Comparative analysis of flavonoids and polar metabolites from hairy roots of Scutellaria baicalensis and Scutellaria lateriflora

Baicalin, baicalein, and wogonin were accumulated in hairy roots derived from Scutellaria lateriflora and Scutellaria baicalensis. The levels of baicalein and baicalin were 6.8 and 5.0 times higher, respectively, in S. baicalensis than in S. lateriflora. A total of 47 metabolites were detected and identified in Scutellaria species by GC-TOF MS. The metabolites from the two species were subjected to principal component analysis (PCA) to evaluate differences. PCA fully distinguished between the two species. The results showed that individual phenolic acids and phenylalanine, precursors for the phenylpropanoid biosynthetic pathway, were higher in S. baicalensis than in S. lateriflora. This GC-TOF MS-based metabolic profiling approach was a viable alternative method to differentiate metabolic profiles between species.     

Comparative Genome Analysis of Scutellaria baicalensis and Scutellaria barbata Reveals the Evolution of Active Flavonoid Biosynthesis

Scutellaria baicalensis (S. baicalensis) and Scutellaria barbata (S. barbata) are common medicinal plants of the Lamiaceae family. Both produce specific flavonoid compounds, including baicalein, scutellarein, norwogonin, and wogonin, as well as their glycosides, which exhibit antioxidant and antitumor activities. Here, we report chromosome-level genome assemblies of S. baicalensis and S. barbata with quantitative chromosomal variation (2n = 18 and 2n = 26, respectively). The divergence of S. baicalensis and S. barbata occurred far earlier than previously reported, and a whole-genome duplication (WGD) event was identified. The insertion of long terminal repeat elements after speciation might be responsible for the observed chromosomal expansion and rearrangement. Comparative genome analysis of the congeneric species revealed the species-specific evolution of chrysin and apigenin biosynthetic genes, such as the S. baicalensis-specific tandem duplication of genes encoding phenylalanine ammonia lyase and chalcone synthase, and the S. barbata-specific duplication of genes encoding 4-CoA ligase. In addition, the paralogous duplication, colinearity, and expression diversity of CYP82D subfamily members revealed the functional divergence of genes encoding flavone hydroxylase between S. baicalensis and S. barbata. Analyzing these Scutellaria genomes reveals the common and species-specific evolution of flavone biosynthetic genes. Thus, these findings would facilitate the development of molecular breeding and studies of biosynthesis and regulation of bioactive compounds.     

不同花色黄芩中dfr基因的克隆及时空表达分析

二氢黄酮醇-4-还原酶(Dihydroflavonol-4-reductase,Dfr)是类黄酮生物合成途径中调控花青素和原花青素合成的关键酶.为探究dfr基因在同一生态环境下不同花色黄芩中的差异,以白色、紫红色和紫色3种花色的黄芩花瓣cDNA为模板,基于同源克隆和RACE技术从中克隆得到3个完整的dfr基因全长序列,...     

酶解法提取黄芩中黄芩素的工艺研究

本文对黄芩药材中黄芩苷的酶解条件和黄芩素的提取工艺进行研究,以样品中黄芩素的含量为指标,采用正交实验对影响酶解的温度、时间和药材粒度进行考察;以黄芩素的提取率为指标,经正交实验优选乙醇浓度、用量、回流次数和时间。得出如下较理想的酶解条件和提取工艺:黄芩药材粉碎至10~20目,加水6倍,于60℃保温10 h;酶解后的黄芩药材加8倍量70%乙醇回流提取2次,每次1 h。黄芩素的提取率大于70%,工艺稳定可行。     

山西省野生黄芩种质资源及植物学性状研究

通过对山西省9个市27个县（区）野生黄芩生长环境及气候条件的调查,发现黄芩在山西省分布范围广泛。山西野生黄芩主要分布于N34?6''19.78''～N40?4''17.71'',E110?0''17.80''～E114?3''13.41'';海拔介于705～1675 m之间,其中以1000～1500 m最为常见;生长区域年均降水量介于400～800 mm之间,年均温度为6.8～14 ℃。群落组成较为单一,其周围主要分布有杂草、灰菜、蒲公英、黄刺玫以及豆科类植株等;少部分伴随有当地优势种植株,其中大同市主要为狼毒、岩青兰,长治市平顺县主要为柴胡、蒲公英等。山西野生黄芩主要分布于半山腰,山顶分布较少,山谷底一般极少分布;山西野生黄芩多分布于半阳坡,少数分布于阳坡,阴坡一般极少分布。气候、海拔、降水量、日照等生态环境的不同以及人为因素是导致山西黄芩分布不均的主要因素。研究结果验证、补充了山西野生黄芩种质资源的数据和资料,为培育性状优良、种源明确的栽培品种提供了依据。     

Molecular characterization of a novel beta-glucuronidase from Scutellaria baicalensis georgi

We cloned a gene encoding Scutellaria beta-glucuronidase (sGUS) that is involved in the initiation of H(2)O(2) metabolism in skullcap (Scutellaria baicalensis). This gene consists of a 1581-nucleotide open reading frame, the deduced amino acid sequence of which contains an ATP/GTP binding site and a leucine zipper motif. sGUS has apparent similarity to the heparan sulfate-metabolizing beta-glucuronidase heparanase but no homology to family 2 beta-glucuronidases. In addition, neither the family 2 glycosylhydrolase signature nor family 2 acid-base catalyst was found in this enzyme. These results suggested that sGUS does not belong to the family 2 beta-glucuronidases. We modified several residues predicted to act as the acid-base or nucleophilic residue of sGUS by site-directed mutagenesis. Mutations at Glu(212) or Glu(329) resulted in much lower k(cat)/K(m) values in the mutants as compared with the wild-type enzyme, indicating that these are the acid-base and nucleophilic residues of the active site, respectively. Moreover, similar site-directed mutagenesis confirmed that Tyr(281) is also involved in the beta-glucuronidase activity. The amino acid sequences of small regions containing these active site residues were conserved in heparanases. As sGUS has various structural characteristics in common with heparanase, we concluded that sGUS and heparanase belong to the same new family.     

Genetic characterization and phytochemical analysis of wild and cultivated populations of Scutellaria baicalensis

Scutellaria baicalensis was collected from four wild and four cultivated populations from different locations in China. Forty-two samples were analyzed using random amplification of polymorphic DNA (RAPD) techniques for genetic profiling, and high performance liquid chromatography (HPLC) techniques to determine the flavonoid content. The selected 23 RAPD primers yielded a total of 838 clear and reproducible bands of which 237 were found to be polymorphic. The wild population exhibited higher polymorphism than that of the cultivated population. The dendrogram generated by the UPGMA method via Nei's genetic distance revealed three distinct genotypes from the cultivated populations and several branches from the wild populations. The contents of baicalin and wogonoside in dried roots of the samples ranged from (w/w) 8.63 to 17.84%, and from 1.99 to 4.21%, respectively, whereas their aglycones, baicalein and wogonin, were within the range of only 0.04-0.23%. The total content of the four flavonoids varied from 9.45 to 26.24%. Comparatively, the cultivated populations contained much higher levels of baicalin and total flavonoids than those in the wild populations. The results from genetic characterization and phytochemical analysis demonstrated that the quality variation of this drug was mainly determined by extrinsic environmental or agricultural factors, rather than by genetic differences. Our findings can be used for the commercial production and germplasm management of this medicinal plant.     

黄芩有效成分提取及对炎症反应的影响

利用常温超高压方法提取黄芩中黄酮类物质,得到黄芩甲醇提取物（SBM）。对该物质抗炎作用研究发现SBM（31．25-500mg／L）可抑制刀豆蛋白A（ConA）诱导的小鼠脾淋巴细胞增殖和脂多糖（LPS）诱导的腹腔巨噬细胞TNFα合成,SBM（125mg／L）的抑制率分别可达96％、99％;SBM（10mg／kg、20mg／kg）也可抑制大鼠棉球性肉芽肿,但不影响IKBα蛋白磷酸化。常温超高压方法可有效提取黄芩中黄酮类物质,黄芩提取物SBM可通过抑制淋巴细胞功能、炎症介质发挥抗炎作用。     

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。     

黄芩组织培养同源四倍体的诱导

应用组织培养技术对黄芩进行多倍体诱导,结果表明：组织培养条件下,在培养基中添加一定浓度和秋水仙素,或者把带有绿色芽点的黄芩愈伤组织经０．２％秋水仙素溶液浸泡一定时间后再进行培养,均可诱发黄芩多倍体的产生,但以后效果较好,诱导率可达４０．０％,通过试管苗根尖染色体显微观察,鉴定出５０多个黄芩同源四倍体,为今后优良品种的选育打下基础。     

Characterization of chemical ingredients and anticonvulsant activity of American skullcap (Scutellaria lateriflora)

American skullcap (the aerial part of Scutellaria lateriflora L.) has been traditionally used by Native Americans and Europeans as a nerve tonic, sedative, and anticonvulsant. However, despite some previous studies, the quality and safety, the bioactive ingredients, and the pharmacological properties of American skullcap are not fully understood. The aims of this study were to characterize the chemical ingredients of American skullcap and to evaluate its anticonvulsant activity. Twelve phenolic compounds including 10 flavonoids and two phenylethanoid glycosides were isolated and identified from American skullcap and used as marker compounds. An HPLC analytic method for analyzing these marker compounds in commercial American skullcap products from different sources was established and validated. The anticonvulsant activity of American skullcap was determined in rat models of acute seizures induced by pilocarpine and pentylenetetrazol. The results from this study indicate that (1) phenolic compounds, especially flavonoids, are the predominant constituents in American skullcap; (2) American skullcap products have similar constituents, but the content and relative proportions of the individual constituents varies widely; and (3) American skullcap has anticonvulsant activity in rodent models of acute seizures.     

黄芩醇提物对副溶血性弧菌抑制机制的研究

从4种常见中药中筛选出对副溶血性弧菌有较好抑制作用的中药,并对其抑制机制进行研究。采用牛津杯法及液体倍比稀释法研究了中药醇提取物对副溶血性弧菌的敏感性;通过生长曲线和杀菌曲线、电镜观察以及SDS-PAGE等方法探讨黄芩醇提物的抑菌作用及机理。结果显示,4种中药醇提物对副溶血性弧菌表现出不同的抑菌活性,其中黄芩醇提物对副溶血性弧菌的抑制作用最强,抑菌圈为22.00±1.00 mm,MIC和MBC均为3.91 mg/m L。经MIC浓度处理后,细菌经过较长一段时间的延迟期,也能进入正常的对数期和稳定期;菌体表面变粗糙,出现裂解,菌液的蛋白含量明显升高,但是菌体的可溶性蛋白变化较小。结果表明,黄芩醇提物对副溶血性弧菌有很强的抑制效果,其作用是破坏细胞膜的完整性导致菌体裂解死亡。     

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。     

野生黄芩内生真菌的分离鉴定及抗菌活性筛选

从野生黄芩的根、茎、叶中分离出97株内生真菌, 其中33株来自生长期植株, 64株来自成熟期植株。经分类鉴定, 隶属于38个属, 优势属为Alternaria, 成熟期的属种多样性高于生长期。将其中的95株内生真菌对14种指示菌进行抑菌试验, 发现其中91株内生真菌对一种或多种指示菌具有抑菌活性, 占分离菌株总数的95.8%。野生黄芩内生真菌对大肠杆菌CGMCC1.1103、枯草芽孢杆菌CGMCC1.769、蜡样芽孢杆菌ATCC1361、致病性大肠埃希氏菌ATCC49105、白色假丝酵母ATCC10231、小孢拟盘多毛孢CNUMB2PM01等6个指示菌菌株抑菌效果较好; 对单核细胞增生李斯特菌ATCC27708、普通变形杆菌ATCC33420、肠炎沙门氏菌ATCC14208、副溶血型弧菌ATCC27519、宋内氏痢疾杆菌ATCC51060、九州镰孢霉CNUMB2FK01等6个指示菌菌株抑菌效果较差; 对深红酵母CGMCC2.282和金黄色葡萄球菌ATCC12600没有抑制作用。     

Antioxidative and anti-inflammatory activities of polyhydroxyflavonoids of Scutellaria baicalensis GEORGI

The active ingredients of 'golden root' of Scutellaria baicalensis GEORGI (Huang-Qin), a valuable traditional Chinese medicine, are polyhydroxyflavonoids, namely baicalein, oroxylin A and wogonin. With the objective of overcoming their poor solubility and to investigate their structure and activity relationships, baicaleinyl 7-O-sulfate was prepared, and extensive comparative antioxidative and anti-inflammatory tests were conducted. All the polyhydroxyflavonoids exhibited significant antioxidative and free-radical scavenging activities. In respect of their nitric oxide (NO) inhibition, wogonin was superior to all the other flavonoids, while oroxylin A was most potent in the inhibition of lipid peroxidation. Wogonin proved to be the most potent (82.9% inhibition, p<0.05) in its anti-inflammatory activity against carrageenan-induced rat hind paw edema. There was a correlation between the in-vivo anti-inflammatory activity and the in-vitro antioxidative activities.     

In vitro production and identification of autotetraploids of Scutellaria baicalensis

The roots of Scutellaria baicalensis are a major traditional Chinese medicine. We report research on induction, characteristics and chemical analysis of polyploid plants of S. baicalensis. Immersing calluses in 0.2% colchicine solution for 12 h prior to culture induced a high number of tetraploid plants. The induction rate reached as high as 40% of treated calluses. More than 50 lines of tetraploid plants were obtained. All tetraploid plants showed typical polyploidy characteristics. Twenty selected tetraploid lines were transferred to the field for determination of morphological characteristics and for chemical assays. Seven elite lines have been selected for further selection and breeding into new varieties for commercial production.     

黄芩种质资源ISSR遗传多样性的分析及评价

采用ISSR分子标记技术对6个野生或栽培居群共147份黄芩种质进行遗传多样性分析和评价。分析结果表明,51个ISSR引物中筛选出18条扩增条带清晰、重复性好和多态性高的引物,共扩增出485条清晰的条带,其中466条具有多态性,平均多态性位点比率为96.08%,平均Nei’s基因多样性指数和Shannon’s信息指数分别为0.244 4和0.388 9,等位基因数（Na）和有效等位基因数（Ne）分别为1.993 8和1.383 9,遗传分化指数G_st=0.122 3,遗传一致度（I）和遗传距离（D）分别为0.951 5和0.050 1,说明收集的黄芩种质资源在总体上具有较高的遗传多样性,不同居群间存在一定的遗传分化和基因交流,遗传变异主要存在于居群内。分子聚类结果表明,同一地区的种质并没有按照收集来源完全聚类,可能与种质不同起源或民间栽培引种有关。在DNA分子水平揭示黄芩种质资源的遗传多样性水平,将为进一步黄芩种质资源评价、保存和新品种选育等利用提供依据。     

Flavones from Scutellaria baicalensis Georgi attenuate apoptosis and protein oxidation in neuronal cell lines

The oxidative modification of proteins plays a major role in a number of human diseases including Alzheimer's disease (AD). Flavones in extracts of Scutellaria baicalensis (SbE) have been reported to have exceptional antioxidant properties. We examined the effects of SbE on neuronal cells exposed to oxidative stress. Neuronal HT-22 cells were exposed to low levels of H(2)O(2) generated from glucose oxidase (GO) under conditions that caused cell death in 24 h. The mechanism of cell death was shown to occur via apoptosis. Flavone extracts (50 microg/ml) protected cells and increased viability to 85+/-5% (P<0.001). The flavones also increased the content of Bcl-2 in the cell, resulted in its phosphorylation, and in contrast decreased the Bax levels. Furthermore, the oxidative-stress-induced protein carbonyl formation was reduced nearly two-fold when cells were pretreated with the flavone extract. Two-dimensional electrophoresis (2-DE) showed that less than 15% of the total visible proteins were oxidized and that the oxidation was specific for certain oxidation-sensitive proteins. These data support the idea that flavones in SbE can attenuate oxidant stress and protect cells from lethal oxidant damage.     

Isolation and characterization of a co-producer of fengycins and surfactins, endophytic Bacillus amyloliquefaciens ES-2, from Scutellaria baicalensis Georgi

An endophytic bacterium was isolated from Chinese medicinal plant Scutellaria baicalensis Georgi. The phylogenetic and physiological characzterization indicated that the isolate, strain ES-2, was Bacillus amyloliquefaciens, which produced two families of secondary metabolites with broad-spectrum antibacterial and antifungal activities. Culture filtrate of ES-2 displayed antagonism against some phytopathogenic, food-borne pathogenic and spoilage bacteria and fungi owing to the existence of antimicrobial compounds. A HPLC-MS analysis showed two series of ion peaks from the culture filtrate. A further electrospray ionization/collision-induced dissociation spectrum revealed that the two series ion peaks represented different fengycin homologues and surfactin homologues, respectively, which had a potential for food preservation and the control of several fungal plant diseases.