Article pubs.acs.org/JAFC
Efficient Production of Isoflavonoids by Astragalus membranaceus Hairy Root Cultures and Evaluation of Antioxidant Activities of Extracts Jiao Jiao,†,‡ Qing-Yan Gai,‡,§,∥ Yu-Jie Fu,*,§,∥ Wei Ma,*,†,⊥ Xiao Peng,§,∥ Sheng-Nan Tan,§,∥ and Thomas Efferth# †
State Key Laboratory of Tree Genetics and Breeding and §Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, Harbin, Heilongjiang 150040, People’s Republic of China ∥ Collaborative Innovation Center for Development and Utilization of Forest Resources, Harbin, Heilongjiang 150040, People’s Republic of China ⊥ School of Pharmaceutical, Heilongjiang University of Chinese Medicine, Harbin, Heilongjiang 150040, People’s Republic of China # Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, University of Mainz, Staudinger Weg 5, 55128 Mainz, Germany S Supporting Information *
ABSTRACT: In this study, Astragalus membranaceus hairy root cultures (AMHRCs) were established as an attractive alternative source for the efficient production of isoflavonoids (IF). A. membranaceus hairy root line II was screened as the most efficient line and was confirmed by PCR amplification of rolB, rolC and aux1 genes. Culture parameters of AMHRCs were systematically optimized, and five main IF constituents were quali−quantitatively determined by LC−MS/MS. Under optimal conditions, the total IF accumulation of 34 day old AMHRCs was 234.77 μg/g dry weight (DW). This yield was significantly higher compared to that of 3 year old field grown roots (187.38 μg/g DW). Additionally, in vitro antioxidant assays demonstrated that AMHRC extracts exhibited antioxidant activities with lower IC50 values (1.40 and 1.73 mg/mL) as compared to those of field grown roots (1.96 and 2.17 mg/mL). Overall, AMHRCs may offer a promising and continuous product platform for naturally derived, high quality and valuable nutraceuticals. KEYWORDS: isoflavonoids, hairy roots, Astragalus membranaceus, response surface methodology, LC−MS/MS, antioxidant activities
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and constraints.10,11 Hairy root cultures (HRCs) produced via Agrobacterium rhizogenes mediated transformations have emerged as an ideal biotechnological system for the production of valuable secondary metabolites that are present in wild-type roots.12−16 HRCs are highly differentiated with the advantages of genetic and biochemical stability, comparable biosynthetic capacity to native plant roots, long-term preservation, and sizable production.12,13,17 They often accumulate relatively high amounts of phytochemicals compared to undifferentiated plant cell/callus cultures.17−19 Moreover, the ability to synthesize their own phytohormones enables fast growth of HRCs in culture media without growth regulators, the presence of which is not desirable in final products.12,15,17 A. membranaceus represents a valuable economic crop, thus culturing hairy roots is an attractive alternative for the production of valuable IF. The present study described a protocol for the development of A. rhizogenes mediated hairy root system in A. membranaceus to produce medicinal IF. A highly productive A. membranaceus hairy root line (AMHRL) was first identified, and then its culture conditions were optimized for potential industrial scale-
INTRODUCTION Astragalus membranaceus root (Radix astragali) is a healthpromoting supplement (typically soups and teas) commonly used in the United States, Europe, and Asian countries for enhancing the immune system.1 The U.S. Dietary Supplement Health and Education Act (DSHEA) (103rd Congress, 1994) has classified Radix astragali as an over-the-counter dietary supplement in the form of beverages or capsules, which can be sold at health food markets.2 Apart from triterpene saponins and polysaccharides, isoflavonoids (IF) are principal active ingredients of Radix astragali, which exhibit various biological effects such as antioxidant, anti-inflammatory, antiviral, antifatigue, hematopoietic, hypolipidemic, neuroprotective, cardioprotective, immunomodulatory, antitumorigenic, and estrogenic activities.3−9 Due to these versatile health benefits, the interest and demand for IF from Radix astragali has been constantly increasing during the past years. The quality and quantity of phytochemicals from wildcollected or field-grown plants is often fluctuating and heterogeneous due to unfavorable environmental conditions such as climatic changes, insect infestation, microbial diseases, etc. Plant tissue culture technology, an alternative system for the standardized production of phytochemicals, can continuously provide high-value medical, food, and health ingredients regardless of geographic, climatic, or environmental variations © 2014 American Chemical Society
Received: Revised: Accepted: Published: 12649
August 8, 2014 December 6, 2014 December 6, 2014 December 7, 2014 dx.doi.org/10.1021/jf503839m | J. Agric. Food Chem. 2014, 62, 12649−12658
Journal of Agricultural and Food Chemistry
Article
DW and total IF (TIF) content (sum amount of CAG, ON, ASG, CA, and FO) were measured and determined, respectively. In order to obtain optimal biomass production and IF accumulation during the culture process, a Box−Behnken design (BBD) was applied to investigate the effects of four independent key variables (culture temperature 24−32 °C, sucrose concentration 2−4%, inoculum size 1−2%, and harvest time 20−40 days) on dependent variables (biomass DW and TIF content). The inoculum size was calculated based on the fresh weight (FW) of hairy roots. Regression analysis was carried out to evaluate the response function as a quadratic polynomial:
up applications in bioreactors. Moreover, this study quali− quantitatively characterized five main IF constituents (calycosin-7-O-β-D-glucoside, ononin, astraisoflavan-7-O-β-D-glucoside, calycosin, and formononetin) from A. membranaceus HRCs (AMHRCs), followed by the evaluation of antioxidant activities of AMHRC extracts. We conclude that AMHRCs were more efficient for production of isoflavonoids compared to field grown roots (AMFGRs) of A. membranaceus.
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MATERIALS AND METHODS
Plant Materials and Reagents. Mature A. membranaceus seeds were generously provided by Heilongjiang University of Chinese Medicine (Harbin, China). Standard compounds including calycosin7-O-β-D-glucoside (CAG), ononin (ON), astraisoflavan-7-O-β-Dglucoside (ASG), calycosin (CA), and formononetin (FO) were purchased from Weikeqi Biological Technology Co. Ltd. (Sichuan province, China). Ascorbic acid (VC), 2,2-diphenyl-1-picryl-hydrazylhydrate (DPPH), linoleic acid, butylated hydroxytoluene (BHT), and β-carotene were obtained from Sigma-Aldrich Co. (Steinheim, Germany). A. rhizogenes LBA9402 strain was obtained from the Key Laboratory of Forest Plant Ecology, Ministry of Education, Harbin, China. The bacterial culture was maintained at 25 ± 1 °C on yeast mannitol broth (YMB) medium. Other reagents of either analytical or optical grade were purchased from Beijing Chemical Reagents Co. (Beijing, China). Ultrapure water was prepared from a Milli-Q system at 18.3 MΩ resistance (Millipore, Bedford, MA). Induction and Establishment of Hairy Roots. Young leaves of 3 week old aseptic plantlets were used as explants for hairy root induction. The excised leaf segments were preincubated on halfstrength Murashige and Skoog (MS/2) solid medium for 2 days prior to the following infection. After that, the explants were immersed into overnight grown bacterial suspensions of A. rhizogenes LBA9402 strain (OD600nm = 0.6−0.8) for 10 min, dry-blotted on sterile filter paper, and incubated in the dark at 25 ± 1 °C on MS/2 solid medium supplemented with acetosyringone (100 μM). After 2 days of cocultivation, the explants were transferred to the hormone-free MS solid medium containing sodium cefotaxim (500 mg/L) to eliminate the residual bacteria. Putative transgenic hairy roots were observed emerging from the wound sites of explants within 13 days of incubation. During subculture every other week, the concentration of antibiotic was gradually decreased and finally omitted, until the bacteria were completely eliminated. Eventually, eight lines (AMHRL I−VIII) among 62 established AMHRLs were selected for the present study. Molecular Characterization of Hairy Roots. Integration of TDNA responsible for hairy root formation was confirmed by PCR analysis using rolB, rolC, aux1, and virD specific primers (see Supporting Information Table S1) as suggested by previous reports.20,21 Genomic DNA was isolated from AMHRL II using a DNeasy Plant Mini Kit (Tiangen, China) following the manufacturer’s instructions. PCR amplification of rolB, rolC, aux1, and virD genes was performed by a S1000 thermal cycler (Bio-Rad, Hercules, CA) according to the following program: initial denaturation at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 59 °C for 50 s, and extension at 72 °C for 50 s, and final extension at 72 °C for 7 min. PCR products were analyzed by electrophoresis on a 2.5% (w/v) agarose−ethidium bromide gel along with 1000 bp DNA marker. Additionally, genomic DNA isolated from A. membranaceus aseptic plantlets and Ri-plasmid (pRi) DNA from A. rhizogenes LBA9402 strain were used as negative and positive controls, respectively. Optimization of Culture Conditions. Once the highly productive AMHRL was identified, a certain amount of hairy roots was transferred into 250 mL Erlenmeyer flasks containing 150 mL of MS liquid medium (pH 5.8) supplemented with 1% casein hydrolysate without NH4NO3, and further incubated on a rotary shaker (100 rpm) at an appropriate temperature in the dark. AMHRCs were harvested by filtration after a certain period of cultivation and dried in a vacuum drier at 60 °C until achieving constant weight. Thereafter, biomass
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