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.

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..

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.

Screening of Anti-Newcastle Disease Virus Ingredients of Phyllanthus urinaria

[ Objective ] The aim of the study was to screen the antiviral ingredients of Chinese herbal medicine Phyllanthus urinaria in order to devel- op new drugs for the treatment of viral diseases of poultry. [ Method ] The whole grass of phyllanthus was respectively decocted in 75% ethanol （E） and pure water （PF）, and then the active ingredients were separately extracted in petroleum ether （PE）, ethyl acetate （EA）, and n-butyl alcohol （BU）. The extracts were inoculated on chicken embryo fibroblast （CEF） to observe their inhibitory effect on cytopathic effect （CPE） of Newcastle disease virus （NDV）, inoculated into chicken embryos to observe the changes of the hemagglutination titer of NDV, and inoculated in chickens to determine the mortality and evaluate their effect of immune protection. [ Result] The E -PE and E -BU extracts, especially E -BU extract, inhibi- ted CPE of NDV on CEF and significantly inhibited the proliferation of NDV in chicken embryo （P〈0.05） ; the survival rate of E-PE and E-BU group was extremely significantly higher than that of ribavirin group and the normal saline control （ P 〈0.01 ）, with E - PE group more higher. [ Condusion] The water extract of phyllanthus is less effective; E -PE extract and E -BU extract should effectively inhibit the CPE and proliferation in chicken embryo of NDV and also have better immune protection against NDV.

Two pyrrole acids isolated from Phyllanthus emblica L.and their bioactivities

An undescribed pyrrole acid,1-(4′-methoxy-4′-oxobutyl)-1 H-pyrrole-2,5-dicarboxylic acid(1)and one known pyr-role acid(2)were isolated from the fruits of Phyllanthus emblica.The structures of these compounds were elucidated via the comprehensive analyses of IR,HRESIMS,1D and 2D spectroscopic data.A series of biological assays revealed that compounds 1 and 2 could inhibit LPS-induced over-production of nitric oxide(NO),interleukin-6(IL-6),monocyte chemotactic protein 1(MCP-1)and tumor necrosis factor-α(TNF-α)by reducing the phosphorylation of extracellular regulated protein kinases(ERK)and c-Jun N-terminal kinases(JNK)in RAW 264.7 cells.Additionally,compounds 1 and 2 were found to reduce lipid deposition and increase the mRNA expression of ATP-binding cassette transporter A1 in oxidized low-density lipoprotein-treated RAW264.7 macrophages.

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.

HPLC指纹图谱结合4种指标含量测定的余甘子质量评价研究

建立余甘子(Phyllanthus emblica L.)HPLC指纹图谱,并对4种化学成分的含量进行测定,为余甘子是否去核使用提供了一定参考。采用CAPCELL PAK C_(18)色谱柱,以乙腈-0.1%磷酸水溶液作为流动相,检测波长273 nm,流速为1.0 mL/min,柱温为30℃,进样量10μL,建立了云南32个不同产地余甘子的指纹图谱。通过对照品比对并结合光谱分析,对共有峰进行鉴定;借助中药色谱指纹图谱相似度评价系统对32批产地的余甘子的图谱进行相似度评价,通过聚类分析(HCA)、主成分分析(PCA)比较两种规格余甘子的差异,寻找差异性成分;并对两种规格余甘子药材4种有效成分含量进行对比研究。32批余甘子指纹图谱中有17个共有峰,指认出其中4个成分,相似度分析均大于0.90,对其进行聚类分析,有3个主成分,可聚为6类。对没食子酸、鞣花酸、柯里拉京、诃子酸定量分析,4个化学成分在各自范围内线性良好(r≥0.9999),精密度、重复性、稳定性试验均符合定量要求,加标回收率为98.77%~100.92%,RSD均小于2.0%(n=9)。含量测定结果显示,有核余甘子中没食子酸、柯里拉京、鞣花酸的含量均高于无核余甘子,而无核余甘子中诃子酸高于有核余甘子。所建立的方法简便、稳定、可靠,HPLC指纹图谱结合多指标成分含量测定可用于余甘子药材质量评价,为余甘子是否去核使用,提供了一定参考。

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.

余甘子(Phyllanthus emblica L.)Vc含量分析

采用2,6-二氯酚靛酚滴定法分析比较了余甘子各器官之间、同品种不同植株之间、不同余甘子品种之间以及与野生资源之间的Vc含量以及不同预处理方法对Vc测定的影响。结果表明,Vc在余甘子各器官中均有分布,以果实与初生幼叶中居多,茎、根中含量较低;皇帝甘与粉甘含量较多,但与其它品种的差异不大;5个野生资源单株Vc含量差异极显著,其中有3个野生单株的果肉Vc含量高于主栽品种粉甘。

余甘(Phyllanthus emblica L.)大小孢子发生及雌雄配子体发育研究

采用常规石蜡切片技术,对平丹1号余甘大小孢子发生及雌雄配子体发育进行研究。结果表明:药壁发育为双子叶型;绒毡层为腺质绒毡层;小孢子母细胞减数分裂时胞质分裂为同时型;产生四面体形四分体;成熟的花粉粒为二细胞型花粉;平丹1号余甘的雌蕊为上位子房,3心皮3室,每室含倒生胚珠2枚,为厚珠心,具珠心喙,胚囊为蓼形;平丹1号余甘部分大孢子母细胞、大孢子及胚囊发育异常,致使不能形成正常的卵细胞,不能进行受精。

Phyllanthus acidus(L.) Skeels and Rhinacanthus nasutus(L.) Kurz leaf extracts suppress melanogenesis in normal human epidermal melanocytes and reconstitutive skin culture

Objective: To determine the effect of extracts from Phyllanthus acidus(P. acidus)(L.) Skeels and Rhinacanthus nasutus(R. nasutus)(L.) Kurz leaves on melanogenesis and the underlying mechanism in normal human epidermal melanocytes(NHEM) and a reconstitutive skin model. Methods: NHEM and a reconstitutive skin model were stimulated with ethanol extracts of P. acidus(L.) Skeels and R. nasutus(L.) Kurz leaves. mRNA expression of microphthalmiaassociated transcription factor(MITF), tyrosinase(TYR), tyrosinase-related protein 1(TYRP1) and dopachrome tautomerase(DCT) were examined by real-time PCR. The melanin content in NHEM was also measured. Moreover, protein levels of tyrosinase were determined using western blot analysis.Results: In NHEM and the reconstitutive skin model, ethanol extracts from P. acidus(at 12.5 and 25.0 μg/mL) and R. nasutus(at 6.25 and 12.50 μg/mL) significantly diminished mRNA expression of MITF, TYR, TYRP1 and DCT in a concentration-dependent manner. P. acidus and R. nasutus extracts also reduced the amount of melanin in α-MSH-stimulated NHEM. Moreover, P. acidus and R. nasutus extracts markedly suppressed tyrosinase at the translational level in the reconstitutive skin model. Conclusions: P. acidus and R. nasutus extracts significantly reduced melanogenesis in NHEM and the reconstitutive skin model, suggesting that P. acidus and R. nasutus extracts can inhibit melanin synthesis through downregulation of MITF, TYR, TYRP1 and DCT. Therefore, the ethanol extracts of P. acidus and R. nasutus contain compounds that have the potential for development as a skin lightening agent for the treatment of hyperpigmentation disorder or melasma.

Development of species-specific SCAR markers for identification of three medicinal species of Phyllanthus

Phyllanthus amarus Schum. & Thonn. has been widely used in traditional medicine in Thailand as an antipyretic, a diuretic, to treat liver diseases and viral infections. Two closely related species, P. debilis L. and P. urinaria Klein ex Willd., with different and less effective medicinal properties, are less commonly used. These three species are similar in morphology and often occur in overlapping populations in nature. The latter two species can easily be mistaken for P. amarus and collected for medicinal uses, which can lead to undesirable results. DNA fingerprints of these species were obtained using RAPD-PCR techniques. RAPD markers specific for each species were identified. Primers for highly specific sequence-characterized-amplified-regions (SCAR) were then designed from nucleotide sequences of specific RAPD markers. These primers efficiently amplified SCAR markers of 408, 501 and 319 bp unique to P. amarus, P. debilis and P. urinaria respectively. This method of plant identification was rapid and highly specific when tested against DNA of several closely related species and was able to amplify specific markers from mixed DNA samples.

Effect of Total Flavonoids from Phyllanthus emblica L. on Tumor Proliferation and Immune Function

[Objectives]To explore the effect of total flavonoids from Phyllanthus emblica L.on tumor proliferation and immune function.[Methods]The effects of total flavonoids of P.emblica L.on the proliferation of 6 different tumor cell lines(human hepatoma cell line HepG-2,human cervical cancer cell line Hela,human gastric cancer cell line SGC7901,human nasopharyngeal carcinoma cell line CNE-2,human lung cancer cell line H460 and human ovarian cancer cell line A2780)were compared by SRB method.The effect of total flavonoids from P.emblica L.on the proliferation of mouse lymphocytes induced by concanavalin(ConA)or lipopolysaccharide(LPS)in vitro was detected by CCK-8 method.[Results]The results of SRB assay showed that compared with the normal group,the total flavonoids of P.emblica L.had obvious inhibitory effect on 6 kinds of tumor cells.Among them,the inhibitory effect on H460 cells and CNE-2 cells was the most significant,and the IC50 was(471.36±50.66),(463.26±40.75)μg/mL,respectively.The high dose group of total flavonoids from P.emblica L.had the same inhibitory effect as the positive drug 5-Fu.The results of CCK-8 assay showed that compared with the blank group,the total flavonoids of P.emblica L.significantly inhibited the proliferation of mouse lymphocytes induced by ConA or LPS(*P<0.05,**P<0.01).[Conclusions]The total flavonoids of P.emblica L.had significant anti-tumor activity.

余甘子斑点病病原菌鉴定、生物学特性及防治药剂筛选

【目的】明确余甘子果实斑点病的病原菌种类,了解其生物学特性,筛选有效的防治药剂,为余甘子果实斑点病的科学诊断和田间防治提供理论依据。【方法】从广东省汕尾市陆河县余甘子产业园种植基地采集发生斑点病的余甘子果实,采用组织分离法对余甘子斑点病病果进行病原菌分离,通过形态学特征和基于多基因(ITS、TUB和EF1-α)的系统发育进化分析对病原菌进行鉴定;采用针刺接种法测定病原菌菌株的致病力;采用菌丝生长速率法测定不同培养条件对病原菌菌丝生长的影响;通过室内毒力测定评估12种常见杀菌剂对病原菌的抑菌活性。【结果】从余甘子病果中分离出1株病原真菌菌株PE3,致病性测定结果表明,该菌株为余甘子斑点病致病菌,其形态特征与间座壳菌Diaporthe phoenicicola相似,且其ITS、TUB和EF1-α基因系统发育进化树分析显示与D.phoenicicola聚在同一分支。生物学特性测定结果显示,菌株PE3菌丝生长的最适温度为25℃,最适p H为7,最适生长碳源为甘油和蔗糖,最适生长氮源为酵母提取物。室内毒力测定结果显示,450 g/L咪鲜胺乳油对菌株PE3菌丝生长的抑制作用最强,抑制中浓度(EC_(50))为0.019 mg/L;其次是8%氟硅唑微乳剂、10%苯醚甲环唑微乳剂和25%吡唑醚菌酯悬浮剂,EC_(50)分别为0.023、0.124和0.194 mg/L;而2%春雷霉素水剂对菌株PE3菌丝生长的抑制效果最差,EC_(50)为402.336 mg/L。【结论】间座壳菌D.phoenicicola是引起余甘子果实斑点病的病原菌,450 g/L咪鲜胺乳油、8%氟硅唑微乳剂、10%苯醚甲环唑微乳剂和25%吡唑醚菌酯悬浮剂能有效防治余甘子果实斑点病,可在余甘子种植中推广应用。

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。

苦味叶下珠提取物对人肝癌细胞株PLC/PRF/5产生HBsAg的影响

实验表明较高浓度(4mg/ml)的叶下珠对人肝癌细胞株PLC/PRF/5细胞株细胞外HBsAg含量有明显的抑制作用,且主要影响HBsAg在该细胞内的合成。叶下珠与阿糖腺苷联合有一定的协同作用。

HPLC法测定叶下珠中没食子酸的含量

目的：建立测定叶下珠中没食子酸的HPLC定量法。方法：采用高效液相色谱法。以SHIMADZUVP-ODS（25mm×4.6mm，5μm）为色谱柱；流动相：甲醇-0．1％磷酸溶液（4：96）；流速：1．0mL／min；检测波长：215砌；结果：没食子酸在0．0214—0．2140μg范围内。浓度与峰面积线性关系良好（r=0．9999）。平均回收率为：100．74％。结论：方法简便、快速准确、灵敏度高，重现性好。可作为叶下珠的含量测定法。

RP-HPLC法同时测定叶下珠中没食子酸、柯里拉京和短叶苏木酚的含量

目的建立反相高效液相色谱法同时测定叶下珠中没食子酸、柯里拉京和短叶苏木酚的含量。方法采用Kromasil C18色谱柱(250 mm×4.6 mm,5μm),流动相为乙腈-体积分数0.1%磷酸水溶液梯度洗脱,流速1.0 mL.min-1,检测波长为280 nm,柱温30℃。结果没食子酸、柯里拉京和短叶苏木酚的质量浓度分别在5.300～106.7 mg.L-1(r=0.999 8)、2.00～40.6 mg.L-1(r=0.999 7)、0.270～5.42 mg.L-1(r=0.999 7)内与峰面积呈良好的线性关系。没食子酸、柯里拉京和短叶苏木酚的平均加样回收率(n=9)分别为97.9%、101.7%、100.2%,RSD分别为1.8%、1.8%、2.7%。结论该分析方法准确可靠,重复性好,为更好地控制叶下珠药材的质量提供方法。