Zuogui Pill Promotes Neurite Outgrowth by Regulating OPN/ IGF-1R/PTEN and Downstream mTOR Signaling Pathway.

AIM AND OBJECTIVE: Zuogui pill (ZGP) is the traditional Chinese medicine for tonifying kidney yin. Clinical and animal studies have shown that ZGP effectively enhances neurologic impairment after ischemic stroke, which may be related to promoting neurite outgrowth. This investigation aimed to prove the pro-neurite outgrowth impact of ZGP and define the underlying molecular pathway in vitro. MATERIALS AND METHODS: The major biochemical components in the ZGP were investigated using UPLC-QTOF-MS. All-trans retinoic acid (ATRA) was employed to stimulate SH-SY5Y cells to develop into mature neurons, followed by oxygen-glucose deprivation and reoxygenation damage (OGD/R). Then the cells were supplemented with different concentrations of ZGP, and cell viability was identified by CCK-8. The neurites' outgrowth abilities were detected by wound healing test, while immunofluorescence staining of ß-III-tubulin was used to label neurites and measure their length. Western blot was employed to discover the changes in protein levels. RESULTS: ZGP improved the cell viability of differentiated SH-SY5Y cells following OGD/R damage, according to the CCK-8 assay. Concurrently, ZGP promoted neurite outgrowth and improved neurite crossing and migration ability. Protein expression analysis showed that ZGP upregulated the expression of GAP43, OPN, p-IGF-1R, mTOR, and p-S6 proteins but downregulated the expression of PTEN protein. Blocking assay with IGF-1R specific inhibitor Linstinib suggested IGF-1R mediated mTOR signaling pathway was involved in the pro-neurite outgrowth effect of ZGP. CONCLUSION: This work illustrated the molecular mechanism underpinning ZGP's action and offered more proof of its ability to promote neurite outgrowth and regeneration following ischemic stroke.

[Characterization and identification of chemical constituents from Schizonepetae Spica based on UHPLC-Q-TOF-MS/MS technique].

The chemical constituents of Schizonepetae Spica were qualitatively analyzed by UHPLC-Q-TOF-MS/MS. An Agilent poroshell 120 SB-C_(18) column(3.0 mm×100 mm, 2.7 µm) was used for gradient elution with 0.1% formic acid water(A)-acetonitrile(B) solution as mobile phase at the flow rate of 0.4 mL·min~(-1) and column temperature of 45 ℃. The data were collected by scanning in positive and negative ion modes, and the compounds were identified by comparison of reference materials and PeakView software. Ninety-seven compounds were identified from Schizonepetae Spica, including 28 flavonoids, 23 phenolic acids, 23 fatty acids, 15 terpenoids, and 8 other compounds. The UHPLC-Q-TOF-MS/MS method established in this study can identify the chemical components of Schizonepetae Spica rapidly, accurately, and comprehensively, and provide a basis for the basic study of pharmacodynamic substances of Schizonepetae Spica.

An integrated approach of high-performance liquid chromatography-mass spectrometry-based chemical profiling, network pharmacology, and molecular docking to reveal the potential mechanisms of Qishen Gubiao granules for treating coronavirus disease 2019.

Qishen Gubiao granules, a traditional Chinese medicine preparation composed of nine herbs, have been widely used to prevent and treat coronavirus disease 2019 with good clinical efficacy. In the present study, an integrated strategy based on chemical profiling followed by network pharmacology and molecular docking was employed, to explore the active components and potential molecular mechanisms of Qishen Gubiao granules in the therapy of coronavirus disease 2019. Using the ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry technique, a total of 186 ingredients corresponding to eight structure types in Qishen Gubiao preparation were identified or structurally annotated with the elucidation of the fragmentation pathways in the typical compounds. The network pharmacology analysis screened 28 key compounds including quercetin, apigenin, scutellarein, luteolin and naringenin acting on 31 key targets, which possibly modulated signal pathways associated with immune and inflammatory responses in the treatment of coronavirus disease 2019. The molecular docking results observed that the top 5 core compounds had a high affinity for angiotensin-converting enzyme 2 and 3-chymotrypsin-like protease. This study proposed a reliable and feasible approach for elucidating the multi-components, multi-targets, and multi-pathways intervention mechanism of Qishen Gubiao granules against coronavirus disease 2019, providing a scientific basis for its further quality evaluation and clinical application.

Integrated analysis of comprehensive metabolomics and network pharmacology to reveal the mechanisms of abelmoschus manihot (L.) medik. in the treatment of cisplatin-induced chronic kidney disease.

Background: Abelmoschus manihot (L.) Medik ("Huangkui" in Chinese, HK) has been widely used for the treatment of kidney diseases. Nephrotoxicity is the side effect of cisplatin (CDDP), which greatly limits its clinical application. Therefore, CDDP could be used to establish the chronic kidney disease (CKD) model. However, the protective effects of HK on CDDP-induced CKD have not been investigated. Purpose: To explore the protective effect and underlying mechanisms of HK on multiple low-dose CDDP-induced CKD in rats by the integrated analysis of serum, kidney, and urine metabolomics and network pharmacology. Methods: The CKD model was induced by multiple low-dose CDDP. Body weight, organ index, serum biochemical, and kidney histology were examined to evaluate the effect of HK. Serum, kidney, and urine were collected and profiled by HILIC/RPLC-Q-TOF/MS-based metabolomics. Potential biomarkers (PBs) were screened according to the criteria of VIP >1, p < 0.01, and FC > 2, and then identified or assigned. The pathway analysis and PBs enrichment were conducted by MetaboAnalyst and ChemRICH. Furthermore, network pharmacology was adopted to dig out the active components and targets. Finally, the results from metabolomics and network pharmacology were integrated to confirm each other. Results: HK could recover the CDDP-induced abnormal pharmacological and metabolic profile changes. A total of 187 PBs were screened and identified from the serum, kidney, and urine metabolomics. Pathway analysis showed that multiple metabolic pathways, mainly related to amino acid and lipid metabolisms, were involved in the nephroprotective effect of HK, and especially, HK could significantly alleviate the disorder of tryptophan metabolism pathway in serum, kidney, and urine. Meanwhile, network pharmacology analysis revealed that 5 components in HK and 4 key genes could be responsible for the nephroprotection of HK, which also indicated that the metabolism of tryptophan played an important role in HK against CKD. Conclusion: HK has a nephroprotection on CDDP-induced CKD, mainly by restoring the dysregulation of tryptophan metabolism. Integrated analysis of serum, kidney, and urine metabolomics and network pharmacology was a powerful method for exploring pharmacological mechanisms and screening active components and targets of traditional Chinese medicine.

Biomarkers of Metabolomics in Inflammatory Bowel Disease and Damp-Heat Syndrome: A Preliminary Study.

Aims: This study aims to investigate the potential biomarkers of inflammatory bowel disease (IBD) and IBD with damp-heat syndrome (IBD-DH) by metabolomics. Methods: Plasma and urine samples were collected from 15 healthy controls and 30 IBD patients, including 15 IBD-DH and 15 IBD with spleen deficiency syndrome (IBD-SD), which was coded as SF8G and SF70 according to the International Classification of Diseases Eleventh Revision (ICD-11) issued by World Health Organization. Pseudotargeted metabolomics method was used based on ultra-high-performance liquid chromatography-high-resolution mass spectrometry and triple-quadrupole mass spectrometry. Results: Under the condition of false discovery rate (FDR) < 0.05, variable importance projection (VIP) > 1.0, and fold change (FC) > 1.5 or < 2/3, we found 57 plasma differential metabolites and 20 urinary differential metabolites in IBD. Then, with area under the curve (AUC) ≥ 0.85 and FC ≥ 2 or ≤ 0.3, 11 potential biomarkers were identified, such as acylcarnitine (ACar 20:4, ACar 18:1, and ACar 20:3), 3-indoleacetic acid, hippuric acid, and dehydroepiandrosterone sulfate, which is related to intestinal microbiota and immune response. However, less obvious differences were observed in IBD-DH when compared with IBD-SD. Under the condition of FDR < 0.2, VIP >1.0, and FC > 1.5 or < 2/3, we identified 16 plasma differential metabolites. In urine samples, IBD-DH and IBD-SD had the same metabolite pattern. With AUC ≥ 0.80, 7 differential plasma metabolites, mainly glycerophospholipids, were identified in IBD-DH. Kyoto Encyclopedia of Genes and Genomes analysis indicated that metabolic pathways, such as citrate cycle and amino acids metabolism, were mainly responsible for the distinction between IBD and healthy controls, whereas glycerophospholipid metabolism perturbation was not only a manifestation of IBD but also an important pathway to distinguish two subtypes defined by traditional medicine, IBD-DH and IBD-SD. Conclusion: In this study, we found that several metabolites of aromatic acids and lipid derivatives could act as potential biomarkers to discriminate IBD from healthy controls. Glycerophospholipids metabolites might be used to differentiate IBD-DH from IBD-SD.

Modified Bu-zhong-yi-qi decoction synergies with 5 fluorouracile to inhibits gastric cancer progress via PD-1/PD- L1-dependent T cell immunization.

Gastric cancer remains the second most common tumor in China. Modified-Bu-zhong-yi-qi decoction (mBYD) as an adjuvant therapy for gastric cancer patients after chemotherapy could significantly prolong the survival time of patients. However, the potential anticancer mechanism for mBYD has not been well characterized. Here, we conducted a comprehensive study of mBYD on a gastric cancer xenograft model with MFC cells in 615 mice and patients. Our results showed that the survival times of the 5-FU + mBYD and mBYD groups were significantly longer than that of the control group. Moreover, the 5-FU + mBYD group had a longer survival time than the 5-FU group. Flow cytometry revealed that the value of CD4+/CD8+ in the mBYD group increased and that the proportions of CD8+PD-1+ T cells and PD-1+Treg cells were decreased when compared to the control group. Compared with the 5-FU group, CD8+PD-1+ T cells and Treg cells were both decreased when 5-FU was combined with mBYD. Further analysis showed that mBYD inhibited PD-L1 expression by the PI3K/AKT pathway in gastric cancer. An in vitro study also showed that mBYD directly promoted the proliferation, activation and cytotoxicity of T lymphocytes. Meanwhile, mBYD reduced the upregulation of CD8+PD-1+ T cells induced by chemotherapy in patients with gastric cancer. In conclusion, mBYD could modulate peripheral immunity and suppress the immune escape of tumors, which may be a promising therapy for gastric cancer.

The effect of Jianpi Yangzheng Xiaozheng Decoction and its components on gastric cancer.

ETHNOPHARMACOLOGICAL RELEVANCE: Jianpi Yangzheng Xiaozheng Decoction (JPYZXZ) is an empirical compound prescription based on the theory of traditional Chinese medicine. JPYZXZ, which is "Qi-invigorating, spleen-strengthening and stasis-removing," can improve the quality of life of gastric cancer patients and prolong their survival; however, the exact mechanism underlying the antitumor effects of this compound is still not clear. AIM OF THE STUDY: The aim of this study is to clearly define the effect of JPYZXZ and its components, Jianpi Yangzheng Decoction (JPYZ) and Xiao Zheng San Jie Decoction (XZSJ), on inhibiting the progression of gastric cancer. MATERIALS AND METHODS: The effect of JPYZXZ and its components on the motility of gastric cancer MGC-803 cells was measured by MTT, adhesion, transwell assays and wound-healing assays. JPYZXZ, JPYZ and XZSJ were administered to 615 mice with gastric cancer xenografts, and their effect on the inhibition of subcutaneous transplantation was analyzed. THP-1 monocyte cells were used to establish tumor-associated macrophage (TAM) models. The polarized state of the TAMs was detected by Flow Cytometry, ELISA and Immunohistochemistry. The mRNA and protein expression of tumor epithelial-mesenchymal transition (EMT) and TAM-related genes was determined by Real-time PCR and Western Blot, respectively. RESULTS: We determined that both JPYZXZ and its components inhibited the progress of gastric cancer in vitro, and JPYZXZ was clearly more effective than JPYZ or XZSJ. The in vivo results demonstrated that the JPYZXZ and XZSJ group exhibited a significant decrease in the tumor weight compared to the control group. Further analysis indicated that JPYZXZ was more active than JPYZ or XZSJ in inhibiting the gastric cancer EMT transformation both in vivo and in vitro. However, JPYZ was more effective compared with JPYZXZ for inducing the phenotypic change in macrophages from M2 to M1. CONCLUSIONS: Our results demonstrate that both JPYZXZ and its components prevent the progress of gastric cancer. JPYZXZ inhibits the gastric cancer EMT more effectively than JPYZ and XZSJ, but JPYZ primarily works to regulate the phenotypic change in macrophages from M2 to M1.

[Screen astragalosides from Huangqi injections by LC-TOF-MS-based mass defect filtering approach].

The samples of Huangqi injection (HI) were analyzed by liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-TOF-MS), and both positive and negative ion modes were employed to obtain the LC-TOF-MS analysis information of chemical compounds in HI. Then the mass defect filtering (MDF) approach, which was developed based on the previously published articles, was utilized to rapidly screen the astragalosides from the obtained LC-TOF-MS data. Each screened astragaloside was confirmed by the presence of no less than 2 quasi-molecular ions. All the screened astragalosides were then tentatively assigned according to the parent ion and daughter ion information. Finally, a total of 62 astragalosides were screened and characterized from the HI samples, including 15 new detected ones. The identification results indicated that acetylation, hydrogenation, dehydrogenation, methoxylation and hydration might be the major conversion reactions involved in the formation of the astragalosides. The LC-TOF-MS-based MDF approach was proved to be a feasible and efficient tool to screen the chemical constituents in complex matrices such as herbal medicines.

Urinary metabolomics reveals the therapeutic effect of HuangQi Injections in cisplatin-induced nephrotoxic rats.

The side effects of cisplatin (CDDP), notably nephrotoxicity, greatly limited its use in clinical chemotherapy. HuangQi Injections (HI), a commonly used preparation of the well-known Chinese herbal medicine Astragali radix, appeared to be promising treatment for nephrotoxicity without compromising the anti-tumor activity of CDDP. In this study, the urinary metabolomics approach using liquid chromatography time of flight mass spectrometry (LC-TOF/MS) was developed to assess the toxicity-attenuation effects and corresponding mechanisms of HI on CDDP-exposed rats. As a result, successive administration of HI significantly recovered the decline of body weight and downregulated the abnormal increase of serum creatinine and urea. HI partly restored the CDDP-induced alteration of metabolic profiling back into normal condition. Totally 43 toxicity-attenuation potential biomarkers were screened and tentatively identified, which were involved in important metabolic pathways such as amino acid metabolism, TCA cycle, fatty acid metabolism, vitamin B6 metabolism and purine metabolism. The results clearly revealed that HI could alleviate CDDP-induced nephrotoxicity and improve the disturbed metabolic balance induced by repeated CDDP exposure. The present study provided reliable evidence for the protective effect of HI on CDDP-induced toxicity with the multi-target pharmacological characteristics.

Tounong Powder () extracts induce G1 cell cycle arrest and apoptosis in LoVo cells.

OBJECTIVE: To further explore the anti-cancer effect of Tounong Powder () extracts (TNSEs) on human colon cancer LoVo cells and examine the possible molecular mechanisms. METHODS: The contents of TNSEs were determined by liquid chromatograph-mass spectrometer (LC-MS) analysis after extraction with water and methanol. Variations of cell morphological features were observed using fluorescence microscopy. Cytotoxicity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell cycle distribution and apoptosis were analyzed using flow cytometry at different TNSE doses (0, 62.5, 125, or 250 µg/mL). Protein expressions of phosphatidylinositol 3-kinase (PI3K), phosphate protein kinase B (p-AKT), phosphate mammalian target of rapamycin (p-mTOR), p-p70s6k1, cleaved caspase-9 and -3 were detected using Western blot analysis. RESULTS: TNSEs induced cell growth inhibition in a concentration- and time-dependent manner. Flow cytometric analysis showed apoptotic cells and cell cycle arrest at the G phase after TNSEs treatment compared with controls. Furthermore, TNSEs significantly down-regulated the proteins PI3K, p-AKT, p-mTOR, and p-p70s6k1, and up-regulated the proteins cleaved caspase-9 and -3 dosedependently, as determined by Western blot. CONCLUSIONS: TNSEs reduced LoVo cell proliferation, and caused apoptosis and cell-cycle arrest in LoVo cells. This effect might be associated with regulation of the PI3K/AKT signaling pathway.

Simultaneous determination of notoginsenoside R1 and ginsenoside Re in rat plasma by ultra high performance liquid chromatography with tandem mass spectrometry and its application to a pharmacokinetic study.

A rapid and high sensitive ultra high performance liquid chromatography with tandem mass spectrometry method for the simultaneous determination of notoginsenoside R1 and ginsenoside Re in rat plasma was developed. The analytes and internal standard, digoxin, were extracted from rat plasma via protein precipitation with methanol and separated on an Phenomenex Gemini C18 column within 2 min. Quantitation was performed on a triple quadrupole mass spectrometer employing electrospray ionization technique, operating in multiple reaction monitoring and positive ion mode. The precursor to product ion transitions monitored for notoginsenoside R1, ginsenoside Re, and internal standard were m/z 955.5→775.5, 969.6→789.1, and 803.6→283.1, respectively. The assay was validated with linear range of 1.9-380 ng/mL for notoginsenoside R1 and 0.5-100 ng/mL for ginsenoside Re. The intra- and interday precisions (RSD%) were within 8.96% for each analyte. The absolute recoveries were greater than 93% for R1 and 96% for Re. Each analyte was stable during all sample storage, preparation, and analytic procedures. The method was successfully applied to a pharmacokinetic study of Xuesaitong dispersible tablets in eight rats.

Systematic screening and characterization of astragalosides in an oral solution of Radix Astragali by liquid chromatography with quadrupole time-of-flight mass spectrometry and Peakview software.

Liquid chromatography with quadrupole time-of-flight mass spectrometry coupled with automated data analysis by Peakview software was employed to systematically screen and characterize the astragalosides in Radix Astragali, a Chinese medical preparation. The separation was performed on a poroshell 120 SB-C18 column equipped in a conventional liquid chromatography system. After being separated using a general gradient elution, the analytes were detected by the triple quadrupole time-of-flight mass spectrometer in both positive- and negative-ion modes. The mass defect filtering function built in the Peakview software was utilized to rapidly screen the potential ions of interest, while some functions of Peakview such as Formula Finder, XIC manager, and IDA Explorer were employed to facilitate the assignment or characterization of the screened astragalosides. A total of 42 astragalosides were screened and tentatively characterized or assigned, and 20 of them were firstly detected in Radix Astragali. According to the screened astragalosides, acetylation, glycosidation, hydrogenation, oxidation, and hydration were considered to be the major secondary metabolic pathways involved in the formation of the astragalosides. The combination of liquid chromatography with quadrupole time-of-flight mass spectrometry and automated Peakview analysis is a feasible and efficient tool to screen and identify the constituents in complex matrices of herbal medicines.

[Study on biomarker of Tripterygium wilfordii in treatment of rheumatoid arthritis based on PK/PD].

To observe the serum samples and the anti-inflammatory effect of Tripterygium wilfordii in treating RA by using the pharmacokinetic-pharmacodynamic model, make a correlation analysis on concentration-time and effect-time curves, and explore RORγt, IL-17, STAT3, IL-6 mRNA transcriptional levels in rats by PCR. Methotrexate, tripterine and high-dose T. wilfordii could down-regulate RORγt, IL-17, STAT3, IL-6 mRNA transcriptional levels in AA rat lymph nodes. The study on PK-PD model showed correlations between inflammatory factors and blood concentration of T. wilfordii. T. wilfordii and its main active constituent tripterine could show the inflammatory effect and treat RA by inhibiting IL-17 cytokine.

Pharmacokinetic profiles of hydroxysafflor yellow A following intravenous administration of its pure preparations in healthy Chinese volunteers.

ETHNOPHARMACOLOGICAL RELEVANCE: Hydroxysafflor yellow A (HSYA), the major active marker compound isolated from Carthamus tinctorius L., has been demonstrated to possess various attractive pharmacological activities. However, there is a lack of information about the complete clinical pharmacokinetic profiles of HSYA following the administration of its pure preparations. The purpose of this study was to fully characterize the pharmacokinetic (PK) properties of HSYA in healthy Chinese volunteers following drip intravenous infusion of injectable powder of pure HSYA (IPPH), a new drug recently approved for the phase I clinical study by China Food and Drug Administration. MATERIALS AND METHODS: 36 healthy subjects of either sex were recruited in this single-center, and open-label, single doses (25, 50, and 75 mg) and multiple doses (50 mg, once daily, 7 consecutive days) study. Plasma samples were analyzed with a validated LC-MS/MS method. Various PK parameters were estimated from the plasma concentration versus time data using non-compartmental methods. RESULTS: After single dose administration of IPPH, the values of AUC(0-t), AUC(0-∞) and C(max) for HSYA were statistically proportional over the dose range of 25-75 mg. After 7 repeated doses of 50 mg IPPH, both C(max) and AUC(0-∞) were significantly decreased, from 3207 to 2959 µg L(-1), and from 12,811 to 12,135 µg h L(-1) respectively, while t(1/2) was significantly prolonged from 3.912 to 4.414 h. The minimum plasma concentrations on day 5, 6 and 7 showed good stability with no significant difference. Both Cmax and AUC of HSYA in male volunteers were generally lower than that in females. IPPH was generally well tolerated in healthy volunteers by either single or multiple dosing. CONCLUSION: HSYA displayed moderately linear PK properties over the doses ranging from 25 to 75 mg of IPPH. Repeated administration of IPPH once daily could not lead to the in-vivo drug accumulation, but significantly affect PK behavior of HSYA. Gender difference should be considered for dosage recommendation in the clinic.

The multi-target capabilities of the compounds in a TCM used to treat sepsis and their in silico pharmacology.

OBJECTIVE: This study aims to explain the mechanisms at the molecular level of a traditional Chinese medicine (TCM) for the treatment of sepsis. METHODS: We first identified 16 targets involved in the sepsis disease network; then we constructed a molecular ligand database and investigated the effects between the ligand database and the sepsis targets using computational biology methods. The results of the calculation were validated with in vitro biological testing against bovine thrombin. RESULTS AND CONCLUSION: We found that multiple active compounds contained in the TCM interact with multiple sepsis-related targets. We predicted several promising compounds for sepsis treatment, and the first 10 compounds were characterised. Among those tested, rosmarinic acid displayed the strongest biological activity in the in vitro activity test with a half-maximal inhibitory concentration (IC(50)) of 85µM. This study demonstrates a novel way of identifying naturally occurring chemical entities as new leads for sepsis treatment.

Oral bioavailability and gender-related pharmacokinetics of celastrol following administration of pure celastrol and its related tablets in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Celastrol is a natural compound extracted from the traditional Chinese medicinal herb, Thunder God Vine (TGV). Owing to its potential anti-inflammatory and antitumor effects, celastrol has been considered as a promising candidate for drug development. AIM OF THE STUDY: To establish a sensitive LC-MS/MS method to investigate the pharmacokinetic properties of celastrol in rats. Key pharmacokinetic issues of celastrol including oral bioavailability, comparative pharmacokinetics between pure compound and tablet preparation, as well as gender-related pharmacokinetic difference are to be addressed for the first time. MATERIALS AND METHODS: Sprague-Dawley rats were administrated an intravenous dose (100 µg kg(-1)) of pure celastrol and an oral dose (1000 µg kg(-1)) of pure celastrol and TGV tablets (corresponding to 534 µg kg(-1) of celastrol), respectively. At different time points, the concentration of celastrol in rat plasma was determined by a sensitive and well-validated LC-MS/MS method. Main pharmacokinetic parameters including area under the plasma concentration-time curve (AUC), maximal plasma concentration (Cmax), the time for maximal concentration (Tmax) and mean residence time (MRT) were estimated by Drug and Statistic1.0 pharmacokinetic software (Chinese Pharmacological Association, Anhui, PR China). Statistical analysis was performed using two one-side t test with p-values less than 0.05 as the level of significance. RESULTS: The standard curve of celastrol showed good linearity in the concentration range of 0.11~54.3 ng mL(-1) in our current method, with acceptable selectivity, precision, recovery, and stability. The oral absolute bioavailability of celastrol significantly increased from 17.06% for pure celastrol to 94.19% for TGV tablets containing equivalent celastrol. After oral administration of TGV tablets, the Cmax and AUC values of celastrol in female rats were (32.03±8.41) µg L(-1) and (379.49±118.19) µg h L(-1), which were significantly higher (p<0.01) than that in males with the values of (14.31±7.33) µg L(-1) and (188.17±92.33) µg h L(-1). CONCLUSION: Celastrol administered orally in the rat was poorly absorbed into the systemic circulation. However, the poor absorption of celastrol could be greatly improved when celastrol-containing TGV tablets orally administered, and thereby the oral bioavailability of celastrol was significantly increased. As for gender difference, female rats showed significantly better absorption of celastrol than males.

Simultaneous characterization of prenylated flavonoids and isoflavonoids in Psoralea corylifolia L. by liquid chromatography with diode-array detection and quadrupole time-of-flight mass spectrometry.

RATIONALE: Prenylated flavonoids and isoflavonoids are widely distributed throughout the plant kingdom, with many biological effects. Psoralea corylifolia, which contains many kinds of prenylated components, has been widely used as a medicinal plant in Asia and India for thousands of years. The goal of this study was to characterize the components in P. corylifolia using a liquid chromatography with diode-array detection and quadrupole time-of-flight mass spectrometry (LC-DAD/Q-TOF-MS) method, and to elucidate the fragmentation behavior of the different prenyl substituent groups and their appropriate characteristic pathways in positive ion mode. METHODS: The calculated accurate masses of the protonated molecules, the fragment ions, the retention behavior, and the data from UV spectra were used for identification of the components in P. corylifolia. RESULTS: A total of 45 compounds, including 43 prenylated components, were identified or tentatively identified in P. corylifolia. Different diagnostic fragment ions and neutral losses were observed in different prenyl substructures: neutral loss of 56 Da (C(4)H(8)) and a fragment ion at m/z 69 (C(5)H(9)(+)) were generated by a prenyl chain; neutral losses of 42 Da (C(3)H(6)), 54 Da (C(4)H(6)), 15 Da (CH(3•)) and 16 Da (CH(4)) were observed in a ring-closed prenyl group; neutral losses of 72 Da (C(4)H(8)O), 60 Da (C(2)H(4)O(2)), 58 Da (C(3)H(6)O) and 18 Da (H(2)O) were detected in a 2,2-dimethyl-3,4-dihydroxydihydropyran ring; neutral losses of 72 Da (C(4)H(8)O), 60 Da (C(3)H(8)O) and 18 Da (H(2)O) were yielded from a 2,2-dimethyl-3-hydroxydihydropyran ring, a 2-(1-hydroxy-1-methylethyl)dihydrofuran ring or a 1-hydroxy-3-methylbut-3-enyl chain. CONCLUSIONS: This method can be applied for analysis of prenylated components in P. corylifolia and other herbal medicines.

Identification of metabolites of Buyang Huanwu decoction in rat urine using liquid chromatography-quadrupole time-of-flight mass spectrometry.

In the present study, rapid resolution liquid chromatography was coupled with quadrupole time-of-flight tandem mass spectrometry (RRLC-Q-TOF-MS) to identify the absorbed components and metabolites in rat urine after oral administration of Buyang Huanwu decoction (BYHWD). After oral administration of BYHWD, urine samples were collected and pretreated by solid phase extraction. The mass measurements were accurate within 5 ppm of error for all the protonated molecules, and subsequent fragment ions offered higher quality structural information for interpretation of the fragmentation pathways of various compounds. A total of 50 compounds were detected in rat urine samples within 20 min, including 12 parent compounds and 38 metabolites. Except for three prototype components (Hydroxysafflor yellow A, Paeoniflorin, and Amygdalin), the metabolites identified mainly came from Radix Astragali, Radix Angelicae Sinensis, and Rhizoma Chuanxiong. The results indicated that glucuronidation and sulfation were the major metabolic pathways of isoflavonoids, while glutathione conjugation, glucuronidation and sulfation were the main metabolic pathways of phthalides. No saponin-related metabolites were detected. The present study provided important structural information on the metabolism of BYHWD. Furthermore, the results of this work have demonstrated the feasibility of the RRLC/ESI-Q-TOF-MS approach for rapid and reliable characterization of metabolites from herbal medicines.

Correlative analysis of metabolite profiling of Danggui Buxue Tang in rat biological fluids by rapid resolution LC-TOF/MS.

In this work, the metabolite profiles of Danggui Buxue Tang (DBT) in rat bile and plasma were qualitatively described, and the possible metabolic pathways of DBT were subsequently proposed. Emphasis was put on correlative analysis of metabolite profiling in different biological fluids. After oral administration of DBT, bile and plasma samples were collected and pretreated by solid phase extraction. Rapid resolution liquid chromatography coupled to time-of-flight mass spectrometry (RRLC-TOFMS) was used for characterization of DBT-related compounds (parent compounds and metabolites) in biological matrices. A total of 142 metabolites were detected and tentatively identified from the drug-containing bile and plasma samples. Metabolite profiling shows that rat bile contained relatively more glutathione-derived conjugates, more saponins compounds and more diverse forms of metabolites than urine. The metabolite profile in plasma revealed that glucuronide conjugates of isoflavonoids, dimmers, acetylcysteine conjugates and parent form of phthalides, as well as saponin aglycones were the major circulating forms of DBT. Collectively, the metabolite profile analysis of DBT in different biological matrices provided a comprehensive understanding of the in vivo metabolic fates of constituents in DBT.

Turbulent-flow chromatography coupled on-line to fast high-performance liquid chromatography and mass spectrometry for simultaneous determination of verticine, verticinone and isoverticine in rat plasma.

A method based on the on-line turbulent-flow chromatography and fast high-performance liquid chromatography/mass spectrometry (TFC-LC/MS) was developed for sensitive and high throughput pharmacokinetic study of traditional Chinese medicines (TCMs). In this method, an on-line extraction column (Waters Oasis HLB) and a fast HPLC column with sub-2 microm particle size (Agilent Zorbax StableBond-C(18), 4.6 mm x 50 mm, 1.8 microm) in a column-switching set-up were utilized. HLB is a reversed-phase extraction column with hydrophilic-lipophilic balanced copolymer (2.1 mm x 20 mm, 25 microm particle size), which will exhibit some turbulent-flow properties at a high-flow rate. The method combines the speed and robustness of turbulent-flow extraction and the sensitivity and separation efficiency of fast HPLC-MS to analyze multiple and trace constituents of TCMs in plasma matrix. This method was successfully applied for pharmacokinetic study of verticine, verticinone and isoverticine, the chemical markers of Fritillaria thunbergii, after oral administration of total steroidal alkaloids extract of F. thunbergii to rats. Each plasma sample was analyzed within 7 min. The method demonstrated good linearity (R>0.999) ranged from 0.505 to 96.0 ng/mL with satisfactory accuracy and precision, and the lower limit of quantifications of verticine, verticinone and isoverticine were estimated to be 0.120, 0.595 and 0.505 ng/mL, respectively. These results indicate that the proposed method is fast, sensitive, and feasible for pharmacokinetic study of TCMs.