高效液相色谱法测定红景天甙含量方法的研究

以高效液相色谱法作为红景天甙含量检测方法, 系统探讨了用超声法从高山红景天根中提取红景天甙用以制备分析样品的方法。确定的最佳提取条件为:以蒸馏水为提取溶剂在60℃下超声提取40 min。     

高效液相色谱法测定红景天甙含量方法的研究

以高效液相色谱法作为红景山甙含量检测方法,系统探讨了用超声法从高山红景天根中提取红景天甙用以制备分析样品的方法。确定的最佳提取条件,以蒸馏水为提取溶剂在６０℃超声提取４０min。     

高效毛细管电泳法测定高山红景天中红景天甙含量的比较研究

采用高效毛细管电泳法对采集于两个野生产地不同居群和两个人工栽培地的高山红景天中红景天甙的含量进行了对比研究,实验结果表明,该方法高效、简便、灵敏、可靠;人工栽培高山红景天和自然生境下高山红景天中红景天甙含量有差异,不同天然居群高山红景天中红景天甙含量也有差异。     

高效毛细管电泳地测定高山红景天中红景天甙含量的比较研究

采用高效毛细管电泳法对采集于两个野生产地不同居群和两个人工栽培地的高山红景天中红景天甙的含量进行了对比研究,实验结果表明,该方法高效,简便,灵敏,可靠;人工栽培高山红景天和自然生境下高山红景天中红景天甙含量有差异,不同天然居群高山红景天中红景天甙含量也有差异。     

大海林地区不同生境高山红景天根部红景天甙含量的差异

在黑龙江省大海林地区海拔1400~1620m的高山上,依据高山红景天的生境于岳桦林内、高山草甸、苔原上选择了6个样地,测定了高山红景天根部的红景天甙含量。生境不同的高山红景天,根部红景天甙的含量有明显差异,红景天甙含量范围为0.101%~0.684%。从测定结果看,光照条件好、土壤肥沃的生境下高山红景天根部的红景天甙含量较高。     

大海林地区不同生境高山红景天根部红景天甙含量的差异

在黑龙江省大海林地区海拔１４００－１６２０ｍ的高山上,依据高山红景天的生境于岳桦林内、高山草甸、苔原上选择了６个样地,测定了高山红景天根部的红景天甙含量。生境不同的高山红景天,根部红景天甙的含量有明显差异,红景天甙含量范围内０．１０１％－０．６８４％。从测定结果看,光照条件好、土壤肥沃的生境下高山红景天根部的红景天甙含量较高。     

温室栽培光强和光质对高山红景天生物量和红景天甙含量的影响

为探讨光强及光质对高山红景天(Rhodiola sachalinensis)生物量和红景天甙含量的影响。于2000年4月至6月在东北林业大学温室内以移栽于大兴安岭加格达奇圃地人工种植生长3a的高山红景天为材料,通过纱布遮荫及遮以不同颜色的滤光膜分别进行了光强、光质控制实验(处理45d)。随着光强的降低,高山红景天全株生物量、根生物量、根的红景天甙含量和产量以及叶中叶绿素a、叶绿素b、总叶绿素的含量均有降低的趋势,但叶绿素含量变化很小,不同光强及对照之间的差异均未达到显著水平。相对光强为67．75％和44．71％的两种处理下的全株生物量、根生物量、根的红景天甙含量和产量差异不显著,它们的全株生物量和红景天甙含量与对照(全光照)的差异也不显著,但根生物量和红景天甙产量与对照的差异显著。当相对光强减弱至31．96％,全株生物量、根生物量、根的红景天甙含量和产量均大幅度下降,根冠比显著增加。4种滤光膜处理均使高山红景天的全株生物量及根生物量显著降低,蓝膜和绿膜处理的降低幅度大于红膜和黄膜处理的。红膜处理的红景天甙的含量和产量均高于对照,但黄膜、蓝膜和绿膜处理的红景天甙含量和产量则低于对照。通过计算去除4种滤光膜的光强因素,仅从光质的作用看。4种滤光膜处理仍是减小了全株生物量和根生物量,红膜和绿膜处理提高了红景天甙的含量和产量,而黄膜处理降低了红景天甙的含量和产量,蓝膜处理几乎没有效果。4种滤光膜处理均使叶绿素含量增加,但只有蓝膜处理的与对照差异显著。红膜处理不仅显著提高根中红景天甙的含量(为对照的3．42倍),而且对根生物量的影响较小(为对照的90．24％)。因而提高了高山红景天根的红景天甙产量,这意味着在生产上可能会有一定的实践意义。     

光强和光质对野外栽培高山红景天生物量和红景天甙含量的影响

为探讨野外条件下光强及光质对高山红景天 (Rhodiola sachalinensis)生物量和红景天甙含量的影响 ,于 2 0 0 1年 5月 8日至 9月 16日在大兴安岭加格达奇的高山红景天种植圃地 ,利用纱布及红色、蓝色和绿色的滤光膜遮光处理 ,对生长 3a和 4 a的高山红景天进行了光强、光质控制实验。与温室实验类似 ,遮荫显著抑制高山红景天根的生长 ,并使红景天甙的含量略有提高。红膜处理使光强大约降低一半 ,但仅从光质的角度而言 ,红膜处理对根的生长影响不大 ,却显著增加了根中的红景天甙含量和产量 ,不过效果不如温室实验明显。绿膜处理未表现出对红景天甙积累的促进作用 ,这与温室实验结果不同。红膜处理不同天数的结果表明 ,处理时间对红景天甙含量提高的程度影响很小。这意味着在野外种植的情况下 ,可以在临近收获的最后一段时间用红膜对高山红景天进行处理 ,这样既可避免红膜处理对高山红景天根生长的抑制 (由于减弱了光照 ) ,又可显著提高根的红景天甙含量 ,从而达到大幅度提高红景天甙产量的目的。     

高山红景天细胞悬浮培养中红景天甙生物合成代谢调控Ⅱ:真菌诱导物的影响

考察了6种真菌诱导物对高山红景天(Rhodiola sachalinensis A. Bor)细胞生长与红景天甙积累的影响。其中以黑曲霉诱导物效果最好。在细胞培养初期添加浓度为10mg(carbohydrate)/L的黑曲霉诱导物能使培养细胞中红景天甙含量提高到0.995％。前体与诱导物调控组合运用最终使红景天甙产量达到167.4mg/L,是对照培养的3.5倍。另外,对真菌诱导物的作用机理也进行了探讨,真菌诱导物添加促进红景天甙的积累应该与激活培养细胞中的苯丙烷类代谢途径有关。     

高山红景天细胞悬浮培养中pH值对红景天甙胞外释放及细胞活性的影响

高山红景天(Rhodiola sachalinensis A.Bor.)细胞悬浮培养中,通过降低培养基pH值能有效地诱导培养细胞中红景天甙的胞外释放。红景天甙的跨膜运输是一个与H~ 对运的动态过程,培养基pH值决定了红景天甙在胞内外含量的分布。细胞组织在pH值大于3的培养基中处理3h以内,对细胞活性的影响不大。将诱导释放处理过的细胞组织转入到新鲜的生产培养基中,细胞仍具有合成红景天甙的能力。     

利用高山红景天培养细胞生物转化外源酪醇生产红景天甙的研究

高山红景天（ＲｈｏｄｉｏｌａｓａｃｈａｌｉｎｅｎｓｉｓＡ．Ｂｏｒ．）培养细胞中,甙元酪醇在细胞生长静止期大量积累,而此时糖基化反应的效率很低,因而红景天甙（ｓａｌｉｄｒｏｓｉｄｅ）产量较低。考虑到培养细胞中酪醇葡萄糖基转移酶的活性在指数生长期达到最高,考察了在指数生长期添加外源酪醇生物转化生产红景天甙的可能性,并探讨了酪醇添加浓度、添加方法及细胞密度对酪醇转化率及红景天甙产量的影响。结果表明,细胞在酪醇浓度为１ｍｍｏｌ／Ｌ的培养基中培养２４ｈ后可使酪醇转化率达到９５％;过高的酪醇浓度（＞３ｍｍｏｌ／Ｌ）对细胞生长及酪醇转化率都有明显抑制作用;通过较低浓度酪醇的３次重复添加,可使细胞密度为６ｇＤＷ／Ｌ、１２ｇＤＷ／Ｌ及１８ｇＤＷ／Ｌ的培养物中的红景天甙产量分别达到１３２０ｍｇ／Ｌ、１７４０ｍｇ／Ｌ和１９８０ｍｇ／Ｌ。     

Production of Salidroside Through Biotransformation of Exogenous Tyrosol in Rhodiola sachalinensis Cell Suspension Cultures

In Rhodiola sachalinensis A. Bot. cell cultures, low yields of salidroside was supposed to be associated with the low efficiency of glucosylation reaction at the stationary phase of cell growth, when large amounts of the substrate, aglycon tyrosol, were accumulated. Considering the activity of tyrosol glucosyhransferase being the highest at the exponential growth phase, the author added exogenous tyrosol into the cultures at this time so as to produce salidroside through biotransformation. The effects of tyrosol concentration, the way of tyrosol addition as well as the cell density on the transformation rate and salidroside yield were investigated. It was found that the transformation rate attained 95 % after cells were incubated in the medium containing 1 mmol/L tyrosol for 24 h. Excess high concentrations of tyrosol in medium ( > 3 mmol/L) caused inhibition of transformation rate and cell growth. By 3 repeated additions of tyrosol in low concentrations, the salidroside yields of 1 320 mg/L, 1 740 mg/L and 1 980 mg/L to the cell densities of 6 g DW/L, 12 g DW/L and 18 g DW/L were obtained respectively.     

The Metabolism of Salidroside to Its Aglycone p-Tyrosol in Rats following the Administration of Salidroside

Salidroside is one of the major phenolic glycosides in Rhodiola, which has been reported to possess various biological activities. In the present study the in vivo deglycosylation metabolism of salidroside was investigated and its aglycone p-tyrosol but not the original salidroside was identified as the main form in rat tissues following the administration of salidroside. After the i.v. administration of salidroside at a dose of 50 mg/kg in rats, salidroside was quantified only in the liver, kidney and heart tissues. The highest level of p-tyrosol was detected in the heart, followed by the spleen, kidney, liver and lungs, in order. Salidroside was detected only in the liver, in contrast, p-tyrosol was detectable in most tissues except the brain, and the kidney tissues contained a significant amount of p-tyrosol compared to the other tissues after the i.g. administration of 100 mg/kg salidroside. The excretion behaviour revealed that the administrated salidroside mainly eliminated in the form of salidroside but not its aglycone metabolite p-tyrosol through urine. After i.v. and i.g. administration in rats, 64.00% and 23.80% of the total dose was excreted through urine in the form of salidroside, respectively. In addition, 0.19% and 2.25% of the dose was excreted in the form of p-tyrosol through urine after i.v. and i.g. administration, respectively. The faecal salidroside and p-tyrosol concentrations were 0.3% and 1.48% of the total dose after i.v. administration, respectively. After the i.g. administration of salidroside, trace salidroside and p-tyrosol were quantified in faeces within 72 h. In addition, the biliary excretion levels of salidroside after i.v. and i.g. administration were 2.86% and 0.02% of the dose, respectively. The obtained results show that salidroside was extensively metabolised to its aglycone p-tyrosol and distributed to various organs and the orginal salidroside was cleared rapidly through urine following the administration of salidroside.     

高山红景天细胞悬浮培养中红景天甙生物合成代谢的调控 Ⅰ:前体的影响

探索了高山红景天(Rhodiola sachalinensis A.Bor)细胞培养中红景天甙生物合成的途径,认为甙元酪醇是经由莽草酸途径生成的。在此基础上研究了酪醇、L-酪氨酸与L-苯丙氨酸三种前体加入对红景天甙生物合成的调控作用。结果表明,酪醇、酪氨酸等前体易被多酚氧化酶氧化成褐色,用与前体浓度为1:1的V。来防止褐化效果显著;浓度为0.5mmol/L的酪醇,酪氨酸及苯丙氨酸在细胞培养15d时添加,使红景天甙含量由0.336％分别提高到1.43％、1.11％、0.85％。     

红景天甙生物合成途径：酪醇合成的起始反应及其糖基化

红景天甙(Salidroside)生源途径分子机制的解析是利用基因工程、代谢工程技术合成目标化合物的基础。糖基化是红景天甙生物合成的最后一步反应。在前期工作中,本课题组率先报道了与红景天甙生物合成相关的3个尿苷二磷酸葡萄糖基转移酶(UGTs)基因,在体外酶学性质研究的基础上,利用根癌农杆菌和发根农杆菌介导分别建立了相关转基因体系,鉴别了红景天甙生物合成最适UGT及植物和毛状根生物反应器系统合成红景天甙的效率差异;酪醇(Tyrosol)是红景天甙糖基化反应的甙元底物分子,其具体的代谢通路及其调控机制仍不明确。针对酪醇生物合成来源主要存在两种观点:一是酪醇可能来自于苯丙烷代谢途径产生的4-香豆酸,该途径起源于苯丙氨酸;二是生物碱代谢途径的中间产物酪胺可能是酪醇生物合成的前体,该途径则起源于酪氨酸。在后续工作中,否定了酪醇来源于苯丙烷代谢途径的可能性,进一步的工作证实酪氨酸脱羧酶(TyrDC)在酪醇生物合成的起始反应中担负着重要功能,酪醇作为一种苯乙烷类化合物衍生物,其生物合成来源于生物碱代谢途径。     

Production of salidroside and tyrosol in cell suspension cultures of Rhodiola crenulata

Salidroside and its aglycone tyrosol are important compounds found in Rhodiola plants. In this study, callus derived from Rhodiola crenulata was induced and grown when explants were incubated on a Murashige and Skoog (MS) medium containing various concentrations of 6-benzyaldenine (BA), naphthalene acetic acid (NAA) and thidiazuron (TDZ). Callus was easily initiated from juvenile leaves in half strength MS medium supplemented with 0.5 mg/L BA and 3.0 mg/L NAA, while full strength MS containing 0.5 mg/L TDZ and 0.5 mg/L NAA was the best for callus subculture and subsequent cell suspension culture. The activities of l-phenylalanine ammonia lyase (PAL) and β-d-glucosidase, two key enzymes in salidroside synthesis, increased at first and subsequently decreased in cell suspension cultures. The salidroside and tyrosol levels in the cell suspension cultures were determined using high-performance liquid chromatography. High levels of salidroside and tyrosol were detected in cell suspension cultures of R. crenulata extracted with 75 % methanol, demonstrating that the biotechnological production of these compounds using plant cell suspension cultures derived from R. crenulata may be an attractive alternative to harvest-based production.     

红景天甙的生物合成及其关键代谢酶研究

红景天甙是红景天属植物的主要药效成分,是一种很有前途的环境适应药物,具有抗疲劳、抗衰老、抗微波辐射、抗病毒及抗肿瘤等特异功效,尤其在军事、航天、运动及保健医学上具重要应用价值,近年来备受关注。本文在介绍了红景天属植物的开发利用价值及资源现状的基础上,重点探索了红景天甙生物合成的可能途径。认为其甙元酪醇是经由莽草酸途径合成,再由UDP葡萄糖基转移酶（Glucosyltransferase）催化葡萄糖和酪醇合成红景天甙,而红景天甙又可能在β-D-葡萄糖苷酶作用下降解为甙元酪醇和葡萄糖。文章阐述了参与红景天甙代谢的上述两个关键酶的研究现状及前景。     

Rewiring central carbon metabolism for tyrosol and salidroside production in Saccharomyces cerevisiae

Metabolic engineering of Saccharomyces cerevisiae for high-level production of aromatic chemicals has received increasing attention in recent years. Tyrosol production from glucose by S. cerevisiae is considered an environmentally sustainable and safe approach. However, the production of tyrosol and salidroside by engineered S. cerevisiae has been reported to be lower than 2 g/L to date. In this study, S. cerevisiae was engineered with a push-pull-restrain strategy to efficiently produce tyrosol and salidroside from glucose. The biosynthetic pathways of ethanol, phenylalanine, and tryptophan were restrained by disrupting PDC1, PHA2, and TRP3. Subsequently, tyrosol biosynthesis was enhanced with a metabolic pull strategy of introducing PcAAS and EcTyrA^M53I/A354V. Moreover, a metabolic push strategy was implemented with the heterologous expression of phosphoketolase (Xfpk), and then erythrose 4-phosphate was synthesized simultaneously by two pathways, the Xfpk-based pathway and the pentose phosphate pathway, in S. cerevisiae. Furthermore, the heterologous expression of Xfpk alone in S. cerevisiae efficiently improved tyrosol production compared with the coexpression of Xfpk and phosphotransacetylase. Finally, the tyrosol yield increased by approximately 135-folds, compared with that of parent strain. The total amount of tyrosol and salidroside with glucose fed-batch fermentation was over 10 g/L and reached levels suitable for large-scale production.     

Use of apple seed meal as a new source of beta-glucosidase for enzymatic glucosylation of 4-substituted benzyl alcohols and tyrosol in monophasic aqueous-dioxane medium

A facile method for enzymatic glycosylation of 4-substituted benzyl alcohols and tyrosol with glucose in a monophasic aqueous-dioxane medium was reported, using a crude meal of apple seed as a new catalyst. The corresponding beta-d-glucosides were synthesized in moderate yields (13.1-23.1%), among which the salidroside was obtained in 15.8% yield.