薯蓣皂甙元ELISA定量分析方法的建立Ⅰ-薯蓣皂甙元全抗原的合成

建立薯蓣皂甙元的ELISA定量分析方法必须先合成薯蓣皂甙元的全抗原。试验利用薯蓣皂甙元3位上的-OH,在DMAP的催化下,薯蓣皂甙元与丁二酸酐反应,生成薯蓣皂甙元丁二酸单酯,用MSI、R1、H NMR1、3CNMR等方法对产物结构进行了表征。用混合酸酐法制备薯蓣皂甙元与牛血清白蛋白的结合物(DG-HS-BSA),碳二亚胺法制备薯蓣皂甙元与卵清蛋白的结合物(DG-HS-OVA),经TNBS测定,每分子结合物连接的薯蓣皂甙元分子数分别为28.0和8.8。     

盾叶薯蓣中薯蓣皂甙元提取新工艺的研究

为研究新工艺提取盾叶薯蓣薯蓣中薯蓣皂甙元的最佳实验指标,以薯蓣皂甙元得率为评价参数,采用6因素5水平的正交实验,用分光光度法对25种提取方法所得到的薯蓣皂甙元进行比较分析。结果表明,硫酸浓度对薯蓣皂甙元提取的影响最大,在实验室条件下,可采用20g样品加甲醇回流提取4h,回流速度为10min／次,用2．5mol／L的硫酸水解6h,120号溶剂汽油回流提取2h,回流速度为15min／次,能清洁快速提取盾叶薯蓣中的薯蓣皂甙元。     

盾叶薯蓣中薯蓣皂甙元不同提取方法的比较

为提高盾叶薯蓣(Dioscorea zingiberensis C.H.Wright)中薯蓣皂甙元得率,在实验室条件下,以薯蓣皂甙元含量为评价指标,采用4因素4水平的正交试验,用高效液相色谱法对16种提取方法所得的薯蓣皂甙元含量进行了比较分析.结果表明,硫酸的浓度对薯蓣皂甙元含量有极显著影响,在实验室条件下,可采用10 g样品加2.0mol·L-1硫酸200 mL水解4 h,水解物用石油醚回流提取5 h,能快速准确提取盾叶薯蓣中的薯蓣皂甙元.     

薯蓣皂甙元氨基酸酯的合成及其动物实验--薯蓣皂甙元衍生物的研究Ⅱ

以薯蓣皂甙元为原料,经过与氨基酸缩合,合成了6个新化合物,甘氨酸薯蓣皂甙元酯(1),DL-组氨酸薯蓣皂甙元酯醋酸盐(2),L-赖氨酸薯蓣皂甙元酯(3),N-L-赖氨酰基甘氨酸薯蓣皂甙元酯醋酸盐(4),N-L-精氨酰基甘氨酸薯蓣皂甙元酯醋酸盐(5),N-DL-组氨酰基甘氨酸薯蓣皂甙元酯醋酸盐(6),并对结构进行了鉴定。同时发现这6个化合物具有抗大鼠实验性心肌梗死作用。     

盾叶薯蓣营养器官薯蓣皂甙元含量的动态变化

利用高效液相色谱(HPLC)法对盾叶薯蓣营养器官特别是根状茎中薯蓣皂甙元含量的动态变化、品种之间的差异以及雌雄株之间的差异进行了研究。结果表明：实生苗根状茎中薯蓣皂甙元的含量,2年生高于1年生;根茎营养繁殖的2年生根状茎中皂甙元的含量高于1年生的含量;花叶品种的含量高于绿叶品种的含量;雄株的含量比雌株的含量高。在地上的缠绕茎和叶中没有检测到薯蓣皂甙元。由根茎繁殖的1年生根状茎前期皂甙元含量增加缓慢,后期增加较快;2年生根状茎盛花期含量最高,开花后期含量最低,随后含量逐渐增加。为此应在花叶品种中选择产量高、抗性强的品种作为栽培品种。合适的采挖期仍以地上缠绕茎枯萎期为宜。     

盾叶薯蓣营养器官薯蓣皂甙元含量的动态变化

利用高效液相色谱(HPLC)法对盾叶薯蓣营养器官特别造根状茎中薯蓣皂甙元含量的动态变化、品种之间的差异以及雌雄株之间的差异进行了研究。结果表明:实生苗根状茎中薯蓣皂甙元的含量,2年生高于1年生;根茎营养繁殖的2年生根状茎中皂甙元的含量高于1年生的含量;花叶品种的含量高于绿叶品种的含量;雄株的含量比雌株的含量高。在地上的缠绕茎和叶中没有检测到薯蓣皂甙元。由根茎繁殖的1年生根状茎前期皂甙元含量增加缓慢,后期增加较快;2年生根状茎盛花期含量最高,开花后期含量最低,随后含量逐渐增加。为此应在花叶品种中选择产量高、抗性强的品种作为栽培品种。合适的采挖期仍以地上缠绕茎枯萎期为宜。     

薯蓣皂甙元的研究进展

我国是薯蓣资源大国,其有效成分薯蓣皂甙元是甾体激素的主要合成原料.对薯蓣皂甙元的鉴定、生物合成、提取、分离以及利用等研究进行综述.     

薯蓣皂甙元丁二酸单酯氨基酸盐的合成及其动物实验——薯蓣皂甙元衍生物的研究I

以薯蓣皂甙元丁二酸单酯为原料,经过与氨基酸缩合,合成了5个新化合物,4—L—(N—丁二酸—2—基)胺基—4—氧代丁酸薯蓣皂甙元酯二钠盐(1),4—(N—乙酸—2—基)胺基—4—氧代丁酸薯蓣皂甙元酯钠盐(2),4—L(N—(5—胍基)戊酸—2—基)胺基—4—氧代丁酸薯蓣皂甙元酯醋酸盐(3),4—(N—(3—咪唑-4—基)丙酸—2—基)胺基—4—氧代丁酸薯蓣皂甙元酯醋酸盐(4),4—(N—戊二酸—2—基)胺基—4—氧代丁酸薯蓣皂甙元酯二钠盐(5),并对其进行了结构鉴定,同时发现这5个化合物对大鼠都具有抗心肌梗死活性。     

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。     

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%。     

Development of media for growth ofDioscorea Deltoidea cells andin vitro diosgenin production: Influence of media constituents and nutrient stress

Summary Callus culture ofDioscorea deltoidea produced diosgenin and sterols during stationary phase. Ammonium nitrate (420 mg Nitrogen/l) as sole nitrogen source supported better growth than a combination of ammonium nitrate and potassium nitrate (totally equivalent to 840 mg Nitrogen/l). The production of diosgenin increased under low phosphate concentration (100 mg/l) whereas high phosphate concentration (240 mg/l) promoted growth. Micronutrients, when used at 1 1/2 strength, enhanced growth and diosgenin production. Depletion of nitrogen increased the diosgenin synthesis by a factor of 2. Adoption of a two stage culture method enhanced the diosgenin production in cultured cells by eight-fold.     

Improved diosgenin production in Dioscorea deltoidea cell cultures by immobilization in polyurethane foam

Dioscorea deltoidea Wall (Dioscoreaceae) cell cultures were entrapped by passive invasion into reticulate polyurethane foam cubes. Immobilization of cells grown in medium containing 3% sucrose reduced the lag phase in growth and thereby reduced the time required to reach maximum diosgenin concentration by 36% compared to cells in suspension culture. Immobilization also increased the total diosgenin produced by 40%. Increased efficiency in diosgenin production was greatest in 3% sucrose; higher concentrations inhibited diosgenin production.     

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...     

Elicitation of diosgenin production in <Emphasis Type="Italic">Trigonella foenum</Emphasis>-<Emphasis Type="Italic">graecum</Emphasis> L. seedlings by heavy metals and signaling molecules

Seedlings of Trigonella foenum-graecum were treated with four heavy metal salts (CdCl_2, CoCl₂, K₂Cr₂O₇ and NiCl₂) to study the effect of heavy metals on growth and diosgenin production. It was found that CdCl₂ increased diosgenin production up to 40-fold and CoCl₂ increased diosgenin production up to 41-fold at concentrations which did not affect growth significantly. But K₂Cr₂O₇ and NiCl₂ were toxic to growth and inhibited diosgenin production. Effect of exogenously applied methyl jasmonate (MeJa) and calcium (Ca²⁺) on diosgenin production in seedlings of T. foenum-graecum was also investigated. MeJa enhanced the production of diosgenin. Maximum increase (10.5-fold) was found at 100 μL L^−l concentration of MeJa. To study the role of Ca²⁺ on diosgenin production, seedlings of T. foenum-graecum were treated with a promoter of Ca²⁺ influx (calcium ionophore A23187), calcium depleted medium, Ca²⁺ channel blocker (verapamil) and antagonist (LaCl₃), a divalent cation chelator (EGTA) and modulator of calcium release (caffeine). All the treatments were compared with a control containing 220 mg L^−l concentration of CaCl₂. The results suggest that the increase in cytosolic Ca²⁺ has an inhibitory role on diosgenin production. However, a calcium chelator or Ca²⁺ channel inhibitors could be used to elicit diosgenin production in this plant.     

Stimulated production of diosgenin in Dioscorea galeottiana cell suspension cultures by abiotic and biotic factors

Dioscorea deltoidea cell suspension cultures were established in modified Murashige and Skoog medium. The diosgenin production increased from 0.10 g^–1 to 3.98 g^–1 dry cell weight when cells were cultivated in the light and in a growth medium limited in phosphate and sucrose. The addition of 1.3 g of autoclaved fungal mycelium of Alternaria tenuis per litre of cell culture growing in the dark induced the production of 0.04 mg diosgenin g^–1 dry cell weight. In both cases, the production of diosgenin was preceded by a transient induction of isopentenyl diphosphate isomerase activity.     

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.     

高效转化黄姜皂苷为薯蓣皂苷元菌株的筛选及转化条件优化

薯蓣皂苷元是甾体激素类药物重要的生产原料,在制药工业中有广泛应用。传统的生产方法是黄姜酸解法,污染严重。为寻找更清洁高效的生产方式,从本实验室保藏的菌株中筛选出一株赤霉菌Gibberellaintermedia WX12(层出镰孢菌Fusarium proliferatum的有性阶段),能将黄姜中的皂苷转化为薯蓣皂苷元。采用统计学实验设计方法对其转化培养基进行研究,优化的转化培养基配方为(g/L):葡萄糖20.6;酵母膏5.0;氯化钠1;磷酸二氢钾3;硫酸锌1.5;黄姜酶解物3。采用以上优化参数,薯蓣皂苷元的得率提高到(31±0.3)mg/g干黄姜,较优化前提高了3倍。这是目前关于赤霉菌转化黄姜中皂苷的首次报道。     

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。