 1847; Bentham, 1996; and

Taubert, 1894).

12
1.2.3 Traditional Use and Ethnopharmacology

Indigofera polyphylla is popularly known as “indigo.” Such a nickname comes from the German

language, meaning “blue pigment,” which is extracted through fermentation by hot infusion of

its leaves and was used in the textile industries to dye yarns. Currently, this extract is processed

by industrial chemical processes, and the use of this plant in the textile industries was abandoned

(jeeju et al.,2004). I. polyphylla may also be related to other popular names such as jiquilite,

tzitzupu, indigo fields, anileira guinea, real anileira, caá-chica, caá-chira, timbó-mrim,

timbozinho, and indigueira. The species is widely used in folk medicine to cure many health

problems, with uses based on infusions and decoctions of different parts of this plant (jeeju et

al.,2004). They are attributed to this plant’s febrifuge, antispasmodic, diuretic, abortive, and

analgesic properties against stomach and urinary problems, jaundice, ulcers and purgative,

sedative, and insecticidal properties (jeeju et al.,2004).

1.2.4 Chemical Constituents

Several studies have identified and isolated some chemical constituents of I. polyphylla,

including flavonoids, alkaloids, coumarins, triterpenoids, and carbohydrates. Early investigations

of the chemical components of I. polyphylla were made by Miller and Smith, 1973, using seed

extract, with a highlight of the rich presence of amino acids and possible toxic effects. According

to the Natural Products Alert and Chemical Abstracts, the phytochemical profile of this species

reveals the presence of alkaloids, polyphenols, terpenoids, steroids, reducing sugars, proteins,

and indigoids (saidana et al.,2008).

Paiva et al., (2008) quantified proteins and natural fibers of this species, showing its use as feed

for ruminants. Isolation of 3-nitropropanoic acid glucose esters is featured by having toxic

13
effects due to its conversion to the 3-nitropropanoic acid, a respiratory toxin that inhibits

mitochondrial enzymes. In addition, D-(+)-pinitol, β-sitosterol, and louisfieserone have been

isolated from this plant. Apart from these isolated compounds, Kamal and Mangla identified,

characterized, and quantified six rotenoids from different parts of I. polyphylla. Preliminary

studies of leaves, seeds, and stems of I. polyphylla demonstrate the presence of polyphenols

(coumarin and chlorogenic acid) and flavonoids (quercetin, rutin, and gallic acid), alkaloids,

triterpenoids, and carbohydrates (Paiva et al., 2008).

The main flavonoids identified and isolated from the methanol extract of I. polyphylla leaves

include quercetin 7-O-β-d-glucopyranoside, quercetin 3-O-[β-d-xylopyranosyl-(1⟶2)-β-d-

galactopyranoside], quercetin 3-O-[α-l-rhamnopyranosyl-(1⟶6)-β-d-glucopyranoside], and

quercetin 3-O-[β-d-glucopyranosyl-(1⟶2)-β-d-glucopyranoside. In addition to these

compounds, indigo and indirubin were also isolated (Paiva et al., 2008).

Pentadecanoic acid, 14-methyl-, n-hexanedecanoic acid, z-[13, 14-epoxy]tetradec-11-en-1-ol

acetate, oleic acid, 9-octadecenoic acid[z]-, 2-hydroxy-1-[hydroxyl methyl], heptanoic acid,

docosyl ester, octadecanoic acid, 7-hydroxy-, 6-octadecenoic acid[z]-, and 8-octadecenoic acid

were also found (Abu-Shanab, 2004).

Chen et al. (2002) using aqueous and ethanol extracts of I. polyphylla identified the following

different phenolic compounds: syringic acid, p-coumaric acid, vanillin, syringaldehyde, salicylic

acid, quercetin, isoliquiritigenin, and formononetin.

The presence of such compounds in the leaf oil of I. polyphylla, (z)-3-hexenyl benzoate, methyl

hexadecanoate, phytol, linoleic acid, methyl linoleate, n-docosane, n-tricosane, was also found.

14
1.2.5 Pharmacological Activity

1.2.5.1 Embryotoxic and Cytotoxic Activity

Leite et al. (1998) investigated the cytotoxic potential of aqueous extracts from leaves of I.

polyphylla in mouse embryos and found that, at high concentrations of the extract, the growth of

the embryos was inhibited, preventing them reaching the final stage of embryogenesis, indicating

that their use in high doses in humans can be harmful.

Vieira et al. (2000) realized in his studies that aqueous extracts of leaves of I. polyphylla by

infusion and maceration in different concentrations (from 6.25 to 50 µg/ml) tested in cell lines of

HEp-2 by the MTT method did not produce any cytotoxic effect (>30 µg/ml) when compared

with the control and DMEM (Dulbecco’s Modified Eagle Medium).

In another study, the indigo ethanol extract purifier of I. polyphylla showed a potent cytotoxic

agent, showing the value of 0.89 for the breast tumor cell line (LM2) and lung tumor cell line

(LP07), clarifying that the extract has cellular responses such as inducing apoptosis [31].

Carli et al. (1998) also observed the cytotoxic effect on cell viability assays with 3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), having inhibitory concentration

(IC50) of 200 μg/ml.

Bioactive compounds of natural origin are essential in antineoplastic therapy, since they show

promising effects on carcinogenesis and contribute to new medicinal interests. Therefore, such

information has great relevance in understanding the use of this species and its promising future.

Vieira et al. (2007) used aqueous I. polyphylla extracts at varying doses (250–1000 mg/ml) in

models for embryotoxicity in the development and oviposition of Aedes aegypti. In the study, a

15
significant repellent effect was found on oviposition and an embryotoxicity was also observed,

slowing the normal growth of the larvae of Aedes aegypti.

The use of plant extracts is an alternative method for insect control, which can minimize the

harmful effects of some insecticides on nontarget insect species, humans, and the environment,

leading to new opportunities for the development of commercial products of natural origin and of

great importance.

1.2.5.2. Antitumor Activity

Vieira et al. (2007) evaluated the effect of aqueous extracts of I. polyphylla processed by

maceration and infusion at a dose of 50 mg/kg on sarcoma 180 and found that both forms had

significant effect on reducing the tumor (62.6 and 64.5%), respectively.

Cancer is still the leading cause of death in the world, and plants used in traditional medicines

may be a potential source of powerful chemopreventive agents because they are enriching source

of beneficial secondary components. There are many plant species that have relevant biological

data in the scientific literature and even commercial use for this purpose. However, the use of I.

polyphylla is still poorly studied, and new studies should investigate the possibility of using as an

antitumor drug.

1.2.5.3. In Vivo Activity against Ectoparasite (Pediculosis capitis)

García et al. (2006) used tincture of I. polyphylla to 5% in the population reduction

of Pediculosis capitis and observed the population reduction of lice mainly in cases of

persistence in patients of 55 years. After 2 days of application, the results confirmed the

16
insecticidal activity of I. polyphylla tincture; this treatment seems to be a valuable and effective

alternative to existing treatments.

1.2.5.4. Antimutagenic Activity

Calvo et al. (1999) evaluated the effect of the ethanol extract of the aerial parts of I. polyphylla in

trials on Salmonella, which showed mutagenic activity, suggesting that such an action is due to

the presence of indigo pigment. Since plants are primary food sources and cure, the natural

products need to be evaluated with regard to their toxicity and dosage because the indiscriminate

use of homemade preparations of plants can be dangerous to health.

1.2.5.5. Antioxidant Activity

Ethanolic extracts of leaves of I. polyphylla stand out with potent antioxidant activity in an

experimental model in vitro with the free radical 2,2-diphenyl-1-picrilidrazil (DPPH), and this

method is a rapid way to measure the antioxidant capacity of compounds, it is based on the

reduction of DPPH in solution, and this action is attributed to the presence of high concentrations

of gallic acid in the extract [30]. Plants contain several phytochemicals, such as phenolic

compounds, which have promising antioxidant activity, mainly in the prevention of chronic

diseases.

1.2.5.6. Hepatoprotective Activity

The aqueous extract of I. polyphylla (50 mg/kg, ip) showed the protective effect on liver tissue of

mice bearing sarcoma 180. Lima et al. (2011), using the purified indigo compound from the

17
leaves of I. suffruticosa, did not observe a reduction in sarcoma 180 tumor but found that liver

cells remained preserved, emphasizing its hepatoprotective effect.

Lima et al. (2011) investigated the antitumor action of indica, a compound extracted from the

leaves of I. polyphylla, on mice bearing sarcoma 180 cell lines and found that such a compound

did not promote a reduction in tumor growth; however, it is found that treatment with indica does

not allow to alter the architecture of the liver cells, suggesting a hepatoprotective effect.

Such an effect is considered of extreme importance since liver diseases have become a global

public health problem, and much of them are a consequence of the prolonged use of chemicals

and drugs.

1.2.5.7. Antimicrobial Activity

Santos et al. (2004) evaluated the antimicrobial activity of ether, chloroform, and acetone

extracts of I. polyphylla against nine strains of Staphylococcus aureus, with minimal inhibitory

concentration (MIC) ranging from 0.78 to 6.25 mg/ml. The methanol extract of the aerial parts

of I. suffruticosa showed a significant effect against Mycobacterium tuberculosis with an MIC of

125 μg/mL, suggesting the presence of an important bactericidal agent (Santos et al., 2004).

The endophytic fungi isolated from I. polyphylla also showed activity against different fungi

such as B. subtilis, S. aureus, E. coli, K. pneumoniae, and P. aeruginosa, with an MIC ranging

from 0.39 to 6.25 mg/ml, emphasizing that this species has a potential to inhibit fungil growth

(Santos et al., 2004).

The aqueous extract of the leaves of I. polyphylla displays significant results against two strains

of Trichophyton rubrum and Microsporum canis, with concentrations of 5 and 10 mg/ml with

18
variation in MIC between 20 and 15 mm, with a similar effect to the standard drug ketoconazole

(MIC −20 mm) (Santos et al., 2004).

The results obtained in these studies suggest that the extracts used had bioactive compounds

responsible for the efficacy of the organic material studied. Not only the plant extracts but also

those produced by microorganisms can be a source for the industrial manufacture of drugs and

useful in the therapy of some microbial infections.

1.2.5.8. Anticonvulsant Activity

The I. polyphylla fluid extract (0.06 g/kg for 10 days) in experimental models of shock promotes

protective effect in both doses administered orally or intraperitoneally on seizures induced by

this model, highlighting the antiepileptic potential of such an extract.

At the concentration of 0.06 g/kg, the fluid extract of I. polyphylla was tested in models of

chronic epilepsy, which reduced the concentration of inhibitory amino acids (glycine and tannin)

and increased the excitatory amino acid (glutamic acid) (Santos et al., 2004).

In models of seizures, the methanol extract of the leaves of I. polyphylla showed significant

activities, and its action is due to the presence of secondary metabolites such as flavonoids and

linalool having an action on the GABAergic system (Santos et al., 2004).

The anticonvulsant effect of the aqueous extract of I. polyphylla was confirmed following

behavior and electrophysiological analyses in rats. The systematic analysis of seizure behavior

and its potentials was also recorded.

The findings of these studies stimulate the continuity of research in the search for new treatments

for epilepsy. Although traditional therapy has good efficacy, it has high toxicity, and about 20–

19
30% of patients who use this therapy are unable to control their seizures appropriately and have

severe side effects. Therefore, new alternatives that reduce clinical manifestations and chronic

conditions become essential.

1.2.5.9. Gastroprotective Activity

Luiz-Ferreira et al. (2004) explored the gastroprotective effect of chloroformic and methanolic

extracts of the aerial parts of I. polyphylla, partitioned with ethyl acetate and administered at a

dose of 100 mg/kg, which significantly inhibited the gastric mucosal lesions induced by ethanol

and nonsteroidal drugs in rats.

In traditional medicine, several plants are used in the treatment of gastric disorders, and from the

evidence presented here, it could be stated that extracts obtained from the aerial parts of I.

polyphylla are interesting sources for the development of a phytotherapeutic formulation to treat

gastric ulcer.

1.2.5.10. Immunostimulatory Activities

Carli et al. (2003) investigated the effect of the ethanol extracts of I. polyphylla on immune

activity in vitro and observed that the extract triggered a high nitric oxide production and

stimulated the synthesis and release of TNF-α, thus triggering the activation of macrophages and

promoting the production of other molecules that improve or restore the responsiveness of the

innate immune system reaction against infections.

20
In another study, using a purified compound of I. polyphylla, the indigo, also in experimental

models in vitro, showed an increase in the production and release of nitric oxide and TNF-

α (Carli et al., 2003).

The immune system is of fundamental importance to every living being, and its malfunction can

cause various biological damages. Drugs that stimulate the production and action of the

components of this system are of great biological relevance, primarily in immunodeficient

individuals who are more susceptible to infections. In this way, the good results obtained for this

species favor even more effective use in several diseases.

1.2.5.11. Anti-Inflammatory Activity

Aqueous extracts of I. polyphylla (250 mg/kg) in inflammation experimental models of mice

showed significant anti-inflammatory effect, with similar action to the commercial standard

drug, acetyl salicylic acid (Shahidi Bonjar, 2004).

In another study, aqueous and ethanolic extracts of leaves of I. polyphylla were used in

experimental models of inflammation induced by lipopolysaccharides (LPS) in macrophages,

and it was possible to observe a significant anti-inflammatory effect (Shahidi Bonjar, 2004).

The prolonged use of anti-inflammatories promotes several biological damages and the search

for new drug therapies that reversibly abuse the harmful effect, and the low cost is still incessant.

The studies already published show that I. polyphylla is a strong candidate to be applied in this

activity.

21
1.2.6. Gynecological Problems/Issues

Yazbek et al. (2005) reported that, in Brazilian folk medicine, leaves and root of I. polyphylla

have been commonly used to prepare tea for inflammatory diseases of the gynecological tract,

mainly organs such as ovaries and/or uterus.

In many places, the main therapeutic resource for the treatment of discomforts and diseases of

gynecological origin is still commonly represented by medicinal plants. However, few studies,

such as (Yazbek et al., 2005), show the promising effect that may resonate on new beneficial

therapeutic approaches and on strengthening the practice of women’s periodic care.

1.2.6.1 Toxicity

Studies using aqueous extracts obtained by infusing leaves of I. polyphylla in acute toxicity in

mice demonstrated the presence of deaths in the tested groups. Some signs of toxicity were noted

after a few hours of intraperitoneal administration from a lower concentration to a higher

concentration tested dose (2400 mg·kg−1): agitation, piloerection, exhaustion, sleepiness,

irritability, and spasms. Also, it was found that the LD50 of the acute toxicity of the aqueous

extract of leaves of I. polyphylla made by infusion administered in different doses in mice

showed no mortality during 72 h of observation (Yazbek et al., 2005).

The methanol extract of leaves of I. polyphylla showed a low toxicity with an LD50 of 1600

mg/kg (ip) in mice. The results exhibit a lower significant change in individual behavioral and

parameters that slight decrease in spontaneous locomotor activity and an increase in breathing

frequency (Yazbek et al., 2005).

22
1.3 Organisation of the Report

The entire report has been divided into four chapters. Chapter one give introduction and

literature review, chapter two gives practical method and materials, chapter three present the

project result while chapter four deals with the evaluation of the overall project, conclusion and

recommendation.

1.4 Limitation of the Study

This project write up is limited or restricted only on the investigation of medicinal and

toxicological properties of I. polyphylla

1.5 Aim and Objectives

The aim of the present study is to evaluate the medicinal and toxicological potential of I.

polyphylla leaves and to give a scientific validation in terms of phytochemical analysis. To

achieve this target the following objectives are laid. They are;

 To extract the phytochemicals from I. polyphylla leaves using solvents viz., methanol,

Acetate Nitrate and distilled water.

 To investigate the antifungil property of I. Bracteolata leaves extract using invitro

bioassay.

 To evaluate the quantitative analysis of flavonoids of the extracts.

 To analyze the toxicological properties of the extracts.

23
 CHAPTER TWO

2.0 LITERATURE REVIEW

A lot of works has been carried out on the study on the Phytochemical, medicinal and toxicological

Properties of natural product which are the source of new chemical diversity and are the choice of

today’s world. The sources of natural product are plants, animals and microorganisms. Among them

plant and its products are more reliable for its renewability and therefore, considered as catalyst for

human welfare. Still, they are the primarily required materials for health care system in some parts of

the world. Therefore, in the past few decades, there is a growing research interest in plants as a

therapeutic agent.

The literature available on herbal medicines dated back to the early age of the Rigveda (4500-1600

BC). The Rig veda mentions 67 herbal drugs, the Yajur veda 81 and Atharua veda about 290.

Charaka Samhita (700BC), first recorded treatise Ayurveda was followed by Sushruta Samhita (600

BC) and both compiled a centuary apart believed to be not later than 900 BC. Charaka Samhita gives

the properties of drugs prepared from indigenous plants, their uses and the methods of their

administration.

Hindu thoughts influenced the Greek medical literature in the fifth and 6 th centuries BC. About

400BC, a Greek named Hippocrates asserted that medicine was a science and art rather than religious

ritual full of imagination and mystery.

24
Picture of Indigofera Polyphylla

25
2.1 Morphology and Anatomy.

Indigofera polyphylla is annual or perennial, herbaceous or woody, and erect to prostrate in

habit. Vegetatively, the leaves may be simple, unifoliolate, or imparipinnately compound, with

the leaflets arranged oppositely or alternately along the rachis. (Gillett 1958) Members of

Indigofera are characterized by a set of characters which, although not necessarily unique

individually, adequately provide for the definition of the genus when considered collectively.

Significant defining characteristics are to be found in vegetative, floral, and fruiting features.

1.) Although other types of hairs may be present, all members of the genus have biramous hairs.

These may be appressed or ascending and equally to unequally two-armed. 2.) The corolla,

typical in basic construction to other papilionoid legumes, is some shade of red and easily

caducous. 3.) Each keel petal has a pouch or spur extending outward from the outer surface. 4.)

A mucro is found tipping each of the anthers with some species having scales extending from the

base of the anther; 5.) The stamens are diadelphous in arrangement. 6.) The seeds of Indigofera

is often squarish and separated from each other in the pod by transverse partitions. 7.) The inside

surface of the pod and sometimes the surface of the seed may exhibit mottling; this spotting is

due to the presence of tannin-containing cells (Gillett 1958).

Metcalfe and Chalk (1950) presented a summary of the anatomical features known in various

species of the genus. In recent years, anatomical studies have primarily focused on the leaf, seed,

and trichome, with particular attention to the trichome. The type of hair that occurs in all species

of Indigofera is the biramous hair.

In addition to the biramous hair, other types of hairs have been noted in species of Indigofera.

Although on occasion apparently simple hairs can be found, native New World species seem to

26
have primarily the biramous type (personal observation), either equally-armed or unequally-

armed.

Certain Old World species, on the other hand, have been found additionally to exhibit other

types, among these being unicellular conical hairs, unicellular cylindrical hairs, uniseriate

cylindrical hairs, uniseriate clavate hairs, uniseriate conical hairs, biseriate cylindrical hairs,

biseriate clavate hairs, multiseriate cylindrical hairs, multiseriate clavate hairs, multiseriate

capitate hairs, and multiseriate hollow-discoid hairs (Vijay Kumar 1988). Biramous hairs have

been noted to be smooth or "beset with tiny projections" (Kuar and Travedi 1984). Electron

microscopic studies of New World species of Indigofera have also shown these projections on

the surface of the hairs (personal observation).

2.1.1 Floral Syndrome.

Another feature that is characteristic of members of Indigofera is the observed floral tripping

syndrome resulting in the release of pollen. Explosive pollen delivery has long been known;

Hildebrand (1866) and Henslow (1867), though not the first to note the phenomenon, were

probably the first to clearly and accurately describe the process. Henslow's words follow: "If any

object, such as a pin, be inserted at the base of the vexillum, to which it will be guided by the

projecting ridges on the claws of the alae, and made to touch the point of insertion of the carina,

the latter immediately springs violently down, and from being in a horizontal position becomes

vertical, by the claw becoming curved at right angles. The alae also fall laterally, having lost

their support. The claw of the carina splits, and detaches itself from the calyx, so that this as well

as the other petals now quickly falls off. In consequence of the sudden jerk thus caused by the

fall of the carina, a cloud of pollen is thrown upwards...." Hildebrand (1866) figured what

happens with the petals when the flower is tripped. Henslow (1867) concluded that "...although

27
self-impregnation may be possible,...yet at the same time by the flowers being so contrived as to

specially load the insects that visit them with pollen, the inference cannot be avoided, that the

intercrossing is the most important end." Henslow's account of the movements of the petals is a

good one and little can be added to those observations. The existence of such a method of pollen

delivery suggests insect pollination (Arroyo 1981).

Arroyo (1981), in a survey of breeding systems and pollination biology in legumes, made several

significant points in this regard. First, both self-compatibility and self-incompatibility can be

found in Indigofera. Second, tripping may be a requirement for pollination in some legumes.

Third (1981), tripping mechanisms are found in a number of legume tribes, possibly as a result of

convergence. Explosive pollen delivery is found not only in Indigofereae but also in

Brongniartieae, Genisteae, and Desraodieae. Lastly, she felt that this explosive technique

represents an advanced means of pollination.

Although I have demonstrated the explosive tripping mechanism in Indigofera flowers, I have

been unable to document that it is caused by insects. Through field observations, I have observed

insects at flowers of four different species of Indigofera (I. spicata Forssk., I_. polyphylla Mill.,

I_. miniata Ort., and I_. lindheimeriana Scheele); at no time have I observed an insect land on a

flower nor have I seen an insect trip a flower. This matter deserves further study.

2.1.2 Cytology.

Cytological work in the genus has established that 2_n=16 and 2ji=32 appear to be the most

common chromosomal numbers (Gupta and Agarwal 1982) although these are not the only

chromosomal numbers found. From such work, a base chromosomal number of x=8 is suggested

but, again, other base numbers are postulated. These (xf=4, x.=6> and 21=7) are felt to be

secondarily derived from x=8 (Gupta and Agarwal 1982). Some work (Bhatt and Sanjappa 1995)

28
has been done on Old World representatives of taxa that are found in the New World; these taxa

conform to the general patterns observed for the genus. The only report dealing specifically with

North American taxa of Indigofera appears to be that of Turner (1996). Turner found _n=8 for

_I. hendecaphylla Jacq.

(=1^ spicata), ii=16 for _I. leptosepala Nutt, ex Torrey & A. Gray (=I.*miniata), _n= 16 for

texana Buckl., and n=8 for I_. lindheimeriana.

2.2 Other Investigative Approaches.

Species of Indigofera have been evaluated for biochemical compounds. Such studies have more

often than not involved widespread, well-known, or economically important species— e.g.,

review of indigo by Fernelius and Renfrew (1993), studies of Indigofera polyphylla [Garcez al^

(1999), Dominguez et al. (1998)] and I_. mlcrocarpa Desv. [Moraes e Souza et al. (1998)], and

the potential toxicity of such species as _I. spicata [Hegarty et al. (1998) and Morton (1989)]. A

few studies on chemotaxonoraic applications of biochemical data exist [Anuradha et al. (1997)

and Bhalla and Dakwale (1998)]. A preliminary paper chromatographic study (see discussion on

methodological use in Radford et al. 1994) using several taxa (I. miniata, 1_. texana, _I.

suffruticosa, and I_. lindheimeriana) in our laboratory was not found to be particularly useful as

a taxonomic tool for distinguishing these species.

Molecular techniques have become important in answering systematic questions in various plant

groups. Data derived from chloroplast DNA mapping studies are being utilized in legume

systematics (Doyle 1987) but, to my knowledge, no such work has been done with the genus

Indigofera. The present taxonomic study of the genus provides the necessary framework for this

type of undertaking.

29
2.3 Phytochemistry

Chatterji and Dutt (1937) isolated Indigoferin and Enneaphyllin from ethyl alcohol, methyl

alcohol, acetone, benzene, chloroform, carbon tetrachloride, water extracts of whole plant of

Indigofera enneaphylla.

Souza et al., (1988) isolated 2-acryl-3methylbenzo(b)furans acrylic acid kirilowin C and

kirilowin D together with four new glucose3-nitropropanoalteskirilowins E-H from methanol

extract of leaves of Indigofera microcarpa.

Garcez et al., (1989) isolated a new nitropropanoyl- glucopyranoside [2,3,4,6-tetra(3-

nitropropanoyl) α-D-glucopyranose] from root and stem of Indigofera polyphylla.

Benn et al., (1992) elucidated the structure of three 3-nitropropanoyl esters of D-glucose such as

1,2,6-tri-O-3-nitropropanoyl-β-D-glucopyranose (karakin) and 2,3,4,6-tetra-O-3-nitropropanoyl-

α-D-glucopyranose,3,4,6-tri-O-3-nitropropanoyl-α- D-glucopyranose from Indigofera linnaei.

Kamal and Mangla (1993) identified and quantified the six rotenoid compounds namely

deguelin, dehydrodeguelin, rotenol, rotenone, tephrosin and sumatrol from Indigofera tinctoria.

Hasan et al., (1993) isolated Kaempferol 7-alloside; Kaempferol 3,7- diarabinoside; Kaempferol

3-arabinoside 7 rhamnoside; Kaempferol 3-rhamnoside 7- arabinosid from methanol extract of

leaves of Indigofera hebepetala.

Hasan et al., (1996) isolated flavanol triglyglycosides, Kaempferol 3-0-α-L-rhamnopyranosyl

(12)-β-D-galactopyranoside-7-0-α-L-arabinofuranoside from n-butanol extract of flowers

Indigofera hebepetala.

Thangadurai et al., (2001a) isolated a new decahydropyridoquinoline, rel-(3S, 5R, 65, 8R, 8aR,

12aR)-8-acetoxy-6-butyl-3-isothiocyanatode-hyropyrido(2,l) quinoline from dichloromethane

extract of air-dried stems of Indigofera longeracemosa.

30
Thangadurai et al., (2001b) isolated a novel xanthene, 3-isopropyl-9a-methyl- 1,2,4a,9a-

tetrahydroxanthene from the stem of Indigofera longeracemosa Chanayath (2002) isolated

Indirubin from water extract of leaves of Indigofera tinctoria.

Thangadurai et al., (2002) isolated a novel abietane diterpenoid compound namely Indigofera

bietone with potential antituberculous and antifungil activity from Indigofera longeracemosa.

Garcez, et al., (2003) isolated 2,3,4,6-tetrakis-0-(3-nitropropanoyl)-α-Dglucopyranose from n-

hexane and ethanol extracts of roots of Indigofera suffruticosa.

Prasad and Chakradhar (2004) isolated a new isoflavone namely 7,8-methylenedioxy-4'-methoxy

isoflavone from the whole plant of Indigofera linnaei.

Rehman et al., (2005) isolated 4’-hydroxy-3’,5,7-trimethoxyflavone; 5-hydroxy-2’,4 ‘,5’,7-

tetramethoxyflavone; 3’,5-hydroxy-4’,6,7-trimethoxyflavanone (eupatorin), 4’,7-dihydroxy-3’-

methoxyflavanone, 3,3’,4’,7-tetrahydroxyflavanone and 3’,4’,5,7 -tetrahydroxyflavanone 3--

tetrahydroxyflavone 3-0-α-L-aranino-side from chloroform soluble fraction of the methanolic

extract of stem of Indigofera hetrantha.

Sharif et al., (2005) isolated Indigin, alkylated xanthene and indigoferic acid along with β-

sitosterol and 3-hydroxybenzoic acid from the chloroform soluble fraction of Indigofera

oblongifolia.

Sharif et al., (2006) isolated seven compounds namely 2β-O-(E)- coumaryllupeol, N-

acetylhomoveratrylamine, ursolic acid, 7-O-methyleriodictyol, β-sitosterol 3-O-B-D-

glucopyranoside and chrysoeriol 7-O-B-D-glucopyranoside from Indigofera oblongifolia.

Singh et al., (2006) identified indigotin compound from the dried aerial part of Indigofera

tinctoria and its chemical structure was established on the basis of physical properties and

spectral data, including NMR.

31
2.4 Pharmacology

Sittie and Nyarko (1998) conducted a safety evaluation of an anti-diabetic plant extract of

Indigofera arrecta in non-diabetic human volunteers.

Esimone et al., (1999) evaluated the antifungil and antifungal activities of the aqueous petroleum

ether, methanol extracts and eight TLC fractions of the methanol extract of Indigofera

dendroides leaves.

Umar Dahot (1999) conducted antifungil and antifungal activity of small protein of Indigofera

oblongifolia leaves against various microorganisms.

Singh et al., (2001) isolated indigtone obtained by fractionation of petroleum ether extract of the

aerial parts of Indigofera tinctoria and showed significant dose related hepatoprotective activity

against CCl4 induced liver injury in rats and mice.

Verma and Suresh (2002) studied phytochemical evaluation of leaf extract of Indigofera

tinctoria and reported the presence of flavonoids, alkaloids, glycosides, terpenoids from

petroleum ether extract and methanolic extract of Indigofera tinctoria.

Amos et al., (2003) reported the contractile effects of the aqueous extract of the leaves of

Indigofera dendroides and studied on the gastrointestinal motility in mice and isolated smooth

muscle preparations obtained from rats and guinea pigs.

Christina et al., (2003) on testing the ethanolic extract of Indigofera aspalathoides in Swiss

albino mice concluded that the plant is having protective effect against Dalton’s Ascitic

Lymphoma.

Leite et al., (2004) studied the aqueous extract of leaves of Indigofera polyphylla for adverse

effects in pre- implantation mouse embryos.

32
Selvam et al., (2004) isolated a new compound indigocarpan and the known compound

mucronulatol from chloroform extracts of Indigofera aspalathoides and were evaluated for cyclo

oxygenase-1 (COX-1) and cyclo oxygenase-2 (COX-2) inhibitory activities and antioxidant

properties.

Abubakar et al., (2006) investigated the neutralizing effects of methanol extracts of Indigofera

pulchra against Naja nigricollis venom to validate traditional claims of usefulness of the plants

in management of poisonous snakebites.

Chakrabarti et al., (2006) elucidated the anti-diabetic potential of ethanolic extract of the whole

plant of Indigofera mysorensis.

Cola-Miranda et al., (2006) investigated the anti-ulcerogenic properties of Indigofera truxillensis

in different models of acute gastric ulcers (100% ethanol, piroxicam 30 mg.kg-1, hypothermic

restraint stress and pylorus ligature) in mice and rats.

Narender et al., (2006) reported anti-dyslipidemic activity of furanoflavonoids such as

hesperetin, hesperidin, naringenin and naringin isolated from Indigofera tinctoria.

Rajkapoor et al., (2006a) evaluated the anti-tumour activity of ethanol extract of stem of

Indigofera aspalathoides in mice against Erlich Achites Carcinoma tumour model.

Rajkapoor et al., (2006b) evaluated the alcoholic extract of stem of Indigofera aspalathoides for

its anti-hepatotoxic activity against CCl4-induced hepatic damage in rats.

2.5 Pharmacognosy

In Pharmacognosy, only scanty work has been carried out in the genus Indigofera L. Rajeshwar

et al., (2013) focussed on the establishment of pharmacognostical standards of Indigofera

barberi stem.

33
Saravana Ganthi et al., (2014) carried out systematic and detailed pharmacognostical studies on

Indigofera aspalathoides which will help in evaluating or assuring the quality of raw drug.

Suvarnalatha et al., (2014) studied the pharmacognostic studies of Indigofera hirsuta for

quantifying and qualifying the drug for better uses in the formulations and to check for its

adulterants.

34
 CHAPTER THREE

MATERIALS AND METHOD

3.1 CHEMICALS/REAGENTS

S/No NAME
1 Distilled Water
2 Potassium hydroxide
3 Hydrogen chloride
4 Mayer’s and Wagner’s reagent
5 Sodium Hydroxide
6 Chloroform
7 Sulfuric acid
8 Acetic Acid
9 Methanol
10 Fehling Solution
11 Iron(III) chloride
12 Acetate Nitrate

3.2 EQUIPMENT AND APPARATUS

S/ NAME MODEL MANUFACTURERS

No
1 Weighing Balance Scont proSPU 202 OHAOS
2 Fume Cupboard HUS4HF Lab. System furniture ltd. UK.
3 Beaker Glass Pyrex England
4 Test tube Glass Pyrex England
5 Measuring cylinder Glass Pyrex England
6 Volumetric Flask Glass Pyrex England
7 Reagent Bottle Glass Merck
8 Filter Paper No.42 Whatmann
9 Funnel --------- -----------

3.3 Sample Collection and Pre-Treatment

Fresh leaves of I. polyphylla were obtained from Isa Kaita College of Education, Dutsinma, Katsina

campus and transported to the Department of Pure and Applied Sciences, Isa Kaita College of Education,

Dutsinma, Katsina State, Nigeria for identification. The sample was washed with distilled water and

dried under the shade at normal room temperature for 21 days. After drying, the plant material

was pounded using mortar and pestle into smaller particles and then blended to powder using an

35
electric blender. The powdered sample were stored in airtight containers and kept under normal

room temperature for further screening.

3.4 Sample Extraction

3.4.1 Preparation of Aqueous extract

Fifty gram of the ground sample underwent extraction by soaking with 300cm 3 of distilled water

for 24hr at room temperature, with occasional shaking. The mixture was filtered and the filtrate

was divided into two portions, one portion was used for phytochemical screening and the other

portion was preserved by drying in an oven at 60-80°C for 2 days to be used for toxicological

analysis (Daniyan and Muhammad, 2008).

3.4.2 Preparation of Methanolic extract

50g of the sample underwent extraction were soaked in 300cm 3 of methanol for 24hr at room

temperature, with occasional shaking. The suspension was filtered and the filtrate was divided

into two portions, one portion was used for phytochemical screening and the other portion was

preserved by drying in an oven at 60-80°C for 2 days to be used for toxicological analysis

(Daniyan and Muhammad, 2008).

3.4.3 Preparation of Acetate Nitrate extract

50g of the sample underwent extraction were soaked in 300cm 3 of acetate nitrate for 24hr at

room temperature, with occasional shaking. The suspension was filtered and the filtrate was

divided into two portions, one portion was used for phytochemical screening and the other

portion was preserved by drying in an oven at 60-80°C for 2 days to be used for toxicological

analysis (Daniyan and Muhammad, 2008).

36
3.5 Phytochemical analysis

The qualitative tests were saponins carried out on the aqueous, methanol and acetate nitrate

extracts of the analytes sample using the method used by Lawal and Dangoggo (2014), as well as

Lawal et al. (2010), for the determination of alkaloids, tannins, saponins, flavonoids, glycosides,

cardiac glycosides, saponin glycosides, volatile oils and steroids, as follows:

3.5.1 Test for alkaloids

Three test tubes occupied with 3cm3 of aqueous, methanol and acetate nitrate extracted samples

and 1cm3 of 10% HCl was added and heated for 20min on a steam bath at 80-95°C. The mixtures

were allowed to cool and filtered. 1cm 3 the filtrates were treated with three drops of Mayer’s and

Wagner’s reagents. Appearance of creamy and reddish brown precipitate indicated the presence

of alkaloids.

3.5.2 Test for flavonoids

The 1cm3 of 10% NaOH was mixed with 3cm3 of aqueous, methanol and acetate nitrate extracted

analytes sample into a test tubes. Appearance of yellowish solution indicated the presence of

flavonoids.

3.5.3 Test for glycosides

A mixture of 5cm3 of aqueous, methanol and acetate nitrate extracted analyte sample and 2.5 cm 3

of 50% H2SO4 were introduced into the test tubes. The mixtures were heated in boiling water for

15min, cooled and neutralized with 10% NaOH. Then, 5ml of Fehling’s solutions (A and B)

were added to the mixtures and heated. Occurrence of brick-red precipitate indicated the

presence of glycosides.

37
3.5.4 Test for cardiac glycosides

The 2cm3 of acetic acid containing traces of FeCl3 and 2cm3 of conc. H2SO4 were subsequently

added to 2cm3 of aqueous, methanol and acetate nitrate extracted analytes in a different test

tubes. A blue-layer appearance indicated the presence of cardiac glycosides.

3.5.5 Test for saponin glycosides

Approximately 2.5cm3 of Fehling solutions (A and B) were added to 2.5 cm 3 of aqueous,

methanol and acetate nitrate extracted analyte sample in a different test tube. The bluish-green

precipitate formed indicated the presence of saponin glycosides.

3.5.6 Test for steroids

One cubic centimeter (1cm3) of the aqueous, methanol and acetate nitrate extracted sample were

added to 2cm3 of chloroform in a different test tube. 2cm 3 of conc. H2SO4 were carefully added

to form a lower layer. Appearance of a reddish brown colour at the interface indicated the

presence of steroids.

3.5.7 Test for volatile oils

The 20% HCl was mixed with 5cm 3 of aqueous, methanol and acetate nitrate extracted analytes

sample and shaken. Indication of white precipitate confirmed the presence of volatile oils.

3.5.8 Test for tannins

Five drops of freshly prepared 10% KOH was added to 1cm 3 of the aqueous, methanol and

acetate nitrate extracted sample. Dirty-white precipitate appeared indicating the presence of

tannins.

38
3.5.9 Test for saponins

Five cubic centimeters (5cm3) of the aqueous, methanol and acetate nitrate extracts were strongly

shaken in a test tube. Formation of large amount of froths that lasted for 30 min confirmed the

presence of saponin.

3.6 Quantitative analysis of flavonoids

The method of Okwu and Josiah (2006) was adopted for the quantitative analyses of flavonoids.

Ten grams (10g) of the dried (ground) sample of I. polyphylla was extracted with 100 cm3 of

80% aqueous methanol at room temperature. The solution was filtered through no.42 filter paper

into weighed crucibles. Then, the crucibles’ contents was evaporated to dryness over water baths

and the final weights were determined. The determination of percentage of flavonoids contents

of the sample was calculated using Equation below;

Loss∈Weight
Flavonoids Content (%) = x 100
Sample Weig h t

3.7 Antimicrobial Susceptibility Test

The agar well diffusion method was used for the antimicrobial susceptibility test. Mueller Hilton

agar was prepared according to manufacturer’s specification. The media were autoclaved and

dispensed into sterile petri-dishes and allowed to gel. Standardized inocula of each fungal isolate

were streaked on the agar plate. Four wells of 6mm each was made in each plate with a central

well for control using a sterile cork borer. The wells were filled with 0.1ml of different

concentrations (400μ/ml, 200μ/ml, 100μ/ml and 50μ/ml) of the extract with the aid of sterile

pipettes per well. Likewise, 400μ/ml, 200μ/ml, 100μ/ml and 50μ/ml of the standard antibiotic

(amoxicillin) were used in separate plates to serve as positive control. While sterile distilled

water was used as negative control on separate plates. The plates were allowed to stand for 15

minutes on a table to allow free diffusion of the extracts. Diameters of zones of inhibition were

39
measured using transparent plastic meter rule after 24 hours of incubation at 37°C (Dahiruet al.,

2013).

2.8 Toxicological analysis

Lethal dose concentration (LD50) determination on the aqueous extract of I. polyphylla was

carried out using the method of Organization for Economic and Cultural Development (OECD,

2001); Rats were grouped into five so that each group had one rat. A rat from each group was

administered a single oral dose of 3000mg/kg of the aqueous extracts of I. polyphylla with a

feeding tube and observed for 48hr. This was repeated one after the other for all the groups. The

controlled group of the rats was administered with distilled water. Symptoms of toxicity such as

increase or decrease in movement, loss of appetite and time of regaining it, body scratching,

nervous sensation, salivation, depression and time of death were recorded. The number of

survivors in each of the groups after 48hr was recorded, respectively.

40
 CHAPTER FOUR

RESULTS AND DISCUSSION

4.0 Results

4.1 Percentage (%) Recovery on Extraction

Table 4.1: Weight of the various extracts of I. polyphylla

Extracts Mass of extract (g/500g) (%) Recovery

Methanol 30 6

Aqueous 12 3

Acetate Nitrate 15 2.3

The percentage recovery of the extract and fractions were shown in Table 4.1. Methanol gave the

highest recovery of 30 g (6 %). Acetate Nitrate gave 15 g (3 %) while Aqueous gave the least

yield of 12 g (2.3 %).

41
4.2 Phytochemical Screening

Table 4.2 Phytochemical Screening of I. polyphylla

Secondary metabolites Methanol Aqueous Acetate Nitrate

Carbohydrates ++ ++ ++

Glycosides + + +

Cardiac glycosides + + +

Saponins + + +

Steroid / triterpene ++ ++ ++

Flavonoid ++ ++ ++

Tannin ++ ++ ++

Alkaloid --- --- ---

KEY + = present - = absent

The methanol extract and the two fractions showed the presence of carbohydrates, glycosides,

and cardiac glycoside (Table 4.2). The extract and the fractions also showed the presence of

saponins, steroids/triterpene, flavonoids and tannins. Alkaloids are absent in all extracts.

42
4.3 Antimicrobial Susceptibility Test

Table 4.3: Antimicrobial Susceptibility Test of the extracts

Test organisms Aqueous Acetate Nitrate Methanol

Candida albicans S S R

Aspergillus fumigatus S S S

Trichophyton rubrum S S S

Epidermophyton floccosum S R S

Microsporum canis R R S

Cryptococcus neoformans S R S

Malassezia furfur S S R

Candida krusei S S S

Candida tropicalis S R S

Keys S= sensitive R= resistant.

The activity of methanol extract and other fractions (Acetate Nitrate and Aqueous), were tested

against both gram positive and gram negative microbes, which are Aspergillus fumigatus,

Trichophyton rubrum, Epidermophyton floccosum, Microsporum canis, Cryptococcus

neoformans, Malassezia furfur, Candida albicans, C. krusei and Candida tropicalis. The results

(Table 4.3) indicate that the microorganisms are all sensitive to aqueous fraction except

Microsporum canis. Four pathogens showed resistance to ethyl acetate namely Epidermophyton

floccosum, Microsporum canis, Candida tropicalis and Cryptococcus neoformans, while two

microorganisms showed resistance to methanol extract which are; Candida albicans and

Malassezia furfur

43
4.4 Zone of Inhibition (mm)

Table 4.4: The Zone of Inhibition (mm) of the Extract and Fractions

Test Organism Diameter of Zone of inhibition (mm)

Methanol Acetate Aqueous Ciprofloxacin

Extract Nitrate 10μg/disc

Aspergillus fumigatus - 20 24 40

Trichophyton rubrum 20 14 23 35

E. floccosum 16 15 25 32

Microsporum canis 17 - 21 34

Cryptococcus neoformans 21 - 19 37

C. albicans - 16 18 33

C. tropicalis 18 - 17 31

C. krusei 18 14 18 30

The zone of inhibition of the extract and the two fractions in Table 4.4 showed that the

chloroform fraction inhibits all the tested microorganisms, methanol extract showed no inhibition

against A. fumigatus and C. albicans, While the ethyl acetate fraction showed no inhibition to

three microorganisms which are: M. canis, C. neoforman and C. tropicalis. The methanol extract

ranged between 16 mm and 21 mm, chloroform fraction ranged between 17 mm and 25 mm,

while the ethyl acetate fraction ranged between 14 mm and 20 mm respectively.

44
4.5 Minimum Inhibitory Concentration (MIC)

Table 4.5: Minimum Inhibitory Concentration (MIC) of the Extracts

Test organism Methanol extract Aqueous Acetate Nitrate

(mg/ml) (mg/ml) (mg/ml)

A. fumigatus - 12.5 6.25

Trichophyton rubrum 6.25 6.25 -

E. floccosum 3.123 3.123 3.123

Microsporum canis 12.5 12.5 -

Cryptococcus neoformans 1.562 6.25 -

C.albicans - 1.562 6.25

C.tropicalis 3.123 3.123 -

C. krusei 1.562 6.25 3.123

Key: - = No activity

The minimum inhibitory concentration (MIC) confirmed that the chloroform fraction inhibit

growth in all of the tested microorganisms (Table 4.5). The (MIC) of methanol extract was

inactive against A. fumigatus, and C. albicans, while the acetate nitrate fraction was not active

against Trichophyton rubrum, Microsporum canis, Cryptococcus neoformans, and C. tropicalis. The

MIC of methanol extract ranged between 1.562 mg/ml and 12.5 mg/ml, aqueous fraction ranged

between 1.562 mg/ml and 12.5 mg/ml, while that of acetate nitrate is between 3.123 mg/ml and

6.25 mg/ml.

45
4.6 DISCUSSION

The extract and the fractions confirmed the presence of saponin, steroids/triterpene, flavonoids,

tannins, carbohydrate and cardiac glycosides. This confirmed that the root part of I. Polyphylla

posseses antimicrobial properties as such, they can be used as drugs traditionally and also for

drug synthesis. The secondary metabolites were also reported to be present in the acetate nitrate

fraction of the stem bark of I. Polyphylla studied by Neuwenger, (2000). According to the report

by the traditional medical practitionals and researches conducted on Indigofera species, shows

that the plant I. Polyphylla has maximum ability to synthesize secondary metabolites which serve

as defensive mechanisms against microorganisms. The presence of terpenoids and glycosides

show the ability of the plant I. Polyphylla to exhibit antifungal activities on some of the fungi

parasites. The confirmation of saponins also demonstrates several biological and

pharmacological importances such as antipyretic, analgetic, anticancer and anti-inflammentory

activities (Mathia, 2010).

The result of the zone of inhibition confirmed that I. Polyphylla extract and the fractions were

active against the tested gram positive and gram negative microorganisms (Cryptococcus

neoformans,C.albicans, A. fumigatus e.t.c). This shows that I. Polyphylla can be used in the

treatment of different types of fungil infections (Tulikk, 2003). The lack of activity of the

fractions to some of the microorganisms may be due to the nature of the microorganism which

poses strong mechanisms that prevent the effect of the active components on them (Kanwal et

al., 2009). The MIC result of the extract and the fractions confirmed that the claim made by the

traditional doctors that the plant is used for treatment of typhoid fever, sexually transmited

diseases like Staphylococcus aureus, Siphillis and gonorrhoea is confirmed. The isolated

46
compounds which are confirmed by the chemical test to be triterpene could be further developed

as a drug for the treatment of infectious diseases.

47
 CHAPTER FIVE

SUMMARY, CONCLUSION AND RECOMMENDATION

5.1 Summary

The plant of Indigofera Polyphylla was collected from Dutsinma, Katsina State; it was properly

identified at the Chemistry Department, Isa Kaita College of Education, Dutsinma. The plant was

air- dried and pulverized. The powdered was extracted using methanol, aqueous and acetate

nitrate. The methanol extract as well as aqueous and acetate nitrate fractions that were subjected

to preliminary phytochemical screening revealed the presence of saponins tannins flavonoids

steroids and triterpenes. The antimicrobial screening of the methanol extract with the aqueous

and acetate nitrate fractions showed a broad spectrum of antifungal activity against tested micro-

organisms.

5.2 Conclusion

Considering the results of phytochemical screening and antifungal studies of I. polyphylla

extract, these results justify that I. polyphylla could be considered as a potential source of

defensive, analgesic, antiviral, antifungal and antifungal mechanisms. These would provide the

medicinal benefits. Although, more research should be carried out on the sub-chronic (long-time

toxicity) in order to have a safe and profitable consumption.

5.3 Recommendations

From data collected, literature reviewed, and field observations, the following recommendations

are offered;

48
 More research should be carried out on the sub-chronic (long-time toxicity) in order to

have a safe and profitable consumption.

 The government should provide more equipment for antifungal analysis so as to obtained

good result when done and more research study should be done in this field as its

beneficiate to the society.

49
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