Bupleurum scorzonerifolium: Systematic research through pharmacodynamics and serum pharmacochemistry on screening antidepressant Q-markers for quality control.

Bupleurum scorzonerifolium (BS) is one of the sources of Bupleuri Radix, which was first recorded in Shennong's classic of materia medica. It has a medicinal history of 2000 years and is now widely used for the treatment of depression clinically. However, the material basis of antidepressant effects is unclear, and the quality evaluation method is lacking. The paper aims to investigate the antidepressant quality markers (Q-markers) of BS by electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC-ESI-Q-TOF-MS). Firstly, the rat depression model was established by using chronic unpredictable mild stress (CUMS) combined with the solitary confinement method to evaluate the pharmacodynamics of BS. After verification of the antidepressant effect of BS, UPLC-ESI-Q-TOF-MS was used to analyze BS and the blood components of BS. A total of 34 components were identified in BS, in which 8 components, including saikosaponin a (SSa), saikosaponin c (SSc), saikosaponin d (SSd), saikosaponin b1 (SSb1), saikosaponin b2 (SSb2), glycyrrhetinic acid, nootkatone and valerenic acid, were detected in serum. SSa, SSc, SSd, SSb1 and SSb2 were found as metabolites, and glycyrrhetinic acid, nootkatone and valerenic Acid were identified as the prototypes in the blood. The depression model of zebrafish was established with reserpine to verify the antidepressant effect of the potential eight active components. The results showed that all these components could markedly improve the depressive behavior of zebrafish, increase the content of 5-HT and reduce the cortisol content. Finally, according to the principles of effectiveness, accessibility and measurability for Q-markers, SSa, SSc, and SSd were confirmed as Q-markers of BS, and the contents of 3 Q-markers in 10 batches of BS from different origins were determined to be 0.0728-1.465%. In addition, the total contents of 3 Q-markers in BS produced in Lindian, Heilongjiang Province, were higher than those in other origins. This paper provided a reliable method for the quality evaluation of BS for depression treatment.

[Study on correlation between color and effective components contents of Glycyrrhiza uralensis].

To explore the correlation between color of Glycyrrhiza uralensis and its quality evaluation,the colors of root bark and transverse section were determined by Precision Color Reader and Visual Analyzer,and the contents of six flavonoids and two saponins in G.uralensis were determined by high performance liquid chromatography（HPLC）.The partial least squares regression（PLSR）method was employed to correlate the colors with component contents in G.uralensis. The results showed that there were no significant differences in the colors of root bark but significant or very significant differences（P<0.05,P<0.01）in the colors of transverse section between the wild and cultivated G. uralensis. Compared with those in the cultivated G. uralensis, the contents of liquiritin, isoliquiritin isoliquiritigenin and the contents of ammonium glycyrrhizinate, glycyrrhetinic acid were obviously significant or remarkably significant in the wild G. uralensis.The correlation results showed that there was a significant or very significant correlation between the colors and the effective component contents. This study provides a scientific basis to evaluate the quality of G.uralensis by color and a new reference for the traditional evaluation methods for Chinese drugs.

Simultaneous Determination of Twenty-Five Compounds in Rat Plasma Using Ultra-High Performance Liquid Chromatography-Polarity Switching Tandem Mass Spectrometry and Its Application to a Pharmacokinetic Study.

An attempt was made to characterize the pharmacokinetic profiles of Qishen Keli (QSKL) that has been widely proved to be effective in clinical practice. A method using ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) for the simultaneous determination of 25 analytes in rat plasma was developed and validated. Satisfactory chromatographic separation was achieved on an ACQUITY UPLC HSS T3 column with gradient elution using mobile phase consisting of 0.02% aqueous formic acid (A) and acetonitrile fortified with 0.02% formic acid (B), and analyte detection was carried out using polarity-switching multiple reaction monitoring mode. Method validation assays in terms of selectivity, linearity, inter- and intra-day variations, matrix effect, and recovery demonstrated the newly developed method to be specific, sensitive, accurate, and precise. Following the oral administration of QSKL at a single dose, the qualified method was successfully applied for pharmacokinetic investigations in sham and model rats. Mild differences occurred for the pharmacokinetic patterns of most components between those two groups, whereas significant differences were observed for glycyrrhizic acid and glycyrrhetic acid. The obtained findings could provide meaningful information for the clarification of the effective material basis of QSKL.

[Determination of 8 components in healthy food for anti-hangover and hepatoprotection by high performance liquid chromatography].

OBJECTIVE: To develop a simple and sensitive high performance liquid chromatographic method for simultaneous determination of catechin hydrate, epicatechin, epigallocatechin, epicatechin gallate, epigallocatechin gallate, dihydromyricetin, glycyrrhizic acid and glycyrrhetinic acid in healthy food for anti-hangover and hepatoprotection, and compare with the capillary electrophoresis method established by our laboratory. METHODS: The samples were ultrasonically extracted by using methanol-water( 4∶ 1, V/V) for 30 minutes and then centrifuged at 10 000 r/min for 10 minutes. The supernatant was filtered and injected into the HPLC system and then separated on a C_(18) column( 5 µm × 250 mm × 4. 6 mm) at 30℃ with gradient elution at a flow rate of 0. 8mL/min. Catechins and dihydromyricetin were detected at the wavelength of 210 nm, glycyrrhizic acid and glycyrrhetinic acid were detected at 250 nm. RESULTS: Under the optimal analytical conditions, the peak area of each analyte and its concentration had agood correlation within the linear range( r ≥ 0. 9996). The limits of detection and quantification of the method were 0. 07-1. 25 µg/g( S/N = 3) and 0. 22-4. 18 µg/g( S/N = 10), respectively. The intra-and inter-day relative standard deviations( RSDs)of the mixed standard solution were 0. 26%-1. 95% and 1. 17%-3. 89%, respectively. The spiked recoveries of the analytes were 86. 15%-98. 61%. CONCLUSION: The established method is sensitive and reliable, and could be used for quality control of the healthy food for anti-hangover and hepatoprotection.

LC-MS/MS as a tool for identification of bioactive compounds in marine sponge Spongosorites halichondriodes (Dendy 1905).

The sensitivity, specificity and selectivity of liquid chromatography/mass spectrometry tandem mass spectrometry (LC-MS/MS) make it an essential tool for the characterization and identification of low molecular compounds such as fatty acids, sterols, cholastane derivatives, nucleosides etc. In the current work, the marine sponge Spongosorites halichondriodes (order Halichondrida, Family Halichondriidae); a particularly rich source of cytotoxic compounds is studied for the initial characterization of bioactive compounds. The composition of ethyl acetate and butanol extracts were subjected to LC-MS and LC-MS/MS. Many novel sterol derivatives compounds which were not reported in any marine sponge mainly belonged to the group of C25-C28 saturated and unsaturated esters like 3ß, 4ß, 7α, 12α-tetrahydroxy-5ß-cholan-24-oic acid methyl ester, 7 α, 12 ß-dihydroxy-5 ß-cholan24-oic acid methyl ester, novel isocoumarin citrinolactone A, a triterpenoid glycyrrhetinic acid as well as other unknown compounds in this species such as nucleoside inosine was identified. Other compound investigated was 3ß, 6ß, 7α-trihydroxy-5ß-cholan-24-oic acid methyl ester. All the sterol ester derivatives are reported here for the first time in marine sponge belonging to family Halichondriidae. However, the literature report supports the occurrence of 3ß-hydroxy sterols which is considered as a biomarker for this family.

[Simultaneous determination of five main index components and specific chromatograms analysis in Xiaochaihu granules].

Reversed phase high performance liquid chromatography with diode array detector was employed for simultaneous determination of five main index components and specific chromatograms analysis in Xiaochaihu granules with a linear gradient elution of acetonitrile-water (containing 0.1% phosphoric acid) as mobile phase. The results showed that five main index components (baicalin, baicalein, wogonoside, wogonin, enoxolone) were separated well under the analytical condition. The linear ranges of five components were 0.518 - 16.576, 0.069 - 2.197, 0.167 - 5.333, 0.009 - 0.297 and 0.006 - 0.270 mg x g(-1), respectively. The correlation coefficients were 0.999 9, and the average recoveries ranged from 95% to 105%. Twelve common peaks were selected as the specific chromatograms of Xiaochaihu granules with baicalin as the reference peak. There were good similarities between the reference and the ten batches of samples. The similarity coefficients were no less than 0.9. The analytical method established is highly sensitive with strong specificity and it can be used efficiently in the quality control of Xiaochaihu granules.

Determination of corosolic acid, a natural potential anti-diabetes compound, in rat plasma by high-performance liquid chromatography-mass spectrometry and its application to pharmacokinetic and bioavailability studies.

A simple, robust, and sensitive high-performance liquid chromatography/mass spectrometric method was developed for the determination of corosolic acid, a potential anti-diabetes substance, in rat plasma using glycyrrhetinic acid as the internal standard (IS). This method involved a liquid-liquid extraction with acetic ether and a subsequent analysis performed on an LC-MS system which contained an electrospray ionization interface. Chromatographic separation was performed using an ODS column, and the mobile phase was composed of methanol and 5 mmol/L ammonium acetate (88 : 12, v/v). Good linearity was observed over the concentration range of 20-10 ,000 ng/mL with a correlation coefficient (r² ≥ 0.995. The method was proved to be accurate and reliable and was applied to a pharmacokinetic study in the rat following intragastric and intravenous administration of corosolic acid.

Identification and quantification of oleanolic acid and ursolic acid in Chinese herbs by liquid chromatography-ion trap mass spectrometry.

A rapid and sensitive method for the identification and quantification of ursolic acid (UA) and oleanolic acid (OA) in Chinese herbs is described. The method combines liquid chromatography (LC) with ion trap-mass spectrometry (IT-MS) detection. The UA and OA standard solution were directly infused into IT-MS for collecting MS(n) spectra. The major fragment ions of UA and OA were confirmed by MS(n) at m/z 455, 407, 391, 377 and 363 in negative ion mode, and m/z 457, 439, 411 and 393 in positive mode, respectively. The possible main cleavage pathway of fragment ions was studied. UA and OA provided good signals corresponding to the deprotonated molecular ion [M - H](-). The method is reliable and reproducible, and the detection limit is 5 ng/mL. The method was validated in the concentration range of 0.04-40 µg/mL; intra- and inter-day precisions ranged from 0.78 to 2.15%, and the accuracy was 96.5-108.2% for UA and OA. The mean recovery of UA and OA was 97.1-106.2% with RSD less than 1.86%. An LC-IT-MS method was successfully applied to determine the UA and OA in nine Chinese herbs.

Quality control of modified xiaoyao san through the determination of 22 active components by ultra-performance liquid chromatography.

A simple, sensitive, and reliable ultra-performance liquid chromatography (UPLC) method has been developed for simultaneous determination of 22 major constituents in modified xiaoyao san (MXS), a multiherbal formula. The chromatographic separation was performed on an ACQUITY UPLC BEH C18 column (150 x 2.1 mm, 1.7 microm, particle size), with an aqueous 0.5% acetic acid and acetonitrile mobile phase gradient. The method was validated for linearity (r2 >0.9937), intraday and interday precision (RSD <8.51%), recovery (91.18-107.73%), LOD (0.02-4.17 ng/mL), and LOQ (0.05-12.50 ng/mL). The established method was successfully applied to quantify the 22 marker compounds in MXS, which provided a useful basis of overall evaluation of the quality of MXS.

An automated method of on-line extraction coupled with flow injection and capillary electrophoresis for phytochemical analysis.

In this study, an automated system for phytochemical analysis was successfully fabricated for the first time in our laboratory. The system included on-line decocting, filtering, cooling, sample introducing, separation, and detection, which greatly simplified the sample preparation and shortened the analysis time. Samples from the decoction extract were drawn every 5 min through an on-line filter and a condenser pipe to the sample loop from which 20-µL samples were injected into the running buffer and transported into a split-flow interface coupling the flow injection and capillary electrophoresis systems. The separation of glycyrrhetinic acid (GTA) and glycyrrhizic acid (GA) took less than 5 min by using a 10 mM borate buffer (adjusted pH to 8.8) and +10 kV voltage. Calibration curves showed good linearity with correlation coefficients (R) more than 0.9991. The intra-day repeatabilities (n = 5, expressed as relative standard deviation) of the proposed system, obtained using GTA and GA standards, were 1.1% and 0.8% for migration time and 0.7% and 0.9% for peak area, respectively. The mean recoveries of GTA and GA in the off-line extract of Glycyrrhiza uralensis Fisch root were better than 99.0%. The limits of detection (signal-to-noise ratio = 3) of the proposed method were 6.2 µg/mL and 6.9 µg/mL for GTA and GA, respectively. The dynamic changes of GTA and GA on the decoction time were obtained during the on-line decoction process of Glycyrrhiza uralensis Fisch root.

Determination of glycyrrhetic acid after consumption of liquorice and application to a fatality.

Besides alcohol and drugs of abuse, several popular foods contain potentially toxic substances and cases of intoxication after consumption of these foods attract notice of forensic toxicology. This is also true for the case of a 34-year-old woman who was suspected to have suffered lethal acute intoxication from eating nothing but liquorice over a period of several months. The liquorice ingredient glycyrrhizin and its metabolite glycyrrhetic acid, which elicits a mineralocorticoid effect, were determined in the sort of liquorice the woman had consumed by using LC-MS/MS. In addition, a fast and sensitive procedure for the quantification of glycyrrhetic acid including a simple sample preparation was developed. The method was proven to be accurate and precise. In a liquorice ingestion experiment, 200 g of liquorice had to be eaten. Afterwards, concentrations of glycyrrhetic acid in the blood of up to 434 ng/ml were measured. Since only traces of glycyrrhetic acid had been found in the blood and stomach content of the deceased woman, the possibility of acute lethal glycyrrhetic acid intoxication could be eliminated. Excluding other causes of death, the woman is believed to have died from a lethal hyperglycemic coma. Nonetheless, the influence of harmful and toxic substances in food should be taken into consideration in special cases.

[Separation and refine of Xiaoyao Pill by macroporous adsorption resin].

OBJECTIVE: To search for the method used in refining Xiaoyao Pill by macroporous adsorption resin, 12 types of macroporous adsorption resin were optimized. METHOD: Static and dynamic adsorption test and de-adsorption test were carried out to screen the best macroporous resin. The single factor test was applied to optimize the manipulation parameters of macroporous resin. RESULT: The macroporous resin D-101-1 possessed the strongest adsorption ability, in addition to an easy de-adsorption property. CONCLUSION: The D-101-1 type macroporous adsorption resin shows better comprehensive adsorption property. It is available for the refine Xiaoyao Pill.

Simultaneous HPLC analysis, with isocratic elution, of glycyrrhizin and glycyrrhetic acid in liquorice roots and confectionery products.

Glycyrrhizin (1), the main active principle of Glycyrrhiza glabra (liquorice) roots, is extensively used in herbal medicines, in pharmaceutical preparations and confectionery products. A feasible and reliable method which allows the simultaneous analysis of 1 and its aglycone, 18beta-glycyrrhetic acid (2), by means of an isocratic HPLC procedure is described. The system uses a C8 column as the stationary phase, and a mixture of acetonitrile, methanol, water and glacial acetic acid as the mobile phase. Good linearity was found in the concentration ranges 1-50 and 0.05-2.50 microg/mL for 1 and 2, respectively. A simple and rapid sample pre-treatment, based on the extraction of the two analytes with a mixture of water and ethanol, was developed for the examination of liquorice confectionery products and root samples. The HPLC method was shown to be appropriate, in terms of precision and feasibility, for the quality control of the analytes in these matrices.

Determination of oleanolic acid in human plasma and study of its pharmacokinetics in Chinese healthy male volunteers by HPLC tandem mass spectrometry.

A highly selective and sensitive HPLC-ESI-MS-MS method was developed for the determination of oleanolic acid in human plasma. The oleanolic acid and glycyrrhetinic acid (internal standard) were recovered from plasma with ethyl acetate liquid-liquid extraction. The organic extracts were dried under a stream of warm nitrogen, reconstituted in mobile phase and injected into a Zorbax-Extend ODS analytical column (150 mm x 4.6 mm i.d., 5 microm), with the mobile phase consisting of methanol-ammonium acetate (32.5 mM) (85:15, v/v) pumped at a flow rate of 1.0 ml/min, and 30% of the eluent was split into a MS system with electrospray ionization tandem mass (ESI-MS-MS) detection in negative ion mode. The tandem mass detection was performed on a Finnigan Surveyor LC-TSQ Quantum Ultra AM tandem mass spectrometer operated in selected reaction monitoring mode. The parent to product ion combinations of m/z 455.4-->455.4 and 469.3-->425.2 at 38 V 1.5 mTorr Ar CID were used to quantify oleanolic acid and glycyrrhetinic acid, respectively. The assay was validated in the concentration range of 0.02-30.0 ng/ml for oleacolic acid when 0.5 ml of plasma was processed. The precision of the assay (expressed as relative standard deviation, R.S.D.%) was less than 15% at all concentrations levels within the tested range and adequate accuracy, and the limit of quantification was 0.02 ng/ml. The established method was applied for the pharmacokinetics study of oleanolic acid capsules in 18 healthy male Chinese volunteers with the mean values of C(max), T(max), AUC(0-48), AUC(0-infinity), t(1/2,) CL/F, and V/F of oleanolic acid after p.o. a single 40 mg dose obtained were 12.12 +/- 6.84 ng/ml, 5.2 +/- 2.9h, 114.34 +/- 74.87 ng h/ml, 124.29 +/- 106.77 ng h/ml, 8.73 +/- 6.11 h, 555.3 +/- 347.7 L/h, and 3371.1 +/- 1,990.1 L, respectively.

The influence of the sennosides on absorption of glycyrrhetic acid in rats.

In the course of our clinical studies of Kampo medicine (traditional Japanese medicines), we observed the pharmacokinetic interactions between two herbs. When Onpito (TJ-8117, Kampo medicine) containing licorice and rhubarb was administered orally to human subjects, we observed that the AUC(0-lim) and Cmax of glycyrrhetic acid (GA) in plasma were lower than those treated with other Kampo medicines containing licorice. In this study, we demonstrate the pharmacokinetic interactions of GA derived from glycyrrhizinic acid (GL) in licorice and anthraquinones derived from rhubarb. To our knowledge, this is the first report to investigate the pharmacokinetic interactions between two herbs. When GL was orally co-administrated to rats with a non-effective dose of sennoside A having purgative activity, the AUC(0-lim) and Cmax of GA decreased. In addition, sennoside A did not affect the metabolism of GL by the intestinal bacteria in vitro. In the examination using an in situ loop of rat colon, the remaining ratio of GA rose drastically by the co-administration of sennoside A, sennidin A and rhein. Observed inhibition activity of these anthraquinones on GA absorption depended on the concentration of the components added. The maximum inhibition ratio was approximately 75% by rhein, 60% by sennoside A and 25% by sennidin A. We conclude that the decrease of the pharmacokinetic parameters of GA in human plasma observed in the clinical study of TJ-8117 is attributable to an interactive action of absorption from the intestinal tract by anthraquinones contained in or derived from rhubarb.

Use of nano-liquid chromatography for the analysis of glycyrrhizin and glycyrrhetic acid in licorice roots and candies.

Glycyrrhizin (G) and glycyrrhetic acid (GA) were separated by using nano-liquid chromatography (nano-LC) in a fused silica capillary packed with RP18 stationary phase (75 microm ID, effective length 33 cm, packed 23 cm) eluting at 300 nL/min in a gradient mode. The mobile phase was a mixture of water-MeOH-MeCN-acetic acid (29:35:35:1, v/v/v/v) that was delivered for one minute and after this was modified by reducing the water content (14:42.5:42.5:1, v/v/v/v). The intra-day and inter-day relative standard deviations (of retention time and peak area) were satisfactory (lower than 2.9 and 4%, respectively). The linearity of the nano-LC method was assessed in the range 0.62-5.00 microg/mL and 80-200 microg/mL for GA and G, with R2 = 0.996 and 0.995, respectively. The licorice was extracted with a mixture of ethanol-water, diluted with the mobile phase, and injected for the analysis.

Analysis and comparison of Radix Glycyrrhizae (licorice) from Europe and China by capillary-zone electrophoresis (CZE).

A simple capillary-zone electrophoresis (CZE) method for the analysis of plant specimens, Glycyrrhiza glabra L., G. uralensisFisch. and G. inflata Bat. (Leguminosae) as well as commercial licorices from Europe and China was developed. Contents of glycyrrhizin (GL), glycyrrhetic acid (GA), glabridin (GLAB), liquiritin (LQ) and licochalcone A (LC(A)) in ethanolic extracts were investigated. Optimum separation was achieved with sodium tetraborate buffer (pH 9.22; 70 mM); voltage, 25 kV. Recovery rate for GL was found to be 101.90+/-2.54%. Adequate correlation was observed between GL contents measured by CZE and HPLC (r=0.977). Advantages over conventional HPLC analysis of Glycyrrhiza species are short analysis time (<15 min), simple running buffer preparation and the none-use of organic solvents. Using the present CZE method, it was demonstrated that (1) G. glabra was distinguished from G. uralensis especially by phenolic compounds GLAB (G. glabra: 0.19+/-0.11%; n=53) and LQ (G. uralensis, 1.34+/-0.34%, n=10); (2) on average, GL contents were higher in Chinese commercial licorices; (3) relatively high LC(A) contents were especially detected in a Chinese commercial licorice (origin estimated as G. inflata); (4) Glycyrrhiza species were also distinguished by applying PCA on the basis of CZE peak area data of GL, GLAB, GA, LQ and LC(A); and (5) liquiritin apioside was found in all samples.

[Studies on the shade-endurance capacity of Glycyrrhiza uralensis].

OBJECTIVE: To study the shade-endurance property of Glycyrrhiza uralensis and provide rationale for the practice of inter-cropping G. uralensis with trees. METHOD: Black shading nets were used to provide five different environments of light intensities (light penetration rates of 100%, 75%, 65%, 50% and 25%, respectively). To assess the shade-endurance capacity of G. uralensis, several aspects were evaluated, including growth characters, physiological and ecological characters, biomass, and chemical contents. RESULT AND CONCLUSION: G. uralensis is a light-favored plant. The growth indices such as plant height, stem diameter, leaves number, root diameter, biomass, and daily average photosynthetic rate (Pn) are highest when light permeation rate is 100%. All these indices decrease when light intensity decreases. However, G. uralensis possesses shade-endurance capacity to some degree; it adapts to the shading environment by increasing the leaf area and chlorophyll contents. Shading has no obvious effect on the absolute light energy utilization rate (Eu) or Fv/Fm ratio. The influence of shading on the chemical contents of G. uralensis is obvious.

Chemical analysis of raw, dry-roasted, and honey-roasted licorice by capillary electrophoresis.

In herbal medicine, licorice is usually processed using a roasting procedure which might modify the chemical compositions in licorice. To test this hypothesis, licorice root samples were roasted under various conditions (with or without honey) and subsequently extracted by refluxing with 95% ethanol. The analysis of chemical compositions of licorice root extracts was achieved by capillary electrophoresis. The running buffer has been optimized to be 50 mM sodium tetraborate (pH 9.01) containing 5 mM beta-cyclodextrin. Thermal decomposition of glycyrrhizin, which was a major ingredient in licorice, was first studied in detail, indicating the conversion of glycyrrhizin to glycyrrhetinic acid. The licorice extracts were then analyzed to indicate the above thermal conversion did occur in the licorice samples. This finding may shed some light on understanding the differences in the therapeutic values of raw versus roasted licorice in herbal medicine.