PLANT PROFILE Tinospora Sinesis Family : Menispermaceae

Synonymes : English Hindi Sanskrit Marathi : Gulancha tinopora, Tinospora : Gulancha. Jungly Giloy, Amrita : Guduchi, Amrita : Gulvel

Distribution : Throughout India in forests. Plant: A large extensively spreading glabours, perennial deciduous twiner with succulent stems; leaves simple; flowers yellow in lax racemes, male flowers in clusters, female flowers usually solitary; fruits drupes, red when ripe. Part uses : Stems, leaves, roots and whole plant. Properties and uses: Plant is widely used in Ayurvedic systems of medicine for its general tonic, anti-diabetic, anti-malarial, anti-allergic and aphrodisiac properties. It is also claimed to be useful in skin diseases, vomiting, anaemia, piles, chronic fever and emaciation.

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The stem is bitter, stomachic, antipyretic and astringent. It is powdered and made into and infusion, used as alterant. Starch from stem and root (Jungly Giloe Satwa) have nutrient property and is used in chronic diarrhea and dysentery. Juice of tehfresh plant is used as a powerful diuretic and is also used in gonorrhea. All part of decoction products are mentioned in Ayurvedic text books for use in joint diseases. The root is known for its anti-stress, anti-leprotic and anti-malarial activities. Various part of plants are shown in fig.

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TINOSPORA SINESIS • The chemical examination of stem of Tinospora Sinesis was carried out. The petroleum ether, chloroform, ethanol extracts of the stem were carried out and their yields were measured. • The alkaloids from the stems of Tinospora Sinesis have been precipitated with Dragendorff reagent. • The alcoholic extract of fresh stems of Tinospora Sinesis was found to contain two bitters (tinosporide and cordifolide), one alkaloid (tinosporin) and δ -sitostrol. • Concentrated alcoholic extracts of fresh stems on fractional distillation reported to have three bitter compounds i.e. tinosporon, tinosporic acid and tinosporal and their structures were established by IR absorption spectra. • Antidiabetic effect of aqueous and alcoholic extracts of the stems of Tinospora Sinesis was reported. • Two compounds were isolated from petroleum ether extract of leaves of Tinospora Sinesis which were identified by TLC on silica gel G plates as octacosanol and β -sitosterol. • From the alcoholic extract of fresh stems, β -sitosterol and a compound named tinosporidine have been isolated. Petroleum ether extract of the leaves gave heptacosanol and a carbonyl compound named cordifolone. • Tinosporide, a diterpene isolated from stems of spectral grounds. 3 Tinospora Sinesis

reported with molecular formula, C23H26O8 and structure was assigned on

A new clerodane furanolactone with molecular formula, C20H22O8 has been isolated from stem and its structure was established by 1H NMR and C13 NMR.

The stem wood of Tinospora Sinesis had yielded a novel 18 norclerodane diterpene O- glucoside which has been named tinosporaside and its structure can be assigned on the basis of NMR studies.

A novel furanoid diterpene glucoside, C26H34O11 was isolated from the hot chloroform extract and its structure was determined by spectroscopic and chemical methods.

Structure of columbin, a diterpenoid furanolactone was established by IR and NMR spectra.

The chemistry and pharmacology of Tinospora Sinesis was studied which includes the constituents which are present in stems, leaves, roots and aqueous extract alcoholic extract gives anti-stress and immunomodulatory activity.

Several glycosides were isolated, as polyacetates, from butanol fraction of stems and structure of 3 new norditerpene furan glycosides, Cordifoliside A, B and C were established by 1D and 2D NMR spectroscopy.

Phenylpropanoid glycosides ad tetra hydro furofuran lignan glycosides were isolated from the plant drugs Tinospora Sinesis and Drypetus roxburghii.

Tinosponone and tinocordioside have been isolated from stem and structure were established by spectroscopic studies and chemical correlation. 4

Cordifoliside D and E were isolated as tetracetates from polar butanol extract and structural elucidations and relative configurations were based on high resolution 1 D and 2D NMR spectroscopy.

The structure of cordioside, isolated from the stem was characterized on the basis of NMR spectroscopy.

Jatrorrhizine, iscolumbin, palmatine, tetrahydro disaccharides were isolated and screened for immunostimulant activity and structure were elucidated on the basis of spectroscopic evidences.

Palmatosides C and F have been isolated as tetracetates from butanol fraction and structure were elucidated by NMR studies.

By using NMR spectra, structure of steroid, isolated from the ethyl acetate extract of aerial parts of Tinospora Sinesis has been established.

A new daucane-type sesquiterpenes, tinocordifolioside and tinocordifolin has been isolated from the stems and their structure were established by detailed spectroscopic studies.

Two photoecdysones viz. ecdysterone, makisterone A and N-trans feruloyltyramine were isolated from butanol fraction of methanol extract of stems and structure were elucidated by NMR studies.

The active principle of Tinospora Sinesis were found to possess anticomplementary and immuno-modulatery activities. Syringin and cordiol inhibited invitro immunohemolysis of antibody coated sheep erythrocytes by guinea pig serum.

Prince, P.S.M. et. al. reported hypolipidaemic and antioxidant activities of Tinospora Sinesis roots in experimental diabetes. 5

Menon, V.P. et. al. reported rats.

hypoglycemic and other related actions

of aqueous extract of Tinospora Sinesis roots in allloxan-induced diabetic

Grover. J.K. et. al. reported anti-hyperglycemic effect of Eugenia jambolana and Tinospora Sinesis in experimental diabetes and their effects on key metabolic enzymes involved in carbohydrate metabolism.

Kar, A., et. al. reported comparative evaluation of hypoglycemic activity of 30 Indian medicinal plant in alloxan-induced diabetic rats which includes Tinospora Sinesis.

Grover, J.K., et. al. reported 45 medicinal plants and their products (active, natural principles and crude extracts) for anti-diabetic potential which includes Tinospora Sinesis.

The structure of tinosporide has been revised from the previously (Ahmad 1978) suggested structure on the basis of its IR, NMR and mass spectra and by chemical correlations.

Guduchisatwa, an Ayurvedic drug derived from Tinospora Sinesis was mainly composed of a 1 → 4 – linked glucan. Chemical and enzymatic studies on the glucan revealed that they have both similarities and differences from amylase.

The stems of Tinospora Sinesis were found to contain magflorine and its biogenetic precursor tembetarine and identified by their methyl derivatives.

The occurrence of quaternary Alkaloids was studied. The components were proterberine bases, berberine and palmatine, with jatrorrhizine, an

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occasional minor constituent, and the apomorphine base, magnoflorine. The presence of choline was also reported. • A new diterpenoid furanolactone has been isolated from the stem of Tinospora Sinesis. Its structure was established by 1H NMR and C13 NMR studies. • Systematic chemical investigation of Tinospora Sinesis yielded a new phenolic lignan was shown by a combination of spectroscopic and chemical methods. • A new clerodene diterpenoid, C20H21O7, has been isolated from stem and its structure was established by spectroscopic means and by comparison with closely related clerodene derivatives. • Chloroform extract of stem yielded new clerodene derivative with molecular formula, C20H22O8 and structure was established by IR spectra.

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OBJECTIVE Diabetes mellitus, a common metabolic disorder known as fast spreading disease with varied etiology and is associated with variety of irreversible complications. The modern management of diabetes, in spite of newer developments, remains unsatisfactory. Presently used antidiabetic drugs have been found to have limitations in therapeutic use, primarily, because or their undesirable and untoward effects. Chlorpropamide like sulphonylureas causes severe cholistatic jaundice and occasionally pulmonary eosinophilia, whereas, hypoglycemia, allergic reactions like leucopenia, thrombocytopenia, agranulocytosis, etc. and cardiovascular complications i.e. stroke and myocardial infarction being common side effects of other sulfonylureas. Biguanides are associated with less severe reactions like anorexia, nausea and abdnominal discomfort. Phenformin is contractindicated in patients with liver, kidney and cardiac diseases because of its ability to produce lactic acidosis. Thiazolidinedione derivatives like rosiglitazone, pioglitazone and

troglitazone are always at a high risk of liver toxicity. They are always prescribed with a constant check on liver toxicity. Thus there is continuous need to develop and antidiabetic drug in view of alarming rise in diabetic patients worldwide. The search for natural sources still leads to drug discovery and drug design, and has established with unexpectedly fast developing biotechnology and biomedical fields. The humanity is turning back towards increasing reliance on herbal medicine.

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Literature survey revealed that the stem of plant Tinospora Sinesis (Menispermaceae) is traditional in Indian system of medicine for treatment of diabetes. The constituents of Tinospora Sinesis stem are Diterpenoids (Clerodane derivative), Alkaloids, Steroids, (β -Sitosterol), Lignan (Phenolic Octacosanol, Heptacosanol and Nonacosa-15-one. Therefore the objective of present study was to screen different extracts for antidiabetic activity and to isolate the constituent responsible fro antidiabetic activity. lignan),

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PLAN OF WORK 1. Procurement of plant material. 2. Drying and size reduction of stems. 3. Extraction with – (a) Petroleum ether (60-80º C) (b) Benzene (c) Chloroform (d) Ethyl acetate 4. Determination of hypoglycemic activity of each extract by using Alloxaninduced diabetic rats. 5. Preliminary phytochemical screening of active extract to isolate the presence of active constituent. 6. Thin layer chromatography of active extract to determine the presence of number of chemical constituents. 7. High performance thin layer chromatography (HPTLC) of active extract to confirm the number of constituents present. 8. Separation of chemical constituents of active extract by using column chromatography. 9. Phytochemical screening of the separated constituents. 10. Screening of hypoglycemic activity of separated constituents by using Alloxan-induced diabetic rats. 11. Physio-chemical studies of separated constituents: (a) Melting point (b) UV spectra (c) IR spectra (d) CHN analysis (e) Mass spectra (f) NMR spectra

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Experimental
LIST OF CHEMICALS AND EQUIPMENTS USED List of Chemicals 1. Petroleum ether (60-80) (LR) 2. Benzene (LR) 3. Chloroform (LR) 4. Ethyl acetate (LR) 5. Methanol (AR) 6. Chloroform (AR) 7. Acetic acid 8. Hydrochloric acid 9. Silica gel G (For TLC) 10. Silica gel (for column chlromatography) (60-120 mesh) 11. GOD / POD Kit

List of Equipments 1. Single pan balance 2. pH meter 3. Centrifuge 4. UV spectrophotometer 5. Soxhlet apparatus 6. Chromatographic column 7. TLC chamber and plates 8. H.P.T.L.C. 9. IR. 10. Melting point apparatus

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PROCUREMENT OF PLANT MATERIAL The plant of Tinospora Sinesis (Wild) was procured form indigenous Drug Market of Nagpur. Identification of the plant material was through an examination of flowering top specimen. Identity of the plant material was confirmed by the outer surface which was grayish brown to almost black in colour, longitudinally wrinkled which showed a soft pale yellow or dirty white surface with marked wedge-shaped structure brought about by radiating medullary rays. The plant material was identified and authenticated from the Department of Botany, Nagpur University Campus, Nagpur. The herbarium sheet and authentication certificate should below : The authentication number is Acc. No. 7/1.

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DRYING AND SIZE REDUCTION Stems were subjected to drying in normal environmental conditions under shade and then subjected for size reduction to coarse power by pulverization. EXTRACTION Powdered stems (250 g) were charged into soxhlet apparatus and successive hot continuous extraction was carried out using following solvents 1. Petroleum ether (60-80º C) 2. Benzene 3. Chloroform 4. Ethyl acetate Powdered stem (5 kg) were extracted with each of the solvent. Each time before extraction with the next solvent, the powdered material was air dried. Each time extract was filtered using suction and each extract was concentrated by distilling the excess of solvent to obtain the crude extractive. The drug was extracted with each solvent until complete extraction was affected (about 40 cycles). EVALUATION OF HYPOGLYCEMIC ACTIVITY OF EACH EXTRACT The determination of blood glucose was most frequently carried out test in the laboratories for screening of hypoglycemic activity and it was done using glucose oxidase / peroxides method. Other methods used were done in three stages. 1. Precipitation of the blood proteins. Reduction, ether of an alkaline cupric copper solution to cuprous oxide, or of alkaline potassium ferricyanide to ferrocyanide.

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Working Reagent Preparation Reagent 2 was mixed with the volume of Regent 1 indicated on the label. The reconstituted reagent was stable up to three months at 2-8º C or one month at 20-25º C. Laboratory Procedure for End point Test Working Reagent Standard Sample Blank 5.0 ml Standard 5.0 ml 5.0 ml Sample 5.0 ml 0.05

Blank, standard and sample were mixed properly and optical density was read after 20 min. incubation at room temperature. Calculations : Glucose (mg/d1) = O.D. Sample ---------------------O.D. (Standard) Where : O.D. = Optical Density
PRELIMINARY PHYTOCHEMICAL SCREENING OF ETHYL ACETATE EXTRACT

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The plant may be considered as a biosynthetic laboratory for a multitude of compounds like alkaloids, diterpenoids, sterols, phenolic lignans, glycosides, etc. that exerts physiological effect. The compounds that are responsible for therapeutic effect are usually the secondary metabolites. A systematic study of a crude drug embraces through consideration of both primary and secondary metabolites derived as a result of plant constituents.

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The ethyl acetate extract was subjected to preliminary phytochemical screening for the detection of various plant constituents. 1) Test for Sterols and Terpenoids i) Salkowski Reaction To a few mg of the residue in 2 ml chloroform, conc. Sulphuric acid (2 ml) was added. The reagents were stirred for few minutes. The red colour was developed in chloroform layer and acid layer gave greenish yellow fluorescence indicating the presence of sterols. ii) Liebermann-Burchard Reaction To a few mg of the residue, chloroform was dissolved, few ml of acetic anhydride and 2 drops of concentrated sulphuric acid from the side of the test tube were added. The greenish transient colour indicates the presence of sterols. The pinkish red colour indicates presence of terpenoids. 2) Test for Alkanoids To a few ml of each extract, 5 ml of 1.5 v/v HCI was added and filtered. Those filtrates were used for testing alkaloids. i) Dragendroff’s Reagent To solution A (17 g of bismuth subnitrate + 200 g tartaric acid + 800 ml distilled water), solution B (160 g potassium iodide + 400 ml distilled water) was added and mixed in 1:1 v/v, from this solution, working standard was prepared by taking 50 ml of this solution and adding 100 g of tartaric acid and making up to 500 ml with distilled water. The above reagent was sprayed on a filter paper and the paper was dried. The sample solution in dilute hydrochloric acid was applied on the paper using a capillary tube.

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Formation of orange red colour indicated the presence of alkaloids in them. ii) Mayers Reagent (Potassium Mercuric Oxide) To 1.38 g of mercuric chloride, 60 ml of distilled water and 5 g of potassium iodide in 10 ml of distilled water were added. The solution mixed and diluted to 100 ml with distilled water. To a little of the extract dilute HCI was added in a watch glass, few drops (excess dissolves the precipitate) of the reagent were added. Formation of cream coloured precipitate showed the presence of alkaloid. iii) Wagner’s Reagent (Iodine – Potassium Iodide) To 1.27 g of iodine & 2 g of potassium iodide, 5 ml of water was added and solution was diluted to 100 ml, to this reagent when a little or the filtrates (i.e. Acid solutions of the extracts) was added, a brown flocculent precipitated which indicate the presence of alkaloids. 3) Test for Saponins Foam Test To a few mg of residue of each extract in a test tube, small amount of sodium bicarbonate and water was added separately and shaken vigorously, stable froth was obtained which indicate the presence of saponins 4) Cardiac Glycosides Keller Killiani Test To a few mg of the residue of the extract, 5 ml alcohol (70%) was added and boiled for two to three minutes and filtered. To the filtrate, 2 ml of water and strong lead acetate solution were added. The solution was filtered and the filtrate was extracted with chloroform. The chloroform layer separated

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and evaporated slowly in a porcelain dish. To the residue, 1 ml of glacial acetic acid containing one drop of ferric chloride was added and this was carefully transferred to the test tube containing 2 ml of sulphuric acid. A reddish brown ring at the junction of two layers indicated positive results for the deoxy sugars and therefore cardiac glycosides. Legal Test To a residue, pyridine and sodium nitroprusside solution was added and make it alkaline. The pink colour indicates the presence of cardiac glycoside. 5) Cyanogenetic Glycosides Guignard Test To 2 g of moist shredded powder stem in a test tube, few drops of chloroform was added to enhance the enzymatic activity. Sodium picrate solution (5 g sodium carbonate, 0.5 g of picric acid, water q.s. 100 ml) was prepared & strip of filter paper were then moistened with this solution. The strips were removed, dried and then inserted between the split cork stopper which was exercised to ensure that the paper strip did not touch the inner sides of the test tube. The test tube was then warmed at 30-35º C for half an hour. Absence of a red color on paper indicated the absence of HCN evolution and hence the absence of cyanogenetic glycosides, 6) Test for Anthraquinone Glycisides Bontrager Test To a little of the extract, 5 ml of 10% sulfuric acid was added and boiled for a few minutes and filtered while hot. The filtrate was cooled and shaken with benzene. The benzene layer was separated and shaken with half of it’s

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Volume of 10% ammonia. Ammonical layer acquired pink colour indicating the presence of anthraquinone. 7) Test of Sugars Mollisch’s Test To a few mg of extracts in test tube, 0.5 ml. of water was added and mixed with two drops of 10% solution of naptol in alcohol, then 1 ml of conc. H 2SO4 through the sides of inclined test tube was added, so that the acid from a layer beneath the aqueous solution at the common surface of the liquids, shake and was added allowed the mixture to stand for two minutes, then 5 ml of water. A dull violet precipitate appeared. It showed the presence of sugar. 8) Test for Lignans To a few mg of extract, 500 ml of conc. HCI and 2% furfuralhehyde were added. The development of red colour indicated the presence of lignans.

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THIN LAYER CHROMATOGRAPHY OF ETHYL ACETATE EXTRACT 1) Preparation of Plate Silica gel G TLC plates were prepared by coating glass plates with slurry composed of 18 g of adsorbent and 45 ml of water. After air drying for 30 min. at room temperature (27º C) Plates were activated in an oven at 105º C for 1 hr. The plates were cleaned from back side and from edges. Before using these plates, they were marked with scale, fixing the distance to be travelled by solvent from using sharp pin. 2) Application of Sample For applying samples on plates, glass capillaries were used. The distance between two spots was kept at minimum of 1 cm. 3) Development of Chromatogram The chromatogram was developed by the ascending technique in which plate was immersed in the developing solvent to a depth of 0.5 cm. The chamber used was lined with sheets of filter paper which dip into the solvent, this ensures that the chamber was saturated with solvent vapour. Development was allowed to proceed until the solvent front has been traveled the required distance (usually 10-15 cm), then the plate was removed from the chamber and solvent front was immediately marked with a pointed object. The plate was dried using heat or a current of air as appropriate. Spray reagents used for visualizing components on chromatogram are benzoyl chloride-sulfuric acid reagent and modified Liberman-Burchard reagent.

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The Rf value was calculated as follows: Rf = Distance travelled by the sample ------------------------------------------Distance travelld by the solvent HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY OF ETHYL ACETATE EXTRACT 1) Selection of Solvent System The solvent system which was developed for TLC was used for HPTLC and it showed same result was obtained. 2) Application of Sample A small quantity of extract was dissolved in ethyl acetate and 5 ul sample was applied on precoated plate with the help of Linomet IV applicator. 3) Development of Chromatogram A rectangular twin trough glass chamber was used in the experiment. To avoid insufficient chamber saturation and the undesirable edge effect, a smooth filter paper was placed in the glass chamber and was allowed to be soaked in the developing solvent. The moistened paper was pressed against the walls of the chamber so that it adheres to the walls. The chamber was allowed to saturate for 15 min. before use. The experiment was carried out at room temperature in diffused day light. Procedure The plate was dipped in a saturated chromatographic chamber containing the solvent system and was allowed to elute upto 8 cm and was air dried. The Spots were scanned in CAMAG TLC scanner-3.

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Chromatographic Condition Following are the chromatographic conditions required to get and effective resolutions by selected mobile phase. Stationary phase Size Developing chamber Mode of application Band size Separation technique Temperature Saturation time Scanning wavelength Scanning mode : HPTLC precoated, silica gel 60 F254 (Merck, Germany : 5 X 10 cm : Twin trough glass chamber : Band : 5 mm : Ascending : 20 ± 5º C : 15 min. : 350 nm : Absorbance / Reflectance

Column Chromatography of Ethyl Acetate Extract 1) Selection of Mobile Phase The solvent system development for TLC and HPTLC was used as mobile phase for column chromatography. Alternation in the composition of the eluting solvent was achieved by adding the second solvent gradually to a reservoir of the first with efficient mixing. This is known as gradient elution which, with proper choice of adsorbent and gradient reduce tailing of the compound on the column. 2) Preparation of the Column The slurry of Silica gel G (60-120 mesh size) was prepared by mixing the adsorbent with the mobile solvent. Solvent poured into the column. A cotton wool was placed at the base of the column and an air bubble inside the

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cotton wool were removed. Small amount of sand poured into the column in order to provide flat base. Slurry of adsorbent poured gradually and allowed to settle. The air entrapped was removed by stirring with glass rod. A filter paper disc was placed above the adsorbent. Sand was added. The excess solvent then run off of until the level falls of 1 cm above the top layer of sand. 3) Preparation and Application of Sample A saturated solution was prepared by dissolving the extract in methanol. 5 ml of sample was applied to the column. 4) Collection of Fractions 5 ml fraction was collected each time.

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SUCCESSIVE SOLVENT EXTRACTION OF STEMS OF TINOSPORA SINESIS On successive solvent extraction of the dried coarse powdered stems of Tinospora Sinesis with different solvents, differing in their polarity, resulted in separation of constituents of different polarity range present in different extracts. The colour, consistency and percentage extractive values were included in table 1. Table No.1: The Colour Consistency and Percentage Extractive Values of Extracts Sr. No. 1 2 3 4 Solvent Petroleum ether (60-80º C) Benzene Chloroform Ethyl Acetate Color and Consistency Greenish yellow, Oily Brown, Semisolid Greenish Brown, Semisolid Dark Brown, Solid % Extractive Value w/w 4.2 0.49 0.20 1.9

SCREENING OF HYPOGLYCEMIC ACTIVITY OF DIFFERENT EXTRACTS All extracts obtained were subjected fro evaluation of hypoglycemic activity in Alloxan-induced diabetic rats. Metformin was taken as standards and administration of Metformin (500 mg/kg) showed significant hypoglycemic activity in Alloxan-induced diabetic rats as compared with control. Among the various extracts, only ethyl acetate extract significantly decreased blood glucose level as compared to the control group. Other extracts did not show significant decrease in blood glucose level.

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Phytochemical examination of ethyl acetate extract: Different test were performed to identify the classes of compounds present in the extract which shows the presence of saponins and sugars. It is shown in table 5. Table 5 Plant constituents 1. Sterols 2. Alkaloid Test a. Salkowski reaction b. Liberman Burchard reaction a. Dragondorf’s reagent Mayer’s reagent 3. Saponin 4. Cardiac glycoside c. Wagner’s reagent a. Foam test a. Keller Killiani test b. Legal test 5. Cyanogenic glycoside 6. Anthraquinone glycoside 7. Flavonoids 8. Sugars c. Kedde’s test a. Guignard test a. Bontrager;s test Magnesium turnings test Mollisch’s test Inference + + +

Table 6. Thin Layer chromatography of ethyl acetate extract Constituent I 1 0.0875 2 0.1586 3 0.1708 4 0.1992 Listed in the order of Rf values Solvent systems: I II III Rf value in solvent system II III 0.1043 0.0163 0.2935 0.7096 0.3114 0.8112 0.3495 0.9154

- Chloroform–formicacid-methanol (4:1:1) - Chloroform-acetic-acid-methanol (5:1:1) - Chloroform-methanol-acetic acid (10:1:1)

PHYTOCHEMICAL SCREENING OF ETHYL ACETATE EXTRACT As the ethyl acetate extract showed hypoglycemic activity, further screening of ethyl acetate extract was performed to isolate active constituents in the extract. The results showed in table 7.

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The ethyl acetate extract showed presence of diterpenoids, as active constituents. Table No. 7: Phytochemical Screening of Ethyl Acetate Extract Sr. No. 1 2. 3 4 5 6 7 8 9 Sterol Alkaloids Terpenoids Cardiac Glycosides Cynogenetic Glycosides Anthraquinone Glycosides Saponins Sugars Lignan Plant Constituents Tests/Reactions i) Salkowaski ii)ibermannBurchard i) Dragendroff’s ii) Wagner’s iii) Mayer’s i) Libermann Burchurd i) Kellar Killani ii) Legal i) Bontrager’s i) Bontrager’s i) Foam i) Mollish i) Methoxylene Group Inference - Ve + Ve - Ve - Ve - Ve + Ve - Ve - Ve - Ve - Ve + Ve - Ve - Ve

+ Ve → Presence of Chemical Constituents. - Ve → Absence of Chemical Constituents THIN LAYER CHROMATOGRAPHY OF ETHYL ACETATE EXTRACT The ethyl acetate extract was subjected to TLC. Various solvent systems were tried, of which the most suitable was Chloroform: Methanol: acetic acid (9:1:0.1). The spots were located by iodine vapour chamber and UV lamp. The ethyl acetate extract showed 5 spots, which indicate the

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COLUMN CHROMATOGRAPHY OF ETHYL ACETATE EXTRACT The constituents were separated by column chromatography using gradient elution technique. The constituents separated were collected in fractions indicated in Table 8. Table No.8 Column Chromatography of Ethyl Acetate Extract Solvent Chloroform : Methanol ( 9 : 1) Chloroform : Methanol (7 : 3) Chloroform : Methanol (5 : 5) Fraction (5 ml) 1-8 9-15 16-19 20-24 25-28 29-.31 32-35 36-40 40-45 Constituents V V, IV IV IV, III III III, II II II, I I Detection Detection on Preparative TLC using ( 9 :1 ) Chloroform : Methanol solvent system

Purification : preparative TLC.

Purification of the constituents obtained was performed by

PHYTOCHEMICAL SCREENING OF SEPARATED CONSTITUENTS The separated constituents were subjected to Liberman-Burchard test for the presence of terpenoids. Of the 5 constituents, constituents 2, 3, 4 and 5 confirmed terpenoids. Table No. 9: Phytochemical Screening of Separated Constituents Sr. No. Constituents 1. 1 2. 2 3. 3 4. 4 5. 5 + ve → Presence of terpenoide. - ve → Absence of terpenoids. Test for Terpenoids - ve + ve + ve + ve + ve

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Silica gel chromatography of ethylacetate extract. Table 10 Solvent Chloroform-methanol (10:1) Chloroform-methanol (7:3) Chloroform-methanol Fractions (5 ml each) 1-17 18-21 2225 26-29 3035 41-45 Constituents None IV IV, III III III, II I II II II II I Detection

(5:5) 46-51 Detection by TLC in solvent system Purification

Purification of the compounds obtained was done by preparative TLC. Physicochemical data of constituents: 1. Melting Point Melting points of the separated constituents were shown in the table 11. Table 11 Sr. No. 1 2 3 4 Constituent 1 2 3 4 Melting point (ºC) 212.4-216.8 198.4-201.8 226.6-228.2 280.1-282.3

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2. U.V. Spectrum of constituents U.V. spectrum was taken by dissolving the constituents in methanol. The absorbance maximas are shown in table 12. Table 12 Sr. No. 1 2 3 4 Constituent 1 2 3 4 Absorbance Maximas (nm) 220 225, 269 222, 257, 370 223, 270, 229

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BIBILIOGRAPHY • • Advances in Pharmacology and Toxicology Volume 7 (2006) Page No El-Shenaway S.M, abdel –Salam O.M. Baluomy A.R, El –Bartram S, Arbid M.S.(2002) . • • 723 . • Kiritikar K.R. Basu b.d.;Indian medicinal plants Vol II ;second edition third reprint international book disrributers Page No 1151 . • Kiritikar K.R. Basu b.d.;Indian medicinal plants Vol II ;sudhindra nath ebasu Ghos, m.n.(1994) .Fundamental of Experiperimental pharmacognosy. Indian Drugs ;November 2005 ;Indian drug manufactures association; Page No 13-14.

M.B.Bahardur gang,allahbad ;Page No. 462. • • Qadry J.S. ;pharmacognosy ; twelth edition; B.S.Shah prakashan . Wallis T. E. Text Book of pharmacognosy; fifth edition ;CBS Publication& Distributers. • • • www.Google.com www.pubmed.com www.siencedirect.vom

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