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..

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.

Ideas and Recommendations for Development of Gauri Gvong Phyllanthus embica Linn Industry

Gauri Gvong Phyllanthus embica Linn is fine variety domesticated by Lujiang Town farmers in Longyang District through many years. It has high economic and ecological benefits and is a medicinal and edible fruit in hot-dry valley regions. Gauri Gvong Phyllanthus embica Linn planting ranks the first place in Baoshan City and even in the whole Yunnan Province. It introduced current situations of Gauri Gvong Phyllanthus embica Linn industry in Longyang District and came up with development ideas,objectives and recommendations.

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。

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.

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。

余甘子叶化学成分没食子酸对人肝癌BEL-7404细胞株凋亡的影响

目的:探讨余甘子叶化学成分没食子酸对人肝癌细胞株BEL-7404凋亡的影响。方法:采用MTT法测定余甘子叶化学成分没食子酸在体外对BEL-7404细胞增殖的影响;普通光学显微镜、倒置相差显微镜和荧光显微镜观察余甘子叶化学成分没食子酸作用BEL-7404细胞后的形态学变化;AnnexinV/PI双标记法流式细胞术检测余甘子叶化学成分没食子酸诱导BEL-7404细胞早期细胞凋亡的情况;Tunel法检测BEL-7404细胞的晚期凋亡情况及其凋亡率。结果:MTT法和显微镜观察显示余甘子叶化学成分没食子酸能不同程度抑制BEL-7404细胞的增殖,可致细胞形态学改变,出现典型的凋亡特征;AnnexinV/PI双标记法检测显示早期凋亡细胞随作用时间的延长而增加,Tunel法检测晚期凋亡细胞,其凋亡率亦随作用时间的延长而增大。结论:余甘子叶化学成分没食子酸能抑制BEL-7404细胞的增殖并诱导其产生凋亡,其对BEL-7404细胞的凋亡影响呈时间依赖性。

HPLC法测定叶下珠中豆甾醇、β-谷甾醇和羽扇豆醇

To establish an HPLC method for separation and determination of stigmasterol,β-sitosterol and lupeol in phyllanthus urinaria L by HPLC.A Eclipse Zorbax XDB-C18 column(4.6 mm×150 mm i.d.,5 μm) was used for chromatographic separation under the conditions with methanol as the mobile phase at flow rate of 0.7 mL/min.The detection wavelength was 210 nm.The results indicated that stigmasterol,β-sitosterol and lupeol could be baseline separated.The linear range of stigmasterol,β-sitosterol and lupeol were 0.4mg/mL～8.8 mg/mL(R2 were 0.9975),0.5～10.0 mg/mL(R2 = 0.9978) and 0.5mg/mL～10.0 mg/mL(R2 = 0.9981),respectively.The mean recoveries of stigmasterol,β-sitosterol and lupeol were 94.4%～95.1%,91.2%～97.9% and 92.4%～104.6 %,respectively.RSD was less than 5%.The method has been applied to monitor the extraction of stigmasterol,β-sitosterol and lupeol in phyllanthus urinaria L by super-critical fluid extraction with satisfactory.

3个余甘子优良无性系在广西贵港市种植表现及品质特性分析

为筛选出适合在广西贵港市种植的余甘子优良无性系,为余甘子良种培育和生产提供科学依据。以野生余甘子为对照,调查和分析“平丹一号”“平丹二号”“平丹三号”3个余甘子优良无性系在广西贵港市的种植表现及品质特性。结果表明,“平丹一号”和“平丹二号”在广西贵港市表现为早熟或中早熟,“平丹三号”为中熟;3个余甘子优良无性系丰产性较好,5年生树单株鲜果产量在46.10~50.95 kg之间,单果质量在9.32~10.54 g之间,都显著高于野生余甘子。不同余甘子优良无性系果实可溶性固形物含量在10.60%以上,果实单宁、维生素C、可溶性糖、可溶性蛋白等内含物含量较高。“平丹一号”“平丹二号”和“平丹三号”在广西贵港市综合表现较好,抗病性较强,可选择培育成良种并加以推广。

叶下珠药材HPLC指纹图谱的比较研究

采用反相高效液相色谱法,对叶下珠药材不同产地、不同采收期、不同部位指纹图谱进行测定和比较分析,并与中成药叶下珠胶囊的HPLC指纹图谱进行了比较研究。结果显示：（1）建立了叶下珠药材的HPLC特征指纹图谱,标定18个共有峰,利用对照品指认4个峰;11批（来源地不同）叶下珠样品的HPLC图谱相似度（相合系数,均值）在0.89～0.99之间。（2）7批不同采收期的叶下珠药材的HPLC指纹图谱相似度在0.94以上,各共有峰的峰面积大多随生长期而增加,至10月5日达到最高,建议叶下珠药材应于每年的10月上旬进行采收。（3）不同部位叶下珠药材HPLC指纹图谱相似度分析发现,叶、果的相似度较高（0.98～0.99）,根、茎的相似度较低（0.86～0.87）,说明根、茎中各成分含量较低,建议采收叶下珠药材的地上部分即可。（4）比较叶下珠胶囊和叶下珠药材的指纹图谱,发现二者的化学成分非常相似,但峰面积差异较大,其差异可能是由于加工过程所致。该研究所建立的HPLC指纹图谱分析方法简便、重现性好,可用于叶下珠药材及中成药的鉴定与质量评价。

叶下珠胶囊的HPLC法指纹图谱研究

目的建立叶下珠胶囊的HPLC指纹图谱质量控制方法。方法采用高效液相色谱法,选用HypersilODSⅡ色谱柱,流动相为乙腈-0.1%磷酸水溶液(梯度洗脱),分析时间65 min,流速0.8 ml/min,检测波长为280nm。结果建立了叶下珠胶囊的HPLC对照指纹图谱,标示了17个共有峰,并初步确定叶下珠胶囊的相似度阈值为0.91。结论本方法可靠、简单,能用于叶下珠胶囊的质量控制。

余甘子叶化学成分没食子酸诱导人肝癌细胞株BEL-7404凋亡机制的研究

目的:探讨余甘子叶化学成分没食子酸诱导人肝癌细胞株BEL-7404产生凋亡的机制。方法:采用PI和Hoechst33342双染色法流式检测对人肝癌细胞株BEL-7404各周期的影响;细胞荧光免疫染色法和Western blot法检测Bax和Bcl-2蛋白表达的变化。结果:PI和Hoechst33342双染色法分析结果表明,余甘子叶化学成分没食子酸作用BEL-7404细胞72 h后,G2/M期细胞数增多,呈现细胞阻滞现象,并伴有大量的细胞凋亡。荧光免疫染色显示,药物作用人肝癌细胞株BEL-7404 72 h后Bax蛋白表达升高,Bcl-2蛋白表达则降低。Western blot法实验亦显示Bax蛋白表达升高。结论:余甘子叶化学成分没食子酸能阻滞G2/M期细胞,并通过上调Bax和下调Bcl-2蛋白表达,导致线粒体膜电位的下降,进而激活Caspase,最终导致细胞死亡,可能是其诱导人肝癌细胞株BEL-7404凋亡的分子机制之一。

基于聚类分析和主成分分析的叶下珠多酚部位HPLC指纹图谱研究

目的:建立基于聚类分析和主成分分析的不同产地叶下珠多酚部位高效液相色谱(HPLC)指纹图谱分析方法。方法:采用HPLC法测定9个不同产地叶下珠多酚部位指纹图谱,对其进行相似度分析、聚类分析和主成分分析,高效液相色谱-质谱(HPLC-MS)指认主要成分。结果:叶下珠多酚部位指纹图谱检出7个共有峰,指认了其中5个共有峰,9个不同产地叶下珠多酚部位相似度达到0.972-0.998,聚类分析和主成分分析从化学成分上说明了9批样品的相似性及差异性。结论:以指纹图谱数据为基础,将聚类分析与主成分分析结合起来进行识别,为叶下珠药材的资源利用提供依据。

HPLC测定余甘子茶中3种多酚成分及抗氧化活性研究

福林酚法测定五个产地的余甘子茶中总多酚(TP)的含量,高效液相色谱法(High performance liquid chromatography,HPLC)分析没食子酸(GA)、柯里拉京(CO)、鞣花酸(EA)的含量,并对其抗氧化活性进行研究。结果表明:TP含量最高的是凉山的余甘子茶,其值为228 mg/g;漳州的余甘子茶中GA、CO、EA含量最高,分别为5.00、28.76、4.30 mg/g;在抗氧化活性研究中,昆明的余甘子茶提取液总还原能力较高,但凉山的余甘子茶提取液对DPPH自由基(DPPH·)和羟自由基(·OH)清除能力较好,EC50分别为7.46μg/mL和0.44 mg/mL,其对自发性肝脂质氧化抑制能力好于其它产地。相比较而言,凉山产地的余甘子茶作为开发天然抗氧化剂更具优势。

余甘子提取物及其复配物改善大鼠的功能性消化不良症状

该研究通过《保健食品功能检验与评价方法(2022年版)》中的促进消化功能检验方法中小鼠小肠运动、大鼠增重、摄食量、食物利用率、胃蛋白酶测定等试验研究了余甘子单方和四种复方的促消化效果。结果表明,余甘子(单方组),余甘子、山楂、茯苓和陈皮(复方1组),余甘子、山楂、白术和麦芽(复方2组),余甘子、山楂、茯苓、白术和陈皮(复方3组),余甘子、山楂、茯苓、白术、麦芽和陈皮(复方4组)小鼠小肠推进率分别提高了65.99%、80.53%、107.91%、67.47%、90.47%(P<0.05,P<0.01,P<0.0001,P<0.05,P<0.001),大鼠胃蛋白酶活性分别增强了89.24%、60.42%、103.07%、74.56%、106.84%(P<0.01,P≥0.05,P<0.01,P<0.05,P<0.01),大鼠胃蛋白酶排出量分别增强了89.30%、60.39%、103.14%、74.69%、106.98%(P<0.01,P≥0.05,P<0.01,P<0.05,P<0.01)。此外,复方4组大鼠食物利用率增加了15.35%(P<0.05),体质量增长、摄食量无显著差异(P≥0.05),其他组别的上述3项指标均无显著影响(P≥0.05)。研究表明余甘子提取物及其复配物可有效改善大鼠功能性消化不良症状。该研究为余甘子及其复配物促消化功能产品的开发和推广提供了相应的理论参考。

基于模糊数学和响应面法优化余甘子果汁饮料生产工艺

以新鲜余甘子果实为原料制备余甘子果汁饮料。在单因素试验的基础上,以综合得分为指标,通过正交试验优化纤维素酶酶解工艺;再在单因素试验的基础上,以综合感官评分为指标,通过响应面法优化余甘子果汁饮料配方。结果表明:最佳酶解工艺为纤维素酶添加量0.15%(以余甘子原汁质量计)、酶解温度55℃、酶解时间40 min;最佳饮料配方为以余甘子果汁饮料质量为基准,余甘子原汁添加量30%、白砂糖添加量11%、食盐添加量0.13%、柠檬酸添加量0.18%,在此条件下制得的余甘子果汁饮料色、香、味佳,综合感官评分为88.10。