[Simultaneous determination of sixteen mycotoxins contaminants in cogon rootstalk by QuEChERS-UPLC-QqQ mass spectrometry].

A method for the simultaneous determination of sixteen mycotoxins in cogon rootstalk was developed using ultra-performance liquid chromatography coupled with triple quadropole mass spectrometry(UPLC-QqQ-MS/MS). The samples were extracted with acetonitrile contained 1% acetic acid and purified by QuEChERS method. The separation was performed on an Agilent Eclipse Plus C18column by gradient elution using methanol and 0.01% aqueous formic acid as mobile phase. The targeted compounds were detected in MRM mode by mass spectrometry with electrospray ionization(ESI)source operated in positive ionization mode. The linear relationships of the sixteen mycotoxins were good in their respective linear ranges. The correlation coefficients(r)ranged among 0.996 2-1.000. The LOQs of the sixteen mycotoxins were between 0.03 and 186.68 µg•kg ⁻¹. The average recoveries ranged from 60.28% to 129.2% with relative standard deviations(RSDs)within 0.29%-11%. The results demonstrated that the proposed method was sensitive and accurate, and suitable for the mycotoxins quantification in cogon rootstalk.

[Determination of 7 flavonol glycosides in Ginkgo biloba reference extract].

Six flavonol glycosides were isolated and calibrated from Ginkgo biloba extract, and then used to calibrate the content in 2 baiches of G. biloba reference extract, so was rutin. RSD values of rutin, kaempferol-3-O-rutinoside, kaempferol-3-O-rhamnoside-2-glu- coside, quercetin-3-O-rhamnop-yranosyl-2-O-(6-O-p-coumaroyl)-glucoside, kaempferol-3-O-rhamnopyranosyl-2-O-(6-O-p-coum-aroyl) - glucoside were around 1.1%-4.6%, nevertheless, RSD values of quercetin-3-O-glucoside and isorhamnetin-3-O-rutinoside were more than 5%. According to the results, the reference extract of G. biloba can be used as the substitute to determine rutin, kaempferol-3-O- rutinoside, kaempferol-3-O-rhamnoside-2-glucoside, quercetin-3-O-rhamnopyranosyl-2-O-(6-O-p-coumaroyl)-glucoside and kaempferol-3-0-rhamnopyranosyl-2-O-(6-O-p-coumaroyl)-glucoside instead of corresponding reference substances. So reference extract in place of single component reference in assay is feasible.

New oleanane-type triterpenoid saponins isolated from the seeds of Celosia argentea.

Three new oleanane-type triterpenoid saponins named celosins H, I, and J were isolated from the seeds of Celosia argentea L. Their structures were characterized as 3-O-ß-D-xylopyranosyl-(1 → 3)-ß-D-glucuronopyranosyl-polygalagenin 28-O-ß-D-glucopyranosyl ester, 3-O-ß-D-glucuronopyranosyl-medicagenic acid 28-O-ß-D-xylcopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 2)-ß-D-fucopyranosyl ester, and 3-O-ß-D-glucuronopyranosyl-medicagenic acid 28-O-α-L-arabinopyranosyl-(1 → 3)-[ß-D-xylcopyranosyl-(1 → 4)]-α-L-rhamnopyranosyl-(1 → 2)-ß-D-fucopyranosyl ester by NMR, MS, and chemical evidences, respectively. In our opinion, celosins H-J could be used as chemical markers for the quality control of C. argentea seeds.

[Metabolic pathway and metabolites of total diterpene acid isolated from Pseudolarix kaempferi].

The preliminary metabolic profile of total diterpene acid (TDA) isolated from Pseudolarix kaempferi was investigated by using in vivo and in vitro tests. Pseudolaric acid C2 (PC2) was identified as the predominant metabolite in plasma, urine, bile and feces after both oral and intravenous administrations to rats using HPLC-UV and HPLC-ESI/MS(n), and demethoxydeacetoxypseudolaric acid B (DDPB), a metabolite proposed to be the glucoside of PC2 (PC2G), as well as pseudolaric acid C (PC), pseudolaric acid A (PA), pseudolaric acid A O-beta-D glucopyranoside (PAG), pseudolaric acid B O-beta-D glucopyranoside (PBG) and deacetylpseudolaric acid A (DPA) originated from TDA could also be detected. It was demonstrated by tests that the metabolism of TDA is independent of intestinal microflora, and neither of pepsin and trypsin is in charge of metabolism of TDA, TDA is also stable in both pH environments of gastric tract and intestinal tract. The metabolites of TDA in whole blood in vitro incubation were found to be PC2, DDPB and PC2G, which demonstrated that the metabolic reaction of TDA in vivo is mainly occurred in blood and contributed to be the hydrolysis of plasma esterase to ester bond, as well as the glucosylation reaction. These results clarified the metabolic pathway of TDA for the first time, which is of great significance to the in vivo active form and acting mechanism research of P. kaempferi.

Simultaneous determination of eleven flavonoid glycosides in ginkgo biloba leaves collected in different seasons by UPLC PDA method.

A new UPLC method was developed for the simultaneous determination of eleven characteristic flavonoid glycosides in Ginkgo biloba leaves. The natural occurrence of flavonoid glycosides in Ginkgo biloba leaves within one vegetative season was investigated for the first time. The analysis was performed on an Agilent ZORBAX Eclipse Plus C18 column (50 mm x 4.6 mm, 1.8 microm), the mobile phase A was acetonitrile, the mobile phase B was 0.4% phosphate aqueous solution in a gradient elution at a flow rate of 0.6 mL x min(-1), the detection was carried out at 360 nm. The result showed that eleven flavonoid glycosides had good linearity with good average recovery, separately. The method was proved to be accurate, rapid and good reproducible for the quality evaluation of Ginkgo biloba leaves, and provide an easy and rapid means for the quantitative analysis of flavonoid glycosides and their content fluctuation with seasons.

[Metabolic pathway and metabolites of pseudolaric acid B].

The metabolic profile of pseudolaric acid B (PB) was investigated by using in vivo and in vitro tests. Pseudolaric acid C2 (PC2) was identified as the specific metabolite of PB in plasma, urine, bile and feces using HPLC and HPLC-ESI/MS(n) after both oral and intravenous administration to rats, and almost no prototype was detected in all kinds of samples. The metabolic behaviors of PB orally administered in rats treated with antibiotics to eliminate intestinal microflora were identical with those in untreated rats, demonstrating that the metabolism of PB is independent of intestinal microflora. PB was stable in 48 h respective incubation with artificial gastric juice and artificial intestinal juice, suggesting that neither pepsin nor trypsin is in charge of metabolism of PB, and also demonstrating that PB is stable in both pH environments of gastric tract and intestinal tract. In vitro research on metabolism of PB in rat liver microsomes incubation revealed that little PB was metabolized and that the proposed metabolites were the demethoxy and demethoxydecarboxy products of the prototype. The amount of metabolites was extremely low compared with the prototype, indicating that liver microsomes are not responsible for the metabolism of PB either. PB was gradually metabolized into PC2 during 1 h in whole blood incubation in vitro, and the metabolic process showed dynamically dependent manner with incubation time. Once absorbed into blood, PB was quickly metabolized into PC2, accordingly, little prototype was detected in all kinds of samples. The metabolism was attributed to the rapid hydrolysis of C-19 ester bond by plasma esterase. These results clarified the metabolic pathway of PB for the first time, which was of great significance to identify the in vivo active form and interpret acting mechanism of the active compounds of P. kaempferi.

Characterization of diterpenoids in the bark of Pseudolarix kaempferi by HPLC-ESI/MSn.

Fragmentation behavior of diterpenoids was investigated by ESI/MSn and the qualitative analysis of diterpenoids in the bark of Pseudolarix kaempferi was performed using high-performance liquid chromatography/ multi-stage mass spectrometry (HPLC-ESI/MSn). The characteristic fragmentation behaviors of the diterpenoids are the cleavages of the lactone ring and C4-O bond. Furthermore, the eliminations of substituent groups at C-18, C-7 and C-8 can also be observed in the MS" (n = 3-4) spectra. For C-4 acetoxy subsititued diterpenoids, [M+Na-60]+ and [M-H-104] are the base peaks of MS2 spectra in the positive and negative ionization modes, respectively. For C-4 hydroxyl subsititued diterpenoids, [M+Na-44]+ and [M-H-62] are the base peaks of MS2 in the positive and negative ionization modes, respectively. For C-18 glucosylated or esterized diterpenoids, [M+Na-44]+ is the base peak of MS2 spectra in positive ionization mode. These fragmentation rules were successfully exploited in the identification of diterpenoids in methanol/water (6:4) extract of P. kaempferi by LC-MS in positive ionization mode. A total of 9 diterpenoids were identified or tentatively characterized, and one of them is reported here for the first time. The described method could be utilized for the sensitive and rapid qualitative analysis of P. kaempferi.

[Fingerprint analysis of Radix Glycyrrhizae by fast HPLC].

The objective of this paper is to develop a fast analysis method to determine fingerprints of Radix Glycyrrhizae from different areas of China for identification and quality control. The experiments were carried out under following conditions: Agilent Eclipse Plus C18 (4.6 mm x 50 mm, 1.8 microm) column, acetonitrile and 0. 05% phosphoric acid solution as the mobile phases with gradient elution, flow rate 1.0 mL x min(-1), analysis time 11 min. The run time of the method was obviously decreased from 36 minutes to 11 minutes compared with routine HPLC method. The cluster analyses of the fingerprints of the 70 samples were performed by SPSS. The results showed that all samples were classified into 2 groups, 59 Glycyrrhiza uralensis as well as 11 G. inflata. Three compounds, liquiritin apioside, liquiritin and glycyrrhiza acid should be considered as effective references for quality control of Radix Glycyrrhizae. This method can be used widely for identification and quality control of Radix Glycyrrhizae.

Simultaneous determination of six major stilbenes and flavonoids in Smilax china by high performance liquid chromatography.

A simple, sensitive and specific HPLC method was developed for simultaneous determination of the six major active constituents in Smilax china, namely taxifolin-3-O-glycoside (1), piceid (2), oxyresveratrol (3), engeletin (4), resveratrol (5) and scirpusin A (6), respectively. The samples were separated on an Aglient Zorbax XDB-C18 column with gradient elution of acetonitrile and 0.02% phosphoric acid (v/v) at a flow rate of 1.0 ml/min and detected at 300 nm. The six target compounds were completely separated within 35 min. All calibration curves showed good linearity (r2>0.999) within test ranges. The reproducibility was evaluated by intra- and inter-day assays and R.S.D. values were less than 3.7%. The recoveries were between 93.7 and 103.0%. The method was successfully applied to the analysis of six constituents in 15 commercial samples of S. china. The results indicated that the developed HPLC assay was readily utilized as a quality control method for S. china.

Simultaneous quantification of six major phenolic acids in the roots of Salvia miltiorrhiza and four related traditional Chinese medicinal preparations by HPLC-DAD method.

A high-performance liquid chromatographic method was applied to the determination of danshensu, protocatechuic aldehyde, rosmarinic acid, lithospermic acid, salvianolic acid B and salvianolic acid A in the roots of Salvia miltiorrhiza and four related traditional Chinese medicinal preparations. The six phenolic acids were simultaneously analyzed with a Zorbax Extend C18 column by gradient elution using 0.026% (v/v) phosphoric acid and acetonitrile as the mobile phase. The flow rate was 1 ml min(-1), and detection wavelength was set at 288 nm. The recovery of the method was in the range of 95.1-104.8%, and all the compounds showed good linearity (r > 0.9997) in a relatively wide concentration range. This assay was successfully applied to the determination of six major phenolic acids in 32 samples. The results indicated that the developed HPLC assay could be readily utilized as a quality control method for S. miltiorrhiza and its related traditional Chinese medicinal preparations.

[Studies on chemical constituents of hairy root of Cassia obtusifolia].

OBJECTIVE: To study the chemical constituents in hairy roots of Cassia obtusifolia. METHOD: The hairy roots of C. obtusifolia were induced with Agrobacterium rhizogenes LBA9402 from cotyledons and cultured in MSO liquid medium. The compounds were isolated by silica gel, polyamide and Sephadex LH-20 column chromatography, and the structures were elucidated by employing chemical and spectral methods RESULT: Eight compounds were isolated from the ethyl acetate fraction of 95% EtOH extract of the transformed roots of C. obtusifolia. They are betulinic acid, chrysophanol, physcion, stigmasterol, 1-hydroxy-7-methoxy-3-methyl-anthraquinone, 8-O-methylchrysophanol, 1-O-methylchrysophanol and aloe-emodin, and aloe-emodin was isolated from the hairy roots of C. obtusifolia for the first time. CONCLUSION: The hairy roots of C. obtusifolia have the ability to synthesize the similar chemical constituents as the original plants.

[Study on influence of processing methods on chemical constituents in Radix Paeoniae Alba].

OBJECTIVE: The influence of processing methods on chemical constituents in Radix Paeoniae Alba was observed. METHOD: A HPLC method was used for analyzing the changes of eight major constituents, namely gallic acid, paeoniflorin sulfonate, catechin, paeoniflorin sulfonate, albiflorin, paeoniflorin, benzoic acid, pentagalloylglucose and benzoylpaeoniflorin, with the three processing procedures of decorticating, boiling and fumigating by burning of sulphur. Analysis was performed using a Zorbax SB-C18 column (4.6 mm x 250 mm, 5 microm) with the mixture of acetonitrile (A) and 0.015% phosphoric acid solution as mobile phase in gradient mode. The detection wavelength was set at 230 nm and the column temperature was at 30 degrees C. RESULT: Except for gallic acid and pentagalloylglucose, the other constituents decreased during procedure of decorticating and boiling. Fumigating by burning of sulphur would produce a new compound, paeoniflorin sulfonate, which was a byproduct from the reaction of paeoniflorin with SO2. CONCLUSION: The significant changes were produced in chemical constituents of Radix Paeoniae Alba during three processing procedures. Therefore, the processing of Radix Paeoniae Alba should be strictly controlled and standardized.

Simultaneous determination of gallic acid, albiflorin, paeoniflorin, ferulic acid and benzoic acid in Si-Wu decoction by high-performance liquid chromatography DAD method.

A high-performance liquid chromatographic method was applied to the determination of gallic acid, albiflorin, paeoniflorin, ferulic acid and benzoic acid in Si-Wu decoction and other 13 combinations of the formula. These five compounds were analyzed simultaneously with a Zorbox SB C-18 column by gradient elution using 0.01% (v/v) phosphoric acid-acetonitrile as the mobile phase. The flow rate was 1 ml min(-1), and detection was set at 230 nm. The recovery of the method was in the range of 94.8-103.1%, and all the compounds showed good linearity (r>0.9995) in a relatively wide concentration range. The result indicated that the content of these five compounds changed after decocting process. The contents of paeoniflorin, albiflorin, ferulic acid and gallic acid increased and that of benzoic acid decreased significantly.

Simultaneous determination of 10 major flavonoids in Dalbergia odorifera by high performance liquid chromatography.

A reversed-phase liquid chromatographic method was developed for the simultaneous quantification of 10 major flavonoids, namely butin, (3R)-4'-methoxy-2',3,7-trihydroxyisoflavanone, liquiritigenin, melanettin, violanone, vistitone, formononetin, dalbergin, sativanone and medicarpin in the heartwood of Dalbergia odorifera, an important traditional Chinese medicine. Samples were extracted with 60% methanol. The optimal conditions of separation and detection were achieved on an Agilent Zorbax SB-C18 column (250 mm x 4.6 mm, 5 microm) with a gradient of acetonitrile and 0.3% (v/v) aqueous acetic acid, at a flow rate of 0.8 ml/min, detected at 275 nm. The complete separation was obtained within 55 min for the 10 target compounds. All calibration curves showed good linearity (r2>0.999) within test ranges. The assay was reproducible with overall intra- and inter-day variation of less than 3%. The mean recovery of the method was 100+/-10%, with R.S.D. less than 5%. The current assay method was considered to be suitable for the quality control of D. odorifera samples and could be readily utilized for the determination of the active principles present in this medicinal herb.

Simultaneous quantification of six major active saponins of Panax notoginseng by high-performance liquid chromatography-UV method.

A simple, sensitive and specific high-performance liquid chromatography-UV (HPLC-UV) method has been developed for the first time to simultaneously quantify the six major active saponins of Panax notoginseng, namely notoginsenoside R1, ginsenoside Rg1, Rb1, Rg2, Rh1 and Rd. Astragaloside IV is used as the internal standard. This HPLC assay was performed on a reversed-phase C18 column with gradient elution of acetonitrile and 0.01% formic acid in 30 min. The method provided good reproducibility and sensitivity for the quantification of six saponins with overall intra- and inter-day precision and accuracy of less than 4.0% and higher than 90%, respectively. This assay is successfully applied to the determination of the six saponins in 23 notoginseng samples. The results indicated that the developed HPLC assay can be readily utilized as a quality control method for P. notoginseng.

The effects of verbascoside on plasma lipid peroxidation level and erythrocyte membrane fluidity during immobilization in rabbits: a time course study.

We studied the changes in the level of plasma lipid peroxidation indicated by malondialdehyde (MDA) level and erythrocyte membrane fluidity expressed by the value of order parameter (S) during single hindlimb immobilization of 21 days using thiobarbituric acid - reactive substances method and spin label electron spin resonance method, respectively. The impacts of verbascoside that has been proved its antioxidative activity on the measured parameters were examined. 11 New Zealand white rabbits were immobilized and divided into two groups. The rabbits in the verbascoside group were administrated with 0.8 mg/kg of verbascoside twice a day orally throughout the immobilization. The rabbits in the placebo group were treated with normal saline. In placebo group, the results showed that the level of MDA significantly increased on day 3, peaked on day 7, and was still significantly higher on day 14 of immobilization, compared with the value measured before immobilization. The value of S reached the highest on day 7 and subsequently lowered but still higher on day 14 than those measured before immobilization. Compared with placebo group, there were lower MDA level (P < 0.05, 0.001, and 0.05 for days 3, 7, and 14, respectively) and higher erythrocyte membrane fluidity (P < 0.05, 0.001, and 0.001 for days 3, 7, and 14, respectively) in verbascoside group. The data indicated that immobilization caused temporal changes of increase in plasma lipid peroxidation and decrease in erythrocyte membrane fluidity. Verbascoside might have the effects to moderate oxidative stress and erythrocyte membrane fluidity during immobilization.

Liquid chromatographic method for determination of four active saponins from Panax notoginseng in rat urine using solid-phase extraction.

Four major active saponins (ginsenosides Rg1, Rb1, Rd and notoginsenoside R1) in Panax notoginseng were determined in rat urine after oral and intravenous administration of total saponins of P. notoginseng (PNS), and the urine samples were treated with solid-phase extraction (SPE) prior to liquid chromatography. A reversed-phase liquid chromatography system with ultraviolet detection and a Zorbax SB-C18 column was used. The within-day and between-day assay coefficients of variation for the four saponins in urine were less than 7% and the recovery of this method was higher than 85%. Using this method, the excretion profile of the drug in rat urine after administration of PNS was revealed for the first time.

SPE-HPLC method for the determination and pharmacokinetic studies on paeoniflorin in rat serum after oral administration of traditional Chinese medicinal preparation Guan-Xin-Er-Hao decoction.

A new HPLC method for the determination of paeoniflorin in rat serum with solid-phase extraction (SPE) for preconcentration is introduced. Paeoniflorin and an internal standard (pentoxifylline) were extracted from serum by means of SPE using cartridges with octadecyl chemically bound phase. The HPLC separation was then performed on a reversed-phase C(18) column using acetonitrile-water (18:82, v/v) as eluting solvent system, and UV detection at 230 nm to measure the analyte with a limit of quantitation about 10 ng ml(-1). The calibration curve for paeoniflorin was linear (r=0.9938) in the concentration range of 10-1200 ng ml(-1), both intra- and inter-day precision of the paeoniflorin were determined and their coefficience of variation did not exceed 10%. The validated method has been successfully applied for pharmacokinetic studies of paeoniflorin from rat serum after oral administration of Guan-Xin-Er-Hao decoction.

[Microbial transformation of sinenxan A, a rich constituent in callus cultures of Taxus].

AIM: To study the microbial transformation of sinenxan A. METHODS: Choose two strains of Fungi (Mucor spinosus AS 3.3450 and Cunninghamella echinulata AS 3.3400) and a strain of bacterium (Proteus vulgaris AS 1.1208) to transform the substrate. RESULTS: Three products were obtained and identified as 10-deacetylsinenxan A1, 6 alpha-hydroxy-10-deacetylsinenxan A2 and 9 alpha-hydroxy-10-deacetylsinenxan A3 respectively. CONCLUSION: Sinenxan A is facile to be transformed by microorganisms, the 10-acetyl group of which is an active group.

[Progress in biotransformation of triptolides and bufadienolides].

The latest results from our research group in the biotransformation of triptolides and bufadienolides were reviewed. The trends in the development of biotransformation in the future were also briefly discussed.