黄芪多糖和板蓝根多糖对鸡胚成纤维细胞增殖和猪细小病毒体外抑制的影响
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摘要
本研究选用中药黄芪与板蓝根作为抗病毒药物,研究了这两种中药多糖成份的免疫增强作用和机理,试验分为四部分:
1.黄芪多糖(APS)和板蓝根多糖(IRPS)的提取及含量测定先用水煎醇沉法分离提取,再用硫酸蒽酮法测定APS、IRPS含量,其含量分别为15.3g/kg和11.3g/kg,提取率分别为85.36%和85.43%。
2. APS和IRPS对鸡胚成纤维细胞(CEF)安全浓度的测定将已提取获得的黄芪和板蓝根多糖用细胞维持培养液分别稀释成系列浓度,与CEF同时或待CEF形成单层后加入到培养板中,通过测定细胞OD值和观察细胞形态的变化判定安全浓度的范围。结果表明,APS和IRPS的最大安全浓度分别为1.50 mg/mL和0.60 mg/mL。
3. APS和IRPS对CEF增殖的影响将APS和IRPS分别稀释成安全浓度范围内的五种浓度,分别与CEF同时或细胞形成单层(CEF培养24 h后)加入培养板,分别在加药后24,36,48,60和72 h,用MTT法测定细胞OD值的动态变化,判定两种多糖对CEF增殖的影响。结果表明,与CEF同时加入时,APS和IRPS均显著促进CEF增殖;CEF细胞形成单层后再加入时,APS和IRPS同样能促进细胞增殖,且具有一定量效和时效关系。
4. APS和IRPS对猪细小病毒的体外抑制作用利用细胞病变(CPE)抑制试验,测定APS与IRPS单独使用及等体积合并时在PK-15单层细胞上对猪细小病毒(PPV)的抑制作用。结果表明,黄芪单独使用时对PPV无明显的抑制作用;IRPS单独使用时,对PPV有明显的抑制作用,最小直接杀灭浓度为0.58 mg/mL,最小阻断浓度为0.28 mg/mL;APS与IRPS等体积合并时,对PPV的抑制作用显著增强,最小直接杀灭浓度提高为0.29 mg/mL,最小阻断浓度提高为0.070 mg/mL。
Astragalus and Isatis root were adopted as antiviral drugs to study the mechanism and immunological enhancement. The experiment was devided into 4 section, the details were summarized as following.
1. Exaction and Determination of APS and IRPS.
The two polysaccharides were extracted by decocting+ethanol precipitating. The contents of two polysaccharides were tested by Vitriol-anthracene ketone. The net contents of APS and IRPS were 15.3 g and 11.3 g and the exactive rates were 85.36% and 85.43%, respectively.
2. Determination of Safe Concentration of APS and IRPS ON CEF
APS and IRPS were diluted with MEM medium into series of concentrations and were added in culture plates in two ways, at the beginning of culture and after CEF monolayer formation. Their maximal safety concentrations to CEF were judged according to OD value and morphological variety of the cells. The results showed that the maximal safety concentrations of APS and IRPS were 1 500 mg/mL and 650 mg/mL respectively.
3. Effects of APS and IRPS on CEF proliferating
APS and IRPS were diluted into five different levels within safety concentration were added into culture plates in two ways described previously. The cells OD were analysed after 24, 36, 48, 60 and 72 hours cultivation for the former groups and for latter groups to judge the effects of APS and IRPS on cell proliferation by MTT method. The results showed that APS and IRPS added with CEF simultaneously in the former groups could stimulate cell proliferation. Cell proliferation was also promoted by APS and IRPS added after CEF monolayer formation. It seemed that there were dosage and time-effect relationships.
4. Inhibitive effect of porcine parvovirus by APS and IRPS in vitro
Inhibitive effect of porcine parvovirus was analysed on pig kidney (PK-15) cell monolayer of antiviral drugs by utilizing the cytopthic effect (CPE) inhibitive experiment when APS and IRPS were used alone and association by equal volumes. It was showed that APS had no obvious inhibitive effect on PPV by single use, but the IRPS had obvious inhibitive effect. the MIC was 0.58 mg/mL, MSC was 0.28 mg/mL; The anti-PPV cutivities of 1:1 mixture of APS and IRPS were significantly higher than that of Radix lsatidis, their MIC was 0.29 mg/mL and their MSC is 0.070 mg/mL.
1.黄芪多糖(APS)和板蓝根多糖(IRPS)的提取及含量测定先用水煎醇沉法分离提取,再用硫酸蒽酮法测定APS、IRPS含量,其含量分别为15.3g/kg和11.3g/kg,提取率分别为85.36%和85.43%。
2. APS和IRPS对鸡胚成纤维细胞(CEF)安全浓度的测定将已提取获得的黄芪和板蓝根多糖用细胞维持培养液分别稀释成系列浓度,与CEF同时或待CEF形成单层后加入到培养板中,通过测定细胞OD值和观察细胞形态的变化判定安全浓度的范围。结果表明,APS和IRPS的最大安全浓度分别为1.50 mg/mL和0.60 mg/mL。
3. APS和IRPS对CEF增殖的影响将APS和IRPS分别稀释成安全浓度范围内的五种浓度,分别与CEF同时或细胞形成单层(CEF培养24 h后)加入培养板,分别在加药后24,36,48,60和72 h,用MTT法测定细胞OD值的动态变化,判定两种多糖对CEF增殖的影响。结果表明,与CEF同时加入时,APS和IRPS均显著促进CEF增殖;CEF细胞形成单层后再加入时,APS和IRPS同样能促进细胞增殖,且具有一定量效和时效关系。
4. APS和IRPS对猪细小病毒的体外抑制作用利用细胞病变(CPE)抑制试验,测定APS与IRPS单独使用及等体积合并时在PK-15单层细胞上对猪细小病毒(PPV)的抑制作用。结果表明,黄芪单独使用时对PPV无明显的抑制作用;IRPS单独使用时,对PPV有明显的抑制作用,最小直接杀灭浓度为0.58 mg/mL,最小阻断浓度为0.28 mg/mL;APS与IRPS等体积合并时,对PPV的抑制作用显著增强,最小直接杀灭浓度提高为0.29 mg/mL,最小阻断浓度提高为0.070 mg/mL。
Astragalus and Isatis root were adopted as antiviral drugs to study the mechanism and immunological enhancement. The experiment was devided into 4 section, the details were summarized as following.
1. Exaction and Determination of APS and IRPS.
The two polysaccharides were extracted by decocting+ethanol precipitating. The contents of two polysaccharides were tested by Vitriol-anthracene ketone. The net contents of APS and IRPS were 15.3 g and 11.3 g and the exactive rates were 85.36% and 85.43%, respectively.
2. Determination of Safe Concentration of APS and IRPS ON CEF
APS and IRPS were diluted with MEM medium into series of concentrations and were added in culture plates in two ways, at the beginning of culture and after CEF monolayer formation. Their maximal safety concentrations to CEF were judged according to OD value and morphological variety of the cells. The results showed that the maximal safety concentrations of APS and IRPS were 1 500 mg/mL and 650 mg/mL respectively.
3. Effects of APS and IRPS on CEF proliferating
APS and IRPS were diluted into five different levels within safety concentration were added into culture plates in two ways described previously. The cells OD were analysed after 24, 36, 48, 60 and 72 hours cultivation for the former groups and for latter groups to judge the effects of APS and IRPS on cell proliferation by MTT method. The results showed that APS and IRPS added with CEF simultaneously in the former groups could stimulate cell proliferation. Cell proliferation was also promoted by APS and IRPS added after CEF monolayer formation. It seemed that there were dosage and time-effect relationships.
4. Inhibitive effect of porcine parvovirus by APS and IRPS in vitro
Inhibitive effect of porcine parvovirus was analysed on pig kidney (PK-15) cell monolayer of antiviral drugs by utilizing the cytopthic effect (CPE) inhibitive experiment when APS and IRPS were used alone and association by equal volumes. It was showed that APS had no obvious inhibitive effect on PPV by single use, but the IRPS had obvious inhibitive effect. the MIC was 0.58 mg/mL, MSC was 0.28 mg/mL; The anti-PPV cutivities of 1:1 mixture of APS and IRPS were significantly higher than that of Radix lsatidis, their MIC was 0.29 mg/mL and their MSC is 0.070 mg/mL.
引文
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