Improving the antioxidant activity and enriching salvianolic acids by the fermentation of Salvia miltiorrhizae with Geomyces luteus

Improving the antioxidant activity and enriching salvianolic acids by the fermentation of Salvia miltiorrhizae with Geomyces luteus Geomyces luteus 发酵丹参实现抗氧化活性的增加和丹酚酸的富集 1 first-author 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:caile@ynu.edu.cnztding@ynu.edu.cn †E-mail:caile@ynu.edu.cnztding@ynu.edu.cn caile@ynu.edu.cn 1 2 1 2 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:caile@ynu.edu.cnztding@ynu.edu.cn †E-mail:caile@ynu.edu.cnztding@ynu.edu.cn caile@ynu.edu.cn Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education, School of Chemical Science and Technology, Yunnan University, Kunming 650091, China Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education, School of Chemical Science and Technology, Yunnan University, Kunming 650091, China Department of Pharmaceutical Sciences, Zhuhai Campus of Zunyi Medical University, Zhuhai 519041, China Department of Pharmaceutical Sciences, Zhuhai Campus of Zunyi Medical University, Zhuhai 519041, China The antioxidant activities and total phenolic content of fermented Salvia miltiorrhiza with fungus Geomyces luteus were investigated. The results revealed that G. luteus fermentation could significantly improve the antioxidant activity and total phenolic content of S. miltiorrhiza . The main antioxidant constituents were characterized by spectroscopic analysis as salvianolic acids. High-performance liquid chromatography (HPLC) quantification also showed the enhanced content of salvianolic acid B after fermentation. The present study suggests that G. luteus fermentations are effective in the S. miltiorrhiza salvianolic acids’ enrichment process. 目的 2 研究真菌固态发酵对丹参抗氧化活性的影响及探索丹酚酸富集的途径。 创新点 2 该研究发现真菌 G. luteus 固态发酵可增加丹参的抗氧化活性及有效地富集丹酚酸B，为丹参更有效的开发利用提供了新思路。 方法 2 采用20种真菌对丹参进行固态发酵，以两种抗氧化模型（二苯代苦味酰基自由基（DPPH·）和2,2’-联氮基双（3-乙基苯并噻唑啉-6-磺酸）二铵盐自由基（ABTS + ·））测定发酵物的抗氧化活性；采用Folin-Ciocaltea比色法测定发酵物的总酚含量；以多种波谱学方法（紫外光谱（UV）、红外光谱（IR）和核磁共振波谱（NMR））和高效液相色谱法（HPLC）表征发酵前后丹参化学成分的变化。 结论 2 抗氧化活性测试和总酚测定结果表明， G. luteus 发酵可有效地增加丹参的抗氧化活性和总酚含量。UV、IR及NMR等波谱表征发现，在丹参经 G. luteus 发酵后，丹酚酸类化合物的含量提高了；同时HPLC测定也发现，丹酚酸B含量显著增加。这些结果表明， G. luteus 发酵可实现丹酚酸B的富集。 Salvia miltiorrhiza Geomyces luteus Salvianolic acids Antioxidant Fermentation 丹参 Geomyces luteus 丹酚酸 抗氧化 发酵 Introduction 1 Salvia miltiorrhiza Bunge (Labiatae), known as Danshen and first recorded in Shennong Bencaojing (an ancient herbalism in China), has long been used to treat cerebrovascular and cardiovascular diseases. It is listed in the Chinese Pharmacopeia. Modern pharmacological studies revealed that S. miltiorrhiza could activate blood circulation to dissipate blood stasis, alleviate pain, and treat inflammatory diseases (Zhou et al., 2005 2005 ), which may be because of its large number of bioactive ingredients, including diterpene, chinone, and phenolic acids (Li, 1997 1997 ). Salvianolic acids, the main polyphenolic acids in S. miltiorrhiza (Sun et al., 2009 2009 ), produce a variety of pharmacological activities such as reducing atherosclerosis, inhibiting formation of low-density lipoprotein, and prohibiting the aggregation of platelets, which has long stimulated scientists’ strong interest (Yang et al., 2010 2010 ). Plant phenolics, particularly phenolic acids, are known to be potent antioxidants, and can effectively scavenge free radicals and cut off radical chain reactions (Duan et al., 2006 2006 ). Previous studies revealed that a certain amount of phenolics possessing significant antioxidant activity exist in S. miltiorrhiza (Zhang et al., 2010 2010 ), and these be vital contributors to the pharmacological activity of S. miltiorrhiza . Traditional Chinese medicine (TCM) has played a significant role in healing for a very long time, but recently scientific evidence regarding the better utilization of the resources of TCM has been needed. Fermented traditional medicine (FTM) has garnered attention for its enhanced biological activity compared to traditional medicine (Wei et al., 2007 2007 ; Okabe et al., 2011 2011 ). Several studies using single herbs and fungi support the availability and efficacy of FTM. Chen and Chen ( 2000 2000 ) had reported that yeast elicitors increased the cryptotanshinone production of the S. miltiorrhiza extracts. Coprinus comatus fermentation enhanced the anti-inflammatory properties of Sophora flavescens (Han et al., 2011 2011 ). In the present study, S. miltiorrhiza was fermented with Geomyces luteus and 19 other fungi. The antioxidant activities of fermented S. miltiorrhiza were evaluated with 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) assays. Subsequently, the total phenolic contents (TPCs) of fermented S. miltiorrhiza were measured. Extensive spectroscopic analysis (ultraviolet (UV), infrared (IR), and nuclear magnetic resonance (NMR)) and the high-performance liquid chromatography (HPLC) method were employed for the characterization and quantitative analysis of the main antioxidant constituents of fermented S. miltiorrhiza . This paper explores a new method to enrich the salvianolic acids in S. miltiorrhiza by microorganism fermentation. Materials and methods 1 Materials, chemicals, and instruments 2 Trolox, salvianolic acid B, ABTS, and DPPH were obtained from J & K Chemical (Beijing, China). Gallic acid and rutin were purchased from Aladdin-Reagent (Shanghai, China). Deuterated methanol (CD 3 OD) and dimethylsulfoxide (DMSO) were obtained from Sigma-Aldrich (Shanghai, China). Deionized water (resistivity ≥18.25 MΩ/cm) was acquired with a Youpu purity water system (Chengdu, China). Acetonitrile and CH 3 OH (HPLC grade) were supplied by Fisher Scientific (NJ, USA). All other reagents were of analytical grade. Roots of S. miltiorrhiza were obtained from Kunming City, China in October 2013. The sample was authenticated by Professor Shu-gang LU at the School of Life Science in Yunnan University. A voucher specimen (No. DS01) has been deposited in the Key Laboratory of Medicinal Chemistry for Natural Resources, Yunnan University, China. Microorganisms and fermentation 2 G. luteus and 19 other fungi ( Talaromyces purpurogenus , Penicillium vancouverense , Alternaria tenuissima , Chaetomium globosum , Penicillium swiecickii , Arthrinium phaeospermum , Stagonosporopsis cucurbitacearum , Arthrinium arundinis , Scytalidium lignicola , Fusarium oxysporum , Clonostachys rogersoniana , Alternaria compacta , Cladosporium cladosporioides , Arthrinium sacchari , Stagonosporopsis chrysanthemi , Botrytis cinerea , Fusarium avenaceum , Helminthosporium maydis , Trichophyton verrucosum ) were inoculated in potato dextrose agar slant media in a constant temperature incubator at 28.0 °C for 7 d. Dried and powdered roots of S. miltiorrhiza (5.0 g) were added to 150 ml Erlenmeyer flask to form the solid fermentation culture medium. A total of 11 ml distilled water was added to the medicine to achieve a moisture circumstance, and autoclave-sterilized at 120 °C for 30 min. Then the mature-inoculated slants were added and incubated with S. miltiorrhiza at 28.0 °C for a month, together with a blank fermentation (without added microbe). Each of the samples had 3 other parallel controls. The original material and microorganism-fermented S. miltiorrhiza were immersed in 50 ml of 80% (v/v) CH 3 OH-H 2 O for 24 h, and the extracts were filtered after ultrasonic treatment for 30 min. The extraction was repeated 3 times. The extracts of the samples and parallel controls were concentrated under vacuum to dryness using a rotary evaporator at 60 °C (Heidolph, Germany). The non-fermented S. miltiorrhiza (original material) and S. miltiorrhiza fermented with G. luteus were respectively obtained as NFS (2.5015 g) and GFS (0.7030 g). DPPH assay 2 The DPPH assay was performed according to the method described by He et al. ( 2015 2015 ). Briefly, DPPH• solution (3.9 ml, 0.075 mmol/L) was mixed with 0.1 ml of the sample at gradient concentrations (10, 20, 25, 30, 35, 40, 50, and 60 μg / ml). The mixture was shaken and rested at 25 °C for 30 min. The absorbance was determined at 515 nm. The DPPH• scavenging effect was calculated using the following equation: inhibition=(1− A s / A o )×100%, where A s is the absorbance of the sample mixture and A o is the absorbance of the mixture without the sample. The 50% absorbance reduction (IC 50 ) was estimated based on a curve relating concentration to the absorbance of a sample. Rutin was used as a positive reference. ABTS assay 2 The ABTS assay was performed according to the method described by Re et al. ( 1999 1999 ). Briefly, 7 mmol/L ABTS solution was mixed with 2.5 mmol/L K 2 S 2 O 8 solution and incubated in the dark for 12‒16 h to achieve an ABTS + • solution. The ABTS + • solution was diluted with phosphate buffer saline (PBS; 200 mmol/L, pH=7.4) to acquire an initial absorbance of 0.70±0.02 at 734 nm. The 100-μl sample extract was added to 3.9 ml of ABTS + • working solution. After resting at room temperature for 6 min, the absorbance was measured at 734 nm. A standard curve was acquired using Trolox solution at different concentrations in the PBS buffer solution. The ABTS + • scavenging effect was evaluated using the following equation: inhibition=(1− A ' s / A ' o )×100%, where A ' s is the absorbance of the sample solution mixed with ABTS + • and A ' o is the absorbance of blank reaction where the sample was replaced by the PBS buffer solution. The antioxidant capacity was obtained by the standard curve and expressed as Trolox equivalent antioxidant capacity (TEAC). Rutin was used as reference standard. Total phenolic content 2 The TPC was measured by the Folin-Ciocalteau method (Gursoy et al., 2009 2009 ). Folin-Ciocalteau reagent was prepared by the method described by Huang et al. ( 2005 2005 ). Briefly, Folin-Ciocalteau reagent (4.5 ml) was mixed with a 1.0-ml proper concentration sample. After 5 min, 3.0 ml of 7.5% (0.075 g/ml) Na 2 CO 3 was added. After resting at room temperature for 30 min, the absorbance was measured at 765 nm. Gallic acid was used as a standard to prepare for a calibration curve (4, 8, 12, 16, 20, and 24 μg). The TPC was expressed as mg gallic acid equivalents/g dry extract (mg GAE/g Ex). Spectroscopic analysis 2 UV spectra of samples (0.05 mg/ml in 50% (v/v) CH 3 OH-H 2 O) were acquired with a Shimadzu UV-Vis 2550 spectrometer (Kyoto, Japan) within the wavelength range of 200 to 600 nm. IR spectra of solid dry samples (0.5 mg) were acquired with a Nicolet Avatar 360 FT-IR spectrometer (Madison, USA) with KBr pellets within the range of 400–4000 cm −1 . 1 H and 13 C NMR spectra were collected using a Bruker AM-500 spectrometer (Karlsruhe, Germany) in CD 3 OD-D 2 O (1:1, v/v). HPLC analysis 2 The quantifications of crude extracts of GFS and NFS were achieved by an HPLC system. NFS (2.00 mg/ml), GFS (1.00 mg/ml), and salvianolic acid B (0.20 mg/ml) were dissolved with 50% CH 3 OH-H 2 O. Before injection, the samples were passed through a filter (0.45 μm). Mobile phase A was 0.1% aqueous formic acid, and mobile phase B was acetonitrile. The recorded HPLC spectrogram was linearly graduated: 0–10.0 min, 10.0%–20.0% (v/v) solvent B; 10.0–27.0 min, 20.0%–33.0% solvent B; 27.0–30.0 min, 33.0%–70.0% solvent B; 30.0–50.0 min, 70.0%–85.0% solvent B. The column temperature was 20 °C, the flow-rate was 1.0 ml/min, the injection volume was 10 μl, and the UV detection wavelength was 280 nm (Qu et al., 2007 2007 ). Statistical analysis 2 The results were expressed as the mean±standard deviation (SD) of three replicates for each sample. Statistical analyses were performed with SPSS Version 19.0 (SPSS Inc., Chicago, IL, USA). A significant difference was assessed at a level of P < 0.05. Results and discussion 1 Antioxidant activity 2 Two different models of S. miltiorrhiza , DPPH and ABTS assays, were used to assess the antioxidant activity of extracts of S. miltiorrhiza fermented with 20 fungi. As observed in Table 1 1 , the GFS (IC 50 , (25.0±0.6) μg/ml) exhibited higher DPPH• scavenging capacity than NFS (IC 50 , (30.9±1.1) μg/ml), but less than the positive control rutin (IC 50 , (4.32±0.15) μg/ml), which implied that the DPPH• scavenging effect of the S. miltiorrhiza was increased after fermentation by G. luteus . Each value is presented as mean±SD (n=3) Sample TPC (mg GAE / g Ex) IC 50 of DPPH• (μg / ml) TEAC of ABTS + • (μmol Trolox / g Ex) NFS 87.9±3.8 30.9±1.1 435.1±15.3 GFS 167.8±4.6 25.0±0.6 1006.6±23.6 Rutin 4.32±0.15 4546.8±79.5 The antioxidant activity of extracts of fermented S. miltiorrhiza was also determined as Trolox equivalents using the ABTS + • assay. As shown in Table 1 1 , the TEACs of ABTS + • of NFS, GFS, and rutin were (435.1±15.3), (1006.6±23.6), and (4546.8±79.5) μmol Trolox / g Ex, respectively. Similar to the results of the DPPH• scavenging effect, the higher ABTS + • scavenging activity was obtained after fermentation by G. luteus . In addition, the results indicated that the GFS showed an ABTS value more than twice as high as that of NFS. In conclusion, the above methods gave similar results that the fermentation by G. luteus could effectively enhance the antioxidant activity of S. miltiorrhiza . Total phenolic content 2 Previous studies have revealed that phenolics are the major bioactive components in S. miltiorrhiza (Zhao et al., 2006 2006 ). In this research, 20 fermentation extracts of S. miltiorrhiza with different fungi were screened for their ability to improve the phenolic content, and the G. luteus fermentation extract of S. miltiorrhiza showed notable improvement in phenolic content. The results revealed that GFS possessed the highest TPC ((167.8±4.7) mg GAE / g Ex), which was about twice as high as that of NFS ((87.9±3.8) mg GAE / g Ex), indicating that some biotransformation might be taking place to improve hydroxyls in the chemical constituents of S. miltiorrhiza . The improvement of phenolic content in S. miltiorrhiza might be responsible for the increase in antioxidant activity. Previous studies have demonstrated the positive correlation between antioxidant constituents and phenolic content in different plant extracts (Socha et al., 2009 2009 ), which suggested that the increased antioxidant activity might result from the increasing of phenolic content after fermentation. Characterization of the antioxidant compounds of S. miltiorrhiza fermented with G. luteus 2 The UV-Vis spectrum was used for the characterizations of NFS and GFS (0.05 mg/ml in H 2 O). As shown in Fig. 1a 1a , the absorption peak at maximal wavelength ( λ max ) 285 nm was the signal of π→π * electron transition of phenolic acid (Fang and Tan, 2005 2005 ; Zhang et al., 2009 2009 ). The Fig. 1b 1b shows that the absorbance ( λ max =285 nm) of GFS was much higher than that of NFS, indicating the higher content of phenolic acid in GFS, which was consistent with the results of the TPC and antioxidant activity. To clarify more biotransformation details of S. miltiorrhiza caused by G. luteus fermentation, the IR spectrum was used for the chemical characterization of GFS compared with NFS. The IR spectrum (Fig. 2 2 ) revealed that the band at 1042 cm −1 was the change angle vibration of O–H and the stretching vibration of C–O–C (Chen et al., 2012 2012 ). The absorption peak at 1042 cm −1 weakened significantly after fermentation, indicating that the hydrolyzation of glucoside and metabolism of saccharides might take place in fermentation processing. More exploration was performed to identify and characterize the major components of GFS and NFS by NMR spectroscopy (Pieri et al., 2011 2011 ). The NMR spectra of GFS indicated the presence of the characteristic signals of salvianolic acids (Figs. 3 3 and 4 4 ). In its 1 H NMR spectrum (Fig. 3a 3a ), δ H 6.0 (d, 1H, J =16 Hz) and 7.1 (d, 1H, J =16 Hz) were assigned to the trans double bond protons of H-7 and H-8, respectively, in unit A of the salvianolic acids (Fig. 5 5 ). Its 13 C NMR spectrum (Fig. 4a 4a ) showed the signals of C-7 and C-8 in unit C at δ C 87.8 and 57.7, respectively. The signals at δ C 38.0, 37.5, 77.8, and 77.1 were assigned to C-7 and C-8 in units B and D, respectively. The chemical shifts at δ C 143.7–148.6 were the typical signals of C-3 and C-4 in units A, B, C, and D, while the signals of δ C 168.9–176.3 were ascribed to C-9 in units A, B, C, and D. Therefore it can be concluded that GFS was mainly composed of salvianolic acids, including salvianolic acids A and B, prolithospermic acid, isolithospermic acid, lithospermic acid, danshensu, and so on (Fig. 5 5 ). Similarly, the NMR spectra of NFS (Figs. 3b 3b and 4b 4b ) showed the presence of the signals of salvianolic acids. However, the NMR spectra of NFS revealed the presence of the obvious saccharide signals at δ H 3.5–4.2, 5.1 and δ C 62.2–78.0, 93.2–105.0, which were not observed in the NMR spectra of GFS. These results implied that G. luteus fermentation caused the deglycosylation of phenolic glycosides in S. miltiorrhiza , which led to the improvement of the TPC and antioxidant activity. Quantification of GFS and NFS 2 The chromatographic profiles of GFS and NFS are shown in Fig. 6 6 . The HPLC quantitative data were acquired from the calibration curve. The content of salvianolic acid B in GFS ((95.2±2.5) mg / g Ex) is nearly twice as high as that in NFS ((59.0±1.2) mg / g Ex). The improvement of salvianolic acid B after fermentation may account for the increased TPC of S. miltiorrhiza . The antioxidant activity of salvianolic acid B was determined using DPPH assay with rutin as the positive control. Salvianolic acid B exhibited significantly high DPPH• scavenging activity (IC 50 , 2.71 μmol/L) and was more effective than rutin (IC 50 , 7.08 μmol/L), which indicated that the enrichment of salvianolic acid B had a significant contribution to the improvement of the antioxidant abilities. Conclusions 1 G. luteus fermentation could significantly improve the antioxidant activity and TPC of S. miltiorrhiza , suggesting that G. luteus fermentation was effective in S. miltiorrhiza processing. This might be attributed to the deglycosylation of phenolic glycosides in S. miltiorrhiza . The main antioxidant constituents were characterized by spectroscopic analysis as salvianolic acids, and the content of salvianolic acid B improved. This study is the first report on the enrichment of salvianolic acids by microbial fermentation. References H Chen , F Chen . 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This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1500264 Project supported by the National Natural Science Foundation of China (No. 81460648) and the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT13095), China 2015-11-04 2016-04-15 2015-12-10 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2016 2016 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 05 17