Study on mechanism of Zishen Pill treating benign prostatic hyperplasia based on serum pharmacochemistry and network pharmacology.

Zishen Pill (ZSP) is a traditional Chinese medicine that is frequently used to treat Benign Prostatic Hyperplasia (BPH), however its specific mechanism of action and active ingredients are yet unknown. We used a combination of serum pharmacochemistry and network pharmacology and a series of biochemical assays to explore the action mechanism of ZSP in the treatment of BPH. The BPH rat model was created using testosterone propionate, and following oral ZSP administration, the components of ZSP in rat serum were detected by UPLC-Q-Exactive Orbitrap/MS method. A "component-target-disease" network and PPI networks were constructed on this foundation. The primary mechanism of ZSP decreasing BPH in rats was studied by KEGG pathway and GO analysis. Finally, the potential pathways and key targets were further verified in vivo by molecular biology and immunological methods. 46 substances were charactered from rat serum, and 164 anti-BPH targets were screened from the database. According to network pharmacology, the primary targets were CASP3, STAT3, JUN, and PTGS2/COX2. Three related pathways (PI3K/Akt signaling pathway, AGE-RAGE signaling pathway and EGFR tyrosine kinase inhibitor resistance) were closely related to the therapeutic effects of ZSP. The findings of molecular biology demonstrated that ZSP may bring Bcl-2, BAX, CASP3, COX2, and 5LOX protein and gene expression in BPH rats appreciably closer to that of normal rats. Additionally, ZSP can lessen the expression of inflammatory cytokines in BPH rats, including VEGF, TNF-α, CCL5, and interleukin. CONCLUSION: The above results suggest that ZSP may reduce BPH through inflammation/immunity and apoptosis/proliferation-related pathways. This study offers a fresh approach to investigate the basic pharmacological effects and mechanism of ZSP in the treatment of BPH.

Flavonoids derived from Anemarrhenae Rhizoma ameliorate inflammation of benign prostatic hyperplasia via modulating COX/LOX pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Flavonoids are the main components of the traditional Chinese medicine Anemarrhenae Rhizoma (dried rhizome of Anemarrhena asphodeloides Bge.), which has been reported to possess activity against inflammation and tumor. AIM OF STUDY: Regulation of the arachidonic acid (AA) cascade through cyclooxygenase (COX) and lipoxygenase (LOX) represent the two major pathways to treat inflammatory of benign prostatic hyperplasia (BPH). In this study, Anemarrhenae Rhizoma flavonoids and its main compounds (mangiferin, neomangiferin and isomangiferin) were investigated for effects on AA metabolism. METHODS: Ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was used to monitor AA metabolites in BPH rats and in PC-3 cells. COX-2 and 5-LOX protein and mRNA levels were measured by Western blot and qPCR, respectively, along with histopathological assessment of prostate tissues. RESULTS: Treatment with flavonoids significantly ameliorated BPH-associated prostate inflammation and inhibited the expression of COX-2 and 5-LOX at the protein and mRNA levels. Quantitative metabolomic analysis of blood plasma showed flavonoids treatment decreased AA levels and its metabolites associated with the COX and LOX pathways. Further exploration of the flavonoid compounds mangiferin, neomangiferin and isomangiferin showed they inhibited AA metabolism to varying degrees in PC-3 cell cultures. CONCLUSION: Anemarrhenae Rhizoma flavonoids act to inhibit BPH-related inflammation in vivo and in vitro by targeting AA metabolism and interfering with COX and LOX pathways. The identification of mangiferin, neomangiferin and isomangiferin as anti-inflammatory components suggests flavonoids interventions represent a promising therapeutic approach for BPH.

The bioactive alkaloids identified from Cortex Phellodendri ameliorate benign prostatic hyperplasia via LOX-5/COX-2 pathways.

BACKGROUND: The bioactive alkaloids identified from Cortex Phellodendri (CP) were highly effective in treating rats with benign prostatic hyperplasia (BPH). Specifically, lipoxygenase-5 (LOX-5) and cyclooxygenase-2 (COX-2) were identified as two primary targets for alleviating inflammation in BPH rats. However, it remains unknown whether the alkaloid components in CP can interact with the two target proteins. PURPOSE: To further identify bioactive alkaloids targeting LOX/COX pathways. METHODS: An affinity-ultrafiltration mass spectrometry approach was employed to screen dual-target LOX-5/COX-2 ligands from alkaloid extract. The structures of bioactive alkaloids were characterized by high-resolution Fourier transform ion cyclotron resonance mass spectrometry. To understand the molecular mechanisms underlying the effects of bioactive alkaloids, the expression levels of LOX-5 and COX-2 in BPH model rats were investigated at both protein and mRNA levels. The LOX-5/COX-2 enzymes activity experiments and molecular docking analysis were performed to fully evaluate the interactions between bioactive alkaloids and LOX-5/COX-2. RESULTS: After comprehensive analysis, the results showed that bioactive alkaloids could suppress the expression of LOX-5 and COX-2 simultaneously to exert an anti-inflammatory effect on the progression of BPH. In addition, the screened protoberberine, demethyleneberberine was found to exhibit prominent inhibitory activities against both LOX-5 and COX-2 enzymes, palmatine and berberine with moderate inhibitory activities. Molecular docking analysis confirmed that demethyleneberberine could interact well with LOX-5/COX-2. CONCLUSION: This study is the first to explore the inhibitory effects of bioactive alkaloids from CP on LOX-5 and COX-2 activities in BPH rats. Our findings demonstrate that the bioactive alkaloids from CP can ameliorate BPH via dual LOX-5/COX-2 pathways, which serves as an efficient approach for the discovery of novel drug leads from natural products with reduced side effects.

Role of Levo-tetrahydropalmatine and its metabolites for management of chronic pain and opioid use disorders.

BACKGROUND: Opioids have been prescribed to reduce suffering from pain and to enhance quality of life. Due to the addictive potential and the lack of other effective alternatives to treat severe acute and chronic pains, opioids remain a serious public health issue. While, opioids directly influence the drug-seeking behavior, tolerance and withdrawal processes, through neuroadaptation, the brain's endogenous opioid system also adapts in the presence of chronic pain and could contribute to the difficulty of treatment. Despite the seemingly obvious interaction between the presence of pain and opioid-abuse, little is known about the underlying mechanisms in the brain. PURPOSE: To review the current understanding of the interaction mechanisms of neurotransmitter circuitries in pain modulation and reward in the brain and the effects of L-tetrahydropalmatine (L-THP) and its metabolites in pain management and opioid use disorder and gain a better insight on the pharmacological profile and in vivo effects of L-THP and its metabolites. METHOD: A detailed literature search on available (preclinical and clinical) studies about the effects of L-THP and its metabolites against drug addiction and chronic pain has been performed. The data was collected using various search engines such as PubMed, ScienceDirect, Google scholar and articles in English up to December 2020 were included in this review. RESULTS: L-THP and its metabolites demonstrated analgesic and anti-addiction effects. Due to their dual pharmacological properties (D1 partial agonist and D2 antagonist) these compounds could be used as molecular tools to provide a better understanding of the interactions between pain and addiction. CONCLUSION: The available data confirms the potential of L-THP and its metabolites to treat both chronic pain and drug addiction. However, further clinical trials are needed to establish safety and efficacy.

Characterization of spirostanol glycosides and furostanol glycosides from anemarrhenae rhizoma as dual targeted inhibitors of 5-lipoxygenase and Cyclooxygenase-2 by employing a combination of affinity ultrafiltration and HPLC/MS.

BACKGROUND: Modulation of the arachidonic acid (AA) cascade via 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2) represent the two major pathways for treatments of inflammation and pain. The design and development of inhibitors targeting both 5-LOX and COX-2 has gained increasing popularity. As evidenced, 5-LOX and COX-2 dual targeted inhibitors have recently emerged as the front runners of anti-inflammatory drugs with improved efficacy and reduced side effects. Natural products represent a rich resource for the discovery of dual targeted 5-LOX and COX-2 inhibitors. By combining affinity ultrafiltration and high-performance liquid chromatography-mass spectrometry (AUF-LC-MS), an efficient method was developed to identify spirostanol glycosides and furostanol glycosides as the 5-LOX/COX-2 dual inhibitors from saponins extract of Anemarrhenae Rhizoma (SEAR). METHODS: A highly efficient method by combining affinity ultrafiltration and high-performance liquid chromatography-mass spectrometry (AUF-LC-MS) was first developed to screen and characterize the 5-LOX/COX-2 dual targeted inhibitors from SEAR. The structures of compounds in the ultrafiltrate were characterized by high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). In addition, in vitro 5-LOX/COX-2 inhibition assays and their dual expression in vivo were performed to confirm the inhibitory activities of the compounds screened by AUF-LC-MS. Molecular docking studies with the corresponding binding energy were obtained which fit nicely to both 5-LOX and COX-2 protein cavities and in agreement with our affinity studies. RESULTS: A total of 5 compounds, timosaponin A-II, timosaponin A-III, timosaponin B-II, timosaponin B-III and anemarrhenasaponin I, were identified as potential 5-LOX/COX-2 dual targeted inhibitors with specific binding values > 1.5 and IC50 ≤ 6.07 µM. CONCLUSION: The present work demonstrated that spirostanol glycoside and furostanol glycoside were identified as two novel classes of dual inhibitors of 5-LOX/COX-2 enzymes by employing a highly efficient screening method of AUF-LC-MS. These natural products represent a novel class of anti-inflammatory agents with the potential of improved efficacy and reduced side effects.

Screening for natural inhibitors of 5-lipoxygenase from Zi-shen pill extract by affinity ultrafiltration coupled with ultra performance liquid chromatography-mass spectrometry.

ETHNOPHARMACOLOGICAL RELEVANCE: Zi-shen pill (ZSP), a traditional Chinese medicine, is widely used for the treatment of benign prostatic hyperplasia (BPH) and has remarkable curative effect. AIM OF THE STUDY: To screen the potential 5-Lipoxygenase(5-LOX) inhibitors from ZSP extract. MATERIALS AND METHODS: A new approach based on affinity ultrafiltration-ultra performance liquid chromatography-mass spectrometry(UPLC-MS) was established and validated. Zileuton and glipizide were chosen as positive and negative control drug, respectively. For better screening result, the concentration of 5-LOX enzyme, incubation temperature and time, pH and ion strength were optimized. In addition, 5-LOX inhibitory assay in vitro and molecular docking technique were used for further verification. RESULTS: 20 compounds were characterized in the ultrafiltrate by high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and 16 ligands showed binding ability to 5-LOX. Among them, six ligands were deduced as high-potential 5-LOX inhibitors with their high specific binding values (>2.0). The inhibitory activities of anemarrhenasaponin I, timosaponin AI, nyasol and demethyleneberberine were confirmed by the 5-LOX inhibitory assay for validating the reliability of affinity ultrafiltration approach and the computer-simulated molecular docking technique further clarified the possible mechanism of action between the active compounds and the 5-LOX active sites.

Characterization and screening of cyclooxygenase-2 inhibitors from Zi-shen pill by affinity ultrafiltration-ultra performance liquid chromatography mass spectrometry.

ETHNOPHARMACOLOGICAL RELEVANCE: Zi-shen pill (ZSP) is a classical Chinese herbal formula used for treatment of benign prostatic hyperplasia (BPH). AIM OF THE STUDY: To characterize and screen cyclooxygenase-2 (COX-2) inhibitors from ZSP extract. MATERIALS AND METHODS: The ZSP extract was incubated with COX-2 and the potential ligands were screened out by affinity ultrafiltration. Celecoxib and glipizide were chosen as positive control and negative control drug, respectively. Affinity ultrafiltration-ultra performance liquid chromatography-mass spectrometry (UPLC-MS) method was used. In addition, in vitro COX-2 inhibitory assay and in silico molecular docking technique were used for further validation. RESULTS: A total of 20 components were discovered and identified from ZSP ultrafiltrate by high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS), among which 8 compounds were deduced as potential COX-2 inhibitors by their high specific binding values (>1.5). Inhibitory activities of demethyleneberberine, palmatine, berberine and timosaponin A-I were confirmed by an enzyme assay of COX-2, which validated the reliability of our approach. Molecular docking simulation investigated potential mechanism of action for these compounds. CONCLUSION: The results revealed that affinity ultrafiltration UPLC-MS could successfully screen out the potential COX-2 inhibitors from complex Chinese herbal formula ZSP extract, indicating that its therapeutic effect on BPH was partly based on the enzyme active ingredients.

Compatibility effects of herb pair Phellodendri chinensis cortex and Anemarrhenae rhizoma on benign prostatic hyperplasia using targeted metabolomics.

Phellodendri chinensis cortex (P. C. cortex) and Anemarrhenae rhizoma (A. rhizoma) herb pair is a core component of traditional Chinese medicines used to treat inflammation and benign prostatic hyperplasia (BPH). The present study was designed to profile the arachidonic acid (AA) metabolomic characteristics in rat plasma and prostate after being treated with P. C. cortex and A. rhizoma as well as their combination. Plasma and prostate samples from sham group, BPH model group, herb pair group and two single herb groups were collected on days 7, 14, 21 and 28. Then, a systemic metabolomic analysis based on UFLC-MS/MS was employed to quantify AA and its cyclooxygenase and lipoxygenase pathway metabolites (15-HETE, 12-HETE, 5-HETE, AA, PGI2 , PGF2α , 8-HETE, PGD2 , PGE2 and LTB4 ). The results demonstrated that BPH led a significant increase of 10 biomarkers in plasma and tissue (p < 0.05). The clusters of herb pair group and single herb groups showed a tendency to return to the initial space, and the AA and its metabolites from those groups were differently downregulated to a healthier level, with the combination of single herbs most obvious. The present study demonstrated that P. C. cortex-A. rhizoma herb pair might produce synergistic or complementary compatibility effects on suppressing inflammatory processes occurring in BPH.

Arachidonic acid metabonomics study for understanding therapeutic mechanism of Huo Luo Xiao Ling Dan on rat model of rheumatoid arthritis.

ETHNOPHARMACOLOGICAL RELEVANCE: Huo Luo Xiao Ling Dan (HLXLD), a traditional Chinese medicine (TCM), is commonly used for the treatment of rheumatoid arthritis (RA). AIM OF THE STUDY: To explore the potential therapeutic mechanism of HLXLD on anti-inflammatory activity. MATERIALS AND METHODS: A metabolomic approach based on UFLC-MS/MS to profile arachidonic acid (AA) metabolic changes was used. The cyclooxygenase (COX) and lipoxygenase (LOX) catalyzed metabolites in plasma were quantified on 7, 14, 21, and 28 days after the rats injected with Complete Freund's adjuvant and orally administrated with HLXLD, methotrexate and dexamethasone in parallel as the positive control drugs. RESULTS: Nineteen metabolites involved in COX and LOX pathways in RA model group were significant increased compared with normal group (P < 0.05), including 12-hydroxyeicosatetraenoic acid (12-HETE), 15-HETE, 8-HETE, leukotriene B4(LTB4), prostaglandin E2 (PGE2), PGI2, PGD2, PGF2α, thromboxane B2 (TXB2), etc. From day 7 to day 28, the trajectory direction of HLXLD group and positive control groups gradually moved towards the initial space, and the concentrations of AA and its metabolites after HLXLD treatment were significantly reduced in dual pathways compared to control groups. CONCLUSION: HLXLD induced a substantial change in the AA metabolic profiles through refrain the expression of COX and LOX. The present investigation also highlights that distinct ingredients of this formula tend to inhibit different target to achieve a therapeutic effect.

Identification of the absorbed components and metabolites of modified Huo Luo Xiao Ling Dan in rat plasma by UHPLC-Q-TOF/MS/MS.

To reveal the material basis of Huo Luo Xiao Ling Dan (HLXLD), a sensitive and selective ultra-high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) method was developed to identify the absorbed components and metabolites in rat plasma after oral administration of HLXLD. The plasma samples were pretreated by liquid-liquid extraction and separated on a Shim-pack XR-ODS C18 column (75 × 3.0 mm, 2.2 µm) using a gradient elution program. With the optimized conditions and single sample injection of each positive or negative ion mode, a total of 109 compounds, including 78 prototype compounds and 31 metabolites, were identified or tentatively characterized. The fragmentation patterns of representative compounds were illustrated as well. The results indicated that aromatization and hydration were the main metabolic pathways of lactones and tanshinone-related metabolites; demethylation and oxidation were the major metabolic pathways of alkaloid-related compounds; methylation and sulfation were the main metabolic pathways of phenolic acid-related metabolites. It is concluded the developed UHPLC-Q-TOF/MS method with high sensitivity and resolution is suitable for identifying and characterizing the absorbed components and metabolites of HLXLD, and the results will provide essential data for further studying the relationship between the chemical components and pharmacological activity of HLXLD.

Simultaneous profiling of eicosanoid metabolome in plasma by UPLC-MS/MS method: Application to identify potential makers for rheumatoid arthritis.

To evaluate the potential relationship between rheumatoid arthritis and arachidonic acid (AA) metabonomics via cyclooxygenase (COX) and lipoxygenase (LOX) pathways, a UPLC-MS/MS method has been developed and validated for simultaneous and quantitative profiling of eicosanoid metabolome in rat plasma. The analytes were extracted from plasma samples by protein precipitation procedure, and then separated on a Shim-pack XR-ODS column with mobile phase A (0.05% formic acid in water, pH=3.3 adjusted with dilute ammonium hydroxide) and mobile phase B [methanol: acetonitrile (20:80, v/v)]. The detection was performed on UPLC-MS/MS system with an electro spray ion source in the negative ion and multiple reaction-monitoring modes. The developed method was optimized to completely separate all twenty-three analytes and three internal standards in 12min. All standard calibration curves were linear and the calibration regression coefficients were ranged from 0.9903 to 0.9992 for all analytes. The recoveries of analytes were all more than 60%. By means of the method developed, the plasma samples from model rats and normal rats had been successfully determined. Results showed that AA and fifteen kinds of metabolites by LOX and COX pathways in model rat plasma were significant higher than those in normal ones(P<0.05), while 5-HpETE and LTD4 in model rat plasma were significantly lower than those in normal ones(P<0.05). The methods demonstrated the changes of eicosanoid metabolome occurring in plasma from rat subjects with rheumatoid arthritis. It could be a powerful manner to diagnostic and/or prognostic values for rheumatoid arthritis.

Arachidonic acid metabolomic study of BPH in rats and the interventional effects of Zishen pill, a traditional Chinese medicine.

Zishen pill (ZSP) is a traditional Chinese medicine (TCM) used to treat benign prostatic hyperplasia (BPH). The study used a metabolomic approach based on UHPLC-MS/MS to profile arachidonic acid (AA) metabolic changes and to investigate the interventional mechanisms of ZSP in testosterone- induced BPH rats. In order to explore the potential therapeutic effect of ZSP, rat models were constructed and orally administrated with ZSP. Plasma and urine samples were collected after four weeks and then eleven potential biomarkers (15-HETE, 12-HETE, TXA2, 5-HETE, AA, PGI2, PGF2α, 8-HETE, PGD2, PGE2 and LTB4) were identified and quantified by UHPLC-MS/MS. The chromatographic separation was carried out with gradient elution using a mobile phase comprised of 0.05% formic acid aqueous solution (pH=3.3) (A) and acetonitrile: methanol (80:20, V/V) (B), and each AA metabolites was measured using electrospray ionization source with negative mode and multiple reaction monitoring. The eleven biomarkers in BPH group rat plasma and urine were significant higher than those in sham group rats. Using the potential biomarkers as a screening index, the results suggest that ZSP can potentially reverse the process of BPH by partially regulating AA metabolism through refrain the expression of cyclooxygenase (COX) and lipoxygenase (LOX). This study demonstrates that a metabolomic strategy is useful for identifying potential BPH biomarkers and investigating the underlying mechanisms of a TCM in BPH treatment.

Ultra-high performance liquid chromatography with tandem mass spectrometry method for the simultaneous quantitation of five phthalides in rat plasma: Application to a comparative pharmacokinetic study of Huo Luo Xiao Ling Dan and herb-pair extract.

A fast, sensitive, and reliable ultra-high performance liquid chromatography with tandem mass spectrometry method has been developed and validated for the simultaneous quantitation and pharmacokinetic study of five phthalides (senkyunolide A, ligustilide, butylidenephthalide, 3-butylphthalide, and levistilide A) in rat plasma after oral administration of Huo Luo Xiao Ling Dan (HLXLD) or Angelica sinensis--Ligusticum chuanxiong herb pair (DG-CX) between normal and arthritis rats. After extraction from blood, the analytes and internal standard were subjected to ultra-high performance liquid chromatography with a Shim-pack XR-ODS column (75 × 3.0 mm(2) , 2.2 µm particles) and mobile phase was composed of methanol and water (containing 0.05% formic acid) under gradient elution conditions, with an electrospray ionization source in the positive ionization and multiple reaction monitoring mode. The lower limits of quantification were 0.192-0.800 ng/mL for all the analytes. Satisfactory linearity, precision, accuracy, mean extraction recovery, and acceptable matrix effect have been achieved. The validated method was successfully applied to a comparative pharmacokinetic study of five bioactive components in rat plasma after oral administration of HLXLD or DG-CX alone, respectively, between normal and arthritic rats. The results showed that there were unlike characters of pharmacokinetics among different groups.

Pharmacokinetic comparison of five tanshinones in normal and arthritic rats after oral administration of Huo Luo Xiao Ling Dan or its single herb extract by UPLC-MS/MS.

A fast, sensitive and reliable ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method has been developed and validated for simultaneous quantitation and pharmacokinetic study of five tanshinones (tanshinone I, tanshinone IIA, tanshinone IIB, dihydrotanshinone I, cryptotanshinone), the bio-active ingredients of Huo Luo Xiao Ling Dan (HLXLD) in rat plasma. After liquid-liquid extraction, chromatographic separation was accomplished on a Shim-pack XR-ODS column (75 × 3.0 mm, 2.2 µm particles) and eluted with a mobile phase consisting of acetonitrile-0.05% formic acid aqueous solution (80:20, v/v) at a flow rate of 0.4 mL/min, and the total run time was 7.0 min. The detection was performed on a triple quadrupole tandem mass spectrometry equipped with an electrospray ionization source in positive ionization and multiple reaction monitoring mode. The lower limits of quantification were 0.050-0.400 ng/mL for all the analytes. Linearity, precision and accuracy, the mean extraction recoveries and matrix effects all satisfied criteria for acceptance. This validated method was successfully applied to a comparative pharmacokinetic study of five bio-active components in rat plasma after oral administration of HLXLD or Salvia miltiorrhiza extract in normal and arthritic rats. The results showed that there were different pharmacokinetic characteristics among different groups. Copyright © 2016 John Wiley & Sons, Ltd.

Metabolites identification of Huo Luo Xiao Ling Dan in rat urine by UPLC coupled with electrospray ionization time-of-flight mass spectrometry.

Huo Luo Xiao Ling Dan (HLXLD), a Chinese herbal formula, is used in folk medicine for the treatment of arthritis and other chronic inflammatory diseases. However, the in vivo integrated metabolism of its multiple components remains unknown. In this paper, an ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS) method was developed for detection and identification of HLXLD metabolites in rat urine at high and normal clinical dosages. The prototype constituents and their metabolites in urine were analyzed. The mass measurements were accurate within 8 ppm, and subsequent fragment ions offered higher quality structural information for interpretation of the fragmentation pathways of various compounds. A total of 85 compounds were detected in high dosages urine samples by a highly sensitive extracted ion chromatograms method, including 31 parent compounds and 54 metabolites. Our results indicated that phase 2 reactions (e.g. glucuronidation, glutathionidation and sulfation) were the main metabolic pathways of lactones, alkaloids and flavones, while phase I reactions (e.g. hydrogenation and hydroxylation) were the major metabolic reaction for coumarins, paeoniflorin and iridoids. This investigation provided important structural information on the metabolism of HLXLD and provided scientific evidence to obtain a more comprehensive metabolic profile.

Simultaneous determination of four secoiridoid and iridoid glycosides in rat plasma by ultra performance liquid chromatography-tandem mass spectrometry and its application to a comparative pharmacokinetic study.

A simple, reliable and rapid ultra-performance liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous quantification of four secoiridoid (gentiopicroside, swertiamarin, sweroside) and iridoid glycosides (loganic acid), the bio-active ingredients in rat plasma. After liquid-liquid extraction, chromatographic separation was accomplished on a Shim-pack XR-ODS column with a mobile phase consisting of methanol and 0.1% formic acid in water. A triple quadrupole tandem mass spectrometry equipped with an electrospray ionization source was used as detector operating both in positive and negative ionization mode and operated by multiple-reaction monitoring scanning. The lower limits of quantitation were 0.25-30 ng/mL for all the analytes. Both intra-day and inter-day precision and accuracy of analytes were well within acceptance criteria (±15%). The mean extraction recoveries of analytes and internal standard (amygdalin) from rat plasma were all >71.4%. The validated method was successfully applied to a comparative pharmacokinetic study of four analytes in rat plasma between normal and arthritic rats after oral administration of Huo Luo Xiao Ling Dan and Gentiana macrophylla extract, respectively. Results showed significant differences in pharmacokinetic properties of the analytes among the different groups.

Simultaneous determination of seven coumarins by UPLC-MS/MS: Application to a comparative pharmacokinetic study in normal and arthritic rats after oral administration of Huo Luo Xiao Ling Dan or single-herb extract.

A simple, sensitive and reliable ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method has been developed and validated for simultaneous quantitation of seven coumarins, the bio-active ingredients of Huo Luo Xiao Ling Dan (HLXLD), in rat plasma. The liquid-liquid extraction method with ether-dichloromethane (2:1, v/v) was used to prepare the plasma samples. Analytes and internal standard (IS) of bifendate were separated on a Shim-pack XR-ODS column (75mm×3.0mm, 2.2µm particles) using gradient elution with the mobile phase consisting of methanol and 0.05% formic acid in water at a flow rate of 0.4mL/min. Detection was performed on a triple quadrupole (TQ) tandem mass spectrometry equipped with an electrospray ionization source in the positive ionization and multiple reaction monitoring (MRM) mode. The lower limits of quantitation (LLOQ) were 0.03-0.25ng/mL for all the analytes. Intra- and inter-day precision and accuracy of the seven analytes were well within acceptance criteria (15%). The matrix effect and the mean extraction recoveries of the analytes and IS from rat plasma were all within satisfaction. The validated method has been successfully applied to compare pharmacokinetic profiles of the seven active ingredients in rat plasma between normal and arthritic rats after oral administration of HLXLD, Angelica pubescens extract and Notopterygium incisum extract, respectively. Results showed that there were remarkable differences in pharmacokinetic properties of the analytes among the different groups.

A UPLC-MS/MS method for simultaneous quantitation of three monoterpene glycosides and four alkaloids in rat plasma: application to a comparative pharmacokinetic study of Huo Luo Xiao Ling Dan and single herb extract.

The objective of this study was to develop a sensitive and reliable ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for simultaneous quantitation of three monoterpene glycosides (paeoniflorin, alibiflorin and oxypaeoniflorin) and four alkaloids (tetrahydropalmatine, corydaline, dehydrocorydaline and berberine), the main active ingredients of Radix Paeoniae Rubra extract (RPE) and Corydalis yanhusuo extract (CYE) in Huo Luo Xiao Ling Dan (HLXLD), and to compare the pharmacokinetics of these active ingredients in normal and arthritic rats orally administrated with HLXLD or RPE/CYE alone. The analytes and internal standard (IS) (geniposide) were separated on a XBridge C18 column (150 × 4.6 mm, 3.5 µm) using gradient elution with the mobile phase consisting of methanol and 0.01% formic acid in water at a flow rate of 0.6 ml/min. The detection of the analytes was performed on Acquity UPLC-MS/MS system with an electrospray ionization and multiple reaction monitoring mode via polarity switching between negative (for monoterpene glycosides) and positive (for alkaloids) ionization mode. The lower limits of quantification were 2.5, 1, 0.5, 0.2, 0.2, 0.02 and 0.01 ng/ml for paeoniflorin, alibiflorin, oxypaeoniflorin, tetrahydropalmatine, corydaline, dehydrocorydaline and berberine, respectively. Intra-day and inter-day precision and accuracy of analytes were well within acceptance criteria (15%). The mean extraction recoveries of analytes and IS from rat plasma were all more than 83.1%. The validated method has been successfully applied to determination of the analytes. Results showed that there were remarkable differences in pharmacokinetic properties of the analytes between herbal formula and single herb group, normal and arthritic group.

Systematic chemical profiling of a multicomponent Chinese herbal formula Huo Luo Xiao Ling Dan by ultra high performance liquid chromatography coupled with electrospray ionization quadrupoletime-of-flight mass spectrometry.

Huo Luo Xiao Ling Dan, a Chinese herbal formula consisting of 11 different herbs, has been used in folk medicine for the treatment of arthritis and other chronic inflammatory diseases. However, the chemical compositions of Huo Luo Xiao Ling Dan are not completely characterized. In the present study, an ultra high performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry method in positive and negative ion modes was employed to identify biochemical constitutes in Huo Luo Xiao Ling Dan. As a result, a total of 76 compounds including alkaloids, monoterpene glycosides, iridoids, phenolic acids, and tanshinones, coumarins, lactones, flavones, and their glycosides, triterpenes, and triterpene saponins were characterized by comparing the retention time and mass spectrometry data with reference standards within 5 ppm error or by reference to the reference literature. These results would provide the basis for a further in vivo study of Huo Luo Xiao Ling Dan and information for potential new drug candidates for treating arthritis and other chronic inflammatory diseases.

Comparative pharmacokinetic study of two boswellic acids in normal and arthritic rat plasma after oral administration of Boswellia serrata extract or Huo Luo Xiao Ling Dan by LC-MS.

Huo Luo Xiao Ling Dan (HLXLD), a Chinese herbal formula composed of 11 different herbs, has been used traditionally for the treatment of arthritis and other chronic inflammatory diseases. However, the pharmacokinetic profile of its anti-inflammatory bioactive compounds has not been elucidated. Boswellic acids are the bioactive compounds with potent anti-inflammatory activity isolated from Boswellia serrate which is one of the 11 herbs of HLXLD. The objective of the study was to compare the pharmacokinetics of the two bioactive bowsellic acids: 11-keto-ß-boswellic acid and 3-O-acetyl-11-keto-ß-boswellic following oral administration of HLXLD or Boswellia serrata extract alone in normal and arthritic rats. An LC-MS method was developed and validated for the determination of 11-keto-ß-boswellic acid and 3-O-acetyl-11-keto-ß-boswellic in the comparative pharmacokinetic study. The results showed that there were significant differences in pharmacokinetic parameters between normal and arthritic groups. Interestingly, the absorptions of two boswellic acids were significantly higher in HLXLD than Boswellia serrata extract alone, indicating the synergistic effect of other herbal ingredients in HLXLD. This comparative pharmacokinetic study provided direct evidence supporting the notion that the efficacy of a complex mixture such as HLXLD is better than that of single components in treating human diseases.