Regulation of isoflavone production in hydroponically grown Pueraria montana (kudzu) by cork pieces, XAD-4, and methyl jasmonate

A mini-hydroponic growing system was employed for seedlings of kudzu vine (Pueraria montana) and contents of isoflavones (daidzein, genistein, daidzin, genistin, and puerarin) from shoot and root parts of seedlings were analyzed quantitatively. In addition, exogenous cork pieces, polymeric adsorbent, XAD-4, and universal elicitor, methyl jasmonate (MeJA), were used to regulate the production of these isoflavones. It was shown that cork pieces up-regulate the production of daidzein and genistein up to seven- and eight-fold greater than the levels obtained for control roots. In contrast, levels of glucosyl conjugates, daidzin and genistin, decrease up to five- and eight-fold, respectively. Cork treatment also induces the excretion of the root isoflavone constituents into the growth medium. Minimal levels of isoflavones are absorbed by the cork pieces. XAD-4 stimulates the production of glucosyl conjugates, daidzin and genistin, in root parts about 1.5-fold greater than that obtained in control roots. These are the highest amounts of daidzin and genistin that are observed (5.101 and 6.759 mg g⁻¹ dry weight, respectively). In contrast to these two adsorbents, MeJA increases the accumulation of isoflavones in shoot rather than in root parts of seedlings, about three- to four-fold over control levels, with the exception of genistein. These studies reveal new observations on the regulation of isoflavone production in hydroponically grown Pueraria montana plants by two adsorbents (cork pieces and XAD-4) and MeJA elicitor.     

葛根的研究与开发现状

阐述了葛属植物的种类和分布,对于葛根的营养保健作用、药理作用及机理,对国内外葛根保健食品的研究开发现状进行了分析。     

Reaction of Toxic Bicyclic Phosphates with Acetylcholinesterases and α-Chymotrypsin

Some toxic bicyclic phosphates (BPs) inhibited acetylcholinesterases (AChEs), but the activity was very weak. Even the most potent inhibitor, 4-nitro BP, inhibited bovine erythrocyte and housefly head AChEs by only 37 and 38 per cent, respectively, at 1.5 mm. Kinetic analysis indicated that the poor inhibitory activity of 4-nitro BP is ascribed not only to the low affinity for AChEs but also to its poor phosphorylating ability. Similar findings were obtained in the case of the reaction of BPs with the serine enzyme α-chymotrypsin. Despite the relatively high reactivity in an alkaline solution, BPs are much less active than other bioactive organophosphorus esters in phosphorylating a general-base-catalyzed hydroxyl group. This fact suggests that the toxic action of BPs does not result from the phosphorylation of a critical site in biological systems.     

野葛异黄酮合酶基因的分离与功能验证

异黄酮是野葛（Pueraria lobata）中的主要活性成分,而异黄酮合酶（IFS）是催化异黄酮生物合成的第一步关键酶,尽管野葛的IFS基因已被分离,但其功能还未得到任何验证。本研究以中国安徽省郎溪县的野葛为材料,利用RT-PCR技术成功克隆到野葛IFS基因,命名为PlIFS,PlIFS开放阅读框大小为1566 bp,编码521个氨基酸,将该基因克隆到GAL1启动子控制下的酵母表达载体pESC-TRP上,得到重组质粒pESC-TRP-PlIFS,通过LiAc/ssDNA/PEG方法将其转化进酿酒酵母（Saccharomyces cerevisiae）WAT11中进行异源表达,并在酵母体内对其活性进行验证,结果显示PlIFS能催化甘草素生成大豆苷元,表现出异黄酮合酶活性特征。荧光定量PCR分析显示,PlIFS基因主要在野葛的根中表达,这与活性物质异黄酮主要在野葛根中的积累模式一致。     

DMSO、Tween-20和Triton X-100对野葛悬浮细胞异黄酮生产的影响

二甲基亚砜(DMSO)、Tween-20和Triton X-100可以增大悬浮培养细胞的细胞膜和液泡膜的透性,促进细胞内次生代谢物的释放,从而影响这些次生代谢产物的产量。为了提高野葛悬浮细胞中异黄酮类化合物的产量,以1％、3％和5％的DMSO、Tween-20和Triton X-100分别处理野葛叶悬浮细胞,结果显示,Tween-20和Triton X-100皆明显促进细胞生物量和葛根素和异黄酮化合物的释放。5％的Triton X-100处理3d,促进细胞产生总异黄酮化合物,增产率达40．6％。     

野葛花中葛花苷的测定方法研究

在硅胶G薄层色谱板上,建立了双波长薄层扫描法测定野葛花中葛花苷的分析方法。薄层分析的展开剂为三氯甲烷一甲醇一冰醋酸(3：1：0．3)．葛花苷在0．484～3．872μg范围内呈良好线性。该方法快速、简便,为野葛花作为中药质量控制提供科学依据。     

蔗糖和光对三裂叶野葛毛状根生长及次生物质产生的影响

研究了蔗糖浓度和光对固体培养的三裂叶野葛毛状根生长及其总异黄酮和葛根素产生的影响。结果表明：在供试的分别添加1%、3%、5%、7%和9%蔗糖的MS固体培养基中,3%蔗糖能促进三裂叶野葛毛状根的生长及其异黄酮类化合物和葛根素的积累;培养20d后,其生物量达到0.48g(DW,干重)/瓶,总异黄酮和葛根素含量分别为25.44mg/g(DW)和11.64mg/g(DW)。与添加3%蔗糖的MS培养基培养的三裂叶野葛毛状根相比,含5%蔗糖的培养基培养的毛状根干重增殖倍数提高了7.0％,而含1％、7％和9％蔗糖的培养基培养的毛状根干重增殖倍数分别下降62.4％、42.8％和65.3％;其总异黄酮含量分别降低574％、13％和33.4％,葛根素含量分别下降47.9％、15.8％和35.1％,但其毛状根培养物的可溶性糖含量则分别增加了0.52、1.45和1.54倍。暗培养30d的毛状根的生物量达到0.83g(DW)/瓶,分别比蓝光和白光培养的毛状根提高37.1%和23.3%。在蓝光和白光下培养的部分毛状根的表面呈淡绿色;但白光处理的毛状根中总异黄酮含量比蓝光和暗培养处理的分别提高了14.7%和19.2%;蓝光抑制毛状根中葛根素含量的积累,白光和暗培养的毛状根培养物中的葛根素含量分别是蓝光处理的1.61倍和1.52倍。     

Fe3+络合萃取法从野葛根中分离葛根素

利用Fe3 + 能够和葛根素生成可溶性络合物的性质建立了一种从中药野葛根中萃取葛根素的新型分离方法。以甲醇冷浸从野葛根中提取葛根总黄酮 ,将其进行水解、中和 ,再给水解葛根总黄酮中加入FeCl3 使葛根素与Fe3 + 络合溶解 ,过滤除去其它不溶性物质 ,用盐酸解聚Fe3 + 葛根素络合物 ,则得葛根素粗品 ,将其重结晶可得葛根素。同时 ,利用分光光度法确定了Fe3 + 葛根素络合物解聚的最佳酸度。利用TLC标准品对照、IR和分光光度法对产品进行了定性和定量分析。该方法从葛根中提取葛根素收率为 1 2 % ,纯度为 96 5 % ,具有操作简便、工艺流程简单 ,容易实现工业化的优点。     

野葛毛状根离体培养与异黄酮生产

用发根农杆菌（Agrobacterium rhizogenes）R1601菌株感染野葛（Pueraria lobata(Willd.)Ohwi）叶片外植体后获得的毛状根,经连续4次继代培养,鲜重增加到最初的6.2倍。PH为5.5的1/2SH液体培养基较适合野葛毛状根的生长。与自然根相比,毛状根在无激素的1/2SH液体培养基中离体培养20d后,鲜重增加22.6倍,释放入培养液中的异黄酮量增加10.6倍。毛状根生产异黄酮的量略高于自然根,且根中含量明显高于愈伤组织及茎中异黄酮的含量。培养液体积影响毛状根的离体生长及异黄酮的释放。     

太白山野葛根中异黄酮和葛根素含量随季节的变化

为了考察太白山野葛根中异黄酮及葛根素含量随季节的变化。采集一年12个月份的野葛根,采用紫外分光光度法测定其中异黄酮含量;以7高效薄层色谱扫描法测定野葛根中游离葛根素及总葛根素含量。结果表明：三月份异黄酮含量最高,为18.53%;四月份游离葛根素及总葛根素含量最高,分别为7.45%和8.93%。对不同季节太白山野葛根中异黄酮、游离葛根素及总葛根素进行定量分析,为这一野生资源的合理采收和开发利用提供了实验依据。