Liquid chromatography with tandem mass spectrometry for the simultaneous determination of baicalein, baicalin, oroxylin A and wogonin in rat plasma

A rapid, sensitive and selective liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the determination of baicalein, baicalin, oroxylin A and wogonin, Scutellaria baicalensis active components in rat plasma was developed. After liquid-liquid extraction with 2-(3,4-dimethoxy-phenyl)-5,7-dihydroxy-chromen-4-one as internal standard, baicalein, baicalin, oroxylin A and wogonin were eluted from an Atlantis C(18) column within 7 min with isocratic mobile phase consisting of methanol and 0.1% formic acid (60:40, v/v). The analytes were detected using an electrospray ionization tandem mass spectrometry in the multiple reaction monitoring (MRM) mode. The standard curves were linear (r=1.000) over the concentration ranges of 5-500 ng/ml for baicalein, wogonin and oroxylin A and 5-5000 ng/ml for baicalin. The coefficients of variation and relative errors of baicalein, wogonin, oroxylin A and baicalin for intra- and inter-assay at three or four quality control (QC) levels were 0.8-6.1% and -4.0 to 5.8%, respectively. The lower limits of quantification for baicalein, wogonin, oroxylin A and baicalin were 5ng/ml using 50 microl of plasma sample. This method was successfully applied to the pharmacokinetic study of baicalein, baicalin, wogonin and oroxylin A after an intravenous administration of Scutellariae radix extract to male Sprague-Dawley rats.     

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.     

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.     

Deep Sequencing of the Scutellaria baicalensis Georgi Transcriptome Reveals Flavonoid Biosynthetic Profiling and Organ-Specific Gene Expression

Scutellaria baicalensis Georgi has long been used in traditional medicine to treat various such widely varying diseases and has been listed in the Chinese Pharmacopeia, the Japanese Pharmacopeia, the Korean Pharmacopoeia and the European Pharmacopoeia. Flavonoids, especially wogonin, wogonoside, baicalin, and baicalein, are its main functional ingredients with various pharmacological activities. Although pharmaological studies for these flavonoid components have been well conducted, the molecular mechanism of their biosynthesis remains unclear in S. baicalensis. In this study, Illumina/Solexa deep sequencing generated more than 91 million paired-end reads and 49,507 unigenes from S. baicalensis roots, stems, leaves and flowers. More than 70% unigenes were annotated in at least one of the five public databases and 13,627 unigenes were assigned to 3,810 KEGG genes involved in 579 different pathways. 54 unigenes that encode 12 key enzymes involved in the pathway of flavonoid biosynthesis were discovered. One baicalinase and three baicalein 7-O-glucuronosyltransferases genes potentially involved in the transformation between baicalin/wogonoside and baicalein/wogonin were identified. Four candidate 6-hydroxylase genes for the formation of baicalin/baicalein and one candidate 8-O-methyltransferase gene for the biosynthesis of wogonoside/wogonin were also recognized. Our results further support the conclusion that, in S. baicalensis, 3,5,7-trihydroxyflavone was the precursor of the four above compounds. Then, the differential expression models and simple sequence repeats associated with these genes were carefully analyzed. All of these results not only enrich the gene resource but also benefit research into the molecular genetics and functional genomics in S. baicalensis.     

Selective fraction of Scutellaria baicalensis and its chemopreventive effects on MCF-7 human breast cancer cells

Based on our previous observation, the whole Scutellaria baicalensis extract (SbE) did not show significant breast cancer cell inhibitory effect. In this study, we isolated a baicalin-deprived-fraction (SbF1) of Scutellaria baicalensis, and baicalin-fraction (SbF3), and evaluated their anti-breast cancer properties using MCF-7 cells. The content of four flavonoids in extract/fractions were determined using high performance liquid chromatography. Analytical data showed that in SbF1, the major constituents are baicalein and wogonin, while SbF3 only contains baicalin. The antiproliferative effects of fractions and SbE were assayed using modified trichrome stain method. SbF1 showed significant antiproliferative effect. Treated with 100 μg/ml of SbF1 for 72 h inhibited MCF-7 cell growth by 81.6%, while in the same treatment concentration, SbF3 increased cell growth by 22.6%. SbF1 was recognized as an active fraction of SbE. The effects of four flavonoids in SbE, scutellarin, baicalin, baicalein and wogonin, were determined, and data showed that baicalein and wogonin significantly inhibited MCF-7 cell growth. In contrast, in certain concentrations, scutellarin and baicalin increased cancer cell growth. The effects of SbF1 on cell cycle and apoptosis were assayed using flow cytometry. SbF1 arrested MCF-7 cells in S- and G2/M-phases, and significantly increased induction of cell apoptosis. These combined phytochemical and biological data provide evidence for further chemopreventive studies of the baicalin-deprived SbE on breast cancer.     

赤霉素GA3调节黄芩组织培养中芽和根的形成

应用组织培养技术对黄芩进行外源激素调控研究。在培养不同时间进行的不同培养基之间的转移培养研究表明,组织培养条件下,在培养基中添加赤霉素,可显著刺激黄芩外植体芽的形成,同时抑制根的生长。在加有GA3的IAA培养基上,GA3显著影响黄芩组织培养物中的黄酮含量。在黑暗条件下,开始在2.5μmol/LIAA培养基中培养6d,随后转移到5μmol/LGA3培养基上培养,黄芩外植体中黄岑苷、黄岑素和汉黄芩苷的含量最高,分别为14.90,2.70和0.54μgmg-1(干重)。     

A novel synthesis of 3-aryl coumarins and evaluation of their antioxidant and lipoxygenase inhibitory activity

A series of coumarin analogues bearing a substituted phenyl ring on position 3 were synthesized via a novel methodology, through an intermolecular condensation reaction of 2-hydroxyacetophenones and 2-hydroxybenzaldehyde, with imidazolyl phenylacetic acid active intermediates. The in vitro antioxidant activity of the synthesized compounds was evaluated using two different antioxidant assays (radical scavenging ability of DPPH stable free radical and inhibition of lipid peroxidation induced by the thermal free radical AAPH). Moreover, the ability of the compounds to inhibit soybean lipoxygenase was determined as an indication of potential anti-inflammatory activity.     

Molecular characterization of flavonoid biosynthetic genes and accumulation of baicalin,baicalein, and wogonin in plant and hairy root of Scutellaria lateriflora

Scutellaria lateriflora is well known for its medical applications because of the presence of flavanoids and alkaloids. The present study aimed to explore the molecular aspects and regulations of flavanoids. Five partial cDNAs encoding genes that are involved in the flavonoid biosynthetic pathway: phenylalanine ammonia lyase (SlPAL), cinnamate 4-hydroxylase (SlC4H), 4-coumaroyl CoA ligase (Sl4CL), chalcone synthase (SlCHS), and chalcone isomerase (SlCHI) were isolated from S. lateriflora. Organ expression analysis showed that these genes were expressed in all organs analyzed with the highest levels correlating with the richest accumulation of wogonin in the roots. Baicalin and baicalein differentially accumulated in S. lateriflora plants, with the highest concentration of baicalin and baicalein detected in the leaves and stems, respectively. Exogenous methyl jasmonate (MeJA) significantly enhanced the expression of SlCHS and SlCHI, and accumulation of baicalin (22.54 mg/g), baicalein (1.24 mg/g), and wogonin (5.39 mg/g) in S. lateriflora hairy roots. In addition, maximum production of baicalin, baicalein, and wogonin in hairy roots treated with MeJA was approximately 7.44-, 2.38-, and 2.12-fold, respectively. Light condition increased the expression level of SlCHS, the first committed step in flavonoid biosynthesis in hairy roots of S. lateriflora after 3 and 4 weeks of development compared to the dark condition. Dark-grown hairy roots contained a higher content of baicalin and baicalein than light-grown hairy roots, while light-grown hairy roots accumulated more wogonin than dark-grown hairy roots. These results may helpful for the metabolic engineering of flavonoids biosynthesis in S. lateriflora.     

一株发酵转化黄芩苷菌株的筛选及鉴定

为实现生物发酵法转化黄芩苷生产活性产物黄芩素,本研究从自然发毛的新鲜黄芩植株根部筛选发酵转化黄芩苷生成黄芩素的菌株,并进行产物分析和菌株鉴定。通过马铃薯葡萄糖培养液(PDB)富集,黄芩药粉培养液初筛,马铃薯葡萄糖琼脂(PDA)平板分离后转入PDB黄芩苷培养液复筛,高效液相色谱法定性及定量分析,得到一株能够高效转化黄芩苷为黄芩素的菌株RM3,该菌在添加0. 1%黄芩苷的PDB培养液中能够将黄芩苷转化为黄芩素,未经任何优化条件下,28℃,150 rpm培养5天后摩尔转化率达到83. 87%。通过对菌落的形态、显微结构观察及ITS序列分析比对,鉴定菌株RM3为青霉菌(Penicillium sp. RM3)。该菌转化黄芩苷生成黄芩素效率高,且清洁环保,可缓解中药资源紧张,满足市场和临床需求,是一株很有前途的黄芩素生产菌。     

Study on the chemical composition and antioxidant activity of extracts from shoot culture and regenerated plants of <Emphasis Type="Italic">Scutellaria altissima</Emphasis> L.

The flavonoid (baicalin, wogonoside, luteolin, luteolin-7-glucoside) and verbascoside contents of Scutellaria altissima in both shoot cultures, and the shoots and roots of micropropagated plants grown in the greenhouse for 12 weeks or in the field for 2 years were determined. The level of secondary metabolites was found to be strongly affected by the age and type of plant organ. A comparative analysis of S. altissima plants propagated in vitro and from seeds revealed no differences in the level of secondary metabolites when plants of the same age were studied. The antioxidant potential of methanolic extracts from shoot cultures, and the shoots and roots of S. altissima plants propagated in vitro, were evaluated using ABTS radical scavenging, FRAP metal reduction power and the lipid peroxidation test, in relation to the content of baicalin, wogonoside, verbascoside, total phenolic and total flavonoid compounds. Extracts from the roots of field-grown regenerated plants at the flowering stage were found to possess the strongest antioxidant activity. Correlation analysis revealed that the antioxidant activity of extracts correlated most closely with their total phenolic content estimated by the Folin-Ciocalteu method.     

Antioxidant activity of anti-inflammatory plant extracts

The antioxidant properties of twenty medical herbs used in the traditional Mediterranean and Chinese medicine were studied. Extracts from Forsythia suspensa, Helichrysum italicum, Scrophularia auriculata, Inula viscosa, Coptis chinensis, Poria cocos and Scutellaria baicalensis had previously shown anti-inflammatory activity in different experimental models. Using free radical-generating systems H. italicum. I. viscosa and F. suspensa protected against enzymatic and non-enzymatic lipid peroxidation in model membranes and also showed scavenging property on the superoxide radical. All extracts were assayed at a concentration of 100 microg/ml. Most of the extracts were weak scavengers of the hydroxyl radical and C. chinensis and P. cocos exhibited the highest scavenging activity. Although S. baicalensis inhibited the lipid peroxidation in rat liver microsomes and red blood cells, the extract showed inhibitory actions on aminopyrine N-demethylase and xanthine oxidase activities as well as an pro-oxidant effect observed in the Fe3+-EDTA-H2O2 system. The results of the present work suggest that the anti-inflammatory activities of the same extracts could be explained, at least in part, by their antioxidant properties.     

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。     

Comparative analysis of bioactive phytochemicals from Scutellaria baicalensis, Scutellaria lateriflora, Scutellaria racemosa, Scutellaria tomentosa and Scutellaria wrightii by LC-DAD-MS

Scutellaria is a geographically widespread and diverse genus of the Lamiaceae family of herbaceous plants commonly known as skullcaps. Scutellaria is used widely as an ethnobotanical herb for the treatment of various ailments ranging from cancers, cirrhosis, jaundice, hepatitis, anxiety and nervous disorders. We used (1) reverse-phase liquid chromatography coupled to a diode array detector (LC-DAD), and (2) multiple reaction monitoring (MRM) using mass spectrometry (LC-MS/MS) to quantify the levels of acteoside, scutellarin, scetellarein, baicalin, baicalein, wogonin, wogonoside, apigenin, chrysin, and oroxylin A in aqueous methanolic extracts of roots, shoots and leaves of S. baicalensis, S. lateriflora, S. racemosa, S. tomentosa and S. wrightii. Our results indicate that both methods (LC-DAD and LC-MS/MS) were robust for the detection of the 10 analytes from Scutellaria extracts although greater sensitivities were achieved using LC-MS/MS in MRM mode. MRM enabled the detection of low levels of analytes which were otherwise undetected using LC-DAD. The baicalin content of S. wrightii roots were 5-fold higher than the commonly used S. baicalensis. Additionally, we also showed that leaves of both S. wrightii and S. tomentosa are good sources of scutellarin compared to S. baicalensis. Our data clearly demonstrated that previously uncharacterized species, S. wrightii and S. tomentosa are both good sources of flavonoids, particularly scutellarin, baicalin, wogonin and baicalein.     

Effects of baicalein and wogonin on drug-metabolizing enzymes in C57BL/6J mice

Effects of baicalein and wogonin, the major flavonoids of Scutellariae radix, on cytochrome P450 (CYP), UDP-glucuronosyl transferase (UGT), and glutathione S-transferase (GST) were studied in C57BL/6J mice. One-week treatment of mice with a liquid diet containing 5 mM baicalein resulted in 29%, 14%, 36%, 28%, and 46% decreases of hepatic benzo(a)pyrene hydroxylation (AHH), benzphetamine N-demethylation (BDM), N-nitrosodimethylamine N-demethylation (NDM), nifedipine oxidation (NFO), and erythromycin N-demethylation (EMDM) activities, respectively. Treatment with a liquid diet containing 5 mM wogonin resulted in 43%, 22%, 21%, 24%, and 35% decreases of hepatic AHH, BDM, NDM, NFO, and EMDM activities, respectively. However, hepatic 7-methoxyresorufin O-demethylation (MROD) activity was increased and decreased by baicalein- and wogonin-treatments, respectively. Similar modulation was observed with caffeine 3-demethylation (CDM) activity. Immunoblot analysis showed that the levels of hepatic CYP2E1 and CYP3A proteins were decreased by both baicalein- and wogonin-treatments. Hepatic CYP1A2 protein level was increased by baicalein but decreased by wogonin. In extrahepatic tissues, renal AHH activity was decreased by wogonin whereas pulmonary AHH, 7-ethoxyresorufin O-deethylation (EROD), and MROD activities were increased by both flavonoids. Both baicalein and wogonin strongly increased CYP1A protein level in mouse lung. Hepatic and renal UGT activities toward p-nitrophenol were suppressed by baicalein- and wogonin-treatments. However, cytosolic GST activity was not affected by flavonoids. These results suggest that ingestion of baicalein or wogonin can modulate drug-metabolizing enzymes and the modulation shows tissue specificity.     

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).     

Protective effect of wogonin on endotoxin-induced lethal shock in D-galactosamine-sensitized mice.

The effects of wogonin, a major flavonoid from Scutellaria baicalensis Georgi, on lipopolysaccharide (LPS)-induced lethal shock in mice was investigated. Wogonin pretreatment prevented the lethal shock in mice injected with D-galactosamine (D-GalN) and LPS, but not in mice injected with a high dose of LPS. Wogonin definitely inhibited the hepatic injury in mice injected with D-GalN, and LPS and reduced the level of circulating tumor necrosis factor (TNF)-alpha. The reduction was more marked in mice injected with D-GalN and LPS compared with that in mice injected with a high dose of LPS. Wogonin pretreatment did not inhibit the lipid peroxidation in mice receiving either D-GalN and LPS or a high dose of LPS. Wogonin inhibited the in vitro production of TNF-alpha and nitric oxide in LPS-stimulated RAW 264.7 cells. The mechanism of the protective effect of wogonin on the lethal shock in mice injected with D-GalN and LPS is discussed.