人肠道菌对黄芩苷的生物转化

本研究利用体外培养人体肠道菌转化黄芩苷,探索转化方法及模型;用醇沉法提取了黄芩苷转化酶,即β-D-葡萄糖醛酸苷酶,并探讨了酶促影响因素;通过高效液相色谱检测产物黄芩素。经实验确定,黄芩苷转化培养液经超声波处理后,在转化液中有黄芩素检出。实验得知,转化酶为胞内酶,该酶的最适反应温度为55℃,最适pH为6.0,Ca2+、Mg2+和Cu2+对酶促反应具有促进作用,而Fe2+则具有抑制作用,Zn2+浓度在l mmol/L时起促进作用,在5 mmol/L时起抑制作用。     

产黄芩苷内生真菌的筛选与鉴定

从药用植物黄芩根、茎、叶和花中分离得到17株内生真菌,其发酵液对10种指示菌进行抑菌活性测定,并通过薄层色谱(TLC)和高效液相色谱(HPLC)对所分离到的内生真菌代谢产物进行分析。结果显示,所分离到的内生真菌中12株至少对一种指示菌有抑菌活性,其中3株(G2、J4和J5)具有较广的抑菌作用。从黄芩茎和花中分离得到的2株内生真菌J1、H3可以在人工培养基中产生黄芩活性成分——黄芩苷,结合菌落形态特征、显微观察及ITS序列分析,初步鉴定这2株菌株均属于青霉菌属。     

兔肠道大豆异黄酮还原菌株的分离鉴定及其转化特性

【目的】从兔新鲜粪样中分离对大豆异黄酮黄豆苷原和染料木素具有转化作用的特定细菌菌株。【方法】在厌氧工作站内对獭兔新鲜粪样进行梯度稀释后涂板,挑取单菌落与底物黄豆苷原和染料木素分别厌氧混合培养,用高效液相色谱检测底物被转化情况。【结果】分离得到一株对大豆异黄酮黄豆苷原和染料木素均具有转化作用的革兰氏阳性严格厌氧细菌菌株AUH-JLR41(KJ188150)。根据产物的高效液相保留时间、紫外吸收图谱和质谱分析结果,将菌株AUH-JLR41代谢底物黄豆苷原和染料木素生成的产物分别鉴定为二氢黄豆苷原和二氢染料木素。经手性高效液相系统检测,产物二氢黄豆苷原和二氢染料木素均呈现两个等面积物质峰,表明这两个产物的对映体过量率均为0。通过转化动态研究发现,菌株AUH-JLR41分别在底物黄豆苷原和染料木素加入48 h和72 h后将底物全部转化为产物,该菌株能转化底物黄豆苷原和染料木素的最大浓度均为0.6 mmol/L。经BLAST比对,菌株AUH-JLR41的16S r RNA基因序列与斯奈克氏菌属菌株Slackia equolifaciens DZE(EU377663)的相似性高达99.6%。【结论】兔肠道分离的斯奈克氏菌属菌株Slackia sp.AUH-JLR41在厌氧条件下能将大豆异黄酮黄豆苷原和染料木素分别还原为二氢黄豆苷原和二氢染料木素。     

一株转化大豆苷元为S-雌马酚菌株的筛选和鉴定

【目的】筛选一株可转化大豆苷元为S-雌马酚的微生物菌株,并对该菌株进行鉴定。【方法】在厌氧条件下采用抗生素抑制非目标菌生长并结合稀释涂平板法进行菌株分离,分离可转化大豆苷元生成S-雌马酚的肠道细菌,并对产物进行结构鉴定。之后通过16S rDNA序列分析,构建该菌系统进化树,结合菌体形态及菌落特征,确立该菌系统发育学地位。【结果】从大鼠肠道内筛选分离到一株可以将大豆苷元转化为S-雌马酚的革兰氏阴性兼性厌氧菌株LH-52(JN861767),16S rDNA序列测序结果 BLAST比对表明该菌株与奇异变形杆菌(Proteus mirabilis)相似度达到了99%,结合形态特征和生理生化实验结果鉴定该菌为奇异变形杆菌。根据HPLC保留时间、质谱、核磁共振等波谱数据分析确定产物为S-雌马酚。【结论】菌株P.mirabilis LH-52为首次筛选到的可转化大豆苷元为S-雌马酚的兼性厌氧菌,相对于文献报道的严格厌氧菌更适合于工业化生产。     

人肠道产柚苷酶菌株分离、鉴定及其对柚皮苷的转化特性

【目的】从健康人肠道微生物菌群中分离能将柚皮苷高效转化为柚皮素的特定细菌菌株,对分离得到的菌株进行菌种鉴定并研究菌株对柚皮苷转化特性,目的是为柚皮素的高效生物合成提供新细菌资源。【方法】取健康人新鲜粪样,在厌氧工作站内培养24 h后进行梯度稀释并涂板,从板上挑取单菌落与底物柚皮苷进行厌氧培养,用高效液相色谱检测底物被转化情况。根据16S rDNA序列及相关生理生化特性分析,对所分离的柚皮苷转化菌进行菌种鉴定,并测定转化菌株对底物柚皮苷的转化动态和转化能力。【结果】从人肠道菌群中分离得到4株能将柚皮苷转化为柚皮素的细菌菌株,分别命名为AUH-JLD3、AUH-JLD7、AUHJLD104和AUH-JLD109。根据16S rDNA序列分析,结合形态学及相关生理生化特性等,将其分别鉴定为布劳特氏菌(Blautia sp.AUH-JLD3)、肠球菌(Enterococcus sp.AUH-JLD7)、拟杆菌(Bacteroides sp.AUHJLD104)和巴氏链球菌(Streptococcus pasteurianus subsp.AUH-JLD109)。转化动态研究结果表明,所分离的4株细菌菌株均能在12 h内将0.2 mmol/L底物柚皮苷转化为柚皮素;转化能力测定结果显示,菌株AUHJLD3、AUH-JLD7、AUH-JLD104及AUH-JLD109能够高效转化底物柚皮苷的最大浓度分别为0.2 mmol/L(平均转化率66.67%)、0.8 mmol/L(平均转化率86.49%)、0.2 mmol/L(平均转化率73.68%)以及1.6 mmol/L(平均转化率93.20%)。【结论】本研究首次从人肠道菌群中分离得到4株能将柚皮苷转化为柚皮素的细菌菌株,其中巴氏链球菌AUH-JLD109对底物柚皮苷转化能力最强。     

一株产虫草素的冬虫夏草内生真菌Bionectria ochroleuca OS17（英文）

虫草素是一种核苷类似物,具有多种药理作用。利用虫草素产生菌发酵的方式获得虫草素,可能是产生虫草素的一种新方法。为筛选能产虫草素的内生真菌,从中国传统的中药冬虫夏草子实体中共分离得到42株真菌,经PDB液体培养后,采用高效液相色谱(HPLC)鉴定其代谢产物。结果表明:菌株OS17的代谢产物中含有虫草素,且虫草素产量为40.16 mg/L。基于形态学特征和ITS序列分析,菌株OS17被鉴定为淡色生赤壳菌(Bionectria ochroleuca)。这是首次从冬虫夏草中分离得到能产虫草素的生赤壳属,表明了内生真菌具有产生多种生物活性物质的潜力。     

干酪乳杆菌对虫草素的生物转化研究

探讨干酪乳杆菌(Lactobacillus casei)对虫草素的生物转化。采用L.casei与虫草素共培养的方法,以虫草素水溶液为底物,接入L.casei活菌体进行厌氧培养,培养液经高效液相色谱(HPLC)和液质联谱(LC/MS)检测后有新产物生成,标记为CA。产物CA由制备液相分离、纯化后经核磁共振波谱(NMR)检测,被确定为3′-脱氧肌苷(3′-Deoxyinosine)。结果表明,L.casei能够对虫草素进行生物转化,可将虫草素C-6位的氨基水解为羟基,生成3′-脱氧肌苷,并在培养72 h时使虫草素转化率达91.2%。     

不同动物肠道优势需氧菌对黄豆苷原转化菌株转化能力的影响

摘要: 【目的】探讨不同动物肠道优势需氧菌对黄豆苷原转化菌株转化能力的影响。【方法】有氧条件下,采用稀释涂布法分别从ICR 小鼠、芦花鸡、长白猪和獭兔等4 种健康动物肠道中分离优势需氧菌,将不同动物的优势需氧菌分别与不同类型黄豆苷原转化菌株进行厌氧混合培养,高效液相色谱检测培养液中黄豆苷原的转化情况。【结果】16S rRNA 基因序列分析,结合形态学及相关理化特性分析表明,分离的22 株优势需氧菌分属埃希氏菌属(10 株) 、变形菌属(5 株) 、肠球菌属(4 株) 、芽胞杆菌属(2 株) 和假单胞菌属(     

真菌产3种β-D- Glucuronidase酶学性质

分离筛选到能代谢甘草酸并产生不同产物的3株菌株,其中Penicillium sp.Li-3转化甘草酸生成GAMG,As-pergillus sp.Li-20生成GAMG和甘草次酸,Aspergillus sp.Li-62生成甘草次酸。对这3株菌表达β-D-葡萄糖醛酸苷酶的酶学性质进行了研究,结果表明:Penicillium sp.Li-3、Aspergillus sp.Li-20和Aspergillus sp.Li-62β-D-葡萄糖醛酸苷酶最适催化pH分别为4.2~4.6,5.8和6.2,最适催化温度为50、45和55℃。Penicillium sp.Li-3表达的β-D-葡萄糖醛酸苷酶Km为0.328μmol/L,Vmax为0.003 5 mmol/(L.min);Aspergillus sp.Li-20β-D-葡萄糖醛酸苷酶Km为3.61 mmol/L,Vmax为0.034 mmol/(L.min);而Aspergillus sp.Li-62β-D-葡萄糖醛酸苷酶Km为0.43 mmol/L,Vmax为0.106 mmol/(L.min)。     

一株产白藜芦醇虎杖内生真菌的分离和鉴定

从药食同源植物虎杖根中分离得到15株内生真菌,在马铃薯葡萄糖液体培养基(PDB)中发酵后对所有内生真菌发酵液进行HPLC检测,有5株菌的发酵液中相关成分与白藜芦醇标准品保留时间一致,确定5株菌能产生白藜芦醇,在此基础上测定了5株菌的生长曲线。其中菌株H8生长最旺盛,白藜芦醇产量最高。应用形态学及ITS序列分析方法对其进行鉴定,确定为拟轮枝镰孢菌Fusarium verticillioides。     

Free radical scavenging and antioxidant activities of flavonoids extracted from the radix of Scutellaria baicalensis Georgi

Free radical scavenging and antioxidant activities of baicalein, baicalin, wogonin and wogonoside, the four major flavonoids in the radix of Scutellaria baicalensis Georgi, were examined in different systems. ESR results showed that baicalein and baicalin scavenged hydroxyl radical, DPPH radical and alkyl radical in a dose-dependent manner, while wogonin and wogonoside showed subtle or no effect on these radicals. Ten micromol/l of baicalein and baicalin effectively inhibited lipid peroxidation of rat brain cortex mitochondria induced by Fe(2+)-ascorbic acid, AAPH or NADPH, while wogonin and wogonoside showed significant effects only on NADPH-induced lipid peroxidation. In a study on cultured human neuroblastoma SH-SY5Y cells system, it was found that 10 micromol/l of baicalein and baicalin significantly protected cells against H(2)O(2)-induced injury. Baicalein was the most effective antioxidant among the four tested compounds in every system due to its o-tri-hydroxyl structure in the A ring. Compared with a well-known flavonoid, quercetin, the antioxidant activity of baicalein was lower in DPPH or AAPH system, but a little higher in those systems which might associate with iron ion. These results suggest that flavonoids in the radix of Scutellaria baicalensis with o-di-hydroxyl group in A the ring, such as baicalein and baicalin, could be good free radical scavengers and might be used to cure head injury associated with free radical assault.     

Effects of endogenous salicylic acid synthesized through PAL and ICS pathway on baicalin and baicalein accumulation in <Emphasis Type="Italic">Scutellaria baicalensis</Emphasis> Georgi

The aim of this study was to investigate the effect of phenylalanine ammonia lyase (PAL) and isochorismate synthase (ICS) on free salicylic acid (FSA) or total salicylic acid (TSA) content, and the effect of endogenous SA on baicalin and baicalein accumulation in Scutellaria baicalensis Georgi, respectively. We amplified partial sequences of PAL and ICS genes in Scutellaria baicalensis Georgi and silenced the two genes with virus-induced gene silence (VIGS) technique, respectively. The influence of gene silence on FSA, TSA, baicalin, and baicalein accumulation in Scutellaria baicalensis Georgi were analyzed, and these parameters were also investigated under high temperature. Results indicated that PAL silence significantly affected the FSA, ICS affected TSA content. FSA significantly affected the baicalin, rather than baicalein content. Our results along with previous studies indicated PAL and ICS were different in the regulation of FSA or TSA synthesis, and FSA and TSA were different in the regulation of baicalin and baicalein synthesis in Scutellaria baicalensis Georgi.     

Flavonoid Production in Transformed Scutellaria baicalensis Roots and Ways of Its Regulation

A root culture of skullcap (Scutellaria baicalensisGeorgi) transformed with pRi T-DNA was initiated by the inoculation of sterile seedlings with Agrobacterium rhizogenes(wild-type strain A-4). The flavonoid concentration in cultured roots comprised 5% of the root dry weight and was maintained essentially constant during a subculture. For four weeks of culturing, the weight of the roots increased by 20–30 times; when the roots were cultured for a longer time and with periodic enrichment of the nutrient medium, their weight increased 50-fold. Skullcap roots were shown to synthesize flavones characteristic of intact roots (wogonin, baicalein, and baicalin). The addition of 0.01–1 mM L-phenylalanine (a precursor of flavonoids) to the nutrient medium affected neither root growth, nor their flavonoid concentration. Root elicitation with 100 M methyl jasmonate for 72 h increased the flavonoid content per flask and per root dry weight by 1.8 and 2.3 times, respectively.     

Baicalein inhibits mitochondrial apoptosis induced by oxidative stress in cardiomyocytes by stabilizing MARCH5 expression

Abnormal mitochondrial fission and mitophagy participate in the pathogenesis of many cardiovascular diseases. Baicalein is a key active component in the roots of traditional Chinese medicinal herb Scutellaria baicalensis Georgi. It has been reported that baicalein can resist cardiotoxicity induced by several stress, but the mechanisms of baicalein operate in the protection of cardiomyocytes need to be researched further. Here we report that baicalein can promote cell survival under oxidative stress by up‐regulating the expression level of MARCH5 in cardiomyocytes. Pre‐treatment cells or mice with baicalein can stabilize the expression of MARCH5, which plays a crucial role in the regulation of mitochondrial network and mitophagy. Overexpressed MARCH5 is able to against H₂O₂ and ischaemia/reperfusion (I/R) stress by suppressing mitochondrial fission and enhancing mitophagy, and then attenuate cells apoptosis. Altogether, our present study investigated that baicalein exerts a protective effect through regulating KLF4‐MARCH5‐Drp1 pathway, our research also provided a novel theoretical basis for the clinical application of baicalein.     

黄芩的结构与黄芩苷含量的关系

应用解剖学、组织化学定位和植物化学技术,研究了一、二年生黄芩（Scutellaria baicalensis Georgi）营养器官的结构特征与黄芩苷积累的关系。结果表明：黄芩各营养器官的结构类似于一般草本双子叶植物,黄芩苷主要分布在各器官的薄壁组织细胞内,其中以维管组织的薄壁细胞为多。器官间含量的不同表现为根〉叶〉茎,这与其结构特征相对应,根中维管组织所占比例最大,其中薄壁组织细胞又占主要部分,故根中黄芩苷含量最高。说明根部是黄芩苷的主要贮存部位,符合黄芩以根入药的传统。不同生长期黄芩苷含量测定结果表明,两年生营养生长前期春季根内含量最高,所以春季采挖为好。     

黄芩过氧化物酶同工酶电泳和抗坏血酸过氧化物酶活性分析

对黄芩（Scutellaria baicalensis Georgi)二倍体和同源四倍体过氧化物酶进行了同工酶电泳分析及抗坏血酸过氧化物酶活性测定。结果表明,黄芩二倍体与同源四倍体过氧化物酶同工酶酶谱一致,但后者的着色程度大于前者,二年生黄芩叶子在快速区Rf为0．494和0．512处出现新的谱带,不同发育阶段和不同组织器官的谱带存在明显差异;根和叶中谱带数最多,花其次,种子最少;试管苗谱带数先减少后增加,并在整个培养过程中出现特征性谱带C。各组织器官抗坏血酸过氧化物酶总活力差异明显,顶芽最高,叶子次之,花最低。黄芩一年生和二年生各个多倍体株系叶子抗坏血酸过氧酶总活力均高于二倍体叶子抗坏血酸过氧化酶总活力。试管苗生长过程中抗坏血酶过氧化物酶活活力的变化与生长趋势一致,表明该酶与植株生长发育紧密相关。     

Purification and characterization of UDP-glucuronate: baicalein 7-O-glucuronosyltransferase from Scutellaria baicalensis Georgi. cell suspension cultures

UDP-glucuronate: baicalein 7-O-glucuronosyltransferase (UBGAT) catalyzes the transfer of glucuronic acid from UDP-glucuronic acid to the 7-OH of baicalein. UBGAT was purified from cultured cells of Scutellaria baicalensis Georgi (Lamiaceae). It was purified 95-fold using various chromatography and chromatofocusing procedures to apparent homogeneity. The Mr was estimated to be 110 kDa by gel filtration chromatography with a 52 kDa subunit by SDS-PAGE. The isoelectric point was pH 4.8. UBGAT was specific to UDP-glucuronic acid as a sugar donor and flavones with substitution ortho- to the 7-OH group such its baicalein (6-OH), scutellarein (6-OH) and wogonin (8-OMe).     

Flavones in genetically transformed <Emphasis Type="Italic">Scutellaria baicalensis</Emphasis> roots and induction of their synthesis by elicitation with methyl jasmonate

The composition and content of flavones were estimated in pRi T-DNA-transformed skullcap (Scutellaria baicalensis Georgi) roots obtained by the inoculation of axenically grown seedlings with a wild A4 strain of the soil bacterium Agrobacterium rhizogenes. It was elucidated by analytical and preparative HPLC of phenolic compounds in the extracts from the pRi T-DNA-transformed roots and also by ultraviolet spectroscopy and ¹H and ¹³C NMR that cultured skullcap roots contained similar basic flavones as intact roots of this plant species, i.e., baicalein and wogonin and corresponding glucuronides, baicalin and wogonoside. The content of these flavones in cultured roots was threefold lower than in the roots of intact five-year-old plants. When skullcap roots were cultured on B5 or Murashige and Skoog medium, the ratios between major flavones changed but their total content remained unchanged. The treatment of three-week-old cultured roots with methyl ether of jasmonic acid (MeJa) doubled the total concentration of major flavones in roots; the content of aglycons, baicalein and wogonin, increased to a greater degree, e.g., by 2.3 and 3.3 times, respectively. The induction of flavone production by elicitors indicates that flavones behave as phytoanticipins because major flavones of skullcap manifest a distinct antimicrobial activity. The results of the short-term treatment of skullcap roots with MeJa show that stress biotic factors can considerably increase the content of physiologically active flavones.Translated from Fiziologiya Rastenii, Vol. 52, No. 1, 2005, pp. 90–96.Original Russian Text Copyright © 2005 by Kuzovkina, Guseva, Kovács, Szöke, Vdovitchenko.