Toxicity Experiment of Polysaccharides from Dicliptera chinensis( L. ) Juss.

[Objectives] To study the acute toxicity,mutation,and feeding experiment of polysaccharides from Dicliptera chinensis( L.)Juss.,and assess its safety. [Methods]In accordance with Procedures for toxicological Assessment on Food Safety,acute toxicity to mice,micronucleus experiment for bone marrow cell in mice,sperm shape abnormality test in mice,and Ames experiment were carried out,and 30 d feeding experiment was detected for rats. [Results] when the maximum tolerance dose( MTD) of mice was higher than 20 g/kg,at polysaccharides from Dicliptera chinensis( L.) Juss. ≤10 g/kg,there was no statistical difference between the control group and mouse bone marrow cell micronucleus and sperm shape abnormality test group; Ames experiment indicated that the number of revertant colonies of different dose groups of polysaccharides from Dicliptera chinensis( L.) Juss. with and without S9 did not exceed two times of the blank control group; rat 30 d feeding experiment indicated that when polysaccharides from Dicliptera chinensis( L.) Juss. ≤ 3. 2 g/kg,rats grew and developed well,and there was no statistical difference with the control group in blood,multiple organ biochemical indicators and liver tissue. [Conclusions]Polysaccharides from Dicliptera chinensis( L.) Juss. are safe and non-toxic chemical compounds and can be used as health products or drugs.

Effects of Polysaccharides from Dicliptera chinensis(L.)Nees.on TLR4/NF-κB Signaling Pathway in Cell Model of Non-alcoholic Fatty Liver

[Objectives]To observe the effects of polysaccharides from Dicliptera chinensis(L.)Nees.on the expression of TLR/NF-κB pathway related proteins in HepG2 cells induced by oleic acid,and to explore the possible mechanism of polysaccharides from D.chinensis(L.)Nees.in the treatment of non-alcoholic fatty liver disease(NAFLD).[Methods]HepG2 cells were induced with oleic acid to establish a non-alcoholic fatty liver cell model.After intervention with 0.25 and 0.5 mg/mL of D.chinensis(L.)Nees.polysaccharides,the ALT and AST activity and TG and TC contents were detected with kits,and the changes in the expression of CDK5,TLR4,p-NF-κB and NF-κB were analyzed using Western-blotting.[Results]In the HepG2 cells induced with oleic acid,the ALT and AST activity increased significantly,the TG and TC contents increased significantly,and the expression levels of CDK5,TLR4 and p-NF-κB proteins up-regulated significantly.In the HepG2 cells intervened with D.chinensis(L.)Nees.polysaccharides,the activity of ALT and AST,the contents of TG and TC,and the expression levels of CDK5,TLR4 and p-NF-κB proteins all reduced significantly.[Conclusions]Polysaccharides from D.chinensis(L.)Nees.may interfere with NAFLD by inhibiting the TLR4/NF-κB pathway.

Ethyl Acetate Fraction of Dicliptera chinensis(L.)Juss.Ameliorates Liver Fibrosis by Inducing Autophagy via PI3K/AKT/m TOR/p70S6K Signaling Pathways

Objective:To investigate the molecular mechanism underlying the anti-hepatic fibrosis activity of ethyl acetate fraction Dicliptera chinensis(L.)Juss.(EDC)in human hepatic stellate cells(HSCs)in vitro and in a carbon tetrachloride(CCl4)-induced hepatic fibrosis mouse model in vivo.Methods:For in vitro study,HSCs were pre-treated with platelet-derived growth factor(10 ng/mL)for 2 h to ensure activation and treated with EDC for 24 h and 48 h,respectively.The effect of EDC on HSCs was assessed using cell counting kit-8 assay,EdU staining,transmission electron microscopy,immunofluorescence staining,and Western blot,respectively.For in vivo experiments,mice were intraperitoneally injected with CCl4(2μL/g,adjusted to a 25%concentration in olive oil),3 times per week for 6 weeks,to develop a hepatic fibrosis model.Forty 8-week-old male C57 BL/6 mice were divided into 4 groups using a random number table(n=10),including control,model,positive control and EDC treatment groups.Mice in the EDC and colchicine groups were intragastrically administered EDC(0.5 g/kg)or colchicine(0.2 mg/kg)once per day for 6 weeks.Mice in the control and model groups received an equal volume of saline.Biochemical assays and histological examinations were used to assess liver damage.Protein expression levels ofα-smooth muscle actin(α-SMA)and microtubule-associated protein light chain 3 B(LC3 B)were measured by Western blot.Results:EDC reduced pathological damage associated with liver fibrosis,downregulated the expression ofα-SMA and upregulated the expression of LC3 B(P<0.05),both in HSCs and the CCl4-induced liver fibrosis mouse model.The intervention of bafilomycin A1 and rapamycin in HSCs strongly supported the notion that inhibition of autophagy enhancedα-SMA protein expression levels(P<0.01).The results also found that the levels of phosphoinositide(PI3 K),p-PI3 K,AKT,p-AKT,mammalian target of rapamycin(mTOR),p-mTOR,and p-p70 S6 K all decreased after EDC treatment(P<0.05).Conclusion:EDC has anti-hepatic fibrosis activity by inducing autophagy and might be a potential drug to be further developed for human liver fibrosis therapy.

狗肝菜总黄酮微波辐射预处理提取工艺及其生物活性研究

为了更深入研究狗肝菜总黄酮的提取工艺,为后续的研究奠定基础,本文采用微波辐射预处理结合传统水浴法对狗肝菜总黄酮进行提取,并以芦丁标准品作为对照品,采用紫外可见分光光度计测定吸光度并分析狗肝菜中总黄酮的含量。以单因素试验为基础,采用响应面优化工艺参数得到狗肝菜总黄酮的最佳提取率。结果显示:微波辐射预处理提取狗肝菜总黄酮的最佳工艺条件为微波功率350 W、微波时间77 s、汽化剂用量4.3 mL,在此条件下狗肝菜总黄酮提取率为8.382‰。在抗肺癌活性细胞试验中,狗肝菜总黄酮提取物的半抑制浓度为9.516 mg/mL,在药物浓度27 mg/mL时,抑制率达到了88.419%。研究结果表明:微波辐射预处理提取狗肝菜总黄酮是可行的,其总黄酮提取物(乙醇提取物)具有一定的抗肿瘤细胞活性,可以为今后的研究提供参考。

狗肝菜多糖减轻D-氨基半乳糖与脂多糖诱导的大鼠急性肝损伤

目的:观察壮药狗肝菜多糖对D-氨基半乳糖(D-GalN)及脂多糖(LPS)所致大鼠急性重症肝炎的影响。方法:将48只雄性Wistar大鼠随机分成模型、狗肝菜多糖高、低剂量和对照组;狗肝菜多糖高剂量组(300mg·kg-1)、低剂量组(150mg·kg-1)在造模前灌胃给药6d,其它各组用生理盐水对照;末次给药后1h除对照组外均在腹腔内注射D-GalN(500mg.kg-1)和LPS(20μg/kg)建立大鼠急性重症肝炎模型,对照组用生理盐水对照。于6h、12h分别观察并计算各组动物死亡率;存活动物于6h行眶后静脉窦采血用于检测血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、肿瘤坏死因子α(TNF-α)、白介素1β(IL-1β);12h摘除眼球采血用于检测血清ALT、AST、TNF-α、IL-1β和一氧化氮(NO);分光光度法检测血清ALT、AST、NO含量,ELISA法检测血清TNF-α、IL-1β水平。12h后全部处死存活动物,肝组织切片观察肝脏病理形态学变化。结果:6h各组动物均存活;与模型组相比,狗肝菜处理组12h死亡率明显低于模型组。模型组血清ALT、AST活力和TNF-α、IL-1β、NO水平均升高,狗肝菜多糖高、低剂量组血清指标均明显低于模型组。模型肝组织大片坏死、淤血,狗肝菜多糖高、低剂量组组织损伤程度明显降低,以高剂量组效果最佳。结论:狗肝菜多糖能减轻D-GalN/LPS诱导的肝损伤,可能通过降低炎性介质TNF-α、IL-1β和NO的水平发挥作用。

狗肝菜的化学成分研究

目的对狗肝菜Dicliptera chinensis全草的化学成分进行研究。方法采用硅胶柱色谱和制备性薄层色谱进行分离、纯化,IR、NMR和MS等方法进行结构鉴定。结果从环己烷萃取部分得到13个化合物,分别鉴定为正三十六烷醇(Ⅰ)、硬脂酸(Ⅱ)、羽扇烯酮(Ⅲ)、羽扇豆醇(Ⅳ)、谷甾烷-4-烯-3-酮(Ⅴ)、豆甾烷-5-烯-7-酮-3β-棕榈酸酯(Ⅵ)、β-谷甾醇(Ⅶ)、齐墩果酸(Ⅷ)、3β,6β-豆甾烷-4-烯-3,6-二醇(Ⅸ)、6β-羟基-豆甾烷-4-烯-3-酮(Ⅹ)、3β-羟基-豆甾烷-5-烯-7-酮(Ⅺ)、去氢催叶萝芙叶醇(Ⅻ)和催叶萝芙叶醇(ⅩⅢ)。结论除Ⅱ化合物和Ⅶ外,皆为首次从该属植物中发现。

狗肝菜中多糖提取工艺的比较研究

目的建立狗肝菜多糖的最佳提取方法和工艺。方法分别选取微波法、超声波法和溶剂回流提取法制备狗肝菜多糖。采用水提醇沉法得到粗多糖,再用Sevag法脱蛋白后,依次采用乙醚、无水乙醇、丙酮洗涤纯化,得精制多糖。比较3种方法所得多糖产率和含量,从中筛选出最佳提取方法。采用最佳提取方法进行正交实验,以筛选出其最佳提取工艺。结果 3种方法得到的多糖产率和含量存在较大差异,其中微波法多糖产率为7.10%,含量为70.10%,超声波法多糖产率为4.66%,含量为61.42%,溶剂回流提取法多糖产率为9.15%,含量为87.90%。狗肝菜的最佳提取方法为溶剂回流提取法,提取工艺最佳为料液比1∶20,提取时间2h,提取2次,温度85℃。结论该提取方法和工艺适合于狗肝菜中多糖的提取。

不同采收季节的狗肝菜中多糖含量测定

目的:考察广西产不同采收季节狗肝菜中多糖的含量。方法:采用蒽酮-浓硫酸比色法测定不同采收季节的狗肝菜中多糖的含量。结果:广西不同采收季节的狗肝菜中多糖含量各不相同,野生品种狗肝菜以6～8月份产的多糖含量最高,栽培品种狗肝菜以6月份产的为最高。结论:本实验研究可为广西产狗肝菜质量控制与种植区域划分研究提供科学依据,促进广西民族药资源开发与利用。

狗肝菜多糖颗粒剂成型工艺的优选

目的:优选狗肝菜多糖颗粒剂的最佳成型工艺条件。方法:以颗粒成型率、流动性与溶化性为考察指标,分别用单因素试验和正交试验优选颗粒剂处方中所用辅料(填充剂与黏合剂)的种类及其用量,并对制得的颗粒剂进行质量检查。结果:最佳成型工艺条件为狗肝菜多糖-甘露醇-3%HPMC水溶液=1∶2.5∶0.035,所制得的颗粒剂粒度、流动性与溶化性等项目检查都符合《中国药典》要求。结论:优选出的狗肝菜多糖颗粒剂成型工艺可行,可为进一步工业化生产提供参考和依据。

不同产地狗肝菜中多糖的含量测定

目的:考察不同产地狗肝菜中多糖的含量。方法:采用蒽酮-硫酸比色法测定不同产地狗肝菜中多糖的含量。结果:在15-105μg范围内,葡萄糖μg数与吸光度A值线性关系良好,平均回收率为99.52%,RSD=2.12%,R2=0.9997。不同产地的狗肝菜中多糖含量为5.77%-31.49%。结论:各产地狗肝菜中多糖含量差异较大;野生品多糖含量高于栽培品;以广西桂林产狗肝菜中多糖含量为最高。