Proteomic analysis of mouse liver for the evaluation of effects of Scutellariae radix by liquid chromatography with tandem mass spectrometry

Scutellariae radix or Scutellaria baicalensis is a medicinal plant that contains major flavonoids such as baicalein, baicalin, wogonin and wogonosides. The present work describes the development of an approach using proteomic analysis of mouse liver to study the effects of prolonged exposure to substances present in chemically standardized Scutellariae radix extracts. Histopathological examination of the mouse liver was compared with the proteome data. The botanical extracts were prepared using pressurized liquid extraction (PLE). A method without isotope labeling was developed, using proteolytic digestion with one- and two-dimensional liquid chromatography with tandem mass spectrometry, and was used to characterize the extent of differential protein expression in mouse liver in response to external factors such as extracts from Scutellariae radix. From the histopathological examination and proteome data, significant changes in the mouse livers were not observed for the low-dose group. The Scutellariae radix extracts at high dose were observed to cause damage at the bile duct and expression change of a number of proteins including some involved in catabolism of triglyceride-rich particles, carbohydrate metabolism, regulators of cell signaling processes, and enzymes involved in biotransformation. Thus, proteomic analysis of liver samples from mice treated with botanical extracts is a promising approach to provide information on any potential toxicity effects of the extracts. The present method also provides another means for comparing proteomes in biological samples such as liver lysates from mice subjected to different treatments.     

Determination of Baicalin in Traditional Chinese Preparation by High Performance Liquid Chromatography with Chemiluminescence Detection

Herbal medicine, a form of complementary and alternative medicine, is becoming increasingly popular in the world1. Scutellariae radix is the root of Scutellariabaica -lensis georgi. The primary active constituent includes baicalin as follows: Clinical studies showed that baicalin exhibited therapeutic functions of antifever, moistening aridity, anti-inflammatory and detoxifying 2 and it is also an anti-abortion agent as well as can scavenge free radicals and against oxidation3. So it is…     

Analysis of baicalein, baicalin and wogonin in Scutellariae radix and its preparation by microemulsion electrokinetic chromatography with 1-butyl-3-methylimizolium tetrafluoborate ionic liquid as additive

Microemulsion electrokinetic chromatography (MEEKC) using 1-butyl-3-methylimidazolium tetrafluoroborate (BMIM-BF4) ionic liquid (IL) as additive was developed for the analysis of baicalin, wogonin and baicalein in Scutellariae radix and its preparation. After conducting a series of optimizations, baseline separation was obtained for the analytes within 5min under the optimum conditions (sodium dodecyl sulfate (SDS) 0.88% (m/v) ethyl acetate 0.8% (v/v) butan-1-ol 0.2% (v/v) and the buffer composition were 25% acetonitrile (v/v), 7.5 mM BMIM-BF4 and 10 mM NaH2PO4, pH 8.2, applied voltage 17.5 kV and detection at 254 nm), the method has been successfully applied to the determination and quantification of the analytes in the extracts of S. radix (cooked), S. radix (raw) and Qingfeiyihuowan which was the preparation including S. radix.     

A chromatographic method for baicalin quantification in rat thalamus

A rapid reversed-phase high-performance liquid chromatographic (rp-HPLC) assay for the determination of baicalin in rat thalamus was developed. This was carried out on a Hypersil -C(18) column using 4-nitro-benzoic acid as the internal standard with a mobile phase of methanol-water-H(3)PO(4) (45:55:0.2, v/v/v). Detection was by UV at 277 nm. The calibration curve for baicalin was linear (r=0.9992) over the concentration range of 0.05--4.0 microg/mL and the limit of detection was 10 ng/mL. The coefficients of variation of intra- and inter-day assays were 2.64, 5.19 and 3.19% and 3.46, 6.21 and 5.58% at concentrations of 0.5, 1.0 and 3.0 microg/mL, respectively. The recoveries of baicalin from rat thalamus were 85.4+/- 5.62, 90.7+/- 2.43 and 89.1+/- 4.75% at concentrations of 0.5, 1.0 and 3.0 microg/mL, respectively. The method was applied to determine the time course of baicalin in rat thalamus, following a single dosage of intravenous administration of Scutellariae radix extract at 90 mg/kg of baicalin to male Wistar rats.     

Simple preparation of baicalin from Scutellariae Radix

A simple, rapid and reproducible method was developed to isolate high-purity baicalin from Scutellariae Radix, the dried roots of Scutellaria baicalensis Georgi. The method involves partition/recrystallization steps without repeated column chromatography or special instruments. Isolated baicalin was characterized by comparisons of TLC, HPLC, IR, MS, and NMR data with an authentic sample. Moreover, beta-glucuronidase hydrolysis of baicalin sequentially yielded glucuronic acid and baicalein as confirmed by co-TLC with authentic samples. The purity of baicalin was more than 97% with yield ca. 8.7% (w/w). The method presented here appears suitable for commercial application.     

Baicalin Represses Type Three Secretion System of Pseudomonas aeruginosa through PQS System

Therapeutics that target the virulence of pathogens rather than their viability offer a promising alternative for treating infectious diseases and circumventing antibiotic resistance. In this study, we searched for anti-virulence compounds against Pseudomonas aeruginosa from Chinese herbs and investigated baicalin from Scutellariae radix as such an active anti-virulence compound. The effect of baicalin on a range of important virulence factors in P. aeruginosa was assessed using luxCDABE-based reporters and by phenotypical assays. The molecular mechanism of the virulence inhibition by baicalin was investigated using genetic approaches. The impact of baicalin on P. aeruginosa pathogenicity was evaluated by both in vitro assays and in vivo animal models. The results show that baicalin diminished a plenty of important virulence factors in P. aeruginosa, including the Type III secretion system (T3SS). Baicalin treatment reduced the cellular toxicity of P. aeruginosa on the mammalian cells and attenuated in vivo pathogenicity in a Drosophila melanogaster infection model. In a rat pulmonary infection model, baicalin significantly reduced the severity of lung pathology and accelerated lung bacterial clearance. The PqsR of the Pseudomonas quinolone signal (PQS) system was found to be required for baicalin’s impact on T3SS. These findings indicate that baicalin is a promising therapeutic candidate for treating P. aeruginosa infections.     

Study of major flavonoids in crude Scutellariae Radix by micellar electrokinetic capillary chromatography

A method of micellar electrokinetic capillary electrophoresis for determining the six flavonoids in Scutellariae Radix (i.e., baicalin, baicalein, wogonin 7-O-glucuronide, wogonin, oroxylin A 7-O-glucuronide, and oroxylin A) has been developed. The buffer solution (pH 7.24) composed of 20 mM sodium dodecyl sulfate (SDS), 20 mM sodium dihydrogen phosphate, and 25 mM sodium borate was found to be the most suitable electrolyte for the separation. The contents of the six flavonoids in crude Scutellariae Radix could easily be determined within about 15 min. On-column UV (254 nm) monitoring allowed the quantitative determination of baicalin. The effects of pH, surfactant concentration, and applied voltage on the migration behavior of the solutes were studied.     

Evaluation of phenyldimethylethoxysilane treated high-performance thin-layer chromatographic plates. Application to analysis of flavonoids inScutellariae radix

Summary The retention and selectivity of flavonoids (baicalin, baicalein, wogonin, oroxylin A) inScutellariae radix have been studies by high-performance thin-layer chromatography on phenyldimethylethoxysilane-treated silica plates. The silica plates treated with phenyl groups were used for physical and chemical analysis. From elemental carbon analysis, the maximum number of bonded phenyl surface groups per gram was calculated to be 0.467×10²¹ (Oginal silica plate: Merck Art. 15109, Silica gel 100 F₂₅₄). With methanol-1/15 M phosphate buffer (pH 6.2) mixtures as mobile phase, baicalin, baicalein, wogonin, and oroxylin A inScutellariae radix were separated. It has been shown that phenyl-treated plates are more suitable for selective separation of baicalin, baicalein, wogonin, and oroxylin A than octadecyl-treated plates.     

Differential distribution of phytochemicals in Scutellariae Radix and Scutellariae Amoenae Radix using microscopic mass spectrometry imaging

Scutellariae Radix, the root of Scutellaria baicalensis Georgi, is widely applied in China for the treatment of fever, ulcer, bronchitis, hepatitis and inflammatory symptoms. Sctuellariae Amoenae Radix, the root of Scutellaria amoena C.H. Wright, is often prescribed as the substitute for Scutellariae Radix. Up to now, no attempt has been made to unveil and compare the localization of phytochemicals in Scutellariae Radix and its succedaneum. This investigation succeeded to look into the differential distribution of natural compounds in Scutellariae Radix and Scutellariae Amoenae Radix using microscopic mass spectrometry imaging. Compounds presenting different distribution modes in two kinds of roots were sorted out, then a quick method for the differentiation between Scutellariae Radix and Scutellariae Amoenae Radix was established. Accumulation sites of baicalein, baicalin, wogonin and wogonoside in Scutellariae Radix were also uncovered using microscopic mass spectrometry imaging. Moreover, the application of matrix assisted laser desorption ionization-quadrupole-time of flight mass spectrometry allowed the on-tissue dissociation of major flavonoids. Overall, the utilization of microscopic mass spectrometry imaging and matrix assisted laser desorption ionization-quadrupole-time of flight mass spectrometry provided a novel perspective for the discovery of natural compounds within medicinal plants.     

黄芩中黄芩苷的亚临界水提取及高效液相色谱分析

建立了黄芩药材中黄芩苷的亚临界水提取 高效液相色谱测定方法。分别考察了温度、压力、提取时间、提取物颗粒度、溶剂比等因素对提取量的影响,并与有机溶剂提取法比较。结果表明,当两者具有相同的提取效果时,亚临界水提取法的提取时间及提取溶剂的消耗量大大减少,避免了使用有机溶剂造成的污染。黄芩苷提取最佳条件为:样品颗粒度80～100目,溶剂比0.2 mL/mg ,5 MPa,130 ℃保持10 min。并通过提取 高效液相色谱联机分析实现了对提取物的实时监测。该技术有望成为从中药材中提取脂溶性成分的有效方法。     

The overall quality control of Radix scutellariae by capillary electrophoresis fingerprint

The qualitative and quantitative analyses are developed for the overall quality control of Radix Scutellariae, a Chinese herb, by capillary electrophoresis fingerprint (CEFP). All experiments are carried out in an uncoated fused silica capillary (75 cm x 75-microm i.d., effective length 63 cm) with a 50 mmol/L sodium borate solution (containing 5% acetonitrile, pH 9.30) under 12 kV. The detection wavelength is set at 280 nm, and naproxen is selected as the referential peak. Some parameters are used to evaluate the similarities of CEFPs. Eight co-eluting peaks are selected as the fingerprint peaks of Radix Scutellariae. The similarities between each of the ten samples and the referential CEFP of Radix Scutellariae are evaluated both qualitatively and quantitatively. Determination results indicate that the baicalin contents from different locations are varied. The methods of CEFP and quantitative analysis are rapid, simple, and accurate, with good repeatability, and can be used for the quality control of Radix Scutellariae.     

Simultaneous determination of the bioactive components in rat plasma by UPLC‐MS/MS and application in pharmacokinetic studies after oral administration of radix Scutellariae extract

A highly sensitive and rapid ultraperformance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method has been developed and validated for simultaneous quantification of the four main bioactive compounds, i.e. baicalin, baicalein, wogonoside and wogonin, in rat plasma after oral administration of Radix Scutellariae extract. Clarithromycin was used as an internal standard (IS). Plasma samples were processed by protein precipitation with methanol. The separation was performed on an Acquity BEH C₁₈ column (100 × 2.1 mm, 1.7 μm) at a flow rate of 0.4 mL/min, using 0.1% formic acid–acetonitrile as mobile phase. The MS/MS ion transit ions monitored were 447.5 → 270.1 for baicalin, 270.1 → 168.1 for baicalein, 461.2 → 284.0 for wogonoside, 284.2 → 168.1 for wogonin and 748.5 → 158.1 for IS. Method validation was performed according to US Food and Drug Administration guidelines and the results met the acceptance criteria. The lower limit of quantification (LLOQ) achieved was 1.13 ng/mL for baicalin, 1.23 ng/mL for baicalein, 0.82 ng/mL for wogonoside and 0.36 ng/mL for wogonin. The calibration curves obtained were linear (r > 0.99) over the concentration range ~ 1–1000 ng/mL. The intra‐ and inter‐day precision was <15% and the accuracy was within ±14.7%. After validation, this method was successfully applied to a pharmacokinetic study of Radix Scutellariae extract.     

Determination of paeoniflorin, ferulic acid and baicalin in the traditional Chinese medicinal preparation Dang-Guei-San by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of paeoniflorin, ferulic acid and baicalin in the traditional Chinese medicinal preparation Dang-Guei-San, which contains Paeoniae Radix, Swertiae Herba, Cnidii Rhizoma and Scutellariae Radix, was established. The samples were separated with a Cosmosil 5C₁₈-AR column by gradient elution with 0.03% (v/v) phosphoric acid- acetonitrile (0 min 96:4, 5 min 84:16, 7 min 82:18, 14–30 min 78:22) as the mobile phase at a flow-rate of 1.0 ml/min, with detection at 245 nm. Methylparaben was used as the internal standard and three equations were derived showing linear relationships between the peak- area ratios of marker components (paeoniflorin, ferulic acid and baicalin) to methylparaben and concentration. The recoveries of paeoniflorin, ferulic acid and baicalin were 27.86, 33.89 and 49.31%, respectively. The repeatability (relative standard deviation) was generally less than 5% (n = 5). The effects of various processes such as concentration by reduced-pressure evaporation, freeze-drying and spray-drying were studied and commercial concentrated herbal preparations containing Paeoniae Radix, Swertiae Herba, Cnidii Rhizoma and Scutellariae Radix were also analysed.     

黄芩苷在纳米MnO2-离子液体修饰电极上的电化学性质及电分析方法研究

用疏水性离子液体1-丁基-3-甲基咪唑六氟磷酸盐([BMIM]PF6)和纳米MnO2作修饰剂,通过壳聚糖(CHIT)的成膜效应,构置了玻碳修饰电极([BMIM]PF6-MnO2-CHIT/GCE).在Britton-Robinson(B-R)缓冲液中研究了黄芩苷在修饰电极上的电化学行为,建立了测定黄芩胶囊中黄芩苷含量的...     

高效液相色谱-质谱联用快速筛选并鉴定黄芩甲醇提取物中抗氧化活性成分

建立了高效液相色谱-质谱联用技术快速筛选和鉴定黄芩甲醇提取物中抗氧化活性成分的方法.在黄芩甲醇提物中加入适量的二苯基三硝基苯肼( DPPH)作为实验组,避光室温反应30 min后直接经液相色谱分析,并与不加入DPPH的空白组进行比较.有抗氧化活性的成分因会与DPPH反应而使峰面积减少,而不具有抗氧化活性的成分峰面积则不变.基于色谱数据可以获得有抗氧化活性组分的相对活性强度,基于质谱数据可获得活性组分的结构信息用于结构鉴定.本方法成功地从黄芩甲醇提取物中筛选并鉴定出了黄芩苷、千层纸素A-7-O-B-D-葡萄糖醛酸苷、汉黄芩苷和千层纸素A4种较强的抗氧化活性成分.本方法利用HPLC-MS技术对复杂体系(如中药提取物)中小分子的分离和鉴定优势,可以直接筛选出黄芩甲醇提取物中的抗氧化活性成分,而不需要繁琐的前期分离和纯化,并且不需要仪器改装工作,有利于实现复杂体系中抗氧化活性成分的高通量筛选.     

Discovery of conversion driven by β-glucuronidase from flavone glycoside to aglycone and application in identifying the raw Scutellariae Radix

Scutellariae Radix (SR), the dried root of Scutellaria baicalensis Georgia, is a famous Chinese materia medica that has been widely employed. Raw Scutellariae Radix (RSR), steamed Scutellariae Radix (SSR), and wine Scutellariae Radix (WSR) are adopted for use in clinical practice. Because of their easily confused appearance, they are always misused. Aiming at this problem, an ultra performance liquid chromatography coupled with photodiode array detector (UPLC-PDA) method was established to survey misuse of the RSR and the processed SR (SSR and WSR) in the market by employing baicalin (BC), wogonoside (WS), baicalein (BN), and wogonin (WN) as quality indicators. Fortunately, β-glucuronidase, which mediates conversion from flavone glycoside to aglycone, was identified in the RSR samples by the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The significant production of BN and WN was witnessed in the RSR samples, which did not occur in the SSR and WSR samples in virtue of the inactivated β-glucuronidase. Besides, the different capacities of β-glucuronidase were evaluated in the tested samples. In general, we provided the first evidence to scientifically identify RSR from SSR and WSR.     

Biliary excretion of metabolites of baicalin and baicalein in rats

Biliary excretion of metabolites of baicalin, present in Scutellariae Radix, was investigated using rats. The bile of rats administered baicalin orally was shown to contain five major metabolites, which were identified as baicalein 6-O-beta-glucopyranuronoside (M1), 6-O-methyl-baicalein 7-O-beta-glucopyranuronoside (oroxylin A 7-O-beta-glucuronide (M2], baicalein 7-O-beta-glucopyranuronoside (M3), 6-O-beta-glucopyranuronosyl- baicalein 7-O-sulfate (M4), and baicalein 6,7-di-O-beta-glucopyranuronoside (M5) on the basis of chemical and spectroscopic evidence. The bile of rats treated with baicalein also contained the above metabolites. Slower biliary excretion of the metabolites after baicalin administration suggested that it was absorbed as baicalein after hydrolysis in the gastrointestinal tract. The total cumulative amounts of the five metabolites excreted in the bile during 30 h after oral administration of baicalin and that of baicalein were approximately 54% and 40% of the doses, respectively. In addition the bilary metabolites of both drugs were shown to be mainly composed of M5 and M4, which have high polarity and large molecular weight.     

Determination of Baicalin by High Performance Liquid Chromatography Coupling with Flow Injection Chemiluminescence Detection

A new method for the determination of baicalin in Scutellariae Radix extract and its preparations by high performance liquid chromatography (HPLC) coupling with flow injection chemiluminescence detection (FIA‐CL) has been developed. The method was based on the chemiluminescence reaction of potassium permanganate with baicalin in nitric acid medium; the CL intensity can be enhanced by formaldehyde. In this study, the conditions of chemiluminescence and chromatography were examined, and the schematic diagram of the HPLC‐FIA‐CL analyzer was optimized. The analytes were separated on Hypersil RP‐C18 columns (100 × 4.6 mm, I.D., 5 μm) by equality elution with 47:53 (v/v) methanol‐0.3% phosphoric acid as a mobile phase at a flow rate of 0.8 mL·min^?1 and a column temperature of 40 °C. Under the optimum condition, the CL intensity was proportional to the concentration of baicalin over the range of 4.10 × 10^?7 ? 6.15 × 10^?5mol·L^?1. The limit of detection (S/N = 3) was 2.79 × 10^?7mol·L^?1 with the relative standard deviation 2.5% (Cs = 6.15 × 10^?6 mol·L^?1, n = 5). The method has been applied to the determination of baicalin in Scutellariae Radix extract and its preparations, and satisfactory results were obtained.     

Development of a combined technique using a rapid one-step immunochromatographic assay and indirect competitive ELISA for the rapid detection of baicalin

A colloidal gold conjugated anti-baicalin monoclonal antibody (anti-BA MAb) was prepared and used in an immunochromatographic assay (ICA) for BA in Scutellariae Radix and Kampo medicines. This competitive ICA uses an anti-BA MAb which shows a high specificity for BA and baicalein. Its advantages include a short assay time (15 min), no dependence on any instrumental systems, and it can detect BA in plant materials and Kampo medicines. The limit of detection for the ICA was found to be around 0.6 μg mL⁻¹of baicalin. Moreover, the usefulness of the combination of indirect competitive ELISA and the ICA using anti-BA MAb as a quality control method was confirmed for analysis of BA in Scutellariae Radix and Kampo medicines with a sufficient sensitivity (200 ng mL⁻¹ to 2 μg mL⁻¹), obtainable in an easy and timely manner.     

A simple isocratic HPLC method for the simultaneous determination of bioactive components of Scutellariae Radix extract

Scutellariae Radix, the dried root of Scutellaria baicalensis Georgi, has been widely used in Asian countries for the treatment of dermatitis, diarrhoea, inflammatory disease and hepatic disease. A simple, sensitive and precise reversed-phase liquid chromatographic method with isocratic elution was developed to simultaneously determine four bioactive compounds in Scutellariae Radix: baicalein, baicalin, wogonin and wogonoside. Chromatographic analysis was performed on a YMC Pack Pro C(8) column (150?×?4.6?mm(2), 3?μm), with a mobile phase of 0.1% formic acid?:?acetonitrile (70?:?30, v/v) at a flow rate of 1.0?mL?min(-1), and UV detection at 280?nm. Linear behaviour was observed over the investigated concentration range (0.25-10?μg?mL(-1)) for all analytes, with a correlation coefficient of >0.997. The intra- and inter-day precisions were <8.07%, and accuracies were 92.3-102.9%. This method was successfully applied for the analysis of marker compounds for the quality control of Scutellariae Radix extract.