A metabolomic analysis of medicinal diversity in Huang-qin (<Emphasis Type="Italic">Scutellaria baicalensis</Emphasis> Georgi) genotypes: discovery of novel compounds

In vitro manipulation of plant regeneration in the Chinese medicinal species Scutellaria baicalensis Georgi (Huang-qin) resulted in 26 chemically distinct germplasm lines. Antioxidant potential, growth rate and concentration of baicalin, baicalein, melatonin, and wogonin were the selective markers used to identify elite lines. Metabolomic analysis of a subset of the most distinct lines revealed that Huang-qin extracts contained over 2,000 compounds including 781 determined to be of putative medicinal importance as determined by a database search, as well as previously unidentified amino-derivatives of baicalin and wogonin. Huang-qin also contained a metabolite with the same net formula as hyperforin, previously thought to be unique to Hypericum perforatum L. Together these results provide new insights into the biochemical complexity of an important medicinal species and demonstrate the power of in vitro manipulation in combination with untargeted metabolomic screening for the production of new germplasm.     

Induced accumulation of triterpenoids in Scutellaria baicalensis suspension cultures using a yeast elicitor

When growth-phase cell suspension cultures of Scutellaria baicalensis were treated with 50 g of yeast elicitor preparation ml^–1, both oleanolic acid and ursolic acid transiently increased in the culture medium rather than in the cells. The maximal triterpenoid concentration was 13.7 mg l^–1 media approx. 35 h after treatment, whereas the maximum concentration was 2.1 mg l^–1 media after about 20 h following treatment with methyl jasmonate. Elicitor treatment also doubled phospholipase A₂ activity (25 pmol mg^–1 min) and the simultaneous treatment of aristolochic acid, a phospholipase A₂ inhibitor, inhibited triterpenoids accumulation as well as phospholipase A₂ activity.     

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。     

黄芩及其成分抗流感病毒感染的实验研究进展

黄芩性味苦寒,可清热燥湿、泻火解毒,历代医籍中记载了黄芩及其配伍方剂治疗肺疾病的功效,现代临床治疗流感的方剂和制剂中黄芩也是常用的一味中药,。黄芩的主要有效成分为黄芩苷、黄芩素和汉黄芩素等,现代研究表明黄芩及其成分对多种流感病毒感染的肺炎动物模型确有效果,其作用机制包括直接抗病毒作用和调节免疫炎症反应两方面。黄芩及其成分通过阻止病毒吸附、抑制病毒基因复制和蛋白合成、抑制神经氨酸酶活性等起到直接的抗病毒作用;通过调节促炎性细胞因子的合成和释放、抑制炎症介质、抗氧化、清除自由基等作用,抑制免疫炎症反应,减轻病理损伤。在调节免疫炎症反应方面的研究总体还偏少,研究也不够系统、深入。今后应加强在调节免疫炎症机制方面的研究,突出中药的特点,阐明黄芩及其成分的药效机制,最终开发出抗流感病毒制剂。     

Identification and production of flavonoids in a cell suspension culture of Scutellaria baicalensis G.

A high yielding cell line of Scutellaria baicalensis G. has successfully been developed to produce flavonoids. Major components of the flavonoids were identified as baicalin and wogonin-7-O-glucuronic acid by a series of instrumental analyses using UV, IR, MASS, and NMR. After 12 days in suspension culture, the cell growth reached 14 g ^DW/l, and baicalin and wogonin-7-O-glucuronic acid were obtained in concentrations of 2.9 g/l and 1.07 g/l, respectively. The culture temperature was found to be an important parameter for improving production yield of the flavonoids. The yield of baicalin was observed to increase to 4.2 g/l by shifting the temperature from 30 °C to 25 °C after 72 h of suspension culture.Abbreviations DW cell dry weight - FW cell fresh weight - 2,4-D 2,4-dichlorophenoxyacetic acid - PSH medium phytohormone added Schenk and Hildebrandt medium - FPM a modified Schenk and Hildebrandt medium for flavonoid production     

Baicalin prevents Candida albicans infections via increasing its apoptosis rate

Background

These experiments were employed to explore the mechanisms underlying baicalin action on Candida albicans.

Methodology and principal findings

We detected the baicalin inhibition effects on three isotope-labeled precursors of ³H-UdR, ³H-TdR and ³H-leucine incorporation into C. albicans using the isotope incorporation technology. The activities of Succinate Dehydrogenase (SDH), cytochrome oxidase (CCO) and Ca²⁺–Mg²⁺ ATPase, cytosolic Ca²⁺ concentration, the cell cycle and apoptosis, as well as the ultrastructure of C.albicans were also tested. We found that baicalin inhibited ³H-UdR, ³H-TdR and ³H-leucine incorporation into C.albicans (P < 0.005). The activities of the SDH and Ca²⁺–Mg²⁺ ATPase of C.albicans in baicalin groups were lower than those in control group (P < 0.05). Ca²⁺ concentrations of C. albicans in baicalin groups were much higher than those in control group (P < 0.05). The ratio of C.albicans at the G0/G1 stage increased in baicalin groups in dose dependent manner (P < 0.01). There were a significant differences in the apoptosis rate of C.albicans between baicalin and control groups (P < 0.01). After 12–48 h incubation with baicalin (1 mg/ml), C. albicans shown to be markedly damaged under transmission electron micrographs.

Innovation and significance

Baicalin can increase the apoptosis rate of C. albicans. These effects of Baicalin may involved in its inhibiting the activities of the SDH and Ca²⁺–Mg²⁺ ATPase, increasing cytosolic Ca²⁺ content and damaging the ultrastructure of C. albicans.     

neo-Clerodane diterpenoids from Scutellaria barbata with cytotoxic activities

Three neo-clerodane diterpenoids, named barbatins A-C (1-3), and the neo-clerodane diterpenoid nicotinyl ester, named scutebarbatine B (4), were isolated from the whole plant of Scutellaria barbata D. Don. Their structures were elucidated by spectroscopic analyses (UV, IR, HRFAB-MS, 1D NMR and 2D NMR). In vitro, compounds 1-4 showed significant cytotoxic activities against three human cancer lines, namely, HONE-1 nasopharyngeal, KB oral epidermoid carcinoma, and HT29 colorectal carcinoma cells, with IC50 values in the range 3.5-8.1 microM.     

Flavonoid production in Scutellaria baicalensis callus cultures

St-20 and St-7 lines were isolated from the stem callus of Scutellaria baicalensis Georgi on indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid media, respectively. The flavonoid content of St-20 line was superior to that of St-7 line. The growth and flavonoid (baicalin, baicalein, wogonin and wogonin-7-0-glucuronide) content in St-20 line were best on Linsmaier-Skoog's basal medium containing 10^-7 M–10^-5 M kinetin. St-20 line showed the same flavonoid content and pattern as the root of parent plant after the culture period of 70 days.     

Chemical composition and antimicrobial activity of the essential oil of Scutellaria barbata

The essential oil of Scutellaria barbata was obtained by hydrodistillation with a 0.3% (v/w) yield and analysed by GC and GC-MS. The main compounds in the oil were hexahydrofarnesylacetone (11.0%), 3,7,11,15-tetramethyl-2-hexadecen-1-ol (7.8%), menthol (7.7%) and 1-octen-3-ol (7.1%). The antimicrobial activity of the oil was evaluated against 17 microorganisms using disc diffusion and broth microdilution methods. The gram-positive bacteria, including methicillin-resistant Staphlococcus aureus, were more sensitive to the oil than gram-negative bacteria and yeasts.     

黄芩的生物学研究进展

药用黄芩为唇形科(Labiatae)黄芩属(Scutellaria)多年生草本植物黄芩(Scutellaria baicalensisGeorgi)的干燥根,是我国大宗中药材之一,药用历史悠久。我们对其原植物黄芩的种质资源、形态结构、生长发育、繁殖方式等方面的研究进展,以及黄芩药材的解剖学研究和显微鉴定等进行了系统考察和总结,可为深入进行原植物黄芩的生物学研究及药用黄芩的真伪鉴别和提高药用部分的质量和产量提供依据和资料。     

Induction of apoptosis in densovirus infected Aedes aegypti mosquitoes

The mechanism of death in densovirus infected mosquitoes remains unexplored. This study investigated the cellular consequences of densovirus infection in Aedes aegypti mosquitoes after a second generation challenge with a densovirus isolated from adult Aedes albopictus mosquitoes in Thailand (AThDNV). Specimens were analyzed by TUNEL assay, fluorescent in situ hybridization (FISH) and a calorimic assay to detect activation of caspase 3-like activity. After challenge, moribund mosquitoes showed considerable evidence of TUNEL positive cells. The caspase 3-like activity assay showed that the presence of TUNEL positive cells was associated with increased levels of activated caspase 3-like activity in AThDNV infected mosquitoes.     

Leishmania infection inhibits cycloheximide-induced macrophage apoptosis in a strain-dependent manner

Activation of apoptosis is one of the most ancient mechanisms to eliminate intracellular infections; the capacity to subvert this programed cell death provides an adaptive advantage to pathogens that persist in an intracellular environment. Leishmania species are obligate intracellular parasites that primarily reside within host macrophages. We demonstrate here that Leishmania infection protects macrophages from cycloheximide-induced apoptosis in a species and strain specific manner. Our data further reveal that Leishmania phosphoglycans and direct contact between parasites and host cells are required for the inhibitory phenotype.     

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。     

Cloning and expression of UDP-glucose: flavonoid 7-O-glucosyltransferase from hairy root cultures of Scutellaria baicalensis

 A cDNA encoding UDP-glucose: baicalein 7-O-glucosyltransferase (UBGT) was isolated from a cDNA library from hairy root cultures of Scutellaria baicalensis Georgi probed with a partial-length cDNA clone of a UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) from grape (Vitis vinifera L.). The heterologous probe contained a glucosyltransferase consensus amino acid sequence which was also present in the Scutellaria cDNA clones. The complete nucleotide sequence of the 1688-bp cDNA insert was determined and the deduced amino acid sequences are presented. The nucleotide sequence analysis of UBGT revealed an open reading frame encoding a polypeptide of 476 amino acids with a calculated molecular mass of 53 094 Da. The reaction product for baicalein and UDP-glucose catalyzed by recombinant UBGT in Escherichia coli was identified as authentic baicalein 7-O-glucoside using high-performance liquid chromatography and proton nuclear magnetic resonance spectroscopy. The enzyme activities of recombinant UBGT expressed in  E. coli were also detected towards flavonoids such as baicalein, wogonin, apigenin, scutellarein, 7,4′-dihydroxyflavone and kaempferol, and phenolic compounds. The accumulation of UBGT mRNA in hairy roots was in response to wounding or salicylic acid treatments. Received: 8 September 1999 / Accepted: 4 October 1999     

The deubiquitinase emperor's thumb is a regulator of apoptosis in Drosophila

We have characterized the gene emperor's thumb (et) and showed that it is required for the regulation of apoptosis in Drosophila. Loss-of-function mutations in et result in apoptosis associated with a decrease in the concentration of DIAP1. Overexpression of one form of et inhibits apoptosis, consistent with et having an anti-apoptotic function; however, overexpression of a second form of et induces apoptosis, indicating that the two forms of et may have competing functions. et encodes a protein deubiquitinase, suggesting it regulates apoptosis by controlling the stability of apoptotic regulatory proteins.     

Angiostrongylus cantonensis: apoptosis of inflammatory cells induced by treatment with mebendazole or/and interleukin 12 in mice

Angiostrongylus cantonensis is the major cause of human eosinophilic meningoencephalitis. ICR mice were infected orally with 35 infective larvae and sacrificed at 4-14 days, 25 days or 32 days post infection (dpi) for pathological and immunocytochemical examinations. In the non-treated group, no apoptosis signal was found in the meninges or parenchyma of the brains (4-14 dpi). Only a few apoptotic cells were noticed at 25 dpi (3%) and 32 dpi (10%). In the groups, the animals were given a single dose of mebendazole (20 mg/kg, per os at various times) or injections of interleukin 12 (IL-12) (10 ng/daily, intraperitoneally), all the animals were sacrificed at 14 dpi; the number of apoptotic cells was increased (17-21%). In the group that received a single dose of mebendazole (4 dpi) in combination with IL-12 injections (4-13 dpi), mild meningitis was observed, and most of the infiltrated inflammatory cells were in the apoptotic program (55%). Taken together, apoptosis of the inflammatory cells (most were eosinophils) could be induced when the infected mice were treated with mebendazole or/and IL-12.     

Disruption of Smad5 gene induces mitochondria-dependent apoptosis in cardiomyocytes

Our previous studies have shown that SMAD5, an important intracellular mediator of transforming growth factor beta (TGF-beta) family, is required for normal development of the cardiovascular system in vivo. In the current study, we reported that the lack of the Smad5 gene resulted in apoptosis of cardiac myocytes in vivo. To further investigate the mechanism of the Smad5 gene in cardiomyocyte apoptosis, the embryonic stem (ES) cell differentiation system was employed. We found that the myotubes that differentiated from the homozygous Smad5ex6/ex6 mutant ES cells underwent collapse and degeneration during the late stages of in vitro differentiation, mimicking the in vivo observation. By electron microscopy, abnormal swollen mitochondria were observed in cardiomyocytes both from Smad5-deficient embryos and from ES-differentiated cells. There was also a significant reduction in mitochondrial membrane potential (Deltapsi m) and a leakage of cytochrome c from mitochondria into the cytosol of myocytes differentiated from Smad5 mutant ES cells. The expression of p53 and p21 was found to be elevated in the differentiated Smad5 mutant myocytes, and this was accompanied by an up-regulation in caspase 3 expression. These results suggest that the Smad5-mediated TGF-beta signals may protect cardiomyocytes from apoptosis by maintaining the integrity of the mitochondria, probably through suppression of p53 mediated pathways.     

Gamma-glutamyltranspeptidase of Helicobacter pylori induces mitochondria-mediated apoptosis in AGS cells

Gamma-glutamyltranspeptidase (GGT) is a novel protein involved in the induction of Helicobacter pylori-mediated apoptosis; however, the signal pathway involved in GGT-induced apoptosis remains unclear. Using DNA recombination techniques, ggt was cloned into pET117b and transformed into Escherichia coli. Recombinant GGT was purified using nickel-affinity resin and was digested by thrombin. Recombinant GGT induced apoptosis in AGS cells in a time-dependent manner, which was confirmed by TUNEL staining, the MTT assay and immunoblot analysis for caspases-9, -3, Bax, Bcl-2, Bcl-xL and cytochrome c release. Activation of caspase-3 and -9 following exposure to GGT increased in a time-dependent manner and upregulation of proapoptotic Bax and a downregulation of antiapoptotic Bcl-2 and Bcl-xL was detected. Apoptotic signals also trigger changes in mitochondria, which lead to a release of cytochrome c into the cytosolic space. The GGT-deficient mutant was not as able to induce apoptosis as the wild-type strain. These results indicate that GGT of H. pylori induces apoptosis via a mitochondria-mediated pathway.     

不同产地黄芩中无机元素含量及其与根际土壤无机元素的关系

药用植物中各无机元素含量的不仅影响药用植物的生长发育,也是药材有效成分的构成因子。通过对全国范围内16个不同产地(即居群)的92个野生黄芩(Scutellaria baicalensis Georgi)样本及其相应的根际土壤中10种无机元素含量的分析,发现不同产地黄芩及其根际土壤无机元素都有很大变异,且不同产地黄芩根际土壤中无机元素的变异远大于黄芩药材中无机元素的变异。总体来看,黄芩中Mg(9级)含量较其他植物含量高;P(1级)、K(2级)、Mn(3级)含量与其他植物相比处处较低水平;黄芩对Sr(富集系数达到3.52)有较强富集。并且通过无机元素分布曲线分析建立了无机元素指纹谱,主成分分析筛选出黄芩主要特征无机元素为Mg、K、Ca、Fe、Zn。本研究还表明,黄芩对各元素的吸收能力受产地的影响较大,提示黄芩对无机元素的吸收与各产地根际土壤无机元素有一定关联性。     

黄芩细胞生长特性及次生代谢产物生产性能的研究

采用4种不同激素组合的MS培养液对黄芩细胞进行悬浮培养．结果表明。在各培养液中黄芩细胞的生长曲线均呈S形。培养周期仅为9d;在M2[MS 2．4-D(0．5mg／L) 6-BA(1．0mg／L)]培养液中,黄芩细胞的总黄酮积累速率、硝酸还原酶活性及过氧化氢酶活性均高于其它处理。是进行黄芩细胞悬浮培养生产总黄酮的最佳培养基配方。