Stimulation of dermal fibroblast collagen synthesis in vitro by saponin enriched extract from soybeans Sarunya Laovitthayanggoon* a, Siripen Jarikasem a, Sarinthip Muensaen a a Pharmaceutical and Natural Products Department, Thailand Institute of Scientific and Technological Research, Pathum Thani, Thailand 12120 *sarunya@tistr.or.th phone +66-25779094 fax +66-25779110 ABSTRACT Soybean (Glycine max L.) is a species of legume native to East Asia, widely grown for its edible bean which has numerous uses. It has been used as a source of human food of high quality protein and other nutrients for hundreds of years. Recently, soyasaponin, one of the significant bioactive constituents from soybeans, has received more attention to be used in skin care cosmetic products due to its relevant beneficial properties including antioxidation, anti-inflammation and water retention. Previous works have also reported anti-wrinkle property of certain saponins of which effect on collagen synthesis is of interest. In this study, saponin enriched extract from soybeans prepared by use of Diaion HP-20 macroporous resin was investigated for in vitro stimulating effect on type 1 collagen synthesis. The human dermal fibroblasts cell line (ATCC CRL-1744) was treated with three different concentrations (50, 100, 200 µg/ml) of the extract for 24 h and then the amount of type 1 collagen was measured using ELISA test kit. The results revealed that the percentage of stimulating effect on collagen synthesis after saponin enriched extract treatment were 8.08 ± 1.23 and 21.12 ± 1.54 at concentrations 100 and 200 µg/ml, respectively while that of a positive control ascorbic acid at 50 µg/ml was 31.08 ± 0.28. The present study demonstrates the collagen synthesis stimulating potential of soysaponins and provides a possibility to develop as anti skin aging agent in cosmetic products. Keywords: Soybean, Saponin, Human dermal fibroblast, Collagen synthesis B-P-088 www.natpro5.psu.ac.th 193
1. INTRODUCTION Skin aging has been suggested to be associated with a decrease in dermis collagen content which clinically manifested on wrinkle and laxity. Collagen deficiency is partly arisen from its reduced synthesis [1]. Presently, substances which possess stimulatory effect on collagen synthesis have received more attention to be used for skin anti-aging cosmetic preparations. Currently, several plant-derived saponins have been reported to possess collagen synthetic property including ginsenoside [2], asiaticoside [3] and astragaloside [4]. Glycine max L. (Legumonosae) or soybean is a well known edible bean crop which has been used as a source of human food of high quality protein and other nutrients for hundreds of years. Moreover soybeans have been reported to contain about 0.17-6.16% of saponins [5]. Interestingly, beneficial effects for skin care of soysaponins have been demonstrated including antioxidation [6] and anti-inflammation [7]. In order to obtain scientific evidence to support a use of soyasaponins for aging skin treatment targeting on collagen alteration, we investigated on its collagenesis enhancing effect in dermal fibroblast monolayer. Fractionation of saponins was performed by column chromatographic technique using Diaion HP-20 adsorptive resin. 2. MATERIALS AND METHODS Plant material Soy beans were purchased from health food shop (Lemon green) in Bangkhen District, Bangkok, in October 2011. They were pulverized into coarse powder, then exhaustedly soxhletted with hexane to give the defatted powder. Extraction and fractionation Defatted powder 1.1 kg was macerated with 11 L of 70% ethanol for 72 h with occasional stirring, then filtered. After removal of ethanol under reduced pressure, 300 ml portion of aqueous solution equivalent to 360 g of powder was passed through Diaion HP-20 column (450 ml) and sequentially eluted with 0, 25, 50 and 95% aqueous ethanol. The fractions were concentrated under reduced pressure and freeze-dried. Saponin enriched extract (1.20 g) was obtained from 50% ethanolic eluate as monitored by TLC. Determination of cytotoxicity and collagen stimulation Cell culture Human dermal fibroblast cells (ATCC CRL-1474) were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum, 2mM L-glutamine and 100 unit/ml penicillin and streptomycin. The cells were incubated in a humidified 5% CO 2 atmosphere at 37 0 C for 72 h. MTT cytotoxicity test [8, 9] The cells were seeded in a 96-well plate at a density of 10 5 cells/ml, and incubated for 24 h. Various concentrations of test samples dissolved in DMSO (200 µl, 500-4000 µg/ml) were added to the cells and incubated for 24 h, and were then washed out. MTT (50 µl, 5 mg/ml) and the medium (150 µl) were added into each well. The cells were then incubated at 37 0 C 5% CO 2 for additional 4 h. The medium containing MTT was discarded, and MTT formazan that had been produced was extracted with 150 µl of DMSO under 15 min agitation. The absorbance was read at 540 nm. The toxicity of extract was indicated by 50% inhibitory concentration (IC 50 ). Determination of collagen synthesis using ELISA [10-12] The cells were seeded in a 6-well plate at a density of 2 10 5 cells/ml, and incubated for 24 h. After that the media were removed and added with different concentrations of extract (50, 100 and 200 µg/ml), incubated continually for 24 h and the extracts were then removed. Attached cells were transferred to a microcentrifuge tube using cell scraper and digested for collagen using pepsin solution in acetic acid (0.1mg/ml 50 mm acetic acid). Finally, the supernatant was detected for human collagen type I using ELISA test kit (Cosmo Bio Co.LTD). Collagen determination was performed by measuring absorbance at 412 nm using microplate reader. Then collagen concentration was calculated by interpolating sample OD value to the standard curve. After that the percentage of collagen stimulation was generated with the following equation: % Collagen stimulation = [(A C - A T )/ A C ] 100 A C = the collagen concentration of control = the collagen concentration of test sample A T www.natpro5.psu.ac.th 194
3. RESULTS The cytotoxicity results showed the %survival of dermal fibroblast cell line, at each concentration compared to control and IC 50 value over the test concentrations of 500-4000 µg/ml. The results showed that the IC 50 value of soyasaponin enriched extract was 2,445.99 ± 0.00 µg/ml, as shown in Figure 1. According to classification of the cytotoxicity for natural ingredients [13], our saponin enriched extract could be classified as potentially non toxic substance. Figure 1. The survival of dermal fibroblast cell line following exposure to soyasaponin enriched extract. The collagen synthesis stimulating potential of soyasaponin enriched extract was investigated by using ELISA assay compared with the positive control ascorbic acid. The results as shown in Figure 2 revealed that the percentage of stimulating effect on collagen synthesis after saponin enriched extract treatment were 0, 8.08 ± 1.23 and 21.12 ± 1.54 at concentrations 50, 100 and 200 µg/ml, respectively. The effect was in a dose dependent manner. Ascorbic acid at 50 µg/ml showed 31.08 ± 0.28% of collagen synthesis stimulating effect. 31.08±0.28 21.12±1.54 8.08±1.23 0.00±0.00 Figure 2. The percentage of collagen stimulation of dermal fibroblast following exposure to saponin extract. 4. CONCLUSIONS www.natpro5.psu.ac.th 195
Saponin enriched extract from soybeans is nontoxic to normal human dermal fibroblast cell line (IC 50 2,445.99 ± 0.00 µg/ml). The extract at the concentration of 100 and 200 µg/ml showed the percentage of collagen stimulatory effect of 8.08 ± 1.23 and 21.12 ± 1.54, respectively. The results demonstrate a collagen synthesis stimulating potential of soyasaponin extract and provide a possibility to develop as skin anti- aging agent in cosmetic products. www.natpro5.psu.ac.th 196
ACKNOWLEDGEMENTS The author would like to gratitude thanks to Thailand Institute of Scientific and Technological Research (TISTR) for the financial support. REFERENCES 1. Joe MJ, Kim SN, Choi HY, Shin WS, Park GM, Kang DW, Kim YK. 2006. The Inhibitory effects of Eckol and Dieckol from Ecklonia stolonifera on the expression of matrix metalloproteinase-1 in human dermal fibroblasts. Biol Pharm Bull. 29(8), 1735-1739. 2. Lee J, Jung E, Lee J, Huh S, Kim J, Park M, So J, Ham Y, Jung K, Hyun CG, et al. 2007. Panax ginseng induces human type I collagen synthesis through activation of Smad signaling. J Ethnopharmacol. 109, 29-34. 3. Lee J, Jung E, Kim Y, Park J, Park J, Hong S, Kim J, Hyun C, Kim YS, Park D. 2006. Asiaticoside induces human collagen I synthesis through TGFbeta receptor I kinase (TbetaRI kinase)-independent Smad signaling. Planta Med. 72(4), 324-8. 4. Chen X, Peng LH, Li N, Li QM, Li P, Fung KP, Leung PC, Gao JQ. 2012. The healing and anti-scar effects of astragaloside IV on the wound repair in vitro and in vivo. J Ethnopharmacol. 15,139(3): 721-7. 5. Xianli Wu, Jie Kang. 2011. Phytochemicals in soy and their health effects, phytochemicals-bioactivities and impact on health, Prof. Iraj Rasooli (Ed.). ISBN: 978-953-307-424-5. 6. Lv FX, Lu ZX. 2001. Research process of bioactive substances in soybean. Food Sci. Technol. 5, 69-71. 7. Kang JH, Sung MK, Kawada T, Yoo H, Kim YK, Kim JS, Yu R. 2005. Soybean saponins suppress the release of proinflammatory mediators by LPS-stimulated peritoneal macrophages. Cancer Lett. 18, 230(2), 219-227. 8. Culture of animal cell: A manual of Basic Technique. Wiley-Less, New York, Freshney RI, USA, 4 th edition. 9. Plumb JA, Milroy R, Kaye SB. 1989. Effects of the ph dependence of 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide-formazan absorption on chemosensitivity determined by a novel tetrazolium based assay. Cancer Res. 49, 4435-4440. 10. Murad S, Grove D, Lindberg KA, Reynolds G, Sivarajah A, Pinnell SR. 1981. Regulation of collagen synthesis by ascorbic acid. Proc Natl Acad Sci USA. 78, 2879-2882. 11. Wha Kim S, Lee IW, Cho HJ, Cho KH, Han Kim K, Chung JH, Song PI, Chan Park K.. 2002. Fibroblasts and ascorbate regulate epidermalization in reconstructed human epidermis. J Dermatol Sci. 30, 215-223. 12. Appling WD, O'Brien WR, Johnston DA, Duvic M. 1989. Synergistic enhancement of type I and III collagen production in cultured fibroblasts by transforming growth factor- and ascorbate. FEBS Lett. 250, 541-544. 13. Ballantyne B, Cawley TJ. 1999. Toxicology update. J. App. Toxicol. 19(4), 291-294. www.natpro5.psu.ac.th 197