Abstract OBJECTIVE To introduce the developing procedure of reference standards of nitrosamines by taking 1-methyl-4-nitrosopiperazine(MNP) as example and discuss the problems needing attention when using nuclear magnetic resonance(NMR) quantitative method. METHODS The structure of the material was confirmed by mass spectrometry, infrared spectrum and nuclear magnetic methods. In terms of quantitative assessment, the purity of the material was analyzed by HPLC, and the water was investigated. The content of MNP was determined by mass balance method, the content of the material was verified by NMR quantitative method. RESULTS MNP was consistent with its structure. In the quantitative NMR investigation, it was found that the use of different internal standards had a great impact on the calibration results. CONCLUSION Since MNP is basic, it is easy to reduce the final determination result by using an acidic internal standard in NMR quantitative analysis. Therefore, it is suggested that an appropriate internal standard should be selected for this kind of sample in the process of NMR quantitative determination.

Aim To investigate the effect of curcumin against high-fat-diet induced C57BL/6J mice bone changes and the correlation between the expression of cathepsin K and curcumin.Methods Curcumin treated C57BL/6J mice had been on high fat diet for 12 weeks.The HE, Alizarin red S staining and Safranin O/fast green staining of femur were employed to evaluate bone microstructure, bone metabolism and bone development.The expressions of cathepsin K were assessed by Western blot and immunohistochemical staining.Results Histopathological results showed that curcumin could improve the destruction of trabecular bone structure, cartilage development and bone calcification.Biomechanical results proved that curcumin could improve the bone strength of the type 2 diabetic mice induced by high fat.The results of immunohistochemistry and Western blot assay indicated that curcumin could significantly inhibit the expression of cathepsin K in bone tissues of mice.Conclusion Curcumin can increase bone strength, improve bone microstructure, and enhance the degree of bone calcification, which may be achieved by inhibiting the expression of cathepsin K.

ObjectiveTo study the effects of tanshinoneⅡA on proliferation of cervical squamous cancer Siha cells; To discuss its possible molecular mechanism.Methods Cervical squamous cancer Siha cells were treated with different doses of tanshinoneⅡA. The effects of tanshinoneⅡA on proliferation of Siha cells were measured by MTT assay and flow cytometry analysis. The effects of tanshinoneⅡA on expression levels of phospho-extracellular regulate kinase (p-ERK) and Cyclin D in Siha cells were measured by Western blot.Results 1×10-5, 5×10-6, 1×10-6, 5×10-7 mol/L tanshinoneⅡA significantly inhibited Siha cell proliferation and such effect could be enhanced by ERα antagonist MPP and attenuated by ERβ antagonist PHTPP. 1×10-5, 5×10-6, 1×10-6 tanshinoneⅡA could significantly decrease the proliferation index of Siha cells. 1×10-5, 5×10-6, 1×10-6, 5×10-7 mol/L tanshinoneⅡA could significantly reduce the protein expression levels of p-ERK and Cyclin D of Siha cells.ConclusionTanshinoneⅡA can inhibit cervical squamous cancer Siha cell proliferation and such effect is realized via estrogen receptor pathway. TanshinoneⅡA plays anti-proliferation roles by reducing the expression levels of p-ERK and Cyclin D.

Osteoporosis is a progressive metabolic disease. In traditional Chinese medicine (TCM), deficiency of the liver, spleen and kidney and blood stasis can induce osteoporosis. The main etiology was kidney deficiency which harmed the liver and spleen and causeqi-blood deficiency and blood stasis. The insufficiency of natural endowment and postnatal malnutrition caused poor nutrition of tendons and vessels, which induced debility of bone. This article discussed the etiology and pathogenesis of TCM understanding on osteoporosis in details. Opportunities and challenges of TCM in osteoporosis treatment were explored. The homology of Chinese medicine and food as well as the long-term of osteoporosis showed that TCM had incomparable superiority in osteoporosis treatment. While, the scientific research methods and reasonable evaluation of TCM safety were important to display its advantages in osteoporosis treatment.

Aim To explore the effects of tanshinone IIA on cell proliferation via G protein-coupled estrogen receptor inductive and regulative pathway in typical es-trogen receptor and G protein-coupled estrogen receptor positive T47D breast cancer cells. Methods The pro-liferation rate of T47 D cells influenced by tanshinone IIA was analyzed by MTT assay. G protein-coupled es-trogen receptor agonist G1 and GPER antagonist G15 were employed as tools. GPER SiRNA was applied to build GPER gene silence T47D cells. GPER expres-sion influenced by tanshinone IIA was measured by Western blot. Results The proliferation rates of T47D cells treated with 1 × 10 -5 mol·L-1 - 1 × 10 -7 mol· L-1 of tanshinone IIA were decreased significantly. Such effects could be attenuated by G1 or enhanced by G15 . Growth of GPER SiRNA transfected T47 D cells were significantly inhibited by 1 × 10 -5 mol·L-1 - 1 × 10 -7 mol·L-1 of tanshinone IIA treating. Result of Western blot showed that tanshinone IIA at 1 × 10 -5 mol· L-1 and 1 × 10 -6 mol · L-1 could induce de-crease of GPER protein expression in T47D cells. Conclusions Tanshinone IIA shows inhibitory effects on proliferation rate of T47 D breast cancer cells via GPER pathway. Tanshinone IIA could perform regula-tive function on GPER expression level in target cells.

This study was aimed to observe the influence of β-sitosterol (BSS) on estrogen receptor (ER) positive the human breast cancer cell line T47D and to study its mechanisms. ER antagonist ICI182 780 was employed to observe the influence on the proliferation. Proliferations of T47D cells influenced by different concentrations of BSS were analyzed by MTT assay. Cell cycle analyses were examined by flow cytometry. The protein expression of cyclin D1 was measured by western blot analysis and cyclin D1 mRNA was quantified by real-time PCR assay. The results showed that BSS in high dose exhibited significant inhibitory effects that were partly antagonized by ICI182 780 and decreased the proliferative index on T47D cells. However, BSS in low dose obviously promoted the proliferation that was completely inhibited by ICI182 780 and increased the proliferative index on T47D cells. The mRNA and protein levels of cyclin D1 were increased in low-dose BSS. The effect was blocked by ICI182 780. It was concluded that BSS in low concentration had phytoestrogenic effect by up-regulating the expression of cyclin D1 via ER pathway.

This study was aimed to compare the antioxidant activity of puerarin and 3 other flavonoid compounds, and to investigate their structure-activity relationship. The intragastric administration of 4 kinds of typical flavonoids compounds (soybean element, puerarin, quercetin and rutin) were given to mice, respectively. The model mice of acute hepatic injury were established with intraperitoneal injection of 0.1% carbon tetrachloride (CCl4) after 7 days. After 18 h fasting, liver tissues were removed. The histomorphology was observed after paraffin sectioning and hematoxylin-eosin staining. The activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT) and alkaline phosphatase (ALP) in serum were detected with automatic biochemical analyzer. The content of malondialdehyde (MDA), the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in liver tissues were detected with homogenization. The pathological results of liver tissues showed that hepatic damages were decreased in all 4 medicine treatment groups compared with the model group, but there were no significant differences among these treatment groups. The results of blood serum bio-chemical analysis showed that compared with the model group, puerarin and quercetin could decrease the activities of ALT, AST and GGT in serum significantly (P < 0.05, orP < 0.01). There were no content changes of ALP. In the soybean element group, only the activities of ALT and AST decreased obviously (P < 0.05, orP < 0.01). There was no obvious change in the serum of mice in the rutin treatment group. The homogenate detection results of liver tissues showed that compared with the model group, quercetin and rutin significantly lowered MDA (P < 0.05), increased SOD and GSH-Px (P < 0.05, orP < 0.01); while soybean element and puerarin only improved GSH-Px levels (P < 0.05, orP < 0.01). It was concluded that the antioxidant capacity of quercetin was better than that of soybean element, puerarin and rutin, which may be related to its structure. Compared with 3 other chemical compounds, quercetin had more polyhydroxies and its polyhydroxies were not glycosylated, which suggested that the structure of quercetin may be closely related to its antioxidation activity.

Objective To determine the efficacy of berberine in the treatment of non-alcoholic steatohepatitis ( NASH) , and to investigate the regulating effect on macrophage phenotype transformation in hepatic tissue on methionine -choline deficiency (MCD) diet induced NASH mice.Methods Fourty male C57BL/6 mice were randomly divided into 4 groups (10 mice per group): the normal group (fed with normal diet), the NASH model group (fed with MCD diet), rosiglitazone treatment group (30mg/kg) and berberine treatment group (150mg/kg).Drugs were adopted in the preventive intervention method for 2 weeks.The hepatic histopathological method was adopted to evaluate the drug therapeutic effect.The serum levels of tumor necrosis factor-α(TNF-α), interleukin(IL)-6, and IL-10 were examined with ELISA method.M1 and M2 phenotype were detected by flow cytometry .Results The results showed berberine improved the degree of hepatic histopathology .Berberine not only reduced the level of TNF-α, but also increased the level of IL-10 in serum on NASH mice significantly ( P <0.05 ) . Flow cytometry data indicated that berberine decreased M 1 type macrophages and increased M 2 type macrophages in liver tissue .The ratio of M1/M2 was significantly decreased in berberine and rosiglitazone treated group ( P <0.01 ) .Conclusion Berberine may improve the hepatic pathological process in MCD diet induced NASH model possibly through modulating macrophage phenotype transformation , i.e.The ratio of M2 type is more than M1 type in hepatic tissue , and increasing anti-inflammatory cytokines .

Aim To isolate and characterize the human circulating fibrocytes from human peripheral blood and explore the effects of curcumin on human circulating fi-brocytes.Methods The cells were isolated and puri-fied by density gradient centrifugation,and identified by flow cytometry and immunocytochemistry .Then , CCK-8 and flow cytometry were used to study the effect of curcumin on the proliferation as well as COL I ex-pression of human circulating fibrocytes,respectively. Results After being isolated the cells expressed CD34,CD45 and COLⅠ,among which 79.7% were both CD45 and collagen I positive,typical of human circulating fibrocytes.Curcumin could exert regulatory effects on proliferation of human circulating fibrocytes. Exposure of the cells to curcumin for as short as 24 hours promoted their growth,while prolonged treatment (72 h ) significantly inhibited cell propagation and downregulated the COLⅠ levels,best manifested at a concentration as high as 20 μmol · L-1 .Conclusion The proliferation of cells and COLⅠexpressions can be effectively inhibited by curcumin with the prolonged action period and high concentrations.

This study was aimed to observe the pharmacodynamics of genistein combined with tripterygium gly-cosides ( GTW ) in the rheumatoid arthritis ( RA ) treatment of ovariectomized rats . SD rats were randomly divid-ed into four groups , which were the sham-operation group , model group , Methotrexate ( MTX ) treatment group ( 2.7 mg/kg ) , and genistein ( 30 mg/kg ) combined with GTW ( 70 mg/kg ) treatment group . There were ten rats in each group . Except the sham-operation group , rats in other groups were ovariectomized and immunized by collagen type II to prepare collagen induced arthritis ( CIA ) model . The intragastric administration of drugs was given once a day for two weeks . The arthritis index , joints swelling degree , and levels of anti-Col II , IL-6 and IL-10 in serum were tested . The results showed that the arthritis index , joints swelling degree , and levels of anti-Col II , IL-6 and IL-10 in serum were all significantly increased in the model group ( P < 0 . 05 or P< 0 . 01 ) . Compared with the model group , the arthritis index and joints swelling degree of both MTX and genistein combined with GTW were significantly decreased . The level of anti-Col II in serum of the MTX treatment group was significantly decreased ( P < 0 . 05 ) . However , there were no significant changes on levels of IL-6 and IL-10 . The levels of anti-Col II and IL-6 in serum of the genistein combined with GTW were significantly decreased ( P < 0 . 05 ) . The level of IL-10 was significantly increased ( P < 0 . 01 ) . It was conclud-ed that genistein combined with GTW can significantly inhibit the pathological progress of CIA on ovariec-tomized rats . Compared with MTX , the genistein combined with GTW received a better regulating effect on the expression of IL-6 and IL-10 . The results suggested that phytoestrogen combined with GTW played a good therapeutic effect on gonadal dysfunction type of RA . It provided novel strategies and experimental evidences on postmenopausal RA treatment in the clinical practice .