Study on anti-inflammatory effect of Shangkehuangshui in vitro and in vivo based on TLR4/TLR2-NF-κB signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Shangkehuangshui (SK) has been traditionally used to treat traumatic injury, soft tissue and bone injury in Foshan hospital of traditional Chinese medicine for more than 60 years, which composed of many Chinese herbs such as Coptis chinensis Franch., Gardenia jasminoides Ellis, Phellodendron chinense Schneid. and etc. SK exhibits heat-clearing and detoxifying, enhancing blood circulation to eliminate blood stasis properties, and demonstrates noteworthy clinical efficacy. Nevertheless, the underlying mechanism remains uncertain. AIM OF THE STUDY: The early study found that SK had good anti-inflammatory effects in acute soft tissue injury model. This research is to verify the anti-inflammatory properties of SK both in vitro and in vivo via TLR4/TLR2-NF-κB signaling pathway, to clarify the underlying mechanisms responsible for the curative effect of SK. METHODS: The RAW264.7 cells inflammatory model was established with lipopolysaccharide (LPS) in vitro. NO and TNF-α, IL-6, IL-1ß were determined with Griess method and ELISA method respectively. The mRNA and protein expression levels of TLR4/TLR2-NF-κB pathway were evaluated by qPCR and Western blot method. In vivo experiment, chronic soft tissue injury rat models were established by tracking gastrocnemius muscle with electrical stimulation, then local appearance and pathological changes were observed and recorded, the contents of inflammatory factors in serum and tissue were performed. Moreover, we also measured and contrasted the expression of TLR4/TLR2-NF-κB related factors. RESULTS: SK effectively inhibited the LPS-induced generation of inflammatory cytokines, including NO, TNF-α, IL-6 and IL-1ß in RAW264.7 cells, and significantly suppressed the expression of TLR4, TLR2, MyD88, IκB, and NF-κB. In vivo, SK remarkably decreased the damage appearance scores after 4 and 14 days of administration and inhibit the quantity of NO and leukocytes present in the serum. Additionally, the inflammatory infiltration in the pathological section was alleviated, myofibrillar hyperplasia and blood stasis were reduced. SK markedly downregulated NO, TNF-α, IL-6 and IL-1ß in injured tissues of rats, also declined the expression of TLR4, TLR2, MyD88, IκB, NF-κB, IL-6, TNF-α and IL-1ß. CONCLUSION: This study revealed that SK had obvious effects of anti-inflammatory actions in vivo and vitro, effectively reduced acute and chronic soft tissue injury in clinical, this might be attributed to inhibit the TLR4/TLR2-NF-κB pathway, further inhibit the expression of downstream relevant pro-inflammatory cytokines.

[Network Meta-analysis of single Hirudo prescriptions in treatment of ischemic cerebrovascular disease].

This study intended to evaluate the efficacy and safety of single Hirudo prescriptions in the treatment of ischemic cerebrovascular disease(ICVD) by frequency network Meta-analysis and traditional Meta-analysis. CNKI, Wanfang, VIP, SinoMed, PubMed, EMbase, and Cochrane Library databases were searched to collect the randomized controlled trial(RCT) of single Hirudo prescriptions for ICVD from the inception of the databases to May 2022. The quality of the included literature was evaluated by Cochrane risk of bias tool. Finally, 54 RCTs and 3 single Hirudo prescriptions were included. Statistical analysis was conducted by RevMan 5.3 and Stata SE 15. Network Meta-analysis showed that in terms of the clinical effective rate, the surface under the cumulative ranking curve(SUCRA) of intervention measures was as follows: Huoxue Tongmai Capsules+conventional treatment>Maixuekang Capsules+conventional treatment>Naoxuekang Capsules+conventional treatment>conventional treatment. Traditional Meta-analysis revealed that in terms of the safety of ICVD treatment, Maixuekang Capsules+conventional treatment had higher safety than conventional treatment alone. According to the network Meta-analysis and traditional Meta-analysis, it was found that conventional treatment combined with single Hirudo prescriptions improved the clinical efficacy of ICVD patients, and compared with that of conventional treatment alone, the incidence of adverse reactions of combined treatment was low and the safety was high. However, the methodological quality of the articles included in this study was generally low and there were large differences in the number of articles on the three combined medication. Therefore, the conclusion of this study needed to be confirmed by subsequent RCT.

Purification Process and In Vitro and In Vivo Bioactivity Evaluation of Pectolinarin and Linarin from Cirsium japonicum.

Pectolinarin and linarin are two major flavone O-glycosides of Cirsium japonicum, which has been used for thousands of years in traditional Chinese medicine. Pharmacological research on pectolinarin and linarin is meaningful and necessary. Here, a process for the purification of pectolinarin and linarin from C. japonicum was established using macroporous resin enrichment followed by prep-HPLC separation. The results show the purity of pectolinarin and linarin reached 97.39% and 96.65%, respectively. The in vitro bioactivities result shows the ORAC values of pectolinarin and linarin are 4543 and 1441 µmol TE/g, respectively, meanwhile their inhibition rate of BSA-MGO-derived AGEs is 63.58% and 19.31% at 2 mg/mL, which is 56.03% and 30.73% in the BSA-fructose system, respectively. The COX-2 inhibition rate at 50 µg/mL of linarin and pectolinarin reached 55.35% and 40.40%, respectively. Furthermore, the in vivo bioassay combining of histopathologic evaluation and biochemical analysis of liver glutamic oxaloacetic transaminase, serum creatinine and TNF-α show pectolinarin can alleviate lipopolysaccharide (LPS)-induced acute liver and kidney injury in mice. Metabolomics analysis shows that pectolinarin attenuates LPS-challenged liver and kidney stress through regulating the arachidonic acid metabolism and glutathione synthesis pathways. Collectively, our work presents a solid process for pectolinarin and linarin purification and has discovered a promising natural therapeutic agent-pectolinarin.

[Mechanism of Liangfu Pills in treatment of functional dyspepsia: based on network pharmacology and experimental verification].

This study aims to explore the potential mechanism of Liangfu Pills in the treatment of functional dyspepsia(FD) based on network pharmacology and molecular docking, and verify the mechanism by animal experiment. The active components of Liangfu Pills were screened from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP), and the targets of Liangfu Pills were predicted by SwissTargetPrediction. The targets of FD were retrieved from GeneCards. On this basis, the common targets of the disease and the pills were yielded and the protein interaction was retrieved based on STRING. The core targets were screened out, followed by Gene Oncology(GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis with DAVID. Finally, molecular docking was carried out with the help of AutoDock Tools to predict the binding degree between the effective components of Liangfu Pills and core targets. A total of 19 active components of Liangfu Pills and 591 FD-related targets were screened out by network pharmacology, of which 253 were common targets of the disease and the prescription. Liangfu Pills was mainly involved in the biological processes of response to drug, negative regulation of transcription, positive regulation of apoptotic process, and cell surface receptor signaling pathway, and the KEGG pathways of hypoxia-inducible factor-1(HIF-1) signaling pathway, serotonergic synapse, tumor necrosis factor(TNF) signaling pathway, cyclic adenosine monophosphate(cAMP) signaling pathway, calcium signal pathway, and inflammatory mediator regulation of transient receptor potential(TRP) channels. The results of molecular docking showed that the key active components of Liangfu Pills had certain binding activity to the targets mitogen-activated protein kinase 1(MAPK1), protein kinase B(AKT1), transient receptor potential cation channel subfamily V member 1(TRPV1), 5-hydroxytryptamine receptor 1 A(HTR1 A), and 5-hydroxytryptamine receptor 2 A(HTR2 A). FD was induced in rats, and then Liangfu Pills was given to FD rats for 7 days. The results showed that Liangfu Pills could significantly relieve the symptoms of FD rats, significantly increase the expression of 5-hydroxytryptamine(5-HT), and down-regulate the expression of TRPV1. Through network pharmacology, molecular docking, and experimental verification, this study proved that Liangfu Pills improved FD through multiple components and multiple targets. The result lays a basis for further research on the mechanism and clinical application of Liangfu Pills in the treatment of FD.

Anoectochilus roxburghii flavonoids extract ameliorated the memory decline and reduced neuron apoptosis via modulating SIRT1 signaling pathway in senescent mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Anoectochilus roxburghii (A. roxburghii) is a precious herb and folk medicine in many Asian countries. It has been used traditionally to treat diabetes, etc., and also used as a dietary therapy to delay senescence. AIM OF THE STUDY: This study was to evaluate the neuroprotective effects of A. roxburghii flavonoids extract (ARF) and whether its effects were due to the regulation of SIRT1 signaling pathway in senescent mice and in D-galactose (D-gal) induced aging in SH-SY5Y cells. MATERIALS AND METHODS: 18-month-old mice were randomly divided into senescent model, low-dose ARF, high-dose ARF and vitamin E group. 2-Month-old mice were as a control group. After 8 weeks treatment, Morris water maze (MWM) was performed. The levels of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), monoamine oxidase (MAO) and acetylcholinesterase (ACh-E) in the cortex were determined. Hippocampus morphologic changes were observed with haematoxylin and eosin (H&E), Nissl, senescence-associated-galactosidase (SA-ß-gal) and terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) staining. Apoptosis-related molecular expressions in the hippocampus were performed by western blotting. Furthermore, after stimulated by EX527 (a SIRT1 inhibitor), the SIRT1-dependent neuroprotective effects of ARF were determined by measuring SRIT1 and p53 expression in SH-SY5Y aging cells induced by D-gal. RESULTS: ARF could significantly ameliorate memory decline in senescent mice and reduce the generations of ROS, MDA and the activities of MAO and ACh-E, while increasing SOD activities in the cortex of aging mice. ARF obviously improved hippocampus pathological alterations, increased the number of Nissl bodies, while reducing senescent and apoptotic cells in senescent mice hippocampus. Further, ARF positively regulated SIRT1 expression, and reduced apoptosis-related molecules p53, p21 and Caspase-3 expression, while increasing the ratio of Bcl-2/Bax. In D-gal-induced SH-SY5Y cells, the effects of ARF on SIRT1 and p53, and the ability of scavenging ROS were mostly abolished after incubation with the EX527. CONCLUSIONS: ARF, in a SIRT1-dependent manner, exerted neuroprotection via modulating SIRT1/p53 signaling pathway against memory decline and apoptosis due to age-induced oxidative stress damage in senescent mice.

[A new phloroglucinol from Dryopteris fragrans and its antibacterial activity in vitro].

A new phloroglucinol was isolated from 50% ethanol extract of Dryopteris fragrans by silica gel column chromatography, Sephadex LH-20 gel column chromatography, thin-layer chromatography(TLC), and preparative liquid column chromatography. On the basis of MS, ~1H-NMR, ~(13)C-NMR, and reference materials, compound 1 was identified as 2,5-cyclohexadien-1-one, 2-{[2,6-dihydroxy-4-methoxy-3-methyl-5-(1-isobutyl)phenyl]methyl}-3,5-dihydroxy-4,4-dimethyl-6-(1-oxobutyl)(1), and named disaspidin BB. Compound 1 was evaluated for its antibacterial activity. The experimental results showed that compared with the commonly used topical antibiotics erythromycin or mupirocin, disaspidin BB exhibited significant antibacterial activities against Staphylococcus epidermidis(SEP), S. haemolyticus(SHA), and methicillin-resistant S. aureus(MRSA)(P<0.05). Additionally, disaspidin BB was sensitive to ceftazidime-resistant SEP1-SEP4, SHA5-SHA7, MRSA8, and MRSA9. The MIC values of disaspidin BB against SEP and SHA were 1.67-2.71 µg·mL~(-1) and 10.00-33.33 µg·mL~(-1) respectively. Disaspidin BB has good antibacterial activities and deserves development as a new anti-infective drug for external use.

Pharmacological Mechanisms of Shangke Huangshui against Skin and Soft Tissue Infection.

BACKGROUND: Skin and soft tissue infections (SSTIs) are a group of common diseases, usually caused by bacteria. Shangke Huangshui (SK) has been widely used to treat various SSTIs diseases for many years, but its mechanism is unclear. Therefore, this study was designed to investigate the anti-infective effect of SK on different skin and soft tissue infection diseases and to explore its underlying mechanism. METHODS: The subcutaneous abscess mouse model, the dermal ulcer rat model, and the infectious soft tissue injury rat model were established by injection of Staphylococcus aureus to evaluate the anti-inflammatory and antibacterial effects of SK. Abscess volume, local appearance score and histological changes, bacterial contents, and hydroxyproline concentration in the skin or soft tissue were analyzed. The levels of NO, TNF-α, IL-1ß, and IL-8 in the serum and tissue were determined by ELISA method. The mRNA expression levels of TLR2, MyD88, TAK1, NF-κB, AP-1, and other genes were measured with qRT-PCR method, and the protein expression of TLR2, MyD88, TAK1, NF-κB, and AP-1 was detected by western blot method. RESULTS: SK had an obvious therapeutic effect on skin and soft tissue infections. It reduced the volume of abscess and promoted the healing of skin ulcer, improved pathological phenomena, such as inflammatory infiltration of skin and soft tissue, reduced the levels of white blood cells and NO in the blood, decreased bacteria contents in the skin and soft tissue. Furthermore, SK decreased the mRNA expression of TLR2, MyD88, TAK1, NF-κB and AP-1, and other related genes and also downregulated the protein expression of TLR2, MyD88, TAK1, NF-κB, and AP-1. CONCLUSION: The experiments provide evidence that SK can treat skin and soft tissue infection diseases based on its comprehensive antibacterial and anti-inflammatory effects. The therapeutic mechanism may be associated with the inhibition of TLR2/MyD88/NF-κB signaling pathway.

[Effects of Huoxiang Zhengqi Oral Liquid on intestinal barrier function in rats with dampness obstructing spleen-stomach syndrome].

The aim of this paper was to investigate the effect of Huoxiang Zhengqi Oral Liquid on intestinal barrier functions in rats with dampness obstructing spleen-stomach syndrome and primarily explore the mechanism. The rat model of dampness obstructing spleen-stomach syndrome was established, and then the modeled rats were randomly divided into the model control group, Huoxiang Zhengqi Oral Liquid high and low dose groups, and natural recovery group according to gender and body weight, with 10 rats in each group. Another 10 rats were taken as blank control group. After each group received the corresponding treatment for 7 days, rat serum was isolated. D-lactic acid content was detected by the MTT method, and diamine oxidase(DAO) activity was detected by the rate method. Colon tissues of the rats were isolated to detect Na~+-K~+-ATPase activity and Ca~(2+)-Mg~(2+)-ATPase activity by phosphate determination method, glutathione peroxidase(GSH-Px) activity was detected by spectrophotometry, catalase(CAT) activity was detected by ammonium molybdate, superoxide dismutase(SOD) activity was detected by hydroxylamine, the expression of occludin protein and ZO-1 protein was detected by immunofluorescence, and the expression levels of occludin protein and ZO-1 protein were detected by Western blot. RESULTS:: showed that low dose Huoxiang Zhengqi Oral Liquid could improve the body weight, diet, stool and urine state of rats with dampness obstructing spleen-stomach syndrome obviously. The D-lactic acid content and the DAO activity in the serum of rats with dampness obstructing spleen-stomach syndrome were reduced obviously. The activities of Na~+-K~+-ATPase, Ca~(2+)-Mg~(2+)-ATPase, GSH-Px, CAT and SOD in rat colon tissues were increased obviously. The occludin proteins and ZO-1 protein expression levels in rat colon tissues were raised obviously. The differences in the above indexes between Huoxiang Zhengqi Oral Liquid group and the model control group were statistically significant(P<0.05). Huoxiang Zhengqi Oral Liquid could effectively restore the intestinal barrier function in rats with dampness obstructing spleen-stomach syndrome and its mechanism may be related to the repair of intestinal mechanical barrier function.

The effect of isoflavaspidic acid PB extracted from Dryopteris fragrans (L.) Schott on planktonic and biofilm growth of dermatophytes and the possible mechanism of antibiofilm.

ETHNOPHARMACOLOGICAL RELEVANCE: Dryopteris fragrans (L.) Schott (D. fragrans), a deciduous perennial herb, has been traditionally used for treatment of various skin diseases in Heilongjiang province of China for many years. Phloroglucinol derivatives extracted from D. fragrans were the most effective fraction against dermatophytes. Isoflavaspidic acid PB is a typically phloroglucinol derivative which extracted from D. fragrans and has been reported to exert anti-fungal activities against several dermatophytes. AIM OF THE STUDY: This study aimed to evaluate anti-fungal and anti-biofilm activity of isoflavaspidic acid PB on planktonic and biofilm growth of dermatophytes and explore possible mechanisms of anti-biofilm. MATERIALS AND METHODS: Minimal inhibitory concentrations (MIC) and minimal fungicidal concentrations (MFC) of isoflavaspidic acid PB against 25 isolates of dermatophytes were determined by the Clinical and Laboratory Standards Institute (CLSI) M38-A2 method. The effects of isoflavaspidic acid PB on dermatophytes biofilm formation and pre-formed biofilm were assessed by 2.3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[carbonyl (phenylamino)]-2H-tetrazolium hydroxide (XTT) assay. Morphology of mature biofilm were observed by Scanning Electron Microscope (SEM). Biomass, exopolysaccharide and ergosterol content of mature biofilm were analyzed by gravimetric analysis, anthranone sulfuric acid method and Ultra Performance Liquid Chromatography (UPLC) assay respectively. RESULT: The MIC and MFC ranges of isoflavaspidic acid PB against 25 isolates of dermatophytes were 20-80 µg/mL and 40-80 µg/mL respectively. Isoflavaspidic acid PB (2 MIC) inhibited not only Trichophyton biofilm formation (54.8% ∼ 81.2%) but also the metabolic activity of mature biofilm (20.7% ∼ 44.2%). The result of SEM showed that isoflavaspidic acid PB (8 MIC) could destroy the morphology of hyphae seriously. Comparing with control group, biomass, exopolysaccharide and ergosterol content of the mature biofilm under isoflavaspidic acid PB (8 MIC) were significantly decreased (P < 0.01). CONCLUSION: Isoflavaspidic acid PB had anti-fungal and fungicidal activities against dermatophytes. Isoflavaspidic acid PB could inhibit the biofilm of Trichophyton. The mechanism might be related to the decline of the biofilm biomass, exopolysaccharide and ergosterol content. These results showed that isoflavaspidic acid PB could be explored for promising anti-biofilm drugs.

Protective Effects of Chaihu Shugan San () on Nonalcoholic Fatty Liver Disease in Rats with Insulin Resistance.

OBJECTIVE: To investigate the protective effects of Chinese medicine formulation Chaihu Shugan San (, CHSGS) on nonalcoholic fatty liver disease (NAFLD) in rats with insulin resistance (IR) and its molecular mechanisms. METHODS: Male Sprague-Dawley rats were randomly divided into six groups: the control group, the model group, Dongbao Gantai group (, DBGT, 0.09 g methionine/kg), CHSGS high-dose group (CHSG-H, 12.6 g crude drug/kg), CHSGS medium-dose group (CHSG-M, 6.3 g crude drug/kg), and CHSGS low-dose group (CHSG-L, 3.15 g crude drug/kg). After establishing the NAFLD rat model and treatment for 8 weeks, total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), free fatty acid (FFA), fasting blood glucose (FBG), fasting insulin (FINS) contents in blood serum, and TC, TG contents in the hepatic homogenate were measured by an automatic biochemical analyzer, and a homeostasis model assessment was applied to assess the status of IR, insulin sensitivity index (ISI), and homeostasis model assessment for insulin secretion (HOMA-IS). The expression levels of adiponectin and leptin mRNA in liver tissue were analyzed by reverse transcription polymerase chain reaction. Pathological changes of livers were observed by hematoxylin-eosin staining of paraffin section. RESULTS: Compared with the model group, the serum levels of TC, TG, FFA, FBG, FINS, IRI, ISI, and the liver levels of TC and TG in CHSG-H, CHSG-M, CHSG-L groups showed significant declines (P<0.01 or P<0.05); the serum levels of HDL-C, HOMA-IS were significantly increased (P<0.01 or P<0.05); the expression of leptin mRNA was dramatically decreased and the expression of adiponectin mRNA was increased in the hepatic tissue (P<0.01 or P<0.05). The fatty deposition of liver cells could also be alleviated. CONCLUSION: CHSGS could up-regulate the expression of adiponectin mRNA and down-regulate the expression of leptin mRNA on the liver, suggesting the CHSGS had positive therapeutic effect on NAFLD in rats with IR.

Anti-myocardial Ischemia Effect and Components of Litchi Pericarp Extracts.

Litchi (Litchi chinensis Sonn.) is a famous fruit in south China, and it is also effective for chest tightness or chest pain, irritability, flatulence, epigastric pain and neuralgic pain, hernia pain and testicular swelling, cough, etc. It is valued because a great amount of polyphenol was found in litchi pericarp. In this paper, we got litchi pericarp pure extract by a simple purification method, then evaluated its activity to clear oxygen free radicals in vitro, and evaluated its myocardial protection effect in vivo through acute myocardial ischemia rat model. The results showed that the pure extract had protective effect on myocardial ischemia injury in a certain dose-effect relationship, which reflected in the electrocardiogram, myocardial pathological morphology and other indicators such as cardiac function enzymes, serum and myocardial antioxidant capacity, and eNOS, Bcl-2 and Bax gene expression. Furthermore, we analyzed the components of pure extract by UPLC-MS, ESI-MS and NMR. The main components of PLPE were procyanidin which were identified as procyanidin B2(1), (-)-epicatechin(2), epicatechin-(4ߠ→ 8,2ߠ→ O â†’ 7)-epicatechin-(4ߠ→ 8)-epicatechin(3), A-type procyanidin trimer(4), B-type procyanidin dimer(5) and procyanidin A2(6).This study proved that litchi pericarp extract may have antioxidant activity and cardioprotection effect. It suggested that litchi pericarp may be good for cardiovascular disease. Copyright © 2017 John Wiley & Sons, Ltd.

Mechanisms of Chinese Medicine Xinmailong's protection against heart failure in pressure-overloaded mice and cultured cardiomyocytes.

Patients with heart failure (HF) have high mortality and mobility. Xinmailong (XML) injection, a Chinese Medicine, is clinically effective in treating HF. However, the mechanism of XML's effectiveness on HF was unclear, and thus, was the target of the present study. We created a mouse model of pressure-overload-induced HF with transverse aortic constriction (TAC) surgery and compared among 4 study groups: SHAM (n = 10), TAC (n = 12), MET (metoprolol, positive drug treatment, n = 7) and XML (XML treatment, n = 14). Dynamic changes in cardiac structure and function were evaluated with echocardiography in vivo. In addition, H9C2 rat cardiomyocytes were cultured in vitro and the phosphorylation of ERK1/2, AKT, GSK3ß and protein expression of GATA4 in nucleus were detected with Western blot experiment. The results showed that XML reduced diastolic thickness of left ventricular posterior wall, increased ejection fraction and fraction shortening, so as to inhibit HF at 2 weeks after TAC. Moreover, XML inhibited the phosphorylation of ERK1/2, AKT and GSK3ß, subsequently inhibiting protein expression of GATA4 in nucleus (P < 0.001). Together, our data demonstrated that XML inhibited the TAC-induced HF via inactivating the ERK1/2, AKT/GSK3ß, and GATA4 signaling pathway.

Two pentasaccharide resin glycosides from Argyreia acuta.

Two new compounds of acutacosides 1 and 2, pentasaccharide resin glycosides were isolated from the aerial parts of Argyreia acuta. The core of the two compounds was operculinic acid A, and they were esterfied at the same position, just one substituent group was linked at C-2 of Rha. The absolute configuration of the aglycone in the two compounds was established by Mosher's method, which was (11S)-hydroxyhexadecanoic acid (jalapinolic acid). Their structures were established by a combination of spectroscopic and chemical methods.

Antinociceptive and anti-inflammatory activities of Schefflera octophylla extracts.

ETHNOPHARMACOLOGICAL RELEVANCE: Schefflera octophylla (Lour.) Harms, a traditional Chinese herb mainly distributed in Southeast Asia, is extensively prescribed to alleviate pain and treat rheumatoid arthritis (RA), influenza, throat swelling, pain, etc. In this paper, the antinociceptive and anti-inflammatory activities of the ethanol extract and its five different polar fractions of this plant were evaluated. Furthermore, the anti-rheumatoid arthritis activity of the ethanol extract and its active fraction (CHCl3 fraction) were evaluated. And the chemical constituents of the CHCl3 active fraction displayed significant antinociceptive and anti-inflammatory activities were investigated. MATERIALS AND METHODS: Antinociceptive and anti-inflammatory activities were investigated by hot plate test, acetic acid-induced abdominal writhing test and formalin test, xylene-induced ear edema test. The anti-rheumatoid arthritis activity was evaluated through the model of adjuvant-induced arthritis (AA) in rats, paw swelling, pain response, arthritis index and histopathological changes of ankle, the levels of TNF-α, IL-1ß, IL-6 and rheumatoid factor (RF) of rats were detected. The chemical constituents of the CHCl3 fraction were isolated using chromatographic techniques. Their structures were elucidated by spectroscopic data analysis. RESULTS: The results showed that the ethanol extract of S. octophylla has significant dose-dependent anti-inflammatory and antinociceptive activities. And its five different polar fractions especially the CHCl3 fraction significantly inhibited the abdominal writhing induced by acetic acid and ear edema induced by xylene, also increased pain threshold in hot plate test in 120 min and reduced ticking times in formalin test. The ethanol extract of S. octophylla and the CHCl3 fraction demonstrated an anti-RA effect in a dose-dependent manner. The levels of TNF-α, IL-1ß and IL-6 in ethanol extract (600 mg/kg) and CHCl3 fraction (300 mg/kg) groups were significantly lower than those of the model group. The chemical constituents study of the CHCl3 fraction from S. octophylla led to six triterpenoids which were identified as taraxerone (1), 3-epi-taraxerol (2), aleuritolic acid (3), 3-oxofriedelan-28-oic acid (4), 3ß,19α -dihydroxy-urs-12-ene- 24,28-dioic acid (5) and asiatic acid (6). Compounds 1-5 were obtained from this plant for the first time. CONCLUSION: This study proved the antinociceptive, anti-inflammatory and anti-rheumatoid arthritis activities of S. octophylla. Triterpenoids obtained from its CHCl3 fraction may be responsible for those activities. These results could support the fact that S. octophylla is used traditionally to cure inflammatory and pain diseases.

[Identification of Moghania philippinensis and Moghania macrophylla].

OBJECTIVE: To study the identification methods of Moghania philippinensis and Moghania macrophylla, and to establish a comprehensive precise discrimination method. METHODS: TLC and HPLC were applied to analyze genistein in the root of Moghania philippinensis and Moghania macrophylla. DNA barcoding establishment was based on ITS2 sequcence. RESULTS: A comprehensive differentiation method for Moghania philippinensis and Moghania macrophylla based on TLC was proposed, which was combined with HPLC for determination of genistein. The plants of Moghania philippinensis and Moghania macrophylla and their related species could be distinguished by DNA barcoding effectively. CONCLUSION: TLC and HPLC profiles of Flemingia Radix provide alternative methods of identification using chemical approach. This integrated chemical and molecular approach allows accurate comprehensive fast identification of Moghania philippinensis and Moghania macrophylla, which avoids the methods limitations on the accuracy of identification. The differentiation methods based on TLC, HPLC and DNA barcoding are simple,which provide a new scientific evidence for the identification of authenticity of Flemingia Radix.

Immunization of mice with recombinant protein CobB or AsnC confers protection against Brucella abortus infection.

Due to drawbacks of live attenuated vaccines, much more attention has been focused on screening of Brucella protective antigens as subunit vaccine candidates. Brucella is a facultative intracellular bacterium and cell mediated immunity plays essential roles for protection against Brucella infection. Identification of Brucella antigens that present T-cell epitopes to the host could enable development of such vaccines. In this study, 45 proven or putative pathogenesis-associated factors of Brucella were selected according to currently available data. After expressed and purified, 35 proteins were qualified for analysis of their abilities to stimulate T-cell responses in vitro. Then, an in vitro gamma interferon (IFN-γ) assay was used to identify potential T-cell antigens from B. abortus. In total, 7 individual proteins that stimulated strong IFN-γ responses in splenocytes from mice immunized with B. abortus live vaccine S19 were identified. The protective efficiencies of these 7 recombinant proteins were further evaluated. Mice given BAB1_1316 (CobB) or BAB1_1688 (AsnC) plus adjuvant could provide protection against virulent B. abortus infection, similarly with the known protective antigen Cu-Zn SOD and the license vaccine S19. In addition, CobB and AsnC could induce strong antibodies responses in BALB/c mice. Altogether, the present study showed that CobB or AsnC protein could be useful antigen candidates for the development of subunit vaccines against brucellosis with adequate immunogenicity and protection efficacy.

[Study on antifungal susceptibility of different extract of Dryopteris fragrans].

OBJECTIVE: To examine the antifungal effect of different extract of Dryopteris fragrans (L.) Schott. in vitro, and screen the effective fraction from those extracts. METHODS: Separated the Dryopteris fragrans extract and got four parts by refluxing extraction,and determined the contents of total phloroglucinol. Disc agar diffusion method and solid agar dilution method were used to determine inhibitory effect. Minimum inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) of different parts of Dryopteris fragrans extract against four strains of common clinical dermatophytes were investigated. RESULTS: The data showed that the contents sequence of total phloroglucinol was in the following order: 95% -ethanol extract > water extract > diethyl ether extract > petroleum ether extract, and the antimicrobial activities against the four dermatophytes were as following order: 95% -ethanol extract > water extract > di-ethyl ether extract > petroleum ether extract. CONCLUSION: The contents of total phloroglucinol in 95% -ethanol extract of Dryopteris fragrans is the highest, and the antifungal activity against dermatophytes in vitro is the strongest. The effective fraction of Dryopteris fragrans is the 95%-ethanol extract.

[Effect of Huangqi gegen decoction (HGD) on TGF-beta1/Smad3 pathway in diabetic cardiomyopathy rats].

OBJECTIVE: To study the effect of HGD on diabetic cardiomyopathy and its mechanism. METHODS: The T2-DM rats model was established by combining high fat diet with STZ. The blood glucose, insulin, myocardial fibrosis and TGF-beta1/Smad3 signaling pathway were observed; TGF-beta1 and Smad3 mRNA expression were detected by RT-PCR method, protein expression detected by immunohistochemical method. RESULTS: HGD obviously reduced fasting blood glucose, insulin, improved insulin resistance, reduced myocardial hydroxyproline contents, lowered cardiac index, significantly inhibited over-expression of TGF-beta1/SMAD3 mRNA and protein in diabetic rats cardiac. CONCLUSION: HGD can obviously prevent experimental diabetic myocardial fibrosis through the regulation effect on TGFbeta1/Smad3 signaling pathway.