Anti – Biofilm Activity and Bioactive Component Analysis Associated With Psoralea Corylifolia Seed Extracts

Plants have been an important source of medicine for thousands of years. As per World Health Organization estimates, up to 80 percent of people still depend on traditional remedies such as herbs for their medicines. Today, Ayurvedic, and Unani physicians utilize numerous species of medicinal plants as a source for their drugs [1]. The twenth century set a trend for introducing a new generation of botanical therapeutics that includes plant derived pharmaceuticals, multicomponent botanical drugs; dietary supplements, functional foods and plant produced recombinant proteins. Many of these products will soon complement conventional pharmaceuticals in the treatment, and prevention of diseases. Psoralea corylifolia Linn. belongs to the family Fabaceae, commonly known as ‘Bakuchi’ by Indian traditional medicine. It is a medicinally important plant, indigenous to tropical and subtropical regions of the world. [2] Several chemical compounds were identified and documented from the Psoralea corylifolia including flavonoids (bavachalcone, bavachinin, bavachin, corylin, and 6-prenylnaringenin etc.), coumarins (psoralidin, psoralen, isopsoralen and angelicin) and meroterpenes bakuchi-ol and 3-hydroxybakuchiol). [3] The Psoralea corylifolia extracts have been reported to possess antibacterial,[4]antifungal,[5] anti-oxidant,[6,7] anti-inflammatory,[8] and immunomodulatory activity[9]. In continuation with the earlier studies and considering the therapeutic efficacy of Psoralea corylifolia in skin diseases a scientific investigation was undertaken to screen the antibiofilm activity of extracts of Psoralea corylifolia seed against major uropathogen. The aim of the present study was to assess and confirm the antibiofilm activity of bakuchi seed extract against catheter associated urinary tract (CAUTI) pathogen, Klebsiella Spp. The in-vitro study was performed on longitudinal section of catheter surface, along with the phytochemical investigation of the most promising extract using TLC profiling and GC/MS analysis.

Klebsiella Spp. was a clinical isolate was obtained from patient suffering from urinary tract infection in local hospital from Mumbai, India. The bacterial culture was further sub-cultured and stored on Tryptone Soy Agar (TSA). Tryptone Soy Broth (TSB) media was used for conducting further experiments.

Methanol and Diethyl ether used for extractions were of HPLC grade, and were purchased from M/S Merck ltd. Mumbai. The Chemicals such as 1% Crystal Violet was obtained from Biolab Diagonostics (I) Pvt. Ltd., 95% Ethanol was prepared from absolute alcohol made by S D Fine Chem Ltd. and phosphate buffer saline (pH 7.3) was prepared in the laboratory itself.

The seeds of Psoralea corylifolia plant were procured from local ayurvedic medicine supplier. Thedried seeds were cleaned and disinfected with 15% H2O2, crushed into powder sample using an electronic blender. The powdered sample was stored in bottle at room temperature prior to subject for an extraction and ensure to use in a day for extraction.

Sterile urethral catheter which was used to test the biofilm was manufactured by Romsons Scientific & Surgical Industries Pvt.ltd. Nonpyrogenic, 15ml High clarity, screw cap Polypropylene Conical tubes, manufactured by BD Falcon were used.

The fine powder of each of the seed (25g), with 250ml of methanol and diethyl ether solvents respectively were taken in a round bottom flask. For successive extraction with these solvents, seed powder was allowed incubate for 48 h with intermittent shaking at room temperature. The liquid extracts so obtained were filtered with whatman filter paper and were filter sterilized. and labeled as M1, D2, respectively. Both extracts were stored at -20°C in air tight bottle and used within 1 week after preparation.

The anti-biofilm activity of two extracts of the bakuchi seeds (M1 and D2) were tested against in catheter tube assay [10] in which 100 µl /ml of bacterial inoculums was inoculated in two tubes containing LB. Respective controls with only LB media as well as LB media with each extract was prepared and was incubated for 96 h at 370C. The amount of biofilm produced in each tube was then quantified and measured. This test was performed in triplicate and the final readings were taken as an average of the three readings. The percentage reduction in biofilm formation was measured using the modified formula [11]

The seed extract with promising broad spectrum biofilm inhibition activity was subjected for phytochemical tests for detection of plant secondary metabolites, tannins, saponins, steroid, alkaloids and glycolsides in accordance with prior studies. [12]

The most promising extract was spotted in preparative TLC plates coated with silica gel G. The plates were developed in TLC chamber previously saturated with Ethyl acetate: Methanol (3:7) solvent system. The solvent was allowed to evaporate and different spots developed were identified by means of UV light at λ max 254 nm and the Rf value (for each spot developed) was calculated. [13]

The most promising extract of Psoralea corylifolia seeds (M1) was subjected for gas chromatography-mass spectrometry analysis with the GC MS–QP 2010 plus system (Shimadzu). The methanol seed extract with most promising activity was subjected for the GC /MS analysis. 2μl of the sample was injected to the GC-MS with the oven temperature programmed as 80°-280°C with increment of 5°C/min with Helium as a carrier gas. The injection size was 0.1 μL, and detector temperature of 2700 C. The eluted peaks were identified using NIST library by comparing with the mass spectral data and retention indices in the literature.

The results obtained in the present study relieved that the tested two extracts of bakuchi seeds possess considerable reduction in biofilm formation on catheter tube. The optical density was measured for all test tubes under investigation. The final biofilm inhibition was calculated by subtracting the readings of the inhibition of biofilm by the two extracts in

percentage reduction of biofilm was calculated and documented. Among the two extract subjected for biofilm inhibition activity on the surface of the urinary catheter tube, methanol extract showed maximum inhibition of biofilm almost (61.35%) as compare to that of diethyl ether extract (52.98%). Methanol extract with the maximum inhibition activity was selected and was subjected for further analysis.

The preliminary phytochemical screening of methanol extracts of the Psoralea corylifolia seeds showed the presence of alkaloids, carbohydrates, flavonoids, glycosides and saponin. However the steroids and terpanoids were absent as represented in table 1. Table 1: Phytochemical analysis of Psoralea corylifolia seed extract

Presence of the secondary metabolites will be held responsible for antibiofilm activity. The retention factors (Rf) of methanol extract in different solvent systems are shown in table 2. The methanol extracts produces four fraction having Rf 0.16, 0.33, 0.44 and 0.91 under ethyl acetate: methanol (3:7) solvent system. The results of TLC indicate that methanol extracts has number of chemical constituents.

methanol extract .A total of 15 compounds were identified in methanol extract of P. corylifolia seeds. The major components identified were, 1-(+) -Ascorbic acid 2, 6-dihexadecanoate (8.76%), Coumarin –Psoralen (7.67%), Angecin- Isopsoralen (6.34%), caryophyllene oxide (2.82%). Several other components in trace amounts were also present. In earlier findings by S. Shreenivasan et al also reported the presence of the phenyl derivative of pyranocoumarin (PDP) in bakuchi oil with a potent antifungal activity against Fusarium species. And antimicrobial activities. [14]

In the present study, the antibiofilm activities of Psoralea corylifolia seed extract in methanol and diethylether extract were explored specifically against major bacterial urinary skin pathogen. The results clearly demonstrate that, the methanol seed extract of P. Corylifolia comprise of a promising antibiofilm activity against selected pathogen as compare to diethylether extract. The phytochemical and TLC analysis of the methanol extract followed by GC- MS screening confirmed the presence of various phytochemicals. Major identified phytochemicals in methanol extract of P. corylifolia seeds are phenol derivatives and coumarin – psoralen, isopsoralen which may be accountable for its antibiofilm activity. In Ayurvedic system of medicine, Psoralea corylifolia seeds are commonly used in various dermatological conditions [15- 19]. The present study supports the use of bakuchi seed in the development of ayurvedic skin formulations. Present study explored the additional benefits associated with its traditional applications. Additional in vitro studies would be needed to justify and further the potential of this extract as an effective biofilm inhibitor in UTIs. Further individual phytochemical evaluation with each identified chemical fractions responsible for these inhibitory activities is essential. Thus, the effect of these extract and its derived molecules could be of considerable interest in future new drug developments.

The required infrastructure support provided at Department of Microbiology, Rishi Biotech for taking up a part of this study is sincerely acknowledged.

1. Narayana Rao and K. Thammanna;Medicinal Plants of Ritual Hills, Department of Garden, 2. Rao G.V., Annamalai T., Kavitha K. and Mukhopadhyay T., Chemical Examination and Biological Studies on the Seeds of Psoralea Corylifolia Linn. Research Journal of Chemical Sciences, Vol. 2(1), 50-58, Jan. 2012; 50-58 3. Kaufman, PB; Duke, JA; Brielmann, H; Boik, J; Hoyt, JE ("A comparative survey of leguminous plants as sources of the isoflavones, genistein and daidzein: Implications for human nutrition and health". Journal of alternative and complementary medicine : 1997 3 (1): 7–12 4. Dev S, Nayak UR, Mehta G. Mono-terpenoids-I, Psoralea corylifolia Linn. - Bakuchiol, A novel monoterpene phenol. Tetrahedron 1973; 29: 1119-1125. 5. Prasad NR, Anand C, Balasubramanian S, Pugalendi KV. Antidermatophytic activity of extracts from Psoralea corylifolia (Fabaceae) correlated with the presence of a flavonoid compound. Journal of Ethno-pharmacology2004; 91: 21-24. 6. Tang SY, Whiteman M, Peng ZF, Jenner A, Yong EL, Halliwell B. Characterization of Antioxidant and Antiglycation properties and isolation of active ingredients from traditional chinese medicines. Free Radical Biology & Medicine 2004; 36(12): 1575-1587. 7. Haraguchi H, Inoue J, Tamura Y, Mizutani K. Inhibition of mitochondrial lipid peroxidation by Bakuchiol, a meroterpene from Psoralea corylifolia. Planta Medica 2000; 66(6): 569-71. 8. Pae HO, Cho H, Oh GS, Kim NY, Song EK, Kim YC, et al. Bakuchiol from Psoralea corylifolia inhibits the expression of inducible nitric oxide synthase gene via the inactivation of nuclear transcription factor- kB in RAW 264.7 macrophages. International Journal of Immuno-pharmacology2001; 1: 1849-1855. 9. Latha PG, Panikkar KR. Inhibition of chemical carcinogenesis by Psoralea corylifolia seeds. Journal of Ethno-pharmacology 1999; 68: 295-298. 10. Mathur T, Singha S, Khan S, Upadhyay DJ Fatma T and Rattan A; Detection of biofilm formation among the clinical isolates of staphylococci: an evaluation of three different screening methods. Indian Journal of Medical Microbiology, 2006; 24 (1): 25-29. Protocols in Microbiology, 2005; 1 (1B)-1. 12. Harbone J.B. Phytochemical Methods – a guide to modern techniques of plant analysis. 3 rd ed. Springer, New Delhi, 2005; 1-32 13. Mukherjee P.K. Quality Control Herbal Drugs–An approach to evaluation of botanicals. Business Horizons Pharma-ceutical Publishers. New Delhi, 2005;131-196. 14. Sangeetha Shrinivasan & D.V.L.Sarda , Antifungal Activity of Phenyl Derivative of Pyranocoumarin from Psoralea corylifolia L. Seeds by Inhibition of Acetylation Activity of Trichothecene 3-O-Acetyltransferase (Tri101), Journal of Biomedicine and Biotechnology Volume 2012, Article ID 310850, pp 1-8 15. Siddhinandan Mishra., Bhiashajya Ratnavali Chaukhambha Publication Varanasi, Edition 2009, Vidangadi Lepa (54/9)– Kushtharogadhikar 16. Siddhinandan Mishra., Bhiashajya Ratnavali Chaukhambha Publication Varanasi, Edition 2009,Pathyadi Lepa (54/12) – Kushtharogadhikar 17. Siddhinandan Mishra., Bhiashajya Ratnavali Chaukhambha Publication Varanasi, Edition 2009, Paradadi Lepa (54/36) – Kushtharogadhikar 18. Siddhinandan Mishra., Bhiashajya Ratnavali Chaukhambha Publication Varanasi, Edition 2009, Avalgujadi Lepa (54/41) – Kushtharogadhikar 19. Siddhinandan Mishra., Bhiashajya Ratnavali Chaukhambha Publication Varanasi, Edition 2009, Panchanana Taila (54/266-268)–Shweta kushthahara, Kushtharakshasa Taila (54/285-289)