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Sandeep Project Defending Final

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A PROJECT ON “Preliminary Phytochemical Screening and Antimicrobial Properties of Azima tetracantha Lam., Schleichera oleosa (Lour.) Oken. And Canarium strictum Roxb.” Guided by: Prof. M.B. Shivanna Director of School of Biological Science and Project Co-Ordinator Dept. of Applied Botany Kuvempu University Presented by: Sandeep Otari M.Sc. IV semester Dept. of Applied Botany Kuvempu University KUVEMPU UNIVERSITY Department of PG Studies and Research in Applied Botany, Jnana Sahyadri, Kuvempu University, Shankaraghatta
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Page 1: Sandeep Project Defending Final

A PROJECT ON

“Preliminary Phytochemical Screening and Antimicrobial Properties of Azima tetracantha Lam., Schleichera oleosa (Lour.) Oken. And

Canarium strictum Roxb.”

Guided by: Prof. M.B. ShivannaDirector of School of Biological Science and Project Co-OrdinatorDept. of Applied Botany Kuvempu University

Presented by:Sandeep OtariM.Sc. IV semesterDept. of Applied BotanyKuvempu University

KUVEMPU UNIVERSITY

Department of PG Studies and Research in Applied Botany,Jnana Sahyadri, Kuvempu University, Shankaraghatta

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Aims and Objectives

To know the phytochemical compounds present in the plants.

To check the antimicrobial activity

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IntroductionMedicinal and aromatic plants constitute a major segment of the flora, which

provides raw materials for use in the pharmaceuticals, cosmetics, and drug industries.

In one of the studies by the World Health Organisation, it is estimated that 80 per cent of the population of developing countries relies on traditional plant based medicines for their health requirements (WHO, 1991).

More than 9,000 native plants have established and recorded curative properties and about 1500 species are known for their aroma and flavour.

The indigenous systems of medicines, developed in India for centuries, make use of many medicinal herbs. These systems include Ayurveda, Siddha, Unani, and any other indigenous practices.

An anti-microbial is a substance that kills or inhibits the growth of microorganisms such as bacteria, fungi, or protozoans. Antimicrobial drugs either kill microbes or prevent the growth of microbes.

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Azima tetracantha Lam.

Vernacular Name: Needle-bush ; bee-sting bush (English). Uppimullu (Kannada), Malayalam : YasankuFamily : Salvadoraceae.Description: Stem bark green on younger branches, turning brown, young twigs sometimes square in cross-section, hairy; characteristic whorls of four long straight spines occur along the length of branches at each of the leaf axils. Leaves Oval to circular, opposite or nearly so, each pair at right angles to the previous and following one; light green, leathery and usually hairy; apex has a sharp tip, margin entire, tapering at both ends, short petiole. Flower Dioecious; light green or yellow, small flower clusters in axils; floral parts in fours; petals recurving, calyx bell-shaped. Seed and fruit Round berry of 1 cm in diameter with a sharp apical tip; fleshy, light-coloured, containing one or two seeds; ripe from summer into the next winter. Medicinal Uses: Toothache and snakebite

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Schleichera oleosa (Lour.) Oken.

Vernacular name: Lac tree, Ceylon oak(English) ; Kusum(Hindi), Chandala(Kannada), Tamil: Kumbadiri Malayalam: Cottilai  Family: Sapindaceae 

The plant has several synonyms like; S. trijuiga, Willd. & Klein., Pistacia oleosa, Lour.

Description: It is a deciduous tree with red heart wood, leaves paripinnate, leaflets opposite, sessile, flower – yellow, male and female flowers in separate trees, echinate, seeds 1-2, oily cotyledons.

Uses: The seed oil, fruits and seeds are used for burns, cholera, fever, rheumatism, pneumonia, sprains, skin disease, wounds, leprosy, inflammation, ulcers oil-alopecia, acne. The bark is astringent and used against skin inflammations and ulcers, while an infusion is taken against malaria (Iwasa, 1997). This is analyzed for the presence of phytochemicals by various scientists but there is no evidence of scientific study on the antimicrobial potential.

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Canarium strictum Roxb. Vernacular names : Tamil: Karunkungiliyam, Karangkunthrikam.  Malayalam: Pantham, Pantappayan, Thelli, Viraka, Thellippayin.  Kannada: Nalla rojanamu. English: Black dammar, Doopa.

Family: BURSERACEAEDescription:Canarium strictum Roxb. is commonly known as black dammer tree, dhup in Sanskrit dhoopavriksha, raaladhoopa, mandadhoopa. A large resinous tree grows up to 40 meters in height. Leaves compound, imparipinnate, alternate, spiral, clustered at twig ends, leaflets 3-9 pair with odd one at apex, increasing in size towards apex; lamina 5-15 x 2.5-7 cm usually oblong, sometimes ovate, apex acuminate, base asymmetric-rounded; serrate or serrulate, coriaceous, rusty tomentose or pubescent beneath, glabrous above. Inflorescence axillary panicles, flowers white. Fruits are oblong drupes, contain 1-3 seeds.Uses: Plant pacifies vitiated kapha, vata, psoriasis, other skin diseases, rheumatoid arthritis, fever cough, epilepsy venereal diseases and asthma. Resin (black dammer) is used part. Chemicals present: Black dammer resin contains (+)- junenol, canarone and epikhusinal. The plant contains a sesquiterpene ketone—canarone. 

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Most people especially in rural areas depend on herbal medicines to treat many diseases including inflammation-related ailments such as rheumatism, muscle swelling, cut wound, accidental bone fracture, insect bites, pains and burn by fire and hot water. Finally 34 species in 32 genera and 22 families were encountered(Nima et al., 2010) during the field survey. Botanical families such as Asteraceae, Euphorbiaceae, Zingiberaceae and Lamiaceae were represented by the highest numbers of species reported in this study. Thirteen plant species, namely: Bombax ceiba, Canarium strictum, Chloranthus erectus, Xanthium indicum,Lycopodium clavatum, Coleus blumei, Batrachospermum atrum, Chlorella vulgaris, Marchantia palmata, Marchantia polymorpha, Eria pannea,Sterculia villosa and Alpinia galanga are reported for the first time for the treatment of inflammation-related diseases. Canarium strictum Roxb. (Burseraceae) is used in the traditional Ayurvedic medicine under the name Raladhupa and black dammar resin. The compound A and Compound B were investigated for antimicrobial (antibacterial and antifungal) activity by using cup plate method or diffusion agar method. The results obtained shows that compound A and compound B possess broad-spectrum antimicrobial activity at concentration of 100μg/ml. The inhibitory effect of each compound is very close and identical in magnitude for Gram- positive, Gram-negative bacteria and fungi. Anti-inflammatory and analgesic activities from compound A and B extracted from Canarium strictum gum resin were studied.(Suruse et al., 2008)

Review of Literature

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HINGE et al.,(1965 and 1966) isolated two new pentacyclic triterpcnes of the taraxastane group from Canarium strictum Roxb. One is a ketol and the other a diol. They have been named as epi-Ψ-taraxastanonol and epi-Ψ-taraxastane diol respectively. And it also contains junenol (III), a new monoethynaid sesquiterpene ketone canarone(IV), a liquid sesquiterpene alcohol epi-khusinol (V), α-amyrin(Ia), β-amyrin(IIa), β-amyrin acetate, Petroleum ether extract of the methanol insoluble portion was found to contain α-amyrin, β-amyrin and 1 I-keto-α=amyrin (Xa). BLACK DAMMAR is a commercial name given to the resin which exudes from the plant Canarium strictum Roxb., a large tree found abundantly in the evergreen forests of South India. Although the timber is soft and of inferior quality, the black nodular lumps of the resin find many uses. It was reported that these compounds have the medicinal properties.

Antipyretic activity in rats using Brewer’s yeast induced Pyrexia and anticancer activity in mice (Nargis et al., 2010). Treatment with A. tetracantha extract at a dose of 100, 200 mg/kg decreased the rectal temperature of the rats in dose dependent manner. This effect was maximal at dose of 200 mg/kg and it caused significant lowering of body temperature (P< 0.01) up to 4 hour after its administration. The antipyretic effect started as early as 1h and the effect was maintained for 4h, after its administration. Both the standard drug paracetamol 25 mg/kg and tested drug A. tetracantha extract were significantly reduced the yeast elevated rectal temperature, at 2nd, 3rd and 4th hour compared to control group.

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The leaves of Azima tetracantha (Salvadoraceae) commonly known as “mulluchangu” is best known for its medicinal properties. A. tetracantha contains significant quantities of the antioxidant(Hepsibha et al., 2010) . Thus the therapeutic property of the leaves of the plant A. tetracanth a can be attributed to the antioxidant principles which scavenge the free radicals responsible for pathological severity. The ethanolic extract of Azima tetracantha leaves were investigated for its ulcer protective activity on aspirin and pylorus ligation and cold restraint stress induced ulcer models (Muthusamy et al., 2010). This revealed that the extract of A. tetracantha had ulcer protective activity comparable with standard drugs ranitidine and omeprazole, which may be mediated by its antioxidant effects. Leaves of A.tetracantha also reported for anthelmintic and antimicrobial properties(Ekbote et al., 2010)

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Novel fatty acids in A. tetracantha seed oil was estimated by Daulatabad et al. (1991), and they found that the plant contains ricinolic acid (9.8%) and cyclopropenoid fatty acid (9.6%) along with normal fatty acids. Maruthi et al., 2000, listed A. tetracantha in their study and says that the peelings of stem is ground with garlic and applied on eczema and old wound. In 2003, Azimine a novel class of macrocyclic dilactones containing a 2,3,6-trisubstituted piperidine skeleton is synthesized for the first time by Taro et al., in their work entitled Enantioselective total synthesis of (+)-Azimine and (+)-Carpaine. Profiling of glucosinolates, flavonoids, alkaloids, and other secondary metabolites in tissues of Azima tetracantha L. (Salvadoraceae) is conducted by Bennett et al., (2004). In conclusion they added that the plant has a simple glucosinolate profile but has additional complexities with the presence of flavonoids and alkaloids. Aqueous extracts from 30 medicinal plants used in Yemeni ethnomedicine were screened by Al-Fatimi, et al. (2007), for antimicrobial activities. The Strong inhibition was exhibited by Azima tetracantha (Fruits). Nandgude, et al. (2007), conducted analgesic activity of various extracts of leaves of Azima tetracantha Lam. The fraction obtained by extraction from Benzene, chloroform and aqueous extract clearly showed a good analgesic activity. Antiulcer activity of A. tetracantha: a biochemical study is conducted by Muthuswamy et al., in 2009.

However there are no works reported upon the preliminary phytochemical analysis of secondary metabolites of the soxhlet extracts of S. oleosa (stem bark) and A. tetracantha (whole plant- leaves, root and stem). Neither there is any studies conducted strictly upon the antibacterial properties of the S. oleosa, nor A. tetracantha, has been analyzed for the antimicrobial properties, hence the present study aims to prove that A. tetracantha and S. oleosa has the antibacterial property.

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Materials and Methods

Plant collection:The plants used in the study were Azima tetracantha (whole plant-leaves, stem

and root), Schleichera oleosa (stem bark) and Canarium strictum (stem bark) and the plant materials were collected from Kuvempu University campus, Shimoga and Tarikere during November (A.tetracantha and S.oleosa) and C.strictum was collected from Hannia, Hosanagar taluk, Shimoga district during September 2010 and the location of the tree was North 13o15’26.47’’ and east 75o03’24.91’’. The plants were identified by using standard manual “Flora of Madras Presidency” by Gamble.

Plants materials of each plant were air dried for about 15-30 days, coarsely powdered and were then used for the extraction by soxhlet apparatus.

Preparation of crude extracts:100 gram of the air-dried and coarsely powdered plants materials were

exhaustively extracted for about 40 cycles (≈18 hour) with petroleum ether in soxhlet apparatus. The petroleum ether extract was separated and evaporated under a fan. The extracted plant material was then air-dried, repacked in soxhlet apparatus and exhaustively extracted with ethyl acetate for about 20 hour (40 cycles) and the above procedure is repeated for the methanol (take ≈24 hour to complete 40 cycles)

Thus obtained extracts were collected separately in clean and dry screw capped bottles, labeled and stored. After each successive solvent extraction the extract yield was recorded. Thus obtained solvent extracts were used to carry out the preliminary phytochemical analysis and antimicrobial assay.

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Preliminary phytochemical Group Test The preliminary phytochemical group test of solvents extracts of the two plants was performed by the standard methods (Goering & Van Soet et al., 1975;Krishnaswamy, 2003; Pederson, 2006; Adeloye et al., 2007; Arunachalam et al.,2009).1.Primary compounds:

1.Test for Carbohydrates:

•Molisch’s Test: To the 2ml of sample 2 drops of alpha- napthol and mixed carefully. Rundown the concentrated sulfuric acid along the walls of the test tube. The purple violet ring at the junction of the two layers which indicates the presence of carbohydrates.

2.Test for Polysaccharides: To 1 ml of test solution, 2 drops of iodine added. Blue coloured solution indicates the presence of polysaccharides.

3.Test for Proteins:•Biuret reaction: biuret reaction can be made to 2ml of test solution and 2ml of 10% sodium hydroxide mix, add 2 drops of 0.1% copper sulphate solution. If violet or pink colour then compounds with two or more peptide bonds give a violet colour with alkaline copper sulphate solution.•Ninhydrin Test: to 4ml of solution which should bear neutral pH add 1ml of 0.1% freshly prepared ninhydrin solution mix the concentration and boil a couple of minutes. Observation of violet or purple colour indicates the presence of proteins.

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4.Test for Lipids: To 2ml of test sample iodine solution was added dropwise. Original iodine

colour disappears which indicates the presence of lipids.

5.Test for Oils:The one drop of sample placed on filter paper and allowed to dry. Clear

greasy spot is observed which indicates the presence of oils.

2.Secondary compounds:1.Test for alkaloids:

1. Mayer’s test: Small quantities of the plant extracts of both the plants were treated with few drops of dilute hydrochloric acid (HCl) and then they were treated with Mayer’s reagent. The formation of yellowish buff coloured precipitate indicated positive test for alkaloids.

2. Wagner’s test: Small quantities of plant extracts were separately treated with few drops of dilute HCl and then with Wagner’s reagent, a reddish brown precipitate developed, suggesting the presence of alkaloids.

3. Dragendroff’s test: In this test addition of 2ml of dragendroff’s reagent and 1ml of diluted hydrochloric acid to the plant extract produced orange precipitate indicating presence of alkaloids.

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2.Test for flavonoids

1. Ferric chloride test: Test solution with few drops of ferric chloride solution shows intense green colour indicating the presence of flavonoids.

2. Shinoda test: For the positive test the test solution with few fragments of magnesium ribbon and concentrated HCl shows pink to magenta red colour.

3. Zinc-Hydrochloric acid reduction test: Test solution with zinc dust and few drops of hydrochloric acid shows magenta red colour.

4. Alkaline reagent test: Test solution taken when treated with sodium hydroxide solution, shows increase in the intensity of yellow colour which becomes colourless on addition of few drops of dilute acid.

5. Lead acetate solution test: Test solution with few drops of lead acetate (10%) solution gives yellow precipitate.

6. Flavonoid test: in this test the plant extract a few magnesium turning when added with concentrated sulphuric acid through sides of test tube produced magenta colour indicating flavones or deep cherry red colour indicating flavonoids.

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3.Test for glycosides

1. Keller – Killiani test: The test solution was treated with few drops of ferric chloride solution and mixed. When concentrated sulphuric acid containing ferric chloride solution was added, it forms two layers, lower layer reddish brown and upper acetic acid layer turns bluish green.

2. Bromine water test: Test solution when dissolved in bromine water gives yellow precipitate.

•Raymond’s test: The test solution when treated with dinitrobenzene in hot ethanolic alkali gives violet colour

•Molisch’s test: In this test 1ml of molisch’s reagent was added to the plant extract and when 1ml concentrated sulphuric acid was poured through the sides of the test tube, a reddish violet ring was formed at the junction of two layers indicating the presence of glycosides.

4.Test for tannins:

1. Ferric chloride test: Test solution is treated with 1ml of 5% ferric chloride solution; formation of greenish black colouration demonstrated the presence of tannins.

2. Gelatin test: Test solution when treated with 1% gelatin solution gives white precipitate.

3. Sodium chloride test: The formation of precipitate with the addition of few drops sodium chloride solution to the extract indicated the presence of tannins.

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5.Test for triterpinoids

Salkowaski test: A few drops of concentrated sulphuric acid was added to the test solution, shaken and allowed to stand, lower layer turns yellow indicating the presence of triterpinoids.

6.Test for saponins:

Foam test: Test solution is treated with water, shaken well for 15 minute, formation of stable foam suggested the presence of saponins.

7.Test for lignins:Furfuraldehyde test: when extract was treated with furfuraldehyde solution(2%), formation of red colour indicated the presence of lignins.

8.Test for phenols 1. Phenol test: Test was done by adding 0.5ml of ferric chloride solution to the plantextract. The presence of phenolics was indicated by the formation of theintense colour in the solution. 2. Ellagic acid test: This test was yielding muddy yellow, olive brown, niger brown or deep chocholate upon reaction of plant extract with few drops of 5% mixture containing glacial acetic acid and 5% (V/V) sodium nitrate solution. 3. Hot water test: In this test, leaf and stem pieces when partially dipped in hot water produced intense colour at the junction.

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9.Test for steroids

Salkowaski test: A few drops of concentrated sulphuric acid was added to the test solution, shaken and allowed to stand, lower layer turns to red indicating the presence of steroids.

In-vitro antimicrobial assay: Antibacterial and antifungal activity will be carried out by inoculating clinical pathogens and plant pathogens (bacterial and fungal species).

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Reference Hedge, J.E. and B.T. Hofreiter (1962) In :carbohydrate chemistry 17(Eds. Whistler, R.L. and

Be Miller, J.N.) Academic Press, New York. Biochemical methods (Second edition). S,Sadasivaram, Ph. D. Director. A. Manjckam, Ph. D. Professors, p.8-9.

Lowry, O.H. Rosebrough, N.J., Farr,A.L. and Randall, R.J. 1951. J.Biol. Chem., 193265.(1951)

Mattoo, R.L. (1970). Inland J. Biochem. 782.

Nima D., Namsa; Hui Tag; M. Mandal; P. Kalita; A.K. Das; An Ethnobotanical Study of Traditional anti-inflammatory Plants Used by the Lohit Community of Arunachal Pradesh, India.(2010)

Suruse P.B. ; Bodele; S.B.; Duragkar N.J.; Kale; M.K.; Anti-inflammatory and Analgesic Activities of Isolateted Compounds from Canarium strictum Gum Resin.(2008)

V. K. HINGE, S. K. PAKNIKAR, K. G. Das, A. K. Bose and S. C. BHATTACHARYYA ;TERPENOIDS-LXXXVI; STRUCTURE OF EPI-Ψ-TARAXASTANONOL AND EPI-Ψ-TARAXASTANEDIOL; National Chemical Laboratory, Poona, India.(1966)

V. K. HINGE, S. K. PAKNIKAR and S. C, BHATTACHARYYA ; TERPENOIDS-LXXI; CONSTITUENTS OF INDIAN BLACK DAMMAR RESIN ; National Chemical Laboratctry, Poona.(1965)

P.B. Suruse, N.J. Duragkar, U.D. Shivhare and S.B. Bodele; Study of Antimicrobial Activity of Canarium strictum Gum Resin.(2010)

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T. Nargis Begum, A. Vijaya Anand and R. Senthil; Antipyretic Activity of Azima tetracantha in Experimental Animals.(2010)

Jigna PAREKH, Sumitra V. CHANDA; In vitro Antimicrobial Activity and Phytochemical Analysis of Some Indian Medicinal Plants; Phytochemical, Pharmacological and Microbiological Laboratory, Department of Biosciences, Saurashtra University, Rajkot.(2006)

B. Thendral Hepsibha, S. Sathiya , C. Saravana Babu , V. Premalakshmi and T. Sekar; In vitro studies on antioxidant and free radical scavenging activities of Azima tetracantha. Lam leaf extracts.(2010)

T. Nargis Begum, M.H. Muhammad ILyas, S. Kalavathy, A. Vijaya Anand, P. Sampath Kumar and R. Senthil; Effects of Ethanolic Leaf Extract of Azima tetracantha Lam. On Ehrlich Ascites Carcinoma Tumour Bearing Mice.(2009)

P Muthusamy, A Jerad Suresh and G Balamurugan; Antiulcer Activity of Azima Tetracantha: A Biochemical Study.(2010)

Maruthi T Ekbote, Ramesh C. K and Riaz Mahmood; Evaluation of Anthelmintic and Antimicrobial activities of Azima tetracantha Lam.(2010)

R S Deshpande, Neelakanta N.T., Naveen Hegde ; CULTIVATION OF MEDICINAL CROPS AND AROMATIC CROPS AS A MEANS OF DIVERSIFICATION IN AGRICULTURE; Agricultural Development and Rural Transformation Centre; Institute for Social and Economic Change; Nagarbhavi, Bangalore-560 072;June 2006

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Thank You


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