A validated HPLC-MS/MS method for determination of genipin-1-o-glucuronic acid in rat plasma after administration of genipin and its application to a pharmacokinetic study.

A sensitive and specific method was developed and validated for the quantitation of one major metabolite of genipin in rats plasma. The major metabolite was isolated from rat bile via semi-preparative HPLC technology and its chemical structure was identified as genipin-1-o-glucuronic acid (GNP-GLU), which was for the first time used as a standard compound for quantitative analysis in rat plasma after administration of genipin. The application of high-performance liquid chromatography-tandem mass spectrometry in negative mode in multiple reaction monitoring mode was investigated. Chromatographic separation was achieved on an Eclipse XDB-C18 column using a mobile phase consisting of water with 0.1% formic acid (A)-acetonitrile (B). The limit of detecation was 0.214 ng/mL and the lower limit of quantification was 0.706 ng/mL. The calibration curve was linear from 1.27 to 3810 ng/mL for plasma samples, with a correlation coefficient of 0.9924. The intra- and inter-day precisions and accuracy were all within 15%. The recoveries of GNP-GLU and puerarin were above 90.0 and 76.2%, respectively. The highly sensitive method was successfully applied to estimate pharmacokinetic parameters of GNP-GLU following oral and intravenous administration of genipin to rats.

[Preparation of 20(S)-Protopanaxadiol-Phospholipid Complex HAP Assemblies].

OBJECTIVE: To study the preparation of 20(S)-protopanaxadiol-phospholipid (20(S)-PPD) complex HAP assemblies. METHODS: 20(S)-PPD phospholipid complex was assembled with the drug carriers of HAP. Effects of technological factors on the assembled amount were investigated, including HAP species, phospholipid complex concentration, ratio of HAP and assembled liquid, and then the preparation technology of 20(S)-PPD phospholipid complex HAP assemblies was determined. RESULTS: The average quality of phospholipid complex assembled was 136. 26 mg/g,the average assembled rate was 5.3% for 20(S)-PPD phospholipid complex HAP assemblies prepared by the determined assembly process. FT-IR showed that 20(S)-PPD phospholipid complex was absorbed in the HAP assemblies, and the hydrogen-bonding effect was the main mechanism of HAP assemblies to assemble and adsorb phospholipid complex. The cumulative release rate in pH 7.4 phosphate buffer of the HAP assemblies indicated that the assemblies had the effect of sustained release. CONCLUSION: The phospholipid complex HAP assembles have the advantages of simple preparation process, and sustaining release effect, which can provide preliminary research foundation for research and development of 20(S)-PPD sustained-release preparations.

Quantification of conjugated metabolites of drugs in biological matrices after the hydrolysis with ß-glucuronidase and sufatase: a review of bio-analytical methods.

Glucuronidation and sulfation represent two major pathways in phase II drug metabolism in humans and other mammalian species. The great majority of drugs, for example, polyphenols, flavonoids and anthraquinones, could be transformed into sulfated and glucuronidated conjugates simultaneously and extensively in vivo. The pharmacological activities of drug conjugations are normally decreased compared with those of their free forms. However, some drug conjugates may either bear biological activities themselves or serve as excellent sources of biologically active compounds. As the bioactivities of drugs are thought to be relevant to the kinetics of their conjugates, it is essential to study the pharmacokinetic behaviors of the conjugates in more detail. Unfortunately, the free forms of drugs cannot be detected directly in most cases if their glucuronides and sulfates are the predominant forms in biological samples. Nevertheless, an initial enzymatic hydrolysis step using ß-glucuronidase and/or sulfatase is usually performed to convert the glucuronidated and/or sulfated conjugates to their free forms prior to the extraction, purification and other subsequent analysis steps in the literature. This review provides fundamental information on drug metabolism pathways, the bio-analytical strategies for the quantification of various drug conjugates, and the applications of the analytical methods to pharmacokinetic studies.

[Study on the extraction process and macroporous resin for purification of Timosaponin B II].

OBJECTIVE: To optimize the extraction process and macroporous resin for purification of Timosaponin B II from Anemarrhena asphodeloides. METHODS: Orthogonal design L9 (34) was employed to optimize the circumfluence extraction conditions by taking the extraction yield of Timosaponin B II as index. The absorption-desorption characteristics of eight kinds of macroporous resins were evaluated, then the best resin was chosen to optimize the purification process conditions. RESULTS: The optimum extraction conditions were as follows: the herb was extracted for 2 times (2 hours each time) with 8.5-fold 50% ethanol at the first time and 6-fold 50% ethanol at the second time. HPD100 resin showed a good property for the absorption-desorption of Timosaponin B II. The optimum technological conditions of HPD100 resin were as follows:the solution concentration was 0.23 mg/mL, the amount of saturated adsorption at 4/5 body volumn (BV) resin, the HPD100 resin was washed with 3 BV water and 6 BV 20% ethanol solution to remove the impurity, then the Timosaponin B II was desorbed by 5 BV ethanol solution. The purity of Timosaponin B II was about 50%. CONCLUSION: The optimized extraction process and purification is stable, efficient and suitable for industrial production.

HPLC-MS/MS method to determine genipin in rat plasma after hydrolysis with sulfatase and its application to a pharmacokinetic study.

A sensitive high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the quantification of genipin in rat plasma after hydrolysis with sulfatase. Genipin could not be detected directly as it could be transformed into other forms such as conjugated-genipin immediately after administration. The conjugated genipin could be hydrolyzed by sulfatase to genipin. The conditions of hydrolysis were investigated. Genipin and the internal standard, peoniflorin (IS), were separated on a reversed-phase column by gradient elution and detected using an electrospray ion source on a 4000 QTrap triple-quadrupole mass spectrometer. The quantification was performed using multiple reaction monitoring with selected precursor-product ion pairs of the transitions m/z 225.0 → 122.7 and m/z 479.1 → 449.1 for genipin and peoniflorin. The assay was linear over the concentration range of 1.368-1368 ng/mL, with correlation coefficients of 0.9989. Intra- and inter-day precisions and accuracy were all within 15%. The lower limit of quantification was 1.368 ng/mL. The recoveries of genipin and peoniflorin were more than 53.3 and 51.2%. The highly sensitive method was successfully applied to estimated pharmacokinetic parameters of genipin following oral and intravenous administration to rats. The absolute bioavailability of genipin was 80.2% in rat, which is the first report.

[Research on the preparative method of Arctigenin].

OBJECTIVE: To research on the preparation of Arctigenin in vitro. METHODS: Took enzyme concentration, time course and substrate concentration as investigation factors, used Box-Behnken design-response surface methodology to optimize the enzyme hydrolysis path of Arctigenin. RESULTS: The best operational path for Arctigenin was as follows: the temperature was 50 degrees C, pH was 4.8, enzyme concentration was 0.44 U/mL, time course was 46.81 min, substrate concentration was 0.29 mg/mL, the conversion rate was 90.94%. CONCLUSION: This research can be regarded as a referencein preparing Arctigenin in vitro.

[Purification of geniposide in the extract fluid of Gardenia jasminoides with macroporous absorption resin].

OBJECTIVE: To optimize the process for purification of geniposide in the extract fluid of Gardenia jasminoides with macroporous absorption resin. METHODS: By comparing the content and transfer ratio of geniposide during the process of purification, we optimized the process for purification of geniposide. RESULTS: The optimal process for purification of geniposide with D301R macroporous absorption resin included the diameter height ratio 1:7.5, the concentration of the extract fluid of Gardenia jasminoides 2:1, the flow rate 1BV/h (1BV represented one column volume), the sample volume 1/3BV. We loaded the sample and left it for 2 hours, afterwards, rinsed the macroporous absorption resin using 2BV water until the solution became colourless. Then we rinsed the macroporous absorption resin with 20% alcohol,and the volume of elution solvent was 2BV. We collected 20% alcohol elution solvent and recycle alcohol using rotating evaporation and dried the rest solution in a vacuum to get the light yellow powder which was the purified geniposide. CONCLUSION: This process is simple and affordable, can be used to refine and purify geniposide in the extract fluid of Gardenia jasminoides Ellis. It provides a guidance for industrial production basis.

[Technology study on extraction and purification of alkaloid from Stemona japonica].

OBJECTIVE: In order to obtain the optimal conditions for separating the alkaloids from the extract of Stemona japonica by selecting appropriate cation exchange resins. METHODS: Seven types of cation exchange resins were evaluated in separating efficiency with measuring the adsorption ratio and eluting ratio of total alkaloids as indices, and the content of total alkaloids from Stemona japonica was determined as an index by spectrophotometry to choose the optimal technological parameters. RESULTS: The optimal result of extraction was obtained as Stemona japonica shattered into thick powder, adding eight times amount of 90% alcohol and refluxing and extracting for 3 h (totally extracting for 3 times). Each gram of D004 cation exchange resin could absorb 0.5003 mg of the total alkaloid, and the desorption ratio was 68.45%. The transfer rate of total alkaloids was 58.70%. the product purity of alkaloids was up to 70%. CONCLUSION: The D004 cation exchange resin can be used for purificating total alkaloids from Stemona japonica and the established procedure is simple and feasible.

[Pharmacokinetics of pueraria for intranasal on spray in rabbits].

OBJECTIVE: To explore pharmacokinetic features of puerarin in pueraria spray and calculate pharmacokinetic parameters according to puerarin of drug-time curve in rabbits. METHODS: The concentration of puerarin in plamsa was determined by HPLC. The methanol was used to sediment protine. The 3P87 program was used to calculate the pharmacokinetic parameters. RESULTS: The vivo course of pueraria in spray could be described by the two compartment model. The main pharmacokinetic parameters of Pueraria spray were: t(l/2(beta)) =0.93 h, CL =44.23 mg x L(-1), AUC = 16.28 mg x h x L(-1), Cmax =5.9 mg x L(-1) and tmax = 0.975 h. CONCLUSION: The study will provide some scientific basises for the quality evaluation and pharmaceutics reformation of pueraria for intranasal.

Advances in extraction and analysis of phenolic compounds from plant materials.

Phenolic compounds, the most abundant secondary metabolites in plants, have received more and more attention in recent years because of their distinct bioactivities. This review summarizes different types of phenolic compounds and their extraction and analytical methods used in the recent reports, involving 59 phenolic compounds from 52 kinds of plants. The extraction methods include solid-liquid extraction, ultrasound-assisted extractions, microwave-assisted extractions, supercritical fluid extraction, and other methods. The analysis methods include spectrophotometry, gas chromatography, liquid chromatography, thin-layer chromatography, capillary electrophoresis, and near-infrared spectroscopy. After illustrating the specific conditions of the analytical methods, the advantages and disadvantages of each method are also summarized, pointing out their respective suitability. This review provides valuable reference for identification and/or quantification of phenolic compounds from natural products.

Simultaneous determination of dihydromyricetin and resveratrol in Ampelopsis sinica (Miq.) W.T. Wang by high-performance liquid chromatography coupled with a diode array detection method.

Yeputaoteng is the dried ground part of Ampelopsis sinica (Miq.) W.T. Wang, which is widely used in traditional Chinese medicine for preventing and treating tumors, chronic nephritis, hepatitis, rubella, traumatic bleeding, stomach heat and vomiting. A simple and reliable method using high-performance liquid chromatography with diode array detection (HPLC-DAD) was developed for the simultaneous determination of dihydromyricetin and resveratrol in Yeputaoteng. The chromatographic analysis was performed on a Dikma C18 column (250 × 4.6 mm, 5 µm) at 30°C with a gradient elution of acetonitrile and 0.1% phosphoric acid at a flow rate of 1 mL/min, and used ultraviolet detection at 292 and 306 nm. The established method was validated in terms of linearity, precision, reproducibility, stability and recovery. The calibration curves showed good linear regression (R(2) > 0.9994). Limits of detection and quantification fell in the ranges of 1.47-2.48 and 2.93-4.97 µg/mL, respectively. The mean recovery of dihydromyricetin and resveratrol was 104.1% [relative standard deviation (RSD): 2.94%] and 100.8% (RSD: 2.80%), respectively. The quantitative results indicated that the HPLC-DAD method can be effectively applied to the quality control of Yeputaoteng and its preparations.

Potential hepatotoxicity of geniposide, the major iridoid glycoside in dried ripe fruits of Gardenia jasminoides (Zhi-zi).

The safety of geniposide, mainly focusing on its hepatotoxicity in rats, was determined by liver enzymes in serum and histopathology ultrastructural preparation. The lethal dose, 50% (LD50) of per oral geniposide was 1431.1 mg kg(-1). The acute toxicity study indicated geniposide at dose of 574 mg kg(-1) or more could cause hepatic toxicity in rats and the hepatotoxicity often appeared at 24-48 h after the oral administration. The hepatotoxicity was associated with oxidative stress with decrease of total superoxide dismutase activity and increase of malondialdehyde concentration in rats' livers. Subchronic toxicity study showed geniposide did not cause hepatotoxicity at the doses of 24.3 and 72.9 mg kg(-1) orally for 90 days in rats. Thus, acute hepatotoxicity of geniposide at high doses was likely to be linked to oxidative stress, while geniposide at normal dose of 24.3 mg kg(-1) or less did not cause hepatotoxicity even in the repeated dosing study.

A convenient and efficient one-pot conversion of peimisine into cyclopamine.

A convenient and efficient one-pot synthesis of cyclopamine from peimisine is described. The key steps involve one-pot hydrazination and subsequent Bamford-Stevens reaction. The mild reaction conditions, high overall yield as well as an easy purification indicate this process can potentially be used for the scale-up preparation of cyclopamine.

Antibacterial constituents from Stemona sessilifolia.

Bioassay-guided fractionation led to the isolation of eight compounds from Stemona sessilifolia. Of the eight isolates, three new bibenzyls, stilbostemins M-O (1-3), and a new tocopherol, 6-methoxy-3,4-dehydro-delta-tocopherol (4) were revealed together with four known compounds 3,5-dihydroxy-2'-methoxy bibenzyl (5), 3,5-dihydroxy bibenzyl (6), beta-tocopherol (7), and gamma-tocopherol (8). Compounds 5, 6, and 8 exhibited strong antibacterial activities against Staphylococcus aureus and S. epidermidis.