Radix Scutellariae Extract Research Articles

Study the interactions between multiple flavonoids and bovine serum albumin by the developed equilibrium dialysis

The therapeutic function of traditional Chinese medicine (TCM) is based on the combination effect of multiple active ingredients. However, the current pharmacological studies mainly focus on the protein binding of the single component from TCM, which is difficult to explain the overall therapeutic mechanism. Thus in this work the equilibrium dialysis method combined with high performance liquid chromatography (HPLC) and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed to study the interactions between multi-components and protein. Firstly, the binding constants of seven different structural types of flavonoids with bovine serum albumin (BSA) were determined. The results showed that the binding affinity of flavones and flavonols with BSA was stronger than that of dihydroflavonoids, and the substitution of glycosides would reduce the binding affinity with BSA. The results of competitive displacement experiment showed that there existed competitive interactions among the four flavonoids (rutin, luteolin, hesperetin and kaempferol). The binding constants of flavonoids to BSA were significantly changed under the condition of multi-components coexistence. Especially, the binding constant of hesperetin to BSA increased from 9.46 × 104 L/mol to 1.49 × 106 L/mol under the coexistence of rutin. The results of fluorescence spectroscopy showed that the reason for competitive binding was that the four flavonoids were mainly bound to the IIA region of BSA. Finally, the method was successfully applied to study the binding of multiple components in Radix Scutellariae (RS) extract with BSA. Five flavonoids in RS extract were identified by UPLC-MS/MS, they had different degrees of binding to BSA, among which oroxylin A had the strongest binding degree. In conclusion, the equilibrium dialysis was reliable and sufficiently accurate for study of the interaction between multi-components or TCM extract and protein, which can provided a theoretical basis for the scientific explanation of the overall treatment mechanism of TCM.

The effect of Wogonoside on the expression of TGF-β1/Smad3 in Mycoplasma pneumoniae pneumonia

Screening active compounds from complex herbal extracts is an important part of the drug mechanism research. The present study describes the establishment and application of a sensitive, convenient and efficient method for screening active ingredients targeting pulmonary fibrosis factor TGF-β1 from Radix Scutellariae extracts based on the surface plasmon resonance (SPR) biosensors. Previous studies have proved that Qin bai qing fei pellet (Qin bai) can inhibit the expression of TGF-β1 in Mycoplasma pneumoniae pneumonia (MPP). Radix Scutellariae is the main component of Qin bai. However, the mechanism of Radix Scutellariae inhibiting TGF-β1 expression remains unexplained. The study investigated the active compound in Radix Scutellariae which was bound with TGF-β1 by SPR and UPLC-Q-TOF-MS techonology. As a result, Radix Scutellariae extracts showed a good combination with the TGF-β1 in vitro. Wogonoside was identified as a bioactive ingredient by UPLC-Q-TOF-MS. The affinity constant of Wogonoside to TGF-β1 was determined by SPR affinity analysis (KD=21.7 μm). Pharmacology assays revealed that Wogonoside strongly inhibited the expression of TGF-β1 and Smad3 in A549 cells infected by Mycoplasma pneumoniae(M.pneumoniae). Wogonoside prevented Mycoplasma pneumoniae infection by inhibiting the expression of TGF-β1, which might be one of the mechanisms for the anti-MPP properties of Qin bai. These results provided a foundation to further explore the mechanisms of Qin bai.

Radix Scutellariae Ameliorates Stress-Induced Depressive-Like Behaviors via Protecting Neurons through the TGFβ3-Smad2/3-Nedd9 Signaling Pathway.

Chronic stress can impair hippocampal neurogenesis, increase neuronal apoptosis, and cause depressive-like behaviors. Our previous studies found that Radix Scutellariae (RS) can rescue the stress-induced neuronal injury, but the mechanism is not clear. Here, we continued to investigate the underlying antidepressant mechanisms of the RS extract. A 7-week chronic unpredictable mild stress (CUMS) procedure was used to establish a murine depression model. 0.75 g/kg or 1.5 g/kg RS was administered daily to the mice during the last 4 weeks. Depressive-like behaviors were evaluated by the sucrose preference test (SPT), forced swimming test (FST), open field test (OFT), and tail suspension test (TST). The neuroprotective effect of RS was evaluated with the expression of hippocampal neuron-related markers and apoptosis-associated proteins by Nissl staining, immunohistochemistry, and western blot. Transforming growth factor-β3 (TGFβ3) pathway-related proteins were detected by western blot. Results showed that RS could ameliorate depressive-like behaviors, increase the expression of the antiapoptotic protein B-cell lymphoma 2 (BCL-2), reduce the expression of the proapoptotic protein BCL-2-associated X (BAX), and increase the number of doublecortin- (DCX-), microtubule-associated protein 2- (MAP2-), and neuronal nucleus- (NeuN-) positive cells in the hippocampus. Moreover, RS could reverse the CUMS-induced decrease of TGFβ3 protein, promote the phosphorylation of SMAD2/3, and increase the expression of downstream NEDD9 protein. These results suggest that RS could exert antidepressant effects via protecting neurons. And the molecular mechanism might be related to the regulation of the TGFβ3-SMAD2/3-NEDD9 pathway.

High dose of baicalin or baicalein can reduce tight junction integrity by partly targeting the first PDZ domain of zonula occludens-1 (ZO-1)

The tight junction (TJ) is the apical-most intercellular junction complex, serving as a biological barrier of intercellular spaces between epithelial cells. The TJ's integrity is maintained by a key protein–protein interaction between C-terminal motifs of claudins (CLDs) and the postsynaptic density 95 (PSD-95)/discs large/zonula occludens 1 (ZO-1; PDZ) domains of ZO-1. Weak but direct interaction of baicalin and its aglycon, baicalein—which are pharmacologically active components of Chinese skullcap (Radix scutellariae)—with ZO-1(PDZ1) have been observed in NMR experiments. Next, we observed TJ-mitigating activity of these flavonoids against Madin-Darby canine kidney (MDCK) II cells with the downregulation of subcellular localization of CLD-2 at TJs. Meanwhile, baicalein—but not baicalin—induced a slender morphological change of MDCK cells' shape from their normal cobblestone-like shapes. Since baicalin and baicalein did not induce a localization change of occludin (OCLN), a “partial” epithelial–mesenchymal transition (EMT) induced by these flavonoids was considered. SB431542, an ALK-5 inhibitor, reversed the CLD-2 downregulation of both baicalin and baicalein, while SB431542 did not reverse the slender morphology. In contrast, the MEK/ERK inhibitor U0126 reversed the slender shape change. Thus, in addition to inhibition of the ZO-1–CLD interaction, activation of both transforming growth factor-β (TGF-β) and MEK/ERK signaling pathways have been suggested to be involved in TJ reduction by these flavonoids. Finally, we demonstrated that baicalin enhanced the permeability of fluorescence-labeled insulin via the paracellular pathway of the Caco-2 cell layer. We propose that baicalin, baicalein, and Radix scutellariae extract are useful as drug absorption enhancers.

Evaluation of potential herb-drug interactions between oseltamivir and commonly used anti-influenza Chinese medicinal herbs

Ethnopharmacological relevanceAccording to Traditional Chinese Medicine theory, influenza is categorized as a warm disease or Wen Bing. The Wen Bing formulas, such as Yin-Qiao-San and Sang-Ju-Yin, are still first-line herbal therapies in combating variant influenza virus. To continue our study on the pharmacokinetic and pharmacodynamic interactions between Wen Bing formulas and oseltamivir (OS), the first-line western drug for the treatment of influenza, further interactions between OS and the eight single herbs and their relevant marker components from Wen Bing formulas were investigated in the current study. Aim of studyTo establish an in-vitro screening platform for investigation of the potential anti-influenza herbs/herbal components that may have pharmacokinetic and pharmacodynamic interactions with OS. Materials and methodsTo screen potential inhibition on OS hydrolysis, 1 μg/mL of OS is incubated with herbs/herbal components in diluted rat plasma, microsomes and human recombinant carboxylesterase 1(hCE1) under optimized conditions. MDCK-WT and MDCK-MDR1 cell lines are utilized to identify potential modification on P-gp mediated transport of OS by herbs/herbal components. Caco-2 cells with and without Gly-Sar inhibition are performed to study the uptake of OS via PEPT1 transporters. Modification on OAT3 mediated transport is verified by the uptake of OS on HEK293-MOCK/HEK293-OAT3 cells. Anti-virus effects were evaluated using plaque reduction assay on H1N1 and H3N2 viruses. Potential pharmacokinetic and pharmacodynamic interaction between OS (30 mg/kg) and the selected herb, Radix Scutellariae (RS), at 300–600 mg/kg were carried out on rats. All samples are analyzed by an LC/MS/MS method for the contents of OS and OSA. A mechanistic PK model was developed to interpret the HDI between OS and RS in rats. ResultsOur developed platform was successfully applied to screen the eight herbal extracts and their ten marker components on metabolic inhibition of OS and modification of OS transport mediated by P-gp, OAT3 and PEPT1. Results from six in-vitro experiments were analyzed after converting raw data from each experiment to corresponding fold-change (FC) values, based on which Radix Scutellariae (RS) were selected to have the most HDI potential with OS. By analyzing the plasma and urine pharmacokinetic data after co-administration of OS with a standardized RS extract in rats using an integrated population pharmacokinetics model, it is suggested that RS could inhibit OS hydrolysis during absorption and increase the absorbed fraction of OS, which leads to the increased ratio of OS concentration versus that of OSA in both rat plasma and urine. Never the less, the anti-virus effects of 2.5 h post-dose rat plasma were not influenced by co-administration of OS with RS. ConclusionA six-dimension in-vitro screening platform has been developed and successfully applied to find RS as a potential herb that would influence the co-administrated OS in rats. Although co-administered RS could inhibit OS hydrolysis during absorption and increase the absorbed fraction of OS, which lead to the increased ratio of OS concentration versus that of OSA in both rat plasma and urine, the anti-virus effect of OS was not influenced by co-administered RS.

Screening anaphylactoid components of Shuang Huang Lian Injection by analyzing spectrum-effect relationships coupled with UPLC-TOF-MS.

Shuang Huang Lian Injection (SHLI) has been used in China for over 30 years as an effective and widely used Chinese herbal prescription to treat acute respiratory infectious. SHLI has, however, caused many severe anaphylactoid reactions. It is important to identify the potential anaphylactoid components of SHLI. Spectrum-effect relationships were used to explore potentially anaphylactoid components. Based on the original herbal formula, honeysuckle, Fructus Forsythiae and Radix Scutellariae extracts were prepared and combined in appropriate proportions. The preparations were then injected into the caudal vein of rats to obtain in vivo serum samples for pharmacological evaluation and fingerprint analysis. The release rate of β-hexosaminidase from RBL-2H3 cells and plasma histamine level was used as the pharmacological index. Chromatographic fingerprint analysis identified 22 common peaks. Regression analysis and correlation analysis were used to calculate the relationships between the peaks and the pharmacological effects and identified peaks 5, 6, 11, 12 and 17 as likely anaphylactoid agents. The correlated peaks were identified by comparing the fingerprints with in vitro samples and reference standard samples and the structure was identified by UPLC-TOF-MS. This study established a prospective method to clarify the anaphylactoid components in SHLI, which would provide guidances for screening anaphylactoid components in other traditional Chinese medicine injections.

The Influence of Compatibility of Rhubarb and Radix Scutellariae on the Pharmacokinetics of Anthraquinones and Flavonoids in Rat Plasma.

Rhubarb-Radix scutellariae is a classic herb pair, which is commonly used to clear away heat and toxin in clinic. The aim of this study was to investigate the influence of compatibility of Rhubarb and Radix scutellariae on the pharmacokinetic behaviors of anthraquinones and flavonoids in rat plasma. Eighteen rats were randomly divided into three groups, and were orally administered Rhubarb and/or Radix scutellariae extracts. A sensitive and rapid UPLC-MS/MS method was developed and validated to determine the concentrations of baicalin, baicalein, wogonside, wogonin, rhein, and emodin in rat plasma. The concentrations of phase II conjugates of flavonoid aglycones and anthraquinone aglycones were also determined after hydrolyzing the plasma with sulfatase. Compared with administration of Radix scutellariae alone, co-administration of Rhubarb significantly decreased the first maximum plasma concentration (C max1) of baicalin, wogonside, and the phase II conjugates of baicalein, wogonin to 46.40, 61.27, 41.49, and 20.50%, respectively. The area under the plasma concentration-time curve from time zero to infinity (AUC0-∞) was significantly decreased from 82.60±20.22 to 51.91±7.46μM·h for rhein and 276.83±98.02 to 175.42±86.82μM·h for the phase II conjugates of wogonin after compatibility. The time to reach the first maximum plasma concentration (T max1) of anthraquinones was shortened and the second peak of anthraquinones disappeared after compatibility. Compatibility of Rhubarb and Radix scutellariae can significantly affect the pharmacokinetic behaviors of characteristic constituents of the two herbs. The cause of these pharmacokinetic differences was further discussed combined with the in vivo ADME (absorption, disposition, metabolism, and excretion) processes of anthraquinones and flavonoids.

Enhancement of solubility and dissolution rate of baicalein, wogonin and oroxylin A extracted from Radix scutellariae

Baicalein, wogonin, and oroxylin A are three major hydrophobic components in the extract of Radix scutellariae with wide spectrum of pharmacological applications. The purpose of this study was to enhance the solubility, dissolution rate and stability of baicalein, wogonin and oroxylin A by solid dispersion (SD) technique. SD of the extract with various polymers was prepared to select the best carrier. Solubility study, chemical stability study and dissolution study were performed to characterize the SD. The solubility of all three components, after forming solid dispersion with povidone K-30 (PVP K-30) was significantly increased in pH 6.8 medium at room temperature. Stability study conducted for 80days elucidated that the SD in powder state was fairly stable without the aid of Vitamin C (VC). VC was required as antioxidant to impart stability to baicalein in aqueous medium. The dissolution test of the SD of three components, admixed with VC at the weight ratio of 1:6 (Radix scutellariae extract: VC, w/w) exhibited faster dissolution rate with 100% release of all components. Pharmacokinetic study of baicalein solid dispersion revealed that AUC and Cmax significantly increased by solid dispersion preparation. Thus, the current developed method, being simple, economical and effective, can be useful for the production of soluble dosage forms of the extract of Radix scutellariae in commercial scale.

An activity-integrated strategy of the identification, screening and determination of potential neuraminidase inhibitors from Radix Scutellariae.

Small molecules isolated from herbal medicines (HMs) were identified as the potential neuraminidase inhibitors which are effective in influenza prevention and treatment. Unfortunately, current available screen methods of small molecules isolated from HMs are inefficient and insensitive. Here a novel Ultra Performance Liquid Chromatography coupled with diode-array detectors and auto-fraction collector / time-of-flight mass spectrometry (UPLC-DAD-FC/Q-TOF-MS) screening method with high efficiency was developed and validated to separate, collect, enrich, identify and quantify potential neuraminidase inhibitors from Radix Scutellariae. The results showed that 26 components with neuraminidase inhibitory activity were identified from Radix Scutellariae extracts. It was also found that the influence of origins on the quality of RS was more than that of cultivated time on the basis of the concentration of the effective components. These results brought novel insights into quality evaluation of Radix Scutellariae. It was demonstrated that new activity-integrated strategy was a suitable technique for the identification, screening and determination of potential neuraminidase inhibitors in herbal medicine and will provide novel potential strategies in other drug screening from herbal medicine.

An improved cell membrane chromatography method for the simultaneous screening of two epidermal growth factor receptor antagonists from radix scutellariae.

A high-expression epidermal growth factor receptor cell membrane chromatography using the silica gel with the average particle size of 3 μm as the stationary phase carrier coupled with high-performance liquid chromatography and mass spectrometry was established for the online screening of epidermal growth factor receptor antagonists from Radix Scutellariae (Huang Qin in Chinese), a traditional Chinese medicine. In this study, the growth factor receptor cell membrane chromatography model using the smaller particle size carrier showed a higher efficiency for simultaneous screening baicalein, another one of the potential epidermal growth factor receptor antagonists from Radix Scutellariae extract besides wogonin, which was found in our previous work. The molecular docking result showed the occupancy site and binding mode of baicalein and wogonin with epidermal growth factor receptor tyrosine kinase were similar to gefitinib. The result of the assay for the in vitro inhibitory activity showed that baicalein and wogonin inhibited the growth of the high-expression epidermal growth factor receptor cell in a dose-dependent manner and even achieved a better inhibition effect than gifitinib in the low-dosage range.

Evaluation of Embryotoxicity of Radix scutellariae Based on Embryonic Stem Test

Purpose: To determine the potential embryotoxicity of Radix scutellariae (RS) extract using an embryonic stem cell test (EST) and to evaluate its effect on the differentiation of mouse embryonic stem (ES) cells.Methods: All the test samples were obtained by water extraction method. The embryotoxicity of RS was assessed with cytotoxicity assays, namely, embryonic stem (ES) cells (IC50ES) and 3T3 fibroblasts (IC503T3), as well as cardiac differentiation inhibition assay (ID50ES). The expression of specific genes and proteins was analyzed by quantitative reverse transcription – polymerase chain reaction (RT-PCR) and Western blot.Results: RS was weakly embryotoxic with IC50ES, IC503T3 and ID50ES of 0.1524, 0.1061, and 0.4169 mg/ml, respectively. Also RS had discordant effects on the expression of tissue-specific genes and proteins in three germ layers, promoting differentiation of the ectoderm (⋆p < 0.05; ⋆⋆p < 0.01) and endoderm (⋆p < 0.05; ⋆⋆p < 0.01; ⋆⋆⋆p < 0.001), while inhibiting mesoderm differentiation (⋆p < 0.05; ⋆⋆p < 0.01; ⋆⋆⋆p < 0.001). The effect of RS on the embryonic development of the three germ layers was concentration-dependent.Conclusion: These results indicate that RS possesses weak embryotoxicity. The variability in the effects of RS may be responsible for its weak embryotoxicity.Keywords: Embryonic stem test, Radix scutellariae, Embryotoxicity, Cardiac differentiation inhibition assay, Ectoderm, Endoderm, Mesoderm

Inhibition of three novel Radix Scutellariae extracts on immediate hypersensitivity.

Radix Scutellariae, a few papers reported its pharmacology activities including alleviate small intestines smooth muscles spasm, sedation, antihypertensive effect. However, the inhibition of its different organic extracts on immediate hypersensitivity has not bee researched. To investigate the anti-immediate hypersensitivity of three extracts including ethanol extracts, acetone extracts, ethyl acetate extracts from Radix Scutellariae, four pharmacological screening model were chose, such as 4-Aminopyridine induced pruritus model, histamine-induced mouse paw edema model, PCA(passive cutaneous anaphylaxis) in ear of mouse, activie cutaneous anaphylaxismouse (mouse ear edema test), furthermore, total IgE level in the sensitized mice serum was evaluated deeply. Ethanol group at 1.42 g/kg and 0.71 g/kg could greatly decrease the licking number to 1.2 and 12.7 respectively; also keep mice paw swelling at 0.29 ml and 0.51 ml at 15 min after injection of histamine. Both ear passive cutaneous allergic reaction and active cutaneous anaphylaxis-ear swelling test demonstrated that ethanol group exhibit great inhibition on immediate hypersensitivity.Low IgE level was found in ethanol group, but high in other two groups. The ethanol extracts exhibits obvious strong inhibition, however, the acetone ones and ethyl acetate showed a little.

Comparative pharmacokinetic investigation on baicalin and wogonoside in type 2 diabetic and normal rats after oral administration of traditional Chinese medicine Huanglian Jiedu decoction

Ethnopharmacological relevanceHuanglian Jiedu decoction (HLJDD) is used traditionally in China for the treatment of diabetes mellitus in clinical practice, which has been proved to be effective. The purpose of this study was to investigate the pharmacokinetic characteristics (especially the area under the curve, AUC) of baicalin and wogonoside in type 2 diabetic rats after oral administration of HLJDD extract and to explore its possible mechanism. Materials and methodsHLJDD extract and Radix scutellariae extract were prepared and the contents of baicalin and wogonoside contained in two extracts were assayed with high performance liquid chromatography (HPLC). Type 2 diabetic rats were induced by high fat diet and intraperitoneal injection of streptozotocin. Pharmacokinetics of baicalin and wogonoside in type 2 diabetic and normal control rats after oral administration of HLJDD extract or Radix scutellariae extract were investigated. Pharmacokinetics of baicalin in type 2 diabetic and normal rats after oral administration of pure baicalin was also investigated. ResultsThe pharmacokinetic parameters (especially AUCs) of baicalin and wogonoside in type 2 diabetic rats after oral administration of HLJDD extract were remarkably different from those in normal rats. And the alterations of the AUCs of baicalin and wogonoside in type 2 diabetic rats after oral administration of Radix scutellariae extract were similar to those after oral administration of HLJDD extract. Moreover, the increase of the AUC of baicalin in type 2 diabetic rats after oral administration of pure baicalin was similar to that after oral administration of HLJDD extract or Radix scutellariae extract. ConclusionThe pharmacokinetic behaviors of baicalin and wogonoside (especially the systemic exposure [AUCs] of baicalin and wogonoside) were significantly altered in type 2 diabetic rats after orally administrated HLJDD extract. And the increased AUCs of baicalin and wogonoside in type 2 diabetic rats after oral administration of HLJDD extract resulted from neither the effects of other herbs contained in HLJDD nor the effects of other components contained in Radix scutellariae. It might result from the effects of the pathological status of type 2 diabetes mellitus.

Comparative metabolism of Radix scutellariae extract by intestinal bacteria from normal and type 2 diabetic mice in vitro

Ethnopharmacological relevanceTraditional Chinese medicine (TCM) has been used in clinical practice for several thousand years. TCM has played an indispensable role in the prevention and treatment of disease, especially the complicated and chronic ones. In TCMs, many ingredients which are known to have biological effects just pass through the gut, they do not get into the bloodstream. Study on interactions of these active ingredients with the intestinal bacteria is very helpful to unravel how TCM works. Radix scutellariae was widely used alone or in combination with other medicinal herbs to the treatment of type 2 diabetes mellitus for a long time in China even in Asia. Additionally, the incidence of type 2 diabetes is closely related to the changes of intestinal flora. In this paper, the metabolism of baicalin in Radix scutellariae extract by normal and type 2 diabetic mice intestinal bacteria were firstly investigated. Materials and methodsUltra performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UPLC/QTOF-MS) technique combined with MetabolynxTM software was used for analysis of the metabolic profile of baicalin in Radix scutellariae extracts by the intestinal bacteria from normal and type 2 diabetic mice. ResultsThe amount of baicalin׳s aglycone (baicalein) in type 2 diabetic mice samples were remarkably more than that in normal mice samples and oroxylin A only existed in type 2 diabetic mice samples. Intestinal bacteria produced not only a small amount of baicalein, but also some conjugates such as hydrogenated baicalin and methylated baicalin. ConclusionsWe proposed that β-d-glucuronidases contributed to the deglycosylation prior to absorption. Intestinal bacteria from pathological state mice produced more baicalein, which was well absorbed contributing to the treatment of type 2 diabetes. Additionally, the pharmacological effects of oroxylin A were associated with type 2 diabetes. Hence, the production of metabolites of baicalin might influence the effects of traditional medicines. Thus the study on the metabolism of baicalin by intestinal bacteria from normal and type 2 diabetic mice was of great importance to understanding the effects of traditional medicines. Furthermore, this work demonstrated the potential of the ultra performance liquid chromatography/quadrupole time-of-flight mass spectrometry approach with MetaboLynx for quite rapid, simple, reliable and automated identification of metabolites of natural products.

Comparative pharmacokinetics of baicalin in normal and the type 2 diabetic rats after oral administration of the Radix scutellariae extract

Radix scutellariae was used alone or in combination with other medicinal herbs in the treatment of type 2 diabetes mellitus in China. At present, the pharmacokinetics of baicalin in type 2 diabetic rats following oral administration of Radix scutellariae extract was investigated. The results showed that the pharmacokinetics (especially AUC) of baicalin in type 2 diabetic rats after oral administration of Radix scutellariae extract was remarkably different from that in normal rats. Then the mechanism which resulted in the increased AUC of baicalin in diabetic rats was investigated from system clearance and presystemic metabolism. And it was found that the increased AUC of baicalin in diabetic rats at least partly resulted from higher production of baicalein in the intestinal tract of type 2 diabetic rats. Moreover, the activity of β-glucuronidase in intestinal mucosa of type 2 diabetic rats was demonstrated to be higher than that in normal rats, which confirmed the results above. In conclusion, the pharmacokinetic behavior of baicalin was significantly altered in type 2 diabetic rats after orally administrated Radix scutellariae extract, which may partly result from the increased activity of intestinal β-glucuronidase under the pathological state of type 2 diabetes mellitus.

Simultaneous determination of baicalin, wogonoside, baicalein, wogonin, oroxylin A and chrysin of Radix scutellariae extract in rat plasma by liquid chromatography tandem mass spectrometry

A liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of baicalin, wogonoside, baicalein, wogonin, oroxylin A and chrysin in rat plasma, using naringin as an internal standard. After acidifying with HCl, plasma samples were pretreated by liquid-liquid extraction with acetone. Chromatographic separation was accomplished on a Hypersil Gold-C(18) analytical column (2.1×150 mm, 5 μm) utilizing a gradient elution profile and a mobile phase consisting of (A) 0.1% formic acid in water and (B) acetonitrile. Detection was performed by multiple reaction monitoring (MRM) mode using electrospray ionization in the positive ion mode. All analytes showed good linearity over the investigated concentration range (r>0.9900). The lower limit of quantification was 0.5 ng/ml for baicalin, wogonoside, wogonin and oroxylin A, and 1.0 ng/ml for baicalein and chrysin. Intra-day and inter-day precisions (RSD%) were less than 15% and accuracy (RE%) ranged from -6.7% to 5.8%. The validated method was successfully applied to investigate the pharmacokinetics of the major flavonoids of Radix scutellariae extract after oral administration to rats.

Comparative pharmacokinetics of baicalin and wogonoside by liquid chromatography–mass spectrometry after oral administration of Xiaochaihu Tang and Radix scutellariae extract to rats

The aim of this study was to compare the pharmacokinetics of baicalin and wogonoside in rats following oral administration of Xiaochaihu Tang (Minor Radix Bupleuri Decoction) and Radix scutellariae extract. Thus, a specific LC–MS method was developed and validated for the determination of these flavonoids in the plasma of rats after oral administration Xiaochaihu Tang and Radix scutellariae extract. Chromatographic separation was performed on a Zorbax SB C 18 column (150 mm × 4.6 mm, i.d.: 5 μm) with 0.1% formic acid in water and acetonitrile by linear gradient elution. Baicalin, wogonoside and carbamazepine (internal standard, I.S.) were detected in select-ion-monitoring (SIM) mode with a positive electrospray ionization (ESI) interface. The following ions: m/ z 447 for baicalin, m/ z 461 for wogonoside and m/ z 237 for the I.S. were used for quantitative determination. The calibration curves were linear over the concentration ranges from 0.1231 to 6.156 μg mL −1 for baicalin and 0.08832 to 4.416 μg mL −1 for wogonoside. The lower limit of detection (LLOD) based on a signal-to-noise ratio of 2 was 0.06155 μg mL −1 for baicalin and 0.04416 μg mL −1 for wogonoside. Intra-day and inter-day precisions (RSD%) were within 10% and accuracy (RE%) ranged from −6.4 to 4.4%. The extraction recovery at three QC concentrations ranged from 74.7 to 86.0% for baicalin and from 71.3 to 83.7% for wogonoside. The plasma concentrations of baicalin and wogonoside in rats at designated time periods after oral administration were successfully determined using the validated method, pharmacokinetic parameters were estimated by a non-compartment model. Following oral administration of Xiaochaihu Tang and Radix scutellariae extract, the t 1/2 of baicalin was 3.60 ± 0.90 and 5.64 ± 1.67, the C max1 was 1.64 ± 0.99 and 5.66 ± 2.02, the t max1 was 0.13 ± 0.05 and 0.20 ± 0.07, the C max2 was 2.43 ± 0.46 and 3.18 ± 1.66, and the t max2 were 6.40 ± 1.67 and 5.66 ± 2.02, respectively. Following oral administration of Xiaochaihu Tang and Radix scutellariae extract, the t 1/2 of wogonoside was 4.97 ± 1.68 and 7.71 ± 1.55, the C max1 was 1.39 ± 0.83 and 1.45 ± 0.37, the t max1 was 0.21 ± 0.20 and 0.17 ± 0.01, the C max2 was 1.90 ± 0.55 and 1.42 ± 0.70, and the t max2 was 5.60 ± 1.67 and 5.20 ± 1.79, respectively. A significant difference ( p < 0.05) was observed for t 1/2, and the elimination of baicalin and wogonoside in Xiaochaihu Tang was increased.

Comparative pharmacokinetics of baicalin after oral administration of pure baicalin, Radix scutellariae extract and Huang-Lian-Jie-Du-Tang to rats

Huang-Lian-Jie-Du-Tang (HLJDT) is an important “heat-clearing” multiherb remedy of traditional Chinese medicine, and Radix scutellariae ( Scutellaria baicalensis Georgi, Labiatae) is a key ingredient herb in it. Baicalin and wogonoside are two main effective ingredients enriched in Radix scutellariae. In the present study, pharmacokinetic differences of baicalin following oral administration of pure baicalin, Radix scutellariae extract, baicalin co-administrated with extract of the other three herbs of HLJDT and HLJDT were investigated in male S.D. rats with approximately the same dose of 200 mg/kg baicalin. The pharmacokinetic comparison of wogonoside was conducted only in Radix scutellariae extract and HLJDT. Plasma concentrations of baicalin and wogonoside were determined using HPLC method. Unpaired Student's t-test was used for statistical comparison. The results indicated that baicalin and wogonoside demonstrated bimodal phenomenon in the plasma profile. Some ingredients in the other three herbs of HLJDT, not in Radix scutellariae itself, had pharmacokinetic interaction with baicalin and wogonoside and hence decreased their systematic exposure level ( p < 0.01). The absorption site of baicalin was preliminary evaluated in rat using in situ absorption in stomach and different intestinal segments and results revealed the existence of double-site absorption of baicalin. The first absorption site was in upper intestinal, probably via directly absorption of baicalin; while the second absorption site was in colon in the form of aglygon.

Comparison of the Metabolism of Baicalin in Rats Orally Administered with Radix scutellariae Extract and Shuang-Huang-Lian Extract

Shuang-Huang-Lian (SHL) is a traditional Chinese formula containing Flos lonicerae, Radix scutellariae (RS) and Fructus forsythiae, and is commonly used for treating acute upper respiratory tract infection, acute bronchitis and light pneumonia. The aim of the present study is to compare the metabolites of baicalin in rats when orally administered with SHL and Radix scutellariae, and try to explore the principle of SHL compatibility. By using LC-MS(n) and HPLC-DAD, the metabolites of baicalin were analyzed from bile, urine and feces of rats dosed with SHL and RS. Our results showed significant difference of baicalin metabolism between RS and SHL. However, baicalein was found to be the main metabolites of baicalin in intestinal tract after oral administration of RS and SHL, glucuronide, glucoside and methylated products were also found in rat urine after administration of either RS or SHL. Meanwhile, several sulphates were found in rat urine after RS administration, but not found after SHL. Among the metabolites of the SHL, potentially there existed a isomerized baicalin and methylated product: 5,7-dihydroxy-6-methoxyisoflavone-7-O-beta-glucopyranuronoside, but without unidentified metabolite M3. Baicalein-6-O-beta-glucopyranuronoside-7-O-beta-glucopyranuronoside and baicalein-6-O-beta-glucose-7-O-beta-glucopyranuronoside were first reported by this study. The major metabolites of baicalin of RS and SHL in rat bile were the same, including baicalin-6-O-beta-glucopyranuronoside-7-O-beta-glucopyranuronoside, baicalin-6-beta-glucopyranuronoside and 6-O-methyl-baicalin-7-O-beta-glucopyranuronoside. Moreover, baicalein-6-O-beta-glucose-7-O-beta-glucopyranuronoside was also first found in rat bile by this study. Although baicalin-6-O-sulfate-7-O-beta-glucopyranuronoside was found in rat bile after RS administration, no sulphated products were found after oral administration of SHL. These differences of baicalin metabolism between RS and SHL indicated that compatibility of medicines could result in the differences of metabolites.