Analysis of Mechanism of Yishen Huashi Granules for Improving Diabetic Nephropathy Based on Metabonomics and Transcriptomics / 中国实验方剂学杂志

ObjectiveIn this study， based on ultra-high performance liquid chromatography-mass spectrometry（UHPLC-MS/MS） and high-throughput transcriptome sequencing technology（RNA-seq）， we investigated the mechanism of Yishen Huashi granules in regulating serum metabolites and renal messenger ribonucleic acid（mRNA） expression to improve diabetic kidney disease（DKD）. MethodSD rats were randomly divided into normal group ， model group and Yishen Huashi granules group， with 8 rats in each group. The rat model of DKD was established by intraperitoneal injection of streptozotocin. Yishen Huashi granules group was given 5.54 g·kg-1·d-1 of Yishen Huashi granules by gavage， and the normal group and the model group were given the same amount of normal saline for 6 weeks. During the experiment， the body weight and blood glucose of rats were monitored， and the rats were anesthetized 24 hours after the last administration， blood was collected from the inferior vena cava， serum was separated， and renal function， blood lipid， and inflammatory indicators were detected. Kidney tissue of rats was fixed in neutral paraformaldehyde， and stained with hematoxylin-eosin（HE）， Masson and periodic acid-Schiff（PAS） to observe the renal pathological changes. UHPLC-MS/MS and RNA-seq were used to identify the changes of serum metabolism and the differences of renal mRNA expression， and real time fluorescence quantitative polymerase chain reaction（Real-time PCR） and Western blot were used to detect the differential mRNA and protein expression in renal tissue to explore the common expression mechanism. ResultCompared with the normal group， rats in the model group showed a decrease in body weight， a significant increase in blood glucose， urinary microalbumin to urinary creatinine ratio（UACR）， blood urea nitrogen（BUN）， cystatin-C（Cys-C）， β2-microglobulin（β2-MG）， interleukin-6（IL-6）， triglyceride（TG） and total cholesterol（TC）， and a significant decrease in total superoxide dismutase（T-SOD）（P<0.01）. After the intervention of Yishen Huashi granules， all the indexes were improved to different degrees in rats（P<0.05， P<0.01）. Compared with the normal group， the model group showed renal mesangial stromal hyperplasia， fibrous tissue hyperplasia and tubular vacuolar degeneration. Compared with the model group， the renal pathology of rats in Yishen Huashi granules group was improved to a certain extent. A total of 14 target metabolites and 96 target mRNAs were identified， the target metabolites were mainly enriched in 20 metabolic pathways， including sphingolipid metabolism， glycerophospholipid metabolism， and the biosynthesis of phenylalanine， tyrosine and tryptophan. The target mRNAs were enriched to obtain a total of 21 differential mRNAs involved in the TOP20 pathways closely related to glycolipid metabolism. A total of 6 pathways， glycerophospholipid metabolism， arachidonic acid metabolism， purine metabolism， primary bile acid biosynthesis， ascorbic acid and uronic acid metabolism， and galactose metabolism， were enriched by serum differential metabolites and renal differential mRNAs， among them， there were 7 differential metabolites such as phosphatidylethanolamine（PE） and 7 differential mRNAs such as recombinant adenylate cyclase 3（ADCY3）. Seven differential metabolites had high predictive accuracy as verified by receiver operating characteristic（ROC） curve， and the results of Real-time PCR and Western blot were highly consistent with the sequencing results. ConclusionYishen Huashi granules can reduce UACR， BUN and other biochemical indexes， correct the disorder of glucose and lipid metabolism， and improve renal function of DKD rats. And its mechanism may be related to the regulation of the level of PE and other blood metabolites， and expression of Phospho1 and other mRNAs in the kidney， of which six pathways， including glycerophospholipid metabolism， may play an important role.

Metabonomic Analysis of Wenfei Huaxian Granules Against Idiopathic Pulmonary Fibrosis in Mice / 中国实验方剂学杂志

ObjectiveTo investigate the changes of differential metabolites in the serum of mice at different stages of bleomycin sulfate（BLM）-induced pulmonary fibrosis modeling and administration， and the mechanism of Wenfei Huaxian granules（WHG）against idiopathic pulmonary fibrosis. MethodMice were randomly divided into control group， control group of 14 days， model group， model group of 14 days， low-dose WHG group and high-dose WHG group. BLM（0.04 U per mouse）was injected into the trachea of mice in the model group， model group of 14 days， low-dose WHG group and high-dose WHG group， and sterile normal saline was injected into the trachea of mice in the control group and control group of 14 days. Mice of low-dose WHG group and high-dose WHG group were given different doses of WHG by gavage every day after injection of BLM， and mice of control group， control group of 14 days， model group and model group of 14 days were given sterile water by gavage every day. The peripheral blood of mice in the control group of 14 days and model group of 14 days were taken to prepare serum after injection of BLM for 14 days， and the peripheral blood and other materials of mice in the other groups were taken after continuous administration for 28 days. The bronchoalveolar lavage fluid（BALF）was collected for leucocyte differential count， the pathological examination and hydroxyproline（HYP）content determination of lung tissues of mice were performed， and the small molecule metabolites in serum samples of mice in each group were determined by ultra-high performance liquid chromatography-mass spectrometry（UHPLC-MS）. Principal component analysis（PCA）and orthogonal partial least squares-discriminant analysis（OPLS-DA）were conducted to screen differential metabolites and their biological functions were analyzed. ResultCompared with the control group， a large number of continuous fibrotic foci appeared in the lung tissue of mice in the model group， the alveolitis score， fibrosis degree score and HYP content increased significantly（P<0.01）， and the total number of leukocytes， macrophages and lymphocytes in BALF increased significantly（P<0.05）. A total of 33 differential metabolites were screened between the control group of 14 days and model group of 14 days， mainly lipid metabolites， which were mainly involved in oxidative damage and inflammatory process. A total of 34 differential metabolites， mainly amino acid metabolites， were screened between the control group and model group， mainly involving nucleic acid damage and inflammatory process. Compared with the model group， the HYP content， fibrosis score and alveolitis score in the lung tissue of mice from high-dose WHG group decreased significantly（P<0.05， P<0.01）， and the total number of lymphocytes in BALF decreased significantly（P<0.05）. Compared with the model group， 27， 40 differential metabolites were identified in the serum of mice from the low-dose WHG group and high-dose WHG group separately. There were totally 9 common differential metabolites between the model group and low-dose WHG group/high-dose WHG group， which mainly involved in the metabolic pathways of inflammation related lipids metabolism， arginine and tryptophan metabolism， and the change trends in low-dose WHG group and high-dose WHG group were significantly back-regulated compared with the model group. ConclusionWHG can alleviate BLM-induced pulmonary fibrosis， collagen deposition and inflammatory reaction in mice， and its anti-fibrotic effect may be related to the adjusting of inflammatory factors， nitric oxide and oxidative stress related metabolic pathways.

Simultaneous determination of seven components and their distribution in Anoectochilus roxburghii by UHPLC-MS/MS / 药学学报

Based on ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), a rapid and simultaneous quantitative method for the measurement of seven components (kinsenoside; rutin; kaempferol-3-O-rutinoside; quercimeritrin; narcissin; isorhamnetin-3-O-glucoside; quercetin) of A. roxburghii was established. The separation was performed over 8.0 minutes on a Waters Acquity UPLC BEH Shield RP18 (2.1 mm × 100 mm; 1.7 μm) with a mobile phase consisting of acetonitrile (A) and 0.1% formic acid water solution (B) with gradient elution at a flow rate of 0.2 mL·min-1; the column temperature was 30 ℃ and the injection volume was 2 μL. Electrospray spray ionization source (ESI source) was used for mass spectrometry, and positive and negative ion modes were detected at the same time. The results showed good linearity (R2 ≥ 0.998 0), with good precision, repeatability and stability, and the average recovery was 97.71%-103.33%. Through cluster heat map and redundancy analysis, we found that kinsenoside was mainly distributed in stems, followed by leaves, and the lowest content in roots. The content of kinsenoside increased significantly in the stems of plants 6 months, but less change was evident in the roots and leaves. Flavonoids and flavonoid glycosides were mainly distributed in leaves. The UHPLC-MS/MS method established in this paper can be used for the quality control of A. roxburghii and provides a reference for establishing a more comprehensive quality detection method for this medicinal.

Characterization of multiple chemical components of GuiLingJi by UHPLC-MS and 1H NMR analysis / 药物分析学报

GuiLingJi(GLJ),a classic traditional Chinese medicine(TCM)formula,is composed of over 20 herbs,according to the Pharmacopeia of the People's Republic of China.Owing to its various activities,GLJ has been used in clinical settings for more than 400 years in China.However,the ambiguous chemical material basis limits the development of studies on the quality control and pharmacological mechanisms of GLJ.Therefore,comprehensive characterization of the multiple chemical components of GLJ is of great significance for the modernization of this formula.Given the great variety of herbs in GLJ,both UHPLC-MS and 1H NMR techniques were employed in this study.In addition,solvent extraction with different polarities was used to eliminate signal interference and the concentration of trace components.A variety of MS analytic methods were also used,including implementation of a self-built compound database,diagnostic ion filtering,mass defect filtering,and Compound Discoverer 3.0 analysis software.Based on the above strategies,a total of 150 compounds were identified,including 5 amino acids,13 phenolic acids and glycosides,11 coumarins,72 flavones,20 triterpenoid and triterpenoid saponins,23 fatty acids,and 6 other compounds.Moreover,13 compounds were identified by 1H NMR spectroscopy.The UHPLC-MS and 1H NMR results supported and complemented each other.This strategy provides a rapid approach to analyzing and identifying the chemical composition of Chinese herbal prescriptions.The current study provides basis for further research on the quality control and pharmacological mechanism of GLJ.

Determination of eight active components of Bufei Huoxue Capsules in rat plasma and their pharmacokinetics by UHPLC-MS/MS / 中国中药杂志

An ultra-high performance liquid chromatography-tandem mass spectrometry(UHPLC-MS/MS) method was established to investigate the pharmacokinetic behaviors of psoralenoside, isopsoralenoside, calycosin-7-glucoside, ononin, psoralen, isopsoralen, methylnissolin, and neobavaisoflavone in rat plasma after oral administration of Bufei Huoxue Capsules. After SD rats were administered with Bufei Huoxue Capsules suspension by gavage, blood samples were collected from the inner canthus at different time points. After protein precipitation, plasma samples were separated on ACQUITY UPLC BEH C_(18) column(2.1 mm×100 mm, 1.7 μm). The mobile phase consisted of acetonitrile(A) and water(B) containing 0.1% formic acid in gradient elution. The positive and negative ions were measured simultaneously in the multi-reaction monitoring(MRM) mode. The pharmacokinetic parameters were calculated and fitted by DAS 3.2.8. Psoralenoside, isopsoralenoside, calycosin-7-glucoside, ononin, psoralen, isopsoralen, methylnissolin, and neobavaisoflavone were detected in the rat plasma after drug administration, with AUC_(0-t) of(3 357±1 348),(3 555±1 696),(3.03±0.88),(2.21±0.33),(1 787±522),(2 295±539),(5.69±1.41) and(3.40±0.75) μg·L~(-1)·h, and T_(max) of(1.56±0.62),(1.40±0.70),(0.21±0.05),(0.25±0.12),(0.26±0.11),(0.34±0.29),(0.74±0.59), and 0.25 h. The method is proved specific and repeatable and is suitable for the determination of psoralenoside, isopsoralenoside, calycosin-7-glucoside, ononin, pso-ralen, isopsoralen, methylnissolin, and neobavaisoflavone in the rat plasma, which can be applied to pharmacokinetic study.

Rapid quantitative analysis of 12 chemical constituents in wild-simulated and cultivated Astragali Radix based on UHPLC-MS / 中草药·英文版

Objective: Astragali Radix (AR) is one of the most widely used traditional Chinese medicines (TCMs) for tonic, which can be divided into wild-simulated and cultivated AR according to its cultivation method. However, whether cultivated AR can replace wild-simulated AR has always been a concern. Methods: In this study, a rapid, highly sensitive and specific analytical method using ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS) was developed to quantitatively measure 12 chemical constituents of AR in the different cultivation methods. Results: AR samples were analyzed with a good linear regression relationship (R

The effect of acute hypobaric hypoxia on bile acid composition in the small intestine of rats / 药学学报

In order to clarify the influence of acute hypobaric hypoxia on the bile acids of the rat small intestine, we used ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) to identify bile acids in the contents of the small intestine from untreated and acute hypobaric hypoxia-treated rats. Thirty-nine bile acids were detected; PCA and OPLS-DA analysis revealed marked differences in the composition of bile acids between the untreated and the acute hypobaric hypoxia groups. Bile acids were screened with VIP > 1, |log2FC| ≥ 1, P < 0.05, and a total of 7 bile acids with significant differences in content between the two groups were obtained, including 5 conjugated bile acids, 2 unconjugated bile acids; in addition, the content of conjugated bile acids has risen in the treated group. This study demonstrated the influence of high-altitude hypoxic environment on bile acid composition and metabolism in rats. All the animal experiments in this study were approved by the 940th Hospital Ethics Committee (approval No: 2020KYLL012).

UHPLC-MS/MS determination of uracil and dihydrouracil in human plasma / 药学实践杂志

Objective To establish an UHPLC-MS/MS method for the determination of uracil (U) and dihydrouracil (UH2) in human plasma. Methods A positive ion detection mode was adopted on the Agilent 6460A mass spectrometer. Chlorouracil was used as the internal standard. 3% bovine serum albumin was used as surrogate plasma matrix. The pretreatment of plasma sample was completed based on liquid-liquid extraction with ethyl acetate. The chromatographic separation was achieved on an Agilent Poroshell 120 SB-Aq (2.1 mm×100 mm, 2.7 μm) column with gradient elution. The mobile phase was 5 mmol/L ammonium acetate aqueous solution and acetonitrile solution. The flow rate was 0.3 ml/min. The column temperature was 30°C. The injection volume was 5 μl. Results The linear range of uracil and dihydrouracil was 10.0-1500.0 ng/ml. Both of uracil and dihydrouracil had good linear relationship with correlation coefficient (r)>0.990. Both of inter- and intra-day precision was <15%. Conclusion The established method is simple, selective, and suitable for the determination of U and UH2 in human plasma.

Determination of 11 main components in Hanshi Yufei decoction, a COVID-19 empirical prescription by UHPLC-MS/MS / 药学实践杂志

Objective To establish a method to determine 11 main components in Hanshi yufei decoction. Methods The method adopted UHPLC-MS/MS with an Agilent ZORBAX SB-C18 (3.5 μm，2.1 mm×150 mm) column. The mobile phase was consisted of 0.2% formic acid plus 10 mmol/L ammonium acetate aqueous solution(A) - acetonitrile(B) and gradient elution (0–0.6 min, 80%–40%A; 0.6–1 min, 40%–30%A; 1–4.3 min, 30%–5%A) at 0.3 ml/min. The column temperature was 40 ℃ and 11 main components including vanillic acid, magnolol, honokiol, wogonin, sophorin, 6-gingerol, citrinin, qianghuo alcohol, nobiletin, nodakenin, and hesperidin were quantified in a multiple reaction monitoring mode. The reserpine was the standard. Results The 11 main components in Hanshi Yufei decoction had a good linear relationship within their concentration range (r>0.98), and the average recovery was 93.11%～111.73%. Conclusion The UHPLC-MS/MS method established in this experiment is easy to operate and has good reproducibility, which provides a laboratory basis for the quality control of Hanshi Yufei decoction.

UHPLC-MS/MS analysis of cAMP and cGMP in rat plasma as potential biomarkers of Yin-Yang disharmony in traditional Chinese medicine / 药物分析学报

Cyclic 3',5'-adenosine monophosphate (cAMP) and cyclic 3',5'-guanosine monophosphate (cGMP) are considered as potential biomarkers for Yin-Yang disharmony in traditional Chinese medicine.However,phosphodiesterase-mediated ex vivo degradation of these molecules in biological samples may result in their underestimation.In the present study,a ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed for determination of cAMP and cGMP in rat plasma,with special consideration of their stability ex vivo.Following precipitation of proteins from plasma samples with 0.4 M perchloric acid,the analytes were chromatographed on a Shimadzu Shim-pack-XR-ODS Ⅱ column with 2.5 mM ammonium acetate and methanol in gradient mode.The MS/MS detection was performed using multiple reaction monitoring in the positive electrospray ionization mode.The lower limit of quantification was 0.27 ng/mL for cAMP and 0.37 ng/mL for cGMP.The method was used to determine the plasma cAMP and cGMP levels in normal and Yin deficiency diabetic rats treated with or without Rehmannia glutinosa.The developed method may be useful for evaluating the regulatory effects of Chinese herbal medicine on the levels of cAMP and cGMP in the body.

Comparative permeability of three saikosaponins and corresponding saikogenins in Caco-2 model by a validated UHPLC-MS/MS method / 药物分析学报

Saikosaponins (SSs) are the main active components extracted from Bupleuri Radix (BR) which has been used as an important herbal drug in Asian countries for thousands of years.It has been reported that the intestinal bacteria plays an important role in the in vivo disposal of oral SSs.Although the deglycosylated derivatives (saikogenins,SGs) of SSs metabolized by the intestinal bacteria are speculated to be the main components absorbed into the blood after oral administration of SSs,no studies have been reported on the characteristics of SGs for their intestinal absorption,and those for SSs are also limited.Therefore,a rapid UHPLC-MS/MS method was developed to investigate and compare the apparent permeability of three common SSs (SSa,SSd,SSb2) and their corresponding SGs (SGF,SGG,SGD) through a bidirectional transport experiment on Caco-2 cell monolayer model.The method was validated according to the latest FDA guidelines and applied to quantify the six analytes in transport medium samples extracted via liquid-liquid extraction (LLE).The apparent permeability coefficient (Papp) determined in this study indicated that the permeability of SGs improved to the moderate class compared to the corresponding parent compounds,predicting a higher in vivo absorption.Moreover,the efflux ratio (ER) value demonstrated an active uptake of SSd and the three SGs,while a passive diffusion of SSa and SSb2.

Pharmacokinetic studies of multi-bioactive components in rat plasma after oral administration of Xintiantai extract and effects of guide drug borneol on pharmacokinetics / 中草药·英文版

Objective: To investigate the in vivo pharmacokinetic characteristics of 17 bioactive components including ginsenoside Rg1, Rb1, Rd, berberine, epiberberine, jatrorrhizine, palmatine, columbamine, coptisine, evodiamine, dehydroevodiamine, rutaecarpine, limonin, hyperin, curcumin, demethoxycurcumin and bisdemethoxycurcumin in rat plasma after oral administration of Xintiantai I extract powder (XI) and Xintiantai I without guide drug borneol extract powder (XI without borneol), and study the compatibility effects of guide drug borneol on the pharmacokinetics. Methods: A UHPLC-MS/MS method was established and fully validated for the comparative pharmacokinetics of 17 bioactive components. The pharmacokinetics parameters of 17 bioactive components after oral administration of XI and XI without borneol were calculated by the software of DAS 3.0 and intercompared. Results: The specificity, linearity, lower limit of quantification (LLOQ), precision, accuracy, extraction recovery rates, matrix effects, and stability of the UHPLC-MS/MS assay were good within the acceptance criteria from FDA guidelines. Guide drug borneol can significantly increase AUC of G-Rd, palmatine, hyperin, curcumin, demethoxycurcumin, bisdemethoxycurcumin and Cmax of 16 bioactive components except for dehydroevodiamine (P < 0.05), decrease Tmax of G-Rd, berberine, columbamin, coptisine, limonin and MRT of 17 bioactive components in XI group (P < 0.05). Conclusion: Guide drug borneol enhanced the absorption of G-Rd, palmatine, hyperin, curcumin, demethoxycurcumin and bisdemethoxycurcumin.

Simultaneous determination of thirteen components in Ainsliaea fragrans by UHPLC-MS/MS / 中草药

Objective: To establish an UHPLC-MS/MS method for rapid and simultaneous determination of the content of 13 components in Ainsliaea fragrans. Methods: The UHPLC-MS/MS method was performed on UPLC BEH C18 (100 mm × 2.1 mm, 1.7 μm) with acetonitrile-water (containing 0.1% formic acid and 5 mmol ammonium formate) as mobile phase for gradient elution. Flow rate was 0.25 mL/min and column temperature was 40 ℃. A triple quadrupole mass spectrometer equipped with electrospray ionization source (ESI) was applied in negative ion mode with the following parameters: ion spray, -4 500 V, ion source temperature, 500 ℃, curtain gas, 344.5 kPa, nubulizer (GS1), 344.5 kPa, heater gas (GS2), 206.7 kPa, and multiple reaction monitoring (MRM) was performed for quantitative analysis of these compounds. Results: Under the optimized MS/MS condition, the linearity ranges of protocatechuic acid, caffeic acid, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, 1,3-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, luteolin, luteoloside and apigenin were 1.099-274.800 ng/mL, 1.006-100.600 ng/mL, 1.080-1 080.000 ng/mL, 83.12-8 312.00 ng/mL, 19.92-996.00 ng/mL, 1.076-269.000 ng/mL, 5.555-555.500 ng/mL, 4.420-4 420.000 ng/mL, 44.76-17 904.00 ng/mL, 48.00-9 600.00 ng/mL, 2.108-210.800 ng/mL, 4.136-413.600 ng/mL, 1.070-107.000 ng/mL (r ≥ 0.9958), respectively, with good linearity. The average recovery rate of thirteen compounds in the samples were in the range of 96.54%-99.75%, and the RSD range was from 0.48% to 0.96%. The RSD values of precision, stability and repeatability test were all less than 3.90%. Conclusion: The method had good repeatability, high specificity, stability and controllability, and could be used for quality control of A. fragrans and its preparations.

Anti-inflammatory activity and identification of the Verbena litoralis Kunth crude extract constituents

Verbena litoralis is a plant popularly known as "gervãozinho-do-campo" in Portuguese. It is traditionally used for stomach, liver and gallbladder problems, and as an anti-inflammatory and anthelmintic. The goal of this study was to determine the chemical composition of the crude extract obtained from the aerial parts of V. litoralis by Ultra High Performance Liquid chromatography coupled with Mass Spectrometry in Tandem (UHPLC-MS/MS); assess the anti-inflammatory activity of ethyl acetate fraction and of crude extract; and verify liver, kidney and pancreas damage. In this study, the chemical composition of the extract was identified via UHPLC/MS/MS, assessing the anti-inflammatory activity of the crude extract and the acetate fraction in an induction model of the granulomatous tissue, as well as given liver, kidney and pancreas damage markers. Chlorogenic acid, luteolin, caffeic acid, apigenin, p-coumaric acid, vanillic acid, ferulic acid and quercetin were quantified in the extract. After the seven-day treatment, the granuloma of the animals treated with the plant extract and fraction presented values very close to the positive control (nimesulide). The V. litoralis crude extract and ethyl acetate fraction show anti-inflammatory activity similar to the nimesulide without evidence of liver, kidney and pancreas damage, which attributes the plant's pharmacological action to the flavonoids found.

Determination of phenolic and triterpenic compounds in Jatropha gossypiifolia L by Ultra-high performance liquid chromatography-tandem mass spectrometric (UHPLC-MS/MS)

This work describes the chemical characterization of extracts of Jatropha gossypiifolia (from Amazonia region) concerning the presence of phenolic and triterpenic compounds using Ultra High Perfomance Liquid chromatography coupled to Mass Spectrometry in Tandem (UHPLC-MS/MS) methods. The studied compounds belong to the most frequently found classes in medicinal plants (triterpenes, flavonoids, flavones, hydroxycinnamic acids, coumarins, catechins and stilbenes), which have been analyzed as chemical and bioactive markers in hydroethanolic and aqueous extracts. Several polyphenolic compounds identified herein are unprecedented in the scientific literature for this species. The chemical markers identified and quantified in the studied extracts of J. gossypiifolia were gallic acid, chlorogenic acid, catechin, caffeic acid, vanillic acid, p-coumaric acid, ferullic acid, rutin, quercitrin, 3-acetylcoumarin, trans-cinnamic acid, quercetin, luteolin, apigenin, kaempferol, chrysin. α-amyrin, β-amyrin and lupeol. As the majority compounds, (+)-catechin, p-coumaric acid, ferulic acid, luteolin, α-amyrin and β-amyrin were found to be present at mg kg-1 levels. J. gossypiifolia extracts presented a high in vitro activity against different reactive oxygen species (hydroxyl, peroxyl, and superoxide anion radicals). Several polyphenolic compound data presented herein are unprecedented in the scientific literature for this plant species. As a result, this plant can be a new source of bioactive molecules for therapeutic purposes.

Analysis of Crystalline Substances in a TCM Hospital Preparation for Treatment of Rheumatism / 中国药学杂志

OBJECTIVE: To analyze the potential adulterations in a traditional Chinese medicine hospital preparation for the treatment of rheumatism. METHODS: The suspicious crystalline substances were isolated under a stereoscopic microscope, and analyzed with GC-MS and UHPLC-MS. GC-MS measurements were preformed on an Agilent HP-5MS column with ion electron impact ionization. UHPLC-MS measurements were preformed with Agilent Zorbax SB-C18 as stationary phase and the mixture of acetontrile-0.1% formic acid as mobile phase under electronic spray ionization mode. RESULTS: Five kinds of chemical drugs were identified from the hospital preparation, including piroxicam, prednisone acetate, indomethacin, ibuprofen and vitamin B2. CONCLUSION: More attention should be paid to the adulteration in some hospital preparations.

Stereoselectivity of the Excretion of R-/S-HPABA in Rats / 中国药学杂志

OBJECTIVE: To develop an UHPLC-MS/MS method for the determination of R-/S-2-(2-hydroxypropanamido) benzoic acid (R-/S-HPABA) in rat urine, feces and bile and apply the method to study the excretion of R-/S-HPABA in rats. METHODS: After liquid-liquid extraction by ethyl acetate,the separation was achieved on a Thermo Syncronis C18 column (2.1 mm×50 mm, 1.7 μm) using mobile phase consisting of solvent A (methanol) and solvent B (0.1% formic acid) at a flow rate of 0.4 mL•min-1. The concentration of R-/S-HPABA after single dose oral administration of 50 mg•kg-1 R-/S-HPABA to rats was detected by UHPLC-MS/MS method. RESULTS: The calibration curve was linear over the range of 0.002-5 μg•mL-1 and the lower limit of quantification (LLOQ) was 0.002 μg•mL-1. The intra-and inter-day RSDs were less than 13.0% and the accuracy (relative error) of R-/S-HPABA was within -7.5%-4.2%. The average recoveries were (87.3±3.4)%-(104.4±7.0)%. After intragastric administration of R-HPABA and S-HPABA at a dose of 50 mg•kg-1, the cumulative amounts of R-HPABA and S-HPABA excreted in the urine (0-48 h) were (536.1±29.7) and (771.7±38.6) μg, the urinary excretion amounted to 4.9% and 7.0% of the dosage, respectively; the cumulative amounts of R-HPABA and S-HPABA excreted in feces (0-48 h) were (3 963.0±345.2) and (4 771.8±355.0) μg, the fecal excretion amounted to 36.0% and 43.4% of the dosage, respectively; the cumulative amounts of R-HPABA and S-HPABA excreted in bile (0-12 h) were (150.6±30.3) and (747.7±89.2) μg, the biliary excretion amounted to 1.4% and 6.8% of the dosage, respectively; the summation of urinary, fecal and biliary excretion amounted to 42.3% and 57.2% of the dosage in rats for R-HPABA and S-HPABA, respectively. The cumulative amounts of S-HPABA excreted in the urine, feces and bile were higher than those of R-HPABA. CONCLUSION: This UHPLC-MS/MS method is suitable for the study of the excretion of R-/S-HPABA in rats. The excretion of the two enantiomers in rats shows significant stereoselectivity difference.

Pharmacokinetics of Achyranthes bidentata on adjuvant arthritis rats by microdialysis and UHPLC-MS/MS / 中国中药杂志

To investigate the " drug-guide" effect of Achyranthes bidentata saponins( ABS) and geniposide( GE) in the treatment on adjuvant arthritis( AA) rats. A UHPLC-MS/MS method for the quantitative determination of GE,zingibroside R1,ginsenoside Ro and chikusetsu saponin Ⅳa in rat blood and joint dialysate was established. After single or combined administration with ABS and GE was given to AA rat model,a microdialysis sampling method for rat joint cavity and jugular vein blood vessels was established to collect microdialysis samples. Waters Acquity HSS C_(18) column was used to separate the above four components,with mobile phase as acetonitrile-0. 1% formic acid water as mobile phase for gradient elution. ESI source was adopted for mass spectra in a negative ion scanning mode. Multiple reaction monitoring( MRM) mode was applied to detect the above four components. The methodological results showed that GE,zingibroside R1,ginsenoside Ro and chikusetsu saponin Ⅳa demonstrated a good linear relationship within the concentration ranges of 2-4 000,16-4 096,14-3 584,23-5 888 μg·L-1 respectively. The precision,accuracy,stability and matrix effect of these four ingredients reached the requirements of quantitative analysis of biological samples. The pharmacokinetic results demonstrated that the combined administration of ABS and GE( 60 mg·kg~(-1)+60 mg·kg~(-1)) can increase the degree of GE in joint cavity distribution,and the AUCjoint/AUCplasmwere twice of that of single administration of GE( 60 mg·kg~(-1)),which indicated that ABS might played a vital role in GE's distribution to joint cavity. Moreover,there was no significant difference between the distribution trend of total three ABS and GE in rats. The pharmacodynamics results showed that the combined administration of ABS and GE has stronger effects on paw swelling,arthritis index and synovial pathomorphology of AA rats than single administration of GE,which suggested that ABS might improve GE's anti-inflammatory effect in AA rats. Based on the above results,ABS has a targeting effect in increasing GE's concentration in joint cavity,with a synergy in efficacy.

Simultaneous determination of six kinds of components in Buyang-Huanwu decoction by UHPLC-MS/MS / 国际中医中药杂志

Objective To develop the UHPLC-MS/MS method for the determination of amygdalin, paeoniflorin, ferulic acid, calycosin glucosidase, quercetin and formononetin in Buyang-Huanwu decoction. Methods Isocratic elution was carried out with mobile phase consisting of methanol- 2 mM ammonium formate. The separation was performed on Agilent ZORBAX SB-C18 maintained at 35 ℃. The flow rate was 200 μl/min, and the injection volume was 2 μl. The mass spectrometer was operated in the positive and negative ionization electrospray (ESI) mode using multiple monitoring (MRM) for analysis of six components. The mass spectrometric conditions were that ion source temperature 400 ℃, dry gas flow 500 L/h, atomization gas flow rate 75.8 Kpa, spray voltage 4000 V, dry gas temperature 400 ℃. Results The amygdalin, paeoniflorin, ferulic acid, calycosin glucosidase, quercetin and formononetin were all analyzed exactly, and the linear ranges were 0.5-32, 0.2-12.8, 0.1-6.4, 0.8-51.2, 0.4-25.6, 0.08-5.12 ng, respectively. The r were 0.9921, 0.9945, 0.9928, 0.9958, 0.9947, 0.9966, respectively. The recoveries of six analytes ranged from 99.21％ to 101.44％ and the relative standard deviations were all below 2.05％. Conclusions A sensitive, accuracy and suitable UHPLC-MS/MS method has been developed, and the method could be applied for the determination of amygdalin, paeoniflorin, ferulic acid, calycosin glucosidase, quercetin and formononetin in Buyang-Huanwu decoction.

Pharmacokinetic Study on Notoginsenoside R1 and Ginsenoside Rg1 in Hushen (Ⅲ) Capsules in Rats / 中国中医药信息杂志

Objective To establish a UHPLC-MS/MS method for the determination of notoginsenoside R1 and ginsenoside Rg1 in rat plasma after oral administration of Hushen (Ⅲ) Capsules; To study its pharmacokinetics. Methods SD rats were given Hushen (Ⅲ) Capsules 2 g/kg and then the blood was collected at time point. Digoxin was used as internal standard. The drug concentrations of notoginsenoside R1 and ginsenoside Rg1 in plasma were determined by UHPLC-MS/MS. And the relative pharmacokinetic parameters were calculated by using DAS2.0 software. Results The linear relationship of notoginsenoside R1 and ginsenoside Rg1 was good (r≥0.997 5). The RSD of intra-day and inter-day precision were less than 13.1%. The results of accuracy, recovery and stability met the requirements for the biology sample analysis. For notoginsenoside R1, the pharmacokinetics parameters T1/2, Tmax, Cmaxin the plasma were (7.86±1.69)h, (4.00±1.04)h, (0.23±0.05)mg/L. For ginsenoside Rg1, the pharmacokinetics parameters T1/2, Tmax, Cmaxin plasma were (4.58±0.95)h, (6.00±0.00)h, (0.32±0.03)mg/L. Conclusion The established UHPLC-MS/MS method is sensitive, accurate and reliable, and is suitable for the pharmacokinetic study of Hushen (Ⅲ) Capsules.