Research Journal of Chemical Sciences ______________________________________________ ISSN 2231-606X Vol. 2(1), 50-58, Jan. (2012) Res.J.Chem.Sci. International Science Congress Association 50 Chemical Examination and Biological Studies on the Seeds of Psoralea Corylifolia Linn. Rao G.V.*, Annamalai T., Kavitha K. and Mukhopadhyay T.Cavin Kare Research Centre, 12, Poonamalle Road, Ekkattuthangal Chennai 60032, INDIA Available online at: www.isca.in (Received 31st October 2011, revised 21st November 2011, accepted 24th November 2011)Abstract The aim of the present investigation was to isolate chemical constituents and study of its biological activity from the seeds of acetone extract of Psoralea corylifolia Linn. The bio-assay guided isolation of acetone extract of seeds yielded five known compounds, -cadinene (1), bakuchiol (2), psoralen (3), isopsoralen (4) and psoralidin (5). The structures of these compounds were elucidated by physical and spectral data (UV, IR, H, 13C NMR and mass). The compound, -cadinene (1) is first report from this plant. Different extracts, fractions and compounds from the seeds were investigated for antimicrobial property. The methanolic, acetone and hexane extracts and isolated compound, bakuchiol (2) of Psoralea corylifolia were tested for antimicrobial studies against three gram positive bacteria and showed positive results. The compound bakuchiol (2) showed an excellent antibacterial activity than its crude extract. Key words: Psoralea corylifolia, bakuchiol, psoralidin, antibacterial studies, tetracycline. Introduction Plants are being used as source of medicine since ages. Many medicinal plants are nature’s gift to human beings to make disease free healthy life. More than 80% of the world population are in poor and less developed countries depends on traditional plant based medicines for their primary health care needs. India is one of the diverse countries in the world where the medicinal plant sector is a part of time-honored tradition that is respected even today. India is richly endowed with a wide variety of plants having medicinal value. These plants are widely used by all sections of the society whether directly as folk medicines or indirectly as pharmaceutical preparation of modern medicine. Over the past five decades focus on plant research has increased all over the world and a large body of evidence has collected to show immense potential of medicinal plants used in various traditional system. Medicinal plants are major source and biodynamic compounds of therapeutic value. Interestingly, Psoralea corylifolia is an endangered plant and medicinally important plant and found in tropical and subtropical region of the world. Its medicinal usage is reported in Indian pharmaceutical codex, Chinese, British and the American pharmacopoeias and in different traditional system of medicines such as Ayurveda, Unani and Siddha. Psoralea corylifolia Linn., is belongs to Fabaceae family. It is an erect annual, 30-180 cm high, found almost throughout India. It is reported to be grown on some extent in Rajasthan and the eastern districts of Punjab, adjoining Uttar Pradesh for its seeds. The fruits of P. corylifolia consist of a sticky oily pericarp, a hard seed coat and kernel. The seeds are used in indigenous medicine as laxative, aphrodisiac, anthelminitic, diuretic and diaphoretic in febrile conditions. The seeds have been specially recommended in the treatment for leucoderma, leprosy, psoriasis and inflammatory diseases of the skin. The seed extracts inhibits the growth of Staphylococus citreus, S. aurues and S. albus including strains resistant to penicillin’s. The seeds posses anthlemintic activity against earth worms, psoralen being the active principle. The essential oil shows a selective activity against the skin Streptococci and used in the treatment of skin affections. The seeds are used locally in the preparation of certain types of medicated oils and incense preparations. The root is useful in the caries of teeth. Previous studies reported the presence of several new and known compounds like, furanocoumarins, prenyl flavonoids, aromatic terpenoids and chromenes. In our continuous interest on the isolation and characterization of bioactive compounds from the indigenous medicinal plants for personal care applications8-18, we have undertaken chemical examination of the seeds of Psoralea corylifolia Linn. In this article we report the isolation and characterization of five known compounds. The crude extracts, its fractions and isolated compound were analyzed for antimicrobial activity. The crude extracts of acetone and methanol showed less activity than hexane fraction and compound showed potent antimicrobial activity against oral care organisms. Research Journal of Chemical Sciences __________________________________________________________ ISSN 2231-606X Vol. 2(1), 50-58, Jan. (2012) Res.J.Chem.SciInternational Science Congress Association 51 Material and Methods General: Melting points reported are uncorrected. The 400 MHz NMR spectra were recorded on a Bruker AMX 400 in CDCl or -DMSO with TMS an internal standard. The 13C NMR spectra were recorded at 100 MHz in CDCl and DMSO. IR spectra were recorded on a Shimadzu IR prestige 21; UV spectra were recorded on Shimadzu UV spectrophotometer; GC-MS were on a Jeol SX 102/DA 6000 mass spectrometer. TLC was performed on pre-coated silica gel 60 F254 plates (Merck) and the spots were visualized by exposure to iodine vapor or spraying with 5% sulphuric acid in methanol followed by heating the plate at 110ºC for 5 min. Plant material: The seeds of P. corylifolia were obtained from bazaar and was authenticated by Dr. P. Santhan, botanist, M/s. Durva Herbal Centre, Chennai. A voucher specimen was deposited in M/s. CavinKare Research Centre, Chennai, India. Extraction and Isolation: Air-dried seeds of P. corylifolia (670g) were crushed and coarsely powdered. The powdered material was successively extracted with acetone followed by methanol to get corresponding extracts 126 g and 30.4 g, respectively. The crude acetone extract suspended in acetone and water (2:8), partitioned with hexane and chloroform to get corresponding fractions 51g and 16.5 g, respectively. The aqueous fraction was concentrated to get 55g of the extract. All three fractions were submitted for biological studies and hexane fraction was found to potent than other fractions. After TLC analysis, the dark brown residue from hexane (51.0 g) was subjected to silica gel vacuum liquid chromatography, eluted with hexane, hexane: ethyl acetate (98:1, 98:2, 95:5, 90:10, 75:25). The homogeneous fractions were combined based on TLC and divided into six major fractions A (1.6 g), B (25.3 g), C (5.5 g), D (2.5g), E (3.7g), F (9.0 g). Fraction A was further purified by silica gel column using hexane and obtained compound 1 as colorless liquid (275mg). Fraction B was found to be mixture of two compounds and was purified by silica gel column using hexane, hexane: chloroform (8:2, 1:1), obtained again mixture of two compounds. Part of the fraction B (2.0g) was purified by silver nitrate impregnated column using hexane as eluent to obtain compound 2 (1.1g) Fraction C was showed one major and several minor spots along with lipid on TLC. It was purified by another small column using hexane: ethyl acetate mixture, followed by crystallization with ethyl acetate to get compound 3 (1.13g) and compound (800mg). Fraction D was found to be solid nature in the flask and was crystallized with hexane: chloroform to obtain compound 5 (0.91g). Fraction E and F were found to be lipids and coloring pigments and did not yield any compound. Results and Discussion Compound 1 (gg-Cadinene)19: Colorless liquid; UV (CHCl, max in nm): 215; IR (KBr, max in cm-1): 2850, 1620, 1400, 980, H NMR (CDCl): 0.98 (6H, d, =5.6Hz, 24-H), 1.65 (3H, s), 2.0- 2.35 (7H, m), 4.88 (1H, s), 4.95 (1H, s), 5.30 (1H, br t), 13C NMR (CDCl): 16.2, 22.5, 28.2, 29.2, 29.9, 34.6, 39.9, 40.3, 49.2, 53.5, 111.5, 124.4, 135.4, 154.6. GC-MS (m/z): 204 [M], 161, 146. Compound 2 (Bakuchiol)20: Light brown colored oil; UV (CHCl, max in nm): 265; IR (KBr, max in cm-1): 3360, 2850, 1620, 1400, 980; H NMR (400 MHz, CDCl): 1.21 (3H, s, H-16), 1.51 (2H, m,H-10), 1.60 (3H, s, H-15), 1.69 (3H, s, H-14), 1.98 (2H, m, H-11), 5.04 (2H, m, H-18), 5.12 (1H, br t, H-18), 5.90 (1H, dd, =17.3, 10.8 Hz, H-17), 6.07 (1H, d, =16.0 Hz, H-8), 6.27 (1H, d, = 16.0 Hz, H-7), 6.78 (2H, d, =8.0 Hz, H-3,5), 7.26 (2H, d, =8.0Hz, H-2,4). 13C NMR (100 MHz, CDCl): 17.6 (C-15), 23.1 (C-16), 23.3 (C-11), 25.6 (C-14), 41.2 (C-10), 42.4 (C-9), 111.8 (C-18), 115.3 (C-3 and 5), 124.7 (C-12), 126.4 (C-7), 127.3 (C-2 and 6), 130.7 (C-1), 131.2 (C-13), 135.8 (C-8), 145.9 (C-17), 154.4 (C-4). GC-MS (m/z): 256 [M], 241, 213, 186, 173, 158, 145, 107. Compound 3 (Psoralen)20: Colorless needles, mp 162-63C; UV (CHCl, max in nm): 245, 290, 327; IR (KBr, max in cm-1): 1720, 1629, 1568, 980; H NMR (400 MHz, CDCl): 6.38 (1H, d, =9.6 Hz, H-3), 6.84 (1H, d, =1.9Hz, H-11), 7.47 (1H, s, H-8), 7.68 (1H, s, H-5), 7.70 (1H, d, =1.9Hz, H-12), 7.80 (1H, d, =9.6 Hz, H-4). 13C NMR (100 MHz, CDCl): 99.7 (C-8), 106.3 (C-11), 114.6 (C-3), 115.3 (C-10), 119.7 (C-5), 124.8 (C-6), 143.9 (C-4), 146.8 (C-12), 152.0 (C-9), 156.3 (C-7), 160.8 (C-2). GC-MS (m/z): 186[M], 158, 130, 102. Compound 4 (Isopsoralen)20: Colorless needles , mp 137-38C; UV (CHCl, max in nm): 246, 296; IR (KBr, max in cm-1): 1721, 1629, 1568, 980; H NMR (400 MHz, CDCl): 6.39 (1H, d, J = 9.6 Hz, H-3), 7.13 (1H, d, J = 1.97Hz, H-11), 7.37 (1H, d, J = 8.5Hz, H-5), 7.43 (1H, d, J = 8.5Hz, H-6), 7.70 (1H, d, J =1.97Hz, H-12), 7.81 (1H, d, J = 9.6 Hz, H-4). 13C NMR (100 MHz, CDCl): 104.0 (C11), 108.7 (C-6), 113.4 (C-3), 114.0 (C-10), 116.8 (C-8), 123.7(C-7), 144.4 (C-4), 145.8 (C-12), 148.3 (C-9), 157.2 (C-7), 160.7 (C-2). GC-MS (m/z): 186 [M], 158, 130, 102. Research Journal of Chemical Sciences __________________________________________________________ ISSN 2231-606X Vol. 2(1), 50-58, Jan. (2012) Res.J.Chem.SciInternational Science Congress Association 52 Table-1 Comparison of MIC values of various organism Sl. No. Extract / Fraction / Compound Tested organisms MIC value (g/mL) Streptococcus mutans - MTCC 497 Actinomyces viscosus - ATCC 15987 Streptococcus sanguis - ATCC 10556 1 Methanolic extract 1250 NA NA 2 Acetone extract �1250 NA NA 3 Hexane fraction 19.53 NA NA 4 Bakuchiol 9.76 - 19.5 9.76 – 19.5 9.76 – 19.5 5 Tetracyclin 0.48 1.95 1.95 * NA = Not analyzed Compound 5 (Psoralidin)21: Pale yellow crystals, mp: 286-88C; UV (CHCl, max in nm): 244, 305, 347; IR (KBr, max in cm-1) : 3440, 3349, 1720, 1629, 1568, 980; H NMR (400 MHz, -DMSO): 1.69 (3H, s), 1.73 (3H, s), 3.28 (2H, d, J = 8.0 Hz), 5.32 (1H, d, J = 8.0 Hz), 6.83 (1H, s), 6.92 (1H, d, J = 8.0Hz), 7.14 (1H, s), 7.54 (1H, s), 7.65 (1H, d, J = 8.0 Hz), 10.00 (1H, br s), 10.68 (1H, br s). 13C NMR (100 MHz, -DMSO): 18.0, 25.9, 27.9, 99.0, 102.3, 102.7, 104.1, 114.2, 115.0, 120.9, 122.1, 126.8, 132.8, 153.2, 156.3, 157.3, 158.0, 159.3, 159.8. GC-MS (m/z): 336[M], 281. Antibacterial studies: Antibacterial activity was evaluated by turbidometric method on oral microorganisms especially the effects on adherent Streptococci sp., and Actinomycess viscosus. The experimental methodology has been well documented and published22. The current test cultures were obtained from MTCC and ATCC. It was found that the methanolic and acetone extract, hexane fractions and bakuchiol () showed significant antimicrobial activity against Oral care bacteria. MIC value of bakuchiol was found to be 9.76-19.5 g/mL while the MIC value of the control, tetracyclin was found to be 0.48-1.95g/mL [table 1]. Conclusion Out of five isolated compounds, -cadinene is the first report from the plant and the remaining four compounds, bakuchiol, psoralen, isopsoralen and psoraledin have already been reported from this plant. Bakuchiol () is an alternative to synthetic compounds. Acknowledgement We thank Mr. C. K. Ranganathan, CMD of CavinKare Pvt. 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Guo J., Weng X., Wu H., LI Q, Bi K., Antioxidants from a Chinese medicinal herb- Psoralea corylifolia, Food Chem., 91(2), 287-292 2005) 22.Erazo S., Gonzalez V., Zaldivar M. and Negrete R., Antimicrobial activity of Psoralea glandulosa L. Int. J. Pharmacognosy, 35(5), 385-387 (1997Figure-1 Compounds from Psoralea coylifolia Research Journal of Chemical Sciences __________________________________________________________ ISSN 2231-606X Vol. 2(1), 50-58, Jan. (2012) Res.J.Chem.SciInternational Science Congress Association 54 Research Journal of Chemical Sciences __________________________________________________________ ISSN 2231-606X Vol. 2(1), 50-58, Jan. (2012) Res.J.Chem.SciInternational Science Congress Association 55 Research Journal of Chemical Sciences __________________________________________________________ ISSN 2231-606X Vol. 2(1), 50-58, Jan. (2012) Res.J.Chem.SciInternational Science Congress Association 56 Research Journal of Chemical Sciences __________________________________________________________ ISSN 2231-606X Vol. 2(1), 50-58, Jan. (2012) Res.J.Chem.SciInternational Science Congress Association 57 Research Journal of Chemical Sciences __________________________________________________________ ISSN 2231-606X Vol. 2(1), 50-58, Jan. (2012) Res.J.Chem.SciInternational Science Congress Association 58