Article pubs.acs.org/JAFC
Innovative Approach to the Accumulation of Rubrosterone by Fermentation of Asparagus filicinus with Fusarium oxysporum Ying Li, Le Cai, Jian-Wei Dong, Yun Xing, Wei-He Duan, Hao Zhou, and Zhong-Tao Ding* Key Laboratory of Medicinal Chemistry for Nature Resource, Ministry of Education, School of Chemical Science and Technology, Yunnan University, Kunming, Yunnan 650091, People’s Republic of China S Supporting Information *
ABSTRACT: Rubrosterone, possessing various remarkable bioactivities, is an insect-molting C19-steroid. However, only very small amounts are available for biological tests due to its limited content from plant sources. Fungi of genus Fusarium have been reported to have the ability to convert C27-steroids into C19-steroids. In this study, Asparagus filicinus, containing a high content of 20-hydroxyecdysone, was utilized to accumulate rubrosterone through solid fermentation by Fusarium oxysporum. The results showed that F. oxysporum had the ability to facilitate the complete biotransformation of 20-hydroxyecdysone to rubrosterone by solid-state fermentation. The present method could be an innovative and efficient approach to accumulate rubrosterone with an outstanding conversion ratio. KEYWORDS: rubrosterone, 20-hydroxyecdysone, biotransformation, Asparagus filicinus, Fusarium oxysporum
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INTRODUCTION iotransformation with microorganisms has been an important approach in the field of applied biocatalysis.1 In recent years, studies on fermentation using microorganisms such as fungi, bacteria, and algae for the production of useful compounds and materials have been widely conducted.2−4 Microorganisms have long played a major role in the generation of new, active, and less toxic bioactive products such as foods (dairy, fish, and meat products) and alcoholic beverages that would be difficult to obtain from either biological systems or chemical synthesis.5 To date, a wide range of applications has been described for the development and use of natural food and additives (antioxidants, flavors, colorants, preservatives, sweeteners, and so on)6 derived from microorganisms because they are more desirable than synthetic ones. Two important examples are the production of L-aspartic and L-malic acids from fumaric acid7 and the transformation of ginseng saponins to ginsenoside Rh2 by human intestinal bacteria8 in the fermentation industry. Additionally, previous studies have reported that the fermentation process could modify a large variety of naturally occurring constituents, such as terpenes, steroids, alkaloids, isoflavones, saponins, phytosterols, and phenols, simultaneously, and have a significant effect on the enhancement of biological activities.9−12 Rubrosterone, 1 (Figure 1), is a 19-carbon metabolite of insect-molting substances in plants. It was separated from Achyranthes rubrofusca for the first time in 196813 and was then isolated from Serratula tinctoria,14 Cyanotis arachnoidea,15 and Taxus yunnanensis.16 Previous studies have revealed that rubrosterone possesses many remarkable bioactivities, including antidiabetic activities, the promotion of eye formation in insects,17 moderate influence on the development of wing disks of cabbage armyworm,18 and significant positive effects on the in vitro growth rate (spermatogenesis) of testes isolated from the diapausing slug moth prepupae.19 In addition, Chihara et al.20 reported that the evagination of mass-isolated imaginal
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© 2015 American Chemical Society
Figure 1. Structures of rubrosterone, 1, and 20-hydroxyecdysone, 2.
disks of Drosophila melanogaster could be induced by rubrosterone in vitro. Hikino et al.21 demonstrated that rubrosterone could promote the in vitro differentiation of cultured eye-antennal disks of Drosophila, and the activity was 106 times stronger than that of ecdysterone and inokosterone. Studies by Otaka et al.22 revealed that rubrosterone had a high stimulating effect on protein synthesis in mouse liver. It is expected that additional interesting biological activities of this substance will be discovered. Unfortunately, only very small amounts of rubrosterone from natural resources worldwide have hitherto been available for biological tests because the quantity of rubrosterone from plant sources is very limited. Furthermore, studies on the synthesis of this compound showed that it is extremely complicated, consisting of 20 steps from dehydroepiandrosterone23 and 7 steps from 3α,5αcycloandrostane-6,17-dione.24 Furthermore, synthesis from βecdysterone is also laborious,21 requiring the use of reagents that are hazardous to health and constituting a serious environmental disposal problem. Consequently, a straightforReceived: Revised: Accepted: Published: 6596
February 25, 2015 July 3, 2015 July 5, 2015 July 5, 2015 DOI: 10.1021/acs.jafc.5b02570 J. Agric. Food Chem. 2015, 63, 6596−6602
Article
Journal of Agricultural and Food Chemistry
Plant Material. A. f ilicinus was collected in Kunming City, Yunnan Province, of China in November 2013 and authenticated by Prof. Shugang Lu of the School of Life Sciences at Yunnan University. A voucher specimen (No. YNU-XBB01) is deposited in the Key Laboratory of Medicinal Chemistry for Natural Resources at Yunnan University. Microorganisms and Fermentation. The plant pathogen fungus F. oxysporum was obtained from the Stains Collection of Yunnan Institute of Microbiology, Yunnan Institute of Microbiology, Yunnan Province, China. F. oxysporum was then activated in PDA (1 L of water, 200 g of potato, 20 g of dextrose, and 15 g of agar) slant culture medium in a constant-temperature incubator at 28 °C for 5 days. The mature plant pathogen fungus was added to the fermentation culture medium containing 5.00 g of A. f ilicinus powder that had been dampened with 8 mL of water, sterilized at 121 °C for 30 min, and then incubated at 28 °C for 30 days. The blank fermented A. filicinus was treated the same as the microorganism-fermented A. filicinus in the absence of plant pathogen fungus. The nonfermented A. f ilicinus was the original material of the herb. Extraction and Isolation. The nonfermented A. f ilicinus (5.00 g), blank fermented A. f ilicinus (5.00 g), and microorganism-fermented A. f ilicinus (5.00 g) were immersed with 80% methanol/H2O (v/v) at a ratio of 1:10 (w/v) for 24 h at room temperature and then exhaustively extracted ultrasonically three times for 30 min each. The extracts were decanted, filtered through Whatman no. 1 filter paper, and concentrated under a rotary evaporator. The nonfermented, blank fermented, and F. oxysporum-fermented A. f ilicinus extracts were obtained as E1 (3.41 g), E2 (3.07 g), and E3 (0.56 g), respectively. E3 was then chromatographed on a silica gel column (column, 200 mm × 20 mm) eluted with chloroform/methanol (30:1, 600 mL) to yield fraction A. Fraction A (42.6 mg) was further subjected to silica gel CC (column, 200 mm × 10 mm) with petroleum ether/acetone (3:1, 200 mL) to yield compound 1 (25.1 mg). E1 was passed through a macroporous resin and then isolated by a combination of RP-C18 silica gel (column, 450 mm × 40 mm) and silica gel CC (column, 200 mm × 20 mm) with a gradient elution mixture of chloroform/ methanol (10:1, 500 mL) for the acquisition of compound 2 (24.7 mg). Structure Elucidation of Rubrosterone and 20-Hydroxyecdysone. Rubrosterone, 1: white crystal (methanol/H2O); mp 245.0− 247.0 °C; HR-ESI-MS m/z 357.1683 [M + Na]+ (calcd for C19H26O5Na [M + Na]+, 357.1672); 1H NMR (400 MHz, pyridined5) δH 0.85 (3H, s, H-18), 1.04 (3H, s, H-19), 1.58 (1H, dd, J = 12.0, 4.8 Hz, H-11), 1.94 (1H, t, J = 12.4 Hz, H-1), 2.28 (2H, m, H-15), 3.05 (1H, dd, J = 13.2, 3.6 Hz, H-5), 3.54 (1H, m, H-9), 4.15 (1H, dt, J = 11.2, 3.2 Hz, H-2), 4.26 (1H, br s, H-3), 6.29 (1H, s, H-7); 13C NMR (100 MHz, pyridine-d5) δC 37.6 (C-1), 67.8 (C-2), 67.8 (C-3), 32.2 (C-4), 51.3 (C-5), 203.1 (C-6), 121.8 (C-7), 162.7 (C-8), 34.8 (C-9), 38.6 (C-10), 19.9 (C-11), 28.7 (C-12), 53.0 (C-13), 79.3 (C14), 33.4 (C-15), 24.5 (C-16), 217.1 (C-17), 17.0 (C-18), 24.3 (C19). The NMR data were compared with the literature,14,15 and compound 1 was determined to be rubrosterone (Figure 1). 20-Hydroxyecdysone, 2: white amorphous powder; HR-ESI-MS m/ z 481.3137 [M + H]+ (calcd for C27H45O7 [M + H]+, 481.3165), 503.2962 [M + Na]+ (calcd for C27H44O7Na [M + Na]+, 503.2985), 983.6036 [2M+Na]+ (calcd for C54H88O14Na [2M + Na]+, 983.6072); 1 H NMR (400 MHz, pyridine-d5) δH 0.97 (3H, s, H-19), 1.11 (3H, s, H-18), 1.30 (6H, br s, H-26, 27), 1.55 (3H, s, H-21), 2.18 (1H, m, H24), 2.87 (2H, br s, H-5, 17), 3.47 (1H, br s, H-9), 3.75 (1H, d, J = 6.4 Hz, H-22), 4.12 (2H, m, H-2, 3), 6.14 (1H, s, H-7); 13C NMR (100 MHz, pyridine-d5) δC 37.2 (C-1), 67.5 (C-2), 67.6 (C-3), 31.8 (C-4), 50.7 (C-5), 203.3 (C-6), 121.1 (C-7), 165.8 (C-8), 33.8 (C-9), 38.1 (C-10), 20.9 (C-11), 31.4 (C-12), 47.5 (C-13), 83.7 (C-14), 31.1 (C15), 20.5 (C-16), 49.5 (C-17), 17.3 (C-18), 23.9 (C-19), 76.4 (C-20), 21.1 (C-21), 77.0 (C-22), 26.8 (C-23), 41.9 (C-24), 69.3 (C-25), 29.5 (C-26), 29.4 (C-27). The evidence reported above, combined with the data reported in the literature,30 led to the assignment of the structure of 20-hydroxyecdysone, 2, as shown in Figure 1. HPLC and HPLC-MS Analysis. The identification and quantitation of crude extracts of nonfermented A. f ilicinus, blank fermented A.
ward and efficient approach to make large amounts of rubrosterone available for biological testing is urgently required. It has been well documented that in the sterol-degrading microorganisms, the ring nucleus and side chain are metabolized by different enzyme systems simultaneously and independently.25 According to previous papers, the microorganism Mycobacterium sp. (NRRL B-3805) was a mutant for the selective cleavage of the sterol side chain to generate C19 and other useful steroid intermediates.26 The microorganism Fusarium lini (ATCC 9593) was also capable of oxidatively catabolizing the 20R,22R-dihydroxy side chain in ponasterone A to yield rubrosterone; incubation of β-ecdysone yielded rubrosterone, as well.27 Nevertheless, this type conversion using F. lini was relatively less efficient (15% of ponasterone A and
