Chenrui Li

Pharmacological effects and pharmacokinetics properties of Radix Scutellariae and its bioactive flavones

Radix Scutellariae is the dried root of the medicinal plant Scutellariae baicalensis Georgi. It exhibits a variety of therapeutic effects and has a long history of application in traditional formulations as well as in modern herbal medications. It has been confirmed that flavonoids are the most abundant constituents and induce these therapeutic effects. Six flavones are proven to be the major bioactive flavones in Radix Scutellariae existing in the forms of aglycones (baicalein, wogonin, oroxylin A) and glycosides (baicalin, wogonoside, oroxylin A-7-glucuronide). All six flavones are pharmacologically active and show great potential in the treatment of inflammation, cancers and virus-related diseases. The current review covers the preparation of the herb Radix Scutellariae, quantification of its major bioactive ingredients, and pharmacological effects of the proposed six bioactive flavones. In addition, this review summarizes the pharmacokinetic profiles of the bioactive flavones reported so far that could be used for further improvement of their pharmacokinetic study. Moreover, due to abundant co-occurring bioactive components in Radix Scutellariae, our review further documents the pharmacokinetic interactions among them.

Contents of major bioactive flavones in proprietary traditional Chinese medicine products and reference herb of radix Scutellariae.

A simple and efficient HPLC/UV method for the simultaneous determination of six bioactive flavones, namely baicalein, baicalin, wogonin, wogonoside, oroxylin A and oroxylin A-7-O-glucuronide, has been developed and applied for their content determination in reference herb and proprietary traditional Chinese medicine (PTCM) products of radix Scutellariae. The chromatographic separation was carried out on a Thermo C(18) column and linear gradient elution was employed with a mobile phase containing acetonitrile and 20 mM sodium dihydrogen phosphate buffer (pH 4.6). All the analytes were detected by PDA detector at a wavelength of 270 nm. Contents of the analytes in radix Scutellariae containing PTCM products in forms of capsule, soft capsule, tablet and dripping pill and the reference herb of radix Scutellariae were analyzed by sonicator extraction with methanol and water mixture (80:20) containing 1 mM HCl for 30 min followed by HPLC analysis. Separation of the six analytes was achieved within 25 min with good linearity (R(2)>0.99). The R.S.D. of both the intra-day and inter-day precision for all the six analytes was below 10.14%. The accuracy at different concentrations was within the range of -7.83 to 4.06%. The extraction recovery was within the range of 89.22-107.33% for all the analytes. Contents of the six flavones were found to vary significantly among different products with glycosides, such as baicalin, wogonoside and oroxylin A-7-O-glucuronide, in much greater quantity than their corresponding aglycones. In addition to baicalin (18.54+/-0.71%, w/w), the commonly used marker compound for radix Scutellariae, wogonoside (3.54+/-0.18%, w/w) and oroxylin A-7-O-glucuronide (2.84+/-0.14%, w/w) also existed in abundant amount in the reference herb. Our findings suggested that wogonoside and oroxylin A-7-O-glucuronide should also be served as the chemical markers together with baicalin for the quality control of herbs and PTCM products of radix Scutellariae.

Identification and quantification of baicalein, wogonin, oroxylin A and their major glucuronide conjugated metabolites in rat plasma after oral administration of Radix scutellariae product

The current study aims to identify and quantify three flavones (baicalein, wogonin and oroxylin A) and their major metabolites (baicalin, wogonoside and oroxylin A-7-O-glucuronide) in rat plasma after oral administration of Radix scutellariae product. A simple HPLC/UV method has been developed to simultaneously determine the three flavones and their major metabolites in rat plasma. The chromatographic separation of the six analytes was achieved by a Thermo C(18) column with linear gradient elution of a mobile phase containing acetonitrile and 20mM sodium dihydrogen phosphate buffer (pH 4.6). All the tested analytes were detected by PDA detector at a wavelength of 320nm. The intra-day and inter-day precision for the current assay of the six analytes was within the range of -2.23% to 15.13% and -10.83% to 6.42%, respectively. All the studied analytes could be efficiently extracted from the rat plasma using HLB cartridge with extraction recoveries above 70% and were stable under different storage conditions. The developed assay method was successfully applied to the pharmacokinetic study of baicalin, wogonoside after oral administration of a commercially available Radix scutellariae containing capsule at a dose of 3.2g/kg to Sprague-Dawley rats. In addition to wogonoside, a new metabolite of wogonin has been identified using LC/MS/MS for the first time.

Comparison of Intestinal Absorption and Disposition of Structurally Similar Bioactive Flavones in Radix Scutellariae

Radix Scutellariae is a commonly used herbal medicine. Baicalein, wogonin, and oroxylin A are three major bioactive flavones in Radix Scutellariae and share similar chemical structures. The intestinal absorption and disposition of baicalein have been systematically investigated by our group before. In this study, the intestinal absorption and disposition of wogonin and oroxylin A were further explored and compared with the profiles of baicalein to find potential structure–activity relationship. Absorptive models including Caco-2 cell monolayer model and rat in situ single-pass intestinal perfusion model as well as in vitro enzymatic kinetic study were employed in the current study. The absorption of baicalein, wogonin, and oroxylin A were favorable with wogonin showing the highest permeability based on two absorptive models. However, three flavones underwent a fast and extensive phase II metabolism. The intestinal metabolism of three flavones exhibited species difference between human and rat. Oroxylin A demonstrated the highest intrinsic clearance of glucuronidation among three flavones. The multidrug resistance proteins might be involved in the efflux of their intracellularly formed conjugated metabolites. The pathway of intestinal absorption and disposition of B, W, and OA was similar. However, the extent of permeability and metabolism was different among three flavones which might be due to the number and position of the hydroxyl group.

Pharmacokinetic interactions among major bioactive components in Radix Scutellariae via metabolic competition.

Baicalein (B), wogonin (W) and oroxylin A (OA) are major components in Radix Scutellariae with similar pharmacokinetic properties. Due to the co‐presence of these three flavones in herbal formulations for Radix Scutellariae, they are likely consumed together. The aim of this study is to investigate whether the pharmacokinetics of individual flavones is influenced by each other and the underlying mechanism of the interaction. Various systems were utilized in the current study including a rat in vivo study, a Caco‐2 cell monolayer model and a rat in situ single‐pass intestinal perfusion as well as in vitro enzymatic kinetics studies. The B, W and OA given singly as well as in a mixture were administered and the corresponding pharmacokinetic parameters were calculated and compared. After co‐administration of the three flavones to rats, OA absorption increased significantly in comparison with when OA was administered alone. Mechanistic studies on the Caco‐2 cell monolayer and rat in situ single‐pass intestinal perfusion models revealed that co‐administration of B, W and OA could significantly enhance their absorption and decrease the extent of phase II metabolism. Further in vitro enzymatic study and a transport study in transfected MDCK cells revealed that metabolic competition rather than membrane transporters might contribute to the pharmacokinetic interactions. The co‐presence of multiple active components would result in metabolic interactions, which may induce further changes in pharmacodynamics. Copyright

Non-linear pharmacokinetics of piperine and its herb-drug interactions with docetaxel in Sprague-Dawley rats.

Piperine (PIP), the major alkaloid component from Piper longum L. and Piper nigrum L., could enhance the bioavailabilities of other drugs including rosuvastatin, peurarin and docetaxel (DOX) via inhibition of CYP3A and P-glycoprotein activity. Nevertheless, the effect of such drug combination usage on the in vivo exposure of PIP has not been investigated due to lack of assay for the simultaneous determination of PIP and other drugs such as DOX. Besides, the reported pharmacokinetics of PIP varied a lot without appropriate bioavailability determined from the same dose. In the current study, an LC/MS/MS method has been developed to simultaneously determine the plasma concentrations of PIP and DOX and further applied to investigate the pharmacokinetics properties of PIP after oral and intravenous administrations as well as the pharmacokinetics interactions between PIP and DOX after their co-administration. A simple protein precipitation method was employed for plasma sample treatment by adding a mixture of methanol and acetonitrile (1:1, v/v) with glibenclamide as internal standard (IS). The LC/MS/MS system consisted of Agilent 6430 series LC pumps and auto-sampler. The chromatographic separation was carried out in 15min on a Waters C18 column (150×3.9mm i.d., 4μm) with a mobile phase containing 0.2% formic acid and acetonitrile (1:1, v/v) at a flow rate of 0.4ml/min. The detection was performed using the positive ion electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode with precursor-to-product ion transitions at m/z 286.1→201.1 for PIP, m/z 830.3→548.9 for DOX and m/z 494.2→369.0 for IS. The method demonstrated good linearity for both PIP and DOX over the concentration range of 2.5-1280ng/ml with LLOD at 2.5ng/ml. The intra-day and inter-day precisions were less than 13.34% and relative error (R.E.) representing accuracy was in the range of -11.38 to 3.15%. The recoveries of PIP, DOX and IS were above 75% and there was no matrix effect. PIP and DOX exhibited good stabilities under various conditions. PIP was administrated via intravenous bolus at 3.5mg/kg and via oral administration at 35mg/kg and 3.5mg/kg, while DOX was intravenously administrated at 7mg/kg to Sprague-Daley rats. The plasma concentrations of PIP and DOX were determined using the above developed and validated method. At the dose of 3.5mg/kg, the bioavailability of PIP was calculated to be 25.36%. Its AUC0→t was unproportionally increased with doses, indicating a potential non-linear pharmacokinetics profile of PIP. It was found that the AUC0→t and C0 of DOX and t1/2 of PIP were significantly increased after their combination use, suggesting potential enhanced bioavailability of not only DOX but also PIP, which may lead to the overall enhanced pharmacological effects.

Enhanced anti-tumor efficacy and mechanisms associated with docetaxel-piperine combination- in vitro and in vivo investigation using a taxane-resistant prostate cancer model

Docetaxel (DTX) is widely used for metastatic castrated resistant prostate cancer, but its efficacy is often compromised by drug resistance associated with low intracellular concentrations. Piperine (PIP) could enhance the bioavailability of other drugs via the inhibition of CYPs and P-gp activities. Thus, we hypothesize a positive effect with the DTX-PIP combination on the anti-tumor efficacy and intra-tumor DTX concentrations in taxane-resistant prostate cancer. ICR-NOD/SCID mice implanted with taxane-resistant human prostate cancer cells were administrated with saline as well as PIP and DTX separately or in combination. The tumor growth was monitored together with intra-tumor concentrations of DTX. The inhibitory effects on CYPs and P-gp were further assessed in mouse liver microsome and MDCK-MDR1 cells. Compared with DTX alone, DTX-PIP combination significantly inhibited the tumor growth (114% vs. 217%, p = 0.002) with corresponding significantly higher intra-tumor DTX concentrations (5.854 ± 5.510 ng/ml vs. 1.312 ± 0.754 ng/mg, p = 0.037). The percentage of DTX metabolism was significantly decreased from 28.94 ± 1.06% to 18.14 ± 2.22% in mouse liver microsome after administration of PIP for two weeks. DTX accumulation in MDCK-MDR1 cell was significantly enhanced in the presence of PIP. Further microarray analysis revealed that PIP inhibited P-gp as well as CYP1B1 gene expression and induced a significant gene expression change relating to inflammatory response, angiogenesis, cell proliferation, or cell migration. In conclusion, DTX-PIP combination significantly induces activity against taxane-resistant prostate tumor. Such effect appeared to be attributed to the inhibitory effect of PIP on CYPs and P-gp activity as well as gene expression changes relating to tumorigenesis and cellular responses.

Efficient brain uptake of piperine and its pharmacokinetics characterization after oral administration

Abstract 1. Piperine, the major biological active component in black pepper has been associated with miscellaneous pharmacological effects, especially on central nervous system. To correlate with its neurological activity, a comprehensive pharmacokinetic profile of piperine in brain, plasma and cerebrospinal fluid after oral administration in rats was investigated in this study. 2. It was noted that piperine could efficiently penetrate and homogeneously distribute into brain with similar pharmacokinetics profiles in each region. In addition, piperine concentrations in brain and plasma were found to be comparable with brain to plasma area under curve extrapolated to infinity (AUC0→∞) ratios of 0.95 and 1.10 for total concentration and unbound concentrations, respectively. Piperine also demonstrated high affinity toward brain tissue (98.4–98.5%) and plasma protein (96.2–97.8%) leading to a brain distribution volume of 36.32 ± 1.40 ml/g brain. Moreover, its efficient membrane permeability (P app values of 5.41  ±  0.40 × 10- 5 cm/s and 4.78  ±  0.16 × 10- 5 cm/s for basolateral to apical and apical to basolateral transport in Caco-2 monolayer model) and limited hepatic metabolism (Clint of 8.15 μl/min/mg) could also contribute to its quick and high extent brain exposure. 3. In summary, this study for the first time demonstrated high brain penetration potency of piperine could be resulted from its high brain tissue affinity and membrane permeability together with its limited liver metabolism.