Chemical composition of the leaves of Azadirachta Indica Linn (Neem)

Chemical composition of the leaves of Azadirachta Indica Linn (Neem) Prashanth G.K 1, G.M. Krishnaiah 2 Department of Chemistry, Sir M. Visvesvaraya Institute of Technology, Hunasamaranahalli, Bangalore-562 157, India. Abstract: The aim of this study was to carry out the phytochemical screening and GC-MS analysis of the leaves of Azadirachta Indica Linn. Phytochemical screening of the aqueous and ethanolic extracts of the leaves revealed the presence of alkaloids, reducing sugars, saponins etc, in them. GC-MS of the ethanolic extract revealed the presence of many compounds in the leaves of Azadirachta Indica Linn. Keywords: Azadirachta indica Linn, Phytochemical screening, GC-MS INTRODUCTION Meliaceae or the Mahogany family is a flowering plant family of mostly trees and shrubs. They are characterized by alternate usually pinnate leaves without stipules and by syncarpous apparently bisexual flowers borne in panicles, cymes, spikes, or clusters. Most species are evergreen, but some are deciduous, either in the dry season or in winter. The family includes about 50 genera and 550 species. Azadirachta indica Linn is a tropical evergreen tree native to India and is also found in other southeast countries. It is a tree in the mahogany family Meliaceae. Azadirachta indica is locally known as Neem. It is a tree in the mahogany family of Meliaceae. It is one of two species in the genus of Azadirachta. It is native to India, Bangladesh, Thailand, Nepal and Pakistan. It grows well in tropical and sub-tropical regions. In India, Neem is known as the village pharmacy because of its healing versatility. The tree has been used in Ayurvedic medicine due to its medicinal properties. Neem is also Prashanth,Krishnaiah Page 21

called arista in Sanskrit- a word that means perfect, complete and imperishable. Fruit, seeds, oil, leaves, roots, bark and almost every part of the tree is bitter and contain compounds with proven antiviral, antiretroviral, antiinflammatory, anti-ulcer and antifungal, antibacterial, anti plasmodial, antiseptic, antipyretic and anti diabetic properties [1-3]. The various parts of this tree have many uses that aptly give Neem its name in Sanskrit- sarva roga nivarini, meaning the curer of all ailments. India has encouraged scientific investigations of Neem tree as a part of its program to revitalize Indian tradition and also to increase commercial interest on Neem [4]. In India, this tree is called Divine tree, Wonder tree, heal all, Materia Medica, Free tree of India, Nature s drugstore, Village Pharmacy, Panacea for all diseases [5-7]. The Neem oil is isolated from its fruits and seed [2, 8-10]. Neem is the most important medicinal plant that has been declared as the Tree of the 21 st century by the United Nations. Thus as the plant possesses immense medicinal properties, the aim of the present study was to identify the phyto-components present in the aqueous and etanolic extracts of the leaves of Neem by qualitative photochemical testing and to identify the compounds present in the ethanolic extract of the leaves by Gas chromatography-mass spectrum (GC-MS) analysis. EXPERIMENTAL DETAILS Plant collection Neem leaves were collected in Hunasamaranahalli, Bengaluru. It was ensured that the plant was healthy and uninfected. Leaves were washed under running tap water to remove any traces of soil particles and other dirt. Then washed with distilled water, air dried and cut in to small pieces and dried for 15 days in shade. Then the leaves were ground and sieved to get fine powder. Prashanth,Krishnaiah Page 22

Preparation of aqueous and ethanolic extracts All the chemicals and reagents used in this study were of analytical grade. The powdered leaves (20g) were extracted successively in double distilled water and absolute ethanol at 50-60 0 C for 18 hours using Soxhlet apparatus. The solvents used were recovered under pressure until dry extracts were obtained. Phytochemical screening Detailed phytochemical examinations were carried out for both the extracts as per the standard methods [11-13]. Tests for Alkaloids To the extract, dilute hydrochloric acid was added, shaken well and filtered. With the filtrate, the following tests were performed. Mayer s reagent test To 3 ml of filtrate, few drops of Mayer s reagent were added along sides of tube. Formation of creamy precipitate indicates the presence of alkaloids. Wagner s test To 2 ml of filtrate, few drops of Wagner s reagent were added in a test tube. Formation of reddish brown precipitate indicates the presence of alkaloids. Hager s test To 2 ml of filtrate, few drops of Hager s reagent were added in a test tube. Formation of yellow color precipitate indicates the presence of alkaloids. Tests for Carbohydrates Molisch test 2 ml of aqueous extract was treated with 2 drops of alcoholic α-naphthol solution in a test tube and then 1 ml of concentrated sulphuric acid was added carefully along the sides of the test tube. Formation of violet ring at the junction indicates the presence of carbohydrates. Prashanth,Krishnaiah Page 23

Barfoed s test 1 ml of extract and Barfoed s reagent were mixed in a test tube and heated on water bath for 2 minutes. Red color due to formation of cupric oxide indicates the presence of monosaccharide. Tests for Reducing Sugars Fehling s test To 1 ml of aqueous extract, 1 ml of Fehling s A and 1 ml of Fehling s B solutions were added in a test tube and heated on a water bath for 10 minutes. Formation of red precipitate indicates the presence of reducing sugar. Benedict s test Equal volume of Benedict s reagent and extract were mixed in a test tube and heated on a water bath for 5-10 minutes. Solution appears green, yellow or red depending on the amount of reducing sugar present in the test solution which indicates the presence of reducing sugar. Tests for Flavonoids Alkaline reagent test The extract was treated with few drops of sodium hydroxide solution separately in a test tube. Formation of intense yellow color, which becomes colorless on addition of few drops of dilute acid indicates the presence of flavonoids. Lead Acetate Test The extract was treated with few drops of lead acetate solution. Formation of yellow precipitate indicates the presence of flavonoids. Tests for Glycosides Borntrager s test To 3 ml of test solution, dilute sulphuric acid was added, boiled for 5 minutes and filtered. To the cold filtrate, equal volume of benzene or chloroform was added and it was shaken well. The organic solvent layer was Prashanth,Krishnaiah Page 24

separated and ammonia was added to it. Formation of pink to red color in ammonical layer indicates the presence of anthraquinone glycosides. Legal s test 1 ml of test solution was dissolved in pyridine. 1 ml of sodium nitropruside solution was added and made alkaline using 10% sodium hydroxide solution. Formation of pink to blood red color indicates the presence of cardiac glycosides. Keller-Killiani test To 2 ml of test solution, 3 ml of glacial acetic acid and 1 drop of 5% ferric chloride were added in a test tube. Carefully 0.5 ml of concentrated sulphuric acid was added by the sides of the test tube. Formation of blue color in the acetic acid layer indicates the presence of cardiac glycosides. Tests for Tannin and Phenolic compounds Ferric chloride test A small amount of extract was dissolved in distilled water. To this solution 2 ml of 5% ferric chloride solution was added. Formation of blue, green or violet color indicates presence of phenolic compounds. Lead Acetate Test A small amount of extract was dissolved in distilled water. To this solution few drops of lead acetate solution were added. Formation of white precipitate indicates the presence of phenolic compounds. Dilute iodine solution test To 2-3 ml of extract, few drops of dilute iodine solution were added. Formation of transient red color indicates the presence of phenolic compounds. Test for Saponin Froth test The extract was diluted with distilled water and shaken in a graduated cylinder for 15 minutes. The formation of layer of foam indicates the presence Prashanth,Krishnaiah Page 25

of saponins. Tests for Protein and Amino acids Ninhydrin test 3 ml of the test solution was heated with 3 drops of 5% Ninhydrin solution on a water bath for 10 minutes. Formation of blue color indicates the presence of amino acids. Biuret test The extract was treated with 1 ml of 10% sodium hydroxide solution in a test tube and heated. A drop of 0.7% copper sulphate solution was added to the above mixture. The formation of violet or pink color indicates the presence of proteins. Tests for Triterpenoids and Steroids: Salkowski s test The extract was treated with chloroform and filtered. The filtrate was added with few drops of concentrated sulphuric acid, shaken and allowed to stand. If the lower layer turns red, sterol is present. Presence of golden yellow layer at the bottom indicates the presence of triterpenes. Libermann-Burchard s test The extract was treated with chloroform. To this solution few drops of acetic anhydride were added, boiled and cooled. Concentrated sulphuric acid was added through the sides of the test tube. Formation of brown ring at the junction of two layers, if upper layer turns green, indicates the presence of steroids and formation of deep red color indicates the presence of triterpenoids. GC-MS analysis of the ethanolic extract of Neem leaves The chemical composition of ethanolic extract of the leaves was analyzed by GC-MS. The analysis was carried out on Jeol spectrometer (Model: Accu TOF GCV). RESULTS AND DISCUSSIONS Prashanth,Krishnaiah Page 26

Results of qualitative phytochemical analysis of the aqueous and ethanolic extracts of Neem leaves The results of qualitative phytochemical analysis of aqueous leaf extract (ALE) and ethanolic leaf extract (ELE) of Neem are given in Table 1. Results indicate the presence of many phyto-components in both the extracts. The results of our present studies are almost in agreement with the results published by the other research groups [14-16]. Sl. Constituent Test Result Sl. Constituent Test Result No. ALE ELE No. ALE ELE 1 Alkaloids Mayer s reagent + + Glycoside Bomtrager s test - + test Wagner s + + Killer-Killiani test + + reagent test Hager s reagent + + 6 Tannins Ferric chloride test + + test and 2 Carbohydrat Molish s test + + Phenolic Lead acetate test + + es Barfoed s test + + compounds Dilute iodine + + solution test 3 Reducing Fehling s test + + 7 Saponins Froth test + + sugars Benedicts test + + 8 Proteins Biuret test - - 4 Flavanoids Alkaline + + and Amino Ninhydrin test - - reagent test acids Lead acetate + + 9 Triterpenoi Salwonski test - - test ds and 5 Glycoside Legal s test - + Steroids Libermann and - - Burchard s test Table 1 Results of Qualitative Phytochemical Screening + is present - is absent GC-MS analysis A typical GC-MS of the ethanolic extract of the leaves of Neem is presented in Fig. 1. Mass spectra of the ethanolic extracts of Neem leaves are depicted in Fig. 2 (a-b). The fragmentation patterns of the mass spectra were compared with those of the known compounds stored in the National Institute of Standards and Technology (NIST) research library. It revealed the presence of (Acetyloxy) acetic acid (C 4 H 6 O 4 ), Hydroxy pivalic acid (C 5 H 10 O 3 ), Phytol (C 20 H 40 O), 4-Cycloocten-1-ol, 8,8 -(iminodi-2,1-phenylene) bis- (C 28 H 35 NO 2 ), Prashanth,Krishnaiah Page 27

1,3-Diphenyl-2-azafluorene (C 24 H 17 N), Lup-20 (29)-2n-3-ol, acetate, (3β)- (C 32 H 52 O 2 ), Germanicol (C 30 H 50 O). Fig. 1 GC-MS of the ethanolic extract of the leaves of Neem Prashanth,Krishnaiah Page 28

Fig. 2 (a-b) Mass spectra of the ethanolic extract of the leaves of Neem Prashanth,Krishnaiah Page 29

CONCLUSION Thus in our present studies, the phytochemical screening of the aqueous and ethanolic extracts of Neem leaves revealed the presence of many phytocomponents. The GC-MS analysis of ethanolic extract showed a number of components. This study may be useful to explore the pharmacological and biosynthetic activity of Neem plant further. ACKNOWLEDGEMENT The authors thank the Management and the Principal of Sir MVIT for the support and encouragement extended towards this work. The authors acknowledge SAIF, IITB for GC-MS analysis. REFERENCES 1. R. Subapriya, S. Nagini, Medicinal properties of Neem leaves: A Review Source, Current Medicinal Chemistry - Anti-Cancer Agents, 5 (2005) 149-156. 2. S.S. El-Hawary, M.E El-Tantawy, M.A. Rabeh, W.K. Badr, Chemical composition and biological activities of essential oils of Azadirachta indica A. Juss, International Journal of Applied Research in Natural Products, 6 (2013) 33-42. 3. Indra Prasad Pandey, Sayed Farooq Ahmed, Suman Chhimwal, Shalini Pandey, Chemical composition and wound healing activity of volatile oil of leaves of Azadirachta indica A. juss, Advances in Pure and Applied Chemistry 62 (2012) 2167-0854. 4. Stix G. Village pharmacy. The neem tree yields products from pesticides to soap. Sci. Am. 266 (1992) 132. 5. Ruchi Tiwari, Amit Kumar Verma, Sandip Chakraborty, Kuldeep Dhama, Shoor Vir Singh Neem (Azadirachta indica) and its Potential for Safeguarding Health of Animals and Humans: A Review, Journal of Biological Sciences 14 (2014) 110-123. 6. A.K. Ghimeray, C.W. Jin, B.K. Ghimire, D.H. Che, Antioxidantactivity and quantitative estimation of Azadirachtin and Nimbin in Azadirachta indica, African Journal of Biotechnology 54 (2009)1684 5315. Prashanth,Krishnaiah Page 30

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