Meenaa Venkataramani et al. / Journal of Pharmacy Research 2012,5(5),2895-2899 Research Article Available online through ISSN: 0974-6943 http://jprsolutions.info Preliminary phytochemical screening and GC-MS profiling of benghalensis (L.)kurz Meenaa Venkataramani* and Sasikumar Chinnagounder1 *PG and Research Department of Biotechnology, Nehru Memorial College (Autonomous), Puthanampatti, Tiruchirapalli, Tamilnadu. 1Head, PG and Research Department of Biotechnology, Nehru Memorial College (Autonomous), Puthanampatti, Tiruchirapalli, Tamilnadu. India Received on:11-02-2012; Revised on: 17-03-2012; Accepted on:19-04-2012

ABSTRACT The present study is focused on the preliminary phytochemical screening of various leaf extracts of and also in analyzing the phytocomponents using Gas chromatography-Mass Spectroscopy. The preliminary phytochemical screening revealed the presence of Coumarins, Sterols, Lignin, Saponins, Flavanoids, Alkaloids, Tannins, Terpenes and Protein in various leaf extracts of the under study and the GC-MS investigation of the chloroform fraction of the plant retrieved a total of 55 components which have been reported and discussed below.

Keywords: Phytochemical screening, Gas Chromatography–Mass Spectroscopy, Hiptage benghalensis.

INTRODUCTION The Indian subcontinent is a vast repository of medicinal that are used also to quench thirst. According to some researches the therapeutic actions of in traditional medical treatments(1).Chemical principles from natural sources this plant may be due to the presence of mangiferin (13), which is known to be have become much simpler and have contributed significantly to the develop- anti-inflammatory, hepatoprotective, antioxidant, and antimicrobial (14),(15). ment of new drugs from medicinal plants (2-3). Biologically active compounds The pharmacological potentials of H.benghalensis Kurz and the various from natural sources have always been of great interest to scientists working phytocomponents attributing to it still remain unexplored. Hence the present on infectious diseases. Research to find out scientific evidence for claims of investigation focuses on the preliminary phytochemical screening of aque- plants used for Indian Ayurvedic system of medicine has been intensified. In ous, ethanol, methanol and chloroform leaf extracts of H.benghalensis col- recent years the thirst to investigate on the bioactive components of the lected from Kolli hills, Tamilnadu and analysis of the phytoconstituents by plants has been incredibly focused on. Chromatographic separation with Gas chromatography and Mass Spectroscopy. mass spectrometry for the chemical characterization and composition analy- sis of botanicals has been growing rapidly in popularity in recent years. MATERIALS AND METHODS:

Even though global herbal resources have a great potential as natural drugs Plant Material and are of great commercial importance, they are very often procured and Aerial parts of the selected plant were collected from the Eastern Ghats, processed without any scientific evaluation, and launched onto the market Tamilnadu. The taxonomic identification and authentication of the plant was without any mandatory safety and toxicology studies (4-5). The lack of phar- certified (PARC/2012/1238) by Prof.Dr.P.Jayaraman, Director, Institute of macological and clinical data on the majority of herbal medicinal products is Herbal Botany, Plant Anatomy Research Centre, Chennai. a major impediment to the integration of herbal medicines into conventional medicinal practice. For valid integration, pharmacological studies must be Extraction of Plant material conducted on those plants lacking such data (6,7,8). The list of many such The collected leaves were dried, coarsely powdered using electrical grinder undocumented, unexploited and uncharacterized plants of medical impor- and were subjected to cold and hot extractions. A definite quantity of the tance have been brought to light by many researchers and one such unex- powder was weighed (1:3) and soaked in the respective solvents for 2 days at plored plant is Hiptage benghalensis. room temperature with intermittent shaking and on the 3rd day, extracts were separated using Whatman No.1 filter paper. The hot extraction of the plant Hiptage benghalensis (L) Kurz. belongs to the family Malphigiaceae. It is a was carried over subsequently and with sufficient volume of various organic large, woody climber found in India, Burma, Malaysia, Thailand, China and solvents in the order of increasing polarity. The collected extracts were used the (9-11). The bark, leaves and flower of H.benghalensis are aro- for further analysis. All chemicals and solvents used for different studies matic. They are useful in conditions of burning sensation, wounds, ulcers, were of analytical grade. inflammations, leprosy, scabies, cough and rheumatism (12). The plant has tremendous therapeutic potential with every part of the plant being used Preliminary Phytochemical Screening of various extracts medicinally. The leaves of H.benghalensis (L.) Kurz are used in treating skin Preliminary phytochemical screening of various extracts was carried out as diseases in Burma and the bark is used to heal wounds in . In India, per the standard textual procedure (16). H.benghalensis (L.) Kurz is widely used to treat cough, asthma, leprosy and Test for Saponins: *Corresponding author. The substance was shaken well with water. Meenaa Venkataramani Test for Tannins: PG and Research Department of Biotechnology, The substance was mixed with basic lead acetate solution. Nehru Memorial College (Autonomous), Puthanampatti, Tiruchirapalli, Tamilnadu. India. Test for Steroids: (Liebermann burchard test) The sample was taken in a test tube. Added few drops of glacial acetic acid,

Journal of Pharmacy Research Vol.5 Issue 5.May 2012 2895-2899 Meenaa Venkataramani et al. / Journal of Pharmacy Research 2012,5(5),2895-2899 , 18-JAN-2012 + 10:26:22 Vm chloroform extract 18 01 12 Scan EI+ 17.40;191 31.24 TIC 100 21.36;43 24.79;88 57 1.34e9 23.36 32.90 43 57

18.45 29.40 43 57 34.46 22.12 57 % 43 27.67 71

16.28 29.69 43 21.12 25.67 69 5.39 43 91 9.09 14.89 32.20 43 6.15 19.16 22.67 7.74 41 12.01 164 99 43 222 43 43 128 1 Time 5.56 7.56 9.56 11.56 13.56 15.56 17.56 19.56 21.56 23.56 25.56 27.56 29.56 31.56 33.56 Chromatogram (x-axis = Retention time; y-axis = % intensity/% abundance/ concentration) Figure1: Chromatogram representing the peak areas of Phytocomponents matched with the NIST Library acetic anhydride and 1ml of concentrated Sulphuric acid was added along the extract of the plant was performed using PerkinElmer Clarus 500 equipped sides of the test tube. with a column type Capillary Column Elite-5 (5%Phenyl 95% dimethylpolysiloxane), with a column length of 30 m /0.25mm and a film Test for Terpenoids: (Salkowshi test) thickness of 250µm respectively. The extracts were diluted (1µl/ml) and 1µl The substance was warmed with tin and thionyl chloride. was taken as the injection volume. They were injected in the split mode with 10:1 ratio. The oven was programmed at 50ºC @ 8ºC/min to 220ºC (2min) @ Test for Flavonoids: (Shinado’s test) 8ºC/min to 290ºC (10min) and the injector was maintained at 290ºC. Helium To the substance, added alcohol, few magnesium turnings and few drops of was used as a carrier gas with a constant flow at 1 ml/min. The ionization concentrated hydrochloric acid and boiled for 5 minutes. voltage was 70eV. The constituents were identified after comparison with those available in the computer library (NIST) attached to the GC-MS in- Test for Coumarin: strument and the results obtained are reported. A small quantity of substance was mixed with few drops of 10% sodium hydroxide. RESULTS AND DISCUSSION: Preliminary phytochemical screening of various leaf extracts of the plant Test for Quinones: Hiptage benghalensis revealed the presence of Coumarins, Sterols, Lignin, The substance was mixed with few drops of concentrated sulphuric acid. Saponins, Flavanoids, Alkaloids, Tannins, Terpenes and Protein (Table – 1). Several investigations have been attributed to study the phytochemical Test for Lignins: compounds in H.benghalensis and it was found that the results of the present The substance was mixed with alcoholic solution of phloroglucinol and added phytochemical analysis conducted in this study are in accordance to those few drops of concentrated hydrochloric acid. previous reports on the plant (17)(18)(19)(20).

Test for alkaloids: Table 1: Phytochemical screening of leaf extracts of Hiptage 1. To the substance few drops of acetic acid was added, followed by benghalensis Dragendroff’s reagent and shaken well.

S.NO Test for Chloroform Methanol Ethanol Water 2. The substance was mixed with little amount of dilute hydrochloric acid extract extract extract extract and Mayer’s reagent. 1 Coumarin + - + + Test for Sugars: 2 Sterol + + + - 3 Quinone - - - - The substance was mixed with anthrone and 1 drop of concentrated Sulphuric 4 Lignin + + - + acid and warmed gently. 5 Saponin + + + - 6 Flavonoid - + + + Test for Proteins: 7 Gum - - - - 8 Alkaloid + + + + The substance was mixed with saturated solution of picric acid. 9 Protein + + + + 10 Terpenoid + + + - Gas Chromatography – Mass Spectroscopy analysis 11 Tannin - + + + The Gas chromatography –Mass Spectroscopy analysis of the chloroform + Positive, - Negative.

Journal of Pharmacy Research Vol.5 Issue 5.May 2012 2895-2899 Meenaa Venkataramani et al. / Journal of Pharmacy Research 2012,5(5),2895-2899

Table 2: Phytocomponents identified in the Chloroform fraction of Hiptage benghalensis by GC-MS

S.No. Peak Name Retention Peak %Peak time area area

1 Name: 5,9-Dodecadien-2-one, 6,10-dimethyl-, (E,E))- 5.39 7370377 0.9609 Formula: C14H24O MW: 208 2. Name: Cyclopentanol, 2-methyl-, acetate, cis- 6.15 3335318 0.4349 Formula: C8H14O2 MW: 142 3. Name: 5-Hepten-2-one, 6-methyl- 7.74 2385087 0.311 Formula: C8H14O MW: 126 4. Name: Pentane, 1-(1-butenyloxy)-, (Z)- 8.49 177112 0.0231 Formula: C9H18O MW: 142 5. Name: D-Limonene 8.7 2713036 0.3537 Formula: C10H16 MW: 136 6. Name: 6-Octen-1-ol, 3,7-dimethyl-, formate 9.09 4675808 0.6096 Formula: C11H20O2 MW: 184 7. Name: 2-Isopropenyl-5-methylhex-4-enal 9.2 638941 0.0833 Formula: C10H16O MW: 152 8. Name: 5-Octen-2-one, 6-methyl- 9.58 444371 0.0579 Formula: C9H16O MW: 140 9. Name: Cyclohexane, 2-ethenyl-1,1-dimethyl-3-methylene- 9.84 444478 0.058 Formula: C11H18 MW: 150 10. Name: 4-Methyl-1,5-Heptadiene 10.23 698409 0.0911 Formula: C8H14 MW: 110 11. Name: Cyclopropane, 1-methyl-2-(1-methylethyl)-3-(1-methylethylidene)-, cis- 10.73 319746 0.0417 Formula: C10H18 MW: 138 12. Name: 2-Cyclohexen-1-one, 4-(1-methylethyl)- 11.12 268670 0.035 Formula: C9H14O MW: 138 13. Name: 7-Oxabicyclo[4.1.0]heptan-2-one, 3-methyl-6-(1-methylethyl)- 11.39 59185 0.0077 Formula: C10H16O2 MW: 168 14. Name: Cyclohexanol, 2,3-dimethyl- 11.77 573751 0.0748 Formula: C8H16O MW: 128 CAS 15. Name: Naphthalene 12.01 3173608 0.4138 Formula: C10H8 MW: 128 16. Name: Benzaldehyde, 2,4-dimethyl- 12.42 93658 0.0122 Formula: C9H10O MW: 134 17. Name: (1R,2R,3R,5S)-(-)-Isopinocampheol 12.72 746939 0.0974 Formula: C10H18O MW: 154 18. Name: 2,6-Octadienal, 3,7-dimethyl-, (E)- 13.25 1320005 0.1721 Formula: C10H16O MW: 152 à-Citral 19. Name: Dihydromyrcene 14.18 460839 0.0601 Formula: C10H18 MW: 138 CAS 20. Name: 3-Hexadecene, (Z)- 15.23 495538 0.0646 Formula: C16H32 MW: 224 CAS 21. Name: Octan-2-one, 3,6-dimethyl- 15.31 1778689 0.2319 Formula: C10H20O MW: 156 CAS 22. Name: Cyclohexene, 3-methyl-6-(1-methylethylidene)- 16 99081 0.0129 Formul+C55a: C10H16 MW: 136 Isoterpinolene 23. Name: 4,8,12-Tetradecatrienal, 5,9,13-trimethyl- 16.39 384485 0.0501 Formula: C17H28O MW: 248 24. Name: Tricyclo[4.4.0.0(2,7)]dec-3-ene-3-methanol, 1-methyl-8-(1-methylethyl)- 16.66 387907 0.0506 Formula: C15H24O MW: 220 25. Name: Phenol, 2,4-bis(1,1-dimethylethyl)- 17.4 45747216 5.9645 Formula: C14H22O MW: 206 26. Name: 6,10-Dodecadien-1-yn-3-ol, 3,7,11-trimethyl- 17.52 7409964 0.9661 Formula: C15H24O MW: 220 27. Name: Farnesol isomer a 17.95 1030268 0.1343 Formula: C15H26O MW: 222

Journal of Pharmacy Research Vol.5 Issue 5.May 2012 2895-2899 Meenaa Venkataramani et al. / Journal of Pharmacy Research 2012,5(5),2895-2899 Sl No Peak Name Retention Peak %Peak time area area 28. Name: Epoxy-linalooloxide 18.01 1236814 0.1613 Formula: C10H18O3 MW: 186 29. Name: 1,6,10-Dodecatrien-3-ol, 3,7,11-trimethyl-, (E)- 18.11 1777816 0.2318 Formula: C15H26O MW: 222 ñ-trans-Nerolidol 30. Name: Dodecanoic acid 18.16 1628495 0.2123 Formula: C12H24O2 MW: 200 31. Name: 6,11-Dimethyl-2,6,10-dodecatrien-1-ol 18.23 1869215 0.2437 Formula: C14H24O MW: 208 32. Name: E-15-Heptadecenal 18.45 28195770 3.6761 Formula: C17H32O MW: 252 33. Name: 1,3-Dioxolan-2-one, 3-methyl-3-(4,8-dimethylnona-3,7-dienyl)-4-methylene- 18.76 1182878 0.1542 Formula: C16H24O3 MW: 264 34. Name: 1H-3a,7-Methanoazulene, 2,3,4,7,8,8a-hexahydro-3,6,8,8- 19.37 860978 0.1123 tetramethyl-, [3R-(3à,3aá,7á,8aà)]- Formula: C15H24MW: 204 à-Cedrene 35. Name: 6,10-Dodecadien-1-yn-3-ol, 3,7,11-trimethyl- 19.52 1776044 0.2316 Formula: C15H24O MW: 220 36. Name: Isopulegol acetate 19.6 796127 0.1038 Formula: C12H20O2 MW: 196 37. Name: Diepi-à-cedrene epoxide 19.84 1729819 0.2255 Formula: C15H24O MW: 220 38. Name: 2,6,10-Dodecatrienal, 3,7,11-trimethyl- 20.78 2928433 0.3818 Formula: C15H24O MW: 220 39. Name: Tetradecanoic acid 21.12 9395291 1.2249 Formula: C14H28O2 MW: 228 40. Name: 1-Octadecene 21.36 51528044 6.7182 Formula: C18H36 MW: 252 41. Name: 3,7,11,15-Tetramethyl-2-hexadecen-1-ol 21.99 11186347 1.4585 Formula: C20H40O MW: 296 42. Name: 2-Pentadecanone, 6,10,14-trimethyl- 22.12 22315114 2.9094 Formula: C18H36O MW: 268 43. Name: 5,9,13-Pentadecatrien-2-one, 6,10,14-trimethyl-, (E,E)- 23.36 59773428 7.7932 Formula: C18H30O MW: 262 44. Name: 7,9-Di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione 23.51 3448206 0.4496 Formula: C17H24O3 MW: 276 45. Name: Hexadecanoic acid, ethyl ester 24.79 106602688 13.8988 Formula: C18H36O2 MW: 284 46. Name: Azuleno[4,5-b]furan-2(3H)-one, 3a,4,6a,7,8,9,9a,9b-octahydro- 25.67 19117560 2.4925 6-methyl-3,9-bis(methylene)-, [3aS-(3aà,6aà,9aà,9bá)]- Formula: C15H18O2.MW: 230 47. .All-trans-Squalene 26.85 2276487 0.2968 Formula: C30H50 MW: 410 48. Name: Eicosane 27.25 11638936 1.5175 Formula: C20H42 MW: 282 CAS 49. Name: Phytol 27.67 41501264 5.4109 Formula: C20H40O MW: 296 50. Name: 9,12-Octadecadienoic acid, ethyl ester 28.69 7610675 0.9923 Formula: C20H36O2 MW: 308 CAS 51. Name: (E)-9-Octadecenoic acid ethyl ester 28.81 29080560 3.7915 Formula: C20H38O2 MW: 310 52. Name: 1-Docosene 29.3 40723956 5.3096 Formula: C22H44 MW: 308 53. Name: Docosane 29.4 44065604 5.7452 Formula: C22H46 MW: 310 54. Name: Tetracosane 31.24 81646200 10.645 Formula: C24H50 MW: 338 55. Name: Tetratriacontane 36.03 93869088 12.2386 Formula: C34H70 MW: 478

Journal of Pharmacy Research Vol.5 Issue 5.May 2012 2895-2899 Meenaa Venkataramani et al. / Journal of Pharmacy Research 2012,5(5),2895-2899 The GC-MS chromotagram of chloroform fraction of H.benghalensis is pre- 7. Veale DJH, Oliver DW, Furman KI. South African traditional herbal sented in Fig1. The study revealed the presence of 55 active phytocomponents medicines used during pregnancy and childbirth, J. Ethnopharmacol. and their retention time (RT), molecular formula and concentration (%) have 1992; 36: 185-191. been presented below. Phytochemical compounds are known to play an 8. Anesini C, Perez C. Screening of plants used in Argentine folk important role to identify the bioactivity of medicinal plants. The medicine for antimicrobial activity, J. Ethnopharmacol. 1993; 39(2): phytocomponents reported from the GC-MS study on the leaf fraction of 119-28. the plant has been first of its kind and all the biological activities related to the 9. Gates RB. Banisteriopsis, Diplopterys (). Flora Neotropica Monographs, 1982; 30: 1–236. plant may be due to its major constituents namely Phenol, 2,4-bis(1,1- 10. Bailey LH, Bailey EZ. Hortus. 3rd ed. Macmillan General Refer- dimethylethyl) (5.9645%) which is reported to possess antioxidant prop- ence, New York, USA. 1976. (21) erty , E-15-Heptadecenal (3.6761%) and a - Cedrene, a sesquiterpene by 11. Bor NL, Raizada MB. Some Beautiful Indian Climbers and , nature (0.1123%) are reported to possess anticancer, antioxidant and antimi- Diocesan Press, Madras, 1954. crobial activity (22-23,24) while Tetradecanoicacid (1.2249%), Octadecene 12. Warrier PK, Nambiar VPK, Ramakutty C. Indian medicinal plants: (6.7182%), Hexadecanoicacid (13.8988%), Octadecanoic acid ethyl ester A compendium of 500 , Vol I–V, Arya Vaidya Sala, Kottakkal, (3.7915%) and Eicosane (1.5175%) have also been reported with antioxidant Orient Longman Ltd, India, 1995. and antimicrobial activities . 13. Finnegan RA, Stephani RA, Ganguli G. Occurrence of mangiferin in Hiptage madablota geartn [J] ,J Pharm Sci. 1968; 57(6):1039– CONCLUSION 1040. Traditional knowledge with its holistic and systematic approach supported 14. Shibnath G, Prasad C, Basumatari, Shanta Bannerjee. The An- by experimental base can serve as an innovative and powerful discovery giosperms or Flowering Plants Ind J Chem . 1996; 35(6): 561–566. engine for newer, safer and affordable medicines. The various bioactive 15. Zhu XM, Song JX., Huang ZZ, Wu YM., Yu MJ. Antiviral activ- ity of mangiferin against herpes simplex virus type 2 in vitro, compounds reported from the GC-MS analysis and subsequent literature Chung Kuo Yao Li Hsueh Pao. 1993; 4: 452–454. evidences of their medicinal activities provide ample proof to the therapeutic 16. Dey BB, Sitaraman MV. Laboratory Manual of Organic Chemis- and pharmacological potential of H.benghalensis which needs to be further try, (Vishwanathan S. Publication, Madras, 1957; pp.120. explored and validated so as to use it as a potential force in the field of health 17. Kumudhavalli MV, Jayakar B, Margret Chandira R, Kumar M, care against many diseases. Saravanan C. Phytochemical and Pharmacological studies on leaves of Hiptage bengalensis (L) Kurz, International Journal of Pharm ACKNOWLEDGEMENT Tech Research. 2010; 2 (1): 1017-1020. We place on record our deep sense of gratitude to the Principal and the 18. Chenthurpandy P, Kalidass C, Mohan VR..Pharmacognostical In- Management, of Nehru Memorial College (Autonomous), for providing nec- vestigation of Hiptage benghalensis (L.) Kurz. (Malpighiaceae), essary equipments and constant encouragement through out the work. I also Phcog.net. 2009; 1: 103-105. dedicate my sincere thanks to Dr.P.Brindha, Associate Dean, Centre for 19. Hiptage benghalensis (L).Kurz. Syn. H.madablota Gaert. (Glos- Advance Research in Indian system of Medicine ,SASTRA University, sary Indian medicinal plants-Chopra and Nayer PID, New Delhi – 1956. p.134. Thanjavur for helping me carry over the GC-MS analysis under her guidance. 20. Biren Shah H, Bharat Patel G, Avani Patel V. Evaluation of Anti- Asthmatic activity of Leaves of Hiptage benghalensis(L) Kurz REFERENCE using various experimental animal models, International Journal of 1. Subhose V, Srinivas P, Narayana A. Basic principles of Pharmaceutical research and Bioscience. 2012; 1(2): 236-247. pharmaceutical science in Ayurveda, Bull Indian Inst Hist Med 21. Ajayi GO, Olagunju JA, Ademuyiwa O, Martin OC. Gas chroma- Hyderbad. 2005; 35(2): 83-92. tography-mass spectrometry analysis and phytochemical screen- 2. Cox PA. Ciba Foundation Symposium 154, Chichester, John Wiley ing of ethanolic root extract of Plumbago zeylanica Linn. Journal & Sons, 40 1990. of Medicinal Plants Research.2011; 5(9): 1756-1761. 3. Cox P, Balick M. Sci American. 1994; 82. 22. Lee YS, Kang MH, Cho YS, Jeong CS. Effects of constituents of 4. Dev S. Ethnotherapeutics and Modern Drug Development; The Amomum xanthioides on gastritis in rats and on growth of gastric potential of Ayurveda, Current Sci. 1997; 73: 909-928. cancer cells. Arch. Pharm. Res. 2007; 30: 436-443. 5. Perumalsamy R, Ignacimuthu S. Antibacterial activity of some 23. Devdutt Chaturvedi. Sesquiterpene lactones: Structural diversity folklore medicinal plants used by tribals in Western Ghats of India, and their biological activities, Opportunity, Challenge and Scope J. of Ethnopharmacol. 2000; 69: 63-71. of Natural Products in Medicinal Chemistry. 2011; 313-334 6. John D. One hundred useful raw drugs of the Kani tribes of 24. Agoramoorthy G, Chandrasekaran M, Venkatesalu V, Hsu MJ. Trivandrum forest division, Kerala, India. Int. J. Crude Drug Res. Antibacterial and antifungal activities of fatty acid methyl esters 1984; 22: 17-39. of the blind-your-eye mangrove from India. Brazilian Journal of Microbiology. 2007; 38: 739-742. Source of support: Nil, Conflict of interest: None Declared

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