Identification of the target protein and molecular mechanism of honokiol in anti-inflammatory action.

BACKGROUND: Searching the targets of natural products is very important for drug discovery and elucidating the mechanism of drug action and disease. Honokiol (HK), as the major active component of Magnolia officinalis Rehder & E.H.Wilson, has been widely used in medicine and cosmetics. Among its bioactivities, its anti-inflammatory activity is particularly impressive. However, the target protein of HK in anti-inflammatory action and its regulatory mechanism are unclear. PURPOSE: Here, we identified the target protein and molecular mechanism of the anti- inflammatory action of HK. METHODS: First, an LPS-induced septic shock model and DSS-induced ulcerative colitis model were used to assess the anti-inflammatory efficacy of HK. Second, the drug affinity responsive target stability, proteomics analysis, thermal shift assays and cellular thermal shift assays were used to identify and validate the target of HK. Finally, western blot, ELISA, LDH immunofluorescence staining, shRNA and LC/MS for L-leucine analysis were performed to determine the mechanism of the anti-inflammatory action of HK. RESULTS: This study revealed that HK significantly alleviated LPS-induced septic shock and DSS-induced ulcerative colitis in vivo, suggesting that HK has significant anti-inflammatory activity. HK treatment dramatically reduced IL-1ß release and caspase-1 activation at different time points, showing that HK could inhibit both NLRP3 inflammasome priming and activation processes in cells. HK also suppressed adaptor apoptosis speck-like protein oligomerization. Mechanistically, SLC3A2 was identified as a direct target of HK in THP-1 cells. HK downregulated SLC3A2 expression by promoting its degradation via proteasome-mediated proteolysis. Further study demonstrated that HK triggered SLC3A2 to suppress NLRP3 inflammasome activation by significantly reducing the content of L-leucine transported into cells and lysosomes to block the mTORC1 pathway. CONCLUSIONS: Our work identified HK as a promising anti-inflammatory drug candidate through the SLC3A2/L-leucine/mTORC1/NLRP3 pathways.

The ethanolic extract of Artemisia anomala exerts anti-inflammatory effects via inhibition of NLRP3 inflammasome.

BACKGROUND: Artemisia anomala S. Moore (Compositae), known as "Nan-Liu-Ji-Nu" in traditional Chinese medicine (TCM), has been used to treat many inflammatory diseases, including enteritis, acute icteric hepatitis, rheumatism, toothache, tonsillitis, and chronic bronchitis, for centuries. Our preliminary studies have demonstrated that the ethanolic extract of A. anomala (EAA) might be with the potential of inhibiting the activation of the NLRP3 inflammasome. However, the anti-inflammatory activity of EAA based on NLRP3 inflammasome inhibition is still unclear. PURPOSE: This work aimed to elucidate the anti-inflammatory mechanism of EAA by inhibiting NLRP3 inflammasome activation. METHODS: Lipopolysaccharide (LPS)-primed bone marrow-derived macrophages (BMDMs) were used to evaluate the inhibitory effects on NLRP3 inflammasome activation. The level of IL-1ß was determined by ELISA. The expression levels of IL-1ß, caspase-1, NLRP3, and ASC were assayed using western blot analysis. ASC oligomerization and speck formation were detected by immunofluorescence microscopy. The measurements of intracellular chloride and potassium were conducted using N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) probe assay and inductively coupled plasma-optical emission spectrometry (ICP-OES), respectively. Mitochondrial reactive oxygen species (mtROS) were examined using the MitoSOX method. Acridine orange (AO) staining was used to detect the permeability of the lysosomal membrane. A DSS-induced ulcerative colitis model was established to evaluate the anti-inflammatory effects of EAA in vivo. Finally, high-performance liquid chromatography (HPLC) was employed to identify and quantify the major constituents of EAA. RESULTS: In BMDMs, EAA significantly inhibited the release of IL-1ß induced by LPS. The mechanistic study revealed that EAA inhibited NLRP3 inflammasome activation by blocking the oligomerization of ASC and suppressed the LPS-induced priming step. Furthermore, EAA protected lysosomes by inhibiting the TAK1-JNK pathway, thereby inhibiting the assembly of downstream NLRP3 inflammasome and the production of IL-1ß. In addition, EAA exerted potent protective effects in an ulcerative colitis model by decreasing the content of colonic IL-1ß and alleviating the process of ulcerative colitis. HPLC analysis identified eight main components of EAA, including isofraxidin (1), quercetin-7-O-ß-D-glucopyranoside (2), apigenin-7-O-ß-D-glucopyranoside (3), 7-methoxycoumarin (4), quercetin (5), luteolin (6), kaempferol (7), and eupatorin (8), Of these compounds, quercetin and kaempferol were found to be the most potent ingredients. CONCLUSION: These findings collectively reveal that EAA exerts anti-inflammatory effects by both suppressing the NLRP3 priming step and protecting lysosomes to inhibit NLRP3 inflammasome activation, suggesting that this traditional herbal medicine might be used to treat NLRP3-driven inflammatory diseases.

Ethanol extract of Liriodendron chinense (Hemsl.) Sarg barks attenuates hyperuricemic nephropathy by inhibiting renal fibrosis and inflammation in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Liriodendron chinense (Hemsl.) Sarg, known as the Chinese tulip tree, has a long history of cultivation and utilization in many Asia countries, especially in China to use in traditional Chinese medicine for expelling "wind and dampness", a term corresponding to rheumatic fever and rheumatoid arthritis. Interestingly, the barks of Liriodendron chinense (Hemsl.) Sarg was also found in folk to treat gout. However, further experimental studies remained to confirm its uric acid-lowering effects. AIM OF THE STUDY: The aim of the study was to evaluate the protective effect of ethanol extract of the barks of Liriodendron chinense (Hemsl.) Sarg (EELC) in a mouse model of hyperuricemic nephropathy (HN) and the involved mechanisms. MATERIALS AND METHODS: EELC at a respective dose of 250 mg/kg/d or 500 mg/kg/d were orally administered to HN mice induced by a mixture of adenine (160 mg/kg/d)/potassium oxonate (2.4 g/kg/d) for 21 days. At the end of the treatment, serum uric acid, kidney functions (serum creatinine, blood urea nitrogen and urine microalbumin), 24-h urine uric acid excretion, as well as kidney pathological changes were investigated by biochemical assay, histopathological score, immunofluorescence and histochemistry, RT-qPCR, and western blotting analysis. RESULTS AND DISCUSSION: Oral administration of EELC significantly lowered serum uric acid level at 500 mg/kg (185.75 ± 15.49 µmol/L of EELC vs. 238.28 ± 20.97 µmol/L of HN model, p < 0.01) in HN mice. EELC at 500 mg/kg also remarkably reduced the levels of serum creatinine (82.92 ± 7.86 µmol/L of EELC vs. 92.08 ± 6.13 µmol/L of HN model, p < 0.0001), blood urea nitrogen (21.50 ± 1.87 mmol/L of EELC vs. 29.40 ± 3.95 mmol/L of HN model, p < 0.001) and urine microalbumin (4.25 ± 0.40 mg/L of EELC vs. 5.95 ± 0.33 mg/L of HN model, p < 0.001) to improve renal function. It also attenuated renal fibrosis, especially the high-dose of EELC. Furthermore, EELC could inhibit the activation of NF-κB, ASK1/JNK/c-Jun, JAK2/STAT3 signaling pathways and reduce the release of pro-inflammatory cytokine TNF-α in the kidneys of HN mice. Additionally, EELC remarkably increased urine uric acid excretion of HN mice, which may be achieved by the upregulation of organic anion transporter 1 (OAT1), OAT3 and ATP-binding cassette subfamily G member 2 (ABCG2) proteins. CONCLUSIONS: EELC alleviated the progression of HN by suppressing the activation of NF-κB, ASK1/JNK/c-Jun and JAK2/STAT3 signaling pathway, reducing the infiltration of inflammatory factors and uric acid accumulation in the kidney.

Synthesis and evaluation of new compounds bearing 3-(4-aminopiperidin-1-yl)methyl magnolol scaffold as anticancer agents for the treatment of non-small cell lung cancer via targeting autophagy.

Magnolol and honokiol are the two major active ingredients with similar structure and anticancer activity from traditional Chinese medicine Magnolia officinalis, and honokiol is now in a phase I clinical trial (CTR20170822) for advanced non-small cell lung cancer (NSCLC). In search of potent lead compounds with better activity, our previous study has demonstrated that magnolol derivative C2, 3-(4-aminopiperidin-1-yl)methyl magnolol, has better activity than honokiol. Here, based on the core of 3-(4-aminopiperidin-1-yl)methyl magnolol, we synthesized fifty-one magnolol derivatives. Among them, compound 30 exhibited the most potent antiproliferative activities on H460, HCC827, H1975 cell lines with the IC50 values of 0.63-0.93 µM, which were approximately 10- and 100-fold more potent than those of C2 and magnolol, respectively. Besides, oral administration of 30 and C2 on an H460 xenograft model also demonstrated that 30 has better activity than C2. Mechanism study revealed that 30 induced G0/G1 phase cell cycle arrest, apoptosis and autophagy in cancer cells. Moreover, blocking autophagy by the autophagic inhibitor enhanced the anticancer activity of 30in vitro and in vivo, suggesting autophagy played a cytoprotective role on 30-induced cancer cell death. Taken together, our study implied that compound 30 combined with autophagic inhibitor could be another choice for NSCLC treatment in further investigation.

Biphenyl-type neolignans from stem bark of Magnolia officinalis with potential anti-tumor activity.

Six new biphenyl-type neolignans (1-6), and eighteen known compounds (7-24) were isolated from the EtOH extract of Magnolia officinalis. Their structures were determined by 1D and 2D NMR, and by HRMS. The anti-tumor activities of the isolated compounds were evaluated on HepG2, HCT-116, H1975 and HUVEC cell lines. Among the isolated compounds, nine compounds (3, 5, 7, 8, 12, 14, 20, 22, and 24) showed moderate cytotoxicities, and compound 23 showed the best cytotoxicity with IC50 value lower than 10 µM.

Herb-sourced emodin inhibits angiogenesis of breast cancer by targeting VEGFA transcription.

Anti-angiogenesis is an important and promising strategy in cancer therapy. However, the current methods using anti-vascular endothelial growth factor A (VEGFA) antibodies or inhibitors targeting VEGFA receptors are not as efficient as expected partly due to their low efficiencies in blocking VEGFA signaling in vivo. Until now, there is still no method to effectively block VEGFA production in cancer cells from the very beginning, i.e., from the transcriptional level. Here, we aimed to find bioactive small molecules to block VEGFA transcription. Methods: We screened our natural compound pool containing 330 small molecules derived from Chinese traditional herbs for small molecules activating the expression of seryl-tRNA synthetase (SerRS), which is a newly identified potent transcriptional repressor of VEGFA, by a cell-based screening system in MDA-MB-231 cell line. The activities of the candidate molecules on regulating SerRS and VEGFA expression were first tested in breast cancer cells. We next investigated the antiangiogenic activity in vivo by testing the effects of candidate drugs on the vascular development in zebrafish and by matrigel plug angiogenesis assay in mice. We further examined the antitumor activities of candidate drugs in two triple-negative breast cancer (TNBC)-bearing mouse models. Furthermore, streptavidin-biotin affinity pull-down assay, coimmunoprecipitation assays, docking analysis and chromatin immunoprecipitation were performed to identify the direct targets of candidate drugs. Results: We identified emodin that could greatly increase SerRS expression in TNBC cells, consequently reducing VEGFA transcription. Emodin potently inhibited vascular development of zebrafish and blocked tumor angiogenesis in TNBC-bearing mice, greatly improving the survival. We also identified nuclear receptor corepressor 2 (NCOR2) to be the direct target of emodin. Once bound by emodin, NCOR2 got released from SerRS promoter, resulting in the activation of SerRS expression and eventually the suppression of VEGFA transcription. Conclusion: We discovered a herb-sourced small molecule emodin with the potential for the therapy of TNBC by targeting transcriptional regulators NCOR2 and SerRS to suppress VEGFA transcription and tumor angiogenesis.

Modulation of LPS-induced inflammation in RAW264.7 murine cells by novel isoflavonoids from Millettia pulchra.

Millettia pulchra is a renowned anti-inflammatory herbal medicine in southeast provinces of China. However, the underlying anti-inflammation mechanism remained incompletely understood. Herein, four new isoflavones, pulvones A-D and eleven reported constituents were isolated from the stems of Millettia pulchra with their structures being elucidated by HRMS and NMR analysis. The anti-inflammatory activities of pulvones A and C were further evaluated due to the better inhibitory activity on nitric oxide production in LPS-stimulated RAW264.7 cells and no obvious cytotoxicity to RAW264.7 cells. Western blot showed that pulvones A significantly decreased the levels of iNOS and COX-2 proteins and pulvones C only decreased the level of iNOS protein. ELISA analysis demonstrated that pulvones A inhibited the production of both interleukin-6 (IL-6) and IL-1ß while pulvones C showed better suppression effect on IL-1ß production in LPS-stimulated RAW264.7 cells. Then, their potential inhibitory effects on NF-κB pathway were tested in LPS-stimulated RAW264.7 cells. Immunofluorescence and western blot assay showed that pulvones A and C reduced the nuclear translocation of NF-κB(p65) and interrupted IκB phosphorylation. The ADP-Glo™ kinase assay showed pulvones A and C could directedly inhibit the IKKß kinase activity with the inhibitory rate of 40%, which were also verified by docking study. Collectively, these results suggested that pulvones A and C's anti-inflammatory effects were relevant to the interruption of NF-κB activation by inhibiting IKKß kinase.

Alkaloids from Black Pepper (Piper nigrum L.) Exhibit Anti-Inflammatory Activity in Murine Macrophages by Inhibiting Activation of NF-κB Pathway.

Black pepper (Piper nigrum L.) has been commonly utilized in food preparation and traditional medicine in several countries. Seven new amide alkaloids, pipernigramides A-G (3, 10, 38, and 41-44), a new piperic ester, pipernigrester A (48), along with 47 known compounds were isolated from the EtOH extract of P. nigrum. The inhibitory effects on nitric oxide (NO) of all compounds were then evaluated. Among the tested compounds, three of them (42-44) significantly inhibited inducible nitric oxide synthase (iNOS)-mediated NO (IC50 = 4.74 ± 0.18, 4.08 ± 0.19, and 3.71 ± 0.32 µM, respectively), and IL-1ß, IL-6, TNF-α, and PGE2 release in RAW 264.7 cells stimulated by lipopolysaccharide. Moreover, 42-44 suppressed IκB degradation and further inhibited the cytosol-nucleus translocation of the p65 subunit by targeting IKK-ß. In the carrageenan-induced paw edema test, 42-44 demonstrated anti-inflammatory effects as well. These results indicate that all three compounds from P.nigrum have the potential anti-inflammatory effects.

Anti-inflammatory Ellagitannins from Cleidion brevipetiolatum for the Treatment of Rheumatoid Arthritis.

Six new ellagitannins, brevipetins B-G (5 and 7-11), and a new phenolic glucoside, brevipetin A (4), along with six known compounds were isolated from the traditional Chinese medicinal plant Cleidion brevipetiolatum. Their structures and absolute configurations were determined by spectroscopic analyses, chemical methods, and TD-DFT-ECD calculations. Compounds 5-11 exhibited NO inhibitory effects with IC50 values of 1.9-8.2 µM, and 9 showed the most potent inhibitory effect (IC50: 1.9 µM). An in vivo anti-inflammatory assessment of 9 showed that it exerts therapeutic effects in both the carrageenan-induced rat paw edema and collagen-induced arthritis (CIA) models at 50 mg/kg oral administration. The enhanced protein and mRNA expression levels of iNOS (inducible nitric oxide synthase) and COX-2 (cyclooxygenase-2) in LPS-stimulated RAW 264.7 cells were dose-dependently suppressed by 9. An anti-inflammatory mechanistic study revealed that 9 suppressed NF-κB activity by inhibiting IκBα phosphorylation and blocking translocation of p65 from the cytosol to the nucleus. Therefore, 9 might have the potential to be developed as a lead compound for relieving rheumatoid arthritis.

Millepachine showed novel antitumor effects in cisplatin-resistant human ovarian cancer through inhibiting drug efflux function of ATP-binding cassette transporters.

Millepachine (MIL), a bioactive natural chalcone from Chinese herbal medicine Millettia pachycarpa Benth, exhibits strong antitumor effects against many human cancer cells both in vitro and in vivo. In this study, we found that MIL significantly inhibited the proliferation of cisplatin-resistant A2780CP cells via inducing obvious G2/M arrest and apoptosis and down-regulating the activity of topoisomerase II protein. We further found that the mechanism by which MIL showed good antitumor effects in cisplatin-resistant human ovarian cancer was associated with inhibiting the expression of ATP-binding cassette transporters in cisplatin-resistant A2780CP cells. Importantly, MIL did not only significantly inhibit the tumor growth in cisplatin-sensitive A2780S xenograft model, with an inhibitory rate of 73.21%, but also inhibited the tumor growth in the cisplatin-resistant A2780CP xenograft model, with an inhibitory rate of 65.68% (p < 0.001 vs. control; p < 0.001 vs. DDP). In addition, MIL did not induce acquired drug resistance in A2780S tumor-bearing mice with an inhibitory rate of 60.03%. The promising in vitro and in vivo performance indicated that MIL exhibited potential significance for drug research and development.

Discovery and synthesis of novel magnolol derivatives with potent anticancer activity in non-small cell lung cancer.

EGFR T790 M accounts for 50% to 60% of cases of non-small-cell lung carcinoma (NSCLC) resistance to the first-generation EGFR tyrosine kinase inhibitors (TKIs). Hence, identifying novel compounds with activity against TKIs resistant is of great value. In this study, twenty honokiol and magnolol derivatives were isolated from the EtOH extract of Magnolia officinalis and the antiproliferative activity was evaluated on HCC827 (19del EGFR mutation), H1975 (L858 R/T790 M EGFR mutation), and H460 (KRAS mutation) cell lines. Among the isolated compounds, piperitylmagnolol (a 3-substituted magnolol derivative) showed the best antiproliferative activity against those three cell lines with the IC50 values of 15.85, 15.60 and 18.60 µM, respectively, which provided a direction for the structural modification of magnolol. Further structural modification led to the synthesis of thirty-one magnolol derivatives, and compounds A13, C1, and C2 exhibited significant and broad-spectrum antiproliferative activity with the IC50 values ranging from 4.81 to 13.54 µM, which were approximately 4- and 8-fold more potent than those of honokiol and magnolol, respectively. Moreover, their aqueous solubility was remarkably improved with 12-, 400- and 105 fold greater than those of honokiol and magnolol. Anti-tumor mechanism research revealed that these three compounds were able to induce cell cycle arrest at G0/G1 phase, cause efficient apoptosis in H1975 cells, and also prevent the migration of HUVECs in a dose-dependent manner through Cdk2, Cdk4, Cyclin E, and Cyclin D1 inhibition as well as up-regulation of cleaved-PARP and cleaved-caspase 3 levels. In in vivo antitumor activity, C2 (10, 30 and 100 mg/kg, po) dose-dependently inhibited the tumor growth in H1975 xenograft model with the tumor inhibition rate of 46.3%, 59.3% and 61.2% respectively, suggesting that C2 is a potential oral anticancer agent deserving further investigation.

Identification, characterization and HPLC quantification of formulation-related impurities of honokiol, an antitumor natural drug candidate in clinical trials.

Natural products and their derivatives have historically been invaluable as a source of therapeutic agents. Honokiol, as a well-known natural product in Chinese herbal medicine Houpu, is finally being studied in a Phase I clinical trial (CTR20170822) in patients with Advanced Non-Small Cell Lung Cancer (NSCLS) in China this year. During the honokiol liposome formulation process, five major impurities were present in the range of 0.05-0.1% based on the HPLC analysis. These five major impurities were obtained from the forced degradation product of honokiol through countercurrent chromatography and prep-HPLC. The structure were elucidated with 1H NMR, 13C NMR, 2D NMR and MS spectral data. The proposed HPLC method was validated for specificity, linearity (concentration range 0.01-1.62, 0.003-0.96, 0.05-7.98, 0.04-6.52, 0.03-5.18 µg/ml for impurities I-V respectively, R2 > 0.9988), accuracy (99.11-100.67%), precision (CV < 1.6%), and sensitivity (LOD 3.3, 0.1, 16.7, 13.3, 10.0 ng/ml for impurities I-V respectively). The validated method was employed in the further study of the honokiol drug substance.

Barbigerone reverses multidrug resistance in breast MCF-7/ADR cells.

Development of agents to overcome multidrug resistance (MDR) is one of the important strategies in cancer chemotherapy, and P-glycoprotein (P-gp) correlates with the degree of resistance. As a naturally occurring isoflavone, whether barbigerone (BA) could reverse MDR, is unknown. In this paper, we evaluated effects of BA on reversing P-gp mediated MDR of adriamycin (ADR)-resistant human breast carcinoma (MCF-7/ADR) cells. BA (0.5 µM) treatment showed strong potency to increase ADR cytotoxicity toward MCF-7/ADR cells. It was also demonstrated that BA time- and dose-dependently increased accumulations of ADR and reduced the efflux in MCF-7/ADR cells, pretreatment of these cells with BA might relocalized ADR to the nuclei. Furthermore, the results also revealed that BA did not affect P-gp, but alter P-gp ATPase activity. Intravenous administration of BA significantly increased anticancer efficacy of ADR to MCF-7/ADR xenograft model in nude mice. These results revealed that BA might reverse P-gp mediated MDR through inhibition of ATPase activity, which indicated a novel use of BA as a potent candidate for cancer chemotherapy.

Separation of caffeoylquinic acids and flavonoids from Asteris souliei by high-performance counter-current chromatography and their anti-inflammatory activity in vitro.

Eleven compounds were successfully separated from Asteris souliei by using a two-step high-performance counter-current chromatography method. The first step involved a reversed phase isocratic counter-current chromatography separation using hexane/ethyl acetate/methanol/water (1:0.8:1:1 v/v/v/v), which produced three fractions, the first two of which were mixtures. The second step used step-gradient reversed-phase counter-current chromatography with hexane/butanol/ethyl acetate/methanol/water (1:0.5:3.5:1:4 v/v/v/v/v) initially followed by hexane/ethyl acetate/methanol/water (1:2:1:2 v/v/v/v) to separate Fraction 1 into seven compounds; and hexane/ethyl acetate/methanol/water (1:1:1:1.2 v/v/v/v) to separate Fraction 2 into three further compounds. The chemical structures of the separated compounds were identified by ESI-MS and NMR spectroscopy (1 H and 13 C). Baicalin (5), eriodictyol (7), apigenin-7-glycoside (8), quercetin (9), luteolin (10), and apigenin (11) showed obvious inhibitory effects on lipopolysaccharide-induced nitric oxide production in RAW264.7 cells at a concentration of 10 µg/mL.

Sample injection strategy to increase throughput in counter-current chromatography: Case study of Honokiol purification.

Counter-current chromatography (CCC) has been widely used as a preparative separation method to purify natural products from plant extracts and fermentation broths. Traditionally, throughput optimization in CCC has focused on sample concentration and sample volume. In this paper sample injection was considered as consisting of three variables: injection flow rate, post-injection flow rate and sample solvent. The effects of these parameters were studied using a honokiol purification from a Magnolia officinalis bark extract as a case study aiming to achieve the highest throughput/yield ratio for greater than 99% purity of this potential anti-cancer drug obtained for submission to the Chinese FDA. An injection method was established that increased the throughput of honokiol by 46.5% (from 3.05g/h to 4.47g/h), and decreased the solvent consumption of mobile phase and stationary phase per gram of honokiol by 40.0% (from 0.68L/g to 0.41L/g) and 48.4% (from 0.40L/g to 0.21L/g) respectively. These results show the importance of understanding the whole injection process when optimizing a given CCC separation.

[In vitro metabolism of anti-tumor compound E7 in different species of liver microsomes].

To investigate the metabolic stability of E7 in liver microsomes of human, Beagle dog, Cynomolgus monkey and SD rats, and compare the metabolic differences between different species. Selective chemical inhibitors were used to determine the effects of different inhibitors on E7 metabolic rate, and predict the main enzymes involved in E7 metabolism in rat liver microsomes. The experimental results showed that the in vitro half-lives (T1/2) of E7 in liver microsomes of human, dog, monkey and rats were 57.75, 69.30, 16.90,30.13 min respectively. Their intrinsic clearance rate was 0.004 8, 0.004 0, 0.016 4 and 0.009 2 mL•min⁻¹â€¢mg⁻¹ respectively. Hence, it could be speculated that the metabolic rate of E7 was similarly slow in human and dog liver microsomes; while it was similarly fast in monkey and rat liver microsomes. There was significant difference in metabolic rate of E7 between different species. The results showed that CYP2E1, CYP2A6, CYP1A2 and CYP2D6 might participate in metabolism of E7, while the contribution of polymorphic CYP3A4 was small.

Separation of flavonoids from Millettia griffithii with high-performance counter-current chromatography guided by anti-inflammatory activity.

Millettia griffithii is a unique Chinese plant located in the southern part of Yunnan Province. Up to now, there is no report about its phytochemical or related bioactivity research. In our previous study, the n-hexane crude extract of Millettia griffithii revealed significant anti-inflammatory activity at 100 µg/mL, inspiring us to explore the anti-inflammatory constituents. Four fractions (I, II, III, and A) were fractionated from n-hexane crude extract by high-performance counter-current chromatography with solvent system composed of n-hexane/ethyl acetate/methanol/water (8:9:8:9, v/v) and then were investigated for the potent anti-inflammatory activity. Fraction A, with the most potent inhibitory activity was further separated to give another four fractions (IV, V, VI, and B) with solvent system composed of n-hexane/ethyl acetate/methanol/water (8:4:8:4, v/v). Compound V and fraction B exhibited remarkable anti-inflammatory activity with nitric oxide inhibitory rate of 80 and 65%, which was worth further fractionation. Then, three fractions (VII, VIII, and IX) were separated from fraction B with a solvent system composed of n-hexane/ethyl acetate/methanol/water (8:1:8:1, v/v), with compound VIII demonstrating the most potent inhibitory activity (80%). Finally, the IC50 values of compound V and VIII were tested as 38.2 and 14.9 µM. The structures were identified by electrospray ionization mass spectrometry and(1)H and (13)C NMR spectroscopy.

Pharmacological effects of the water fraction of key components in the traditional Chinese prescription Mai Tong Fang on 3T3-L1 adipocytes and ob/ob diabetic mice.

Mai Tong Fang (MTF), a Chinese herbal combination, has been used for the treatment of diabetic nephropathy in traditional medical clinics in China. However, the anti-adipogenic and anti-hyperglycemic effects of MTF have not been fully elucidated, so this study explored these pharmacological activities in 3T3-L1 adipocytes and ob/ob mice, respectively, of the water fraction of milkvetch root, salviae miltiorrhizae and mulberry as key components of MTF. MTF was found to inhibit adipogenesis and triglyceride accumulation in 3T3-L1 adipocytes. Oral administration of MTF in ob/ob mice for 8 weeks, exhibited positive controls on blood glucose and body weight, and further improved glucose tolerance according to an oral glucose tolerance test. Importantly, MTF extract alleviated fat deposition and ballooning degeneration in liver tissue and blocked the increase of adipocyte size in adipose tissue from treated ob/ob mice. These results indicated that the extract of key components in the traditional Chinese prescription MTF continue a potent anti-adipogenic and glucose-lowering agent.

Millettia pachycarpa exhibits anti-inflammatory activity through the suppression of LPS-induced NO/iNOS expression.

The present study was designed to investigate the in vitro and in vivo anti-inflammatory activity of flavonoids isolated from Millettia pachycarpa Benth. The seeds of M. pachycarpa Benth were extracted with ethanol and subjected to chromatographic separation for the isolation of bioactive compounds. Their structures were elucidated by spectroscopic methods. The anti-inflammatory activity of the compounds was investigated by evaluating the inhibition ability of NO production, iNOS activity and iNOS protein expression induced by LPS-stimulated RAW264.7 macrophages in vitro and the carrageenan-induced hind paw edema model in vivo. Molecular docking simulation was also employed to obtain the binding parameters in the binding pocket of iNOS. Thirteen compounds (1-13) were isolated from Chinese herbal medicine M. pachycarpa Benth. Among them, 4-hydroxylonchocarpin (6) and deguelin (7) exhibited remarkable inhibitory rates of 66.5% and 57.7%, respectively, compared with that of 52.5% of indomethacin in LPS-induced macrophages cells. 4-hydroxylonchocarpin (6) with low toxicity (IC50 > 100 µm) exhibited better inhibitory effects to positive control of 1400W on iNOS activity at the concentration of 10 µm. Western blot assay revealed that 4-hydroxylonchocarpin (6) inhibited iNOS protein expression in RAW264.7 cells and molecular docking simulation showed that 4-hydroxylonchocarpin (6) fit well into the binding pocket of iNOS. In the carrageenan-induced paw edema model, our data revealed that the anti-inflammatory potential of 4-hydroxylonchocarpin (6) at 10 mg/kg showed comparable inhibitory ability to indomethacin at 5 h while a higher concentration of 4-hydroxylonchocarpin (6) at 50 mg/kg showed higher inhibitory activity than indomethacin, which was further confirmed by plasma levels of nitrite. The overall results suggest that 4-hydroxylonchocarpin (6) might be used as a potential therapeutic agent for inflammation-associated disorders.

Bioactivity-guided isolation of anti-inflammation flavonoids from the stems of Millettia dielsiana Harms.

Bioactivity-guided isolation of the EtOAc extract of the stems of Millettia dielsiana Harms yielded two new isoflavones together with nine known ones. Their structures were elucidated by analysis of the spectroscopic data including 2D NMR. All of the isolates were evaluated for their potential to inhibit the LPS-induced production of nitric oxide and TNF-α in murine macrophage RAW 264.7 cells. Among the tested compounds, Millesianin C (1) had the most potent anti-inflammatory effect decreasing NO production similar to that of dexamethasone and decreasing TNF-α secretion better than that of dexamethasone. Their structure-activity relationship was also analyzed.