PHYTOCHEMICAL ANALYSIS AND ANTIBACTERIAL ACTIVITY OF

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ISSN 2249-9717 Original Article PHYTOCHEMICAL ANALYSIS AND ANTIBACTERIAL ACTIVITY OF AZADIRACHTA INDICA A JUSS. B. VINOTH, R. MANIVASAGAPERUMAL* AND M. RAJARAVINDRAN. Department of Botany, Annamalai university, Annamalai Nagar 608 002, Tamilnadu, India *Corresponding author Email:[email protected] Received 22 August 2012; accepted 06 September 2012 Abstract Azadirachta indica A Juss. is a very useful traditional medicinal plant in the sub-continent and each part of the tree has some medicinal properties. The plant is native to Asia, but has now naturalized in West Africa and is widely cultivated in Nigeria as an ornamental as well as medicinal plant. Fresh leaves of the plant were collected, dried, homogenized and extracted using 95% Ethanol, Methanol and Acetone. Phytochemical analysis gave positive results for steroids, triterpinoids, reducing sugars, alkaloids, phenolic compounds, flavonoids and tannins. This study aimed at screening the active components and the antibacterial effects of the Ethanol, Methanol and Acetone. Leaf extract of Azadiracta indica contains pharmacologically active constituents that may be responsible for its activity against P. aeroginosa, S. aureus, E. coli and S. typhi. Therefore, the use of Neem plant in our community for treating diverse medical ailments especially infectious diseases is highly justified. © 2011 Universal Research Publications. All rights reserved Key words: Azadiracta indica, Phytochemical analysis, Antibacterial, Gram Pasitive and Negative Bacteria. (Mahesh and Satish et al., 2008). Almost every part of the INTRODUCTION Azadirachta indica (Meliaceae) commonly known tree is bitter and finds application in indigenous medicine. as neem is native of India and naturalized in most of Neem extract has been reported to have antidiabetic, tropical and subtropical countries is of great medicinal antibacterial and antiviral activity (Kirtikar and Basu, value and distributed wide spread in the world. The 1987). Almost every part of the tree has been in use since Chemical constituents contain many biologically active ancient times to treat a number of human ailments and compounds that can be extracted from neem, including also as a household pesticide. The extract from bark, alkaloids, flavonoids, triterpenoids, phenolic compounds, leaves, fruits and root have been used to control leprosy, Carotenoids, steroids and ketones. Azadirachtin is actually intestinal helminthiasis and respiratory disorders in a mixture of seven isomeric compounds labeled as children (Chattopadhyay etial., 1993). Flavonoids, flavonoazadirachtin A-G and azadirachtin E is more effective glycosides, dihydrochalocones, tannins and others are also (Verkerk et al., 1993). Other compounds that have a important constituents of bark, leaves, fruits and flowers of biological activity are salannin, volatile oils, meliantriol neem. The biological activities and medicinal properties of and nimbin (Jacobson et al., 1990; Ahana et al., 2005). neem have recently been reported (Venugopal and Neem leaf is effective in treating eczema, ringworm, acne, Venugopal, 1994). anti-inflammatory, antiheperglycemic properties and it is Natural drugs have been a part of the evolution of used to heal chronic wounds , diabetic food and gangrene human, healthcare for thousands of years. Nowadays nearly developing conditions. It is believed to remove toxins from 88% of the global populations turn to plant derived the body, neutralize free radicals and purify the blood. It is medicines as their first line of defence for maintaining used as anticancer agent and it has hepato-renal protective health and compacting diseases. One hundred and nineteen activity and hypolipidemic effects (Fitoterapia part I and secondary plant metabolites derived from plants are used part II). globally as drugs, 15% of all angiosperms have been Medicinal plants have been found useful in the investigated chemically and of that 74% of cure of a number of diseases including bacterial diseases. pharmacologically active plant derived components were Medicinal plants are a rich source of antimicrobial agents discovered (Raja and Ramanathan, 2009). Plants are rich in

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a wide variety of secondary metabolites such as tannins, terpenoids, alkaloids, flavonoids, etc. which have been found In-vitro to have medicinal properties. Pharmacological studies have accepted the value of medicinal plants as potential source of bioactive compounds (Biswas and Chattopadhyay 2002). Phytochemicals from medicinal plants serve as lead compounds in antimicrobial discovery (Chakravarthy et al., 1985; Ebi et al., 2000; and Cohen et al., 2002). MATERIALS AND METHODS Collection of plant materials: The experiment was conducted in the year 2011 in the college laboratory. Leaves were collected from the Azadirachta indica tree in the college campus. It was ensured that the plant was healthy and uninfected. The leaves were washed under running tap water to eliminate dust and other foreign particles and to clean the leaves throughly and dried. Preparation of leaf extracts: 20-30 grams of fresh leaves were boiled with 200 mL of solvent for 1 hour. The extract was filtered using Whatmann filter paper No. 1 and then concentrated in vacuum at 40°-50°C using a rotary evaporator. Evaporation of solvent in the rotary evaporator affords a crude extract of the soluble components and these extracts were subjected to the qualitative phytochemical analysis and antibacterial studies. Phytochemical Analysis: The extracts were analyzed by the following procedures (Talukdar and Choudhary 2010). To test for the presence of the alkaloids, saponins, tannins, Terpenoids, flavonoids, glycosides, volatile oils and reducing sugars Saponins: Saponins were detected using the froth test. 1g of the sample was weighed into a conical flask in which 10ml of sterile distilled water was added and boiled for 5 minutes. The mixture was filtered and 2.5 ml of the filtrate was added to 10ml of sterile distilled water in a test tube. The test tube was stoppered and shaken vigorously for about 30 seconds. It was then allowed to stand for half an hour. Honeycomb froth indicated the presence of saponins. Tannins: To a portion of the extract diluted with water, 3-4 drops of 10% ferric chloride solution is added. A blue color is observed for gallic tannins and green color indicates for catecholic tannins. Reducing Sugars To 0.5ml of plant extracts, 1ml of water and 5-8 drops of Fehling’s solution was added and heated over water bath. Brick red precipitate indicates the presence of reducing sugars. Glycosides: 25ml of dilute sulphuric acid was added to 5ml extract in a test tube and boiled for 15 minutes, cooled and neutralized with 10%NaOH, then 5ml of Fehling solution added. Glycosides are indicated by a brick red precipitate. Alkaloids: 2ml of extract was measured in a test tube to which picric acid solution was added. An orange coloration indicated the presence of alkaloids.

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Flavonoids: 4ml of extract solution was treated with 1.5 ml of 50% methanol solution. The solution was warmed and metal magnesium was added. To this solution, 5-6 drops of concentrated hydrochloric acid was added and red color was observed for flavonoids and orange color for flavones. Volatile oils: 2ml of extract was shaken with 0.1ml dilute NaOH and a small quantity of dilute HCl. A white precipitate is formed if volatile oils are present. Terpenoids: Four milligrams of extract was treated with 0.5 ml of acetic anhydride and 0.5 ml of chloroform. Then concentrated solution of sulphuric acid was added slowly and red violet color was observed for terpenoid. Ethanol Extract: Azadirachta indica leaves (100 g) were ground into fine powder (Himal Pauel Chhetri et al., 2008) using a stainless-steel grinder, and deep in100% ethanol (200 mL) for overnight. The ethanol fraction was separated using sterile muslin cloth and filter through sterile Whatman filter paper (no. 02). The filtered extract was concentrated by a rotary film evaporator. Acetone Extract: For preparation of Acetone extract ground plant sample (100 g) was added in Acetone respectively (200ml each case) and left for overnight at room temperature (Puri et al., 1995). The extracts were separated using sterile muslin cloth and filter through sterile Whatman filter paper (no. 02). Methanol Extract: Ten grams of dried plant material was extracted with 100 ml of methanol kept on a rotary shaker for 24 h. Thereafter, it was filtered and centrifuged at 5000 g for 15 min. The supernatant was collected and the solvent was evaporated to make the final volume one-fifth of the original volume (17). It was stored at 4°C in airtight bottles for further studies. Source of microorganisms: The organisms used were Escherichia Coli, Pseudomonas aeroginosa, Staphylococcus aureus and Salmonella typhii. The organisms were obtained from the Microbial Lab of Department of Microbiology, A.V. C. College, Mannampandal, Mayiladuthurai, Tamilnadu, India. Determination of Antibacterial Activity: The antibacterial activity of the leaf extracts was determined using agar well diffusion method by following the known procedure. Nutrient agar was inoculated with the given microorganisms by spreading the bacterial inoculums on the media. Wells were punched in the agar and filled with plant extracts. Control wells containing neat solvents (negative control) were also run parallel in the same plate. The plates were incubated at 37°C for 18 hours and the antibacterial activity was assessed by measuring the diameter of the zone of inhibition. The antibacterial potential of the different extracts was evaluated by comparing their zones of inhibition. RESULT The antibacterial activity of Acetone, Ethanol and

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Table 1: Antibacterial activity of Acetone, Ethanol and Methanol extract of Azadirachta indica medicinal plants against human pathogens. Plants Azadirachta indica

Extracts Acetone Methanol Ethanol

Escherichia Coli 17 16 24

Zone of inhibition (mm) Pseudomonas aeroginosa Staphylococcus aureus 15 18 17 12 20 19

Salmonella typhii 16 20 30

“A” – Acetone, “M” – Methanol, “E” – Ethanol. Table 2: Qualitative Phytochemical Analyses of Acetone, Ethanol and Methanol extracts of Azadirachta indica Leaf. Solvents used for extraction Acetone Methanol Ethanol

Alkaloid

Reducing sugar

Flavonoid

-

+ + +

+

Saponin +

+

Tannin

Volatile oil

Glycoside

Terpenoids

+

-

+ + -

+ -

(+) indicates presence while (–) indicates the absence of the components Methanol extracts was investigated using agar well alkaloids, tannins, flavonoids, terpenoids, saponins diffusion method, against the selected human pathogens Glycoside and compounds reducing were present in the such as Escherichia Coli, Pseudomonas aeroginosa, extracts Staphylococcus aureus and Salmonella typhii. All the The ethanol, chloroform and aqueous extract examined extract showed varying degrees of antibacterial showed considerable activity against Salmonella typhii, activities against the pathogens. The Phytochemical test The ethanol extract was more active than the standard was done to find the presence of active chemical against Salmonella typhii. Previous study conducted by constituents such as glycosides, alkaloids, tannins, (Ben Gueddeur et al., 2002) suggests that the essential oil flavonoids, terpenoids, saponin, reducing sugar and volatile of O. majorana posses antibacterial activity. The work oil. conducted by (Farooqi and Sreeramu, 2004) reveals that the Table-1 showed the antibacterial activity of leaves of marjoram have antibacterial activity against Ethanol extract of Azadirachta indica showed maximum Escherichia Coli, Pseudomonas aeroginosa, Staphylozone of inhibition (30mm) against Salmonella typhii, coccus aureus and Salmonella typhii. similarly antibacterial followed by Escherichia Coli (24mm), Pseudomonas activity of ethanol, chloroform and water extract of aeroginosa (20mm) and Staphylococcus aureus (19mm). Marrubium vulgare, was further assessed against, The antibacterial activity of Acetone extract of Azadirachta Salmonella typhii, Staphylococcus aureus, Escherichia coli indica showed maximum zone of inhibition (18mm) and Pseudomonas aeruginosa, were recorded (Al-Bakri against Staphylococcus aureus, followed by Escherichia et al., 2006). Coli (17mm), Salmonella typhii (16mm) and Pseudomonas The presence of these phytochemical components aeroginosa (15mm). The antibacterial activity of Methanol may be responsible for the observed antimicrobial activity extract of Azadirachta indica showed maximum zone of of the plant leaf extract. This findings conforms to the inhibition (20mm) against Salmonella typhii, followed by report of (Anyanwu and Dawet, 2005) in which similar Pseudomonas aeroginosa (17mm), Escherichia Coli constituents was found to exhibits antiprotozoal and (16mm) and Staphylococcus aureus (12mm). antibacterial activities. Flavonoid has also been reported to The phytochemical analysis of plant extracts using have greater potential benefit to human Health (Jouad Acetone, Ethanol and Methanol was showed in Table - 2. et al., 2001). From the phytochemical analysis catecholic reducing sugar Imaran khan et al., 2010 studied that were found in Azadirachta indica in the solvents such as phytochemical analysis of Azadiracta indica leaves by Acetone, Ethanol and Methanol. The Ethanol extract of using different solvent such as Petroleum ethar, Azadirachta indica showed the presence of flavanoids, chloroform, methanol show the presence of triterpenes, saponins, tannin, reducing sugar were found in presence of glycosides and fatty acids. Other phytochemicals studied in Ethanol extract. Reducing sugar, glycosides were observed this analysis were absent in all extract of leaves. only in Acetone extract of Azadirachta indica. In plant all Antibacterial activity of Azadiracta indica was analyzed by extracts found glycosides except in Ethanol extract of previous workers showed that the chloroform extract of Azadirachta indica. Saponin were observed in the Acetone leaves possess significant activity, than petroleum ethar and and Ethanol extract of Azadirachta indica. Terpenoids were methanol extracts. Early studies proved ethanol as the most observed only Methanol extract of Azadirachta indica. The efficient solvent for extracting broad spectrum of Acetone, Ethanol and Methanol all extract of Azadirachta antibacterial compounds from plants. indica showed the absences of alkaloid and volatile oil. Himal paudel chhetri et al., 2008 reported that the ethanolic extract of Azadiracta indica whole plant shows DISCUSSION The findings of the preliminary Phytochemical presence of flavonoids and tannins only. Similarly the investigations and the results of antibacterial activity were extract of Azadiracta indica is active against E.coli depicted in the respective Tables. The preliminary followed by Staphylococcus aureus. Earlier observation phytochemical tests performed were of qualitative type and done by (Srinivasan et al., 2001) also showed the from the phytochemical investigations it was observed that antifungal and antibacterial activity of A. indica.

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The methanolic extract of bulbs of Allium cepa showed pronounced activity (23mm) against Bacillus subtilis and Pseudomonas aeruginosa, high activity (20mm) against Proteus vulgaris, while inactive against Staphylococcus aureus, Escherichia coli and Salmonella typhi. The onion bulbs contains numerous organic sulfur compounds, including trans-S-(1- propenyl) cysteine sulfoxide, S–methyl– cysteine sulfoxide, S– propylcysteine sulfoxide and cycloalliin; flavonoids; phenolic acids; sterols including cholesterol, stigma sterol, b-sitosterol; saponins; sugars and a trace of volatile oil composed mainly of sulfur compounds. Although Allium, Azadirachta and Aloe extracts did not show any activity against Staphylococcus aureus (Nima, and Mossa, 1983; and Enkeblia, N.; Dahmouni, S, et al., 2005). Kumar et al., 2006 studied the antibacterial of dichloromethane: methanol (1:1 v/v) extracts of Vitex negundo against different bacterial strains. Their finding conclude that none of the micro organisms including the bacterial strains like B.subtilis, S.aureus, S.epidermidis, E.coli, and P.aeruginosa were inhibited by dichloromethane: methanol extracts. Ahmad et al., 1998 studied the antibacterial activity of the V.negundo while plant of hexane, alcoholic and aqueous extracts against B.subtilis, E.coli, Proteus vulgaris, S.typhimurium, P.aeruginosa and S.aureus had no activity. (Valasraj et al., 1997) studied the antibacterial activity of ethanol extracts of V.negundo leaf using agar dilution method against four bacteria B.subtilis, S.epidermidis, E.coli and P.aeruginosa. Panda et al., 2009 studied the antibacterial activity of V. negundo on bark and leaf of petroleum ether, chloroform, methanol and aqueous extracts against B.subtilis, S.aureus, S.epidermidis, S.typhimurium, P.aeruginosa, V.cholerae, and V.alginolyteus had little activity but inhibition was measured including disc and cup that measures 6mm indicates low activity moreover less concentration of extract was taken which dose not give accuracy of results. The negative results obtained against Gramnegative bacteria were not unexpected since this class of bacteria is usually more resistant then Gram-positive bacteria (Tomas-Barberan et al., 1988). Similar results were also obtained from agar cup method. E. coli was completely inhibited by all the extracts of both leaf and bark. S. aureus was the second most inhibited bacteria with most of the extracts. Ethanol and methanol extracts of the leaves were most active inhibiting agent against both Gram-positive and Gram-negative bacteria. On other hand petroleum ether and chloroform extracts had better antibacterial activity against all Gram-positive bacteria. However, S. epidermidis and B. subtilis were inhibited completely by petroleum ether and chloroform extracts of bark as well as ethanol and methanol extracts of leaves. Infection caused by P. aeruginosa are among the most difficult to treat with conventional antibiotics (Levison Jawetz et al., 1992). The growth of P.aeruginosa was partially inhibited by petroleum ether and ethanol extracts of leaves and bark and moderately inhibited by chloroform, methanol and aqueous extracts. So the plant V.

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negundo can be used as a source which could yield drugs that could improve the treatment of infection caused by this organism. B. subtilis is the common bacteria found in most natural environments including soil, water plant and animal tissues. Napoleon et al., 2009 also reported Enterobactor spp, S.aureus, P.aeruginosa, S.typhi and E.coli to be sensitive to ethanol, chloroform and aqueous extract of Moringa olifera leaf at concentration of 200 mg/1. phytochemical analysis were similar report of our results. Maluventhan viji et al., 2010 studied that ethanol, chloroform and aqueous extract of Cardiospermum halicacabum leaves shows the presence of flavonoids, tannins, steroids and glycosides, which were similar to our results. Antibacterial activity of Cardiospermum halicacabum was studied by same workers reported that ethanol extract was active against Steptococcus aureus followed by Salmonella typhi, E. coli & P. aeroginosa. It is also related to our results. Results obtained from this study, indicate that the plant extracts showed the strongest antibacterial activity than the commercially available antibiotics. For example, Ciprofloxacin showed the maximum zone of inhibition (34mm) against Streptococcus sp. but the methanol extract of Eucalyptus (Eucalyptus globolus) and the methanol extract of Butterfly Pea (Clitoria ternatea) showed the maximum zone of inhibition (42mm and 36mm) against Streptococcus sp. The phytochemical analysis showed the presence of tannins, glycosides, flavonoids, reducing sugars and saponin, were present in some of the plant extracts. CONCLUSION It may be concluded from this study that Azadirachta indica leaf extract has antibacterial activity against dental pathogens. It is expected that using natural products as therapeutic agents will probably not elicit resistance in microorganisms. This can explain the rationale for the use of the plant in treating infections in traditional medicine. The plant could be a veritable and cheaper substitute for conventional drugs since the plant is easily obtainable and the extract can easily be made via a simple process of maceration or infusion. It is essential that research should continue to isolate and purify the active components of this natural herb and use in experimental animals. ACKNOWLEDGEMENT Grateful thanks goes to the Department of Botany, A.V.C. College for providing the facilities for research work. The authors also thank Prof. V. Karunanithi and Mrs. Sheela for providing the invaluable help during the research work. REFERENCES 1. Al-Bakri AG, Afifi FU. Evaluation of antimicrobial activity of selected plant extracts by rapid XTT colorimetry and bacterial enumeration. J Microbiol Methods. 2007 Jan;68(1):19-25. Epub 2006 Jul 10. 2. Anyanwu, G. I. and Dawet, A. (2005). Pharmacological and phytochemical screening of Hyptis suaveolens poit (Lamiaceae) for bioactivity in rodents.

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Source of support: Department of Botany, A.V.C. College, Tamilnadu, India; Conflict of interest: None declared

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