See discussions, stats, and author profiles for this publication at: http://www.researchgate.net/publication/51185646 Production of 8-Prenylnaringenin from Isoxanthohumol through Biotransformation by Fungi Cells ARTICLE in JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY · JUNE 2011 Impact Factor: 2.91 · DOI: 10.1021/jf2011722 · Source: PubMed CITATIONS READS 9 16 7 AUTHORS, INCLUDING: Feng Chen Yachen Dong Xi'an Jiaotong University Zhejiang University 208 PUBLICATIONS 2,131 CITATIONS 9 PUBLICATIONS 25 CITATIONS SEE PROFILE SEE PROFILE Hui Ni Jimei University 38 PUBLICATIONS 121 CITATIONS SEE PROFILE Available from: Yachen Dong Retrieved on: 18 November 2015 ARTICLE pubs.acs.org/JAFC Production of 8-Prenylnaringenin from Isoxanthohumol through Biotransformation by Fungi Cells † † ‡ § † † † || † Ming-liang Fu, Wei Wang, , Feng Chen, Ya-chen Dong, Xiao-jie Liu, Hui Ni, , and Qi-he Chen*, † Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310058, People's Republic of China ‡ Institute of Quality and Standard for Agriculture Products, Zhejiang Academy of Agriculture Sciences, Hangzhou 310021, People's Republic of China § Food Science and Human Nutrition, Clemson University, Clemson, South Carolina 29634, United States School) of Bioengineering, Jimei University, Xiamen 361021, People's Republic of China ABSTRACT: 8-Prenylnaringenin (8PN), which presents in hop, enjoys fame as the most potential phytoestrogen. Although a number of health effects are attributed to 8PN, few reports are available about the production of it. In this work, screening of fungi to efficiently transform isoxanthohumol (IXN) into 8PN was designed. The biotransformation of IXN was significantly observed in Eupenicillium javanicum, Cunninghamella blakesleana, and Ceriporiopsis subvermispora under five kinds of transformation conditions. As a comparative result of IXN transformation, E. javanicum was the optimal biocatalyst to produce 8PN. Transformation caused by growing precultured fungal mycelia, a process designated as G2, was a favorable condition for IXN transformation in view of the yield of 8PN. The possible transformation pathway of 8PN bioproduction is postulated in this work. The construction of fungus and transformation mode derived from the current work is viable and an alternative procedure for 8PN formation. KEYWORDS: 8-Prenylnaringenin, isoxanthohumol, biotransformation, identification ’ INTRODUCTION advantages such as high stereo- and regioselectivity, as well as milder reaction conditions, simple operation procedures, and Hops (Humulus lupulus L.) are a commonly used material in the 14,15 brewing industry to provide flavor and bitterness to beer. In recent environmental safety. In the earlier study, IXN can be years, hops have proved to be a very rich source of prenylated- converted into 8PN by intestinal microbiota, and Eubacterium fl fl limosum was identified to be capable of this bioconversion avonoids and derivatives. The most important prenylated avo- 16 noids in hops are xanthohumol (XN), isoxanthohumol (IXN), process. However, E. limosum is a kind of rigid anaerobic and 8-prenylnaringenin (8PN) (Figure 1), and they are now bacterium, and it is rather inconvenient to use anaerobic bacteria receiving more and more attention because of their well-known in the biotransformation. In some literature, some fungi were found to possess demethylation enzmyes, such as Aspergillus sp.,17 biological and pharmacological activities. XN, the main prenyl- 18 19 20 flavonoid of hops (0.1À1.0% on dry weight), is a broad-spectrum Mortierella sp., Cunninghamella echinulata, and Eupenicillium sp. ff It was indicated that the demethylation activity may have a relation- cancer chemopreventive agent. It shows di erent inhibition 17,21 mechanisms at the initiation, promotion, and progression stages ship with cytochrome P450. Therefore, the aim of this work is to 1 fl isolate a suitable fungus to produce8PNfromIXN,soastosearch of carcinogenesis. Although XN is the major prenl avonoid in ffi hops, its content is rather low in beer due to the thermal for more possible and e cient ways to obtain 8PN. isomerization during the brewing process. The product of thermal isomerization is IXN, a prenylatedflavonoid that has a lower antic- ’ MATERIALS AND METHODS ancer activity than XN. 8PN in hops shows a potential estrogenic Reagents. activity,2 which enjoys fame as the most potential phytoestrogen Standard IXN was purchased from J&K Scientific Ltd. (Shanghai, China). 8PN was purchased from Sigma-Aldrich Chemical Co. isolated until now. In vivo and in vitro studies show that 8PN has a Ltd. (St. Louis, MO). Acetonitrile and methanol used in high-performance considerably higher activity than some other famous phytoestro- fl 3,4 liquid chromatography (HPLC) analysis was chromatographic grade. gens, such as soy iso avones and genistein. Heyerick's research Water was prepared in a Milli-Q water purification system (Millipore, shows that at a low dosage (100 μg daily), 8PN still has an alleviation ff ff 5 Bedford, MA). Other chemicals used in the study were analytical grade. e ect on women who su er menopausal discomforts. Because of its Microorganisms. Eupenicillium javanicum AS 3.5706, Mortierella strong estrogenic activity, 8PN is considered a more natural ramanniana AS 3.3413, Mortierella isabellina AS 3.3410, Cunninghamella alternative to hormone replacement therapy (HRT).6 Additionally, 7,8 blakesleana AS 3.910, and Aspergillus niger AS 3.429 were obtained from 8PN shows some other bioactivities, such as anticancer, osteo- China General Microbiological Culture Collection Center (CGMCC). 9 10 11 porosis prevention, inhibiting angiogenesis, and antioxidant. Ceriporiopsis subvermispora ACCC 31513 was bought from Agricultural At present, 8PN can be easily synthesized by chemical ways from IXN or naringenin,12,13 but some issues still remain, Received: March 22, 2011 including complex operation, safety, pollution, and production Accepted: June 2, 2011 cost. As compared with chemical methods, biotransformation Revised: June 2, 2011 is a useful tool to produce biologically active compounds with Published: June 02, 2011 r 2011 American Chemical Society 7419 dx.doi.org/10.1021/jf2011722 | J. Agric. Food Chem. 2011, 59, 7419–7426 Journal of Agricultural and Food Chemistry ARTICLE that, culture broth was centrifuged at 4 °C at a speed of 10000 r/min for 15 min. The collected cultured cells were washed with sterilized water three times. Then, the washed wet mycelia were collected and stored at 4 °C for the following experiments. Three types of systems were designed in this study for the resting-cell transformation based on different liquid transformation broth. The suspension cells (3.0 g wt) were cultivated in 30 mL of 2% glucose (type R1), 0.2 M, pH 7.2, phosphate buffer (type R2), and 0.2 M, pH 7.2, phosphate buffer (0.5% Tween 80 was added, type R3), respectively. Meanwhile, 0.1 mL of prepared substrate solution was added into the flasks. Subsequently, the cultures were incubated for 6 days at 28 °C with a shaken speed of 120 r/min. Preparation and Isolation of 8PN from Transformation Figure 1. Structures of XN (a), IXN (b), and 8PN (c). Broth. After incubation, the culture broth was centrifuged at 3000 rpm for 30 min. Then, the supernatant was extracted three times by equivalent Culture Collection of China (ACCC). Bjerkandera adusta CFCC 88349 volumes of ethyl acetate, all of the organic layers were combined, and the and Gloeophyllum trabeum CFCC 86019 were bought from China extracted solutions were concentrated in vacuum at 35 °C. Residues were Forestry Culture Collection Center (CFCC). Armillaria luteo-virens Sacc dissolved in methanol for HPLC determination of IXN and 8PN. QH (CGMCC 1884) was obtained from the Qingzang Plain, China, was Simultaneous Analysis of IXN and 8PN by Means of HPLC. bred after several natural mutations, and was stored in our lab. The fungi Samples were analyzed on a Waters 2695 HPLC system, which was equipped were maintained on potato dextrose agar (PDA) slants at 4 °C. with two Waters 510 pumps (Waters, Milford, MA), a sample injector Preparation of IXN. IXN was prepared according to the reported (Rheodyne, Cotati, CA) with a 20 μL loop, and a Waters 996 photodiode procedure13 with minor modification. Spent hops that were extracted by array detector. Evaluation and quantificationweremadeonaMillennium supercritical carbon dioxide were used to prepare XN. The powder (300 g) chromatography data system (Waters). The column used was a reversed- was extracted with 1.5 L of ethanol under reflux extraction (400 W and phase Symmetry C18 (4.6 mm  250 mm, 4 μm). The mobile phase was 40 °C) for 2 h. The mixture was filtered with 0.22 μm film of Type GV acetonitrile (solvent A) and 1% formic acid (solvent B) with a linear solvent (Millipore), and then, the residue was repeatedly extracted twice (total gradient, starting on injection, from 40 to 100% A over 20 min, then followed 1.5 L of ethanol). The filtrate was combined, and extract solution was by 100% A for 5 min. The flow rate was 0.8 mL/min, and the detection evaporated to dryness by rotary vaporization at 30 °C. The crude XN wavelength was 288 nm. All of the analysis was performed at a was purified by high-speed countercurrent chromatography (HSCCC) temperature of 30 °C. The standard curve of IXN and 8PN was y = 2 2 using n-hexaneÀethyl acetateÀmethanolÀwater (5:5:4:3) as the sol- 35494x À 5034.7 (R = 0.9988), and y = 27390x À 129.15 (R = vent system. After HSCCC purification, the purity of XN was up to 96%. 0.9993), respectively. Figure 2a showed the chromatograms of standard IXN was prepared from XN by dissolving in 1% NaOH solution and IXN and 8PN, and the extraction samples after transformation by E. ° stirring at 0 C for 2 h. Then, 50% H2SO4 was used to acidify the reaction javanicum AS 3.5706 were also identified (Figure 3a). Routine sample mixture. It produced a light yellow precipitate, and after filtration and calculations were made by the comparison of the peak areas with that of washing several times with water, the product was dried by Christ alpha the standard curve.