8种传统中药药对抗氧化协同机制的研究
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摘要
中草药常以方剂的形式广泛应用于功能保健食品中,很多中草药在配伍使用时表现出比单独使用更好的药理功能,但这种协同效应的药理学机制至今仍不明确。研究发现,中药的药理学效应与其抗氧化特性有着密切的关系。因此,本论文应用DPPH自由基清除能力试验研究了黄芪、白芍、白术、甘草、当归、大黄6种常用中草药组成的8种传统药对(黄芪-当归、黄芪-白术、黄芪-白芍、黄芪-甘草、黄芪-大黄、白芍-甘草、白芍-白术、白芍-当归)的抗氧化活性。研究结果表明,这8种传统药对配伍之后的DPPH自由基清除能力均显著高于各自单药的理论加和(P<0.05),这表明8种药对在配伍使用时均具有良好的抗氧化协同效应。
多酚和黄酮类化合物是中草药中一类重要的抗氧化物质。因此,我们进一步分析了8种药对及其单药中多酚、黄酮的含量,并对药对配伍前后多酚、黄酮含量的变化率与各药对抗氧化能力(DPPH自由基清除能力)的增强率之间的相关性进行了分析,发现药对中黄酮类化合物的变化率与其抗氧化能力的增强率之间具有显著的相关性(Spearman相关系数为RREAC-RIFC=0.559, P=0.024),因此,黄酮类化合物可能是对8种药对的协同效应起主要作用的物质。
为了进一步分析药对配伍后的协同效应,我们选择了8种药对中在DPPH自由基清除试验中协同效应最好的3种药对(黄芪-甘草、黄芪-白芍和黄芪-白术)进行了羟基自由基和超氧阴离子自由基清除能力分析,结果发现,3种药对在羟基自由基和超氧阴离子自由基清除能力方面同样具有较显著的协同效应,进一步分析发现,黄芪-白术药对的羟基自由基和超氧阴离子自由基清除能力(0.2 g/mL)均显著高于黄芪和白术两个单药的清除能力。
在黄芪-白芍药对抑制氯化镉引起的MRC-5细胞活性降低的研究中,氯化镉(CdCl2,20μmol/L)能够显著引起MRC-5细胞的活性下降(P<0.05),而黄芪(10-3g/mL)对CdCl2引起的细胞活性下降具有显著的抑制作用,且其抑制效果较强于白芍(10-3g/mL)。黄芪-白芍(10-3g/mL)的抑制效果高于白芍却弱于黄芪的抑制效果。
在研究黄芪-白芍药对CdCl2引起的MRC-5细胞氧化损伤的保护作用中发现,CdCl2(20μmol/L)能够显著降低细胞的Mn-SOD、Cu/Zn-SOD和GSH-Px活性(P<0.05),增加MDA含量;白芍可通过诱导GSH-Px和Cu/Zn-SOD活性增强和降低MDA含量来抑制CdCl2引起的氧化损伤;黄芪对Mn-SOD、Cu/Zn-SOD和GSH-Px三者都具有显著的诱导作用,但对Mn-SOD的诱导效应显著高于白芍,对GSH-Px的诱导效应却显著低于白芍,对MDA含量的影响不显著;黄芪-白芍药对对Mn-SOD、GSH-Px和Cu/Zn-SOD三者活性均具有显著的诱导作用,尤其是对Cu/Zn-SOD的诱导效应显著高于两种单药,且黄芪-白芍药对处理组的MDA含量显著低于黄芪处理组和白芍处理组中MDA含量的理论加和(3.22<(6.85+2.66)/2;P<0.05),这表明两者配伍后在抑制CdCl2引起的酶活降低和氧化损伤方面具有较好的协同作用。这从细胞水平进一步验证了黄芪-白芍药对在抗氧化作用方面具有良好的协同效应,为进一步研发中草药来源的抗氧化功能食品奠定了理论基础。
Many Chinese therapeutic herbs widely used in functional food in the form of formula, and many of them are demonstrated significantly better pharmacological effects when used in the combination than when used alone. However, the pharmacological mechanism for this synergism is still not well understood. Researches show that pharmacologic effects of herbs are closely relevant to their antioxidant properties. Therefore, in the present study, the antioxidant activities of six common herbs ((Paeonia lactiflora (PL), Atractylodes macrocephala (AMA), Angelica sinensis (AS), Astragalus membranaceus (AME), Glycyrrhiza uralensis (GU) and Rheum officinale (RO)), which were historically combined into eight traditional Chinese herb pairs (TCHPs) (AME-AS, AME-AMA, AME-RO, AME-GU, AME-PL, PL-AS, PL-AMA and PL-GU), were investigated in vitro by assessing the 1,1-diphenyl-2-picryl hydrazine (DPPH)-radical scavenging abilities of the herbs. The results of this study showed that all eight TCHPs had a significantly larger scavenging capacity than would be expected from the theoretical sum of those of the respective constituent herbs (P<0.05).
As phenolic and flavonoid compounds are excellent antioxidant in herbs, the changes in the total phenolic and flavonoid contents were also investigated. Our study showed a significant correlation between the rate of enhancement in antioxidant capacity and the rate of increase in flavonoid content ( the Spearman correlation coefficients were: RREAC-RIFC=0.559, P=0.024). Thus, the flavonoids are likely responsible for the synergistic effects present in the eight TCHPs.
In order to further study the synergistic effect, we select three TCHPs (AME-GU, AME-PL and AME-AMA pairs) which showed a better synergistic effect in the DPPH scavenging assay for the hydroxyl radical and superoxide radical anion scavenging assays. Results demonstrated similar conclusions in hydroxyl radical and superoxide radical anion scavenging assays. Interestingly, the AME-AMA combination had a significantly higher superoxide anion (0.2 g/mL) and hydroxyl radical scavenging ability than the AME or AMA.
During the assay of the effect of AME-PL on decrease of MRC-5 cell viability induced by CdCl2, we found that 20μmol/L CdCl2 can significantly result the reduction of MRC-5 cell viability (P<0.05) which can be inhibited by 10-3g/mL AME, 10-3g/mL PL have the weaker inhibited capacity and AME-PL have the middle capacity.
We found in the assay of protective effect of the AME-PL on oxidative injury induced by CdCl2 in MRC-5 cells that CdCl2 can reduce the activities of Mn-SOD ,Cu/Zn-SOD and GSH-Px (P<0.05) and increase the content of MDA, while PL can increase the Cu/Zn-SOD and GSH-Px activities and have lower MDA content. AME can enhance all the activities of Mn-SOD, Cu/Zn-SOD and GSH-Px but have no significantly effect on MDA content. AME-PL can not only enhance all three enzymes activities especially Cu/Zn-SOD but also decrease the MDA content which lower than the theoretical sum of those of AME and PL (3.22<(6.85+2.66)/2, P<0.05). This also demonstrate the excellent synergistic effect of AME-PL on the basis of cell level, which lay a theoretical foundation for the further studies on the herbs-source functional foods.
多酚和黄酮类化合物是中草药中一类重要的抗氧化物质。因此,我们进一步分析了8种药对及其单药中多酚、黄酮的含量,并对药对配伍前后多酚、黄酮含量的变化率与各药对抗氧化能力(DPPH自由基清除能力)的增强率之间的相关性进行了分析,发现药对中黄酮类化合物的变化率与其抗氧化能力的增强率之间具有显著的相关性(Spearman相关系数为RREAC-RIFC=0.559, P=0.024),因此,黄酮类化合物可能是对8种药对的协同效应起主要作用的物质。
为了进一步分析药对配伍后的协同效应,我们选择了8种药对中在DPPH自由基清除试验中协同效应最好的3种药对(黄芪-甘草、黄芪-白芍和黄芪-白术)进行了羟基自由基和超氧阴离子自由基清除能力分析,结果发现,3种药对在羟基自由基和超氧阴离子自由基清除能力方面同样具有较显著的协同效应,进一步分析发现,黄芪-白术药对的羟基自由基和超氧阴离子自由基清除能力(0.2 g/mL)均显著高于黄芪和白术两个单药的清除能力。
在黄芪-白芍药对抑制氯化镉引起的MRC-5细胞活性降低的研究中,氯化镉(CdCl2,20μmol/L)能够显著引起MRC-5细胞的活性下降(P<0.05),而黄芪(10-3g/mL)对CdCl2引起的细胞活性下降具有显著的抑制作用,且其抑制效果较强于白芍(10-3g/mL)。黄芪-白芍(10-3g/mL)的抑制效果高于白芍却弱于黄芪的抑制效果。
在研究黄芪-白芍药对CdCl2引起的MRC-5细胞氧化损伤的保护作用中发现,CdCl2(20μmol/L)能够显著降低细胞的Mn-SOD、Cu/Zn-SOD和GSH-Px活性(P<0.05),增加MDA含量;白芍可通过诱导GSH-Px和Cu/Zn-SOD活性增强和降低MDA含量来抑制CdCl2引起的氧化损伤;黄芪对Mn-SOD、Cu/Zn-SOD和GSH-Px三者都具有显著的诱导作用,但对Mn-SOD的诱导效应显著高于白芍,对GSH-Px的诱导效应却显著低于白芍,对MDA含量的影响不显著;黄芪-白芍药对对Mn-SOD、GSH-Px和Cu/Zn-SOD三者活性均具有显著的诱导作用,尤其是对Cu/Zn-SOD的诱导效应显著高于两种单药,且黄芪-白芍药对处理组的MDA含量显著低于黄芪处理组和白芍处理组中MDA含量的理论加和(3.22<(6.85+2.66)/2;P<0.05),这表明两者配伍后在抑制CdCl2引起的酶活降低和氧化损伤方面具有较好的协同作用。这从细胞水平进一步验证了黄芪-白芍药对在抗氧化作用方面具有良好的协同效应,为进一步研发中草药来源的抗氧化功能食品奠定了理论基础。
Many Chinese therapeutic herbs widely used in functional food in the form of formula, and many of them are demonstrated significantly better pharmacological effects when used in the combination than when used alone. However, the pharmacological mechanism for this synergism is still not well understood. Researches show that pharmacologic effects of herbs are closely relevant to their antioxidant properties. Therefore, in the present study, the antioxidant activities of six common herbs ((Paeonia lactiflora (PL), Atractylodes macrocephala (AMA), Angelica sinensis (AS), Astragalus membranaceus (AME), Glycyrrhiza uralensis (GU) and Rheum officinale (RO)), which were historically combined into eight traditional Chinese herb pairs (TCHPs) (AME-AS, AME-AMA, AME-RO, AME-GU, AME-PL, PL-AS, PL-AMA and PL-GU), were investigated in vitro by assessing the 1,1-diphenyl-2-picryl hydrazine (DPPH)-radical scavenging abilities of the herbs. The results of this study showed that all eight TCHPs had a significantly larger scavenging capacity than would be expected from the theoretical sum of those of the respective constituent herbs (P<0.05).
As phenolic and flavonoid compounds are excellent antioxidant in herbs, the changes in the total phenolic and flavonoid contents were also investigated. Our study showed a significant correlation between the rate of enhancement in antioxidant capacity and the rate of increase in flavonoid content ( the Spearman correlation coefficients were: RREAC-RIFC=0.559, P=0.024). Thus, the flavonoids are likely responsible for the synergistic effects present in the eight TCHPs.
In order to further study the synergistic effect, we select three TCHPs (AME-GU, AME-PL and AME-AMA pairs) which showed a better synergistic effect in the DPPH scavenging assay for the hydroxyl radical and superoxide radical anion scavenging assays. Results demonstrated similar conclusions in hydroxyl radical and superoxide radical anion scavenging assays. Interestingly, the AME-AMA combination had a significantly higher superoxide anion (0.2 g/mL) and hydroxyl radical scavenging ability than the AME or AMA.
During the assay of the effect of AME-PL on decrease of MRC-5 cell viability induced by CdCl2, we found that 20μmol/L CdCl2 can significantly result the reduction of MRC-5 cell viability (P<0.05) which can be inhibited by 10-3g/mL AME, 10-3g/mL PL have the weaker inhibited capacity and AME-PL have the middle capacity.
We found in the assay of protective effect of the AME-PL on oxidative injury induced by CdCl2 in MRC-5 cells that CdCl2 can reduce the activities of Mn-SOD ,Cu/Zn-SOD and GSH-Px (P<0.05) and increase the content of MDA, while PL can increase the Cu/Zn-SOD and GSH-Px activities and have lower MDA content. AME can enhance all the activities of Mn-SOD, Cu/Zn-SOD and GSH-Px but have no significantly effect on MDA content. AME-PL can not only enhance all three enzymes activities especially Cu/Zn-SOD but also decrease the MDA content which lower than the theoretical sum of those of AME and PL (3.22<(6.85+2.66)/2, P<0.05). This also demonstrate the excellent synergistic effect of AME-PL on the basis of cell level, which lay a theoretical foundation for the further studies on the herbs-source functional foods.
引文
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