Xuelian injection ameliorates complete Freund's adjuvant-induced acute arthritis in rats via inhibiting TLR4 signaling.

Background: Xuelian injection (XI), a classic preparation extracted from Saussureae Involucratae Herba, has been clinically used to manage rheumatoid arthritis (RA) for nearly twenty years in China. However, the underlying anti-RA mechanism of XI remains unclear. In this study, complete Freund's adjuvant (CFA)-induced acute arthritic model was used to examine the anti-RA effects of XI in vivo. The molecular mechanisms of this action were further investigated using lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Methods: XI and XI freeze dried powder were characterized by UPLC analysis. CD68 and TLR4 expression in the ankle joints was measured by immunohistochemistry. The secretion of inflammatory mediators was detected by ELISA. The expression levels of TLR4 involved components were measured by Western blotting. The localization of transcription factors was measured by immunofluorescence assay. Results: XI treatment ameliorated arthritic symptoms induced by CFA in the ankle joints of rats. The serum levels of inflammatory mediators, including TNF-α, MCP-1, and Rantes were decreased by XI treatment. The elevation of CD68 and TLR4 levels in ankle joints caused by CFA was suppressed by XI treatment. Moreover, XI treatment inhibited the secretion of nitric oxide and prostaglandin E2 in LPS-treated RAW264.7 macrophages. The expression of their enzymes iNOS and COX-2 was also decreased after XI treatment. The production of inflammatory mediators, including TNF-α, IL-6, IL-1ß, MCP-1, MIP-1α, and Rantes was reduced by XI treatment in LPS-stimulated RAW264.7 cells. The phosphorylation of p38, JNK, ERK, TBK1, IKKα/ß, IκB, p65, c-Jun, and IRF3 was reduced after XI treatment. Additionally, the expression levels of nuclear proteins of p65, c-Jun, and IRF3 were inhibited by XI treatment. Conclusions: Taken together, XI possesses potential anti-RA effect and the underlying mechanism may be closely associated with the inhibition of TLR4 signaling. Our findings provide further pharmacological justifications for the clinical use of XI in RA treatment.

Sichen Formula Ameliorates Lipopolysaccharide-Induced Acute Lung Injury via Blocking the TLR4 Signaling Pathways.

Purpose: Sichen (SC) formula is a classic prescription of Tibetan medicine. Due to its potential anti-inflammatory effect, the SC formula has been clinically used to treat respiratory diseases for many years in the Chinese Tibet region. The present study aimed to investigate the anti-inflammatory effect of SC and explore the underlying mechanisms. Methods: SC formula was characterized by HPLC analysis. The acute lung injury (ALI) mouse model was induced by direct intratracheal lipopolysaccharide (LPS) instillation, and bronchoalveolar lavage fluid (BALF) and lung tissues were collected. Meanwhile, RAW264.7 macrophages were stimulated by LPS. The contents of inflammatory mediators in the culture medium were determined by ELISA. Protein levels were determined by immunohistochemical staining or Western blotting. Nuclear localization of NF-κB, AP-1, and IRF3 was performed using immunofluorescence and Western blotting. Results: In the LPS-induced ALI mouse model, SC treatment suppressed the secretion of inflammatory mediators (TNF-α, IL-6, IL-1ß, MCP-1, MIP-1α, and RANTES) in BALF. SC treatment hindered the recruitment of macrophages. SC treatment also inhibited the expression of CD68, p-p65, and TLR4 in the lung tissue. In the LPS-exposed RAW264.7 cells, the cell viability was not changed up to 400 µg/mL of SC. SC concentration-dependently suppressed the production of nitric oxide, prostaglandin E2, TNF-α, IL-6, MCP-1, MIP-1α, and RANTES in LPS-challenged RAW264.7 cells. The expression levels of iNOS, COX-2, p-p38, p-JNK, p-ERK, p-TBK1, p-IKKα/ß, p-IκB, p-p65, p-c-Jun, and p-IRF3 were decreased after SC treatment. Moreover, the nuclear translocation of p65, c-Jun, and IRF3 was also blocked by SC treatment. Conclusion: SC treatment inhibited the inflammatory responses in LPS-induced ALI mouse model/RAW264.7 macrophages. The underlying mechanism of this action may be closely associated with the suppression of TLR4 signaling pathways. These research findings provide further pharmacological justifications for the medicinal use of SC in the management of respiratory diseases.

Schisandrin B mitigates hepatic steatosis and promotes fatty acid oxidation by inducing autophagy through AMPK/mTOR signaling pathway.

BACKGROUND: Schisandrin B (Sch B), which inhibits hepatic steatosis caused by non-alcoholic fatty liver disease (NAFLD), is one of the most active dibenzocyclooctadienes isolated from Schisandra chinensis (Turcz.) Baill with various pharmacological activities. In this study, the role of Sch B-induced autophagy in lipid-lowering activities of Sch B was examined and the underlying mechanisms were elucidated. METHODS: Free fatty acid (FFA)-stimulated HepG2 cells and mouse primary hepatocytes (MPHs) and high-fat diet (HFD)-fed mice were used as NAFLD models. The role of Sch B-induced autophagy in lipid-lowering effects of Sch B was assessed using ATG5/TFEB-deficient cells and 3-methyladenine (3-MA)-treated hepatocytes and mice. RESULTS: Sch B simultaneously active autophagy through AMPK/mTOR pathway and decreased the number of lipid droplets in FFA-treated HepG2 cells and MPHs. Additionally, siATG5/siTFEB transfection or 3-MA treatment mitigated Sch B-induced autophagy and activation of fatty acid oxidation (FAO) and ketogenesis in FFA-treated HepG2 cells and MPHs. Sch B markedly decreased hepatic lipid content and activated the autophagy through AMPK/mTOR pathway in HFD-fed mice. However, the activities of Sch B were suppressed upon 3-MA treatment. Sch B upregulated the expression of key enzymes involved in FAO and ketogenesis, which was mitigated upon 3-MA treatment. Moreover, changes in hepatic lipid components and amino acids may be related to the Sch B-induced autophagy pathway. CONCLUSION: These results suggested that Sch B inhibited hepatic steatosis and promoted FAO by activation of autophagy through AMPK/mTOR pathway. Our study provides novel insights into the hepatic lipophagic activity of Sch B and its potential application in the management of NAFLD.

Tibetan Medicine for Diabetes Mellitus: Overview of Pharmacological Perspectives.

Diabetes mellitus (DM) and its complications pose a major public health threat which is approaching epidemic proportions globally. Current drug options may not provide good efficacy and even cause serious adverse effects. Seeking safe and effective agents for DM treatment has been an area of intensive interest. As a healing system originating in Tibet, Traditional Tibetan Medicine (TTM) has been widely used by Tibetan people for the prevention and treatment of DM and its complications for hundreds of years. Tibetan Materia Medica (TMM) including the flower of Edgeworthia gardneri (Wall.) Meisn., Phyllanthi Fructus, Chebulae Fructus, Huidouba, and Berberidis Cortex are most frequently used and studied. These TMMs possess hypoglycemic, anti-insulin resistant, anti-glycation, lipid lowering, anti-inflammatory, and anti-oxidative effects. The underlying mechanisms of these actions may be related to their α-glucosidase inhibitory, insulin signaling promoting, PPARs-activating, gut microbiota modulation, islet ß cell-preserving, and TNF-α signaling suppressive properties. This review presents a comprehensive overview of the mode and mechanisms of action of various active constituents, extracts, preparations, and formulas from TMM. The dynamic beneficial effects of the products prepared from TMM for the management of DM and its complications are summarized. These TMMs are valuable materia medica which have the potential to be developed as safe and effective anti-DM agents.

miR-320a in serum exosomes promotes myocardial fibroblast proliferation via regulating the PIK3CA/Akt/mTOR signaling pathway in HEH2 cells.

MicroRNAs (miRNAs/miRs) serve an important role in the pathogenesis of chronic heart failure (CHF). A number of reports have illustrated the regulatory effect of serum exosomal miRNA on myocardial fibrosis. The present study aimed to investigate the expression of miR-320a in serum exosomes, as well as the effect of miR-320a on myocardial fibroblast proliferation. Serum exosome samples from 10 patients with CHF and 5 healthy volunteers were obtained and characterized. mRNA and protein expression levels were measured via reverse transcription-quantitative PCR and western blotting, respectively. The content of soluble growth stimulation expressed gene 2 (sST2) was determined via ELISA. HEH2 cell viability and apoptosis were detected by performing MTT assays and flow cytometry, respectively. The results demonstrated that serum miR-320a expression levels and sST2 content were significantly increased in patients with CHF compared with healthy controls, and the expression of serum miR-320a was significantly correlated with clinical CHF indexes. miR-320a expression levels were significantly increased in exosomes isolated from patients with CHF compared with those isolated from healthy controls. Phosphoinositide-3-kinase catalytic α polypeptide gene (PIK3CA) expression levels and sST2 content were increased in HEH2 cells following transfection with miR-320a mimics compared with NC-mimic, whereas miR-320a inhibitor displayed contrasting effects by reduced the cell viability and apoptosis in myocardial fibroblasts compared with the NC-inhibitor group. The protein expression levels of collagen I, collagen III, α-smooth muscle actin, phosphorylated (p)-mTOR (ser 2448)/mTOR, p-Akt (ser 473)/Akt, p-Akt (thr 308)/Akt and PIK3CA were significantly increased in miR-320a mimic-transfected HEH2 cells compared with the NC-mimics groups. By contrast, miR-320a inhibitor notably downregulated the expression levels of these proteins compared with the NC-inhibitor group. Collectively, the results of the present study demonstrated that miR-320a promoted myocardial fibroblast proliferation via regulating the PIK3CA/Akt/mTOR signaling pathway in HEH2 cells, suggesting that serum exosomal miR-320a may serve as a potential biomarker for the diagnosis of CHF.

Improved Understanding of the High Shear Wet Granulation Process under the Paradigm of Quality by Design Using Salvia miltiorrhiza Granules.

BACKGROUND: High shear wet granulation (HSWG) is a shaping process for granulation that has been enhanced for application in the pharmaceutical industry. However, study of HSWG is complex and challenging due to the relatively poor understanding of HSWG, especially for sticky powder-like herbal extracts. AIM: In this study, we used Salvia miltiorrhiza granules to investigate the HSWG process across different scales using quality by design (QbD) approaches. METHODS: A Plackett-Burman experimental design was used to screen nine granulation factors in the HSWG process. Moreover, a quadratic polynomial regression model was established based on a Box-Behnken experimental design to optimize the granulation factors. In addition, the scale-up of HSWG was implemented based on a nucleation regime map approach. RESULTS: According to the Plackett-Burman experimental design, it was found that three granulation factors, including salvia ratio, binder amount, and chopper speed, significantly affected the granule size (D50) of S. miltiorrhiza in HSWG. Furthermore, the results of the Box-Behnken experimental design and validation experiment showed that the model successfully captured the quadratic polynomial relationship between granule size and the two granulation factors of salvia ratio and binder amount. At the same experiment points, granules at all scales had similar size distribution, surface morphology, and flow properties. CONCLUSIONS: These results demonstrated that rational design, screening, optimization, and scale-up of HSWG are feasible using QbD approaches. This study provides a better understanding of HSWG process under the paradigm of QbD using S. miltiorrhiza granules.

Characterization and comparison of Re-Du-Ning aerosol particles generated by different jet nebulizers.

Inhalation therapy is the first-line therapy for the treatment of respiratory diseases. Re-Du-Ning inhalation solution (RIS) is an aerosol derivative from the Re-Du-Ning injection and has been clinically used to treat respiratory diseases like pneumonia for more than twenty years in China. However, the aerosolization and inhalation performances of RIS using different nebulizers have not been characterized, which may affect the therapeutic effects of RIS on respiratory diseases. We investigated the inhalation performances of RIS using five different nebulizers utilizing Spraytec, breath simulator of BRS 2000 and NGI techniques. We tested 5 different types of jet nebulizer, using RIS and an adult breathing pattern, to determine the difference in aerosol delivery over time. The particle size distribution of RIS was monitored by a Spraytec laser particle sizer. Fine particle fraction (FPF) and mass median aerodynamic diameter (MMAD) for RIS were measured using NGI. Aerosol deposited on the filter was analysed using HPLC. Nebulization time was much longer for the Pari Boy SX (red) nebulizer than for the other nebulizers, with the minimum delivery rate (DR) and the maximum total delivered dose (TDD) and total exhalation dose (TED). Nebulization time for Pari Boy SX (blue) was the lowest, with the highest DR and the lowest TDD and TED. Furthermore, the aerodynamic particle size of RIS was much larger for the Pari blue and Pari LC Plus than other nebulizers. Pari red produced the smallest aerodynamic particle size of RIS in these five nebulizers. In addition, a good linear relationship was found between MMAD and D 50 in these five nebulizers. The results demonstrated that Pari Boy SX (red) delivered most slowly and produced the smallest aerodynamic particle size of the RIS aerosols, which may be applied to manage lower respiratory diseases. Moreover, Pari LC Plus and Pari Boy SX (blue) emitted quickly and generated larger aerodynamic particle size of RIS aerosols, which could be used to treat upper respiratory diseases. A good linear relationship between MMAD and D 50 showed Spraytec could be a reliable technique for the development, evaluation and quality control of aerosol particles of inhalation solution preparations.

Schisandra Chinensis Lignans Suppresses the Production of Inflammatory Mediators Regulated by NF-κB, AP-1, and IRF3 in Lipopolysaccharide-Stimulated RAW264.7 Cells.

Schisandra Fructus (SF) is a traditional Chinese herb used in the treatment of inflammatory disorders like hepatitis. One of the main anti-inflammatory components of SF is the lignans. However, the underlying anti-inflammatory mechanism of Schisandra Chinensis lignans (SCL) remains unclear. This study aims to investigate the effects of SCL on inflammatory mediators in lipopolysaccharide-stimulated RAW264.7 cells and explore the underlying mechanism. The production of nitric oxide (NO) was determined by Griess reaction. ELISA was used to determine cytokine levels and chemokines secretion. To estimate protein levels and enzyme activities, we employed Western blotting. Nuclear localization of NF-κB, AP-1, and IRF3 was detected using immunofluorescence analyses. The results showed that SCL significantly reduced the release of inflammatory mediators, including NO and PGE2, which may be related to down-regulation of iNOS and COX-2 expression. The production of cytokines and chemokines was suppressed by SCL treatment. SCL also decreased the phosphorylation of IKKα/ß, IκB-α, Akt, TBK1, ERK, p38, JNK, NF-κB (p65), AP-1 (c-Jun), and IRF3 in RAW264.7 macrophages activated with LPS. The nuclear protein levels and nuclear translocation of AP-1, NF-κB and IRF3 were suppressed by SCL. These results indicated that SCL suppressed the IKKα/ß/NF-κB, MAPKs/AP-1 and TBK1/IRF3 signaling pathways in LPS-stimulated RAW264.7 macrophages.

Dingchuan tang essential oil inhibits the production of inflammatory mediators via suppressing the IRAK/NF-κB, IRAK/AP-1, and TBK1/IRF3 pathways in lipopolysaccharide-stimulated RAW264.7 cells.

BACKGROUND: Dingchuan tang (asthma-relieving decoction), a formula of nine herbs, has been used for treating respiratory inflammatory diseases for >400 years in the People's Republic of China. However, the mechanisms underlying the anti-inflammatory action of dingchuan tang is not fully understood. This study aims to investigate the effects of Dingchuan tang essential oil (DCEO) on inflammatory mediators and the underlying mechanism of action. MATERIALS AND METHODS: DCEO was extracted by steam distillation. Lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages were used as the cell model. Production of nitric oxide (NO) was determined by the Griess test. Protein secretion and mRNA levels of inflammatory mediators were measured by the enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. Protein levels were examined by Western blot. Nuclear localization of nuclear factor-kappa B (NF-κB) was detected using immunofluorescence analyses. RESULTS: DCEO significantly reduced LPS-triggered production of NO and prostaglandin E2 (PGE2) and decreased protein and mRNA levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). LPS induced upregulation of protein and mRNA levels of cytokines (interleukin-1ß [IL-1ß], interleukin-6 [IL-6], tumor necrosis factor-α [TNF-α]), and chemokines (monocyte chemoattractant protein-1 [MCP-1], chemokine [C-C motif] ligand 5 [CCL-5], and macrophage inflammatory protein [MIP]-1α) were suppressed by DCEO treatment. Phosphorylation and nuclear protein levels of transcription factors (activator protein-1 [AP-1], NF-κB, interferon regulatory factor 3 [IRF3]) were decreased by DCEO. Protein levels of phosphorylated IκB-α, IκB kinase α/ß (IKKα/ß), phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), TGF ß-activated kinase 1 (TAK1), TANK-binding kinase 1 (TBK1), extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38), and c-Jun N-terminal kinase (JNK) were lowered by DCEO. Moreover, degradation of interleukin-1 receptor-associated kinase 1 (IRAK1) and IRAK4 induced by LPS was inhibited by DCEO treatment. CONCLUSION: Suppression of the interleukin-1 receptor-associated kinase (IRAK)/NF-κB, IRAK/AP-1 and TBK1/IRF3 pathways was associated with the inhibitory effects of DCEO on inflammatory mediators in LPS-stimulated RAW264.7 macrophages. This study provides a pharmacological justification for the use of dingchuan tang in managing inflammatory disorders.

An ethanolic extract of the aerial part of Siegesbeckia orientalis L. inhibits the production of inflammatory mediators regulated by AP-1, NF-κB and IRF3 in LPS-stimulated RAW 264.7 cells.

Herba Siegesbeckiae (HS, the dried aerial part of Siegesbeckia orientalis L.) is a commonly used traditional Chinese medicinal herb for treating inflammatory diseases. HS has been reported to exert anti-inflammatory effects by inhibiting the MAPKs and NF-κB pathways, the downstream effectors of TLR4 signalling. This study aims to further investigate the involvement of TLR4 signalling cascades in the effects of an ethanolic extract of HS (HS for short) on inflammatory mediators in murine macrophages. HS was extracted using 50% ethanol. Lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages were used as the cell model. ELISA was used to detect cytokine/chemokine secretion. Real time-PCR and immunoblotting were used to examine mRNA and protein levels, respectively. We observed that HS dose-dependently inhibited the secretion of PGE2, MCP-1, MIP-1α and RANTES, and down-regulated mRNA levels of iNOS, COX-2, IL-1ß, IL-6, TNF-α, mPGES-1, MCP-1, MIP-1α and RANTES in LPS-stimulated RAW264.7 cells. HS did not affect the protein levels of TAK1, TBK1, PI3K, Akt, IKK, c-Jun, c-Fos and IRF3, while, dose-dependently decreased levels of their phosphorylated forms. The protein levels of IRAK1 and IRAK4 were upregulated, while those of TRAF6 and TRAF3 were downregulated by HS. Moreover, the nuclear protein levels of AP-1, NF-κB and IRF3 were dose-dependently decreased by HS. These results indicate that suppression of the IRAK4/MAPKs/AP-1, IRAK4/MAPKs/NF-κB, IRAK4/PI3K/NF-κB and TRAF3/TBK1/IRF3 pathways is associated with the inhibitory effects of HS on inflammatory mediators in LPS-stimulated RAW264.7 cells. This study provides a pharmacological basis for the clinical application of this herb in the treatment of inflammatory disorders.

Comparison of the chemical profiles and inflammatory mediator-inhibitory effects of three Siegesbeckia herbs used as Herba Siegesbeckiae (Xixiancao).

BACKGROUND: Herba Siegesbeckiae (HS, Xixiancao in Chinese) is a commonly used traditional Chinese medicinal herb for soothing joints. In ancient materia medica books, HS is recorded to be the aerial part of Siegesbeckia pubescens Makino (SP) which is also the only origin of HS in the 1963 edition of the Chinese Pharmacopeia (ChP). The aerial parts of Siegesbeckia orientalis L. (SO) and Siegesbeckia glabrescens Makino (SG) have been included as two additional origins for HS in each edition of ChP since 1977. However, chemical and pharmacological comparisons among these three species have not been conducted. METHODS: An HPLC with diode array detector (HPLC-DAD) method combined with similarity analysis, hierarchical cluster analysis (HCA) and principal component analysis (PCA) was developed for comparing the fingerprint chromatograms of the three species. The inhibitory effects of the three species on NO production and IL-6 secretion in LPS-stimulated RAW264.7 macrophages were compared. RESULTS: Fingerprint chromatograms of the three species showed different profiles, but had 13 common peaks. Results from HCA and PCA of the common peaks demonstrated that all 14 herbal samples of the three species tended to be grouped and separated species dependently. The extents of inhibition on NO production and IL-6 secretion of the three species were different, with SG being the most and SP the least potent. CONCLUSIONS: Both chemical profiles and inflammatory mediator-inhibitory effects of the three species were different. These findings provide a chemical and pharmacological basis for determining whether the three species can all serve as the origins of HS.

Investigation of Ginkgo biloba extract (EGb 761) promotes neurovascular restoration and axonal remodeling after embolic stroke in rat using magnetic resonance imaging and histopathological analysis.

EGb 761 is a standardized natural extract from Ginkgo biloba leaf that has shown neuroprotective effects after ischemic stroke. This study aimed to use magnetic resonance imaging (MRI) to noninvasively evaluate whether EGb 761 promotes neurovascular restoration and axonal remodeling in a rat model of focal cerebral ischemia. Male Sprague-Dawley rats were subjected to permanent right middle cerebral artery occlusion (MCAO) and treated with EGb 761 (60 mg/kg) or saline intragastrically once daily for 15 days starting 6 h after MCAO. Functional recovery was analyzed using beam walking test. Multi-parametric MRI was applied to examine the alterations of gray-white structures, intracranial vessels, cerebral perfusion and axonal integrity, and followed with histological studies. Furthermore, the protein expression of axonal remodeling related signaling pathways including protein kinase B (AKT)/ glycogen synthase kinase-3ß (GSK-3ß)/ collapsin response mediator protein 2 (CRMP2) and NogoA/NgR were detected by Western blotting analysis. Multi-parametric MRI demonstrated that EGb 761 significantly reduced infarct volume, alleviated gray and white matter damage, and enhanced collateral circulation, cerebral perfusion and axonal remodeling. Histological examinations supported the MRI results. EGb 761 treatment facilitated behavioral recovery and amplified endogenous neurogenesis. Notably, treatment with EGb 761 significantly increased the levels of p-AKT, p-GSK-3ß and decreased the expression of p-CRMP2. In addition, EGb 761 treatment up-regulated the expression of growth associated protein 43 (GAP-43) and suppressed the activation of axonal growth inhibitory molecules NogoA and NgR. These findings indicated that EGb 761 enhanced neurovascular restoration, amplified endogenous neurogenesis and promoted axonal regeneration, which in concert may contribute to gray-white matter reorganization and functional outcome after stroke.

Sensitization of melanoma cells to alkylating agent-induced DNA damage and cell death via orchestrating oxidative stress and IKKß inhibition.

Nitrosourea represents one of the most active classes of chemotherapeutic alkylating agents for metastatic melanoma. Treatment with nitrosoureas caused severe systemic side effects which hamper its clinical use. Here, we provide pharmacological evidence that reactive oxygen species (ROS) induction and IKKß inhibition cooperatively enhance nitrosourea-induced cytotoxicity in melanoma cells. We identified SC-514 as a ROS-inducing IKKß inhibitor which enhanced the function of nitrosoureas. Elevated ROS level results in increased DNA crosslink efficiency triggered by nitrosoureas and IKKß inhibition enhances DNA damage signals and sensitizes nitrosourea-induced cell death. Using xenograft mouse model, we confirm that ROS-inducing IKKß inhibitor cooperates with nitrosourea to reduce tumor size and malignancy in vivo. Taken together, our results illustrate a new direction in nitrosourea treatment, and reveal that the combination of ROS-inducing IKKß inhibitors with nitrosoureas can be potentially exploited for melanoma therapy.

Standardization of the manufacturing procedure for Pinelliae Rhizoma Praeparatum cum Zingibere et Alumine.

ETHNOPHARMACOLOGICAL RELEVANCE: Pinelliae Rhizoma (PR), the dried tuber of Pinellia ternata (Thunb.) Breit., is a traditional Chinese medicinal herb. It is commonly used for treating cancer, cough and phlegm. To treat cancer, Chinese medicine practitioners often use raw PR; while to treat cough and phlegm, they usually use Pinelliae Rhizoma Praeparatum cum Zingibere et Alumine (PRZA, raw PR processed with ginger juice and alumen as adjuvant materials). Currently, the producing protocol of PRZA varies greatly among different places in China. This study aims to standardize the manufacturing procedure for PRZA. We also evaluated the impact of processing on the bioactivities and chemical profile of raw PR. MATERIALS AND METHODS: We used the orthogonal design to optimize the manufacturing procedure of PRZA at bench scale, and validated the optimized procedure in pilot-scale production. The MTT assay was used to compare the cytotoxicities of raw PR and PRZA in hepatocellular carcinoma HepG2 cells. Animal models (ammonia liquor-induced cough model and phenol red secretion model) were used to compare the antitussive and expectorant effects of raw PR and PRZA, respectively. The chemical profiles of raw PR and PRZA samples were compared using a newly developed ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UPLC/Q-TOF-MS) method. RESULTS: The standardized manufacturing procedure for PRZA is as follows: soak raw PR in water until the center of the cut surface is devoid of a dry core, after that, boil the herb in water (for each 100kg raw PR, 12.5kg alumen and 25L freshly squeezed ginger juice are added) for 6h, and then take out and dry them. The cytotoxicity of PRZA was less potent than that of raw PR. Intragastric administration of raw PR or PRZA demonstrated antitussive and expectorant effects in mice. These effects of PRZA were more potent than that of raw PR at the dose of 3g/kg. By comparing the chemical profiles, we found that six peaks were lower, while nine other peaks were higher in PRZA than in raw PR. Six compounds corresponding to six individual changed peaks were tentatively identified by matching with empirical molecular formulae and mass fragments. CONCLUSION: The manufacturing procedure for PRZA was standardized. This protocol can be used for PRZA industrial production. The bioactivity assay results of raw PR and PRZA (produced using the standardized protocol) support the common practice for the clinical applications of these two decoction pieces. Moreover, raw PR and PRZA showed different chemical profiles. Further studies are warranted to establish the relationship between the alteration of chemical profiles and the changes of medicinal properties caused by processing.

Ultra-performance liquid chromatography-quadrupole/time-of-flight mass spectrometry analysis of the impact of processing on toxic components of Kansui Radix.

BACKGROUND: Kansui Radix (Gansui in Chinese), the dried tuber of Euphorbia kansui, is a Chinese medicinal herb commonly used for the treatment of oedema and ascites with dyspnea. Because of its toxic nature, the herb is usually processed with vinegar to reduce the toxicity. A report has shown that the contents of toxic terpenoids in Gansui decreased after processing with vinegar. However, comprehensive comparison of the chemical profiles between vinegar-processed and raw Gansui has not yet been conducted. METHODS: An ultra-high-performance liquid chromatography in conjunction with ultra-high resolution quadrupole time-of-flight mass spectrometry (UHPLC UHD Q-TOF MS/MS) method was developed for the analysis of chemical profiles of vinegar-processed and raw Gansui in this study. RESULTS: Results showed that processing with vinegar caused conspicuous chemical changes. Among the altered components, 11 toxic terpenoids, 3-O-benzoyl-13-O- dodecanoylingenol/20-O-benzoyl-13-O-dodecanoylingenol, kansuinine D, kansuinine A, 3-O-benzoyl-13-O-dodecanoylingenol/20-O-benzoyl-13-O-dodecanoylingenol, 3-O- benzoylingenol/20-O-benzoylingenol, 20-O-(2'E,4'Z-decadienoyl)ingenol/20-O-(2'E,4'E- decadienoyl)ingenol/3-O-(2'E,4'Z-decadienoyl)ingenol/3-O-(2'E,4'E-decadienoyl)ingenol, 3-O-(2'E,4'Z-decadienoyl)-20-deoxyingenol,3-O-(2'E,4'Z-,ecadienoyl)-5-O-acetylingenol,3-O-(2'E,4'Z-decadienoyl)-20-O-acetylingenol,3-O-(2,3-dimethylbutanoyl)-13-O-dodecanoylingenol, were tentatively identified. The contents of most of these terpenoids were obviously decreased after processing with reductions of 6.66-95.25%. CONCLUSION: Our findings could help us understand the chemical basis for the toxicity reduction of Gansui afforded by processing with vinegar. Further investigations are warranted to establish the relationship between processing-induced chemical changes and the reduction of toxicity of Gansui.

Comparison of the toxicities, activities and chemical profiles of raw and processed Xanthii Fructus.

BACKGROUND: Although toxic, the Chinese medicinal herb Xanthii Fructus (XF) is commonly used to treat traditional Chinese medicine (TCM) symptoms that resemble cold, sinusitis and arthritis. According to TCM theory, stir-baking (a processing method) can reduce the toxicity and enhance the efficacy of XF. METHODS: Cytotoxicities of raw XF and processed XF (stir-baked XF, SBXF) were determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in normal liver derived MIHA cells. Nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) mRNA expression were measured by the Griess reagent and quantitative real-time PCR, respectively. The chemical profiles of XF and SBXF were compared using an established ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UPLC/Q-TOF-MS) method. RESULTS: SBXF was less toxic than XF in MIHA cells. Both XF and SBXF had anti-inflammatory effects as demonstrated by their abilities to reduce nitric oxide production as well as inducible nitric oxide synthase mRNA expression in lipopolysaccharide-stimulated RAW 264.7 macrophages. Interestingly, the anti-inflammatory effects of SBXF were more potent than that of XF. By comparing the chemical profiles, we found that seven peaks were lower, while nine other peaks were higher in SBXF than in XF. Eleven compounds including carboxyatractyloside, atractyloside and chlorogenic acid corresponding to eleven individual changed peaks were tentatively identified by matching with empirical molecular formulae and mass fragments, as well as literature data. CONCLUSION: Our study showed that stir-baking significantly reduced the cytotoxicity and enhanced the anti-inflammatory effects of XF; moreover, with a developed ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry method we differentiated XF and SBXF by their chemical profiles. Further studies are warranted to establish the relationship between the alteration of chemical profiles and the changes of medicinal properties caused by stir-baking.

ERK/GSK3ß signaling is involved in atractylenolide I-induced apoptosis and cell cycle arrest in melanoma cells.

Novel agents need to be developed to overcome the limitations of the current melanoma therapeutics. Atractylenolide I (AT-I) is a sesquiterpene compound isolated from atractylodis macrocephalae rhizoma. Previous findings demonstrated that AT-I exhibited cytotoxic action in melanoma cells. However, the molecular mechanisms of AT­1's anti-melanoma properties remain to be elucidated. In the present study, the cell cycle-arrest and apoptosis-promoting effects as well as the ERK/GSK3ß signaling-related mechanism of action of AT-I were examined. B16 melanoma cells were treated with various concentrations of AT-1 (50, 75 and 100 µM) for 48 or 72 h. Cell cycle and apoptosis were analyzed by flow cytometry. Protein expression levels were detected by western blot analysis. AT-I treatment induced G1 phase arrest, which was accompanied by increased p21 and decreased CDK2 protein expression levels. Apoptosis was observed after AT-I treatment for 72 h, which was accompanied by activated caspase­3 and ­8. AT-I treatment significantly decreased phospho-ERK, phospho-GSK3ß, c-Jun and increased p53 protein expression levels. Lithium chloride (LiCl, 5 mM), a GSK3ß inhibitor, treatment alone did not increase the apoptosis of B16 cells, while pretreatment with LiCl markedly reversed AT-I-induced apoptosis. Additionally, AT-I-induced G1 phase arrest was partially reversed by LiCl pretreatment. In conclusion, ERK/GSK3ß signaling was involved in the apoptotic and G1 phase arrest effects of AT-I in melanoma cells.

Inhibition of STAT3 signalling contributes to the antimelanoma action of atractylenolide II.

Our previous studies showed that atractylenolide II (AT-II) has antimelanoma effects in B16 melanoma cells. In this study, we investigated the involvement of STAT3 signalling in the antimelanoma action of AT-II. Daily administration of AT-II (12.5, 25 mg/kg, i.g.) for 14 days significantly inhibited tumor growth in a B16 xenograft mouse model and inhibited the activation/phosphorylation of STAT3 and Src in the xenografts. In B16 and A375 cells, AT-II (20, 40 µm) treatment for 48 h dose-dependently reduced protein expression levels of phospho-STAT3, phospho-Src, as well as STAT3-regulated Mcl-1 and Bcl-xL. Overexpression of a constitutively active variant of STAT3, STAT3C in A375 cells diminished the antiproliferative and apoptotic effects of AT-II. These data suggest that inhibition of STAT3 signalling contributes to the antimelanoma action of AT-II. Our findings shed new light on the mechanism of action underlying the antimelanoma effects of AT-II and provide further pharmacological basis for developing AT-II as a novel melanoma chemopreventive/chemotherapeutic agent.

A herbal formula consisting of Rosae Multiflorae Fructus and Lonicerae Japonicae Flos inhibits inflammatory mediators in LPS-stimulated RAW 264.7 macrophages.

ETHNOPHARMACOLOGICAL RELEVANCE: A herbal formula (RL) consisting of Rosae Multiflorae Fructus (Yingshi) and Lonicerae Japonicae Flos (Jinyinhua) has been traditionally used to treat inflammatory disorders. This study aims to investigate the anti-inflammatory mode and mechanism of action of the ethanol extract of RL so as to provide a pharmacological basis for the use of RL in treating inflammatory diseases. MATERIALS AND METHOD: RL consisting of Yingshi and Jinyinhua (in 5:3 ratio) was extracted using absolute ethanol. We investigated its effects on nitric oxide (NO), interleukin-6 (IL-6), tumour necrosis factor (TNF)-α, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), mitogen-activated protein kinases (MAPKs) and nuclear factor κB (NFκB) in mouse RAW 264.7 macrophages activated with lipopolysaccharide (LPS). RESULTS: RL could decrease the secretion of NO, IL-6 and TNF-α into the culture medium and the cellular protein levels of iNOS and COX-2, which were associated with the reduction of the phosphorylation/activation of JNK and p38, and the inhibition of the transcriptional activity of NF-κB. CONCLUSIONS: The present study demonstrated an inhibitory effect of RL on the inflammatory mediators regulated by the NF-κB and MAPK signalling pathways in LPS-stimulated RAW 264.7 macrophages, providing a pharmacological basis for RL in the control of inflammatory disorders.