From Cleome Gynandra and Cleome Viscosa

ANTIBACTERIAL ACTIVITIES OF PHYTOCHEMICAL MIXTURE EXTRACTED FROM CLEOME GYNANDRA AND CLEOME VISCOSA

STUDENTS MINI PROJECT

Submitted to Tamil Nadu State Council for Higher Education

R. RAJAKUMARI, II M.Sc.

DEPARTMENT OF ZOOLOGY QUEEN MARY’S COLLEGE (AUTONOMOUS) CHENNAI - 600 004 MARCH - 2018

ACKNOWLEDGEMENT

I thank the Almighty for giving me the health, strength and courage, which enable me to successfully complete this research project.

I am grateful to our principal Dr. (Mrs.) S. Santhi, Queen Mary’s College (Autonomous), Chennai-600 004, for providing the facilities to carry out my research work.

My heartfelt thanks and gratitude to Dr. (Mrs.) Malathi, E. Associate Professor and Head, Department of Zoology, Queen Mary’s College (Autonomous), Chennai-600 004, for their valuable suggestions during the study period.

I articulate my deep felt thanks to Dr. (Mrs.) S. Santhi, Assistant Professor, Department of Zoology, Queen Mary’s College (Autonomous), Chennai-600 004. She has been a constant source of inspiration with her valuable guidance, encouragement and critical suggestions throughout the course of the study. I am grateful to all the Staff Members of Department of Zoology, Queen Mary’s College, Chennai for their valuable inputs and encouragements.

I wish to express my heartfelt gratitude to my father Mr. S. Ravichandiran, my mother Mrs. R Umaiyal, and especially all my family members for their moral support, blessings, encouragement and for being the source of inspiration who were the wind beneath my wings at every step in all endeavors.

I also extend my heart full thank to my classmates and friends for their help and cooperation during the tenure of the work. I wish to thank the Lab Assistants, Department of Zoology, Queen Mary’s College (Autonomous), Chennai-600 004 for their timely assistance.

I extend my sincere thanks to Mr A. Gnanavel, Lab. Technician, Kumaran Hospital, Chennai for providing the bacteria culture samples.

With immense pleasure, I thank Mrs. D. Evangeline and Mrs. P. Nijanthi, Ph.D. Research Scholars for their generous help, support and cooperation during the research work.

I wish to express my heartfelt gratitude to Mr. C. Palanisamy, SHV Energy Private Limited, Chennai – 600 017 for having given me the inputs and helping me in preparing the project report.

I sincerely thank Tamil Nadu State Council for Higher Education, Lady Willington College Campus, Chennai for providing me the opportunity by funding this mini project.

R. RAJAKUMARI

Dr S. SANTHI, Department of Zoology, Research Supervisor Queen Mary’s College (Autonomous), Chennai - 600 004.

CERTIFICATE

This is to certify that the Students Mini Project entitled “Antibacterial

Activities of Phytochemical Mixture Extracted from Cleome gynandra and

Cleome viscosa” submitted to Tamil Nadu State Council for Higher Education is a bonafide record of research work done by R. RAJAKUMARI (Reg.

No.1655006), ІІ M.Sc., Department of Zoology, Queen Mary’s College

(Autonomous), Chennai during 2017 - 2018 and this work is carried out under my guidance and supervision.

Research Supervisor Head of the Department

(Dr. S. Santhi)

DECLARATION

R. RAJAKUMARI, B.Sc. Department of Zoology, Queen Mary’s College (Autonomous), Chennai- 600 004.

I, hereby, declare that this Students Mini Project entitled “Antibacterial

Activities of Phytochemical Mixture Extracted from Cleome gynandra and

Cleome viscosa” submitted to Tamil Nadu State Council for Higher Education is a record of independent research work carried out by me under the guidance of

Dr. (Mrs) S SANTHI, Assistant Professor, Department of Zoology, Queen Mary’s

College (Autonomous), Chennai - 600 004, during the period 2017 - 2018

Place: Chennai (R. RAJAKUMARI)

Date: 16- 03- 2018

CONTENTS

PAGE S. No. TITLES No. LIST OF TABLES LIST OF FIGURES LIST OF PLATES 1.0 INTRODUCTION 1 1.1 Cleome gynandra (L.) 3 1.1.1. Scientific Classification 3

1.1.2. Medical Importance 4

1.2. Cleome viscosa (L.) 4 1.2.1. Scientific Classification 4

1.2.2. Medical Importance 5 1.3. Phytochemical and Antibacterial Activity 6

2.0 OBJECTIVES 7 3.0 REVIEW OF LITERATURE 8 4.0 MATERIALS AND METHODS 10 4.1 Collection of Plants 10 4.2 Preparation of Aqueous Leaf Extract 10 4.3 Phytochemical Screening of C. gynandra and C. viscosa 10 4.3.1 Detection of Alkaloids (Wagner’s Test) 12 4.3.2 Detection of Reducing Sugar (Fehling’sTest 12 4.3.3 Detection of Glycosides, (Borntrager’s Test) 12

4.3.4 Detection of Protein (Biuret’s Test) 12 4.3.5 Detection of Amino Acid (Ninhydrin Test) 12 4.3.6 Detection of Saponins (Foam Test) 13 4.3.7 Detection of Steroids (Salkowski Tests) 13 4.3.8 Detection of Tannins (Ferric chloride reagent test) 13

4.3.9 Detection of Flavonoids (Ferric chloride reagent test) 13 4.3.10 Detection of Anthraquinone (Borntrager’s reaction) 16 4.4 Selection of Microorganisms 16 4.5 Antibacterial Activity Assay 16 4.5.1 Preparation of Inoculum 16 4.5.2 Agar Disc Diffusion Method 16 4.6 Statistical Analysis 17

5.0 RESULTS AND DISCUSSION 18 Results of Phytochemical Screening of Cleome 5.1 gynandra and Cleome viscosa 18 5.2 Results of Antibacterial Activities of C. Gynandra and C. Viscosa 20 6.0 SUMMARY 25 7.0 CONCLUSION 27 8.0 REFERENCES 28

LIST OF TABLES

TABLE PAGE TITLE No. No. Phytochemical Screening of Aqueous Leaf Extract of C. gynandra 1. 19 and C. viscosa

2. Antibacterial Activity of Leaf Extract of C. gynandra and 21 C. viscosa

LIST OF FIGURES

FIGURE PAGE TITLE No. No.

Zone of Inhibition (Mean) of C. gynandra and C. Viscosa 1. Against Gram Negative Bacteria Klebsiella pneumonia and 23 Gram Positive Bacteria Staphylococcus aureus

LIST OF PLATES

PLATE PAGE TITLE No. No.

11 1. Cleome gynandra

11 2. Cleome viscosa

Phytochemical Analysis of C. gynandra and C. viscosa 14 3. Aqueous Leaf Extract

Phytochemical Analysis of C. gynandra and C. viscosa 15 4. Aqueous Leaf Extract Zone of Inhibition of C. gynandra and C. Viscosa 22 5. Against Gram Negative Bacteria Klebsiella pnumonia Zone of Inhibition of C. Gynandra and C. Viscosa Against 22 6. Gram Positive Bacteria Staphylococcus aereus

1. INTRODUCTION

India has a flora that is widely distributed throughout the country which is one of the richest with enormous medicinal and aromatic plants. It constitutes 11% of total known flora having medical property (Sati et al., 2010). Herbal drugs have been the basis of treatment and cure for innumerable diseases and functional condition in traditional systems practiced as

Ayurveda, Unani and Siddha. Currently, multiple drug resistance microorganisms have been developed due to indiscriminate use of commercial antimicrobial drugs generally used in the treatment of infectious disease (Bhaskarwar et al., 2008) thus additional therapy is needed for the future. At present, there has been a lot of attention focused on producing medicines and products that are natural. Plants are good sources for new, safe, eco friendly and renewable drugs.

Plants are used medically in several countries and are a basis for many effective and prevailing drugs (Srivastava et al., 2008). A number of plants with substantial antimicrobial activity have been stated in different traditional works (Jones et al., 1996 and Satish et al.,

1999). Numerous medicinal plants are used daily in Ayurvedic practices. In India more than

7000 medicinal plants are recognized. According to a report of World Health Organization, more than 80% of world’s populations depend on traditional medication for the primary healthcare requirements (Umamaheswari et al., 2008). The fewer availability and high rate of new generation antibiotics needs substitute medicines with antimicrobial activity (Poovendran et al.,

2011). Therapeutic plants represent a ridiculous source of antimicrobial mediators.

Green plants represent a reservoir of effective chemotherapeutic agent and can provide valuable sources of natural pesticides. Bio pesticides have been suggested as an effective

substitute for chemicals. Majority of medicinal plant species are rich in bio-molecule contents which can cope up with health hazard. Recently, antibacterial activities of many plants species have been reported by Pandey and Mishra (2010).

The vast majority of modern medications were derived originally from ancient herbal traditions. There are numerous natural products of plants which shows an antifungal, antibacterial and antiprotozoal activities that could be used either systemically or locally

(Cowman, M.M. 1999).

Many of the medicinal plants used in traditional medicine are readily available in rural areas which are relatively cheaper than modern medicine. Nowadays many of the medicines are acquired from natural products or partial synthetic byproducts of natural sources and used in the customary systems of medicine (Sukanya et al., 2009). Most of these published works come from many countries that are still in practice and the herbal medicines are used for treatment of various diseases for practical and economic reasons (Nascimento et al., 2000).

Many plants are used by native groups to satisfy their food needs, nutrition and health and found a source of extra income and employment (Sandhya et al., 2006; FAO, 2012 and

Visweswari et al., 2013). Usually, the plants consist of metabolites that deliberates to them, have more medicinal advantages that are at the basis of their consumption by people (Zwenger and

Basu, 2008; Visweswari et al., 2013 and Adjatin et al., 2013). The various parts of the plants are conventionally used for the treatment of several complaints and as an antidote for snake bites and scorpion stings (Uma Devi, 2001). The secondary metabolites are the bioactive substances in plants. Most promising targets in search for such biologically active compounds are plants used

in folk medicines. Medicinal plants, with their wide variety of chemical constituents, offer a promising source of new antimicrobial principle (Cooper, 2004 and Patwardhan et al., 2005).

Several leaves extract have been found to have antimicrobial activity against various microorganisms (Kim et al., 2004). The presence of phytochemical constituents in medicinal plants made them useful for healing as well as for curing of human diseases (Nostro et al., 2000).

Plants are rich in the most essential bioactive substances like alkaloids, flavonoids, glycosides, phenols, steroids, tannins, terpenoids, and volatile oils (Cowman, 1999). Recent evidences from various pharmaceutical companies indicate that for certain ailments natural products still represent an extremely valuable source for the production of new chemical entities. Current estimates suggest that, in many developing countries, a large proportion of the population relies heavily on traditional practitioners and medicinal plants to meet primary health care needs (Abu

Rabia, 2005). Hence, the present study is focused on the antibacterial activity of the mixed phytochemicals obtained from the plants Cleome gynandra and Cleome viscosa.

1.1 Cleome gynandra (L.)

1.1.1. Scientific Classification

Kingdom : Plantae Clade : Angiosperms Clade : Eudicots Clade : Rosids Order : Brassicales Family : Cleomaceae Genus : Cleome Species : C. gynandra

Cleome gynandra is commonly called as African cabbage, Cat's whiskers, Spider wisp,

Stinkweed and Nalvelai (Tamil name) used as a green vegetable as well as therapeutic plant. It grows as a weed in paddy fields, road sides and even in grass lands. In India, it grows naturally everywhere and various species of Cleome can be found in all states. It is an annual wildflower native of Africa but commonly present in many sub - tropical and tropical parts of the world. It is an erect, branching plant of 25 to 60 cm height and its leaves have 3 - 5 oval - shaped leaflets.

The flowers are white and its seed is sphere shaped and brown in colour. Both the leaves and flowers are edible. Like mustard greens, the leaves have a strong bitter and peppery flavor.

1.1.2. Medical Importance

Leaf sap may be used as pain-relieving, mainly for headaches. Essence from minced young leaves is squeezed into ears, nostrils and eyes to treat epileptic fits and ear ache. Mixture of boiled leaves and roots is administered to enable childbirth in pregnant women, treat arthritis, stomach - ache, constipation, conjunctivitis, severe thread - worm infection, relieve chest pains, malaria, piles, rheumatism, tumour and also the leaves have anti-inflammatory properties. In

India the plants are usually used as an anti helmentic and are applied externally above the wounds to avoid the sepsis.

1.2. Cleome viscosa (L.)

1.2.1. Scientific Classification

Kingdom : Plantae

Clade : Angiosperms

Clade : Eudicots

Clade : Rosids

Order : Brassicales

Family : Cleomaceae

Genus : Cleome

Species : C. viscosa

Cleome viscosa (Linn.) is generally known as yellow spider or Asian spider flower. It is a sticky plant with yellow flowers and long slender pods enclosing seeds, which are called as

Nayikkadugu and found in waste places of the larger parts of India. The genus is distributed throughout the tropical and warm temperate countries of the word. It consists of 180 families and about 180 to 200 species are of herbaceous annual or perennial plants and shrubs. This sticky plant attains a height of 80 to120 cm and it is used as fodder plantLeaves are sessile, compound, trifoliate, alternate, spiral, stalked, foliate in shape and contain glandular hairs on both sides and are used for cooking as green vegetables. Flowers are pedicellate, bisexual, single, auxiliary, stalked, and yellow colour with four free petals. Seeds are used for making cattle cake.

1.2.2. Medical Uses

C. viscosa is used in various illnesses like diarrhea, fever, inflammation, liver diseases, bronchitis, skin diseases, and malarial fever (Henty and Pritchard, 1975). The sap is useful in piles, backache and ear ache, pain-relieving, antipyretic and anti-diarrhea, anti-helminthic, healing the wounds and ulcer (Nadkami, 1982). It is used to alleviate migraine, vomiting, diphtheria, vertigo, headache, pneumonia, septic ears, and stomach ailments (Kokwaro, 1993 and

Gessler, 1994).

1.3. Phytochemical and Antibacterial Activity

Phytochemicals are biologically active substances which include alkaloids, oxalates, tannins, saponins, essential oils, cardiac glycosides, anthraquinones, terpenoids and flavonoids which could act as an anti-nutritional and anti - bacterial agents. The data collected in the present work will be useful in the production of new drugs of pharmaceutical importance.

2. OBJECTIVES

The objectives of the present study is

 To identify the indigenous medicinal plants species from Thiruvallur and Villupuram

Districts, Tamil Nadu, India.

 To prepare the aqueous leaf extracts with different ratios for the study.

 To determine the presence of various phytochemicals in the aqueous leaf extracts of C.

gynandra and C. viscosa.

 To select the suitable gram positive bacteria and gram negative bacteria for evaluating

the antibacterial activities of the secondary metabolites in the aqueous leaf extracts

using disc diffusion method.

 To analyses the results by using statistical methods and to drive a conclusion.

3. REVIEW OF LITERATURE

Herbal medicine is the foundation for primary health care in the developing countries because of better cultural acceptability, compatibility with human body and lesser side effects.

However, there is a drastic increase in the usage of herbal medicine was found in last few years from the developed countries (Vethanarayanan et al., 2012). Cleome gynandra and Cleome viscosa plants are available in abundance during the rainy season whereas, in the dry season, C. viscosa becomes scarce but C. gynandra remains available in gardens.

Dansi et al., (2008) stated that it is important to do a comparative phytochemical evaluation of C. gynandra and C. viscosa to rightly appreciate their increased use in the prevention or treatment of certain illness. Accurate knowledge of the habitat, ecological requirements and usefulness of a plant species is the cornerstone of any program focusing on the conservation and domestication of this species. These studies are necessary for a better knowledge of the species in order to valorize, promote and conserve them for the benefit of rural populations.

Therefore, the qualitative screening of phytochemicals and antioxidant properties of C. gynandra has been reported in India and other countries (Bala et al., 2011; Aparadh et al., 2012;

Meda et al., 2013 and Annadurai et al., 2014). Anbazhagi (2009) worked on preliminary phytochemical screening of the powdered leaf of C. gynandra and revealed the presence of

Carotenoids, Cardiac glycosides, Cyanogenic Glycosides, Flavonoids, Saponins, Triterpenes, sugars, Tannins etc.

Packialakshmi and Oviya (2014) identified alkaloid, tannins, flavonoids, fixed oil and fats, gum and mucilage, sterols as phytochemical comstituents and investigated zone of inhibition for the invitro antimicrobial (bacteria and fungi) activity of seed extracts of plant

Cleome viscosa. The ethanol extract of seeds of plants have showed the significant antimicrobial activity. The zone of inhibition is found to be (12mm to 23 mm) bacteria and fungi.

Ahouansinkpo et al., (2016) carried out the qualitative phytochemical screening on C. gynandra and C. viscosa using leaves extracts, revealed the presence of tannins, flavonoids, anthocyanin, leuco-anthocyanin, steroids, mucilage, reducing compounds and quinone derivatives which are varied depends upon the plant species.

Shanmuganathan and Karthikeyan (2016) studied the preliminary phytochemical screening of aqueous extracts of in vivo and in vitro shoots of C. gynandra and observed the presence of alkaloids, flavoinoids, phenols, saponins, tannins, terpenoids, anthroquinones, glycosides,. aminoacids and carbohydrates.

Rajaselvam and Basil Rose (2016) investigated the antimicrobial analysis of different extracts by agar well diffusion method and using four solvents as ethanolic extracts, acetone extracts, aqueous extracts, and chloroform extracts against five bacterial pathogen such as Gram positive- Bacillus subtilis, Staphylococcus aureus and Gram negative - Escherichia coli,

Pseudomonas aeruginosa, and Proteus mirabilis . The study reported that among the extract ethanolic extracts were found to possess highest antimicrobial activity against E.coli, Proteus mirabilis and Pseudomonas aeruginosa. Acetone and chloroform extracts showed moderate inhibitory potency and no inhibitory activity was observed when tested in the aqueous extract.

4. Materials and Methods

4.1. Collection of Plants

The plant samples of Cleome gynandra and Cleom viscosa were collected from the nearby areas of Thiruvallur and Villupuram Districts, Tamil Nadu, India. The fresh leaves were separated and washed thoroughly under running tap water and dried under shade at room temperature for one week. After drying, it was powdered and used for the present study. The powdered samples were kept in the refrigerator till further analysis (Plate 1 and 2)

4.2. Preparation of Aqueous Leaf Extract

Aqueous leaf extract was prepared by mixing 20 grams of powdered leaf with 100 ml of double distilled water. The mixture was boiled for 15 minutes and filtered through Whatman filter paper No.1. The aqueous filtrate was stored in an air tight container at 4oC in the refrigerator. The different ratios of extract such as A- sample pure C.gynandra; B- sample pure

C.viscosa; A+B (1:1); A+B (3:1) and A+B (1:3) were prepared and used for antibacterial activity. Another method of leaf extract was prepared by using Soxhlet extractor for the purpose of phytochemical analysis.

4.3. Phytochemical Screening of Cleome gynandra and Cleome viscosa

All the extracts of C.gynandra and C. viscosa were analysed for the presence of phytochemicals such as alkaloids, reducing sugars, glycosides, proteins, aminoacids, saponins, steroids, tannins, flavonoids and anthraquinones according to method described by Harborne,

(1973); Baker and Thormasberg, (1983); Trease and Evans (1989) and Sofowora (1993); Sahm and Washington, (1990); Brindha et al., (1991) (Plate 3 and 4) .

Plate 1 Cleome gynandra

Plate 2 Cleome viscosa

4.3.1. Detection of Alkaloids (Wagner’s Test)

Three ml of aqueous leaf extract was diluted with hydrochloric acid. To this, add few drop of Wagner’s reagent (Solution of Iodine in Potassium Iodide). Formation of reddish brown precipitate indicates the presence of alkaloids.

4.3.2. Detection of Reducing Sugar (Fehling’sTest)

One ml of Fehling solution A and Fehling solution B was mixed and then heated on a water bath for 1minute. To this, equal volume of aqueous extract was added and then heated for

5 – 10 minutes. Appearance of yellow to brick red colour indicates the presence of reducing sugar.

4.3.3. Detection of Glycosides, (Borntrager’s Test)

Boil the aqueous leaf extract with 1ml of diluted sulphuric acid in a test tube for 5minutes and filter while hot. Cool the filtrate and shake with equal volume of chloroform then separate the lower layer of chloroform and shake it well with half volume of dilute ammonia. Formation of rose pink to red colour in the ammoniacal (lower phase) layer indicates the presence of glycoside.

4.3.4. Detection of Protein (Biuret’s Test)

To 3 ml of aqueous leaf extract, 1 ml of 4% sodium hydroxide and 1ml of 1% copper sulphate was added. Formation of violet or pink colour indicates the presence of proteins.

4.3.5. Detection of Amino Acid (Ninhydrin Test)

To 1 ml of aqueous plant extract, 2 drops of 5% Ninhydrin (in butanol) solution was added. The mixture was heated over a water bath for 10 minutes and allowed to cool. Formation of purple or blue colour indicates the presence of amino acids.

4.3.6. Detection of Saponins (Foam Test)

To small quantity of leaf extract, 2ml of water was added and shaken well. Persistence of foam produced for 10 minutes indicates the presence of saponins.

4.3.7. Detection of Steroids (Salkowski Tests)

To 1ml of leaf extract, 10 ml of chloroform was dissolved and then added equal volume of concentrated sulphuric acid from the walls of the test tube and then shake well. Formation of reddish blue colour in the upper layer and green fluorescence in the acid layer indicates the presence of steroids.

4.3.8. Detection of Tannins (Ferric chloride reagent test)

To small quantity of aqueous leaf extract, few drops of 5% ferric chloride are added.

Appearance of dark green colour or deep blue colour indicates the presence of tannin.

4.3.9. Detection of Flavonoids (Ferric chloride reagent test)

The extract was treated with few drops of sodium hydroxide solution separately in a test tube. Formation of intense yellow colour, which becomes colourless on addition of few drops of dilute acid indicates the presence of flavonoids.

Plate 3 Phytochemical Analysis of C. gynandra and C. viscosa Aqueous Leaf Extract

Steroids Reducing sugar Reducing

1: 3 1: 1 3: 1 1: 3 1: 1 3: 1

Alkaloids Anthra quinones Anthra

1:1 1:3 3:1 1:3 3:1 1:1

Plate 4 Phytochemical Analysis of C. gynandra and C. viscosa Aqueous Leaf Extract

Flavonoids Saponins

1:1 3:1 1:3 1:1 3:1 1:3

Tannins

1:1 3:1 1:3

4.3.10. Detection of Anthraquinone (Borntrager’s reaction)

Take 1 ml of the leaf extract in a test tube, 20 ml of chloroform was added. This was heated in steam bath for 5 minutes. The extract was filtered while hot and allowed to cool. To the filtrate, an equal volume of 10% ammonia solution was added and shaken well. Appearance of bright pink colour indicates the presence of anthraquinones.

4.4. Selection of Microorganisms

Klebsiella pneumoniae (gram negative), Staphylococcus aureus, (gram positive) were collected from Microbiology Testing Laboratory, Kumaran Hospital, Kilpauk, Chennai - 600 010 and used for the study of antimicrobial activity. The bacterial cultures maintained on slants consisting of nutrient agar medium and 24 hours cultures were used in the antibacterial activity.

4.5. Antibacterial Activity Assay

Antibacterial activity of both the aqueous leaf extracts of C. gynandra and C.viscosa were tested using agar disc diffusion method (Kartig et al., 1991).

4.5.1. Preparation of Inoculum

Stock cultures were maintained at 4°C on slant of nutrient agar. Active cultures for experiments were prepared by transferring a loop of cells from the stock cultures to test tubes containing nutrient broth for bacteria that were incubated at 24 hours at 37ºC. The assay was performed by agar disc diffusion method.

4.5.2. Agar Disc Diffusion Method

Antibacterial activity of sample was determined by disc diffusion method on Muller Hinton agar (MHA) medium (Muller and Hinton, 1941). The Muller Hinton Agar medium was weighed as 3.8gms and dissolved in 100ml of distilled water and 1gm of agar was added. Then the medium is kept for sterilization. After sterilization the media was poured in to sterile petri plates and were allowed to solidify for 1hour. After the medium was solidified, the inoculums were spread on the solid plates with sterile swab moistened with the bacterial suspension of gram positive bacteria (Klebseilla pneumoniae) and gram negative bacteria ( Staphylococcus aureus).

Discs were prepared with 20 µl sample of respective concentrations (500 µg, 250 µg, 125 µg,

62.5 µg and 31.2 µg). 20 µl of Amikacin for the negative control and 10 µl (10 µg) of Pencillin for the positive control was taken and placed on MHA plates. These plates were incubated for 24 hours at 37ºc .Then the microbial growth was determined by measuring the diameter of zone of inhibition.

4.6. Statistical Analysis

The triplicate results of antibacterial activities were analysed using mean, standard deviation and standard error.

5. RESULTS AND DISCUSSION

The aqueous leaf extracts of different ratios of both the Cleome gynandra and Cleome viscosa were analysed qualitatively for the presence of phytochemicals and also tested the antibacterial activities of such leaf extracts against gram positive (Klebsiella pneumoniae) and gram negative bacteria (Staphylococcus aureus) by using disc diffusion method.

5.1. Results of Phytochemical Screening of Cleome gynandra and Cleome viscosa

The qualitative analysis of phytochemical study reported that the presence of alkaloids, reducing sugars, saponins, steroids, flavonoids, tannins and anthraquinones whereas the amino acid, protein and glycosides were not observed in the aqueous leaf extract (Table 1). The previous studies for phytochemical compound of various water extracts of C. gynandra and C. viscosa found very significant (Mishra, 2011; Anbazhagi, 2009; Deepa Shree and Shubha Gopal,

2014 ) while the absence of glycosides compound was contrary to the results of the present study. Presence of saponins was contrary to the result of Adjatin et al., (2013) and

Shanmuganathan and Karthikeyan (2016). This variation may be associated to agro-ecological conditions, varieties, extraction methods and solvents used (Shanmuganathan and Karthikeyan,

2016).

Presence of higher doses of water soluble tannins in some plants were identified as responsible for the reduction of growth rate and protein digestibility in laboratory animals and also esophageal cancer and stated that the dose of the anti - nutritional compound is reduced before preparation of the medicines (Shils et al., 2006). Whereas Beelen et al., (2006) and

Emebu and Anyika, (2011) indicated that the low concentration of a vegetable tannin is accompanied with positive effects on digestion. According to Park et al., (2008) the flavonoids are mostly active in maintaining good blood flow and have a high antioxidant or anti- radical, anti-proliferative and anti-carcinogenic potential and can inhibit the growth of prostate tumours

(Tomofuji et al., 2009 and Jane et al., 2012).

Table 1 Phytochemical Screening of Aqueous Leaf Extract of C. gynandra and C. viscosa

Cleome Cleome A+B A+B A+C S.No. Phytochemicals gynandra (A) viscosa (B) 1 : 1 3 : 1 1 : 3 1 Alkaloids + + + + + 2 Reducing Sugars + + + + + 3 Proteins - - - - - 4 Amino Acids - - - - - 5 Saponins + + + + + 6 Glycosides - - - - - 7 Steroids + + + + + 8 Flavonoids + + + + + 9 Tannins + + + + + 10 Anthraquinones + + + + + Note: + Positive; - Negative

The presence of alkaloids in the leaf extracts of all the samples of C. gynandra and C. viscosa of the present study agreed with the result of Jane et al., (2012) and Koche et al., (2010).

Jane et al., (2012) and Koche et al., (2010) reported that the alkaloids are phytochemicals, the most effective in therapeutic uses and also stated that alkaloids have anti-malarial, anti-microbial and analgesic activity, which could justify the prescription of crushed leaves of C. gynandra to cure jaundice and other infection. Due to the presence of different chemical components found in

these plants, which are having the pharmacological properties, their regular consumption would help prevent or cure various diseases (Adjatin et al., 2013).

5.2. Results of Antibacterial Activities of C. gynandra and C. viscosa

The antimicrobial activities of different concentrations of aqueous extracts of Cleome gynandra and Cleome viscosa leaves against different bacterial strain such as gram positive

(Klebsiella pneumoniae) and gram negative bacteria (Staphylococcus aureus) was evaluated by the presence or absence of inhibition zone and zone diameters (mm).

It was observed that the antimicrobial effect of aqueous leaf extract varies from one plant to another and also in different concentrations within the same plant. From the results obtained the extract of Cleome gynandra showed higher activity in 1000 µg aqueous extract against gram negative bacteria K. pneumonia (8mm) than C.viscosa (4mm) followed by 7.6mm for 62.5 µg and 7.3 for 125 µg. Rajaselvam and Basil Rose ( 2016) reported that the aqueous extract of

C.gynandra showed no antibacterial activity. The result found in the present study was contradictory to the above result. Whereas the inhibition zone of positive control (Amikacin) against K.pneumoniae and S. aureus was higher (11.6mm and 14 mm respectively) when compared to the negative control Pencillin (8.6 mm and 12 mm) (Table 2, Plate 5 and 6).

The results of inhibitory zone for different ratios of leaf extract showed that when compared to all, the 1:1 ( C. g. and C. v.) ratio of aqueous leaf extracts showed highest activity against K. pneumoniae followed by 1:3 (1 C. g. and 3 C.v.) ratio. However the lowest inhibitory zones (3.6 mm) and no antibacterial activities were noted for all the ratios against S. aureus. This result was more significant with the result of Rajaselvam and Basil Rose ( 2016), stated that less antibacterial activity of C. gynandra was observed against Staphylococcus aureus.

Table 2 Antibacterial Activity of Leaf Extract of C. gynandra and C. viscosa

Antibacterial Activity of Leaf Extract of C. gynandra and C. viscosa

Zone of Inhibition in mm

Micro Amikacin Pencilline Leaf 1000 µg 500 µg 250 µg 125 µg 62.5 µg Extract Organism (- control) (+ control)

C. gynandra K.pneumoniae 8.0 ± 1.41 7.0 ± 1.41 6.3 ± 1.53 7.3 ± 1.47 7.0 ± 1.41 14.7 ± 1.78 8.7 ± 1.08

(2.0) (2.0) (1.53) (2.08) (2.0) (2.25) (1.53)

S.aureus 3.3 ± 1.08 0 4 ± 1.41 0 0 14 ± 1.41 12 ± 1.41

(1.53) (0) (2.0) (0) (0) (2.0) (2.0)

K.pneumoniae 4.3 ± 1.08 5.3 ± 1.08 4.3 ± 1.08 6.3 ± 1.00 5.33 ± 1.08 27 .0 ± 1.41 10.33 ± 1.08 C. viscosa (1.53) (1.53) (1.53) (1.52) (1.52) (2.0) (1.53)

S.aureus 0 4.33 ± 1.08 0 0 0 21.0 ± 1.42 12.0 ± 1.42

(0) (1.53) (0) (0) (0) (2.0) (2.0)

C. g.+ K.pneumoniae 15 ± 0.71 12.67 ± 1.78 10 ± 0.71 8.67 ± 0.82 9 ± 1.41 15.67 ± 1.78 9 ± 0.71 C.v.(1:1) (1.0) (2.52) (1.0) (1.15) (2.0) (2.52) (1.0)

S.aureus 0 0 0 0 0 15 .0 ± 0.71 13.0 ± 1.41

(0) (0) (0) (0) (0) (1.0) (2.0)

C. g.+ K.pneumoniae 0 4.23 ± 1.08 0 3.0 ± 1.41 5.67 ± 1.78 13.67 ± 1.99 8.67 ± 1.78 C. v.(3:1) (0) (1.53) (0 (2.0) (2.52) (2.52) (2.52)

S.aureus 3 ± 1.42 3.67 ± 1.78 3 ± 1.41 0 0 12.66 ± 1.78 14.67 ± 1.78

(2.0) (2.52) (2.0) (0) (0) (2.52) (2.52)

C. g.+ K.pneumoniae 4 ± 1.41 5.33 ± 1.08 7 ± 1.41 5 .0 ± 1.41 3.33 ± 1.08 15.67 ± 1.78 5.67 ± 0.82 C. v.(1:3) (2.0) (1.53) (2.0) (2.0) (1.53) (2.52) (1.15)

S.aureus 0 3.67 ± 1.78 0 3.0 ± 1.41 0 15.0 ± 1.41 14.67 ± 1.78

(0) (2.57) (0) (2.0) (0) (2.0) (2.52)

Values are Mean of Triplicates ± Stand Error Values in Parentheses indicates Standard Deviation

Plate 5 Zone of Inhibition of C. gynandra and C. Viscosa Against Gram Negative Bacteria Klebsiella pneumoniae

A C. g. C. v. B 1 1 - C -C 2 1 2 - C + C + C

3 5 5 4 4 3

C.g.+C.v .(3:1) C.g.+C.v.(1:3) C.g.+C.g.(1:1) 1 -C 1 1 -C - C 2 +C 2 2 5 +C + C 5 5 3 4 3 4 3 4

C D E

Plate 6 Zone of Inhibition of C. Gynandra and C. Viscosa Against Gram Positive Bacteria Staphylococcus aureus

Fig. 1 Mean Zone of Inhibition of C. gynandra and C. viscosa Against Gram Negative Bacteria Klebsiella pneumoniae and Gram Positive Bacteria Staphylococcus aureus

15.0 30.0

)

.) 25.0 C.g. 10.0 (C.v 20.0 15.0 5.0 10.0 5.0

0.0 0.0

Zone of inhibition in mm inhibition of Zone Zone of inhibition in ( mm inhibition of Zone

K.pneumoniae S.aureus K.pneumoniae S.aureus

15.0

20.0

)

C.v)

15.0 10.0

C.g + + C.g C.g + 1C.v + C.g 10.0 5.0 5.0

0.0 0.0

Zone of inhibition in mm (1 mm in inhibition of Zone Zone of inhibition in mm (3 mm in inhibition of Zone

K.pnumoniae S.aereus K.pnumoniae S.aereus

20.0

15.0

10.0

5.0

0.0

Zone of inhibition in mm (1C.g + 3 C.v) 3 + (1C.g mm in inhibition of Zone K.pneumoniae S.aureus

The antibacterial activity observed in Cleome gynandra was due to the presence of some phytochemical substances. Masotti et al., (2003) and Angioni et al., (2006) observed that the antimicrobial effect of plant extract varies from one plant to another in different regions of the world. It may be due to several factors such, as the effect of climate, soil composition, age, on the quality and quantity, composition of extracted product and different bacterial strains.

6. SUMMARY

 The plant samples of Cleome gynandra and Cleome viscosa were collected from the

nearby areas of Thiruvallur and Villupuram Districts, Tamil Nadu, India.

 The fresh leaves were separated and washed thoroughly under running tap water and

dried under shade at room temperature for one week. After drying, it was powdered and

used for this study.

 The aqueous leaf extracts of different ratios of both the Cleome gynandra and Cleome

viscosa were analysed qualitatively for the presence of phytochemicals.

 The qualitative analysis of phytochemical study reported that the presence of alkaloids,

reducing sugars, saponins, steroids, flavonoids, tannins and anthraquinones whereas, the

amino acid, protein and glycosides were not observed in the aqueous leaf extract.

 The antibacterial activities were studied by using disc diffusion method against gram

negative (Klebsiella pneumoniae) and gram positive bacteria (Staphylococcus aureus).

 The extract of Cleome gynandra showed higher activity in 1000 µg aqueous extract

against K. pneumonia (8mm) than C.viscosa (4mm) followed by 7.6mm for 62.5 µg and

7.3 for 125µg.

 The inhibition zone of positive control (Amikacin) against K. pneumonia and S. aureus

was highest (11.6mm and 14 mm respectively) when compared to the negative control

Penicillin (8.6 mm and 12 mm).

 The results of inhibitory zone for different ratios of leaf extract showed that compared to

all, 1:1 ( C. g. and C. v.) ratio of aqueous leaf extracts showed highest activity against K.

pneumonia followed by 1:3 (1 C. g. and 3 C.v.) ratio.

 The lowest inhibitory zones (3.6 mm) and no antibacterial activities were noted for all the

ratios against S. pneumonia.

7. CONCLUSION

Cleome gynandra and Cleome viscosa contain varying amounts of phytochemicals which are having therapeutic properties. When compared to single plant, the combined aqueous extract showed more antibacterial activity. There were no previous literatures for combined activities of both C. gynandra and C. viscosa against bacteria. Thus it is the first preliminary study on the phytochemical and antibacterial activities of different ratios of aqueous leaf extracts of Cleome genus but this aspect needs further study to analyse the antibacterial activities of combined medicinal plant extracts using different solvents.

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