Ind. Eng. Chem. Res. 2010, 49, 8279–8281
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Content Increase of Spirostanol Saponins during Enzymatic Hydrolysis of Dioscorea zingiberensis C. H. Wright Yue’e Peng, Yanxin Wang,* Zhihua Yang, Jianguo Bao, and Hongxia Peng School of EnVironmental Studies and MOE Key Laboratory of Biogeology and EnVironmental Geology, China UniVersity of Geosciences, Wuhan 430074, People’s Republic of China
Yan Hong Zhuxi Chuangyi Diosgenin Co., Ltd., Zhuxi 442300, Hubei, People’s Republic of China
Dioscorea zingiberensis C. H. Wright (DZW) has been used as a traditional Chinese medicinal plant for the extraction of diosgenin, which is a starting material for the semisynthesis of drugs of steroidal hormones such as progesterone and testosterone. In this study, the changes in contents of three spirostanol saponins, i.e., zingiberensis newsaponin, deltonin, and prosapogenin A of dioscin, during enzymatic hydrolysis of DZW were monitored. The results show that xylanase leads to a 212% increase in the content of spirostanol saponins, as compared with that in the raw DZW rhizome, while the other three enzymes induced an ∼22% increase. Therefore, xylanase hydrolysis of DZW would be a promising approach to enhance the content of spirostanol saponins, as well as the diosgenin yield. 1. Introduction
2. Experimental Section
Dioscorea zingiberensis C. H. Wright (DZW), which is a traditional Chinese medicinal plant, has been used for plant molluscicides1 and contraceptives.2 Diosgenin derived from this plant has been used as the starting material for the semisynthesis of drugs of steroidal hormones such as progesterone and testosterone.3 Diosgenin precursors are steroidal saponins consisting of nonsugar aglycone (e.g., diosgenin) coupled to different sugar chains.4 Acid hydrolysis had been the traditional industrial process to transform saponins to diosgenin5,6 before we proposed the saccharification-membrane retrieval-hydrolysis (SMRH) process of cleaner production, both to achieve maximum utilization of resources and to meet the requirements of national standard for diosgenin wastewater discharge. By adding the step of enzymatic hydrolysis of starch that accounts for 13%-18% of DZW rhizomes to obtain soluble sugar before acid hydrolysis, the SMRH process was successfully pilot tested in Shiyan of Hubei Province. Via the hydrolysis of starch or cellulose to soluble sugar, more saponins could be released from the plant cell of the DZW, which could also enhance the microbial transformation and acid hydrolysis of saponins to diosgenin.7,8 However, no work has been done to evaluate the effect of enzymes on yield of steroidal saponins. In this paper, the changes in relative concentrations of three spirostanol saponins during enzymatic hydrolysis of DZW were monitored. The following four types of enzymes were selected for our study: R-amylase, exo-1,4-R-glucosidase, cellulase, and endo-1,4-R-xylanase. The first two enzymes were used in our SMRH process to transform starch to sugar,7 and the cellulase was proposed in an alternative process of diosgenin production to degrade cellulose.8 Xylanase has always been used together with cellulase to catalyze the hydrolysis of xylan, which is the major component of hemicellulose in plant cell walls, to xylose.9 * To whom correspondence should be addressed. Tel.: +86-2767883998. E-mail:
[email protected].
2.1. Solvents and Reagents. Solvents and reagents were purchased from AOPU (Wuhan, PRC), and were of analytical grade or HPLC grade. R-Amylase (from Bacillus licheniformis), exo-1,4-R-glucosidase (from Aspergillus niger), cellulase (from Trichoderma Vride G), and endo-1,4-R-xylanase (from Penicillium) purchased from KAYON (Shanghai, PRC) were biological reagents. Diosgenin was purchased from Sigma (USA). 2.2. Plant Materials. The fresh rhizome of DZW was collected in October 2008 at Shiyan City, Hubei Province, PRC. The samples were transported to the laboratory, cut into slices 0.5 cm thick, dried at 75 °C to a constant weight, ground to pass a 0.2 mm sieve, and stored at 2-8 °C before analysis. 2.3. Enzymatic Hydrolysis of DZW. Dry rhizomes powder (10 g) of DZW and water (40 mL) were transferred into four beakers for mixing. R-Aamylase (1 mL) was added into beaker No. 1, which was then capped with a watch glass and placed in a water bath (4 h, 95 °C). Exo-1, 4-R-glucosidase (0.1 g) was added into beaker No. 2 (pH 4.2), which was then placed in a water bath (84 h, 65 °C). Cellulase (0.1 g) was added into beaker No. 3 (pH 5.0), which was then placed in a water bath (88 h, 50 °C). Xylanase (0.1 g) was added into beaker No. 4 (pH 5.0), which then placed in a water bath (96 h, 50 °C). The conditions of enzymatic hydrolysis were according to the references that the producers provided. The mixtures were collected and dried at 75 °C for analysis. 2.4. Analysis of Spirostanol Saponins in DZW. Extraction of DZW plant material for spirostanol saponins analysis was accomplished by weighing 50 mg of plant material into a 10mL screw top test tube equipped with Teflon-lined caps. Five milliliters (5 mL) of methanol were added to each test tube and placed in an ultrasonic cleaner (45 kHz, 35 °C) for 20 min. The methanol extract then passed through a 0.45-µm syringe filter, and 0.5 mL was then transferred to a 1 mL vial for analysis using an Agilent 1100 series high-performance liquid chromatography (HPLC) system equipped with a variable-wavelength detector and an Agilent HC-C18 column (250 mm × 4.6 mm i.d., 5 µm). The injection volume for all samples was 20 µL.
10.1021/ie1001923 2010 American Chemical Society Published on Web 07/28/2010
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Figure 1. Structures of diosgenin precursors isolated from DZW: 1 ) zingiberensis newsaponin, 2 ) deltonin, and 3 ) prosapogenin A of dioscin.
The mobile phase consisted of CH3OH-H2O (90:10, v:v). Saponins were monitored at 203 nm, at a flow rate of 1.0 mL/ min and a column temperature of 35 °C. 3. Results and Discussion Three spirostanol saponins were separated from DZW and identified by LC-ESI-MSn and 1H and 13C NMR: zingiberensis newsaponin (saponin 1), deltonin (saponin 2), and prosapogenin A of dioscin (saponin 3). Their structures are shown in Figure 1. Saponins 1 and 2 were previously isolated from the stem bark of Balanites roxburgii10 and DZW,11 and saponin 3 was isolated from Trillium kamtschaticum Pall,12 and DZW.13 Obviously, saponins 2 and 3 were derived from saponin 1 by losing one and two glucose units, respectively. According to the results of previous work, saponin 1 exhibits the activity of insect antifeedant,14,15 and saponin 2 has strong antifungal capabilities;16 saponin 3 may potentially have wide applications, because of its strong antifungal and anticancer activity.17,18 The contents of saponins 1-3 were analyzed using HPLC. The three saponins and diosgenin were sequentially eluted at 6.3, 6.8, and 7.2 min and 16.3 min, respectively (see Figure 2). The total amount of the three saponins (TCS) increased during the xylanase hydrolysis of DZW, up to 26.2 µg/mg at 84 h from 8.6 µg/mg in the raw sample. Figure 3 shows that the saponins 1-3 amounts were increased up to 6.5 (1), 14.8 (2), and 4.8 (3) µg/mg, respectively, from 4.4 (1), 3.0 (2), and 1.2 (3) µg/ mg in the untreated DZW sample (see Figure 3). In other words, TCS increased substantially during xylanase hydrolysis. By contrast, the other three enzymes showed slight effect on TCS, which increased up to 10.0-11.0 µg/mg from initial content of 8.6 µg/mg (Figure 3). The slight increase may be partially attributed to the release of saponins from cellulose or other substances. However, because of the fact that many enzymes (such as β-glucosidase and endogenous glycosidase) could transform furostanol glycosides to spirostanol glycoside,16,19,20 and that there are furostanol saponins in fresh DZW,21,22 the increased amount of saponins was possibly from the transformation of furostanol glycosides. The increase in spirostanol saponins content may help increase the yield of diosgenin in diosgenin factories, because the conversion from furostanol to diosgenin was less than that from the spirostanol saponins during the acid hydrolysis of saponins.23 The conversion from spirostanol saponins to diosgenin involves only the detachment of the sugar moiety at the C-3 position, whereas the conversion of furostanol saponins involves separation of the sugar moiety at the C-26 position
Figure 2. HPLC chromatograms of spirostanol saponins extract from raw and enzyme-treated DZW rhizomes. Peak labels are defined as follows: 1 ) zingiberensis newsaponin, 2 ) deltonin, 3 ) prosapogenin A of dioscin, and 4 ) diosgenin.
Figure 3. Changes in the amounts of the three steroidal saponins and their total amounts during the enzymatic hydrolysis of DZW.
and cyclization of the F-ring.24 Besides, xylanase could hydrolyze hemicellulose in plant cell walls9 of DZW to expose more saponins to the acid, to increase the yield of diosgenin in production process. 4. Conclusions In recent years, enzymatic hydrolysis has been applied to transform starch and part of the cellulose contained in Dioscorea zingiberensis C. H. Wright (DZW) to soluble sugar or ethanol in diosgenin cleaner production processes.7,8,25 Therefore, monitoring changes in the amount of steroidal saponins during enzymatic hydrolysis of DZW has become practically important. For the four enzymes tested in this study, the total amount of the three spirostanol saponins increased during enzymatic
Ind. Eng. Chem. Res., Vol. 49, No. 17, 2010
hydrolysis of the DZW: a 212% increase is observed after xylanase hydrolysis of the DZW rhizome for 84 h, and an ∼22% increase is observed after hydrolysis using the other three enzymes. Xylanase showed tremendous capability to improve the spirostanol saponins content, and it has great potential to increase the yield of diosgenin if applied in practice. Besides, for the different and important bioactivities of saponins 1-3, direct extraction of the three saponins to obtain a new product from DZW could be an alternative approach for diosgenin production. More-detailed work is still needed to monitor their changes and understand the controlling factors under continuous operation conditions in a full-scale diosgenin factory that adopts cleaner production processes. Acknowledgment We are grateful to Zhuxi Chuangyi Diosgenin Co. Ltd., (Hubei, PRC) for providing the DZW samples. The research work was supported by National Natural Science Foundation of China (No. 40830748) and National Key Technologies R & D Program (No. 2006BAB04A14-2). Literature Cited (1) Yuan, Y.; Xu, X. J.; Dong, H. F.; Jiang, M. S.; Zhu, H. G. Transmission control of schistosomiasis japonica: implementation and evaluation of different snail control interventions. Acta Tropica 2005, 96, 191. (2) Unny, R.; Chauhan, A. K.; Joshi, Y. C. A review on potentiality of medicinal plants as the source of new contraceptive principles. Phytomedicine 2003, 10, 233. (3) Chen, H.; Wang, C.; Chang, C. T. Effects of Taiwanese yam (Dioscorea japonica Thunb Var. pseudojaponica Yamamoto) on upper gut function and lipid metabolism in Balb/c mice. Nutrition 2003, 19, 646. (4) Oleszek, W.; Bialy, Z. Chromatographic determination of plant saponinssAn update (2002-2005). J. Chromatogr., A 2006, 1112, 78. (5) Rothrock, J. W.; Hammes, P. A.; Mcaleer, W. J. Isolation of diosgenin by acid hydrolysis of saponin. Ind. Eng. Chem. 1957, 49, 186. (6) Mishra, S. P.; Gaikar, V. G. Recovery of diosgenin from Dioscorea rhizomes using aqueous hydrotropic solutions of sodium cumene sulfonate. Ind. Eng. Chem. Res. 2004, 43, 5339. (7) Wang, Y. X.; Liu, H.; Bao, J. G.; Hong, Y.; Yang, Z. H.; Zhang, C. X. The saccharification-membrane retrieval-hydrolysis (SMRH) process: A novel approach for cleaner production of diosgenin derived from Dioscorea zingiberensis. J. Clean. Prod. 2007, 16, 1133. (8) Huang, W.; Zhao, H. Z.; Ni, J. R.; Zuo, H.; Qiu, L. L.; Li, H. The best utilization of D-zingiberensis C. H. Wright by an eco-friendly process. Bioresour. Technol. 2008, 99, 7407. (9) Li, Y.; Liu, Z. Q.; Zhao, H.; Xu, Y. Y.; Cui, F. J. Statistical optimization of xylanase production from new isolated Penicillium oxalicum ZH-30 in submerged fermentation. Biochem. Eng. J. 2007, 34, 82.
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(10) Jain, D. C. Antifeedant active saponin from Balanztes Roxburghlz stem bark. Phytochemistry 1981, 26, 2223. (11) Qi, S. S.; Dong, Y. S.; Zhao, Y. K.; Xiu, Z. L. Qualitative and quantitative analysis of microbial transformation of steroidal saponins in Dioscorea zingiberensis. Chromatographia 2009, 69, 865. (12) Nohara, T.; Miyahara, K.; Kawasaki, T. Steroid saponins and sapogenins of underground parts of Trillium kamtdchaticum pall. II. Pennogenin- and kryptogenin 3-O-glycosides and related compounds. Chem. Pharm. Bull. 1975, 23, 872. (13) Zhu, J. B.; Guo, X. J.; Fu, S. P.; Zhang, X. L.; Liang, X. M. Characterization of steroidal saponins in crude extracts from Dioscorea zingiberensis C. H. Wright by ultra-performance liquid chromatography/ electrospray ionization quadrupole time-of-flight tandem mass spectrometry. J. Pharm. Biomed. Anal. 2010, 53 (3), 462 (DOI: 10.1016/j.jpba.2010.05.019). (14) Jain, D. C. Antifeedant active saponin from balanites foxburghi stem bark. Phytochemistry 1987, 26, 2223. (15) Jain, D. C.; Tripathi, A. K. Insect feeding-deterrent activity of some saponin glycosides. Phytother. Res. 1991, 5, 139. (16) Jin, J. M.; Liu, X. K.; Teng, R. W.; Yang, C. R. Enzymatic degradation of parvifloside. Acta Bot. Sin. 2002, 44, 1243. (17) Sautour, M.; Mitaine-Offer, A.-C.; Lacaille-Dubois, M.-A. The Dioscorea genus: A review of bioactive steroid saponins. J. Nat. Med. 2007, 61, 91. (18) Takechi, M.; Tanaka, Y. Structure-activity relationships of synthetic diosgenyl monoglycosides. Phytochemistry 1991, 30, 2557. (19) Inoue, K.; Shimomura, K.; Kobayashi, S.; Sankawa, U.; Ebizuka, Y. Conversion of furostanol glycoside to spirostanol glycoside by β-glucosidase in Costus speciosus. Phytochemistry 1996, 41, 425. (20) Yang, D. J.; Lu, T. J.; Hwang, L. S. Effect of endogenous glycosidase on stability of steroidal saponins in Taiwanese yam (Dioscorea pseudojaponica yamamoto) during drying processes. J. Agric. Food Chem. 2009, 113, 155. (21) Liu, C. L.; Chen, Y. Y. Isolation and identification of protosaponins from fresh rhizomes of Dioscorea Zingiberensis Wright. Acta Bot. Sin. (in Chin.) 1985, 27, 68. (22) Tang, S. R.; Wu, Y. F.; Pang, Z. Identification and isolation of steroidal saponins from Dioscorea Zingiberensis Wright. Acta Bot. Sin. (in Chin.) 1983, 25, 556. (23) Yang, D. J.; Lu, T. J.; Hwang, L. S. Isolation and identification of steroidal saponins in Taiwanese yam cultivar (Dioscorea pseudojaponica Yamamoto). J. Agric. Food Chem. 2003, 51, 6438. (24) Drapeau, D.; Sauvaire, Y.; Blanch, H. W.; Wilke, C. R. Improvement of diosgenin yield form Dioscorea deltoidea plant cell cultures by use of a non-traditional hydrolysis method. Planta Med. 1986, 52, 474. (25) Zhu, Y. L.; Huang, W.; Ni, J. R. A promising clean process for production of diosgenin from Dioscorea zingiberensis C. H. Wright. J. Clean. Prod. 2010, 18, 242.
ReceiVed for reView February 5, 2010 ReVised manuscript receiVed July 10, 2010 Accepted July 13, 2010 IE1001923
