J. Microbiol. Biotechnol. (2017), 27(11), 1961–1970 https://doi.org/10.4014/jmb.1706.06028 Research Article Review jmb Antioxidative and Antiaging Activities and Component Analysis of Lespedeza cuneata G. Don Extracts Fermented with Lactobacillus pentosus S Joon Seob Seong, Song Hua Xuan, So Hyun Park, Keon Soo Lee, Young Min Park, and Soo Nam Park1* Department of Fine Chemistry, Cosmetic R&D Center, Cosmetic Industry Coupled Collaboration Center, Seoul National University of Science and Technology, Seoul 01811, Republic of Korea Received: June 12, 2017 Revised: September 6, 2017 Lespedeza cuneata G. Don is a traditional herb that has been associated with multiple biological Accepted: September 14, 2017 activities. In this study, we investigated the antioxidative/antiaging activities and performed First published online an active component analysis of the non-fermented and fermented (using Lactobacillus September 15, 2017 pentosus) extracts of Lespedeza cuneata G. Don. The antioxidative activities of the fermented *Corresponding author extract were higher than those of non-fermented extracts. The elastase inhibitory activity, Phone: +82-2-970-6451; inhibitory effects on UV-induced MMP-1 expression, and ability to promote type I procollagen Fax: +82-2-972-9585; E-mail: [email protected] synthesis were investigated in Hs68 human fibroblasts cells. These tests also revealed that the fermented extract had increased antiaging activities compared with the non-fermented extract. A component analysis of the ethyl acetate fractions of non-fermented and fermented extracts was performed using TLC, HPLC, and LC/ESI-MS/MS to observe changes in the components before and after fermentation. Six components that were different before and after fermentation were investigated. It was thought that kaempferol and quercetin were converted S upplementary data for this from kaempferol glucosides and quercetin glucosides, respectively, via bioconversion with the paper are available on-line only at http://jmb.or.kr. fermentation strain. These results indicate that the fermented extract of L. cuneata G. Don has potential for use as a natural cosmetic material with antioxidative and antiaging effects. pISSN 1017-7825, eISSN 1738-8872 Copyright© 2017 by Keywords: Lespedeza cuneata G. Don, Lactobacillus pentosus, fermentation, antioxidative activity, The Korean Society for Microbiology antiaging activity and Biotechnology Introduction materials are present in the form of glycosides, which generally have lower antioxidative and antiaging activities There has been a growing interest in beauty in accordance than the aglycone structures in the skin. Recently, studies with the economic development in Korea. Korea’s cosmetics have focused on converting the glycosides of natural industry has thus been growing rapidly with the influence extracts into active materials by using fermentation and of the Korean Wave, including K-dramas and K-pop. bioconversion technologies [1-3]. These processes can Recently, cosmetic materials developed by Korea’s unique reduce the size and polarity of the molecules, thereby differentiated cosmetics and convergence integration improving their skin absorption and efficacy [4-6]. Previous technology have greatly contributed to enhancing the studies have shown that fermented Melissa officinalis and country’s global competitiveness. In particular, the Lavandula angustifolia extracts have higher antioxidative development and application of new biomaterials for and cytoprotective properties than their non-fermented cosmetic use have recently been conducted by introducing counterparts [7, 8]. Other studies using these fermentation fermentation and bioconversion technologies to herbal and bioconversion technologies to improve the functional medicines or natural plant extracts based on oriental herbs. efficacy of extracted components for antiaging effects and Most of the natural plant extracts used as cosmetic whitening of the skin have recently been actively conducted November 2017 ⎪ Vol. 27⎪ No. 11 1962 Seong et al. in the field of cosmetics [9, 10]. Varian Cary 50 (Varian, Australia), and a 6-channel LB9505 LT When skin is exposed to ultraviolet light, various reactive system (Berthold Technologies, Germany) was used for the 1 chemiluminescence assay. A pH meter (Hanna, Korea) was used oxygen species (ROS), such as singlet oxygen ( O2), superoxide •- in this study. For the component analysis, a high-performance anion radical (O2 ), hydrogen peroxide (H2O2), hydroxyl radical (•OH), alkoxyl radical (•OR), and hydroperoxyl liquid chromatography (HPLC) system (Shimadzu, Japan) and a C18 analytical column (5 μm, 250 mm × 4.6 mm) (Shimadzu) were radical (•OOR), are produced in the skin [11]. These ROS used, and LC/ESI-MS/MS (Applied Biosystems, USA) was carried not only oxidize and damage the lipids, proteins, and DNA out at the National Instrumentation Center for Environmental present in the skin, but also accelerate skin aging, as Management (Nicem, Korea). The 1,1-diphenyl-2-picrylhydrazyl evidenced by wrinkle formation, through the cleavage and radical (DPPH), ethylenediaminetetraacetic acid (EDTA), luminol, abnormal crosslinking of matrix constituent proteins such Triton X-100, and NP-PEG (natural products-polyethylene glycol, as collagen or elastin. In addition, these ROS increase the 2-aminoethyl diphenylborinate) were purchased from Sigma- expression of matrix metalloproteases (MMPs) that degrade Aldrich (USA), and H2O2 was purchased from Dae Jung Chemical collagen and elastin by activating signaling cascades via & Metals (Korea). We purchased the FeCl3·6H2O from Junsei Chemical the phosphorylation of ERK, p38, and JNK kinases and the Co. (Japan). The materials (Na2HPO4·12H2O, NaH2PO4·2H2O, NaCl, AP-1 transcription factor, and may also accelerate skin aging trizma base, HCl, and H2SO4) used to prepare buffer solutions, by inhibiting the synthesis of procollagen [12]. Therefore, and the solvents ethanol (EtOH), methanol (MeOH), ethyl acetate the development of a material that inhibits the expression (EtOAc), and n-hexane, were of extra-pure grade. The reference compounds (+)-α-tocopherol (1,000 IU vitamin E/g), L-ascorbic of MMPs by inhibiting the production of excess ROS acid, oleanolic acid, all-trans retinol, quercetin, kaempferol, generated in the skin, or that promotes the synthesis of isovitexin, quercitrin, avicularin, and juglanin, and the substrate procollagen, is important in functional cosmetic research. N-succinyl-(Ala)3-p-nitroanilide, were obtained from Sigma- Lespedeza cuneata G. Don of the family Leguminosae is a Aldrich. The human Hs68 foreskin fibroblast cell line ATCC CRL- perennial and herbaceous legume widely distributed in Korea, 1635 was purchased from the ATCC (USA). The 60 F254 (0.2 mm) China, Japan, and Australia. As a folk remedy, L. cuneata G. thin-layer chromatography (TLC) aluminum sheet gels were Don extracts have been used to enhance liver and kidney purchased from Merck (USA). The whole plant of L. cuneata function, remove extravasated blood, and calm swelling [13]. G. Don was collected from Yangnyeong market, Korea, in Most of the flavonoid components in L. cuneata G. Don, such November 2015 and was identified by Prof. Soo Nam Park. A as isoorientin, avicularin, and trifolin, were found to exist voucher specimen was deposited at the department of Fine as glycosides [14]. A study on L. cuneata G. Don extracts has Chemistry, Seoul National University of Science and Technology. reported that the ethanol extracts possess antioxidative and Extraction and Fractionation antimicrobial activities [15]. However, no research on the A 100 g portion of the dried whole parts (except the roots) of development of materials from this plant using fermentation L. cuneata G. Don was cut finely and dissolved in 4 L of 70% EtOH or bioconversion technologies has been reported. for 1 week; the resulting powder was obtained by filtration and Therefore, in this study, we measured the free radical vacuum evaporation drying. The ethyl acetate fraction from 70% 3+ and ROS scavenging activities in a Fe -EDTA/H2O2 system, ethanol extracts was obtained by three fractionation cycles and determined the elastase inhibitory effect using human vacuum evaporation drying. Hs68 fibroblasts, assessed the UV-induced inhibition of MMP-1 expression and the promotion of type I procollagen Preparation of the Fermented Extracts synthesis, and performed a component analysis of The fermented extract of L. cuneata G. Don was obtained from unfermented L. cuneata G. Don extracts and extracts obtained GFC Co., Ltd. (Korea). Fermented lactic acid strains (Lb. pentosus) following fermentation by Lb. pentosus. We hypothesized were isolated from several microorganisms by selecting acid- that the extracts of the fermentation product would have producing strains using bromophenol blue [16], isolating strains producing β-glucosidase by the esculin agar method [17], and higher antioxidative and antiaging activities compared with selecting strains with active glucosidase by the ρ-nitrophenyl-β-D- the unfermented extracts, and would thus have greater glucopyranoside (ρNPG) method [18]. (The results of β-glucosidase potential for cosmetic applications. activity in Lb. pentosus are presented in Fig. S1.) Lb. pentosus, which is a high β-glucosidase-producing strain, was selected on Materials and Methods the basis of these studies, and the homology of Lb. pentosus D79211 was confirmed to be 99% by a 16S rRNA gene sequencing Materials analysis using colony PCR