野老鹳草（Geranium carolinianum L.）抗乙肝病毒作用及化学组分研究
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摘要
老鹤草为我国传统中药,2005版中国药典收载的老鹤草为栊牛儿苗(Erodiumstephanianum Wild.)、老鹤草(Geranium wilfordii Maxim.)、野老鹤草(Geraniumcarolinianum L.)的干燥地上部分。具有祛风湿、通经络、止泻痢的功能。在我国老鹤草品种较多,分布较广,资源丰富。野老鹤草(Geranium carolinianum L.)在民间作为习用药材应用较多,也有其水煎剂初步临床治疗乙肝病例的报导,但其作用的物质基础及化学成分迄今末见详细报道,在抗乙肝病毒(HBV)方面更没有系统研究。为研究野老鹳草在抗HBV方面的作用,本文采用天然药物化学、药理学、分析化学、分子生物学等手段,对野老鹳草进行了抗乙肝病毒有效部位的系统研究。在生物活性指导下分离提取了总黄酮提取物,并首次发现该总黄酮提取物具有良好的、独特的体内外抑制乙肝病毒的效果。从抗病毒有效部位中分离得到15个化合物,除金丝桃苷外,其它化合物均为首次从野老鹳草中分离得到,其中槲皮素—3—O—β—D—(6″—没食子酰基)半乳糖苷、牡荆苷为首次从老鹳草属植物中分离得到。本文还探讨了黄酮类化合物抗HBV的构效关系,并首次报道了老鹳草素和金丝桃苷体外对HBsAg和HBeAg较强的抑制作用。
一、野老鹳草抗乙肝病毒有效部位的分离与确定
1、对野老鹳草及其抗乙肝病毒有效部位的确认
从大量中草药的水煎剂中,通过体外试验发现野老鹳草具有较强的抑制HBsAg和HBeAg的作用。采用有机溶媒提取分离,发现野老鹳草乙酸乙酯提取物具有较强的抑制乙肝病毒HBsAg和HBeAg分泌的能力,进一步明确乙酸乙酯提取物的主要成分以黄酮为主。
2、对野老鹳草分离纯化黄酮的工艺进行确认
正交实验结果的直观分析和方差分析,显示酒精提取时最佳提取野老鹳草黄酮工艺条件为回流时间1 h,提取次数3次,药材与溶剂比1:8,乙醇浓度为50%。又通过对影响大孔树脂吸附及解吸的各种因素进行研究,发现D—101大孔吸附树脂较适合分离纯化野老鹳草总黄酮,其分离纯化总黄酮的工艺为:药液浓度以生药计1.0 g/ml,吸附速度为1.0 ml/min,洗脱速度为1 ml/min,洗脱溶剂为70%乙醇,5倍柱床体积的70%乙醇可将总黄酮基本洗脱完全。该工艺总黄酮收率高,成本低,操作简便,避免使用有机溶剂,安全,适宜大规模生产使用。
通过比较野老鹳草乙酸乙酯提取物和大孔树脂D—101分离提取物的抗乙肝病毒活性和细胞毒性,发现乙酸乙酯提取物细胞毒性较强,而大孔树脂D—101的提取物细胞毒性相对较弱,抑制乙肝病毒HBsAg和HBeAg分泌的能力较强,有希望开发成抗乙肝病毒新药。
二、野老鹳草总黄酮提取物抗乙肝病毒药效学试验
1、野老鹳草总黄酮提取物对HBV整合的肝癌细胞HepG_2 2.215细胞半数毒性浓度TC_(50)为152.13μg/ml(10 d)。
2、HepG_22.2.15细胞模型试验显示,当细胞培养液中野老鹳草总黄酮提取物浓度为1 00μg/ml、50μg/ml、25μg/ml、12.5μg/ml和6.25μg/ml时,药物作用10天对HBsAg抑制率分别达到了69.90%、30.54%、12.02%、8.92%和3.40%,IC_(50)为68.39μg/ml,治疗指数(TI)为2.22;对HBeAg抑制率分别达到了59.01%、39.43%、23.93%、16.95%和12.39%,IC_(50)为72.06μg/ml,治疗指数(TI)为2.11。在100μg/ml浓度下,药物作用10天,经Southern Blot检测野老鹳草总黄酮提取物对细胞HBV-DNA的抑制率达到了38.56%。同样条件下荧光定量实时-PCR(FQ-PCR)显示对HBV DNA拷贝数的抑制为35.9%。以上表明野老鹳草总黄酮体外能抑制乙肝病毒,并显示量效关系。
3、体内试验:野老鹳草总黄酮提取物经鸭乙型病毒性肝炎模型试验,结果显示对血清病毒DNA有明显的抑制。口服给药10天,三个不同的给药剂量112 mg/kg、56 mg/kg、28 mg/kg对血清病毒DNA和肝脏病毒的抑制有剂量依赖关系。经dotblot检测,112 mg/kg、56 mg/kg、28 mg/kg的剂量给药10天,对血清病毒DNA的抑制分别为69.00%、50.57%、和41.30%,ED_(50)为47.54 mg/kg;停药3天后,抑制率分别维持在57.84%、32.63%、和21.24%,ED_(50)为91.69 mg/kg。相比对照药物拉米夫定,停药后的反跳不明显。野老鹳草总黄酮提取物对肝脏病毒DNA也有较好抑制,经Southern Blot检测,112 mg/kg的剂量用药10天对肝脏病毒DNA的抑制率为45.37%。经病理切片观察,野老鹳草总黄酮对鸭乙肝病毒引起的肝脏水样变性有明显的减轻效果,经口服给药112 mg/kg和56mg/kg 5天就可以看到血窦,在停药3天后,仍可见到血窦,未见脂肪肝病变。
三、活性部位化学组分的分离、分析及抗病毒活性研究
从野老鹳草总黄酮提取物中分离得到金丝桃苷(hyperoside)、陆地棉苷(hirsutrin)、槲皮素(quercetin)、槲皮素—3—O—β—D—(6″—没食子酰基)半乳糖苷[quercetin-3-O-β—D—(6″—galloyl)galactoside]、鞣花酸(ellagic acid)、没食子酸(gallic acid)、山奈酚(kaempferol)、杨梅素(myricetin)、原儿茶酸(protocatechuic acid)、柯里拉京(corilagin)、老鹳草素(geraniin)、山奈酚—7—O—鼠李糖苷(kaempferol-7-O-rhamnoside)、槲皮甙(quercitrin)、山奈酚-7—O—α—L—呋喃阿拉伯糖苷(kaempferol-7-O-α—L—arabinofuranoside)、牡荆苷(Vitexin)等15个化合物。其中除金丝桃甙外,其它化合物为首次从野老鹳草中分离得到,且槲皮素—3—O—β—D—(6″—没食子酰基)半乳糖苷、牡荆苷为首次从老鹳草属植物中分离得到。
通过比较所分离化合物体外抗乙肝病毒的效果,首次发现老鹳草素、金丝桃甙体外有较强的抑制乙肝病毒的作用,鞣花酸也具有较强的体外抑制乙肝病毒HBsAg和HBeAg分泌的效果,以上三个化合物的活性可用于解释野老鹳草总黄酮抗乙肝病毒的效果。并且第一个发现黄酮甙元B环上羟基的位置和数目影响其抗乙肝病毒的活性,活性大小依次为杨梅素＞槲皮素＞山奈酚。黄酮母核3位羟基上糖取代基的不同显著影响抗乙肝病毒的活性,其中3位上含有半乳糖的黄酮苷具有较强的抑制乙肝病毒活性的效果。生物活性研究发现酚羟基的数目影响抗乙肝病毒的效果,如老鹳草素和柯里拉京具有非常强的体外抗乙肝病毒的效果。
建立了高效液相色谱法(HPLC)测定野老鹳草总黄酮中金丝桃甙含量的方法,为进一步质量标准的研究奠定了基础。
Laoguancao,which is a traditional Chinese medicine,has driving away wind-dampness and opening channels and collaterals effects,and in clinic curing symptoms of wind-dampness flaccidity,spasm numbness and unconsciousness. Geranium carolinianum L.is used as Laoguacao in folk.It has been reported that an aqueous extract of this plant can improve the clinical symptoms of HBV infected patients.Therefore it was supposed that Geranium carolinianum L.might possess the activity to inhibit the replication of HBV and the expression of viral antigens. However,its direct anti-viral avtivity against HBV has not been reported before.
In this study,the antiviral activity of the total flavonoids of Geranium carolinianum L.against HBV was investigated.To our knowledge,this is the first report of the anti-HBV effects of the total flavonoids extract from G carolinianum L.Our observations suggest that the total flavonoids have anti-HBV activity and the potential to be developed as an alternative or complementary anti-HBV agent.In order to identify the active ingredients,15 compounds were isolated and purified from total flavonoids fraction of G.carolinianum L.by chromatography on silica gel column, sephadex LH-20 chromatogram,including ellagic acid,geraniin,hyperin.Geraniin inhibited HBsAg and HBeAg secretion by more than 85.8%and 63.7%,respectively, at the non-cytotoxic concentration of 200μg/ml.The inhibitions of HBsAg and HBeAg secretion by geraniin were higher than the inhibition by the positive control Lamivudine,33.5%and 32.2%respectively,at the same concentration.This is the first report of the anti-HBV effects of geraniin and hyperin,the active substances derived from G.carolinianum L.The presence of the anti-HBV compounds may account for the effectiveness of this folk medicine in the treatment of HBV infections.In addition, relationship between structure and anti-HBV activity of isolated flavonoids was first reported.
Part 1 Separation and confirmation of anti-HBV effective part of G.carolinianum L.
1.It has been confirmed that G.carolinianum L.has anti-HBV activities from many herbs.The ethanol extract of Geranium carolinianum L.was subjected to sequential extractions with different organic solvents.The extracts were assayed for anti-HBV activities.The ethyl acetate fraction was found to contain the highest level of anti-HBV activity and further confirmed to have many flavones.
2.Separation and purification of total flavonoids from G.carolinianum L.by macroporous adsorption resin.Through orthogonal experiments of normal reflux extraction,crude extract was obtained from G.carolinianum L.by reflux extraction using 50%ethanol for three times,1:8 of material to ethanol.It were compared that the ability of adsorption and separation total flavonoids in 5 different type of macroporous resin by means of static adsorption test and dynamic test,it showed that the total ability of low pole D-101 resin was superior to the other macroporous resin. All process conditions influencing on the process of separation and purification total flavonoids with D-101 resin were investigated in detail.The optimal technological conditions were:the concentration of the sample of G.carolinianum L., 1.0g/ml(amount of crude drug),the maximum adsorption capacity for total flavonoids, 21.23mg/g;the optimal column ratio of diameter to height,1:12;the flow velocity of adsorption,1 ml/min;the eluting reagent,70%ethanol(5 times of the volume of the resin);eluting velocity,1 ml/min.The technology of separating and purifying total flavonoids from G.carolinianum L.with D-101 was stable and effective.
Part 2 In vitro and in vivo anti-hepatitis B virus activities of total flavonoids from Geranium carolinianum L.
1.In pharmacological experiment,MTT assay was using to detect the toxicity of the prolifer of HepG_22.2.15 cells treated with total flavonoids from G.carolinianum L.(TFGC) at varied concentration.The TC_(50) is 152.13μg/ml.There was no significant difference of cell viability between TFGC-treated groups whose concentrations were below 100μg/mL
2.In vitro:Treatment of HepG_2 2.2.15 cells with TFGC at various concentrations (100μg/mL,50μg/mL,25μg/mL,12.5μg/mL and 6.25μg/mL for 10 days resulted in a significant reduction of Hepatitis B surface antigen(HBsAg) and Hepatitis B e antigen(HBeAg) secretion in HepG_2 2.2.15 in a dose-dependent manner,with IC_(50) values of 68.39μg/ml and 72.06μg/ml respectively.TFGC was more potent than Lamivudine(3TC) for inhibiting both HBsAg and HBeAg secretion.
To further confirm the anti-HBV activity of TFGC in HepG_2 2.2.15 cells,the HBV DNA levels were evaluated after TFGC treatment by Southern Blot.Consistent with the inhibitory effects on HBsAg and HBeAg secretion,Treatment of HepG_2 2.2.15 cells with TFGC at various concentrations for 10 days resulted in the reduction of the intracellular HBV DNA levels in a dose-dependent manner.TFGC(100μg/ml) reduced intracellular HBV DNA level by 38.56%.By Flurescence Quantity PCR assay, the inhibit dosage curve of TFGC to intracellular HBV DNA of HepG_22.2.25 showed "S" shape during 10 days monitering.The inhibition ratio is 35.9%.
3.In vivo:The duck DHBV model represents a suitable and a widely used system for the study of in vivo activity of anti-HBV agents as well as their toxicity.We fed the ducks with TFGC at various concentrations(112 mg/kg,56 mg/kg,28 mg/kg)(i.g.) once daily for 10 days,no toxicity was observed.To examine the in vivo anti-HBV activity of TFGC,we first checked the plasma DHBV DNA levels of the infected ducks with and without treatment.TFGC reduced plasma and liver DHBV DNA levels in a dose-dependent manner.At dosage of 112 mg/kg,TFGC significantly inhibited DHBV DNA levels in the duck plasma.Densitometric quantitation of the dot blots revealed decrease in DHBV DNA levels(57.07%,69.00%and 57.84%for Day 5,10 of the TFGC treatment and 3 days alter the cessation of treatment respectively,n-6) in ducks treated with 112 mg/kg TFGC,as compared with the untreated ducks.Moreover, the rebound of the DHBV DNA levels in TFGC treated ducks was to a less extent as compared with the 3TC treated group.No significant differences were observed in the DHBV DNA levels of any of the controls.
To further confirm the in vivo anti-HBV effect of TFGC in ducks,the DHBV DNA levels in the livers(obtained at days 5,10 of PPGC treatment and 3 days after the treatment was stopped) were examined by Southern hybridization analysis.Consistent with the inhibitory effect on the plasma DHBV DNA level,TFGC treatment dose-dependently reduced the DHBV DNA levels in the liver.Densitometric analysis of the autoradiographic signals indicated 26.96%,45.37%,and 29.12%inhibition (days 5,10 during the treatment and 3 days after the cessation of the treatment respectively) due to the TFGC treatment(112 mg/kg),whereas 3TC-treated(200 mg/kg) groups resulted in 43.98%,59.74%,and 40.06%inhibition at these three data points.
To evaluate the pathological changes,liver sections from the above treatment groups were examined under the light microscope.In the control group,significant edemas could be observed in the endoplasmic reticulum,indicating the DHBV expression.Samples treated with TFGC,on the other hand,exhibited dose-dependent improvement in the edema formation.It is worth noting that TFGC at 112 mg/kg resulted more significant improvement than 3TC at 200 mg/kg.
Part 3:Chemical constituents of total flavonoids and their anti-HBV activities
15 compounds have been isolated from the total flavonoids of Geranium carolinianum L.Their structures were deduced on the basis of their spectral data and identified as hyperoside、hirsutrin、quercetin、quercetin-3-O-β-D-(6"-galloyl)galactoside]、ellagic acid、gallic acid、kaempferol、myricetin、protocatechuic acid、corilagin、geraniin、kaempferol-7-O-rhamnoside、quercitrin、kaempferol-7-O-α-L-arabinofuranoside、Vitexin.Except hyperin,other compounds were isolated from this plant species for the first time.quercetin-3-O-β-D-(6"-galloyl)galactoside] and vitexin were isolated from the genus Geraniaceae for the first time.
The structure activity relationship of structure and flavonoids anti-HBV effect were studied so as to find some regularities,which may provide the foundation for their exploitation and utility.Hydroxyl groups in the ring B were main active groups anti-HBV.The glucoside contributed to anti-HBV effects,which varied with sort of the glucoside.The phenolic hydroxyl compounds usually have anti-HBV activity
The HPLC was developed to determine the content of hyperin in total flavonoids of Geranium carolinianum L.The content of hyperin is more than 2.5%in the total flavonoids.
一、野老鹳草抗乙肝病毒有效部位的分离与确定
1、对野老鹳草及其抗乙肝病毒有效部位的确认
从大量中草药的水煎剂中,通过体外试验发现野老鹳草具有较强的抑制HBsAg和HBeAg的作用。采用有机溶媒提取分离,发现野老鹳草乙酸乙酯提取物具有较强的抑制乙肝病毒HBsAg和HBeAg分泌的能力,进一步明确乙酸乙酯提取物的主要成分以黄酮为主。
2、对野老鹳草分离纯化黄酮的工艺进行确认
正交实验结果的直观分析和方差分析,显示酒精提取时最佳提取野老鹳草黄酮工艺条件为回流时间1 h,提取次数3次,药材与溶剂比1:8,乙醇浓度为50%。又通过对影响大孔树脂吸附及解吸的各种因素进行研究,发现D—101大孔吸附树脂较适合分离纯化野老鹳草总黄酮,其分离纯化总黄酮的工艺为:药液浓度以生药计1.0 g/ml,吸附速度为1.0 ml/min,洗脱速度为1 ml/min,洗脱溶剂为70%乙醇,5倍柱床体积的70%乙醇可将总黄酮基本洗脱完全。该工艺总黄酮收率高,成本低,操作简便,避免使用有机溶剂,安全,适宜大规模生产使用。
通过比较野老鹳草乙酸乙酯提取物和大孔树脂D—101分离提取物的抗乙肝病毒活性和细胞毒性,发现乙酸乙酯提取物细胞毒性较强,而大孔树脂D—101的提取物细胞毒性相对较弱,抑制乙肝病毒HBsAg和HBeAg分泌的能力较强,有希望开发成抗乙肝病毒新药。
二、野老鹳草总黄酮提取物抗乙肝病毒药效学试验
1、野老鹳草总黄酮提取物对HBV整合的肝癌细胞HepG_2 2.215细胞半数毒性浓度TC_(50)为152.13μg/ml(10 d)。
2、HepG_22.2.15细胞模型试验显示,当细胞培养液中野老鹳草总黄酮提取物浓度为1 00μg/ml、50μg/ml、25μg/ml、12.5μg/ml和6.25μg/ml时,药物作用10天对HBsAg抑制率分别达到了69.90%、30.54%、12.02%、8.92%和3.40%,IC_(50)为68.39μg/ml,治疗指数(TI)为2.22;对HBeAg抑制率分别达到了59.01%、39.43%、23.93%、16.95%和12.39%,IC_(50)为72.06μg/ml,治疗指数(TI)为2.11。在100μg/ml浓度下,药物作用10天,经Southern Blot检测野老鹳草总黄酮提取物对细胞HBV-DNA的抑制率达到了38.56%。同样条件下荧光定量实时-PCR(FQ-PCR)显示对HBV DNA拷贝数的抑制为35.9%。以上表明野老鹳草总黄酮体外能抑制乙肝病毒,并显示量效关系。
3、体内试验:野老鹳草总黄酮提取物经鸭乙型病毒性肝炎模型试验,结果显示对血清病毒DNA有明显的抑制。口服给药10天,三个不同的给药剂量112 mg/kg、56 mg/kg、28 mg/kg对血清病毒DNA和肝脏病毒的抑制有剂量依赖关系。经dotblot检测,112 mg/kg、56 mg/kg、28 mg/kg的剂量给药10天,对血清病毒DNA的抑制分别为69.00%、50.57%、和41.30%,ED_(50)为47.54 mg/kg;停药3天后,抑制率分别维持在57.84%、32.63%、和21.24%,ED_(50)为91.69 mg/kg。相比对照药物拉米夫定,停药后的反跳不明显。野老鹳草总黄酮提取物对肝脏病毒DNA也有较好抑制,经Southern Blot检测,112 mg/kg的剂量用药10天对肝脏病毒DNA的抑制率为45.37%。经病理切片观察,野老鹳草总黄酮对鸭乙肝病毒引起的肝脏水样变性有明显的减轻效果,经口服给药112 mg/kg和56mg/kg 5天就可以看到血窦,在停药3天后,仍可见到血窦,未见脂肪肝病变。
三、活性部位化学组分的分离、分析及抗病毒活性研究
从野老鹳草总黄酮提取物中分离得到金丝桃苷(hyperoside)、陆地棉苷(hirsutrin)、槲皮素(quercetin)、槲皮素—3—O—β—D—(6″—没食子酰基)半乳糖苷[quercetin-3-O-β—D—(6″—galloyl)galactoside]、鞣花酸(ellagic acid)、没食子酸(gallic acid)、山奈酚(kaempferol)、杨梅素(myricetin)、原儿茶酸(protocatechuic acid)、柯里拉京(corilagin)、老鹳草素(geraniin)、山奈酚—7—O—鼠李糖苷(kaempferol-7-O-rhamnoside)、槲皮甙(quercitrin)、山奈酚-7—O—α—L—呋喃阿拉伯糖苷(kaempferol-7-O-α—L—arabinofuranoside)、牡荆苷(Vitexin)等15个化合物。其中除金丝桃甙外,其它化合物为首次从野老鹳草中分离得到,且槲皮素—3—O—β—D—(6″—没食子酰基)半乳糖苷、牡荆苷为首次从老鹳草属植物中分离得到。
通过比较所分离化合物体外抗乙肝病毒的效果,首次发现老鹳草素、金丝桃甙体外有较强的抑制乙肝病毒的作用,鞣花酸也具有较强的体外抑制乙肝病毒HBsAg和HBeAg分泌的效果,以上三个化合物的活性可用于解释野老鹳草总黄酮抗乙肝病毒的效果。并且第一个发现黄酮甙元B环上羟基的位置和数目影响其抗乙肝病毒的活性,活性大小依次为杨梅素＞槲皮素＞山奈酚。黄酮母核3位羟基上糖取代基的不同显著影响抗乙肝病毒的活性,其中3位上含有半乳糖的黄酮苷具有较强的抑制乙肝病毒活性的效果。生物活性研究发现酚羟基的数目影响抗乙肝病毒的效果,如老鹳草素和柯里拉京具有非常强的体外抗乙肝病毒的效果。
建立了高效液相色谱法(HPLC)测定野老鹳草总黄酮中金丝桃甙含量的方法,为进一步质量标准的研究奠定了基础。
Laoguancao,which is a traditional Chinese medicine,has driving away wind-dampness and opening channels and collaterals effects,and in clinic curing symptoms of wind-dampness flaccidity,spasm numbness and unconsciousness. Geranium carolinianum L.is used as Laoguacao in folk.It has been reported that an aqueous extract of this plant can improve the clinical symptoms of HBV infected patients.Therefore it was supposed that Geranium carolinianum L.might possess the activity to inhibit the replication of HBV and the expression of viral antigens. However,its direct anti-viral avtivity against HBV has not been reported before.
In this study,the antiviral activity of the total flavonoids of Geranium carolinianum L.against HBV was investigated.To our knowledge,this is the first report of the anti-HBV effects of the total flavonoids extract from G carolinianum L.Our observations suggest that the total flavonoids have anti-HBV activity and the potential to be developed as an alternative or complementary anti-HBV agent.In order to identify the active ingredients,15 compounds were isolated and purified from total flavonoids fraction of G.carolinianum L.by chromatography on silica gel column, sephadex LH-20 chromatogram,including ellagic acid,geraniin,hyperin.Geraniin inhibited HBsAg and HBeAg secretion by more than 85.8%and 63.7%,respectively, at the non-cytotoxic concentration of 200μg/ml.The inhibitions of HBsAg and HBeAg secretion by geraniin were higher than the inhibition by the positive control Lamivudine,33.5%and 32.2%respectively,at the same concentration.This is the first report of the anti-HBV effects of geraniin and hyperin,the active substances derived from G.carolinianum L.The presence of the anti-HBV compounds may account for the effectiveness of this folk medicine in the treatment of HBV infections.In addition, relationship between structure and anti-HBV activity of isolated flavonoids was first reported.
Part 1 Separation and confirmation of anti-HBV effective part of G.carolinianum L.
1.It has been confirmed that G.carolinianum L.has anti-HBV activities from many herbs.The ethanol extract of Geranium carolinianum L.was subjected to sequential extractions with different organic solvents.The extracts were assayed for anti-HBV activities.The ethyl acetate fraction was found to contain the highest level of anti-HBV activity and further confirmed to have many flavones.
2.Separation and purification of total flavonoids from G.carolinianum L.by macroporous adsorption resin.Through orthogonal experiments of normal reflux extraction,crude extract was obtained from G.carolinianum L.by reflux extraction using 50%ethanol for three times,1:8 of material to ethanol.It were compared that the ability of adsorption and separation total flavonoids in 5 different type of macroporous resin by means of static adsorption test and dynamic test,it showed that the total ability of low pole D-101 resin was superior to the other macroporous resin. All process conditions influencing on the process of separation and purification total flavonoids with D-101 resin were investigated in detail.The optimal technological conditions were:the concentration of the sample of G.carolinianum L., 1.0g/ml(amount of crude drug),the maximum adsorption capacity for total flavonoids, 21.23mg/g;the optimal column ratio of diameter to height,1:12;the flow velocity of adsorption,1 ml/min;the eluting reagent,70%ethanol(5 times of the volume of the resin);eluting velocity,1 ml/min.The technology of separating and purifying total flavonoids from G.carolinianum L.with D-101 was stable and effective.
Part 2 In vitro and in vivo anti-hepatitis B virus activities of total flavonoids from Geranium carolinianum L.
1.In pharmacological experiment,MTT assay was using to detect the toxicity of the prolifer of HepG_22.2.15 cells treated with total flavonoids from G.carolinianum L.(TFGC) at varied concentration.The TC_(50) is 152.13μg/ml.There was no significant difference of cell viability between TFGC-treated groups whose concentrations were below 100μg/mL
2.In vitro:Treatment of HepG_2 2.2.15 cells with TFGC at various concentrations (100μg/mL,50μg/mL,25μg/mL,12.5μg/mL and 6.25μg/mL for 10 days resulted in a significant reduction of Hepatitis B surface antigen(HBsAg) and Hepatitis B e antigen(HBeAg) secretion in HepG_2 2.2.15 in a dose-dependent manner,with IC_(50) values of 68.39μg/ml and 72.06μg/ml respectively.TFGC was more potent than Lamivudine(3TC) for inhibiting both HBsAg and HBeAg secretion.
To further confirm the anti-HBV activity of TFGC in HepG_2 2.2.15 cells,the HBV DNA levels were evaluated after TFGC treatment by Southern Blot.Consistent with the inhibitory effects on HBsAg and HBeAg secretion,Treatment of HepG_2 2.2.15 cells with TFGC at various concentrations for 10 days resulted in the reduction of the intracellular HBV DNA levels in a dose-dependent manner.TFGC(100μg/ml) reduced intracellular HBV DNA level by 38.56%.By Flurescence Quantity PCR assay, the inhibit dosage curve of TFGC to intracellular HBV DNA of HepG_22.2.25 showed "S" shape during 10 days monitering.The inhibition ratio is 35.9%.
3.In vivo:The duck DHBV model represents a suitable and a widely used system for the study of in vivo activity of anti-HBV agents as well as their toxicity.We fed the ducks with TFGC at various concentrations(112 mg/kg,56 mg/kg,28 mg/kg)(i.g.) once daily for 10 days,no toxicity was observed.To examine the in vivo anti-HBV activity of TFGC,we first checked the plasma DHBV DNA levels of the infected ducks with and without treatment.TFGC reduced plasma and liver DHBV DNA levels in a dose-dependent manner.At dosage of 112 mg/kg,TFGC significantly inhibited DHBV DNA levels in the duck plasma.Densitometric quantitation of the dot blots revealed decrease in DHBV DNA levels(57.07%,69.00%and 57.84%for Day 5,10 of the TFGC treatment and 3 days alter the cessation of treatment respectively,n-6) in ducks treated with 112 mg/kg TFGC,as compared with the untreated ducks.Moreover, the rebound of the DHBV DNA levels in TFGC treated ducks was to a less extent as compared with the 3TC treated group.No significant differences were observed in the DHBV DNA levels of any of the controls.
To further confirm the in vivo anti-HBV effect of TFGC in ducks,the DHBV DNA levels in the livers(obtained at days 5,10 of PPGC treatment and 3 days after the treatment was stopped) were examined by Southern hybridization analysis.Consistent with the inhibitory effect on the plasma DHBV DNA level,TFGC treatment dose-dependently reduced the DHBV DNA levels in the liver.Densitometric analysis of the autoradiographic signals indicated 26.96%,45.37%,and 29.12%inhibition (days 5,10 during the treatment and 3 days after the cessation of the treatment respectively) due to the TFGC treatment(112 mg/kg),whereas 3TC-treated(200 mg/kg) groups resulted in 43.98%,59.74%,and 40.06%inhibition at these three data points.
To evaluate the pathological changes,liver sections from the above treatment groups were examined under the light microscope.In the control group,significant edemas could be observed in the endoplasmic reticulum,indicating the DHBV expression.Samples treated with TFGC,on the other hand,exhibited dose-dependent improvement in the edema formation.It is worth noting that TFGC at 112 mg/kg resulted more significant improvement than 3TC at 200 mg/kg.
Part 3:Chemical constituents of total flavonoids and their anti-HBV activities
15 compounds have been isolated from the total flavonoids of Geranium carolinianum L.Their structures were deduced on the basis of their spectral data and identified as hyperoside、hirsutrin、quercetin、quercetin-3-O-β-D-(6"-galloyl)galactoside]、ellagic acid、gallic acid、kaempferol、myricetin、protocatechuic acid、corilagin、geraniin、kaempferol-7-O-rhamnoside、quercitrin、kaempferol-7-O-α-L-arabinofuranoside、Vitexin.Except hyperin,other compounds were isolated from this plant species for the first time.quercetin-3-O-β-D-(6"-galloyl)galactoside] and vitexin were isolated from the genus Geraniaceae for the first time.
The structure activity relationship of structure and flavonoids anti-HBV effect were studied so as to find some regularities,which may provide the foundation for their exploitation and utility.Hydroxyl groups in the ring B were main active groups anti-HBV.The glucoside contributed to anti-HBV effects,which varied with sort of the glucoside.The phenolic hydroxyl compounds usually have anti-HBV activity
The HPLC was developed to determine the content of hyperin in total flavonoids of Geranium carolinianum L.The content of hyperin is more than 2.5%in the total flavonoids.
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