薄层色谱法鉴定天麻胶囊中的天麻素

为了建立天麻胶囊中主要有效成分天麻素的快速鉴定方法,根据天麻素的理化特性,使用乙醇和甲醇提取天麻胶囊中的天麻素,使用薄层色谱法进行鉴定,并与高效液相色谱法的分析结果进行比较。结果表明,薄层色谱法的鉴定结果与高效液相色谱法的检测结果一致,能较准确地鉴别天麻胶囊的真伪。本研究结果表明薄层色谱法能快速简便、准确灵敏地检测天麻胶囊中的有效成分,可作为法定鉴定方法的补充,对天麻胶囊实施快速初筛。     

天麻特异DNA序列的克隆及其在天麻鉴定中的应用

应用改进的RAPD方法测定了名贵中药材天麻基因组DNA指纹图谱;通过选择和回收各种天麻种群共有和优良种群特有的DNA片段,加以克隆、测序和生物信息学分析,证明其中5个DNA序列是未报道的,已被美国基因数据库收录,并运用高效液相色谱技术测定了天麻样本的有效成分天麻素含量。运用PCR技术研究了这些DNA序列在9个天麻种群中的分布及其与天麻素含量的关系。结果表明这5个DNA序列在这些天麻种群中的分布各不相同,其中DNA序列1是所研究的全部天麻种群共有、而其伪品没有的特异DNA分子标记;DNA序列2可能与天麻的天麻素含量高有关。这些DNA标记序列可用于天麻的真伪鉴别、品种鉴定和优选优育等。     

湖北天麻GAP基地不同品种天麻最佳采收期研究

对湖北两个天麻GAP(good agricu ltural practice)基地不同品种的天麻进行了天麻素含量的动态检测和生态条件及产量的考察。结果表明,天麻在5~9月份生长迅速,从10月下旬开始逐渐停止生长,进入11月份天麻产量达到最大;采用RP-HPLC法测定,11~12月份采收的样本天麻素含量最高。据此,确定了湖北GAP基地天麻的最佳采收期:兴山基地的乌天麻、红乌天麻和乌红天麻,广水基地的红天麻均以11月份采收为宜;兴山基地的红天麻以12月份采收为宜。     

麻黄不同炮制工艺对含量影响

目的:探究麻黄不同炮制方法对麻黄有毒成分含量的影响。方法:分别采用蒸制、清炒、酒炙和醋炙这四种炮制方法对麻黄进行炮制,并测定其不同炮制品的有毒成分生物碱的含量,比较该四种炮制方法对其有毒成为生物碱含量影响。结果:麻黄的各炮制品的生物碱的含量从高到低:清炒醋炙酒制蒸制。结论:蒸制炮制工艺在降低药物的毒性效果优于清炒炮制法、酒炙炮制法及醋炙炮制法,可以作为麻黄的优选炮制方式。     

不同蜜环菌菌株对红天麻农艺性状、产量及质量的影响

为研究不同蜜环菌菌株对菌材的侵染情况以及对红天麻主要农艺性状、产量及有效成分含量的影响,筛选适宜黔西北地区天麻生长的野生蜜环菌菌株。采用不同蜜环菌菌株培养菌材,并与红天麻伴栽,观察记录不同蜜环菌栽培种对青冈菌材的侵染情况以及不同处理红天麻的农艺性状,检测天麻的有效成分含量。不同蜜环菌栽培种对青冈菌材的侵染情况不同,对红天麻农艺性状、产量及质量的影响也不同。其中菌株MHJ-1满袋时间最短、上菌速度最快,对菌材的侵染效果最好,且能显著提高红天麻产量,每平方米鲜天麻平均产量为（8.78±0.38）kg,比对照菌株提高5%;其伴栽的天麻醇溶性浸出物含量、天麻素含量、天麻素与对羟基苯甲醇总含量均最高,分别为（29.24±0.43）%、（0.946±0.012）%、（1.140±0.009）%,且极显著高于对照菌株,仿野生栽培试验表现出良好的伴栽效果。结果表明菌株 MHJ-1与红天麻的伴栽效果最好,适宜用于黔西北地区红天麻种植。     

炮制加工对天麻化学成分影响的研究进展

天麻作为著名名贵药材和药食同源试点品种,其产量和消费量逐年上升,炮制加工对其化学成分及活性的影响成为诸多研究者关注的重点。本文从炮制加工(以蒸制和干制为代表)对天麻营养成分(如淀粉、蛋白质和维生素等)、功效成分(如腺苷、多酚及其酚苷和活性多糖等)及天麻微观结构的影响视角深入详细分析了天麻最近5年的研究进展,结果证实在加工过程中天麻化学成分的变化与其微观结构有一定的关联性,揭示了炮制加工影响天麻化学成分及其微观结构的内在原因,最后总结了天麻开发利用现状,为天麻的精细加工及多功能化研发提供了基础与依据。     

天麻及其活性成分药效作用的实验研究

天麻属兰科植物,从天麻中提炼出的化学成份有天麻素、天麻苷元、香荚兰醇、香荚兰醛、天麻醚苷、对羟基苯甲醛、柠檬酸、琥珀酸等[1]。其中活性成份含量最高的有效单体成份是天麻素(化学名为对羟甲基苯-β-D-吡喃葡萄糖苷,又称天麻苷)[2]。天麻素在非酶体系中稳定,在肝、肾、脑组织匀浆中天麻素可被代谢为天麻苷元。在组织中的代谢,以肾最快,脑次之,肝最小;在脑组织中,天麻素在小脑、丘脑、脑桥与延脑区域代谢速度快于皮层、纹状体和海马区[3]。有关天麻及其活性成分药效作用的实验研究较多,可见天麻有效成分的开发利用远无止境,但是某些作用机制尚有待深入研究和阐明,本文对有关研究文献综述如下。     

基于HPLC-MS的西藏天麻的化学成分分析与品质鉴定

目的:天麻具有许多药理作用和重要开发价值,通过探索天麻的未知化学成分和鉴定西藏天麻种群的品质,为深入研究、开发和利用西藏优良天麻种群提供科学技术支持。方法:本研究采用HLPC-MS(液质联用)技术测定西藏天麻的化学成分;采用高效液相色谱技术测定西藏6个天麻种群的生化指纹图谱,利用天麻素峰面积、数学公式和SPSS软件,计算和比较西藏6个天麻种群的平均天麻素含量和各种化学成分分离峰的总面。结果:从天麻块茎中发现了33个未报道的化学物质;西藏天麻的生化指纹图谱具有7个较大的共有特征分离峰,分别位于1.854、2.759、7.279、7.591、8.500、9.557、10.753min;根据生化指纹图谱特征可将它们分为三个主要类型;凡是带有一型生化指纹图谱的天麻个体和种群往往含有更高的天麻素和更大的分离峰总面积,品质更优良。结论:西藏天麻种群3和种群6以一型生化指纹图谱为主,其天麻素含量最高,分离峰总面积最大,品质最优良,具有重要研究、开发和保护价值。本研究成果对于天麻的化学成分分析与鉴定、天麻种群和品质鉴定与评价以及西藏天麻的品种选优、资源保护与利用具有重要指导意义和科学价值。     

天麻初加工方法对主要成分含量的影响及加工方法的化学模式识别

为了阐明天麻初加工对其主要成分的影响,采用HPLC法测定酚类含量,试剂盒法测定多糖含量。并应用HCA、PLS-DA和GS-SVM对不同样品进行化学模式识别分析,寻找差异性成分对初加工方法进行追溯。结果表明:与对照组相比,不同初加工对酚类和多糖影响显著,发汗、蒸制和煮制使天麻素和巴利森苷类增加,对羟基苯甲醇和对羟基苯甲醛降低;酒制使天麻素增加,其他成分均不同程度降低。发汗和煮制使多糖增加,蒸制多糖减少,酒制多糖无显著变化。HCA和PLS-DA对不同样品分类有效,天麻素和多糖为追溯标志性成分。采用GS-SVM建立判别模型,交叉验证和预测正确率分别为97.37%和100%。因此,以天麻主要活性成分综合考察不同加工方法对天麻质量的影响,符合中药的特点、体现中药的特征;化学模式识别可作为追溯天麻不同加工方法的有效手段。     

基于HPLC—MS的西藏天麻的化学成分分析与品质鉴定

目的：天麻具有许多药理作用和重要开发价值,通过探索天麻的未知化学成分和鉴定西藏天麻种群的品质,为深入研究、开发和利用西藏优良天麻种群提供科学技术支持。方法：本研究采用HLPC．MS（液质联用）技术测定西藏天麻的化学成分;采用高效液相色谱技术测定西藏6个天麻种群的生化指纹图谱,利用天麻素峰面积、数学公式和SPSS软件,计算和比较西藏6个天麻种群的平均天麻素含量和各种化学成分分离峰的总面。结果：从天麻块茎中发现了33个未报道的化学物质;西藏天麻的生化指纹图谱具有7个较大的共有特征分离峰,分别位于1．854、2．759、7．279、7．591、8．500、9．557、10．753min;根据生化指纹图谱特征可将它们分为三个主要类型;凡是带有一型生化指纹图谱的天麻个体和种群往往舍有更高的天麻素和更大的分离峰总面积。品质更优良。结论：西藏天麻种群3和种群6以一型生化指纹图谱为主,其天麻素含量最高,分离峰总面积最大,品质最优良,具有重要研究、开发和保护价值。本研究成果对于天麻的化学成分分析与鉴定、天麻种群和品质鉴定与评价以及西藏天麻的品种选优、资源保护与利用具有重要指导意义和科学价值。     

De novo biosynthesis of Gastrodin in Escherichia coli

Gastrodin, a phenolic glycoside, is the key ingredient of Gastrodia elata, a notable herbal plant that has been used to treat various conditions in oriental countries for centuries. Gastrodin is extensively used clinically for its sedative, hypnotic, anticonvulsive and neuroprotective properties in China. Gastrodin is usually produced by plant extraction or chemical synthesis, which has many disadvantages. Herein, we report unprecedented microbial synthesis of gastrodin via an artificial pathway. A Nocardia carboxylic acid reductase, endogenous alcohol dehydrogenases and a Rhodiola glycosyltransferase UGT73B6 transformed 4-hydroxybenzoic acid, an intermediate of ubiquinone biosynthesis, into gastrodin in Escherichia coli. Pathway genes were overexpressed to enhance metabolic flux toward precursor 4-hydroxybenzyl alcohol. Furthermore, the catalytic properties of the UGT73B6 toward phenolic alcohols were improved through directed evolution. The finally engineered strain produced 545 mg l⁻¹ gastrodin in 48 h. This work creates a new route to produce gastrodin, instead of plant extractions and chemical synthesis.     

Gastrodin Inhibits Glutamate-Induced Apoptosis of PC12 Cells via Inhibition of CaMKII/ASK-1/p38 MAPK/p53 Signaling Cascade

Previous research demonstrated that glutamate induces neuronal injury partially by increasing intracellular Ca²⁺ concentrations ([Ca²⁺]_i), and inducing oxidative stress, leading to a neurodegenerative disorder. However, the mechanism of glutamate-induced injury remains elusive. Gastrodin, a major active component of the traditional herbal agent Gastrodia elata (GE) Blume, has been recognized as a potential neuroprotective drug. In the current study, a classical injury model based on glutamate-induced cell death of rat pheochromocytoma (PC12) cells was used to investigate the neuroprotective effect of gastrodin, and its potential mechanisms involved. In this paper, the presence of gastrodin inhibits glutamate-induced oxidative stress as measured by the formation of reactive oxygen species (ROS), the level of malondialdehyde (MDA), mitochondrial membrane potential (MMP), and superoxide dismutase (SOD); gastrodin also prevents glutamate-induced [Ca²⁺]_i influx, blocks the activation of the calmodulin-dependent kinase II (CaMKII) and the apoptosis signaling-regulating kinase-1 (ASK-1), inhibits phosphorylation of p38 mitogen-activated kinase (MAPK). Additionally, gastrodin blocked the expression of p53 phosphorylation, caspase-3 and cytochrome C, reduced bax/bcl-2 ratio induced by glutamate in PC12 cells. All these findings indicate that gastrodin protects PC12 cells from the apoptosis induced by glutamate through a new mechanism of the CaMKII/ASK-1/p38 MAPK/p53-signaling pathway.     

Gastrodin inhibits allodynia and hyperalgesia in painful diabetic neuropathy rats by decreasing excitability of nociceptive primary sensory neurons

Painful diabetic neuropathy (PDN) is a common complication of diabetes mellitus and adversely affects the patients' quality of life. Evidence has accumulated that PDN is associated with hyperexcitability of peripheral nociceptive primary sensory neurons. However, the precise cellular mechanism underlying PDN remains elusive. This may result in the lacking of effective therapies for the treatment of PDN. The phenolic glucoside, gastrodin, which is a main constituent of the Chinese herbal medicine Gastrodia elata Blume, has been widely used as an anticonvulsant, sedative, and analgesic since ancient times. However, the cellular mechanisms underlying its analgesic actions are not well understood. By utilizing a combination of behavioral surveys and electrophysiological recordings, the present study investigated the role of gastrodin in an experimental rat model of STZ-induced PDN and to further explore the underlying cellular mechanisms. Intraperitoneal administration of gastrodin effectively attenuated both the mechanical allodynia and thermal hyperalgesia induced by STZ injection. Whole-cell patch clamp recordings were obtained from nociceptive, capsaicin-sensitive small diameter neurons of the intact dorsal root ganglion (DRG). Recordings from diabetic rats revealed that the abnormal hyperexcitability of neurons was greatly abolished by application of GAS. To determine which currents were involved in the antinociceptive action of gastrodin, we examined the effects of gastrodin on transient sodium currents (I(NaT)) and potassium currents in diabetic small DRG neurons. Diabetes caused a prominent enhancement of I(NaT) and a decrease of potassium currents, especially slowly inactivating potassium currents (I(AS)); these effects were completely reversed by GAS in a dose-dependent manner. Furthermore, changes in activation and inactivation kinetics of I(NaT) and total potassium current as well as I(AS) currents induced by STZ were normalized by GAS. This study provides a clear cellular basis for the peripheral analgesic action of gastrodin for the treatment of chronic pain, including PDN.     

Gastrodin Ameliorates Memory Deficits in 3,3′-Iminodipropionitrile-Induced Rats: Possible Involvement of Dopaminergic System

3,3′-Iminodipropionitrile (IDPN), one of the nitrile derivatives, can induce neurotoxicity, and therefore cause motor dysfunction and cognitive deficits. Gastrodin is a main bioactive constituent of a Chinese herbal medicine (Gastrodia elata Blume) widely used for treating various neurological disorders and showed greatly improved mental function. This study was designed to determine whether administration of gastrodin attenuates IDPN-induced working memory deficits in Y-maze task, and to explore the underlying mechanisms. Results showed that exposure to IDPN (150 mg/kg/day, v.o.) significantly impaired working memory and that long-term gastrodin (200 mg/kg/day, v.o.) could effectively rescue these IDPN-induced memory impairments as indicated by increased spontaneous alternation in the Y-maze test. Additionally, gastrodin treatment prevented IDPN-induced reductions of dopamine (DA) and its metabolites, as well as elevation of dopamine turnover ratio (DOPAC + HVA)/DA. Gastrodin treatment also prevented alterations in dopamine D2 receptor and dopamine transporter protein levels in the rat hippocampus. Our results suggest that long-term gastrodin treatment may have potential therapeutic values for IDPN-induced cognitive impairments, which was mediated, in part, by normalizing the dopaminergic system.     

Relationships Among Genetic Makeup, Active Ingredient Content, and Place of Origin of the Medicinal Plant Gastrodia Tuber

Gastrodia tuber and its component gastrodin have many pharmacological effects. The chemical fingerprints and gastrodin contents of eight Gastrodia populations were determined, and the genomic DNA polymorphism of the populations was investigated. Genetic distance coefficients among the populations were calculated using the DNA polymorphism data. A dendrogram of the genetic similarities between the populations was constructed using the genetic distance coefficients. The results indicated that the genomic DNA of Gastrodia tubers was highly polymorphic; the eight populations clustered into three major groups, and the gastrodin content varied greatly among these groups. There were obvious correlations among genetic makeup, gastrodin content, and place of origin. The ecological environments in Guizhou and Shanxi may be conducive to evolution and to gastrodin biosynthesis, and more suitable for cultivation of Gastrodia tubers. These findings may provide a scientific basis for overall genetic resource management and for the selection of locations for cultivating Gastrodia tubers.     

Gastrodin relieves inflammation injury induced by lipopolysaccharides in MRC‐5 cells by up‐regulation of miR‐103

The beneficial function of gastrodin towards many inflammatory diseases has been identified. This study designed to see the influence of gastrodin in a cell model of chronic obstructive pulmonary disease (COPD). MRC‐5 cells were treated by LPS, before which gastrodin was administrated. The effects of gastrodin were evaluated by conducting CCK‐8, FITC‐PI double staining, Western blot, qRT‐PCR and ELISA. Besides this, the downstream effector and signalling were studied to decode how gastrodin exerted its function. And dual‐luciferase assay was used to detect the targeting link between miR‐103 and lipoprotein receptor‐related protein 1 (LRP1). LPS induced apoptosis and the release of MCP‐1, IL‐6 and TNF‐α in MRC‐5 cells. Pre‐treating MRC‐5 cells with gastrodin attenuated LPS‐induced cell damage. Meanwhile, p38/JNK and NF‐κB pathways induced by LPS were repressed by gastrodin. miR‐103 expression was elevated by gastrodin. Further, the protective functions of gastrodin were attenuated by miR‐103 silencing. And LRP1 was a target of miR‐103 and negatively regulated by miR‐103. The in vitro data illustrated the protective function of gastrodin in LPS‐injured MRC‐5 cells. Gastrodin exerted its function possibly by up‐regulating miR‐103 and modulating p38/JNK and NF‐κB pathways.     

紫花曼陀罗悬浮培养细胞转化对羟基苯甲醛生产天麻素

利用紫花曼陀罗细胞悬浮培养转化外源对羟基苯甲醛合成天麻素,并应用多种色谱技术进行分离纯化,根据转化产物的理化性质和光谱数据分析鉴定结构。实验表明,紫花曼陀罗细胞成功将对羟基苯甲醛转化为天麻素(Ⅱ),同时也得到了由对羟基苯甲醛生成天麻素的转化中间体对羟基苯甲醇(Ⅰ)。在培养基中添加0.1mg/L的水杨酸能显著提高细胞对外源对羟基苯甲醛的糖基化率,而保持气升式发酵罐(25-L)罐内压力为低压(0.001MPa)也能提高细胞对外源对羟基苯甲醛的糖基化率。实验证明,紫花曼陀罗细胞悬浮培养能有效转化对羟基苯甲醛合成天麻素。     

Inhibition of tyrosinase by gastrodin: An integrated kinetic-computational simulation analysis

We studied the inhibitory effect of gastrodin on tyrosinase using inhibition kinetics and computational simulation. Gastrodin reversibly inhibited tyrosinase in a mixed-type manner with K_i = 123.8 ± 20.2 mM. Time-interval kinetics revealed the inhibition to be a first-order process with mono- and bi-phasic components. Using AutoDock Vina, we calculated a binding energy of ?6.3 kcal/mol for gastrodin and tyrosinase, and we performed a molecular dynamics simulation of the tyrosinase–gastrodin interaction. The simulation results suggested that gastrodin interacts primarily with histidine residues in the active site. A 10-ns molecular dynamics simulation showed that one copper ion in the tyrosinase active site was responsible for the interaction with gastrodin. Our study provides insight into the inhibition of tyrosinase by the hydroxyl groups of gastrodin. A combination of inhibition kinetics and computational calculations may help to confirm the inhibitory action of gastrodin on tyrosinase and define the mechanisms of inhibition.     

Gastrodin prevents homocysteine-induced human umbilical vein endothelial cells injury via PI3K/Akt/eNOS and Nrf2/ARE pathway

In this study, we investigated the protective effects of gastrodin (Gas) against homocysteine-induced human umbilical vein endothelial cell (HUVEC) injury and the role of the phosphoinositide 3-kinase (PI3K)/threonine kinase 1 (Akt)/endothelial nitric oxide synthase (eNOS) and NF-E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathways. We stimulated cells with homocysteine (1 mmol/L, 24 hours) and tested the effects of gastrodin (200-800 μg/mL) on cell viability and the production of malondialdehyde (MDA), lactate dehydrogenase (LDH) and reactive oxygen species (ROS). Then, Nrf2 distribution in the cytoplasm and nucleus as well as the expression of enzymes downstream of Nrf2 was determined. Furthermore, we analysed the expression of bax, bcl-2 and cleaved caspase3, and assessed the involvement of the PI3K/Akt/eNOS pathway by Western blots. Finally, we tested the vasoactive effect of gastrodin in thoracic aortic rings. The results showed that gastrodin decreased MDA, LDH and ROS production and increased cell viability, NO production and relaxation of thoracic aortic rings. Moreover, the protective effects of Gas on NO production and relaxation of thoracic aortic rings were blocked by L-NAME but enhanced by Cav-1 knockdown, and MK-2206 treatment abolished the effect of Gas on the ROS. In addition, treatment with gastrodin increased Nrf2 nuclear translocation, thus enhancing the expression of downstream enzymes. Finally, gastrodin increased the expression of PI3K, p-Akt, and eNOS and decreased Cav-1 protein expression. In conclusion, our study suggested that gastrodin may protect HUVECs from homocysteine-induced injury, and the PI3K/Akt/eNOS and Nrf2/ARE pathways may be responsible for the efficacy of gastrodin.