Network pharmacology and experimental validation to reveal the anti-hepatitis B virus pharmacological mechanism of Phyllanthus urinaria L.

Background:To explore the pharmacological mechanism of the anti-hepatitis B virus of Phyllanthus urinaria L.through network pharmacological analysis and experimental validation.Method:The active ingredient,target of action and target of action related to hepatitis B were clarified by searching the herb group identification,GeneCards and OMIM databases,and the protein interaction relationship was obtained by using the String database,and the protein interaction network map was constructed by using Cytoscape software.We also performed gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis of key targets of the anti-hepatitis B action of Phyllanthus urinaria L.and predicted the core targets and pathways of Phyllanthus urinaria L.anti-hepatitis B.The main targets predicted by network pharmacology were then validated by HepG2.2.15 cell experiments.Results:By searching active ingredient targets and hepatitis B disease targets,a total of 19 active ingredients and 64 related targets of action were retrieved from Phyllanthus urinaria L.,and a total of 51 common targets were obtained by mapping the obtained hepatitis B disease targets and drug targets.protein protein interaction network analysis indicated that targets including TNF,JUN,AKT1,IL-10,IL-1B,CAT,HMOX1,NFE2L2,and CASP3 and other targets may be the core targets.gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that the treatment of hepatitis B by Phyllanthus urinaria L.mainly included inflammation and oxidation-related processes,and the signaling pathways mainly included fluid shear stress and atherosclerosis,VEGF,and hepatocellular carcinoma.The results of the in vitro test showed that after the action of different concentrations of the extracts of the Phyllanthus urinaria L.in the safe concentration range on cells HepG2.2.15,HBsAg,HBeAg and hepatitis B virus DNA levels were significantly inhibited,and NFE2L2 and HMOX1 were affecting hepatitis B virus transcription and replication by regulating the oxidative stress response.Conclusion:Using an integrated network pharmacology approach,this study revealed the active components and potential targets of Phyllanthus urinaria L.for the treatment of the hepatitis B virus,providing a theoretical basis for the research and clinical application of Phyllanthus urinaria L..

Suppressed effects of Phyllanthus urinaria L.ethyl acetate extract on hepatitis B virus both in vitro and in vivo

Background:Phyllanthus urinaria L.(P.urinaria)extract(PUE)has been used to inhibit hepatitis B virus(HBV).However,the underlying mechanism remains unclear.To investigate which PUE fractions and main components lead to against HBV and approach the relevant molecular mechanisms.Methods:P.urinaria was extracted with water,and then the decoction was extracted by petroleum ether,ethyl acetate,and n-butanol in turn.The HepG2.2.15 cell was treated with aqueous fraction,petroleum ether fraction,ethyl acetate fraction and n-butanol fraction,gallic acid(GA,C7H6O5)and corilagin(CL,C27H22O18),respectively.The medium was collected for hepatitis B surface antigen(HBsAg)and hepatitis B e antigen assays.Cell counting kit-8 method was used to identify cell proliferation.Also,the levels of cellular oxygen consumption,reactive oxygen species,and reduced glutathione were detected.The HBV modeling mice were treated with ethyl acetate fraction,entecavir and physiological saline,respectively.The serum was collected for HBsAg and inflammatory cytokines assays.Liver tissue metabolites were screened by LC-MS/MS method.Results:The ethyl acetate fraction(EAF)of P.urinaria could significantly inhibit HBV secretion in HepG2.2.15(P<0.05).Furthermore,two main constitutes in ethyl acetate fraction,GA and CL,could significantly inhibit HBV secretion and reduced cell proliferation(P<0.05).Also,GA and CL could increase cellular oxygen consumption,intracellular superoxide anions level,superoxide dismutase level and glutathione depletion.Compared with the Modeling group,EAF significantly decreased the expression levels of HBsAg,IL-1β,IFN-α(P<0.05).LC-MS/MS analysis results showed that EAF dramatically up-regulate hydroxyproline,maltotriose,betaine and down-regulate glutathione disulfide,taurocholate,taurochenodeoxycholate(P<0.05).Kyoto Encyclopedia of Genes and Genomes results show that the differential metabolites were mainly enriched in ATP-binding cassette transporters pathway.Conclusions:P.urinaria exhibits suppressed effects on HBV by modulating reactive oxygen species formation or metabolomics both in vitro and in vivo.These data indicate that P.urinaria may be an alternative therapeutic agent for the treatment of HBV-related hepatitis.

Progress in Pharmacological Research of Phyllanthus urinaria

Phyllanthi Urinariae Herba is dry grass of Phyllanthus urinaria,and has the effects of calming liver,clearing heat,diuresis and detoxification in traditional Chinese medicine. Studies have shown that the effects of anti-hepatitis B virus,hepatoprotective,anti-tumor,antipathogenic microorganism,anti-oxidation,and anti-thrombosis were good and worthy of further research and development. The research progress on the pharmacological effects of P. urinaria was reviewed in order to provide a reference for the rational development of this product.

Neuroprotective effect of Phyllanthus urinaria extract on ischemic brain injury in rats

Objective:To investigate the neuroprotective effect of Phyllanthus urinaria on ischemic stroke and its primary mechanism.Methods:Adult SD rats were selected as the research object,and the right middle cerebral artery infarction rat model was established by the modified suture method(MCAO).Observe the neurological deficit score at 24h,48h and 72h after the model is successfully prepared,then TTC staining method to detect the area of cerebral infarction,the content of superoxide dismutase(SOD),nitric oxide(NO)and endothelial NOS(eNOS)in brain tissue;Immunofluorescence method was used to detect the expression of Caspase-3 positive cells in brain tissue;Western blot method was used to detect the expression of PI3K and AKT protein in brain tissue.72 experimental animals were randomly divided into 4 groups,sham operation group,model group(MCAO),extract of Phyllanthus urinaria low dose group(PuL5 g/kg),high dose group(PuL10 g/kg),and make MCAO model after 7 days of continuous administration,and continue to infuse the medicine once/d until the material is obtained.Results:No neurological deficits in the sham operation group,The 24h,48h and 72h of the modelling showed that the neurological impairment of the two doses of extract of Phyllanthus urinaria and the MCAO group was more severe than that of the sham operation group(P<0.01),however,with the prolongation of the modeling time,the neurological function scores of the two doses of extract of Phyllanthus urinaria were lower than those of the MCAO group,the most significant at 72h(P<0.01);The infarct size of the rats in the two dose groups of extract of Phyllanthus urinaria was lower than that of the MCAO group(P<0.01),and there was no dose dependence between the two groups,the content of SOD in the MCAO group was reduced,and the content of NO and eNOS was increased than the sham operation group(P<0.05),Compared with the MCAO group,the two administration groups significantly increased the content of SOD,decreased the content of NO and eNOS(P<0.05);Although the expression of Caspase-3 positive cells in the two administration groups was higher than that in the sham operation group,it was significantly lower than that in the MCAO group(Plow<0.05,Phigh<0.01);The expression of PI3K and AKT protein in the brain tissue of the MCAO group was significantly lower than that of the sham operation group(P<0.05),but the expression of PI3K and AKT protein in brain tissue of extract of Phyllanthus urinaria in high and low dose groups was significantly higher than that in MCAO group(P<0.05).Conclusion:Extract of Phyllanthus urinaria can improve neurological damage and cerebral infarction area in rats,and reduce the expression of Caspase-3 positive cells in MCAO rats,and then increase the expression levels of PI3K and AKT proteins to protect ischemic brain injury.

The anti-HIV-1 activity of polyphenols from Phyllanthus urinaria and the pharmacokinetics and tissue distribution of its marker compound, gallic acid

Objective:To investigate the in vitro anti-HIV-1 activities and its associated mechanism of action of an extract isolated from Phyllanthus urinaria (P.urinaria) and to develop an HPLC test method for detecting gallic acid (GA) in plasma and tissues to study its pharmacokinetics and tissue distribution in rats.Methods:An extract of P.urinaria was isolated and purified by phytochemistry and chromatography techniques.The anti-HIV-1 activities and toxicities of the extract and its component GA were determined in human T lymph cells (MT-4) by theMTTr method.The mechanism of its anti-HIV-1 action was studied to examine the in vitro binding of its components with HIV-1 target proteins by Biacore technique.The pharmacokinetics and tissue distribution of GA were investigated after oral administration of polyphenol extract (PE) and pure GA in rats.The concentrations of GA in plasma and tissues were determined by HPLC.Results:The PE and GA isolated from P.urinaria had anti-HIV-1 activities with IC50s of 0.61 μg/mL and 0.76 μg/mL,respectively.The Biacore study indicated that PE and GA interacted with HIV-1 RT,gp120,and P24.The pharmacokinetic parameters Tmax,Cm ax,AUC0-t,and T1/2 for GA were (60.0 ± 3.0) minutes,(2.87 ± 0.50) μg·mL-1,(343.5 ± 11.2) mg·min·L-1,and (113.3 ± 9.3) minutes while the parameters for GA in the PE were (10.0 ± 1.3) minutes,(3.89 ± 0.90) μg·mL-1,(394.7 ± 14.0) mg· min· L-1,and (81.7 ± 4.1) minutes,respectively.GA was detected in rat lungs,liver,kidneys,heart and spleen.Conclusion:APE isolated from P.urinaria containing GA has anti-HIV-1 activities.GA is quickly absorbed and slowly eliminated in rats after oral administration.The pharmacokinetics of GA administered as a PE is desirable,and it is widely distributed in the main tissues of lung and liver.Both its properties and anti-HIV-1 activities make it of interest for further studies.

叶下珠黄化植原体和丛枝植原体的分子鉴定

2022年8月在海南省文昌市一个槟榔黄化病园内,分别发现叶片黄化和丛枝症状的叶下珠,前期检测均为植原体感染。为了明确叶下珠黄化植原体和丛枝植原体的分类鉴定,本研究通过克隆16S rDNA基因和核糖体蛋白(rp)基因,并进行基因序列一致性、系统发育树及虚拟RFLP等分析。结果显示:克隆获得叶下珠黄化植原体16S rDNA片段1246 bp,rp基因1212 bp;叶下珠丛枝植原体16S rDNA片段1827 bp,rp基因1240 bp。基因序列一致性显示,叶下珠黄化植原体与丛枝植原体的16S rDNA基因序列均与16SrⅠ组的植原体一致性高达98%以上,与槟榔黄化植原体海南株系的16S rDNA序列一致性达100%;而二者的rp基因序列均与rpⅠ组的植原体一致性高达99%以上。系统发育树分析显示,叶下珠黄化植原体和丛枝植原体16S rDNA基因均与翠菊黄化组(16SrⅠ)植原体聚于一个大分支,且与16SrⅠ-B亚组的槟榔黄化植原体聚于同一个小分支,亲缘关系接近;而rp基因均与翠菊黄化组(rpⅠ)植原体集聚于一个大分支,且与rpⅠ-B亚组的翠菊黄化植原体聚于同一个小分支,亲缘关系接近。虚拟RFLP分析显示,叶下珠黄化植原体和丛枝植原体的16S rDNA基因序列虚拟RFLP图谱与16SrⅠ-B的洋葱黄化植原体的参考图谱相同,且相似系数为1.00。综上表明,叶下珠黄化植原体和丛枝植原体均属于16SrⅠ-B亚组成员。研究结果可对采用铲除槟榔黄化病中间寄主的防控手段提供理论依据。

Liver metabolomics study reveals protective function of Phyllanthus urinaria against CCl_4-induced liver injury

Phyllanthus Urinaria L.(PUL) is a traditional Chinese medicine used to treat hepatic and renal disorders. However, the mechanism of its hepatoprotective action is not fully understood. In the present study, blood biochemical indexes and liver histopathological changes were used to estimate the extent of hepatic injury. GC/MS and LC/MS-based untargeted metabolomics were used in combination to characterize the potential biomarkers associated with the protective activity of PUL against CCl_4-induced liver injury in rats. PUL treatment could reverse the increase in ALT, AST and ALP induced by CCl_4 and attenuate the pathological changes in rat liver. Significant changes in liver metabolic profiling were observed in PUL-treated group compared with liver injury model group. Seventeen biomarkers related to the hepatoprotective effects of PUL against CCl_4-induced liver injury were screened out using nonparametric test and Pearson's correlation analysis(OPLS-DA). The results suggested that the potential hepatoprotective effects of PUL in attenuating CCl_4-induced hepatotoxicity could be partially attributed to regulating L-carnitine, taurocholic acid, and amino acids metabolism, which may become promising targets for treatment of liver toxicity. In conclusion, this study provides new insights into the mechanism of the hepatoprotection of Phyllanthus Urinaria.

Extract from Phyllanthus urinaria L.Inhibits Hepatitis B Virus Replication and Expression in Hepatitis B Virus Transfection Model in Vitro

Objective： To explore the effects of the extract from Phyllanthus urinaria L. on hepatitis B virus （HBV） replication and expression in HBV transient transfection model in vitro. Methods： The eukaryotic expression plasmid pHBVI.1, which contains l.l-fold-overlength genome of HBV, was transfected into the human hepatoma cell line, HepG2, to establish and assess the HBV transient transfection model. The extract from Phyllanthus urinaria L. was prepared in different concentrations and methyl thiazolyl tetrazolium was used to detect the maximum nontoxic concentration of the drug. The extract from Phyllanthus urinaria L. were added into the transfected cell, at the concentrations of 0.8, 0.2 and 0.05 g/L, respectively. Four days after drug application, enzyme-linked immuno sorbent assay was used to detect the concentration of HBsAg in the supernatants, Southern blot was applied to analyze HBV DNA level, and Western blot was used to detect the expression of HBcAg in cells. Results： After the transfection of plasmid pHBVI.1 into HepG2 cells, the concentration of HBsAg in supernatants was increased obviously as compared with that of the normal cells （P〈0.05）, and all expected HBV replicative intermediates were confirmed by Southern blot analysis, which ensured the successful establishment of the HBV transient transfection model. After the application of drugs at the concentrations of 0.8 and 0.2 g/L, the level of HBsAg was obviously decreased in the supernatants, as compared with that of the virus group （P〈0.05）; Southern blot showed that the level of HBV rc DNA, ds DNA, ss DNA was obviously reduced compared with that of the virus group （P〈0.01）; Western blot revealed that the expression of HBcAg in the drug group was obviously inhibited, as compared with that of the virus group （P〈0.01）. Conclusions： The extract from Phyllanthus urinaria L. obviously inhibited replication and expression of HBV in HBV transfected cell lines in vitro, thus exerting distinctive anti-HBV effects.

Efficacy of Early Treatment on 52 Patients with Preneoplastic Hepatitis B Virus-Associated Hepatocellular Carcinoma by Compound Phyllanthus Urinaria L.

Objective:To observe the change in the number of antibodies of preneoplastic hepatocellular carcinoma(HCC) using early treatment by Compound Phyllanthus Urinaria L.(CPUL) on patients with preneoplastic hepatitis B virus(HBV)-associated HCC.Methods:A total of 102 cirrhosis patients with regenerative or dysplastic nodules whose sera were tested positive for at least one of these six proteins(five up-regulated genes URG4,URG7,URG11,URG12 and URG19,and one down-regulated gene DRG2) were assigned randomly to two groups using continual random codes by SPSS software.Fifty-two patients were in the treatment group and 50 patients were in the control group.CPUL was used in the treatment group for 3 years,while the control group did not receive any treatment.The changes in HBV-DNA level,number of antibodies,and hepatocarcinogenesis occurred were observed.Patients who did not develop HCC were followed up for another 2 years.Results:HBV-DNA levels decreased >2log in 22.2%(10/45) of patients in the treatment group in contrast to only 5.0%(2/40) of patients in the control group(P=0.0228).The number of antibodies that were tested positive in the treatment group(1.08± 1.01)was significantly lower compared with the control group(2.11 ±1.12) after 24 months of drug treatment(P<0.01).Both the positive rates of anti-URG11(33/52) and anti-URG19(31/52) were over 60%at baseline in the two groups,and were decreased to 48.1%(25/52) and 46.2%(24/52) respectively at 36 months of drug treatment,while the rates increased to 68.0%(34/50) and 66.0%(33/50) respectively(P=0.0417,P=0.0436) in the control group.The positive rate of anti-DRG2 was increased to 55.8%(29/52) at 36 months of drug treatment,while in the control group was decreased to 36.0%(18/50,P=0.0452).Among the 102 patients who developed HCC,2 were in the treatment group and 9 were in the control group,meaning that a significant difference between the two groups(P=0.0212).In11 patients who developed HCC,anti-URG11 and anti-URG19 were always positive,while anti-DRG2 was negative.Patients newly developing HCC were 6(20.0%) in the control group,and only one(2.5%) in the treatment group(P=0.0441) during 2-year follow-up after the end of the treatment.Conclusions:Anti-URG11,anti-URG19 and antiDRG2 could be used as early markers in the prediction of the therapeutic efficacy of CPUL in treating preneoplastic HCC.CPUL is useful in preventing or delaying the development of HBV-associated cirrhosis to HCC.

叶下珠饮片质量标准优化研究

目的:优化叶下珠饮片的质量标准。方法:采用薄层色谱(TLC)法对叶下珠饮片进行定性鉴别;采用电子显微镜观察叶下珠饮片粉末及横切面特征;采用高效液相色谱法测定没食子酸、柯里拉京的含量;参照2020年版《中华人民共和国药典》测定叶下珠饮片中水分、总灰分、酸不溶灰分和醇溶性浸出物的含量。结果:TLC分析中,叶下珠饮片与对照药材在相近位置均显示紫红色斑点。显微鉴别中,饮片粉末、横切面所含各种组织结构均可作为鉴别依据。含量分析中,没食子酸的线性回归方程为Y=15068X-14509(r=0.9998),含量为0.0110%~0.2582%;柯里拉京的线性回归方程为Y=38265X-7032(r=0.9999),含量为0.1267%~0.3291%;水分、总灰分、酸不溶灰分及醇溶性浸出物含量分别为9.49%~11.29%、6.02%~9.92%、0.64%~1.67%、15.73%~30.25%。结论:该质量标准可为准确、全面控制叶下珠饮片质量提供技术支持。

Triterpenoids from Whole Plants of Phyllanthus urinaria

Objective To study the chemical constituents of whole plant of Phyllanthus urinaria and their biological activity.Methods The chemical constituents were isolated and purified by repeated column chromatography over silica gel,Rp-C18（reverse phase）,MCI,and Sephadex LH-20,as well as semi-preparative HPLC.NMR spectroscopic analyses were used for the structure identification.In this case,the inhibitory rate of NO production of the isolated triterpenoids was evaluated.Results Seven triterpenoids,identified as28-norlup-20（29）-ene-3,17β-diol（1）,betulin（2）,β-betulinic acid（3）,3-oxofriedelan-28-oic acid（4）,oleanolic acid（5）,3R-E-coumaroyltaraxerol（6）,and 3R-Zcoumaroyltaraxerol（7）,were isolated and identified from the whole plants of P.urinaria.Compounds 1-5 exerted inhibitory effects against NO production in LPS-induced RAW264.7 mouse macrophages with the inhibitory rate of NO production ranging from 4.0%to 27.8%at the concentration of 25 μmol/L.Conclusion To the best of our knowledge,this is the first report of compounds 1-4,6,and 7 from the family Euphorbiaceae.Compounds 1-5 exhibited inhibitory effects against NO production in LPS-induced RAW264.7 mouse macrophages.

叶下珠的有效部位和药理作用研究进展

叶下珠是临床常用治疗癌症的中药材,其发挥作用的有效部位包括水提物、醇提物、叶下珠生物碱、叶下珠多糖等。除显著的抗肝癌、肺癌、胃癌、乳腺癌等抗癌作用,叶下珠还具有抗病毒、抗氧化、抑菌、抗炎等药理作用。结合近5年的研究成果,文章对上述叶下珠有效部位和药理作用进行分析整理,同时提出今后关于叶下珠研究的建议,以此反映最新的叶下珠研究成果,为叶下珠临床应用提供理论依据,也为研究者下一步工作提供参考。

叶下珠有效成分提纯工艺和质量控制研究进展

叶下珠主要成分包括黄酮类、酚酸类、木脂素类、生物碱类及多糖等,具有清热解毒、利水消肿、明目消积的功效,以及抗菌、抗氧化、抗病毒、抗血栓、提高脾细胞活性、长期使用毒副作用小的特点。临床上,叶下珠联合其他药物治疗乙型肝炎、脂肪肝效果理想。由于药效明确,开发利用前景良好,关于叶下珠各类成分提取利用和质量控制方面的研究及技术不断革新。为提供叶下珠药材开发利用的参考依据,本研究结合相关文献,综述了叶下珠部分有效成分的提纯工艺和近10年来质量控制方面的研究,发现(1)对于叶下珠中总酚类的提取,超声法适用于工业化生产,而柯里拉京的提取方面,膜过滤技术和柱色谱分离技术最有望用于工业生产;其他成分的提取工艺目前主要以传统方法为主,新方法处于实验探索阶段。(2)尽管有关叶下珠药理作用的研究较多,但是单个活性成分的构效关系研究较少,不利于进一步开发新药。(3)质量控制多集中在含量测定和指纹图谱方面,对重金属和33种农残的研究未见报道;市场上仍存在部分易混品常见的有苦味叶下珠、珠子草等,目前仍缺乏简单有效的鉴别手段。

基于网络药理学探讨芪珠消癥方抗肝细胞癌的机制及PI3K/Akt途径验证

目的探讨芪珠消癥方治疗肝细胞癌(Hepatocellular Carcinoma,HCC)的潜在作用机制。方法分别通过5个中药数据库与2个疾病数据库筛选芪珠消癥方活性成分、作用靶点与HCC疾病靶点,运用网络药理学技术构建芪珠消癥方与HCC关系网络,并通过蛋白互作网络、通路富集分析、分子对接预测机制。采用实验验证芪珠消癥方含药血清及其关键成分槲皮素(Quercetin,QCT)对HCC干预作用,并检测QCT对磷脂酰肌醇3激酶/蛋白激酶B(PI3K/Akt)途径的影响。结果芪珠消癥方与HCC关系网络提示芪珠消癥方主要通过QCT、汉黄芩素等关键活性成分调控HCC的相关靶点。蛋白互作网络、通路富集分析显示芪珠消癥方可能通过AKT1、丝裂原活化蛋白激酶3(MAPK3)等关键靶点与PI3K/Akt、乙型肝炎、凋亡等重要信号途径发挥抗HCC作用。分子对接显示AKT1蛋白与QCT亲和力较好;实验结果表明,芪珠消癥方含药血清可有效地抑制HepG2细胞增殖并促进其凋亡,关键活性成分QCT以浓度相关的方式抑制HepG2细胞的增殖、克隆形成、迁移能力,促使细胞凋亡,并可降低磷酸化蛋白p-PI3K、p-Akt的表达。结论芪珠消癥方治疗HCC的主要机制包括以QCT、汉黄芩素为代表的多活性成分,以PI3K/Akt、乙型肝炎、凋亡代表的多途径的协同调节作用。

基于CNKI叶下珠研究文献计量学及可视化分析

目的:分析目前中国已发表的与中草药叶下珠研究相关的文献特征及研究现况。方法:在中国知识资源总库(CNKI)中检索1966年1月31日至2021年10月19日收录的叶下珠相关研究文献,运用文献计量学方法及CiteSpace软件从多方面进行分析。结果:纳入文献606篇,34%的文献受到了各级科研基金的资助;文献数量在2003年后迅速增长;文献发表最多的研究机构及期刊为广州中医药大学和《中西医结合肝病杂志》;发文量最多的团队是以李常青为核心的研究团队;文章内容多倾向于对疾病的治疗作用,且关键词分析显示研究方向集中在肝脏相关疾病治疗。结论:通过多方面分析,目前叶下珠的研究热点集中在探索肝脏相关疾病的的中药分子机制。旨在为叶下珠研究者提供一些信息,进而加快叶下珠研究的发展。

叶下珠水提物对裸鼠人肝癌移植瘤HBx和VEGFR3表达的影响

目的探讨乙型肝炎病毒x基因(hepatitis B virux x gene,HBx)和血管内皮生长因子受体3(vascular endothelial growth factor receptor 3,VEGFR3)基因与乙型肝炎相关肝癌的相关性,以及叶下株水提物(aqueous extract of phyllanthus urinaria L,AEP)对HBx和VEGFR3蛋白表达的干预作用。方法将60只Balb/c-nu裸鼠随机分为10组。分别复制转染HBx、氯霉素乙酞转移酶(chloramphenicol acetyltransferase,CAT)和VEGFR3基因的HepG2肝癌细胞的裸鼠皮下移植瘤模型,用AEP进行干预,以生理盐水、环磷酰胺(cyclophosphamide,CTX)作为对照,共治疗6周。观察模型复制后不同时间各组裸鼠的移植瘤生长状况,采用酶联免疫吸附试验检测各组裸鼠血清甲胎蛋白(alpha fetal protein,AFP)水平,采用Western blot方法检测移植瘤组织中HBx和VEGFR3蛋白表达水平。结果肝癌移植瘤模型复制成功。各组裸鼠体质量均随着时间显著增长。实验结束时,接种HepG2-HBx细胞的各组中,AEP处理组肿瘤质量显著低于CTX处理组(P<0.05),其AFP水平显著高于NS处理组(P<0.05),其移植瘤组织中HBx表达水平显著低于NS组(P<0.05)。接种相同肝癌细胞的各组中,AEP处理组VEGFR3表达水平最低,但与CTX组VEGFR3表达水平比较,差异无统计学意义(P>0.05);AEP组和CTX组VEGFR3表达水平显著低于NS组(P<0.05)。对于相同的处理因素,接种HepG-HBx细胞和HepG-CAT细胞的裸鼠移植瘤组织中VEGFR3表达水平均显著低于接种HepG-VEGFR3细胞的裸鼠(P<0.01)。结论 AEP通过直接抑制HBx蛋白和VEGFR3蛋白表达,及通过抑制HBx蛋白表达而间接抑制VEGFR3蛋白表达,达到对肝癌移植瘤生长的抑制作用。

复合酶在叶下珠有效成分提取中的应用

[目的]探索复合酶辅助提取叶下珠有效成分的工艺条件,为叶下珠的高效利用提供依据。[方法]以水为提取溶剂,以总黄酮提取得率为考察指标,选择料液比、提取温度、提取时间、纤维素酶浓度和果胶酶浓度等5因素4水平,用正交试验优选其最佳工艺条件。[结果]复合酶法提取叶下珠有效成分的最佳提取工艺为:提取时间2 h,料液比1∶25(g∶ml),提取温度40℃,纤维素酶浓度1.4 g/L,果胶酶2.0 g/L。在此条件下,黄酮的提取率为1.589%。试验所考察的5个因素中,对提取率影响的主次顺序为:提取温度>果胶酶浓度>料液比>提取时间>纤维酶浓度。[结论]复合酶法提取省时、高效、低碳耗且提取率高,是一种对环境友好的方法。

叶下珠化学成分及其抗乙肝病毒活性的研究

对大戟科叶下珠属植物叶下珠的干燥全草进行了化学成分及其抗乙肝病毒活性的研究，共分得１１个化合物，其中２个新化合物为去氢诃子次酸甲酯、短叶苏木酚酸甲酯。通过抗病毒、抗乙肝表面抗原及保肝试验。

叶下珠提取物对酪氨酸酶的抑制和抗氧化作用

叶下珠为传统民间草药,具有平肝清热、利水解毒之功效.以叶下珠为研究对象,经乙醇提取,溶剂极性递增分离法制备获得叶下珠植物成分,测定了叶下珠植物对酪氨酸酶的抑制作用,以及运用1,1-二苯基-2-苦苯肼(DPPH)自由基法检测其清除自由基的能力.结果表明:叶下珠乙酸乙酯提取物和正丁醇提取物对酪氨酸酶二酚酶有较强抑制作用,IC50值分别达到0.26和0.49mg/mL,动力学研究表明:叶下珠乙酸乙酯提取物和正丁醇提取物对酪氨酸酶抑制作用均是可逆的,其抑制类型为混合型抑制作用;叶下珠乙酸乙酯提取物和正丁醇提取物对DPPH自由基具有一定的清除作用,在较低的浓度下有较高的清除效果,IC50值分别为0.012和0.008mg/mL,说明叶下珠植物具有开发成天然酪氨酸酶抑制剂和抗氧化剂的潜力.

不同产地叶下珠及其联合用药抗鸭乙型肝炎病毒的实验研究

本文应用重庆麻鸭乙型肝炎动物模型进行了不同产地中草药叶下珠及其联合中药苦参及抗菌药环丙沙星的抗鸭乙型肝炎病毒（ｄｕｃｋｈｅｐａｔｉｔｉｓＢＶｉｒｕｓ，ＤＨＢＶ）疗效初探，连续用药１个月后，结果表明：广西叶下珠、云南叶下珠能使鸭血清中鸭乙型肝炎病毒脱氧核糖核酸（ＤＨＢＶＤＮＡ）滴度下降（Ｐ＜０．０５），而重庆叶下珠无此作用（ｐ＞０．０５）；云南叶下珠停药后有血清ＤＨＢＶＤＮＡ回升现象；广西叶下珠与苦参合用，无明显协同作用，但与环丙沙星合用，似有增强抗病毒的作用（Ｐ＜０．０５）。