1

ANTIBACTERIAL ACTIVITY IN MEDICINAL PLANTS

Dissertation Submitted to the University of Kerala For the partial fulfillment of the requirements for the award of Bachelors degree in Biotechnology

By Iype Praveen Thomas , Unnimaya .S , Vandana Balakrishna , NeethuNath.R.S , Sreelekshmi.S , Laya Johnson ,Sherin .P.L, Sumita Muralidhar.

Department of Botany and Biotechnology Mar Ivanios College Bethany Hills Trivandrum 15 2011-2012 Thiruvananthapuram-15

CERTIFICATE
This is to certify that this dissertation entitled ANTIBACTERIAL ACTIVITY IN MEDICINAL PLANTS is an authentic record of the studies and research work carried out by Mr. /Ms under my supervision and guidance for the partial fulfillment of the requirements for the degree of Bachelor of Science in Biotechnology (Restructured) of the University of Kerala and that no part of this dissertation has been presented earlier for any degree or diploma in this or any Universities.

MrsThressiamma.Mathew Head of the department

Dr.Bindhu.T.Alex Supervising teacher

Examiners: 1. 2.

Declaration
We hereby declared that this dissertation entitled ANTIBACTERIAL ACTIVITY IN

MEDICINAL PLANTS is an authentic record of the orginal research work carried out by us under the supervision of Dr. Bindhu . T. Alex, Department of Botany & Biotechnology, Mar Ivanios College, Thiruvananthapuram and that not part of this dissertation has been previously presented for any degree or diploma or similar title of this or any other Universities. Iype Praveen Thomas (62436) Unnimaya.S Neethu Nath .R.S Sreelekshmi.S Laya Johnson Sherin .P.L Sumita Muralidhar (62434) (624 ) (62435) (624 ) (62425) (62412 ) Vandana Balakrishna (62413 )

ACKNOWLEDGEMENT

4 We are very much obliged to Dr. Bindhu .T.Alex , Assistant Professor, Department of Botany & Biotechnology, Mar Ivanios College, Nalanchira for suggesting this topic and also for guiding us throughout the course of this dissertation. We also have the good fortune to be able to draw upon the devotion, deep knowledge and mature experience of his for the completion of this study. We wish to express our heartfelt gratitude and indebtedness to Mrs. Thressiamma Mathew, Head of the Department, Department of Botany & Biotechnology, Mar Ivanios College, Nalanchira for providing the infrastructure facilities for this dissertation. We take this occasion to thank Ms. Fathima Zehra (Department of Botany & Biotechnology) for being with us during this work. We express our heartfelt thanks to Mr. R.Surendran, Laboratory Assistant (Department of Botany & Biotechnology for the help he had rendered in the laboratory. We are grateful to all our classmates for their co-operation and suggestion. Moreover we are greatly indebted to our parents for their encouragement and support. Above all we are deeply indebted to The God Almighty who always remains as the helping hand behind us in all crises.

Iype Praveen Thomas (62436) Unnimaya.S Neethu Nath .R.S Sreelekshmi.L Laya Johnson Sherin .P.L Sumita Muralidhar (62434) (624 ) (62433) (624 ) (62425) (62412 ) Vandana Balakrishna (62413 )

CONTENTS

INTRODUCTION

OBJECTIVE

REVIEW OF LITERATURE

MATERIALS AND METHODS

RESULTS AND DISCUSSION

SUMMARY AND CONCLUSION

BIBLIOGRAPHY

INTRODUCTION

Medicinal plants are an integral component of ethenoveterinary medicine. Medicinal plants, since time immemorial, have been used virtually all cultures as a source of medicine. The widespread use of herbal remedies and healthcare preparations, as those described in ancient texts, and obtained from commonly used traditional herbs and medicinal plants, has been traced to the occurrence of natural products with medicinal properties. The use of traditional medicine and medicinal plants in most developing countries, as a normative basis for the maintenance of good health, has been widely observed.

As defined by WHO, health is a state of complete physical, mental and social wellbeing and not merely the absence of disease or infirmity.Medicine, in several developing countries, using local traditions and beliefs, is still the mainstay of healthcare. The practice of traditional medicine is widespread in China, India, Japan, Pakistan, Srilanka and Thailand. In China, about 40% of the total medicine consumption is attributed to traditional tribal medicines. In Thailand, herbal medicines make use of legumes encountered in the Caesalpiniaceae, the Fabaceae and the Mimosaceae.

Ethno botanical information is leading to the discovery of novel phytopharmaceuticals and other phytoproducts.This trend has made their commercialization a necessity.Therfore industries based on medicinal and aromatic plants have been established allover the world with a view to manufacture the so-called green products to satisfy the growing demand. The development of formulation of drugs of plant origin involves botanical identification of the vegetable drug,cultivation and post harvest procedures, extraction procedures, standardization of extracts and pharmaceutical formulations.This means that the phytotherapeutics are in the hands of personnel from different disciplines. The production of phytotherapeutics and drugs from the plants needs the cooperation of a big team of horticulturists, botanists,

8 ecologists,taxonomists,phytochemists,pharmacists,pharmocologists,pharmaceutical specialists, marketing and distribution specialists.

HISTORY OF MEDICINAL PLANTS

As early as 3000BC,the ancient Egyptians put much confidence in plants for curing many diseases.Until now the same confidence is still existing among the contemporary Egyptians and a turn backto remedy by herbsis now becoming global rather than regional or national request.This is strongly favoured,since the natural drugs have little more or no side effects as do the chemically synthesized medications.

Pharmacopoeial wild medicinal plants: These plants are used in folk medicine since a long time ago.Recent and modern studies on these plants proved the occurance of active principles in them. Their pharmacological activity has been investigated.They are among the pharmacopoeial drugs in in different pharmaopoeias;either in thevArab countries or abroad.

Plants used in folkmedicine:There are numerous plant species which are collected from the field to be sold in the Attarin:or the herb seller shops. Plants of potential medicinal value:Many plant species were investigated for their active constituents.These has been done depending on the information of the folk use of these plants,or in species with relatives of species,genera or the same family,known from other countries to have active constituents.

9 Medicinal plants are an important health and economic component of the floras in the developed as well as developing countries.Increasing worldwide interest in herbal remedies expanding reliance of local health care of traditional remedies,and a renewed interest in the development of pharmaceuticals from plant sources have greatly increased trade in medicinal plant materials.Important populations of medicinal plants are found not only in the regions and ecosystems with high biological diversity but also in less diverse floras and in floristic communities that are not a common focus of conservation efforts.

The production of medicinal and aromatic plants requires an inderstanding of plant growth,ecology,business,economics,law,conservation and a lot of subjects related to tillage and gathering plants.While development such as machinery,fertilizers and pesticides have helped farmers meet demands for quality materials at affordable prices,the balance with farming costs,and labour compel societies to set directions and establish limits.

Plants produce a diverse range of bioactive molecules,making them a rich source of different types of medicines.Higher plants,as sources of medicinal compounds, have continued to play a dominant role in the maintainance of human health in ancient times. Over 50% of all modern clinical drugs are of natural product origin and natural products play an important role in drug development programs in the pharmaceutical industry. The relatively lower incidence of adverse reactions to plant preparations compared to modern conventional pharmaceuticals,coupled with their reduced cost, is encouraging,both the consuming public and national health care institutions to consider the plant medicines as alternatives to synthetic drugs. Plants with possible antimicrobial activity should be tested against an appropriate microbial model to confirm the activity to ascertain the parameters associated withit.The effects of plant extracts on bacteria have been studied by a very large number of researchers in different parts of the world. Much work has been done on ethno medicinal plants in India.

Infectious diseases account for approximately one half of all deaths in tropical countries. In industrialized nations, despite the progress made in the understanding

10 of microbiology and their control,incidents of epidemics due to drug resistant micro organisms and the emergence of hither to unknown disease causing microbes,pose enormous public health concerns.Historically plants have provided a good source of antiinfective agents;emetine,quinine,and berberine remain highly effective instruments in the fight against microbial infections.Phytomedicines derived from plants have shown great promise in the treatment of intractable infectious diseases including opportunistic AIDS.

Historically,plants have provided a source of inspiration for novel drug compounds,as plant derived medicines have made large contributions to human health and well being.Their role is twofold in the development of new drugs: (1)they may become the base for the development of a medicine,a natural blueprint for the development of new drugs or (2) a phytomedicine to be used for the treatment of disease.

The first generation of plant drugs were usually simple botanicals employed in more or less their crude form.Several effective medicines used in their natural stale such as chinchona,opium,belladonna and were selected as therapeutics agents based on empirical evidence of their clinical application by traditional societies from different parts of the world.Folllowing the industrial revolution,a second generation of plant based drugs emerged based on scientific processing of the plant extracts to isolate their active constituents.

The second generation phytopharmaceutical agents were pure molecules and some of the compounds were even more pharmacologically active than their synthetic counterparts. Notable examples were Quinine from Chinchona,reserpine from Rauvolfia and more recently taxol from Taxus species.These compounds differed from the synthetic therapeutic agents only in their origin. They follow the same method of development and evaluation as other pharmaceutical agents.In the development of Third Generationphytotherapeutic agents a top-bottom approach is usually adopted.This consists of conducting a clinical evaluation of the treatment modalities and therapy as administrated by

11 traditional doctors or as used by the community as folk medicine.This evaluation is followed by acute and chronic toxicity studies in animals.Studues should,when applicable,includes cytotoxicity studies. It is only if the substance has an acceptable safety index would it be necessary to conduct detailed pharmacological/biochemical studies.

PRESENT USE OF PLANTS

It is estimated that today,plant materials are present,in or have provided the models for 50%Western drugs.Many commercially proven drugs used in modern medicine were initially used in crude form in traditional or folk healing practices,or for other purposes that suggested potentially useful biological activity.The primary benefits of using plant derived medicines are that they are relatively safer than synthetic alternatives,offering profound therapeutic benefits and more affordable treatment.

NEEM For thousands of years the beneficial properties of Neem (Azadirachta indica A. Juss) have been recognized in the Indian tradition. Each part of the neem tree has some medicinal property. Biswas et al (2002) have recently reviewed the biological activities some of the neem compounds, pharmacological actions of the neem extracts, clinical study and plausible medicinal applications of neem along with their safety evaluation. Neem has two closely related species: A. indica A. Juss and M. azedarac, the former is popularly known as Indian neem (margosa tree) or Indian lilac, and the other as the Persian lilac. Neem has been extensively used in ayurveda, unani and homoeopathic medicine.The Sanskrit name of neem tree is Arishtha meaning 'reliever of sickness' and hence is considered asSarbaroganibarini. The tree is still regarded as 'village dispensary' in India. The importance of the neem tree has been recognized by US National Academy of Sciences, which published a report in 1992 entitled 'Neem a tree for solving global problems'.

12 More than 135 compounds have been isolated from different parts of neem and several reviews have also been published on the chemistry and structural diversity of these compounds. The compounds have been divided into two major classes: isoprenoids (like diterpenoids and triterpenoids containing protomeliacins, limonoids, azadirone and its derivatives, gedunin and its derivatives, vilasinin type of compounds and C- secomeliacins such as nimbin, salanin and azadirachtin ) and non-isoprenoids, which are proteins (amino acids) and carbohydrates (polysaccharides), sulphurous compounds, polyphenolics such as flavonoids and their glycosides, dihydrochalcone, coumarin and tannins, aliphatic compounds, etc. Various parts of the neem tree have been used as traditional Ayurvedic medicine in India. Neem oil and the bark and leaf extracts have been therapeutically used as folk medicine to control leprosy, intestinal helminthiasis, respiratory disorders, constipation and also as a general health promoter. Its use for the treatment of rheumatism, chronic syphilitic sores and indolent ulcer has also been evident. Neem oil finds use to control various skin infections. Bark, leaf, root, flower and fruit together cure blood morbidity, biliary afflictions, itching, skin ulcers, burning sensations and pthysis. Oil from the leaves, seed and bark possesses a wide spectrum of antibacterial action against Gram-negative and Gram-positive microorganisms, including M. tuberculosis and streptomycin resistant strains. In vitro, it inhibits Vibrio cholerae ,Klebsiella pneumoniae, M. tuberculosis and M. pyogenes. Antimicrobial effects of neem extract have been demonstrated against Streptococcus mutans and S. faecalis. Neem extract Its effective to cure ringworm, eczema and scabies. Lotion derived from neem leaf, when locally applied, can cure these dermatological diseases within 3-4 days in acute stage or a fortnight in chronic case. A paste prepared with neem and turmeric was found to be effective in the treatment of scabies in nearly 814 people100. Neem leaf extract has been prescribed for oral use for the treatment of malaria by Indian ayurvedic practitioners from time immemorial. Recently, a clinical trial has been carried out to see the efficacy of neem extract to control hyperlipidemia in a group of malarial patients severely infected with P. falciparum. The lipid level, especially cholesterol, was found to be lower during therapy when compared to non-malaria patients. Reports are available regarding

13 the use of neem to treat patients suffering from various forms of cancer. One patient with parotid tumour and another with epidermoid carcinoma have responded successfully when treated with neem seed oi1. NIM- 76, a refined product from neem oil, was studied in 10 human volunteers, where intravaginal application before sexual intercourse could prevent pregnancy with no adverse effect on vagina, cervix and uterus. The data suggested that intrauterine treatment is safe. KACHOLAM Kacholam ( Kaempferia galanga L). is a highly useful medicinal plant. Its rhizomes (tubers) are used in indigenous medicines as stimulant, expectorant, diuretic, carminative etc. Decoction of rhizomes is useful for curing dyspepsia, headache, rheumatism and malaria. For clearing nasal obstructions, rhizomes may be boiled in oil may be applied externally. Digestive power can be enhanced by using kacholam tubers. Larvicidal and anticancerous principles are extracted from tubers. The tubers enhance digestion, remove bad odour of mouth and are used as masticatory for chewing along with betel and arecanut . Kacholam, also known as Sugandhavacha, Chandramulika or Sidhul is a rhizomatous perennial plant, the rhizomes of which yield an essential oil. The oil is utilized in the manufacture of perfumes and in curry flavouring. It is also employed in cosmetics, mouth- washes, hair tonics and toiletries. The pungent, hot, sharp, bitter and aromatic rhizomes find an important place in indigenous medicine as stimulant, expectorant, diuretic and carminative. It promotes digestion and cures skin diseases, piles, phantom tumors, coughs, oedema, fever, epilepsy, splenic disorders, wounds, asthma and rheumatism. The rhizomes are used for protecting clothes against insects and are eaten along with betel and arecanut as a masticatory. The rhizomes and leaves are attached to neck- laces and added to bath water for perfume.Karcura (Kacholam) is a reputed remedy for all diseases caused by the morbidity of vata and kaphaand is especially useful in respiratory ailments like cough, bronchitis and asthma. The drug is reported to be acrid, hot, bitter and aromatic. It cures skin diseases, wounds and splenic disorders. It promotes digestion, removes bad odour of the mouth and destroys pathogenic organisms (Aiyer and Kolammal, 1964). The officinal part is the rhizome and it is a constituent of a variety of Ayurvedic preparations like Dasamularista, Valiya rasnadi kasaya, kaccoradi churna, Asanaeladi taila. Valiya narayana taila, etc. The identity of the plant sources of the drug

14 Karcura is a matter of debate. The source of Karcura in Kerala in the recent times has been Kaempferia galanga (Sivarajan and Balachandran, 1994).

The rhizomes and root-stocks are good for dyspepsia, leprosy, skin diseases, rheumatism, asthma, cough, bronchitis, ulcers, helminthiasis, fever, malarial fever, splenopathy, cephalalgia, inflammatorytumour, nasal obstruction, halitosis, strangury, urolithiasis, and haemorrhoids. The leaves are used for pharyngodynia, ophthalmia, swellings, fever and rheumatism . The tubers reduced to powder and mixed with honey are given in case of coughs and pectoral infections. The oil in which they are boiled is useful in applying to the stoppages of the nasal organs . Kaempferia galanga is reported to have great export potential . The tuberous rhizome of kacholam contains an alkaloid, starch, gum, fatty matter with a fragrant liquid essential oil and a solid white crystalline substance and mineral matter. The rhizome possesses a camphoraceous odour with somewhat bitter aromatic taste resembling that of Hedychium spicatum.The essential oil is reported to contain over 54 components of which the major constituents are ethyl-trans-p-methoxy, cinnamate 16.5%, pentadecane 9%, 1,8-cineole 5.7%, g-carene 3.3%, and borneol .Insectididal constituents from Kaempferia galanga were isolated by .Cyclohexane oxide derivatives and diterpenes were isolated by . Rhizome yields essential oil, which has antifungal activity. Ethyl-p-Methyl O-trans-cinnamate is the main compound in root . p-Methoxycinnamic acid and its methyl and ethyl esters have been isolated from the essential oil . Essential oil from rhizomes contain npentadecane, ethyl-p-methoxy cinnamate, ethyl cinnamate, carene, camphene, borneol, p-methoxystyrene, pmethoxy cinnamate, pmethoxy- trans-cinnamic acid and cinnamaldehyde. Rhizome is cytotoxic. Tuber is stimulant, expectorant, diuretic and carminative . TULSI Tulasi is perhaps the most sacred plant of India nest to kamala the lotus. Particularly, the leaves of Tulasi are used to worship Visnu. It is interesting to note that the plants sacred to Sankara, Ganesha and Visnu are bilva, durva and tulasi, which alleviate vata, pitta and kapha doshas, respectively. There are innumerable references of Tulasi in Indian mythology. The ancient Ayurvedic scriptures have mentioned the plant in the management of several diseases. The plant is supposed to have a purifying influence by liberating ozone and also is said to repel

15 the mosquitoes. Thus, in many parts of India, the plant is grown in the courtyard, traditionally and worshipped daily as a necessary ritual for family will-being. Ayurvedic texts categorise it as kasaghna alleviates cough, svedala induces sweating, ajirna nasaka mitigates indigestion and agnimandya nasaka alleviates anorexia (Bhavaprakas) The plant grows all over India up to 2000 meters height. It is grown in houses, temples and gardens. An erectannual grows 0.5-1.5 meters in height and has red or purple quadrangular branches. The leaves are opposite, about 2-4 cm long, margins entire or toothed, hairy on both the surfaces, dotted with minute glands and are aromatic. The flowers tiny, purple and inflorescence is a long spike or 12-14 cm in length. The fruits are small, smooth nut lets, reddish grey in color. Botanically, Tulasi is known as Ocimum sanctum and it belongs to family Lamiaceae. The leaves contain an essential oil, which contains eugenol, eugenal, carvacrol, methylchavicol, limatrol and caryophylline. The seeds contain oil composed of fatty acids and sitosterol. The roots contain sitosterol and three triterpenes A, B, and C. The leaves also contain a steroid ursolic acid and n-triacontanol. Eugenol (70.5), its methyl ether (4.8), nerol (6.4), caryophyllene (7.5), terpinen 4-(0.4), decylaldehyde (0.2), selinene (0.4), pinene (0.4), camphene (2.0) and a-pinene (3.5%) identified in essential oil by GC. Tulasi is pungent and bitter in taste, pungent in the post digestive effect and has hot potency. It alleviates kapha and vata doshas, but slightly aggravates the pitta dosha. It possesses light and dry attributes. On the contrary the seeds are oily and slimy in attributes and have a cold potency. Tulasi is a stimulant, aromatic herb and effectively reduces the fever.(Bhavaprakash) The seeds, leaves and the roots of Tulasi have great medicinal value. It is used both, internally as well as externally. Tulasi has mild antiseptic, analgesic properties and it relieves the swellings also. Hence, it beneficial, externally, in various skin diseases. The paste of leaves works well, with marica powder, when applied topically in ringworm infestations. The dressing with the pulp of its leaves effectively controls the infections and hastens the healing of chronic infected wounds. The leaves when chewed mitigate the infections of the gums. Instillation of fresh juice of the leaves into ears is an effective domestic medicament for ear aches. The massage with the leaves juice improves the circulation beneath the skin and augments the sensation in the skin. In the headache due to sinusitis, the instillation of juice in the nose

16 facilitates the secretions of kapha and relieves the headache. The dried powder of the leaves can be inhaled, like a snuff, for the same purpose. Internally, Tulasi is used on many occasions, as a vehicle or an adjuvant. It enhances the bioavailability of the medicine or acts synergistically, respectively. It is also used in number of Ayurvedic proparations for bhavana, to potentate their effects, namely in Tribhuvana kurti, Caturbhuja rasa etc. Tulsi is salutary to increase the appetite and improve digestion. It has a mild laxative as well as vermicidal action, hence is rewarding in worm infestations. It is a good blood purifier and is helpful in the diseases of the blood and heart. A tea prepared with the leaves of Tulasi is a common domestic remedy for cold, cough, milk indigestion, diminished appetite and malaise. Tulasi has specified actions on the respiratory system-pranavaha srotasa. It effectively liquefies the phlegm due to its hot and sharp attributes. It gives excellent results in cough due to kapha, allergic bronchitis, asthma and eosinophilia. Combined with honey, the juice works well to control the hiccup. In tubercular cough, Tulasi is also beneficial. It is an effective panacea for fever, especially of kapha type, while given with honey and marica fruit powder. In such conditions it effectively controls colds and reduces pain. Tulasi juice works as amapacana, meaning it digests and destroys ama the toxins. The seeds of tulasi being cool and sweet along with its slimy-(picchila) attribite, are beneficial in dysuria. The khira or pudding, prepared from the seeds, is useful to boost the energy in debility, especially due to pitta dosha. In diarrhea associated with bleeding, the seeds soaked overnight in water to which a teaspoon of sugar is added, and taken in the morning. ANTIMICROBIAL ACTIVITY There is a continuous and urgent need to discover new antimicrobial compounds with diverse chemical structures and novel mechanisms of action because there has been an alarming increase in the incidence of new and re-emerging infectious diseases. Another big concern is the development of resistance to the antibiotics in current clinical use. In recent years, drug resistance to human pathogenic bacteria has been commonly reported from all over the world. In the present scenario of emergence of multiple drug resistance to human pathogenic organisms, this has necessitated a search for new antimicrobial substances from other sources including plants. Higher plants produce hundreds to thousands of diverse chemical compounds

17 with different biological activities. The antimicrobial compounds produced by plants are active against plant and human pathogenic microorganisms. It is expected that plant extracts showing target sites other than those used by antibiotics will be active against drug-resistant microbial pathogens. Antibiotics provide the main basis for the therapy of microbial (bacterial and fungal) infections. Since the discovery of these antibiotics and their uses as chemotherapeutic agents there was a belief in the medical fraternity that this would lead to the eventual eradication of infectious diseases. However, over use of antibiotics has become the major factor for the emergence and dissemination of multidrug resistant strains of several groups of microorganisms. The worldwide emergence of multidrug resistant Escherichia coli and many other -lactamase producers has become a major therapeutic problem Thus, in light of the evidence of rapid global spread of resistant clinical isolates, the need to find new antimicrobial agents is of paramount importance. However, the past record of rapid, widespread emergence of resistance to newly introduced antimicrobial agents indicates that even new families of antimicrobial agents will have a short life expectancy .For this reason, researchers are increasingly turning their attention to herbal products, looking for new leads to develop better drugs against MDR microbe strains The antimicrobial efficacy attributed to some plants in treating diseases has been beyond belief. It is estimated that local communities have used about 10% of all flowering plants on Earth to treat various infections, although only1% have gained recognition by modern scientists. During the last ten years the pace of development of new antimicrobial drugs has slowed down while the prevalence of resistance(especially multiple) has increased astronomically .The increase in number of antibiotic resistant bacteria is no longer matched by expansion in the arsenal of agents available to treat infections. Literature reports and ethnobotanical records suggest that plants are the sleeping giants of pharmaceutical industry. They mayprovide natural source of antimicrobial drugs that will/or provide novel or lead compounds that may be employedin controlling some infections globally.

18

Review OF LITERATURE

Ethanol extracts of 19 Malaysian traditional vegetables (locally known as ulam) belonging to 15 families were screened for antimicrobial and cytotoxic activities. Seven extracts, i.e. Anacardium occidentale, Garcinia atroviridis, Sesbania grandiflora, Barringtonia racemosa, Polygonum minus, Kaempferia galanga and Etlingera elatior displayed cytotoxic

19 activity against the HeLa (human cervical carcinoma) cell line with CD 50 values in the range of 1030 g/ml.( M.M. Mackeen et al ., 1997). Ocimum sanctum the sacred 'Tulsi' finds diverse uses in the indigenous system of Medicine. The leaves of the plant have been used as an expectorant, diaphoretic, anticancer, antihelminthic, antiseptic, analgesic and tonic rejuvinator. Dry leaves are used in fungal infections, the fresh juice of the leaves are used in the treatment of bronchitis, otitis media, and skin diseases. Antibacterial activity of ether extract of the leaves against Escherichia coli, Staphylococcus aureus, and Mycobacterium tuberculosis have been reported.( Geeta et al .,2001). The antimicrobial effects of extracts of neem seed (Azadirachta indica A. Juss.) were investigated using microbial growth inhibition assays.( E. Coventry et Al.,2001). The antibacterial activity of Karanj (Pongamia pinnata) and Neem (Azadirachta indica) seed oil in vitro against fourteen strains of pathogenic bacteria was assessed (BASWA Manjula et al.,2001 ). Fifty four plant extracts (methanol and aqueous) were assayed for their activity against multi-drug resistant Salmonella typhi. Strong antibacterialactivity was shown by the methanol extracts of Aegle marmelos, Salmalia malabarica, Punica granatum, Myristica fragrans, Holarrhena antidysenterica, Terminalia arjuna and Triphal (mixture of Emblica of fi cinalis, Terminalia chebula and Terminalia belerica) ( Rani P et al .,2004 ) Ocimum sanctum fixed oil showed good antibacterial activity against Staphylococcus aureus, Bacillus pumilus and Pseudomonas aeruginosa, where S. aureus was the most sensitive organism.(Singh PP et al ., 2005). Screening was done of some plants of importance in the Ayurvedic system of traditional medicine used in India to treat enteric diseases. Volatile oil of dried rhizome of Kaempferia galanga obtained by water distillation was determined for its chemical components using gas . Antimicrobial activity of the volatile oil was tested against various microbes using agar disc diffusion method (S Tewtrakul et al.,2005). The immunomodulatory activity of the neem-bark extract has been reported. The medicinal and industrial uses of various parts of neem tree and the compounds isolated have been reviewed. (Wafaa A. Helmy et al ., 2007 ). .. The antibacterial activity of guava (Psidium guajava) and neem (Azadirachta indica) extracts against 21 strains of foodborne pathogens were determined. Guava and neem extracts showed higher antimicrobial activity against Gram-positive bacteria compared to Gram-negative bacteria except for V. parahaemolyticus, P. aeroginosa, and A. hydrophila.( M.D. Mahfuzul Hoque et al ., 2007). Basil or tulasi (Ocimum sanctum L.) is considered as a sacred plant and its various medicinal properties have been mentioned in

20 ancient medicinal text, Ayurveda. Different parts of this plant are used for treatment of various ailments. The activity of essential oils obtained from fresh and dried leaves of O. sanctum against clinical isolates of enteric bacteria i.e., Escherichia coli, Shigella sp. and Salmonella typhi and yeast (Candida albicans) was compared.( S. Mondal et al .,2007)

For evaluating antibacterial activity, both aqueous and organic solvent methanol was used. The plants screened were Ocimum sanctum, Jatropha gossypifolia, Boerhavia diffusa, Azadirachta indica, Solidago virgaurea, and Commelina benghalensis. The antibacterial activity was assessed against six bacterial strains--Pseudomonas testosteroni, Staphylococcus epidermidis, Klebsiella pneumoniae, Bacillus subtilis, Proteus morganii, Micrococcus flavus.( Nair R et al .,2007). Essential oil obtained by hydrodistillation and two different solvent extractions(petroleum ether and ethanol) from five Zingiberaceae species: ginger (Zingiber officinaleRoscoe.), galanga (Alpinia galanga Sw.), turmeric (Curcuma longa L.), kaempferia (Boesenbergia pandurata Holtt.) and bastard cardamom (Amomum xanthioides Wall.) was characterized,(Krittika Norajit et al.,2007). Based on local use of common diseases and Ethnobotanical knowledge, an attempt has been made to assess the antibacterial properties of selected medicinal plants.VIZ. Ocimum sanctum (Tulsi), Origanum majorana (Ram Tulsi), Cinnamomum zeylanicum (Dalchini), and Xanthoxylum armatum (Timur), for potential antibacterial activity against 10 medically important bacterial strains, namely Bacillus subtilis, Bacillus cereus, Bacillus thuringiensis, Staphylococcus aureus, Pseudomonas spp, Proteus spp, Salmonella Typhi, Escherichia coli, Shigella dysentriae, Klebsiella pneumoniae. The antibacterial activity of ethanol extracts was determined by agar well diffusion method. ( Bishnu joshi et al ., 2008) . The enhancement of hydrophilicity of DC air and oxygen plasma treated cotton fabric and its effect on the antimicrobial efficacy when treated with neem leaf extract (K Vaideki et al .,2009).

Silver nanoparticles are known to have bactericidal effects. A new generation of dressings incorporating antimicrobial agents like silver nanoparticles is being formulated to reduce or prevent infections. Apart from being environmentally friendly process, use of Neem leaves extract might add synergistic antibacterial effect of Neem leaves to the biosynthesized

21 nanoparticles. With this hypothesis the biosynthetic production of silver nanoparticles by aqueous extract of Neem leaves and its bactericidal effect in cotton cloth against E. Coli were studied (Tripathi et al.,2009).Neem (Azadirachta indica) is an Indian tree well known for its several pharmacological activities, including antimicrobial activity. More than 300 composites have already been isolated and azadirachtin (AZA) is its main active component. Neem leaves hydroalcoholic extracts were prepared by percolation in 96% ethanol different concentrations (50%, 60%, 70%, 80% and 90% (v/v)).( Priscila D. Alves et al .,2009).

In recent years this interest to evaluate plants possessing antibacterial activity for various diseases is growing . Based on local use of common diseases and Ethnobotanical knowledge, an attempt has been made to assess the antibacterial properties of selected medicinal plants.VIZ. Ocimum sanctum (Tulsi), Origanum majorana (Ram Tulsi),Cinnamomum zeylanicum (Dalchini), and Xanthoxylum armatum (Timur), for potential antibacterial activity against 10 medically important bacterial strains, namely Bacillus subtilis, Bacillus cereus, Bacillus thuringiensis, Staphylococcus aureus, Pseudomonas spp, Proteus spp, Salmonella Typhi, Escherichia coli, Shigella dysentriae, Klebsiella pneumonia (Bishnu Josh et al.,2009). The ethnobotanical efficacy of Indian medicinal plants; Achyranthes aspera, Artemisia parviflora, Azadirachta indica, Calotropis gigantean, Lawsonia inermis, Mimosa pudica, Ixora coccinea, Parthenium hysterophorus and Chromolaena odorata were examined using agar disc diffusion method against clinical bacteria (Escherichia coli and Staphylococcus aureus) and phytopathogenic bacteria (Xanthomonas vesicatoria and Ralstonia solanacearum). Leaves were extracted using different solvents such as methanol, ethanol, ethyl acetate and chloroform.( S. L. Sukanya et al.,2009). Tulsi (Ocimum sanctum) extract has an antimicrobial activity against Streptococcus mutans and the concentration of Tulsi (Ocimum sanctum) extract among the 15 concentrations investigated has the maximum antimicrobial activity was determined . (Agarwal P et al.,2010).

. Tulsi has been tested against a variety of microorganisms like Candida albicans, Staphyloccus aureus, enteric pathogens, Klebisella, Escherichia coli and Proteus. It has also

22 demonstrated antigonorrheal efficacy against multiresistant strains of Neisseria gonorrhea and clinical isolates of beta lactamase-producing methicillin-resistant Staphylococcus aureus.( Pooja Agarwal et al .,2010). Shoots and roots were induced from axillary buds of Kaempferia galanga when cultured on Murashige and Skoog (MS) medium supplemented with NAA + BAP (0.1mg + 1.0 mg/l). Liquid detergent (2%) + Alcohol (70%) + Mercuric chloride (0.1%) used for surface sterilization of explants.Rhizomes were developed after four months of transferring in to earthen pots. The methanolic extracts of both micropropagated plant rhizome and natural plant rhizome were screened for antimicrobial activities against two gram ve and two gram +ve pathogenic bacteria i.e. Escherichia coli,Salmonella typhi, Bacillus subtilis and Staphylococcus aureus.( HANUMANTHARAJU N et al.,2010). Four medicinal plants (Quercus infectoria, Kaempferia galanga, Coptis chinensis and Glycyrrhiza uralensis) as well as one traditional Thai treatment for aphthous ulcers based on these four plants were tested for antimicrobial activity.( Mekseepralard C et al.,2010). Four medicinal plants (Quercus infectoria, Kaempferia galanga, Coptis chinensis and Glycyrrhiza glabra) as well as one traditional Thai treatment for aphthous ulcers based on these four plants were tested for antimicrobial activity. MIC values for a range of bacteria and Candida albicans were determined, with both type strains and clinical isolates being used. Antioxidant activity was determined using the ABTS radical scavenging assay (Kamkaen et al.,2010).

Neem has been extensively used in Ayurveda, Unani and homoeopathic medicine and has become a cynosure of modern medicine. The antibacterial activity of neem seed oil was assessed in vitro against 14 strains of pathogenic bacteria. The present study was to find out the antibacterial activity of neem leaves against human pathogenic bacteria, including Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Micrococcus luteus, Bacillus subtilis, Enterococcus faecalis and Streptococcus faecalis.( Saradhajyothi KOONA et al.,2011). The antibacterial activity of aqueous extracts , chloroform extract ,alcohol extract and oil obtained from leaves of Ocimum sanctum against the selected bacteria was observed ( Poonam Mishra et al .,2011). Antimicrobial activity in leaf extract of neem (Azadirachta indica) against human pathogenic bacteria. E.coli, Staphylococcus aureus, Pseudomonas aeruginosa , Salmonella typhimurium, Bacillus pumilus. Antimicrobial activities of alcoholic extracts of neem leaves were used.( Maragathavalli, S et al.,2012). Kaempferia galanga L. (KG), commonly known as cekor is one of those precious medicinal herbs of Zingiberaceae that are still included in un-

23 utilized herbs inspite of the variety of useful pharmacological properties it possesses. Extracts of KG have anti-inflammatory, analgesic, nematicidal, mosquito repellent, larvicidal, vasorelaxant, sedative, antineoplastic, antimicrobial, anti-oxidant, antiallergic and wound healing properties.( Muhammad Ihtisham Umar et al.,2011). Volatile oil from the rhizomes of Kaempferia galanga was isolated and characterized by Gas ChromatographyMass Spectroscopy. The antimicrobial activity of oil was tested against human and plant pathogenic bacteria and fungi. (P.A. Mary Helen et al .,2011).

24

MATERIALS AND METHODS

MATERIALS
COLLECTION OF PLANT MATERIAL

25
Fresh leaves of three different plants Neem ( Azadirachta indica A. Juss ) , Kacholam (

Kaempferia galanga L ), Tulsi ( Ocimum sanctum ) free from disease were collected from
different areas. The leaves were washed thoroughly 2-3 times with running water and once with

sterile distilled water. The material was chopped into small pieces and then air dried on a sterile
blotter under shade for 20-30 days.

PREPARATION OF PLANT EXTRACTS 10 g of each powdered plant material of Neem , Kacholam and Tulsi were defatted with petroleum ether and extracted in a soxhlet extraction apparatus with 100% ethanol. The solvent extracts were concentrated under reduced pressure and preserved at 5C in airtight bottle until further use. TEST MICROORGANISMS USED FOR ANTIMICROBIAL STUDIES Bacterial cultures of of both gram negative and gram positive - were obtained from the culture collection centre. Different bacterial strains used for the antimicrobial studies were ,
Staphylococcus aureus (S.aureus) -

It is a facultative anaerobic Gram

-positive coccal bacterium. It is frequently found as part of the normal skin flora on the skin and nasal passages. S. aureus can cause a range of illnesses, from minor skin infections, such as pimples, impetigo, boils(furuncles), cellulitis folliculitis, carbuncles, scalded skin syndrome, and abscesses, to life-threatening diseases such as pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock syndrome (TSS), bacteremia, andsepsis. Its incidence ranges from skin, soft tissue, respiratory, bone, joint, endovascular to wound infections. It is still one of the five most common causes of nosocomial infections and is often the cause of postsurgical wound infections.
Escherichia coli (E.coli) it is a Gram-negative, rod-shaped bacteriumthat is commonly found

in the lower intestine of warm-blooded organisms (endotherms). Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for people making product recalls. The harmless strains are part of the normal flora of the gut, and can benefit their hosts by producing vitamin K2, and by preventing the establishment of pathogenic bacteria within the intestine.

26
Klebsiella pneumoniae is a Gram-negative, non-

motile, encapsulated, lactose fermenting, facultative anaerobic, rod shaped bacterium found in the normal flora of the mouth, skin, and intestines. It is clinically the most important member of the Klebsiella genus of Enterobacteriaceae. K. pneumoniae can cause the disease Klebsiella pneumonia. They cause destructive changes to human lungs inflammation and hemorrhage with cell death (necrosis) that sometimes produces a thick, bloody, mucoid sputum (currant jelly sputum). Typically these bacteria gain access after a person aspirates colonizing oropharyngeal microbes into the lower respiratory tract.
Pseudomonas fluorescens (P.fluorescens) -

is a common Gram-negative, rod-

shaped bacterium. It belongs to the Pseudomonas genus. P. fluorescens is an unusual cause of disease in humans, and usually affects patients with compromised immune systems (e.g., patients on cancer treatment). . Muller Hinton agar Beef infusion - 300 ml

Caesin hydrolysate - 17.5 g Starch Agar - 25 g - 1.5g

Distilled water 1000 ml The above constituents werw added one by one and the final volume was made up to 1000 ml with distilled water . Then the pH was adjusted to 7 at 25Ccusing 0.1 Naoh/HCL . It was autoclaved and was poured into sterile petridishes and was allowed to solidify . It was stored at 4C. Nutrient Broth Nutrient broth was used for the preparation of inoculum Peptone 5g

27 Beef extract 3g Nacl 5g The above constituents were added one by one and the final volume was made up to 1000 ml with distilled water . Then the pH was adjusted to 7 at 25c using0.1 NaoH / HCL. The bacteria were maintained on nutrient broth (NB) at 37C. Streptomycin was used as a reference . It was purchased and was dissolved in 2ml of distilled water.

DETERMINATION OF ANTIMICROBIAL ACTIVITY WITH WELL DIFFUSION METHOD In order to determine the biological significance and ability of the plant part, the antibacterial activity was determined by agar well diffusion method. Muller Hinton agar plates (MHA) were prepared as follows : Overnight grown different species of bacteria such as E-coli, Staphylococcus aureus, Klebsiella pneumoniae and Pseudomonas fluorescens were swabbed. Wells of approximately 10mm was bored used a well cutter and di concentrations of plant extracts (30,60l ) was added, the zone of inhibition was measured after overnight incubation and compared with that of standard antibiotics, DMSO(Dimethylsulfoxide) was used as negative control and streptomycin was used as reference. The antimicrobial activity was determined by measuring the zone of inhibition after overnight incubation and compared with standards.

28

RESULTS AND DISCUSSION

RESULTS

29

The antimicrobial activity of the extracts and their potency was quantitatively assessed by the presence or absence of inhibition zone and zone diameter, respectively as given in Table 1.

Organism

Sample

Measurement of zone of

Inhibition(cm)

Streptomycin E Coli DMSO

2.1

1.1

Kacholam

1.3

Tulsi

1.4

Neem

1.2

30

Streptococcus

Streptomycin

5.5

DMSO

1.5

Kacholam

1.7

Tulsi

1.5

Neem

2.0

Staphylococcus Streptomycin 2.7

DMSO

1.2

Kacholam

1.3

Tulsi

1.2

Neem

1.3

31

Streptomycin

5.5

Klebsiella

DMSO

1.5

Kacholam

1.7

Tulsi

1.5

Neem

2.0

32

DISCUSSION

Plants are important source of potentially useful structures for the development of new chemotherapeutic agents. Many reports are available on the antiviral, antibacterial, antifungal, anthelmintic, antimolluscal and anti-inflammatory properties of plants. Some of these observations have helped in identifying the active principle responsible for such activities and in the developing drugs for the therapeutic use in human beings. Emergence of multi-drug resistance in human and animal pathogenic bacteria as well as undesirable side effects of certain antibiotics has triggered immense interest in the search for new

33 antimicrobial drugs of plant origin..

In the present study , antimicrobial activity of plant extracts was determined by agar well diffusion assay using Streptomycin as a positive control. The antimicrobial spectrum was evaluated against E. coli, Pseudomonas, Staphylococcus, Klebsiella. The zone of inhibition was more in wells corresponding to extracts of Neem (Azadirachta indica ) when compared with other two extracts (Kaempferia galanga and Ocimum sanctum) . Study suggests potent antibacterial activity of all three plant extract which can find its application in therapeutic regimens, confirming the great potential of bioactive compounds and are useful for rationalizing the
use of this plant in primary health care. In vivo data may be helpful in determining the real potential usefulness of this plant for the treatment of infectious diseases.

34

BIBLIOGRAPHY

1. Thomson, W. A. R., Medicines from the Earth. propilis and antimicrobial drugs. Microbiol. Res., Maidenhead, United Kingdom. McGraw-Hill Book, 1978 , 158: 353-357. 2. Stockwell, C., Natures pharmacy. London, O. Goslinka, 2004. Antimicrobial activity of United Kingdom. Century Hutchinson Ltd. isocytisoside and extracts of Aquilegia vulgaris L. 1988. 3. Gerhartz, W., Y.S. Yamamota, F.T. Campbell, Lett. Appl. Microbiol.,1985, 39: 93-97.

35 4. Kroschwitz, J. I. and M. Howe-Grant, 1992. Kirk- Kandha tribe of Orissa, India. J. Ethnopharmacol,1992, 102: 319-325 5. Newman, D . J., G.M. Cragg and K.M. Snader, , 2006 , 17: 215-234. 6. Srivastava, J. , J. Lambert and N. Vietmeyer, antimicrobial activity of Anogeissus latifolia. Medicinal plants: An expanding role in development. J. Ethnopharmacol.,2008, 106: 57-61. 7. Uniyal, S. K., K.N. Singh, P. Jamwal and B. Lal, Aloe ferox and Withania somnifera inhibit ,Traditional use of medicinal plants among the tribal Candida albicans and Neisseria gonorrhoea. communities of Chhota Bhangal, Western Himalayan. African J. Biotechnol., 2006,7: 12-15. 8. M.M. Mackeen, A. M. Ali, S.H. El-Sharkawy, M.Y. Manap, K.M. Salleh, N.H. Lajis and K. Kawazu , 1997, Vol. 35, No. 3 , Pages 174-178. 9. Geeta, D. M . Vasudevan, R. Kedlaya, S .Deepa, M .Ballal, Department of Biochemistry, Manipal Institute of Medical Sciences, India, 2001 , 55 , 434-438. 10. Rani. P., Khullar N, Department of Biotechnology, Antimicrobial evaluation of some medicinal plants for their anti-enteric potential against multi-drug resistant Salmonella typhi ,2004 Aug ;18(8):670-3.

11. Wafaa A. Helmy, Hassan Amer and Nefisa M. A., EL-Shayeb, Biological and Antimicrobial Activities of Aqueous Extracts from Neem Tree (Azadirachta indica A. Juss., Meliaceae), Journal of Applied Sciences Research, 3(10): 1050-1055, 2007. 12. Baswa M, Rath , C .C, , Dash S .K, Mishra RK, Antibacterial compounds in neem, 2001;105(412):183-9. 13 .Supinya Tewtrakul, Supreeya Yuenyongsawad, Sopa Kummee and Latthya Atsawajaruwan, Chemical components and biological activities of volatile oil of Kaempferia galanga Linn, Songklanakarin, J. Sci. Technol., 2005, 27(Suppl. 2) : 503-507 .

36 14.Mahfuzul Hoque M.D., Bari ,M .L., Inatsu .Y ., Juneja V.K, Kawamoto S., Antibacterial activity of guava (Psidium guajava L.) and Neem (Azadirachta indica A. Juss.) extracts against foodborne pathogens and spoilage bacteria, 2007 Winter;4(4):481-8.

15. S. Mondal, S.C,. Mahapatra, B.R., Mirdha, S.N, . Naik , Antimicrobial activiries of essential oils obtained from fresh and dried leavesof Ocimum sanctum (L.) against enteric bacteria and yeast.

16. Nair R, Kalariya T, Chanda . S., Antibacterial activity of some plant extracts used in folk medicine., 2007;7(3-4):191-201.

17. Bishnu joshi , Sunil Lekhak, Anuja Sharma, Antibacterial Property of Different Medicinal Plants: Ocimum sanctum, Cinnamomum zeylanicum, Xanthoxylum armatum and Origanum majorana, Kathmandu university , J. of science, vol. 5, No. I, january, 2009, pp 143- 150. 143.

18. E. Nithya , R. Radhai , R. Rajendran, S. Jayakumar , K. Vaideki, Enhancement of the antimicrobial property of cotton fabric using plasma and enzyme pre-treatments , Volume 88, 15 April 2012, Pages 986991.

19.Tripathi, A., Chandrasekaran, N. Raichur, A.M., Mukherjee, A., Antibacterial Applications of Silver Nanoparticles Synthesized by Aqueous Extract of Azadirachta Indica (Neem) Leaves, Journal of Biomedical Nanotechnology, Volume 5, Number 1, February 2009 , pp. 93-98(6).

20. Priscila D. Alves, Maria G. L., Brando , Elzria A. Nunan, Cristina D. Vianna-Soares, Chromatographic evaluation and antimicrobial activity of Neem (Azadirachta indica A. Juss., Meliaceae) leaves hydroalcoholic extracts , Rev. bras. farmacogn.,2009, vol.19 no.2b.

21. Pooja Agarwal, L. Nagesh, Murlikrishnan , Evaluation of the antimicrobial activity of various concentrations of Tulsi (Ocimum sanctum) extract against Streptococcus mutans: An in vitro study , Indian journal of dental research, 2010 , Volume : 21 , Issue : 3 ,Page : 357-359.

37 22. Wiley Online Library ,Phytotherapy ResearchVolume 24, Issue 10, Article first published online: 1 JUN 2010. 23. Mekseepralard, Chantana, Kamkaen, Narisa , Antimicrobial and Antioxidant Activities of Traditional Thai Herbal Remedies for Aphthous Ulcers , 24 , October 2010, 1514-1519.

24. Hanumantharaju .N., Shashidhara. S . Rajasekharan .P., E ., Rajendra C. E ., comparative evaluation of antimicrobial and antioxidant activities of Kaempferia galanga for natural and micropropogated plant , 2010 ,Vol 2, Suppl 4. 25. Saradhajyothi Koona, Subbarao Budida, Antibacterial Potential of the Extracts of the Leaves of Azadirachta indica Linn , Not Sci Biol, 2011, 3(1)65-69. 26. Muhammad Ihtisham Uma r, Mohammad Zaini Bin Asmawi , Amirin Sadikun, Rabia Altaf and Muhammad Adnan Iqbal, Phytochemistry and medicinal properties of Kaempferia galanga L. (Zingiberaceae) extracts , African Journal of Pharmacy and Pharmacology Vol. 2011 ,5(14), pp. 1638-1647, 15 October.

27. Almas K , The antimicrobial effects of extracts Azadirachta indica (Neem) and Salvadora persica (Arak) chewing sticks, Indian J Dent Res ,1999,10:23-26.

28. Bandyopadhyay U, Biswas . K, Chatterjee R, Bandyopadhyay D, Chattopadhyay I, Ganguly C .K ., Chakraborty T, Bhattacharya K, Banerjee R .K., Gastroprotective effect of neem (Azadirachta indica) bark extract: possible involvement of H+-K+-ATPase inhibition and scavenging of hydroxyl radical. Life Sci, 2002 ,71:2845-2865. 29. Barry A .J ., Garcia F, Thrupp L .D ., Interpretation of sensitivity test results. American Journal of Clinical Pathology , 1970, 53:140.

30. Baswa M, Rath C .C., Dash S.K ., Mishra ,R .K ., Antibacterial activity of Karanj (Pngamia pinnata) and neem (Azadirachta indica) seed oil: a preliminary report, Microbios , 2001,105:183-189.

38 31. Bhomick B .N ., Choudhary B .K., Antifungal activity of leaf extracts of medicine plants on Alternaria alternata. Indian Botanical Reporter,1982, 1:164-165.

32. Chopra R.N., Nayar S.L., Chopra I.C., Glossary of Indian Medicinal plants, Council of Scientific and Industrial Research, New Delhi, 1956.

33. Fujiwara .T ., Takeka T. O., Gihara Y, Shimizu M, Nomura T, Tomuta Y , Studies on the structure of Polysaccharides from the bark of Medica Azardichta, Chemical Pharmaceu Bull , 1982 ,30:4025-4030.

34. Ganguli S , Neem: A therapeutic for all seasons. Current Science,2002, 82(11):1304.

35. Jagannadh J.H., Radhika. M , Antimicrobial emulsion (coating) based on biolymer containing neem (Melia azadirachata) and Turmeric (Curcuma longa) extract for wound healing,Biomed Mater Eng,2006, 16(5):329-336.

36. Jente .R, Jakupovic J, Olatunji G.A ., A Cembranoid diterpene from Cleome viscose, Photochemistry 1990,29(2):666-667.

37. Khattak S .G ., Gilani S .N., Ikram M , Antipyretic studies on some indigenous Pakistani Medicinal Plants, J Ethnopharmacol ,1985,14:45-51.

38. Nimri L.F., Meqdam M.M., Alkofahi A , Antibacterial activity of Jordanian medicinal plants, Pharmacological Biology ,1999,37(3):196-201.

39. Nostro A, Germano M.P., Angelo V.D., Marino A, Channatelli M.A., Extraction methods and bio-autography for evaluation of medicinal plant antimicrobial activity. Letters in Applied Microbiology ,2006,30:379-384.

39

40. Okpanyi S.N., Ezeukwk G.C , Anti-inflammatory and antipyretic activities of Azadirachta indica, Planta Medica ,1981,41:34-49.

41. Perez C, Paul Mand Bazeque P , Alta Biomedical Group Experiences,1990, 15:113.

42. Pillai N.R,, Santhakumari G , Toxicity studies on nimbidin, a potential antiulcer drug, Planta Medica,1984, 50:146-148.

43. Rao D.V.K., Singh K, Chopra P, Chabra P.C., Ramanujalu G , In vitro antibactericidal activity of neem oil ,Indian J Med Research , 1986, 84:314-316.

44. Rochankij S, Thebraranonth Y, Yenjal .C , Nimbolide a constituent of Azadirachta indica inhibits Plasmodium falciparum in culture. South East Asian J Trop Medi Public Health,1985, 16:66-72.

45. Sairam M, Ilanazhagan G, Sharma SK, Dhaniraj S.A., Suresh B, Parida M.M., Jana A.M., Devendra K, Selvamurthy W , Antimicrobial activity of a new vaginal contraceptive NIM-76 from neem oil (Azadirachata indica), J Ethnopharmacol ,2000, 71(3):377-382.

40

41

Satyavati GV, Raina MK, Sharma M (1976). Medicinal Plants of India. Vol. I.

Saxena K (1997). Antimicrobial screening of selected medicinal plants from India. J of Ethanopharmacology 58(2):75-83.

Shahidi Bonjar GH, Aghighi S, Kamiri Nik A (2004). Antimicrobial and Antifungal Survey in Plants used in Endogenous Herbal-Medicine of South East Regions of

Iran. Journal of Biological Sciences 4(3):405-412.Koona, S. and S. Budida / Not Sci Biol, 2011, 3(1):65-69 69

42

Subapriya R, Nagini S (2005). Medicinal properties of neem leaves: a review. Curr Med Chem Anticancer Agents 5(2):149-146.

Sultana B, Anwar F, Przybylski R (2007). Antioxidant activity of phenolic components present in barks of Azadirachta indica, Terminalia arjuna, Acacia nilotica, and Eugenia jambolana Lam. trees. J Food Chem 104:1106-1114.

Tella A (1977). The effects of Azadirachta indica in acute Plasmodium berghei malaria. Nigerian Medical Journal 7:258-263.

Thompson EB, Anderson CC (1978). Cardiovascular effects of Azadirachta indica extract. J Pharmaceu Sci 67:1476-1478.

Shukla R, Singh S, Bhandari CR (1973). Preliminary clinical trial on antidiabetic actions of Azadirachta indica. Medicine and Surgery 13:11-12.

Singh PP, Junnarkar AY, Reddi GS, Singh KV (2005). Azadirachta indica neuro-ycho pharmacological and antimicrobial studies. Fitoterapia 58:233-238.

43

Sinha KC, Riar SS, Tiwary RS, Dhawan AK, Bhadhan J, Thomas P, Kain AK, Jain RK (1984). Neem oil as a vaginal contraceptive. Indian J Med Research 79:131-136.

Srivastava A, Shukla Kumar YN (2000). Recent development in plant derived antimicrobial constituents- a Review. J Med Arom Pl Sci 20:717-72.

Stainer RY, Ingraham JL, Wheelis ML, Painter PR (1986). The relations between structure and function in prokaryotic cells, p. 145-182. In: Hall P (Eds.)

44