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.