Phytoehemical Studies OH Momordica spp. Linn, and Extraction and Isolation of Charantin from the fruit of M.charantia L.

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1 MM Jour. Myan. Acad. Arts & Sc 2005 Vol. HI. No. 4(ii) Botany Phytoehemical Studies OH Momordica spp. Linn, and Extraction and Isolation of Charantin from the fruit of M.charantia L. Sanda Hlaing*, Htin Aun fc Kyaw" Abstract The genus Momordica belong to the family Cucurbitaceae. In Union of Myanmar 5-species of the genus Momordica was found Preliminary phytochemical test was carried out on the fruits of 5-species. Extraction and isolation of the active steriodal glucoside charantin compound was done on the fruit of M.charantia L. The compound charantin was confirmed by thin layer chromatography, melting point, and U.V, IR spectroscopic methods. Introduction The present era. can not ignore the important role of plant kingdom in solving the human health problems. Most of the countries have been explored and disseminated the traditional medicine knowledge of the plants so that directly or basically useful information could be postulated for the effective drug development. Myanmar has a long history of health care system by medicinal plants as a national heritage. For promotion and development of traditional medicine various categories of research work such as (a) identification of plants (b) chemical analysis (c) bio-activity tests are needed. In the present research work the genus Momordica L. is selected because it is famous in Myanmar as foods and also as medicine. Now a day, chemical compound charantin which is isolated from the fruit of M.charantia L. showed the activity on diabetes, and other compounds present are also active on tumor and HIV infections. * Lecturer, Department of Botany, University of Dawei, Dawei ** Deputy Director, Pharmaceutical Research Dept., Myanma Scientific & Technological Research Department

2 226 Jour. My an. Acad. Am & Sc Vol. in. No. 4(ii) Botany Materials and Methods In this research work, plants material were collected from Yangon and Bago division. The collected specimens were identified at Botany Department, Yangon University and chemical studies were carried out at the Laboratory of Pharmaceutical Research Department, Myanmar Scientific & Technological Research Department. The commercial grade reagents from BDH, MERCK, etc. were used except ethanol and petroleum ether (b.p C) which are local products. These local reagents were distilled for two times before use. Chemical Studies Preliminary Fhytochemical examination of the fruits of genus Momordica L. species Fresh unripe fruits of M.chararitia L, M.dioica Roxb., M.subangulata Blume., M. cochinchinensis Spreng. and M.muricata L were collected from Yangon & Bago division and dried in the hot air oven at the temperature of 50 C. Dried material were grinded to get powder and stored in an air tight containers. Preliminary phytochemical examination for the fruits was carried out (Central Council of Research in Unani Medicine 1987, Trease and Evens 1980). Test for Alkaloids 1 g of powdered plant material was boiled with dilute hydrochloric acid and filtered. The filtrate was subsequently divided into three portions and tested with alkaloid reagents, viz Mayer's reagent, modified DragendorfPs reagent and Sodium picrate solution. Treatment with the above-mentioned alkaloid reagents furnished alkaloid precipitate, indicating the presence of alkaloids in the plant material' Test for Glycosides 1 g of powdered plant material were boiled with distilled water for about 10 minutes, allowed to cool and filtered. The filtrate was treated with 10

3 JO ; SLJ. Jour. Myan. Acad. Arts & Sc 2005 Vol. III. No. 4(ii) Botany 227 % lead acetate solution. Precipitation took place on the addition of the reagent indicating the presence of glycosides in the plant material. Test for Saponin Glycosides 0.5 g powdered plant material was introduced into a test tube and shaken vigorously with distilled water for a few minutes. Marked frothing which lasted for about half an hour took place, indicating the presence of saponin glycosides. Test for Cyanogenic Glyccsides 1 g of powdered plant material was mixed with distilled water and introduced into a test tube. A few^drops of concentrated sulphuric acid was added and sodium picrate paper trapped in a neck with a cork. Sodium picrate paper did not change in colour brick-red, indicating the absence of cyanogenic glycosides. Test for Reducing Sugar 1 g of powdered plant material was boiled with dilute sulphuric and filtered. The filtrate was then neutralized with dilute sodium hydroxide solution. When the resulting solution was treated with Benedict's solution, it furnished brick-red precipitates, indicating the presence of a reducing sugar. Test for Steroids Tne powdered plant material 1 g was extracted with benzene and the solvent was removed by distillation under reduced pressure. When the benzene extract was treated with acetic anhydride and concentrated sulphuric acid, it furnished a green colour indicating the presence of steroids in the plant materials.

4 228 Jour. Myan. Acad. Arts & Sc Vol. III. No. 4(ii) Botany Test for Phenolic Compounds The powdered plant material 1 g was boiled with distilled water and filtered. When the filtrate was treated with neutral ferric chloride solution, it gave blue colouration, indicating the presence of phenolic groups. Test for a-amino Acids 1 g of powdered fruits were boiled with water and filtered. An aqueous portion was drop onto a filter paper, allowed to dry and sprayed with ninhydrin reagent. The filter paper was allowed to dry at room temperature and then kept in an oven at 110 C for a few minutes. Pink spots were observed on the filter paper showing the presence of a-amino acid. Test for carbohydrates 1 g of dried powdered fruit was boiled with distilled water and filtered. The filtrate was introduced into a test tube and a few drops of 10% a-naphthol was added and shaken. The test tube was then inclined at an angle of 45 and concentrated sulphuric acid was added slowly along the side of the test tube. A red ring was formed between the two layers, showing the presence of carbohydrate in the plant material. Test for Acidic or Basic Compounds 1 g of powdered plant materials was extracted with distilled water and filtered. When the filtrate was treated with bromocresol green indicator solution, the resultant solution turned blue indicating the presence of organic basic, when the resultant solution did not turn blue or red it is neutral. Test for Tannins 1 g of powdered plant material was boiled with water and the filtrate was divided into two portions.

5 Jour. My an. Acad. Arts & Sa 2005 Vol. III. No. 4(ii) Botany 229 (a) A few drops of ferric chloride test solution was added to the filtrate, a deep blue colour was produced indicating the presence of tannins. (b) A few drops of lead subacetate solution was added to the filtrate. Precipitates were formed, revealing the presence of tannins. Thus, preliminary phytochemical tests have indicated the presence of alkaloids, glycosides, reducing sugars, saponin glycosides, steroids, phenolic compounds, a-amino acid, carbohydrates, organic base or neutral and tannins. The test has shown the absence of cyanogenic glycosides in the plant materials. The result was shown in table 1. Table (1) Preliminary Phytcchemical Test on the fruits of genus Momordica L. Type of product Test reagent Observation M, M 2 Plants M 3 M 4 M 5 1. Alkalodis Mayer's reagent White ppt Dragendorff s reagent Sodium picrate solution Orange ppt Yellow ppt 2. Reducing Sugars Benedict's solution Brick-red ppt 3. Saponin Distilled water Marked frothing - 4. Cyanogenic glycosides Cone: S/A & Sodium picrate paper No colouration Glycosides 10% lead acetate White ppt 6. Steroids Jenzene, Acetic anhydride, and cone:s/a Green colour 7. Phenolic groups FeCl 3 solution Blue colour 8. a-amino acids ^inhydrin reagent *ink colour 9. Carbohydrates 0%a-Naphthol,Conc: S/A Red ring

6 230 Jour. Myatu AcadL Arts & Sc 2005 Vol. III. No. 4(ii) Botany Type of product Test reagent Observation Mi Plants M 2 M 3 M 4 Mj 10. Organic acidic Bromocresol green blue -> Basic basic N N N basic or basic. yellow - Acidic No colour change -> N 11. Tannin (a) Ferric chloride test solution deep blue colour.. (b) lead subacetate solution Brown ppt Mi M 2 M 3 M4 M 5 = M.charantinh. = M.dioica Roxb. = M.cochinchinensis Spreng. = M.subangulata Blume = M.muricata L. = Present - = absent N = Neutral

7 Jour. Myan. Acad. Arts <ft 5c Vol. III. No. 4(ii) Botany 231 Extraction and Isolation ofcharantin Unripe fruit Powder (100 g) I Pet.ehter(b.p60-80C) 4, 600 ml fat (rejected) Marc 80% EtOH 500 ml EtOH Sol" M^ I KOH solution (phio) basifiedsol" Ether Ether jextract distillation Crude Charantin I crystallization with EtOH { Charantin Unripe fruit of Momordica were cut into small slices and dried in the oven at the temperature of 50 C. The dried material were powdered by means of pulverizer. 100 g of unripe fruit powder were mixed with 600 ml of Pet.ether (bp. 60"-80'C) and refluxed for (6) hours and then filtered. The marc was repeated again for (6) hours with Petether and filtered. The marc was mixed with 80% alcohol and extracted at reflux temperature for (6) hours and then filtered. The filtrate was basified with potassium hydroxide solution till phio and kept for (48) hours. The resulting solution was diluted with water and extracted with diethyl ether. The diethyl ether portion was washed with water, and then with dilute hydrochloric acid, and again with water. Anhydrous sodium sulphate was added to the diethyl ether portion and kept over night. The ether was filtered and concentrated to get residue (crude charantin). The residue was dissolved in minimum amount of alcohol and kept in the refrigerator. The crystal was filtered, and the crystals was recrystallized with ethyl alcohol. J i

8 232 Jour. Myan. Acad. Arts & Sc Vol. HI. No. 4(ii) Botany Identification of the Compound Melting point Charantin is a non-nitrogenous neutral substance, and the melting point is 272 C. The melting points of crude charantin and purified charantin were determined by means of capillary method. The result were shown in the table (2). Table (2) Melting point of crude and purified charantin Crystal Crude charantin Purified charantin Observed C C Reported C C Thin Layer Charomatograpay The purified Charantin crystal was applied on the silica gel G thin layer chromatogram. The plate was developed in methanol and benzene, solvent systems at the ratio of 2:8. Then the plate was dried and the spot was visualized with iodine vapour. The Rf value of purified charantin was 0.5. Ultraviolet absorption spectroscopy The wave length of maximum absorption for the charantin compound was measured by using ultraviolet absorption spectrophotometer which X max was 206 nm.

9 Jour. Myan. Acad. Arts & Sc Vol. III. No. 4(ii) Botany 233 i? CO nc 5 O o Vi $? w CO (TJ s z w c m h-?i S o o o EE9H X921 f N N n N 00 N- to O i 'N w n n N (O (B (O u'; 1 cr. CM «o <O C) <- C 9 fi 6 N fl s a to in ic o i- < o 8> O! t- i- (M (N <3> p» CO re m I" CN Xfl (\ n Q9 <O v t S B? E t «- 55 O 00 -r- r- n CM p o 5 = I {J»8 <S> fo C} 30 f"^ U^ (O CO CO O! t- T" CO CO h- CO CM o CO q. -c5 if a; 5 *n (P C7 f*^ <o «S- in in CO N. o en t- co so ss CO 0) 00 O o CO J o 6 o CO

10 234 Jour. Myan. Aca<L Arts & Sc Vol. III. No. 4(ii) Botany Infrared spectroscopy The infrared spectra of charantin showed the presence of conjugated C=C band at 1639 cm" 1, C-O stretching band at 1263, exocyclic methyl band at 907 cm' 1 and hydroxyl group band at 3435 cm" 1. The infrared spectra of charantin compound was shown in Figure No 1. FTIR Absorption Peaks and their Assignment of Compound Charantin is shown in table (3). Table (3) FTIR Absorption Peaks and Their Assignment of Compound Charantin No. Wave Vibrational mode Functional group number UC-H of alcohol & acid UC-H of vinylidene uc=c of alkene 5CH of vinylidene \ y"*i y~*i v H H UC-H of methyl and methylene CH CH of methyl and methylene CH CH of gemdimethyl group uc-o of saturated secondary alcohol >/ \ /H X CH 3

11 Jour. My an. Acad. Arts & Sc Vol. III. No. 4(ii) Botany 235 Discussion Five species of the genus Momordica L. have been studied in this research. Most of the species are wild grown and some species are cultivated for their edible fruits. The preliminary phytochemical examination was carried out on the fruits of five species. The test indicated that all the fruits showed the presence of alkaloid, glycosides, reducing sugar, saponin glycoside, steroids, phenolic compound, a-aminoacid, carbohydrates, organic base or neutral and tannin. The tests shown that the cyanogenic glycoside was absent from all fruits. The active compound charantin was extracted from the fruits of M.charantia L. The compound charantin showed activity on diabetes. According to the literature, the charantin is a mixture of equal parts of P sutisterol glucoside and 5, 25-stigmastadien.3 3-ol glucoside (A.A Olaniyi, 1975). The melting point of the isolated compound was identical with the melting point of charantin. The thin layer chromatogram showed a single spot so that the isolated compound was pure. The maximum absorption of isolated charantin compound was found at 206 nm. According to IR spectrum, the isolated compound showed all the functional group of charantin. So that the isolated compound was charantin. Conclusion The fruit and leaves of M.charantia L.(Bitter Melon) are used to reduce the levels of blood sugar. For centuries, bitter Melon has been used as food and medicine in Asia, as therapeutic remedy in a variety of illnesses such as leukaemia, diabetes, asthma, insect bites, maladies and stomach problems. According to literature M.charantia L. fruit also show anti-hfv properties. M.charantia L. fruits has shown the presence of charantin compound which can control diabetes (Lestile Tylor, 2002). Furthermore investigation, some species of the genus Momordica L. fruits and leaves also show effect on diabetes and also have antimicrobial activity.

12 236 Jour. Myan. Acad. Arts & Sc Vol. III. No. 4(ii) Botany References Backer, L.H. & Bakhuizen, A.F. (1963). Van Den Brink. Flora of Java.Vol. l.n.v.p. Noordhoff Groningen, The Netherlands. Burkill, L.H. (1935). A Dictionary of the Economic Products of Malay Penisula. Vol II. Grown agent for the Oxford University Press, London. Harbone, J.B. (1989). Phytochemical Methods. Chapman and Hall, London. Hooker, J.D. (1879). Flora of British India. Vol II. L. Reeve and Company England. Hundley, H.G. (1978). List of Trees, Shrubs,Herbs and Principal Climbers, etc. 4th Rev.ed. Swe Daw Oo Press, Mayangon. Hutchinson, J. (1967). The Genera of Flowering Plants. Vol II. The Clarendon Press, Oxford. Kirtikar, K.R & Basu, B.D Indian Medicinal Plants. Vol II 2nd ed. The Prabasi Press Calcutta. Kirtikar, K.R. & Basu, B.D. (1938).' Indian Medicinal Plants. Vol II. 2nd ed. The Prabasi Press, Calcutta. Roxburgh, W. (1832). Flora Indica (or) Description of Indian Plants. New Delhi, India. Tylor, L. (2002). Herbal secrets of the Rainforest, 2 nd Edition, Sage Press, Internet. Trease & Evens. (1978). PharamaGognosy. 11 th ed. Bailliere Tindall. London. Wagner, H. & Blaedt, S. (1996). Plant Drug Analysis. Thin Layer Chromatography Atlas. Springer. Berlin. Physiochemical Standards of Unani Formulations. (1986). Part I Delhi. Internet, Indian Medicinal Plants /product /momordica. htm. The Journal of Natural Products, July-August, Vol 38, No.4., The Lloyd Library and Museum and The American Society of Pharamacognosy. The Indian Journal of Pharamacy, (1966). April, No.4, Vol 28, The Indian Pharamaceutical Association.