Effect of oxymatrine on the replication cycle of hepatitis B virus in vitro

AIM:To determine the antiviral mechanism or target of oxymatrine against hepatitis B virus(HBV).METHODS:HepG2.2.15 cells were incubated with culture medium containing 500 μg/mL of oxymatrine for 2 and 5 d.The surface antigen of HBV(HBsAg) and e antigen of HBV(HBeAg) in supernatant were determined by ELISA.HBV DNA in supernatant,and intracellular covalently closed circular DNA(cccDNA),relaxed circular DNA(rcDNA) and pregenomic RNA(pgRNA) were quantif ied by specif ic real-time polymerase chain reaction(PCR) or reverse transcription(RT)-PCR.RESULTS:Treatment with oxymatrine for 2 d and 5 d reduced the production of HBV by the cell line,as indicated by the decline of HBsAg(22.67%,t = 5.439,P = 0.0322 and 22.39%,t = 5.376,P = 0.0329,respectively),HBeAg(55.34%,t = 9.859,P = 0.0101 and 43.97%,t = 14.080,P = 0.0050) and HBV DNA(40.75%,t = 4.570,P = 0.0447 and 75.32%,t = 14.460,P = 0.0047) in the supernatant.Intracellular cccDNA was also markedly reduced by 63.98%(t = 6.152,P = 0.0254) and 80.83%(t = 10.270,P = 0.0093),and intracellular rcDNA by 34.35%(t = 4.776,P = 0.0413) and 39.24%(t = 10.050,P = 0.0097).In contrast,intracellular pgRNA increased by 6.90-fold(t = 8.941,P = 0.0123) and 3.18-fold(t = 7.432,P = 0.0176) after 500 μg/mL of oxymatrine treatment for 2 d and 5 d,respectively.CONCLUSION:Oxymatrine may inhibit the replication of HBV by interfering with the process of packaging pgRNA into the nucleocapsid,or inhibiting the activity of the viral DNA polymerase.

Inhibition of hepatitis B virus by oxymatrine in vivo

AIM To investigate the anti-HBV effect ofoxymatrine (oxy) in vivo.METHODS HBV transgenic mice were producedby micro-injection of a 4.2kb fragmentcontaining the complete HBV genomes.Expression level of HBsAg and HBcAg in thetransgenic mice liver was determined byimmunohistochemical assay.RESULTS Four groups (6 mice in each group)were injected intraperitoneally with oxy at thedosage of 100,200, and 300 mg/kg or with salineonce a day for 30 days. Both HBsAg and HBcAgwere positive in livers of all the six mice in thecontrol group (injected with saline), and werepositive in livers of two mice in 100 mg/kg groupand 300mg/kg group. In 200mg/kg group,HBsAg and HBcAg were negative in livers of allthe six mice. Based on the results, 200 mg/kg isthe ideal dosage to explore the effect of oxy atdifferent time points. According to the oxytreatment time, mice were divided into fourgroups: 10 d, 20 d, 30 d and 60 d (4 mice in eachgroup). Each mouse underwent liver biopsy twoweeks before the treatment of oxy. Down-regulation of HBsAg and HBcAg appeared aftertreatment of oxymatrine for 10 d and 20 d, Dane-like particles disappeared after the treatment ofoxy for 20d under electron microscopy,however, the expression level of HBsAg andHBcAg returned to normal 60 d later after oxytreatment.CONCLUSION oxymatrine can reduce thecontents of HBsAg and HBcAg in transgenic miceliver, longer treatment time and larger dosagedo not yield better effects.

Role of DOR-β-arrestin1-Bcl2 Signal Transduction Pathway and Intervention Effects of Oxymatrine in Ulcerative Colitis

This study was aimed to investigate the role of the delta-opioid receptor （DOR）-β-arrestinl-Bcl-2 signal transduction pathway in the pathogenesis of ulcerative colitis （UC） and the intervention effects of oxymatrine on UC. Forty Sprague-Dawley rats were divided into nor- mal group, model group, oxymatrine-treated group and mesalazine-treated group （n=10 each） at ran- dom. The rat UC model was established by intra-colonic injection of trinitrobenzene sulfonic acid in the model group and two treatment groups. The rats in oxymatrine-treated group were subjected to intramuscular injection of oxymatrine [63 mg/（kg·day）] for 15 days, and those in mesalazine-treated group given mesalazine solution [0.5 g/（kg·day）] by gastric lavage for the same days. Animals in normal group and model group were administered 3 mL water by gastric lavage for 15 days. On the 16th day, after fasting for 24 h, the rats were sacrificed for the removal of colon tissues. The expres- sion levels of DOR, β-arrestinl and Bcl-2 were determined in colon tissues by immunohistochemistry and real-time quantitative polymerase chain reaction （RT-PCR）, respectively. It was found that the expression levels of DOR, [3-arrestinl and Bcl-2 protein and mRNA were significantly increased in the model group as compared with the other groups （P〈0.05）. They were conspicuously decreased in both mesalazine-treated and oxymatrine-treated groups in contrast to the model group （P〈0.05）. No statistically significant difference was noted in these indices between mesalazine- and oxyma- trine-treated groups （P〉0.05）. This study indicated that the DOR-β-arrestinl-Bcl-2 signal transduc- tion pathway may participate in the pathogenesis of UC. Moreover, oxymatrine can attenuate the de- velopment of UC by regulating the DOR-β-arrestin 1-Bcl-2 signal transduction pathway.

Inhibitory effect of oxymatrine on hepatocyte apoptosis via TLR4/PI3K/Akt/GSK-3β signaling pathway

AIM To evaluate the effect of oxymatrine(OMT) on hepatocyte apoptosis in rats with lipopolysaccharide(LPS)/D-galactosamine(D-Gal N)-induced acute liver failure(ALF). METHODS LPS/D-Gal N was used to establish a model of ALF in rats. To evaluate the effect of OMT, we assessed apoptosis by transmission electron microscopy, and the pathological changes in the liver by light microscopy with hematoxylin and eosin staining. An automated biochemical analyzer was used to measure serum alanine aminotransferase(ALT) and aspartate aminotransferase(AST). Enzyme-linked immunosorbent assay was used to determine the levels of tumor necrosis factor(TNF)-α and interleukin(IL)-1β. Western blotting was used to detect protein levels in liver tissues. Streptavidin peroxidase immunohistochemistry was used to observe expression of Toll-like receptor(TLR)4, active caspase-3, Bax and Bcl-2. RESULTS All rats in the normal control and OMT-pretreated groups survived. The mortality rate in the model group was 30%. OMT preconditioning down-regulated apoptosis of hepatocytes and ameliorated pathological changes in liver tissue. The levels of AST, ALT, TNF-α and IL-1β in the model group increased significantly, and were significantly reduced by OMT pretreatment. OMT pretreatment down-regulated expression of TLR4 and active caspase-3 and the Bax/Bcl-2 ratio, and upregulated expression of P-AktSer473(Akt phosphorylated at serine 473) and P-GSK3βSer9(glycogen synthase kinase 3β phosphorylated at serine 9) induced by LPS/D-Gal N. CONCLUSION OMT inhibits hepatocyte apoptosis by suppressing the TLR4/PI3K/Akt/GSK-3β signaling pathway, which suggests that OMT is an effective candidate for ameliorating acute liver failure.

Oxymatrine reduces neuroinflammation in rat brain A signaling pathway

Cerebral neuroinflammation models were established by injecting 10μg lipopolysaccharide into the hippocampus of male Sprague-Dawley rats. The rats were treated with an intraperitoneal injection of 120, 90, or 60 mg/kg oxymatrine daily for three days prior to the lipopolysaccharide injection. Twenty-four hours after model induction, the hippocampus was analyzed by real-time quantitative PCR, and the cerebral cortex was analyzed by enzyme-linked immunosorbent assay and western blot assay. The results of the enzyme-linked immunosorbent assay and the real-time quantitative PCR showed that the secretion and mRNA expression of the pro-inflammatory cytokines interleukin-113 and tumor necrosis factor-a were significantly decreased in the hippocampus and cerebral cortex of model rats treated with oxymatrine. Western blot assay and real-time quantitative PCR analysis indicated that toll-like receptor 4 mRNA and protein expression were significantly decreased in the groups receiving different doses of oxymatrine. Additionally, 120 and 90 mg/kg oxymatrine were shown to reduce protein levels of nuclear factor-KB p65 in the nucleus and of phosphorylated IKBa in the cytoplasm of brain cells, as detected by western blot assay. Experimental findings indicate that oxymatrine may inhibit neuroinflammation in rat brain via downregulating the expression of molecules in the toll-like receptor 4/nuclear factor-KB signaling Dathwav.

Effects of Oxymatrine on the Apoptosis of Human Esophageal Carcinoma Eca109 Cell Line and Its Mechanism

The effects of Oxymatrine （Oxy） on the proliferation and apoptosis of human esophageal carcinoma Ecal09 cell line and the mechanism were investigated. The human esophageal carcinoma Eca 109 celis were cultured in vitro. The Oxy-induced apoptosis of Eca 109 cells was assayed by using flow cytometry. The expressions of p-ERKII2, Cyclin D1, p21^waf/cipl, Bax and Bcl-2 were detected by Western blot. Flow cytometry revealed that Oxy could induce the apoptosis of Eca l09 cells. Western blot showed that Oxy of different concentrations suppressed the expressions of p-ERK1/2, Cyclin D1 and Bcl-2, but up-regulated the expression of p21waf/cip1 and Bax, and the ratio of Bax/Bcl-2 was increased. It was suggested the Oxy could induce the apoptosis of Eca l09 cells, which might be related to the upregulation of p21waf/cip1 and the downregulation of p-ERK1/2, Cyclin D1 and p21^waf/cip1. The possible pathway may be related to Bcl-2/Bax.

Oxymatrine Improves TNBS-induced Colitis in Rats by Inhibiting the Expression of NF-κB p65

Inflammatory bowel disease is thought to be regulated by the balance between Th1 and Th2 cytokines secreted by T cells, and NF-κB p65 also plays a predominant role in the intestinal inflammation. We evaluated the potency of oxymatrine, one of active components of Sophora Root, in inhibiting the immune responses and inflammation in 2,4,6-trinitrobenzene sulfonic acid （TNBS）-induced colitis. The inflammation was markedly ameliorated in the oxymatrine-treated rats. The level of IL-2 was increased and that of IL-10 was decreased in colon tissue in the rat model, which was reversed by the treatment of oxymatrine. Moreover, the elevated expression of NF-κB p65 in colon tissue in the model was also improved by oxymatrine treatment. Our results suggest that oxymatrine might be beneficial for the abnormal immune responses and inflammation by regulating the unbalance of Th1 and Th2 cytokines secretion and inhibiting the expression of NF-κB p65 in colon tissue.

Effect of oxymatrine on interferon-gamma and tumor necrosis factor-alpha serum levels in an experimental rat model of autoimmune encephalomyelitis

BACKGROUND： Studies have demonstrated that experimental autoimmune encephalomyelitis （EAE） onset correlates with increased interferon-v （IFN-γ） and tumor necrosis factor-α （TNF-α） expression. Oxymatrine （OM） has been shown to inhibit autoimmune responses, but there are no reports showing that it could prevent the development of EAE. OBJECTIVE： To observe the effect of OM on serum levels of IFN-γ and TNF-α in a rat model of EAE.DESIGN, TIME AND SETTING： A randomized, controlled, animal study was performed at the Experimental Animal Center of Henan Academy of Chinese Medicine and at the Key Disciplines Laboratory Clinical Medicine of Henan Province between July and December 2008. MATERIALS： OM was purchased from Chia-tai Tianqing Pharmaceutical, China; complete Freund＇s adjuvant was purchased from Sigma, USA. METHODS： Forty female Wistar rats were randomly assigned to four groups： EAE model （M）, low-dose OM treatment （OM-L）, high-dose OM treatment （OM-H）, and normal control （N, no immunization）, with 10 rats in each group. EAE was established in the M, OM-L, and OM-H groups following immunization with Guinea pig spinal cord homogenate and complete Freund＇s adjuvant. The M and N groups were intraperitoneally injected with normal saline （6.7 mL/kg per day）, the OM-L group received an intraperitoneal injection of OM （100 mg/kg per day）, and the OM-H group received OM （150 mg/kg per day）. MAIN OUTCOME MEASURES： At 16 days after immunization, the degree of histopathological changes in the spinal cord was assessed by hematoxylin-eosin stanining. Enzyme-linked immunosorbent assay was used to detect serum levels of IFN-γ, and radioimmunoassay was utilized to determine serum TNF-α level. Neurological scores were measured on a daily basis according to a 0-5 scale. RESULTS： Daily injections of OM, both high and low doses, resulted in decreased neurological scores in EAE rats （P〈0.01）, as well as reduced cellular infiltration in the spinal cord and decreased levels of serum IFN-γ and TNF-α （P〈 0.01）. CONCLUSION： OM reduced the onset and severity of EAE, which correlated with decreased IFN-γ and TNF-α expression.

Antifungal activities of matrine and oxymatrine and their synergetic effects with chlorthalonil

The EC50 values of matrine and oxymatrine against five forest pathogenic fungi （Fusarium oxysporum, Valsa pini, Cladosporium oxysporum, Sphaeropsis sapinea, Marssonina brunnea） were examined by bioassay methods. The results demonstrated that matrine and oxymatrine had strong inhibitory activities to the conidium germination of the tested fungi. The ECso values of matrine for inhibiting the conidium germination of Marssonina brunnea, Cladosporium oxysporum, Sphaeropsis sapinea were 123μg·mL^-1, 272 μg·mL^-1, 1133 μg·mL^-1, respectively, and the EC50 values of oxymatrine for inhibiting the conidium germination of Fusarium oxysporum, Sphaeropsis sapinea were 532μg·mL^-1, 601μg·mL^-1, respectively. The hyphal growth of the fungi was also significantly inhibited by matrine and oxymatrine. The ECs0 values of matrine inhibiting the conidium germination of Sphaeropsis sapinea, Valsa pini, Fusarium oxysporum were 428μg·mL^-1, 535 μg·mL^-1, 592 μg·mL^-1, respectively. The EC50 values of oxymatrine inhibiting the conidium germination of Valsa pini, Fusarium oxysporum were 323, 618μg·mL^-1, respectively. In the synergetic tests the ECs0 values of the mixtures of thiophanate methyl （or chlorthalonil） and matrine （or oxymatrine） were lower than 34 μg·mL^-1 while their co-toxicity coefficients were significantly higher than 1130 It indicated that the mixture of the alkaloids and the chemical had potential practical utilization in controlling certain forest fungal diseases.

Synthesis of N-Succinyl-chitosan(Suc-Chi) and Preparation of Oxymatrine(OM)/N-Succinyl-chitosannanoparticles

Oxymatrine （OM）/N-succinyl-chitosan （Suc-Chi, with a degree of substitution being 0. 32） was synthesized via the ring-opening reaction of succinic anhydride with chitosan in dimethyl sulfoxide. OM-loaded Suc-Chi nanoparticles were prepared by an ionotropic gelation process and OM was quantified via the HPLC method： The influences of the initial OM concentration on the nanoparticle characteristics and OM release behavior were evaluated. The nanoparticles were found to have a mean diameter within a range of 267-392 nm, a positive surface charge, and a zeta potential in the range of 19-27 inV. The formulation with an initial OM concentration of 100μg/mL provided the highest loaded capacity（0. 77% ） and the highest extent of the released OM （68% at 24 h）, suggesting the possibility to achieve a therapeutic dose. According to the data obtained, this Suc-Chi-based nanotechnology will open up new and interesting prospects for the development of new anticancer drugs.

Evaluation of pharmacokinetics underlies the collaborated usage of lamivudine and oxymatrine in beagle dogs

Combinational therapy of lamivudine and oxymatrine has been employed in the battle against hepatitis B virus in clinical setting. However, the pharmacokinetic behavior of the drug or active metabolism in intravenous/oral co-administration regime is poorly investigated. Herein,we evaluated the pharmacokinetic characteristic through a tailor-designed 3 way crossoverLatin square experiment in adult male beagle dogs. Six dogs were randomly treated by intravenous administration of lamivudine(2.5 mg/kg), oxymatrine(15 mg/kg) and combinational dosage, named as intravenous regime. Meanwhile the other six dogs were orally administrated with lamivudine(2.5 mg/kg), oxymatrine(15 mg/kg) and combinational dosage,named as oral regime. The pharmacokinetic feature in simultaneous oral treatment appeared to have no significant difference when compared with individual administration,even including matrine, the active metabolite of oxymatrine. In intravenous regime, the main pharmacokinetic parameters of simultaneous administration were nearly consistent with intravenous regime remedy. The collaborated application of lamivudine and oxymatrine contributed to non-distinctive pharmacokinetic fluctuations of beagle dogs in intravenous/oral regime, compared with individual employment, which established a vital base for the clinical co-administration against hepatitis B. Furthermore, the present study demonstrated that the determination of pharmacokinetics between combinational and individual therapy might assist in the development of drug compatibility in clinical therapy.

Effects of Oxymatrine on the NF-kappa B expression of HaCaT cells

Objective： To study the effect of oxymatrine on the expression of nuclear factor-kappa B（NF-K B） of Human benign epidermal keratinocytes line（HaCaT cells）. Methods：HaCaT cells were cultured with different concentration of Oxymatrine（10 μ g/ml, 50 μ g/ ml and 100 μg/ml） for 24 h, then 10.5 mol/L Substance P was added to the cells. After 30 min, NF-K B expression in the cells was observed by immunocytochemistry, NF-K B P65 protein expression was evaluated by Western blot, and the mRNA expression of NF-K B P65 was evaluated by reverse transcription polymerase chain reaction（RT-PCR）. The 10.5 mol/L Substance P and culture medium were added to the Substance P group and control group, respectively. Results：In control group, expression rate of positive cells, the expressions of protein and mRNA of NF-K B were all low. In Substance P group, when 10μ mol/L Substance P was added, the expressions were all increased（P 〈 0.05）. But in Oxymatrine groups, the expression rate of positive cells, the expressions of protein and mRNA were all descended in a concentration-dependent manner（P 〈 0.05 or P 〈 0.01）. Conclusion：Oxymatrine can down-regulate the expression of NF-K B of the HaCaT cells and may play an important role in regulating anti-inflammation and immunity.

Inhibitory Effect of Oxymatrine on Quartz-induced Secretion of TNF-α by the Pulmonary Alveolar Macrophages in the Fibroblast Proliferation

To study the inhibitory effect of oxymatrine （OM） on quartz-induced secretion of TNF-α in the fibroblast proliferation, a given amount of quartz powder and OM of different concentrations were put into the media of pure culture containing macrophages. After 24 h of the culture, the TNF-α in the media was measured by double-antibody sandwich ELISA. The TNF-α （10 ng/mL） and OM of different concentrations were added into the media containing the fibroblasts of the 4th generations from neonate rats. The y values of cAMP and cGMP in fibroblasts were determined by the radioimmunoassay and the concentrations of cAMP and cGMP were calculated according to standard curve. The intracellular Ca^2＋ was determined by flow cytometry and cell proliferation was detected by MTT. Our results showed that at the concentrations between 200 μg/mL-1600 μg/mL, OM inhibited the secretion of TNF-α by alveolar macrophages （AM） in a dose-dependent manner. Especially, there were significant differences, to various degrees, in the inhibitory effect of OM between the concentration range of 800 μg/mL-1600 μg/mL and the concentration of 10 ng/mL TNF-α. When compared with 10 ng/mL TNF-α, OM of different concentrations could dose-independently increased the level of intracellular cAMP and decreased the level of cGMP, thereby raising the ratio of cAMP/cGMP and lowering the concentrations of intracellular Ca^2＋. Moreover, OM of 800 μg/mL had the strongest inhibitory effect on cell proliferation and at this concentration, the cAMP/cGMP was highest and Ca^2＋ was at the lowest level. We are led to conclude that OM can antagonize the damaging effect of quartz on the membrane of AM and the effect of TNF-α promoting the proliferation of fibroblasts. It achieves its inhibitory effect on the promoting effect of TNF-α on fibroblast proliferation by elevating the cAMP level and decreasing the release of Ca^2＋.

Differentially expressed genes identified by microarray analysis following oxymatrine treatment of hepatic stellate cell

AIM: To investigate the effects of oxymatrine on the gene expression profile of hepatic stellate cell (HSC) and provide novel insights into the mechanism of oxymatrine against hepatic fibrosis. Methods: HSC was isolated from normal SD by in situ perfusion of collagenase and pronase and density Nycodenz gradient centrifugation. MTT colorimetry was used to study the effect of oxymatrine on the proliferation of HSC. Total RNA and mRNA of quiescent HSC, culture-activated HSC and oxymatrine treated HSC were extracted. Effect of oxymatrine on HSC gene expression profile was detected by oligonucleotide microarray analysis with Affymetrix gene chip rat U230A. Differentially expressed genes were annotated with Gene Ontology (GO) and analyzed with Kyoto encyclopedia of genes and genomes (KEGG) pathway using the Database for Annotation, Visualization and Integrated Discovery. Results: Oxymatrine could inhibit the proliferation of HSC in a dose-dependent manner. A total of 4641 differentially expressed genes were identified by cDNA chip between activated and quiescent HSC, among which 2702 genes were upregulated, and 1939 genes were down-regulated in activated HSC. cDNA microarray uncovered downregulation of 56 genes in response to oxymatrine, the representative genes including alpha 2 type I procollagen, alpha-1 type I collagen, tissue inhibitor of metalloproteinase 1, interleukin 1 beta, early growth response 1, chemokine ligand 2, chemokine ligand 1, CTGF, TGFβ1. The most enriched GO terms included response to wounding, inflammatory response, cell migration, cell motility, wound healing, TGFβ receptor signaling pathway. KEGG pathway analysis revealed that oxymatrine affected the ECM-receptor interaction, focal adhesion, cytokine-cytokine recaptor interaction, TGFβ signaling pathway, MAPK signaling pathway. There were 37 genes upregulated significantly following oxymatrine treatment. The most enriched GO terms included oxidation reduction, negative regulation of lipoprotein oxidation, regulation of lipoprotein oxidation, steroid metabolic process, regulation of lipase activity. Six genes were confirmed with QPCR, consistent with microarray. Conclusion: The mechanism of oxymatrine in inhibiting liver fibrogenesis is associated with multi-genes and multi-pathways regulation.

Effects of oxymatrine on calcium channels and GABA release in mice under neuropathic pain condition

OBJECTIVE To investigate effects of oxymatrine,an alkaloid from Sophora flavescens Ait.,on high-voltage dependent calcium channel and inhibitory neurotransmitter GABA under neuropathic pain condition.METHODS The partial sciatic nerve ligation(PSNL)was executed on C57/BL6 mice to produce neuropathic pain.Oxymatrine(150 mg·kg-1)was administrated intraperitoneally to PSNL mice.Mechanical hindpaw withdral threshold(MWT)was measured under Von-Frey filament stimulation with up-and-down method.In brain tissue,GABA concentration was measured with ELISA.Change of GABAAreceptor protein expression,N-type calcium channel(Cav2.2)and L-type calcium channel(Cav1.3)protein expressions were detected with Western-blot;intracellular calcium concentration was measured in cultured cortical neurons with Fluo-3/AM fluorescent probe.RESULTS Compared to saline,oxymatrine significantly increased ED50 of MWT on PSNL mice(P<0.05).GABA concentration and GABAAreceptor protein level in brain tissue were decreased in PSNL mice,while administration of oxymatrine increased both GABA concentration and GABAA receptor expression.Intracellular calcium concentration was increased in cultured cortical neurons by oxymatrine treatment,but this phenomenon was not seen under calcium-free condition.Protein expression of Cav2.2,but not Cav1.3,was found to be decreased in the brains of PSNL mice and to be restored to a normal level with oxymatrine administration.CONCLUSION Oxymatrine has analgesic effect on PSNL-induced neuropathic pain in mice.This phenominon relates to the increase of GABA release,GABAAreceptor expression,and also the restoration of expression level of Cav2.2 but not Cav1.3 in brain tissues,which suggesting that Ca2+ flow through Cav2.2 calcium channel may be the key point underlying oxymatrine analgesia.

Determination of Matrine and Oxymatrine in Radix Sophorae Flavescentis by Resonance Rayleigh Scattering, Second-order Scattering and Frequency Doubling Scattering Technique

In 0.1 mol/L HCl medium, 12-tungstophosphoric（TP） acid reacted with matrine（Mat） and oxymatrine（Oxy） to form an ion-association complex. As a result, the new spectra of resonance Rayleigh scattering（RRS）, second-order scattering（SOS） and frequency doubling scattering（FDS） appeared and their intensities were enhanced greatly. The maximum scattering wavelengths of RRS, SOS and FDS were located at 370, 670 and 390 nm, respectively. The in-crements of scattering intensity were directly proportional to the concentration of Mat and Oxy in a certain range. Based on this, the method for the determination of matrine and oxymatrine has been established. It has been applied to the determination of matrine and oxymatrine in samples of Radix sophorae flavescentis with satisfactory result. The reaction mechanism and reasons of RRS enhancement were discussed.

Simultaneous determination of berberine,matrine and oxymatrine in traditional Chinese medicines by using nonaqueous capillary electrophoresis

A rapid method for the simultaneous determination of berberine(BBR),matrine(MT)and oxymatrine(OMT)by nonaqueous capillary electrophoresis(NACE)was developed.Optimum separation of the analytes was obtained on a 50cm×50μm i.d.fused-silica capillary using a non-aqueous buffer system of 70mM ammonium acetate,7.0% acetic acid and 10% acetonitrile at 25kV and 20℃.The relative standard deviations(R.S.D.)of the migration times and peak areas of the three active components were 0.06%-0.20% and 0.12%-3.41% for berberine,0.11%-0.60% and 0.74%-1.63% for matrine,0.15% and 0.45% for oxymatrine,respectively.Detection limits of berberine,matrine and oxymtrine were 0.18μg/mL,4.08μg/mL and 4.16μg/mL,respectively.In the tested concentration range,good linear relationships(0.9992 for berberine,0.9988 for matrine and 0.9988 for oxymatrine)were observed.The linear calibration ranges were 0.45-360.0μg/mL for berberine,8.16-408.0μg/mL for matrine and 20.8-416.0μg/mL for oxymatrine.This method has been successfully applied to the phytochemical analysis of alkaloids extracts from two commonly used traditional Chinese herbal drugs:Sophora flavescens Ait.(Kushen)and Cortex phellodendri chinensis(Huangbai)and their medicinal preparations.

Effects and Mechanism of Oxymatrine on Proliferation,Invasion and Migration of Hepatocellular Carcinoma Cells

[Objectives] To observe the effects of oxymatrine on the proliferation,invasion and migration of hepatocellular carcinoma cells,and to explore its possible molecular mechanism.[Methods]The hepatocellular carcinoma cell line Hep G2 was randomly divided into the negative control group and the low,medium and high concentration groups of oxymatrine.The negative control group was added to the cell culture medium,and the low,medium and high concentration groups of oxymatrine were added to the oxymatrine and cell culture medium with the final concentration of 1.0,2.0 and 4.0 mg/m L.The proliferation of each group was measured by MTT assay,and the inhibition rate of cell proliferation was calculated.The invasion and migration ability of each group was determined using Transwell chamber assay.The expression of E-cadherin and CD44 mRNA in each group was detected using Real-time PCR,while the expression of E-cadherin and CD44 protein was detected using Western blotting method.[Results] The inhibition rate of cell proliferation in the high concentration group of oxymatrine was higher than that in the medium and low concentration groups,and the inhibition rate of cell proliferation of medium concentration group was higher than that in the low concentration group.In the cell invasion and cell migration experiments,the number of transmembrane cells of low,medium and high concentration groups of oxymatrine was less than that in the negative control group(P < 0.05),the number of transmembrane cells of high concentration groups of oxymatrine was less than that in medium and low concentration groups,and the number of transmembrane cells of medium concentration group was less than in that of the low concentration group(P < 0.05).Compared with the negative control group,the expression of E-cadherin mRNA and protein increased in the low,medium and high concentration groups of oxymatrine,while the expression of CD44 mRNA and protein decreased(P < 0.05).[Conclusions] Oxymatrine has the effect of inhibiting the proliferation,invasion and migration of hepatocellular carcinoma cell line Hep G2 in vitro.The higher the concentration,the more significant the effect will be.The mechanism is possibly connected with the up-regulation of E-cadherin expression and down-regulation of CD44 expression.

Quantitative Analysis of Matrine and Oxymatrine in Sophora flavescens Extract and Its Biopesticides by UPLC

Successive cartridge clean-up method for the simultaneous determination of matrine and oxyma- trine in biopesticides containing Sophora flavescens extract was developed and validated by UPLC. The clean-up method was established with ENVI-Carb (0.5 g) and C18 SPE (0.5 g) cartridges for the bioactive alkaloid in biopesticides from S. flavescens, and the eluate was analyzed to quantify the matrine and oxymatrine by UPLC. The developed method was validated, and the recovery and LOQ of both materials were 105.0% and 103.6%, and 0.050 and 0.684 mg·kg-1, respectively. Of the twenty one samples, the total content of matrines were analyzed by using the developed method and the result showed the developed successive clean-up method could contribute to the manufacture and control of biopesticides including matrines, and can be ap- plied to the method development for the analysis of alkaloid materials in biopesticides.

Effect of oxymatrine on the p38 mitogen-activated protein kinases signalling pathway in rats with CCl4 induced hepatic fibrosis

Background Recent studies have suggested that p38 mitogen-activated protein kinases （MAPK） signalling pathway plays an important role in hepatic fibrosis. This study explored the antifibrotic effect of oxymatrine on tetrachloromethane induced liver fibrosis in rats and its modulation on the p38 MAPK signalling pathway. Methods One hundred and twenty healthy male Sprague-Dawley rats were randomly assigned to six groups： normal （n=20）, induced fibrosis （n=20）, colchicine （n=20） and three treatment groups of oxymatrine （n=20x3）. We obesrved changes in deposition of collagen, hyaluronic acid （HA）, laminin （LN）, collagen type IV （CIV）, procollagen III （PCIll） and hydroxyproline （Hyp）, a-smooth muscle actin （α-SMA） and phosphor-p38 （pp38）. Results The relative indicators of changes in histopathology, HA, LN, CIV, PCIII, Hyp, a-SMA and pp38 were raised significantly in the induced fibrosis group （P〈0.01 vs normal group）. The semiquantitative hepatic fibrosis staging scores of middle dose group and high dose group were decreased （P 〈0.05 and P 〈0.01 respectively vs the induced fibrosis group）, as was the average area of collagen in rats＇ liver, the concentrations of serum HA, LN, CIV, PCIII and liver tissue homogenate Hyp. The gene expression of α-SMA mRNA was considerably decreased in the treated animals, as was the protein espression of pp38 protein. Conclusions Oxymatrine is effective in reducing the production and deposition of collagen in the liver tissue of experimental rats in ways which relate to modulating the fibrogenic signal transduction via p38 MAPK signalling pathway.