Evaluation of the Anti-Hepatitis B Virus Activity of Compound Huzhang Prescription in vivo and in vitro
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摘要:
目的 研究复方虎杖方体内外抗乙肝病毒作用。 方法 体外实验采用CCK-8染色法检测复方虎杖方水提物、醇提物对HepG2.2.15细胞株的抗增殖活性,采用化学发光微粒子免疫检测法测定HBsAg、HBeAg含量,采用实时荧光定量PCR法检测HBV-DNA含量;体内实验采用血清斑点杂交法(Dot blot)测定给药5、10 d后及停药3 d后鸭血清中的DHBV-DNA含量。 结果 体外结果显示,复方虎杖方水提物、醇提物能有效抑制HepG2.2.15细胞增殖活性,IC50分别为51.28 mg/mL和46.78 mg/mL;复方虎杖方能有效抑制HepG2.2.15细胞分泌HBsAg、HBeAg,具有明显的剂量依赖关系,醇提物对HBeAg分泌的抑制作用明显强于水提物;复方虎杖方醇提物能有效抑制HepG2.2.15细胞HBV-DNA复制,但水提物效果不明显。体内结果显示,复方虎杖方能有效抑制鸭血清中DHBV-DNA复制,且能显著抑制停药后的病毒反跳。 结论 复方虎杖方体内和体外均有一定的抗乙肝病毒作用,研究为该方的开发应用奠定了基础。 Abstract: OBJECTIVE To investigate the anti-hepatitis B virus (HBV) effect of Compound Huzhang Prescription in vivo and in vitro.METHODS In vitro, CCK-8 staining method was used to determine the anti-proliferative activity of Compound Huzhang Prescription against HepG2.2.15 cell lines expressing HBV, chemiluminescent microparticle immunoassay method was used to determine hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) levels, and fluorogenic quantitative PCR method was used to determine HBV-DNA level. In vivo, Dot blot assay was used to investigate the content of DHBV-DNA in serum at the 5th, 10th day after administration and the 3th day after drug withdrawal.RESULTS The results showed that water extract and alcohol extract of Compound Huzhang Prescription had a good inhibitory effect on the growth of HepG2.2.15 cells, with a half inhibitory concentration (IC50) of 51.28 mg/mL and 46.78 mg/mL, respectively. Compound Huzhang Prescription significantly inhibited HBsAg and HBeAg expressions and it showed a significant dose effect relationship. The inhibition of alcohol extract was stronger than that of water extract. The alcohol extract had an inhibitory effect on HBV-DNA replication, but the effect of water extract was not obvious. Compound Huzhang Prescription effectively inhibited DHBV-DNA replication, and significantly inhibited the virus rebound after drug withdrawal in vivo.CONCLUSION Compound Huzhang Prescription shows a certain anti-hepatitis B virus effect in vivo and in vitro, which lays a foundation for the development and application.-
Key words:
- Compound Huzhang Prescription /
- HepG2.2.15 cells /
- DHBV-DNA /
- HBsAg /
- HBeAg /
- anti-HBV activity
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表 1 复方虎杖方提取物对HepG2.2.15的增殖抑制作用(x±s,n=5)
组别 浓度/ (mg·mL-1) 醇提物 水提物 A 抑制率/% A 抑制率/% 空白对照组 - 1.625±0.088 - 1.607±0.067 - 拉米夫定组 0.10 1.052±0.067** 29.1 1.052±0.067** 29.1 复方虎杖方组 0.39 1.564±0.046 3.8 1.572±0.088 2.2 0.78 1.554±0.071 4.4 1.542±0.084 4.0 1.56 1.496±0.058* 7.9 1.532±0.079 4.7 3.12 1.486±0.076* 8.5 1.513±0.042* 5.9 6.25 1.457±0.086** 10.3 1.489±0.087* 7.4 12.5 1.314±0.077** 19.1 1.402±0.058**# 12.8 25 1.103±0.099** 32.1 1.110±0.080** 30.9 50 0.766±0.040** 52.8 0.822±0.076** 48.9 100 0.543±0.024** 66.6 0.624±0.055**## 61.1 注:与空白对照组比较,*P<0.05,**P<0.01;与醇提物组比较,#P<0.05,##P<0.01。 表 2 复方虎杖方提取物对HepG2.2.15细胞分泌HBsAg、HBeAg的抑制作用(x±s, n=3)
组别 浓度/ (mg·mL-1) HBsAg HBeAg 含量/ (×10-6 U·mL-1) 抑制率/% 含量/ (×10-6 U·mL-1) 抑制率/% 空白对照组 - 40.65±3.53 - 143.27±2.97 - 拉米夫定组 0.05 27.67±0.15**## 31.93 84.05±5.70**## 41.33 醇提物组 6.25 16.71±2.22** 58.89 108.77±3.99** 24.08 12.5 10.88±1.02** 73.23 105.20±1.89** 26.57 25 7.58±0.24** 81.35 90.98±7.43** 36.50 50 2.68±0.28** 93.41 27.64±2.90** 80.71 100 2.64±0.11** 93.51 24.47±2.09** 82.92 水提物组 6.25 13.01±1.17** 68.00 94.97±7.43** 33.71 12.5 10.15±0.11** 75.03 92.93±8.24** 35.14 25 8.92±0.49**# 78.06 96.79±2.50** 32.44 50 5.24±0.36**## 87.11 91.67±8.75**## 36.02 100 4.22±0.23**## 89.62 84.64±6.57**## 40.92 注:与空白对照组比较,**P<0.01;与醇提物组比较,#P<0.05,##P<0.01。 表 3 复方虎杖方在HepG2 2.2.15细胞培养中对HBV-DNA含量的影响(x±s,n=3)
组别 浓度/ (mg·mL-1) HBV-DNA/ lg(copies·mL-1) 抑制率/% 空白对照组 - 5.493±0.096 - 拉米夫定组 0.05 3.200±0.164**# 41.74 醇提物组 6.25 4.993±0.141* 9.10 12.5 4.453±0.138** 18.93 25 4.099±0.154** 25.38 50 3.736±0.203** 31.99 100 3.601±0.255** 34.44 水提物组 6.25 5.658±0.106## - 12.5 5.342±0.100## 2.75 25 5.315±0.198## 3.24 50 5.059±0.094**## 7.90 100 5.342±0.100## 2.75 注:与空白对照组比较,*P<0.05, **P<0.01;与醇提物组比较,#P<0.05,##P<0.01。 表 4 复方虎杖方对体内DHBV-DNA的影响(x±s)
组别 剂量/ (g·kg-1) n 灰度值 抑制率/% D0 D5 D10 P3 D5 D10 P3 病毒对照组 - 7 878 484±141 937 1 059 275±154 578 993 071±141 462 1 012 491±178 209 -24.5 -17.6 -18.3 醇提物组 45 6 801 851±18 090 658 557±116 629* 679 085±261 114 411 827±292 410* 18.0 15.3 48.6 15 6 801 643±50 351 753 145±186 445 655 449±208 628 5 017 168±215 876* 5.5 17.9 37.7 5 6 824 137±18 037 448 840±283 521* 461 559±245 227* 260 467±193 381** 45.8 43.8 68.3 拉米夫定组 0.2 4 841 472±30 679 114 331±47 396** 211 554±84 580** 441 942±357 087 86.6 75.0 48.2 注:与D0比较,*P<0.05,**P<0.01。 -
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