D i s s e r t a t i o n

ASSESSMENT OF ANTIMICROB IAL SUSCEPTIBILITY AND PHYTOCHEM ICAL SCREENING OF TERMINALIA CHEBULA FR UITSsubmitted in partial fulfillment of the requirement for the award of degree of Master of Science(Biotechnology)

byGargee Bareth Department of biotechnology Mahila PG Mahavidhyalaya Jodhpur affiliated to Jai Narain Vyas University Jodhpur, RajasthanResearch conducted at

Certifica te

This is to certify that Gargee Bareth is a regular student of M.Sc. Final Biotechnology 2011-2012 Mahila P.G. Mahavidhyalaya, Jodhpur. It is further certified that she has completed her project work at Centre for Microbiology and Bio-Technology Research and Training Institute Bhopal ,Madhya Pradesh.

Dr. Nishi Mathur Head of Department Biotechnology

Declaratio n

I hereby declare that the project work titled, Assessment of antimicrobial susceeptibility and phytochemical screening of Terminalia Chebula fruits, submitted to Jai Narain Vyas University, Jodhpur (Rajasthan), is a record of an original work done by me under the guidance of Abhishek Gupta institute, Bhopal, Madhya Pradesh. This project work is submitted in the partial fulfillment of the requirements for the award of the degree of Master of Science in Biotechnology. The results embodied in this thesis have not been submitted to any other University or Institute for the award of any degree or diploma. of the Centre for Microbiology and Bio-technology (CMBT) research and training

Gargee Bareth

Content s

Sections1.

Page6-12 13-24 24-29 25-39 41-56

Introduction

2. Methodology 3. Review of Literature 4. Conclusion 5. References

5

Introductio n

Man,

ev er desi rous of

kn owledg e,

has already explored many things, far the

but m re and great er still remains co ncealed; perhaps reserv ed for o work in remote pleasu re co untries and m ke a m ny a disco veries for

dist ant generations, who shall pros ecute the examination of their creators and co nvenience of life

(Carl Linnaeus, 1707-1778)

Ethnobotany, is totality, is virtually a new field of research, and if this field is invest igated thoroughly and sy st ematica lly, it will yield resu lts of great value to the ethn ologist s, arch aeologist s, anthropologist s, plantgeograph ers and ph armacologist s etc.

Tho ugh et hnob otany

provides

several approa ches in plant

research es,

here only the resources which help in medicinal are plant-research mentioned.

Concern has been expressed about the rising prevalence of pathogenic microorganisms which are resistant to the newer or modern antibiotics that have been produced in the last three decades worldwide (Cohen, 199 ; Nascimento et al, 2000). Coincidentally, the last decade has also witnessed increasing intensive

studies on extracts and biologically active compounds isolated from plant species used for natural therapies or herbal medicine (Nascimento et al, 2000; Rios and Recio, 2005). For over thousands of years, natural plants have been seen as a valuable source of medicinal agents with proven potential of treating infectious diseases and with lesser side effects compared to synthetic drug agents (Iwe et al, 1999). However, the problem posed by the high cost, adulteration and increasing side effects of synthetic drugs coupled with their inadequacy in diseases treatment found more especially in the developing countries cannot be over emphasized (Shariff, 2001).

Herbs are staging a comeback and herbal renaissance is happening all over the globe. The herbal products today symbolise safety in contrast to the synthetics that are regarded as unsafe to human and environment. Although herbs had been priced for their medicinal, flavouring and aromatic qualities for centuries, the synthetic products of the modern age surpassed their importance, for a while. However, the blind dependence on synthetics is over and people are returning to the naturals with hope of safety and security.

Over three-quarters of the world population relies mainly on plants and plant extracts for health care. More than 30% of the entire plant species, at one time or other, were used for medicinal purposes. It is estimated that world market for plant derived drugs may account for about Rs.2,00,000 crores. Presently, Indian contribution is less than Rs.2000 crores. Indian export of raw drugs has steadily

grown at 26% to Rs.165 crores in 1994-95 from Rs.130 crores in 1991-92. The annual production of medicinal and aromatic plants raw material is worth about Rs.200 crores. This is likely to touch US $1150 by the year 2000 and US $5 trillion by 2050.

It has been estimated that in developed countries such as United States, plant drugs constitute as much as 25% of the total drugs, while in fast developing countries such as China and India, the contribution is as much as 80%. Thus, the economic importance of medicinal plants is much more to countries such as India than to rest of the world. These countries provide two third of the plants used in modern system of medicine and the health care system of rural population depend on indigenous systems of medicine.

Of the 2,50,000 higher plant species on earth, more than 80,000 are medicinal. India is one of the worlds 12 biodiversity centres with the presence of over 45000 different plant species. Indias diversity is unmatched due to the presence of 16 different agro-climatic zones, 10 vegetation zones, 25 biotic provinces and 426 biomes (habitats of specific species). Of these, about 15000-20000 plants have good medicinal value. However, only 7000-7500 species are used for their medicinal values by traditional communities. In India, drugs of herbal origin have

been used in traditional systems of medicines such as Unani and Ayurveda since ancient times. The Ayurveda system of medicine uses about 700 species, Unani 700, Siddha 600, Amchi 600 and modern medicine around 30 species. The drugs are derived either from the whole plant or from different organs, like leaves, stem, bark, root, flower, seed, etc. Some drugs are prepared from excretory plant product such as gum, resins and latex. Even the Allopathic system of medicine has adopted a number of plant-derived drugs which form an important segment of the modern pharmacopoeia. Some important chemical intermediates needed for

manufacturing the modern drugs are also obtained from plants (Eg. diosgenin, solasodine, b-ionone). Not only, that plant-derived drug offers a stable market worldwide, but also plants continue to be an important source for new drugs.

Traditional systems of medicine continue to be widely practised on many accounts. Population rise, inadequate supply of drugs, prohibitive cost of treatments, side effects of several allopathic drugs and development of resistance to currently used drugs for infectious diseases have led to increased emphasis on

the use of plant materials as a source of medicines for a wide variety of human ailments. Global estimates indicate that 80% of about 4 billion population can not afford the products of the Western Pharmaceutical Industry and have to rely upon the use of traditional medicines which are mainly derived from plant material. This fact is well documented in the inventory of medicinal plants, listing over 20,000 species.

In spite of the overwhelming influences and our dependence on modern medicine and tremendous advances in synthetic drugs, a large segment of the world population still like drugs from plants. In many of the developing countries the use of plant drugs is increasing because modern life saving drugs are beyond the reach of three quarters of the third worlds population although many such countries spend 40-50% of their total wealth 4 on drugs and health care. As a part of the strategy to reduce the financial burden on developing countries, it is obvious that an increased use of plant drugs will be followed in the future.

Among ancient civilisations, India has been known to be rich repository of medicinal plants. The forest in India is the principal repository of large number of medicinal and aromatic plants, which are largely collected as raw materials for manufacture of drugs and perfumery products. About 8,000 herbal remedies have been codified in Ayurveda. The Rigveda (5000 BC) has recorded 67 medicinal plants, Yajurveda 81 species, Atharvaveda (4500-2500 BC) 290 species, Charak Samhita (700 BC) and Sushrut Samhita (200 BC) had described properties and

uses of 1100 and 1270 species respectively, in compounding of drugs and these are still used in the classical formulations, in the Ayurvedic system of medicine. Unfortunately, much of the ancient knowledge and many valuable plants are being lost at an alarming rate. With the rapid depletion of forests, impairing the availability of raw drugs, Ayurveda, like other systems of herbal medicines has reached a very critical phase. About 50% of the tropical forests, the treasure house of plant and animal diversity have already been destroyed. In India, forest cover is disappearing at an annual rate 1.5mha/yr. What is left at present is only 8% as against a mandatory 33% of the geographical area. Many valuable medicinal plants are under the verge of extinction. The Red Data Book of India has 427 entries of endangered species of which 28 are considered extinct, 124 endangered, 81 vulnerable, 100 rare and 34 insufficiently known species (Thomas, 1997).

Terminalia chebulla belongs to the familyCombretaceae. commonly known as belleric myrobalan. It is routinely used as traditional medicine by tribal folk of visakhapatnam district, to get remedies from several ailments such as fever, cough, diarrhea, skin diseases and oral thrush. Chemical substances of sitosterol, gallic acid, ethyle gallate, galloyl glucose, a new triterpene, the belleric acid and chebulagic acid have been isolated from fruits of T. bellerica. Fruit extract of T. bellerica produced fall in blood pressure of rats at a concentration of 70 mg/kg body weight (5). But reports on antimicrobial activity of T. bellerica were scanty, particularly on these strains of microorganisms and their biochemical processes. Therefore an attempt has been made to study the antimicrobial activity

of the crude and methanol extract of T. bellerica dry fruit on certain pathogenic microorganisms.

Terminalia chebula is also known as Haritaki. Many big trees (more than 100 species) belong to the genus Terminalia; they are found in different tropical regions of the world, and all of them belong to the family Combretaceae. The genus of this plant comes from the Latin word terminus, as the leaves of the tree are located at the end of the branches.

The trees that belong to this genus are known as a good secondary source of metabolites: Cyclic triterpenes and derivatives, flavonoids, tannins and other aromatic metabolites. Some of these substances are antifungal, antibacterial, anticarcinogen and hepatoprotective.

Te r m i n a l i a c h e b u l a

Systematic position Kingdom: Plantae. Division: Magnoliophyta. Class: Magnoliopsida. Order: Myrtales. Family: Combretaceae. Genus: Terminalia. Species: chebula Rtz.

Fig. 1: Terminalia chebula fruit

The major bio-active constituents of the fruit are tannins, anthraquinones, chebulinic acid, chebulagic acid, chebulic acid, ellagic acid and gallic acid. The other minor compounds include corilegin, -D-glucogallin, glucose and sorbitol. Polyphenolic compounds, triterpene glycosides, terchebulin, punicalagin, terflavin A, flavonoids, reducing sugars and starch are other constituents of the fruit. Terpenene glycosides, arjungenin and arjunglucoside-I. 18 amino acids and a small quantity of phosphoric, succinic, syringic and quinic acids.

Geographic Distribution:Terminalia Chebula as a species of terminalia, native to southern Asia from India and Nepal east to southwestern China and south to sri Lanka, Malaysia and Vietnam. In M.P. it is mainly found in the Districts of Jabalpur, Betul, Bhopal, Gwalior, Sagar, Ujain.

Macroscopic identification:A large tree, young branchlets, leaf buds, and leaves with long, soft, shining, rust colored, sometimes silvery hair. Flowers are dull, white or yellowish in color with a strong offensive smell. Fruits are ovoid, wrinkled and ribbed longitudinally.

Botanic Description:It is deciduous tree growing to 30 m tall, with a trunk up to 1 m diameter. It has rounded crown and spreading branches. The bark is dark brown with some longitudinal cracks. Leaves are ovate and elliptical, with two large glands at the top of petiole. The fruit or drupe is about 1-2 inches in size. It has five lines or five ribs on the outer skin. Flowers bisexual, white or yellow with an offensive Fruit is green when unripe, and yellowish grey when ripe. Fruits are collected from January to April. Fruit formation starts from November to January.

Parts Used:Dried fruits, immature fruits, mature fruits myrobalans and galls, mostly the outer skin of fruits.

Preparations:Powder, paste, and decoction

Medicinal Value:This tree yelds smallish, ribbed and nut-like fruits which are picked when still

green and then pickled, boiled with a little added sugar in their own syrup or used in preserves or concoctions. It is regarded as a universal panacea in the Ayurvedic Medicine and in the Traditional Tibetan medicine. It is reputed to cure blindness and it is believed to inhibit the growth of malignant tumors. The dry nuts peel is used to cure cold-related nagging coughs. Its fruit has digestive, anti- inflammatory, anthelmentic, cardiotonic, aphrodisiac and restorative properties and is additionally beneficial in flatulence, constipation, piles, cough and colds. It is used extensively in the preparation of many Ayurvedic formulations for infectious diseases such as chronic ulcers leucorrhoea, pyorrhea and fungal infections of the skin, it is used to prevent aging and impart longevity, immunity and body resistance against disease.

Pharmacological action :Alterative, astringent, expectorant, anti-inflammatory, anodyne, cardio tonic, aperients, antiseptic, anti pyretic, anti emetic, tonic.

Chemical Composition:-

Fruits contains tannin up to 30 tannic acid, chebulic acid3-5%, chebulinic acid 30%, ellagotannic, gallic acid, ellagic acid etc. Resin and a purgative principle of the nature of anthraquinone and sennoside are also present.

Extraction:Various extracts have been prepared from the powdered fruits. It contains a constituent which ahs a wide anti- bacterial and antifungal spectrum it is very effective against various human pathogens.

Aim and objectives:Keeping in mind the medicinal importance of terminalia chebula were analyzed with the following objectives:

Petroleum ether extraction and Hydroaceton extraction and Phytochemical estimation of Terminalia chebula seed.

To study antimicrobial activity of extract.

Comparative analysis between pet ether extract and hydroaceton extract against pathogenic bacteria.

Methodolo gy

1. EXTRACTIONREQUIRMENTS: terminalia chebula fruit. Grinder Methanol Beaker Water bath Hot air oven Acetone Distilled water Soxhlet apparatus

1.1

Hot ExtractionSolvent Methanol/Acetone

Roots of carica papaya were collected locally from Bhopal (M.P.), India. Roots were washed; air dried under shade and powdered with the help of Grinder

at PBRI, Bhopal.

Powdered root were weighed and packed in soxhlet. Solvent used for soxhletion was mixture of methanol and acetone in the ratio of 70:30 respectively. Extraction was continued at the temperature of 50C till clear solvent was observed in siphon tube. Extract was concentrated in water bath at 40C. Concentrated extract was dried at 40C in Hot air oven. Dried extract was powdered and packed in an air tight container.

2. INVESTIGATION

PHYTOCHEMICAL

Requirements: Acetic acid, Calcium oxalate, Mercuric chloride, Potassiumiodide, Mercury, Nitric acid, - Napthol, Butanal, Phloroglucin, Hydrochloric acid Tannic acid, Sodium chloloride, Sulphuric acid, Cobalt chloride Sodium hydroxide, Copper sulphate, Lead acetate, Cystine, Chloroform, Glacial acetic acid, Gelatine, Ferrous sulphate, Methanol, Acetone, Sodium hydroxide, Calcium Choloride, etc.

Table 1 S . N o . 1. A . Test for carbohydrate Molish test To 2.3 Ml. aqueous extract, added few drops of napthol solution in alcohol. Shake and Violet ring of formed at the junction of two liquids B . 1 ) C . 1 ) Test for pentose sugar Mix equal amount of test solution and HCl. Heated added a crystal of pholoroglucinol Test for Hexose sugars Tollen's phoroglucinol test for galactose Mix 2-3 ml conc. HCl and 4 ml 0.5% phloroglucinol add 1.2 ml test solution heated. b Cobalt chloride test Yellow and red color appear Red color appears Test Observation

add conc. H2SO4 from sides of the test tube.

)

Mixed 3 ml test solution with 2 ml. cobalt chloride boil and cool add few drops NaOH solution

Solution appears greenish blue

D . 1 ) 2 ) 2. a ) b )

Test for on-reducing polysaccharides (starch) Iodine test Mix 3 ml test solution and few drop of dilute iodine solution. Tannic acid test for starch with 20% tannic acid, test solution` Test for protein Biuret test To 3 ml test solution add 4% NaoH and few drops of 1% CuSO4 solution Millions test Mixed 2ml. test solution million reagent. Violet color appears White ppt. wash ppt turn brick red. White ppt is formed Solution turn black or brownish Blue color appears PPT

c ) d )

Xanthoprotein test Mixed 3 ml. test solution with 1 ml. conc.H2SO4 Test for protein containing sulphur Mixed 5 ml test solution 2 ml 40% NaOH and 2 drops 10% lead acetate solution boiled.

3.

Test for Amino acid Test for cysteine

Black ppt of

To

5 ml. test solution add few drops of

lead sulphate in formed

40% NaoH and 10% lead acetate and 4. solution boiled. Test for steroid Salkowski reaction To 2 ml. of extract added 2 ml. chloroform and 2 ml conc. H2SO4 shake well.

Chloroform layer appear red and acid layer show greenish yellow fluorescence. Reddish brown color appears at junction of the two liquid layer and upper layer appears bluish green Separate the organic solvent added ammonia.

5.

Test for glycosides Test for deoxysugar (Keller - Killiani test) To 2ml extract added glacial acetic acid. One drops 5% fecl3 and conc. H2SO4.

6. 1 )

Test for anthroquinone glycosides Borntrager's test for anthroquione glycoside To 2ml. extract added dil. H2SO4 boiled and filter 2 cooled filtrate added equal volume benzene or chloroform shake well.

Ammonia cal layer turn pink and 2 ) Modified C.glycoside To 2 ml extract added 5% fecl3 and 5 ml. dilute HCL. Heated for 5 min. in boiling after water bath cooled and added benzene or any organic solvent shake well. Borntragers test for read. Separate organic solvent layer add equal volume dilute ammonia ammonical layer show pinkish red color. 7. 1 ) 2 ) Test for Flavonoids To small quantity of reduce added lead acetate solution. Addition of increasing amount of sodium hydroxide to the residue show. Yellow color ppt. in formed Yellow coloration which decolourism after addition of acid 8. 1 Test for alkaloids Mayer's test Ppt

) 2 ) 9.

2.3 ml. filtrate with few drops Mayer's reagent. Wagner's test 2-3 ml. filtrate with few drops Wagner's reagent Test for Tannic and phenolic compound To 2-3ml. of aqueous or alcoholic extract, added few drops of following reagent. 5% Fecl3 solution Lead acetate solution Gelatin solution Acetic acid solution Dilute iodine solution Dil. potassium per magnate solution Test for organic acid Confirmatory test for oxalic acid To 2ml. test solution added few drops 5% add few drops 5% lead acetate. To 2ml. test solution and few drops 1% KMnO4 and dilute H2SO4 White ppt. forms, Color of KMnO4 disappear immediately deep blue black color White ppt. White ppt. red color solution red color Discoloratio n reddish brown ppt.

1 ) 2 ) 3 ) 4 ) 5 ) 6 ) 10. 1 ) a ) b )

2

confirmatory test for malic acid

) 2-3 ml test solution added 2-3 drops 40% 11. 1 ) a ) 12. Test for chloride To about 5 to 7 ml filtrated added 3 to 5 ml. lead acetate solution. Fecl3 solution Test for inorganic acid Test for sulphate With lead acetate reagent gives appears yellowish

White ppt. soluble in NaOH White ppt soluble in hot water in observed

13. 1 ) 2 )

Test for Carbonate With dilute acid liberate with mercuric chloride solution CO2 Produces a brownish red ppt. white ppt in formed

14.

Test for nitrate With solution of ferrous sulphate yield no brown color but if sulphuric acid in added.

Brown color is produced at the junction of two liquid

15. 1

Test for Coumarins Glycosides Coumarin glycosides have aromatic odour

) s 2 ) 3 )

Alcoholic extract when made alkaline

Shows blue or green fluorescence It shows yellowish green fluorescence

Take moistened dry powdered in test tube cover test tube with filter paper soaked in dilute NaOH keep in water bath after sometime expose filter paper to U.V. light.

3. Antimicrobial Activity of Extract Microbial StrainsS. aurious P. aroginosa Aspergillus niger Candida albicans

RequirementsNutrient Broth Distilled water Laminar Air Flow Incubator Petri plate (sterilized) Test tubes (sterilized) Micropipettes Hole puncher(sterilized)

3.1 Well diffusion method

The agar well diffusion method technique (Bauer, et al 1966) was used to determine the antibacterial activity of the plant extracts. 1ml of the different standardized organisms were introduced separately and thoroughly mixed with 30ml of molten nutrient agar each in a sterile Petri dish and allowed to set then labeled. A sterile 8mm cork borer was used to punch hole. The wells that were formed were filled with different concentrations of the extract which were labeled accordingly; 50mg/ml, 37.5mg/ml, 25mg/ml, 12.5mg/ml. The plates were then left on the bench for 1 hour for adequate diffusion of the extracts and incubated at 37C for 48hours in upright condition. After incubation, the diameter of the zones of inhibition around each well were measured to the nearest millimeters along two axis i.e. 90 to each other and the mean of the two reading were then calculated.

3.2 Disc diffusion methodThe disc diffusion method (Bauer et al., 1966) was used to test antimicrobial activity of the extractives against 6 bacteria. Solutions of known concentration (g/ml) of the test samples were made by dissolving measured amount of the samples in calculated volume of solvents. Dried and sterilized filter paper discs (6mm diameter) were then impregnated with known amount of the test substances using micropipette and the residual solvent were completely evaporated. Discs containing the test material were placed on nutrient agar medium uniformly seeded with the test microorganisms.

Standard disc of kanamycin (30g/disc) and blank discs (impregnated with solvent followed by evaporation) were used as positive and negative control, respectively. These plates were then kept at low temperature (4C) for 24 h to allow maximum diffusion. These were a gradual change of test material concentration in the media surrounding the discs. The plates were then incubated at 37C for 24 h to allow maximum growth of the organisms. The test material having antimicrobial activity inhibited the growth of the microorganisms and a clear, distinct zone of inhibition was visualized surrounding the medium. The antimicrobial activity of the test agents was determined by measuring the diameter of zone of inhibition expressed in millimeter.

Review of LiteratureGhaisas et.al (2010) experimented on ethanolic extract of stem bark of bauhinia variegate which shows immunomodulatory activity . ethanolic extract of the effect of stem bark of bauhinia variegate on the primary and secondary antibody responses was evaluated by the humoral antibody response for a specific immune response . the effect of ethanolic extract of stem bark of bauhinia variegate on the phagocytic activity was evaluated by the carbon clearance test and neutrophils adhesion test for a non specific immune response.teh data was analysed by one way ANOVA followed by multiple comparison tests . Sharfifar et.al (2009) studied the immunomodulatory activity of aqueous extract of achillea whilhemsti c.koch in mice.they evaluated immunomodulatory activity of aqueous extract of achillea whilhemsti on body weight ,relative organ weight, delayed type of hyper sensitivity and haemagglutination titer in female swiss albino mice. In a result ,achillea whilhemsti showed a stimulatory effect on both humoral and cellular immune functions in mice. kim et.ai (2009) studied immunomodulatory activity of ginsan , a polysachharide of paax ginseng on dendritic cells .according to their experiment ginseng had little effect on production by dendritic cells of IL-12 and TNF-,as measured by ELISA. to examine the maturation of inducing activity of ginseng,they

measured the surface expression levels of the maturation markers MHC class on dendritic cells . Chantima et.al (2009)did scanning electron microscopic study of in vitro effects of craica papaya linn on the death and the tengumental surface of the trematode. they treated worms with aqueous extracts at different concentrations .the SEM( scanning Electron Microscopy ) observation on the tegumental surfaces of the death worms treated the aqueous extracts showed loss of spines specifically around that oral sucker and posterior of the body , curving at the edge spines ,blebing and rupturing at the body surface. Ayutunde et.al (2008)studied papaya) seed acute and chronic toxicity of pawpaw(carica to Nile tilapia oreochromis niloticus

powder

(linne1757)fingerlings.according to them toxicity reaction exhibited by the fish includes erratic movement , air gulping , loss of reflexe, discoloration ,molting, loss of cale and haemorrage.results of the tests provided baseline information and established safe limits of using carica papaya seed powder as an antifrtility agent in controlling excessive breeding of tilapia in fish arm.

Rani and Khullar (2007) worked on Antimicrobial evaluation of some medicinal plants for their anti-enteric potential against multi-drug resistant Salmonella typhi. Screening was done of some plants of importance in the Ayurvedic system of traditional medicine used in India to treat enteric diseases. Fifty four plant extracts (methanol and aqueous) were assayed for their activity against multi-drug

resistant Salmonella typhi. Strong antibacterial activity 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). Moderate antimicrobial activity was shown by Picorhiza kurroa, Acacia catechu, Acacia nilotica, Cichorium intybus, Embelia ribes, Solanum nigrum, Carum copticum, Apium graveolens, Ocimum sanctum, Peucedanum graveolens and Butea monosperma.

Lu et.al (2007) reported immunomodulatory activity of aqueous extract of actinidia macrosperma . immunomodulatory activity of actinidia macrosperma were examined using positive group normal group and drug treated group .body weight , tumor weight , humoral, cellular and non specific immune responses were determined by relative assays . Fakeye et.al (2007) carried out this experiment and observed that effects of coadministration of extract of carica papaya linn on activity of two oral hypoglycemic agents.in this work they investigate activity of metforin and glimpiride in an animal model .Experimental factorial design was used to evaluate the individual and interaction influfences of three variables i.e. nature (N), dose administered (C)and duration of administration employed on blood glucose of diabetic rats on administration of ethanolic extract of carica papaya and two to test for glycemic agents , metforin and gimipiride.unpaired t-test was used significant differences due to the administration of the combination.

Elmahmood et.al (2007)l studied on the antibacterial activity of root extracts of carica papaya L.they extracted the bioactive compounds of root extracts of carica papaya l. using water and organic solvents , and were investigated for antibacterial against some pathogenic bacteria using the cup plate agar diffusion method .the minimum inhibitory concentration (MIC)and minimum bactericidal concentration (MBC) were observed. Thakur et.al (2006)studied immunomodulatory activity of chlorophytam borivilianum sant .f.. In their experiment they used ethanolic extract root and its sapogenin .they determined effect of azathioprene induced myelosupression and administration of extracts on hematological and serological parameters .they observed in vivo phagocytosis by carbon clearance method was after treatment with extracts. Tan et.al (2004) experimented on immunomodulatory and antibacterial effects of some traditional Chinese medicinal herbs .they reviewed reports on seven Chinese herbs which had shown their immunomodulatory and antimicrobial activities .while some of these herbaceous plants have a direct inhibitory effect on microbial organisms , they observed that each plant has at least one compound that selectively modulates of cells of the immune system .the successful derivation of pure bioactive compounds supports the traditional practice of using these plants to stimulants the immune system. Almora et.al (2004)worked on evaluation of volatiles from ripening papaya . in this experiment they investigated in four on the chemical composition of

papaya ,especially regarding volatile components analyses. Payan et.al (2004)l reported that

in four ripening

stages

.ripening was characterized sensorialy ,as well as through physical and chemical

papaya transplant growth is affected by a

trichoderma based stimulator . they conducted this research work to determine the effect of a trichoderma based plant stimulator on papaya seeding growth papaya seeding were grown from seed in Styrofoam peat moss . trichoderma based stimulator was applied as a drench evaluated at the different rates the variable were time from sowing at seedling emergence, shoot height ,

concentration of nitrogen in nitrate and potassium in leaf sap, leaf area , root dry weight , shoot dry weight ,and time from emergence to readiness for transplanting. Daswani et. al (2002) reported immunomodulatory activity of septilin,

polyherbal preparation .this study was under taken to evaluate the effect of septilin on different arms of the immune system .the experimental animals were evaluated for immunological function by studying weight gain, resistance against E .coli sepsis, haemogram, phagocytic activity of PMN cells and reticuloendothelial system, delayed hypersensitivity to oxazolne and the plaque forming cells response of splenic cells lymphocytes to sheep erythrocytes. Desai et.al (2002) reported the immunomodulatory activity according to earlier studied of tinospora

cardifolia With antioxidant activity in cell free system .they explained that dry stem crude extract of tinospora cardifolia contained poly clonal B cell mitogen, G1-4A . dry stem crude also enhanced

immune response in mice. In order to explore the possibility of using G1-4A/ partially purified immunomodulator to modulate radiation from this plant was examined against reactive oxygen induced and nitrogen immunosupression ,the antioxidant effect of partially purified immunomodulator species,generated by photosensitization/peroxynitrate. Haung et.al (2002)experimented on immunomodulatory effects and mechanisms of palnt alkaloid tetrandrine in autoimmune disease.they used several combination of disease modifying antirheumatic drugs to treat the immune effector cells and damaged target organs which are characterized by autoimmune diseases.such a combination treatment strategy not only provides synergistic effects but also reduces side effect from individual drug.

Conclusio n

1. Extraction Yeild:Extraction Yeild for seed was found to be higher for hydroaceton Extract. The total % of yield for soxhlet (solvent-300ml) and maceration (solvent- 500ml) was (soxhlet- 13.33 %) and (maceration- 7.2%) respectively. The total % of yield was as follow Table no. 1 S . N o . 1 . Table no. 3 2. Phytochemical investigation The results of phytochemical screening reveals alkaloids steroids and saponins were found to be present in hydroalcoholic extracts of fruit of T.chebula (Table 2) Hydroaceton Extract 13.33 Extraction % yeild

A Phytochemical Compound Alkaloids Flavonoids Saponins Steriods Glycosids Hydroaceton Extract + + + + +

B

Figure 4: Zone of Inhibition shown by hydraceton extract of T.chebula against S.aureus and P. aroginosa. A. S. aureus B P.aroginosa. C D

Figure. 4: Zone of Inhibition shown by hydraceton extract of T.chebula on A.niger and C.albicans. C. A.niger D C.albicans. 3. Antibacterial Activity Zone of inhibition were tested for concentration ranging from 25mg/ml to 100mg/ml (25mg/ml, 50mg/ml, 75mg/ml, 100mg/ml) and all extract exhibited antibacterial activity against strains. The zone of inhibition for different extract is reported in Graph no. - 4 The strongest zone of inhibition was shows against the Styphylococcus aurius for hydroaceton extracts at 17mm (100mg/ml) and P.aroginosa for Hydroaceton extract at 10mm (50mg/ml).

33

Table no. 4 Concentration of Extract 1 0 0 m g / Microbial Strain 25m g/ml 50mg /ml 75mg/ ml m l 1 7 0 . 3.35 S.aurius P.aroginosa 1.25 0.47 5.20 .40 2.50 .40 7.750. 64 3.250. 28 4 0 1 0 . 3 7 0

0.28

. 2 5

4. Antifungal Activity Zone of inhibition were tested for concentration ranging from- to- (25mg/ml, 50mg/ml, 75mg/ml, 100mg/ml) and all extract exhibited antifungal activity against strains. The zone of inhibition for different extract is reported in Graph no. - 5 The Extract was found to be active against gram positive bacteria. The strong as inhibition were obtained against the A.niger for Hydroaceton extracts at 5.87 mm (100 mg/ml) and Candida albicans for Methenol:Acetone at 11.12mm (100mg/ml).

Table no. 5 Concentration of Extract 1 0 0 m g / Microbial Strain 25m g/ml 50mg/ ml 75mg/ ml m l 5 . 8 7 0 . Aspergillu s nige r Candid a albican 10.4 0 s 3.370 .47 3.37 0.47 30.64 6.370 .47 8 5

8.620 1

. 4 7

. 1 1

2 0 . 6 2

List of Graph Graph no. 1 Graph no. 2 India recently increased research in Traditional Herbal Medicines following scientific confirmation of their effectiveness in treating conditions for which they were traditionally prescribed. The present investigation has explored the use of one such plant, T.chebula, endemic in the whole India, for treating infectious diseases. The antimicrobial and antifungal screening showed interesting activities at all concentrations: Every extract was active. Plant extracts are generally a crude mixture of no active and active compounds and their Zone of inhibition must be interpreted accordingly. The authors noted that Zone of inhibition of less than 50mg/ml should be interpreted as strong antifungal potential. Zone of inhibition obtained in this study are within the range of what is considered significant for plants and even purified extracts. The study clarified a complex ethnopharmacological picture in terms of

extraction. Firstly, results showed the importance of extraction, the method of extraction can also change the results. For the antimicrobial activity, the plant extracts were active against both Gram-positive and Gram-negative bacteria. Activity depends from the bacterial strain, the plant organ, their maturation state and the nature of the extraction. Styphylococcus cohni the microorganism against which your extracts were most active should be correlated to disease cause by that microorganism Osteomyelitis. Activity cannot be imputed to one family of phytochemical only (or its absence). Alkaloids are commonly found to have antimicrobial properties; therefore their presence in roots could account for the activity of this organ. However, alkaloids cannot be solely responsible for the activity. Flavonoids (detected in all the seeds extracts) are known to be synthesized by plants in response to microbial infection (Fogliani et al., 2005); therefore their potential in vitro antimicrobial effectiveness against a wide array of microorganisms should not come as a surprise. The same could be said about most phytochemicals detected by the qualitative chemical analysis and known from works in other plant species to have antimicrobial pharmacological activity Concerning the antifungal activity, the present study showed zone of inhibition significant for crude plant extracts against all strains. The potent activity against candida albicans is particularly welcomed due to the fungus frequency to cause infections, with most of clinical fungal infections due to this strain. The equally strong activity against aspergillus niger opens the possibility to use the extracts either alone or in combination with other antifungal drugs. New active drugs are

particularly needed as triazoles are generally met with resistance and the few alternative treatments, such as Caspofungin, are limited by their low oral bioavailability. Like for the antimicrobial activity, the antifungal activity may be attributed, possibly in combination, to various phytochemicals detected during the extracts chemical screening and which are known to cause damage to cell membranes, causing leakage of cellular materials and ultimately the microorganisms death.

Conclusion: Further studies are required for isolation of active components from the fruit extract and to confirm the mechanism of action of the extract that may be responsible for the antimicrobial activity of extract. A detailed study on dose selection is also a parameter that has to covered in further studies. With all these wide spectrum antibacterial and antifungal properties, Terminalia chebula fruit. can be considered an effective antimicrobial agent to treat infectious diseases. This plant demonstrated activity against some bacteria and fungi prevalent in dermatology, abdominal and pulmonary infections. The study supported scientifically the ethnopharmacological use of the plant as an antimicrobial and antifungal agent and could account for some of the variations observed in the ethnopharmaceutical preparation methods. Therefore, the use of

this plant as antimicrobial agent is validated by the results obtained in this work. Further studies are ongoing to identify the chemical compounds of these antimicrobial extracts.

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