Biotic elicitors enhance diosgenin production in Helicteres isora L. suspension cultures via up-regulation of CAS and HMGR genes

In an attempt to find an alternative and potent source of diosgenin, a steroidal saponin in great demand for its pharmaceutical importance, Helicteres isora suspension cultures were explored for diosgenin extraction. The effect of biotic elicitors on the biosynthesis of diosgenin, in suspension cultures of H. isora was studied. Bacterial as well as fungal elicitors such as Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae and Aspergillus niger were applied at varying concentrations to investigate their effects on diosgenin content. The HPLC based quantification of the treated samples proved that amongst the biotic elicitors, E. coli (1.5%) proved best with a 9.1-fold increase in diosgenin content over respective control cultures. Further, the scaling-up of the suspension culture to shake-flask and ultimately to bioreactor level were carried out for production of diosgenin. During all the scaling-up stages, diosgenin yield obtained was in the range between 7.91 and 8.64 mg l−1, where diosgenin content was increased with volume of the medium. The quantitative real-time PCR (qRT-PCR) analysis showed biotic elicitors induced the expression levels of regulatory genes in diosgenin biosynthetic pathway, the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and cycloartenol synthase (CAS), which can be positively correlated with elicited diosgenin contents in those cultures. The study holds significance as H. isora represents a cleaner and easy source of diosgenin where unlike other traditional sources, it is not admixed with other steroidal saponins, and the scaled-up levels of diosgenin achieved herein have the potential to be explored commercially.     

正交试验法优选薯蓣皂苷元萃取条件研究

目的:为明确薯蓣皂苷元的最佳提取工艺,我们采用超临界CO2萃取技术,通过正交试验优化穿山龙中薯蓣皂苷元的提取工艺。方法:探讨萃取压力、萃取温度、分离Ⅰ压力、分离Ⅰ温度等因素对薯蓣皂苷元收率的影响。确定了薯蓣皂苷元的最佳萃取条件。结果:穿山龙中薯蓣皂苷元超临界萃取的最佳条件为萃取压力30 MPa,萃取温度50℃,分离Ⅰ压力13 MPa,分离Ⅰ温度40℃。结论:超临界CO2萃取法提取薯蓣皂苷元工艺简单,安全有效。     

从盾叶薯蓣组培苗中高压酸解制备薯蓣皂苷元

采用正交试验法对盾叶薯蓣(Dioscorea zingiberensis)组培苗中薯蓣皂苷元的高压酸解制备工艺进行了研究。以薯蓣皂苷元的含量作为评价指标,选用正交表L16(45),以样品用量、硫酸浓度、提取时间为因素,设计了3因素4水平的正交试验。结果表明:高压酸解提取薯蓣皂苷元的最佳工艺条件为:样品用量25 mg、硫酸浓度0.5 mol/L、提取时间2 h,在此条件下提取物中薯蓣皂苷元的平均含量为9.12 mg/g。     

石斛干品基因组DNA的提取与RAPD分析

市场中药干品的药性差异一直是影响中药标准化的瓶颈,而检测技术相对落后是导致这一现象的主要原因。DNA分子水平检测的困难是药材干品的基因组DNA难以提取。本文以铁皮石斛(Dendrobium candidum)干茎为材料,采用了四种方法从干品石斛中提取基因组DNA。结果表明,采用改良的CTAB法可从石斛干品尤其是干茎皮中提取质量较高的基因组DNA,其分子量大于23kb,以此DNA为模板进行不同引物的PCR扩增可获得清晰的RAPD条带。该研究初步建立了石斛干品合适的RAPD技术体系。     

Simultaneous analysis of diosgenin and sarsasapogenin in Asparagus officinalis byproduct by thin‐layer chromatography

Introduction – Asparagus officinalis L. has several biological activities including antifungal, antiviral and antitumoral activities due to the steroidal saponins. Normally diosgenin and sarsasapogenin are analysed separately by thin‐layer chromatography or high‐performance liquid chromatography (HPLC‐UV or HPLC‐ELSD), which is time‐consuming and expensive, so we need to find a rapid solution to this problem. Objective – To develop a sensitive, rapid and validated TLC method for simultaneous detection and quantification of diosgenin and sarsasapogenin. Methodology – Samples were prepared by extraction of A. officinalis with 70% aqueous ethanol to get steroidal saponins, and then hydrolysed using 36 mL 2 m hydrochloric acid for 3 h. The hydrolysis product was extracted with chloroform, and then analysed by TLC, the results of which were verified by HPLC and HPLC‐MS. Results – The retention factor (R_f) of diosgenin and sarsasapogenin on TLC plate were 0.49 and 0.6, respectively. After calculation from the regression equation of the standard curve, the contents of diosgenin and sarsasapogenin in the A. officinalis extract were 0.27–0.46 and 0.11–0.32%, respectively. Conclusion – The study showed that thin‐layer chromatography can be applied for the determination of diosgenin and sarsasapogenin in the oldest tissue of A. officinalis, and also can be conducted for screening of sapogenin in other plant or extracts. Copyright © 2010 John Wiley & Sons, Ltd.     

少根根霉原变种发酵生产薯蓣皂苷元

从穿龙薯蓣Dioscorea nipponica中分离得到的一少根根霉原变种Rhizopus arrhizus var. arrhizus菌株,能实现薯蓣皂苷的生物转化。用该菌株发酵穿龙薯蓣D. nipponica生产薯蓣皂苷元,采用高效液相色谱法测定薯蓣皂苷元的含量,其总得率可达3.00%。运用该菌株发酵制备薯蓣皂苷元,操作简单,环保,且得率高。     

Three-liquid-phase extraction of diosgenin and steroidal saponins from fermentation of Dioscorea zingibernsis C. H. Wright

Diosgenin is an important starting material in the steroidal hormone industry. The yield of diosgenin obtained from the fermentation of Dioscorea zingibernsis C. H. Wright (DZW) by Trichoderma harzianum is higher than that typically obtained from acid hydrolysis. In this paper, the extraction of steroids in the culture broth was studied. A novel three-liquid-phase system (TLPS) consisted of petroleum ether, ethanol, ammonium sulphate and water was used to separate diosgenin and steroidal saponins in the culture broth. The partition behaviors of various steroidal saponins, diosgenin and glucose were investigated. From this, an optimized TLPS was obtained, which composed of 30% ethanol (w/w), 17% (NH₄)₂SO₄ (w/w) and 40% (w/w) petroleum ether. In the optimized TLPS, almost all of the diosgenin was extracted into the top phase giving a recovery of 97.24%, whereas the steroidal saponins were mainly extracted into the middle phase, with recoveries of zingibernsis newsaponin, deltonin and diosgenin-diglucoside reaching almost 100%. The recoveries of trillin and diosgenin-triglucoside were 96.03% and 98.82%, respectively. Glucose tended to remain in the bottom phase, giving a recovery of 72.01%. The three-liquid-phase extraction (TLPE) successfully resulted in the simultaneous separation of diosgenin, untransformed steroidal saponins and glucose.     

Influence of Soil Variation on Diosgenin Content Profile in Costus speciosus from Indo-Gangetic Plains

Costus speciosus is a rich source of commercially important compound Diosgenin, distributed in different regions of India. The present investigation was aimed to quantify diosgenin through High Performance Thin Layer Chromatography in 34 germplasms of Costus speciosus and also to identify the superior sources and to correlate the macronutrients of rhizospheric soil. The starch content varied in microscopic examination and correlated inversely (r=−0.266) with diosgenin content. Findings revealed that the extraction process with acid hydrolysis yielded higher diosgenin content (0.15–1.88 %) as compared to non-hydrolysis (0.009–0.368 %) procedure. Germplasms from Uttar Pradesh (NBCS-4), Jharkhand (NBCS-39) and Bihar (NBCS-2) were identified as elite chemotypes based on hierarchical clustering analysis. The phosphorous content of respective rhizospheric soil correlated positively (r=0.742) with diosgenin content. Findings of present study are useful to identify the new agrotechniques. The elite germplasms can also be used as quality planting material for large scale cultivation in order to assure a sustained supply to the herbal drug industry.     

从葫芦巴种子中提取薯蓣皂甙元方法的研究

为探明葫芦巴种子中药用活性成分薯蓣皂甙元提取的最佳方法,设置了不同的酸解液浓度及酸解时间以了解葫芦巴粉酸解的适宜条件;并在酸化工艺前将脱脂的葫芦巴粉进行自然发酵或接种曲霉发酵以提高薯蓣皂甙元的提取率。结果表明葫芦巴粉酸解液最佳浓度为15%的硫酸溶液,最适宜的酸解时间为6 h。在酸化工艺前增加发酵工艺,可以显著提高有效成份薯蓣皂甙元的提取率,接霉菌发酵比直接酸化提高62%,自然发酵比直接酸化提高48%。     

菌株YM32139转化黄山药生产薯蓣皂苷元的研究

选用菌株YM32139采用固体转化方式对中药材黄山药的根状茎进行微生物转化研究。根据形态、培养特征将菌株YM32139鉴定为微紫青霉(Penicillium janthinellum)。采用硅胶色谱技术对转化产物进行分离纯化,分离到化合物I;通过波谱分析鉴定单体结构,确定化合物I为薯蓣皂苷元(diosgenin)。通过薄层层析和高效液相色谱分析表明微紫青霉(YM32139)将黄山药中原来有效成分主要转化为化合物I(薯蓣皂苷元),测定其在转化产物中的百分含量为20.85%;同时表明转化后化合物种类增多。     

生物合成薯蓣皂素的途径设计及关键酶分析

薯蓣皂素是一种天然甾体皂苷元,可作为数百种类固醇药物的前体,具有重要药用价值.目前工业生产薯蓣皂素主要依赖化学提取法,因此该法依赖植物材料和耕地且对环境有害.随着代谢工程和合成生物学的发展,生物合成法受到广泛关注.文中综述了生物合成薯蓣皂素的代谢途径和关键酶,并在酿酒酵母Saccharomyces cerevisiae...     

Effects of diosgenin on myometrial matrix metalloproteinase-2 and -9 activity and expression in ovariectomized rats

Diosgenin, a traditional Yam extraction, has been used in hormone replacement for menopausal women. We aimed to investigate the influences of diosgenin administration upon the MMP-2 and -9 activity and expression and reproductive hormones of ovariectomized (OVX) rats, a model of menopausal status. Seven-week old female Wistar rats with bilateral OVX or sham operation (controls) were divided and administered different dosages of diosgenin (0, 10, 50, or 100 mg/kg/day) for 8 weeks. Serum was then sampled for progesterone (P4) and estradiol (E2) assay and uterine horns harvested. Myometrial MMP-2 and -9 activity and expression were surveyed and myometrial collagen expression was also assayed. The results show higher body weight in OVX rats across the 8 weeks post surgery and no significant differences were noted among OVX or Sham rats with diosgenin supplements. There were lower P4 and E2 concentrations in OVX rats compared to Sham rats, and higher P4 concentration of Sham rats post diosgenin supplement. MMP-2 and -9 mRNA expression and activity was lower in OVX rats, although higher MMP-2 and lower MMP-9 activity/mRNA expression was observed in OVX rats post diosgenin supplementation. Collagen mRNA expression was higher in OVX rats compared to Sham controls, and diosgenin administration decreased collagen mRNA expression in OVX rats. In conclusion, diosgenin is associated with gelatinase expression and collagen metabolism in OVX rats. Diosgenin administration can partially reverse the effects of OVX upon MMP functions and hormone status. Adequate diosgenin supplement might modulate myometrial gelatinase expression and collagen metabolism in menopausal subjects.     

黄姜皂素提取工艺研究

以黄姜为原料,研究皂素提取的生产工艺。通过对几种工艺的比较,确定一种先分离黄姜中纤维素及淀粉,再经过酸水解、中和提取皂素的方法。该法黄姜皂素的平均得率为传统方法的86.56%,用酸量仅为传统方法的9.62%,同时分离得到46.44%的纤维素和32.75%的淀粉。该法资源综合利用率明显提高,并大幅度减少废水的生成,减少环境污染,节约原料、能源,生产周期也明显缩短。     

Microbial transformation of diosgenin and its precursor furostanol glycosides

Microorganisms have been screened for biotransformational activity against diosgenin or its precursor furostanol glycosides obtained from fenugreek seed (Trigonella foenum-graecum). Using diosgenin as the substrate, Cunninghamella elegans and Aspergillus nidulans produced some androstenes when α,α′-dipyridyl was supplemented to the transformation medium. Also, using the glycosides as a substrate, Rhizopus sp. produced diosgenin in >90% yield from the glycoside. The parameters were optimized to increase the yield of the androstenes.     

Direct Biotransformation of Dioscin into Diosgenin in Rhizome of Dioscorea zingiberensis by Penicillium dioscin

Diosgenin is an important precursor for synthesis of more than 200 steroidal hormone medicines. Rhizome of Dioscorea zingiberensis C. H. Wright (RDZ) contained the highest content of diosgenin in Dioscorea plant species. Diosgenin is traditionally extracted by acid hydrolysis from RDZ. However, the acid hydrolysis process produces massive wastewater which caused serious environment pollution. In this study, diosgenin extraction by direct biotransformation with Penicillium dioscin was investigated. The spawn cultivation conditions were optimized as: Czapeks liquid culture medium without sugar and agar (1,000 ml) + 6.0 g dioscin/6.0 g DL, 30 °C, 36 h; solid fermentation of RDZ: mycelia/RDZ of 0.05 g/kg, 30 °C, 50 h; the yield of diosgenin was over 90 %. Spawn cultivation was crucial for the direct biotransformation. In the spawn cultivation, amount and ratio of dioscin/DL were the key factors to promote biotransformation activity of P. dioscin. This biotransformation method was environment-friendly, simple and energy saving, and might be a potential substitute for acid hydrolysis in diosgenin extraction industry.     

Diosgenin contents and DNA fingerprint screening of various yam (Dioscorea sp.) genotypes

In addition to the importance of many Dioscorea species (yams) as starchy staple food, some representatives are known and still used as a source for the steroidal sapogenin diosgenin, which, besides phytosterols derived from tall-oil, is an important precursor for partial synthesis of steroids for pharmaceutical research and applications. While in edible yams the diosgenin content should be as low as possible, a high yield of the compound is preferable for cultivars which are grown for the extraction of sterols. In the past, miscalculations and insufficiently precise techniques for quantification of diosgenin prevailed. Therefore we set out to re-evaluate the steroid content of a world collection of Dioscorea species, using leaves as sample material. We optimized diosgenin quantification techniques and fingerprinted the whole collection with the DNA amplification fingerprinting (DAF) technique. Total diosgenin contents ranged from 0.04 to 0.93% of dry weight within the collection. Several Dioscorea cultivars can be characterized via their DAF fingerprint patterns.     

The best utilization of D. zingiberensis C.H. Wright by an eco-friendly process

An eco-friendly process for the best utilization of D. zingiberensis C.H. Wright tubers was developed. In the first stage, cellulose and ethanol were recovered by physical separation, multi-enzymes hydrolysis with yeast fermentation, and in the second stage diosgenin was separated using ethanol-modified supercritical carbon dioxide extraction. The new approach could not only recover 95% of diosgenin production, 95% of ethanol and 75% of cellulose, but also efficiently reduce 88% of COD in wastewater compared with the conventional method, which only extract diosgenin with discharging 80,000mg/l of COD into public sewers. The research indicates that the proposed system could be a clean and technological-efficient alternative to conventional processing of D. zingiberensis C.H. Wright tubers in industry.     

Process optimization for the production of diosgenin with Trichoderma reesei

Based on the response surface methodology, an effective microbial system for diosgenin production from enzymatic pretreated Dioscorea zingiberensis tubers with Trichoderma reesei was studied. The fermentation medium was optimized with central composite design (3⁵) depended on Plackett–Burmann design which identified significant impacts of peptone, K₂HPO₄ and Tween 80 on diosgenin yield. The effects of different fermentation conditions on diosgenin production were also studied. Four parameters, i.e. incubation period, temperature, initial pH and substrate concentration were optimized using 4⁵ central composite design. The highest diosgenin yield of 90.57% was achieved with 2.67% (w/v) of peptone, 0.29% (w/v) of K₂HPO₄, 0.73% (w/v) of Tween 80 and 9.77% (w/v) of substrate, under the condition of pH 5.8, temperature 30 °C. The idealized incubation time was 6.5 days. After optimization, the product yield increased by 33.70% as compared to 67.74 ± 1.54% of diosgenin yield in not optimized condition. Scale-up fermentation was carried out in a 5.0 l bioreactor, maximum diosgenin yield of 90.17 ± 3.12% was obtained at an aeration of 0.80 vvm and an agitation rate of 300 rpm. The proposed microbial system is clean and effective for diosgenin production and thus more environmentally acceptable than the traditional acid hydrolysis.     

黄姜中薯蓣皂甙元的提取及定性定量分析

对黄姜中提取的薯蓣皂甙元进行红外和紫外可见波谱扫描,与标准品谱图对比分析,可知提取出的皂甙元与薯蓣皂甙元标准品有相同的结构。用分光光度法测定黄姜中薯蓣皂甙元的含量,以香荚兰醛-高氯酸-冰醋酸的加入顺序,在80℃水浴中加热15min,显色30min后,在542nm处测定吸收值能达到最佳值,最后得出皂甙元含量为2.27%,RSD为1.1%,回收率在97.2%~98.8%之间。