Identification of phase-II metabolites from human serum samples after oral intake of a willow bark extract.

BACKGROUND: Willow bark (Salicis cortex) is a herbal medicinal drug used to treat fever and pain, such as headaches and lower back pain. Until now, it has not been fully understood which compounds are responsible for the efficacy of the drug. PURPOSE: Although salicylic acid is known as a metabolite of salicylic alcohol derivatives of willow bark in vivo, it has been shown in previous studies that its concentration is too low to account for the overall efficacy of Salicis cortex. The aim this study was to broaden the knowledge regarding phenolic phase-II metabolites after oral intake of a willow bark extract. STUDY DESIGN/METHODS: Serum samples of a human pharmacokinetic study (Salicis cortex extract intake corresponding to 240 mg of total salicin, 10 volunteers, 12 h fasting time, controlled diet low in phenolics, and 12 blood withdrawals over a period of 24 h) were analyzed by LC-ESI-MS. A library of 142 possible metabolites associated with salicylic alcohol derivatives, flavonoids, and proanthocyanidins was used to characterize possible metabolization products. Their structures were confirmed by LC-ESI-MS experiments with reference compounds after a cleavage reaction using glucuronidase and sulfatase as well as by LC-MS/MS experiments. RESULTS: In the serum samples, phase-II metabolites of naringenin (2x glucuronides, 2x sulfates, 2x mixed glucuronide-sulfates), eriodictyol (3x glucuronides, 1x sulfate), taxifolin (1x sulfate), catechin (1x sulfate, 1x mixed glucuronide sulfate), ferulic acid (1x sulfate), hydroxyphenyl-propionic acid (1x sulfate), dihydroxyphenyl-valerolactone (1x sulfate), saligenin (1x glucuronide, 1x sulfate), salicylic acid (1x sulfate, 1x unconjugated, 1x salicyluric acid), and catechol (1x glucuronide, 1x sulfate) were characterized. Because taxifolin, dihydroxyphenyl-valerolactone, ferulic acid, and hydroxyphenyl-propionic acid could not be detected in the willow bark preparation, they could be metabolization products of genuine flavanones and flavan-3-ols as well as coumaric acid or C-ring cleavage products of flavonoids, which were present in the extract. No phase-II metabolites of procyanidins and no genuine flavonoid glycosides were detected in all serum samples. CONCLUSION: This is the first study to identify human metabolites of flavonoids, proanthocyanidins and salicylic alcohol derivatives of Salicis cortex beside salicylic acid or catechol. For the most characterized metabolites, anti-inflammatory activity has been described in the literature, and the present results are an important step in understanding the anti-inflammatory efficacy of willow bark in vivo.

Simultaneous determination of ferulic acid and gastrodin of Tianshu Capsule in rat plasma by ultra-fast liquid chromatography with tandem mass spectrometry and its application to a comparative pharmacokinetic study in normal and migraine rats.

Tianshu Capsule, consisting of Ligusticum chuanxiong Hort and Gastrodia elata Blume, is a widely used Traditional Chinese Medicine preparation for the treatment of migraine. Ferulic acid and gastrodin are main active constituents in Ligusticum chuanxiong Hort and Gastrodia elata Blume, and have been used as marker components for quality control of Tianshu Capsule. In this study, a selective, sensitive, and reliable ultra-fast liquid chromatography with tandem mass spectrometry method was developed for simultaneous determination of ferulic acid and gastrodin in rat plasma using geniposide as internal standard. The plasma samples were extracted by protein precipitation with methanol after acidification and separated on a Shim-Pack XR-ODS C18 column (75 × 3.0 mm, 2.2 µm) using gradient elution with a mobile phase consisting of water (containing 0.1% formic acid) and acetonitrile at a flow rate of 0.6 mL/min. Detection was performed on 3200 QTRAP mass spectrometry equipped with turbo ion spray source in negative ionization mode. Validation parameters were within acceptable ranges. The validated method was applied to compare the pharmacokinetic profiles of ferulic acid and gastrodin in normal and migraine rats. Our results showed that there were remarkable differences in the pharmacokinetic properties of the analytes between the normal and migraine groups.

Pharmacokinetic comparative study of gastrodin after oral administration of Gastrodia elata Bl. extract and its compatibility with the different indigents of Ligusticum chuanxiong Hort. to rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Da Chuan Xiong Decoction Compound preparation (DCXDCP) is a classic TCM formula of an aqueous extract made from Chuanxiong Rhizoma (Ligusticum chuanxiong Hort., umbelliferae) and Tianma Rhizoma (Gastrodia elata Bl., Orchidaceae). Gastrodin (GAS), a bioactive component of tianma, its pharmacokinetic (PK) behavior significantly changed after oral administration of DCXDCP compared with the extract of tianma. However, little is known about how the ingredients of chuanxiong influenced on the PK of GAS. AIM OF THE STUDY: To study the possible PK behavior differences of GAS after individually oral administration of tianma extract and tianma extract mixed with different active ingredients of chuanxiong to rats, as well as explore whether there were some herb-herb interactions. MATERIALS AND METHODS: Different DCXDCP suspensions were prepared by mixing tianma extract with different active ingredients of chuanxiong. The rats were randomly assigned to six groups and were orally treated with different DCXDCP. At different predetermined time points after administration, the concentrations of GAS in the rat plasma were determined using HPLC, and the main PK parameters were investigated. RESULTS: The results showed that tetramethylpyrazine had no significant effects on the PK parameters of GAS (p>0.05), whereas ferulic acid (FA), total phenolic acids and total alkaloids significantly increased AUC0-∞ (p<0.05). In general the observed changes in the PK parameters of GAS in DCXDCP could be closely related to the total phenolic acids and total alkaloids. CONCLUSION: It could be shown that total phenolic acids and total alkaloids present in Ligusticum chuanxiong in addition to other components not tested yet play an important role in affecting the PK of gastrodin in DCXDCP.

Comparative pharmacokinetics of gastrodin in rats after intragastric administration of free gastrodin, parishin and Gastrodia elata extract.

ETHNOPHARMACOLOGICAL RELEVANCE: Gastrodia elata Blume, a traditional Chinese herb, was widely used against convulsant, vertigo, paralysis, epilepsy, tetanus, asthma and immune dysfunctions. Gastrodin is one of the major bioactive components of G. elata and it is known for its anticonvulsive, anti-inflammatory, antiepileptic and neuroprotective effects. MATERIALS AND METHODS: An ultra high performance liquid chromatography-fluorescence detection (UHPLC-FLD) method was developed to determine gastrodin in rat plasma. Gastrodin and Thiamphenicol (internal standard, IS) were extracted from rat plasma by immediately protein precipitation. The pharmacokinetics of gastrodin in rats by following differently administered types was studies: intragastric administration of gastrodin (100mg/kg), parishin (116 mg/kg, with the same mole of gastrodin moiety) and G. elata extract (2.3g/kg, with the same mole of gastrodin moiety). Non-compartmental pharmacokinetic profiles were constructed using the software of WinNonlin (Phoenix, version 6.3), and the pharmacokinetic parameters were compared using unpaired Student's t-test. RESULTS: The results showed that the pharmacokinetic parameters, including Cmax, Tmax, AUC0-∞, t1/2, MRT, Vd, CL, were quite different among the three types of gastrodin administration. The administration of parishin and G. elata extract, which either could convert to gastrodin in vivo or contained free gastrodin and abundant gastrodin conjugates, gave rise to higher elimination half-life (t1/2) and mean residence time (MRT) values for gastrodin compared to free gastrodin administered. CONCLUSION: The comparison of the pharmacokinetics of gastrodin among three different administered types of gastrodin in rats suggested that administration of parishin or G. elata extract in clinic may result in a longer duration time of action than that of the administration of free gastrodin. The results may provide some guidance for the clinical applications of parishin and G. elata.

Pharmacokinetic study of Gastrodia elata in rats.

The pharmacokinetics of parishin, gastrodin, Gastrodia elata extract and Rhizoma Gastrodiae capsule was investigated by intragastric and/or intravenous administration to rats. Parishin was metabolized into nine metabolites after intravenous administration, and the area under the curve (AUC0-∞) of parishin and its metabolites (except parishin G and parishin E) increased nonlinearly from 72.5 to 220 mg/kg. When combining regression equation with the AUC0-∞ and dose of gastrodin injection, the percent conversion of parishin to gastrodin was obtained as 50 %. Based on multi-active metabolites of parishin in vivo, integrated pharmacokinetic mode was established. It is notable that each metabolite from parishin shares the similar metabolic process at three dosages of parishin and the bioavailability of parishin was approximately 14 %. The integrated pharmacokinetic mode was successfully applied to evaluate the holistic pharmacokinetics of gastrodin injection, G. elata extract and Rhizoma Gastrodiae capsule. The results showed that the holistic pharmacokinetics of gastrodin injection and G. elata extract was closed to that of gastrodin, but for parishin and Rhizoma Gastrodiae capsule, integrated pharmacokinetic parameters were more suitable to evaluate its holistic pharmacokinetics. Graphical abstract Pharmacokinetic study of Gastrodia elata in rats.

[Pharmacokinetic study on combined application of gastrodin and puerarin in rats].

To establish a HPLC method for simultaneously determining plasma concentrations of gastrodin (Gas) and its metabolites hydroxybenzyl alcohol (HBA), puerarin (Pur) and internal standard (IS) p-hydroxyphenylethanol (Tyr) in rats and studying the pharmacokinetic process and interactions of gastrodin and puerarin after single and combined intravenous injection and oral administration. With Tyr as the internal standard, plasma samples were processed with methanol for protein precipitation, supernatant was dried with N2, and residues were re-dissolved with acetonitrile-0.05% phosphoric acid (20: 80). Chromatography was carried out on an Agilent ZORBAX SB-Aq C18 column (4.6 mm x 250 mm, 5 µm), with acetonitrile-0.05% phosphoric acid as the gradient mobile phase for the gradient elution. The UV detector wavelength was set at 221 nm for Gas HBA and IS and 250 nm for Pur. After the single or combined administration of Gas and Pur, their plasma concentrations in rats were detected. WinNonlin 5.2 pharmacokinetic software and SPSS 17. 0 software were used to respectively calculate pharmacokinetic parameters of each group, make a statistical analysis and compare the pharmacokinetic processes of Gas and Pur after the single or combined administration. According to the results, the absolute recoveries between low, media and high concentrations of Gas, HBA and Pur and IS as well as Tyr were more than 77.20%, with a good linearity (r > 0.999 6, n = 5) for Gas, HBA and Pur within concentration ranges of 0.10-101, 0.03-7.58 and 0.05-5.98 mg xL ('1) respectively. The lower limits of quantification for Gas, HBA and Pur were 0.10, 0.03, 0.05 mg x L(-1), respectively. Their in-ra-day and inter-day precisions were less than 12% with the accuracy between 85. 1% -1 10. %. All of the three substances and IS were stable during the whole analysis process. The findings showed significant differences in the main in vivo pharmacokinetic parame-ers in rats (AUC, C.(max) T,½ T.(max) MRT) after the single and combined administration of Gas and Pur. Either after the oral adminis-ration or after the intravenous injection, parameters showed a lower clearance rate ( L) longer mean residence time ( RT) and higher relative bioavailability, especially after the oral administration. Specifically, the relative bioavailability of the combined oral ad-inistration of Pur was 10. 7 times of that of the single administration, while that of Gas was 1. times of that of the single administra-ion. The combined administration of Gas and Pur can promote the absorption, decrease the elimination rate and prolong the mean resi-ence time. The method is simple and accurate and can be applied in the simultaneous determination of plasma concentrations of Gas, HBA and Pur in rats and the pharmacokinetic studies.

Relative bioavailability of gastrodin and parishin from extract and powder of Gastrodiae rhizoma in rat.

A rapid, sensitive and reliable UHPLC-ESI-MS/MS method was developed for simultaneous determination of gastrodin and parishin in rat plasma. The LLOQ of the two analytes were 1.00×10(-1) and 8.30×10(-5)µg/mL, respectively. The intra-day and inter-day precision were all less than 10% of the relative standard deviation (RSD), whilst the accuracy were all within ±15% of the relative error (RE). The proposed method was successfully applied for pharmacokinetics study on the two analytes in rats after oral administration of Gastrodiae rhizoma (GR) extract and powder at low, medium and high dosages. Blood samples were collected from the suborbital vein at predetermined time points and were precipitated using methanol. Chromatographic separations were carried out on a Kinetex XB-C18 column (2.1mm×150mm, 1.7µm) with a gradient mobile phase of acetonitrile-water with 0.1% formic acid as a modifier. The pharmacokinetic parameters of the two analytes in rats were obtained and the relative bioavailability of gastrodin and parishin in two formulations were calculated. The results indicated that higher bioavailability was obtained when low dosage of GR powder was used, whereas, higher bioavailability values were obtained when medium and high dosages of GR extract were used.

Simultaneous determination of gastrodin and puerarin in rat plasma by HPLC and the application to their interaction on pharmacokinetics.

Gastrodin (Gas) and puerarin (Pur) are bioactive substances derived from traditional Chinese medicine Gastrodia elata and Radix Puerariae, respectively, which were often used together in Chinese clinical prescriptions. Their injections were used in combined way for treatment of some cardiocerebrovascular diseases in clinic, especially for vertigo due to vertebrobasilar ischemia. In this paper, interaction of gastrodin and puerarin in rat plasma pharmacokinetics via intragastic (i.g.)/intravenous (i.v.) administration was investigated. A reliable HPLC method was developed for simultaneous determination of Gas and Pur in rat plasma with a linear range of 0.101-101 µg/mL for Gas and 0.0500-5.98 µg/mL for Pur (r(2)>0.993). The LLOQ, LOD of Gas and Pur were determined to be 0.101, 0.0486 µg/mL, and 0.05, 0.0245 µg/mL, respectively. The intra-day and inter-day precision were all less than 12.0%, whilst the accuracy were all within 96.4±6.00%. The proposed method has been successfully applied to the pharmacokinetic study of the analytes in rats after i.g./i.v. administration of Gas and Pur alone or combined with each other (i.g.: 40 mg/kg Gas, 400 mg/kg Pur; i.v.: 20 mg/kg Gas, 20 mg/kg Pur). Blood samples were collected from retinal vein plexus of rats at predetermined time points and plasma containing the internal standard tyrosol (IS) were precipitated by methanol and chromatography was carried out on a C(18) column with a gradient mobile phase of ACN-H(2)O with 0.05% phosphoric acid as a modifier. The pharmacokinetic profiles of combined administration were found to be distinct from those of given alone. The C(max), T(max), T(1/2), MRT of Gas administrated alone or combined with Pur via i.g. were 21.7 µg/mL, 0.250 h, 2.81 h, 0.830 h and 18.4 µg/mL, 0.550 h, 0.970 h, 1.37 h, respectively, of Pur administrated alone or combined with Gas via i.g. were 0.490 µg/mL, 1.95 h, 1.33 h, 2.10 h and 2.01 µg/mL, 0.570 h, 4.00 h, 5.10 h, respectively. The relative oral bioavailability of Pur in combined administration was 10.7 times as much as that of single administration, whilst 1.52 folds in Gas. These results indicate that co-administration of Gas and Pur is a promising combination to gain higher bioavailability and it is suggested that doctors pay more attention to the dosages of the two when simultaneously using both of them.

[UPLC-MS analysis of constituents of dachuanxiong fang active parts absorbed into blood].

OBJECTIVE: To study the constituents absorbed into blood following the intragstric administration of Dachuanxiong Fang active parts(DCXF) to the experimental migrainous model rats induced by nitroglycerin. METHOD: The UPLC-MS was used as the analytic method. The DCXF, Chuanxiong active parts, Tianma active parts, gastrodin, ferulic acid and senkyunolide I were used as the control samples. The information on the total ion chromatogram, mass chromatogram and the mass spectrogram were synthetically analyzed to confirm the constituents absorbed into blood. RESULT: Ten of the DCXF constituents were detected in the rats plasma post the intragastric administration of DCXF, in whitch four including gastrodin came from Tianma active parts and six including ferulic acid and senkyunolide I from Chuanxiong active parts. CONCLUSION: The findings abtained from the study can provide the useful information for the determination of bioactive substances of the DCXF.

Pharmacokinetics comparative study of a novel Chinese traditional herbal formula and its compatibility.

ETHNOPHARMACOLOGICAL RELEVANCE: Da Chuan Xiong Decoction Compound preparation (DCXDCP), the formulation of a classical Chinese prescription recorded in "Xuanminglunfang", was clinically employed to treat migraine's disease. AIM OF THE STUDY: In order to investigate the influence of compatibility on the pharmacokinetics of the active ingredient gastrodin (GAS), the comparative evaluations on pharmacokinetics of DCXDCP with various combinations of its constituent herbs in plasma after oral administration were studied. MATERIALS AND METHODS: The rats were randomly assigned to four groups and orally administered with different prescription proportion of Gastrodia elata Bl. and Ligusticum chuanxiong Hort. (1:0; 1:0.25; 1:2.1; 1:4.2), respectively. At different predetermined time points after administration, the concentrations of GAS in rat plasma were determined by using HPLC, and main pharmacokinetic parameters were investigated. RESULTS: The results showed that the pharmacokinetic parameters, AUC and C(max) of GAS were dramatically different (p<0.05) after oral administration of G. elata Bl. and the different combinations of its constituent herbs. CONCLUSIONS: These indicated that the compatibility effects of other ingredients present in DCXDCP could affect the pharmacokinetics of the prescription.

Studies of the toxicological potential of capsinoids, XII: pharmacokinetic study of capsinoid-containing CH-19 Sweet extract in rats.

Pharmacokinetics of the main capsinoid components of CH-19 Sweet extract (capsiate, dihydrocapsiate, and nordihydrocapsiate) were investigated in rats receiving a single gavage dose of extract containing 10 or 100 mg of capsinoids per kilogram in medium-chain triglyceride. Resultant blood levels of these capsinoids and a capsinoid metabolite, vanillyl alcohol, were measured in portal vein and systemic blood. Capsinoids were never detected. Portal compartment vanillyl alcohol concentrations and area under the plasma concentration versus time curve increased approximately with dose, whereas the time to maximum concentration of vanillyl alcohol was independent of dose (30 minutes post dosing), suggesting that precipitation in the stomach or intestines was unlikely. Vanillyl alcohol levels were just barely detectable in systemic plasma (5 minutes post dosing). Significant levels of vanillyl alcohol conjugates, sulfate, and glucuronide were detected in the systemic blood. Given that the orally administered capsinoids were never detected in the portal vein or systemic circulation, these compounds must be broken down (chemically or enzymatically) to vanillyl alcohol.

[Pharmacokinetics of gastrodin from compound Tianma granule in rats].

To study the influence of the compatibility of ophiopogonis tuber and Chinese magnoliavine fruit with gastrodia rhizome on the pharmacokinetics of gastrodin in rat, three dosages of compound Tianma granule extract (equivalent to gastrodin 50, 100, 200 mg x kg(-1)) and one dosage of Tianma extract (equivalent to gastrodin 100 mg x kg(-1) were administered to rats by intragastric administration separately. Plasma samples were collected at different times and treated with methanol and acetonitrile to precipitate protein. The contents of gastrodin in plasma were determined by HPLC method. The mean plasma concentration-time curves of different medication administration teams were processed with WinNonlin 5.2.1 pharmacokinetic software. The pharmacokinetic parameters of different medication administration teams were analyzed with SPSS statistics 17.0 software. The results indicated that the in vivo kinetic process of gastrodin was fitted to first-order absorption un-compartment model at low, middle dosages and zero-order absorption un-compartment model at high dosage of compound Tianma granule extract. By comparison with the pharmacokinetic parameters of gastrodin (100 mg x kg(-1)) in Tianma extract, the significant decrease for Cmax and significant increase for MRT0-infinity in compound Tianma granule extract indicated that the compatibility of ophiopogonis tuber and Chinese magnoliavine fruit with Gastrodia rhizome can delay the absorption, reduce the elimination rate and prolong the action time of gastrodin in vivo.

Determination and pharmacokinetics of gastrodin and p-hydroxybenzylalcohol after oral administration of Gastrodia elata Bl. extract in rats by high-performance liquid chromatography-electrospray ionization mass spectrometric method.

In this report, a high-performance liquid chromatography-electrospray ionization mass spectrometric (HPLC-MS) method was used to determine gastrodin (GAS) and p-hydroxybenzylalcohol (HBA) in rat plasma after oral administration of Gastrodia elata Bl. (Chinese name: Tianma) extract. Up to 200 microl of plasma containing GAS, HBA and pyromucic acid (as internal standard, IS) were deproteinized with six volumes of methanol. Calibration curves showed linearity within the concentration range tested 2.00-200.00 microg/ml for GAS and 0.832-104.00 microg/ml for HBA in plasma with a correlation coefficient (r) greater than 0.9997. The limit of quantification of 2.00 and 0.83 microg/ml for GAS and HBA had been achieved, respectively. The intra-day and inter-day precisions of the method were determined to be less than 17.82% for GAS and 10.21% for HBA. The recoveries were in the range of 91.12-108.64% with RSD less than 7.80% for GAS and 92.91-106.14% with RSD less than 4.30% for HBA. Evidence showed that a rapid, simple and reproducible LC-MS assay was established to determine GAS and HBA in rat plasma.

Effects of gavage versus dosed feed administration on the toxicokinetics of benzyl acetate in rats and mice.

Effects of gavage versus dosed feed administration on the toxicokinetics of benzyl acetate were studied in male F344 rats and B6C3F1 mice. Benzyl acetate was rapidly hydrolysed to benzyl alcohol and then oxidized to benzoic acid. After gavage administration of benzyl acetate in corn oil at 500 mg/kg (rats) and 1000 mg/kg (mice), high benzoic acid plasma concentrations were observed. In contrast, much lower benzoic acid plasma concentrations were found after dosed feed administration at about 615 mg/kg/day for rats and about 850 mg/kg/day for mice. Results show that although the daily doses of benzyl acetate are comparable, bolus gavage administration effectively saturated the benzoic acid elimination pathway whereas dosed feed administration did not. In contrast, hippuric acid plasma concentrations were similar after both gavage and dosed feed administration due to the depletion of the glycine supply pool. Study results could explain the different toxicity and carcinogenicity responses of benzyl acetate observed in 2-yr chronic gavage and dosed feed studies.