三种传统配伍中药抗氧化特性的研究
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摘要
研究发现,自由基对机体的氧化损伤与人体的多种疾病有着紧密的关系。中草药是抗氧化成分的重要来源,许多研究发现中草药的药理作用与抗氧化活性成分有关。我国有丰富的中草药资源,其中有201种中药因其安全性比较明确已被列为可直接应用到保健品或食品中。传统中药源功能食品常以复方形式出现,药对是复方的基本单位。因此,筛选和分析药对中的抗氧化活性物质及药理作用,对开发中药源食品具有重要的理论和实际意义。
据文献记载黄芪、甘草和白芍均有较强的抗氧化功能。本论文研究了这三种中药及其药对不同有机溶剂提取物抗氧化活性的协同增效作用;研究了黄芪甘草药对不同溶剂提取物的抗氧化活性变化特性,分析了黄芪甘草药对体外保护细胞氧化损伤的生理机制,并初步对其中的抗氧化物质基础进行了初步鉴定。主要研究结果如下:
1、黄芪、甘草和白芍中药不同有机溶剂提取物抗氧化活性的研究发现,中药配伍之后,在清除DPPH自由基能力上并不是单药的量的加和,配伍后清除能力都有不同程度的增加。并且清除能力的强弱受提取溶剂的极性影响,极性更大的溶剂萃取得到的中药提取物其清除能力更强;白芍-甘草、黄芪-甘草清除自由基能力均与其总酚含量存在较好的线性关系,表明总酚物质在本研究的中药抗氧化方面起了重要作用。
2、黄芪甘草药对不同极性萃取相抗氧化活性的研究发现,不同极性溶剂的萃取物的总酚、黄酮含量和抗氧化能力不同;药对中乙酸乙酯相的总酚酮含量和抗氧化能力最强,表明乙酸乙酯萃取物中总有效成分的含量较高;黄芪甘草配伍与单药的理论加和值比较呈现出更好的抗氧化活性;药对的协同作用与其总酚和黄酮物质的含量增加有关,尤其是总酚类物质的增量;经筛选得出配伍后抗氧化能力最强以及增效最好的萃取相为黄芪甘草药对的乙酸乙酯相(EA-AG)。
3、EA-AG对H_2O_2导致的MRC-5细胞氧化损伤具有一定的保护作用,能显著提高细胞的SOD和GSH-Px酶活性,降低H_2O_2引起的MDA的积累。这表明EA-AG还可能通过诱导细胞内抗氧化酶活性减轻H_2O_2的氧化胁迫伤害。初步研究发现,EA-AG在5 mg/L ~ 160 mg/L浓度范围内,对HepG_2细胞有抑制作用,且呈现明显的浓度依赖关系。这表明EA-AG具有一定的抑制癌细胞增殖的作用。
4、通过HPLC-DPPH法分析黄芪甘草配伍前后抗氧化成分的变化,从乙酸乙酯提取物中筛选出了9个潜在的抗氧化成分。经HPLC-DAD和HPLLC-MS/MS结构鉴定分析可知,其中大部分都是酚类、类黄酮物质。
总之,黄芪甘草配伍并结合适当溶剂萃取方法可以获得更高抗氧化能力的中药提取物,为今后开发功能因子明确的中药源功能食品及寻找优良的天然抗氧化剂或膳食补充剂提供了新的思路。
Recent studies suggested that a variety of diseases of human was related to free radical oxidative damage. Traditional Chinese herbs were an important source of antioxidant agents, and many studies indicated that the pharmacological effects of herbs were relevant to their antioxidant components. China had abundant resources of herbs, 201 kinds of herbs among which had been applied directly to health care products or food for its definite safety. Traditional herbal functional foods often presented in the form of formula, in which the herb pair was the basic unit. Therefore, it was important in theoretical and practical to develop herbal health protection food by investigating the antioxidant activity of substances and pharmacological of herb pairs.
According to literature records, Astragalus membranaceus, Glycyrrhiza uralensis and Paeonia lactifora all had strong antioxidant activities. In this study, antioxidant synergies on different solvent extracts of the three herbs and herb pairs were discussed. The antioxidant activity changes and the physiological mechanisms on protecting cells from oxidative injury and of AG extracts were analyzed. And the pilot study on the basis of antioxidant substances was identified. The major results were as follows:
1. In the study on antioxidant activity of different organic solvent extracts of Astragalus membranaceus, Glycyrrhiza uralensis and Paeonia lactifora,we found that the increase of DPPH free radical scavenging capacity was not only in the amount after combining the herbs. And DPPH free radical scavenging capacity was influenced by the polarity of solvents. More polar solvent extraction of herbs presented stronger capacity. There was a good linear relationship between the free radical scavenging capacity and total phenol content of extracts in PG or AG. It indicated that total phenolic substances had played an important role in the study on antioxidant activity of herbs.
2. According to the study on antioxidant activity of different polar extractions of AME, GU and herb pair,we found that total phenol, flavonoid content and antioxidant capacity were different in various polar extractions. It had the highest total phenol, flavonoid content and antioxidant capacity in ethyl acetate extraction of AG, indicating that the content of total active ingredients were higher in ethyl acetate extraction. The extracts of herb pair showed a better antioxidant activity compared with their respective theoretical sum. The synergy of AG was related to the total phenolic/flavonoid increments, especially that of total phenolics. From the study, we could obtain that EA-AG had the strongest antioxidant capacity and the best synergy efficiency after combining.
3. EA-AG could have a protective effect on H2O2 induced oxidative damage in MRC-5 cells. EA-AG could significantly improve the SOD and GSH-Px activity, and reduce the accumulation of MDA induced by H2O2. It indicated that EA-AG might also reduce oxidative stress damage of H2O2 by inducing the activity of intracellular antioxidant enzymes. Preliminary study found that EA-AG in the concentration range of 5 mg / L ~ 160 mg / L could inhibit the multiplication of HepG2 cells, and it also showed a clearly concentration-dependent manner. This indicated that EA-AG had a certain inhibition of the multiplication of cancer cell.
4. According to changes of HPLC-DPPH analysis before and after antioxidant of AG, we selected 9 kinds of potential antioxidant substances from EA-AG , which proved to be phenols and flavonoids through HPLC-DAD and HPLLC-MS/MS structural identification analysis.
In short, the different polar solvent-extracted method of AME and GU combination could obtain a higher antioxidant capacity. These data provided some useful information for the Chinese health protection food cooked with medicinal herbs, and also offered a theoretical basis for further development and potential application of new, natural antioxidant-containing food materials in functional foods.
据文献记载黄芪、甘草和白芍均有较强的抗氧化功能。本论文研究了这三种中药及其药对不同有机溶剂提取物抗氧化活性的协同增效作用;研究了黄芪甘草药对不同溶剂提取物的抗氧化活性变化特性,分析了黄芪甘草药对体外保护细胞氧化损伤的生理机制,并初步对其中的抗氧化物质基础进行了初步鉴定。主要研究结果如下:
1、黄芪、甘草和白芍中药不同有机溶剂提取物抗氧化活性的研究发现,中药配伍之后,在清除DPPH自由基能力上并不是单药的量的加和,配伍后清除能力都有不同程度的增加。并且清除能力的强弱受提取溶剂的极性影响,极性更大的溶剂萃取得到的中药提取物其清除能力更强;白芍-甘草、黄芪-甘草清除自由基能力均与其总酚含量存在较好的线性关系,表明总酚物质在本研究的中药抗氧化方面起了重要作用。
2、黄芪甘草药对不同极性萃取相抗氧化活性的研究发现,不同极性溶剂的萃取物的总酚、黄酮含量和抗氧化能力不同;药对中乙酸乙酯相的总酚酮含量和抗氧化能力最强,表明乙酸乙酯萃取物中总有效成分的含量较高;黄芪甘草配伍与单药的理论加和值比较呈现出更好的抗氧化活性;药对的协同作用与其总酚和黄酮物质的含量增加有关,尤其是总酚类物质的增量;经筛选得出配伍后抗氧化能力最强以及增效最好的萃取相为黄芪甘草药对的乙酸乙酯相(EA-AG)。
3、EA-AG对H_2O_2导致的MRC-5细胞氧化损伤具有一定的保护作用,能显著提高细胞的SOD和GSH-Px酶活性,降低H_2O_2引起的MDA的积累。这表明EA-AG还可能通过诱导细胞内抗氧化酶活性减轻H_2O_2的氧化胁迫伤害。初步研究发现,EA-AG在5 mg/L ~ 160 mg/L浓度范围内,对HepG_2细胞有抑制作用,且呈现明显的浓度依赖关系。这表明EA-AG具有一定的抑制癌细胞增殖的作用。
4、通过HPLC-DPPH法分析黄芪甘草配伍前后抗氧化成分的变化,从乙酸乙酯提取物中筛选出了9个潜在的抗氧化成分。经HPLC-DAD和HPLLC-MS/MS结构鉴定分析可知,其中大部分都是酚类、类黄酮物质。
总之,黄芪甘草配伍并结合适当溶剂萃取方法可以获得更高抗氧化能力的中药提取物,为今后开发功能因子明确的中药源功能食品及寻找优良的天然抗氧化剂或膳食补充剂提供了新的思路。
Recent studies suggested that a variety of diseases of human was related to free radical oxidative damage. Traditional Chinese herbs were an important source of antioxidant agents, and many studies indicated that the pharmacological effects of herbs were relevant to their antioxidant components. China had abundant resources of herbs, 201 kinds of herbs among which had been applied directly to health care products or food for its definite safety. Traditional herbal functional foods often presented in the form of formula, in which the herb pair was the basic unit. Therefore, it was important in theoretical and practical to develop herbal health protection food by investigating the antioxidant activity of substances and pharmacological of herb pairs.
According to literature records, Astragalus membranaceus, Glycyrrhiza uralensis and Paeonia lactifora all had strong antioxidant activities. In this study, antioxidant synergies on different solvent extracts of the three herbs and herb pairs were discussed. The antioxidant activity changes and the physiological mechanisms on protecting cells from oxidative injury and of AG extracts were analyzed. And the pilot study on the basis of antioxidant substances was identified. The major results were as follows:
1. In the study on antioxidant activity of different organic solvent extracts of Astragalus membranaceus, Glycyrrhiza uralensis and Paeonia lactifora,we found that the increase of DPPH free radical scavenging capacity was not only in the amount after combining the herbs. And DPPH free radical scavenging capacity was influenced by the polarity of solvents. More polar solvent extraction of herbs presented stronger capacity. There was a good linear relationship between the free radical scavenging capacity and total phenol content of extracts in PG or AG. It indicated that total phenolic substances had played an important role in the study on antioxidant activity of herbs.
2. According to the study on antioxidant activity of different polar extractions of AME, GU and herb pair,we found that total phenol, flavonoid content and antioxidant capacity were different in various polar extractions. It had the highest total phenol, flavonoid content and antioxidant capacity in ethyl acetate extraction of AG, indicating that the content of total active ingredients were higher in ethyl acetate extraction. The extracts of herb pair showed a better antioxidant activity compared with their respective theoretical sum. The synergy of AG was related to the total phenolic/flavonoid increments, especially that of total phenolics. From the study, we could obtain that EA-AG had the strongest antioxidant capacity and the best synergy efficiency after combining.
3. EA-AG could have a protective effect on H2O2 induced oxidative damage in MRC-5 cells. EA-AG could significantly improve the SOD and GSH-Px activity, and reduce the accumulation of MDA induced by H2O2. It indicated that EA-AG might also reduce oxidative stress damage of H2O2 by inducing the activity of intracellular antioxidant enzymes. Preliminary study found that EA-AG in the concentration range of 5 mg / L ~ 160 mg / L could inhibit the multiplication of HepG2 cells, and it also showed a clearly concentration-dependent manner. This indicated that EA-AG had a certain inhibition of the multiplication of cancer cell.
4. According to changes of HPLC-DPPH analysis before and after antioxidant of AG, we selected 9 kinds of potential antioxidant substances from EA-AG , which proved to be phenols and flavonoids through HPLC-DAD and HPLLC-MS/MS structural identification analysis.
In short, the different polar solvent-extracted method of AME and GU combination could obtain a higher antioxidant capacity. These data provided some useful information for the Chinese health protection food cooked with medicinal herbs, and also offered a theoretical basis for further development and potential application of new, natural antioxidant-containing food materials in functional foods.
引文
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Brand-Williams W, Cuvelier ME, & Berset C, Use of a free radical method to evaluate antioxidant activity. Lebensm Wiss Technol, 1995, 28(1): 25-30.
Cai YZ, Luo Q, & Sun M, et al, Antioxidant activity and phenolic compounds of 112 traditional Chinese medicinal plants associated with anticancer. Life Sciences, 2004, (74): 2157-2184.
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Lee SE, Hwang HJ, & Ha JS, et al., Screening of medicinal plant extracts for antioxidant activity. Life Sciences, 2003, (73): 167-179.
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Jia ZS, Tang MC, & Wu JM, The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chemistry, 1999, (64): 555-559.
Jackson KM, & Deleon M, Dibenzoylmethane induces cell cycle deregulation in human prostate cancer cells. Cancer Letters, 2002, (178): 161-165.
Kanazawa M, Satomi Y, & Mizutani Y, et al., Isoliquiritigenin inhibits the growth of prostate cancer. European Urology, 2003, (43): 580-586.
Kimura M, Inoue H, & Kazuhiro Hirabayashi K, et al., Glycyrrhizin and some analogues induce growth of primary cultured adult rat hepatocytes via epidermal growth factor receptors. European Journal of Pharmacology,2001, (431): 151-161.
Kulisic TA, Radonic V, & Katalinic MM, Use of different methods for testing antioxidative activity of oregano essential oil. Food Chemistry, 2004, (85): 633-640.
Lee SE, Hwang HJ, & Ha JS, et al., Screening of medicinal plant extracts for antioxidant activity. Life Sciences, 2003, (73): 167-179.
Leung YK, Ng TB, & Ho JW, Transcriptional regulation of fosl-1 by licorice in rat clone 9 cells. Life Sciences, 2003, (73): 3109-3121.
Li P, Tabibi SE, & Yalkowsky SH, Combined Effect of Complexation and pH on Solubilization [J]. Pharm. Sci, 1998, (87): 1535-1537.
Li R, Fu TJ, & Ji YQ, et al., A Study of the Roots of Astragalus Mongholicus and Astragalus Membranaceus by High Performance Liquid Chromatography-Mass Spectrometry [J]. Chinese Journal of Analytical Chemistry, 2005, (33): 1676-1680.
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Ma T, Cao YL, & Bai H, et al., The protective effect of the extracts of Glyccyrrhiza radix on the liver injury induced by pentachloronitrobenzene. J Shenyang Phamaceutical Univ, 2002, (19): 275-277.
Miller NJ, Rice-Evans C, & Davies MJ, et al., A novel method for measuring antioxidant capacity and its application to monitoring the antioxidant status in premature neonates.[J]. Clin Sci, 1993, (84): 407-412.
Mitsuyama S, Role of oxidative stress in angiotensin II-induced heart failure [J]. Nippon Rinsho, 2007, 65, Suppl 4(8): 243-250.
Moure A, Cruz JM, & Franco D, et al., Natural antioxidants from residual sources [J]. Food Chemistry, 2001, (72): 145-171.
Okuda T, Antioxidant in herbs: poyphenols (1999). In Packer L, Midori H, & Toshikazu Y.
Park JM, Kim SD, Lee WM, Cho JY, Par HJ, Kim TW, Cho NHe, Kim SK, & Rhee MH, In vitro anti-oxidative and anti-inflammatory effects of solvent-extracted fractions from Suaeda asparagoides. Pharmazie, 2007, (62): 453-458.
Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, & Rice-Evans C, Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic Biol Med, 1999, 26(9-10): 1231-1237.
Rice-evens C, Halliwell B, & Lunt GG, Free Radicals and Oxidative Stress: Environment, Drugs and Food Additives. 1995, London: Portland Press.
Robin van den Berg, & Guido R M M Haenen, Applicability of an improved Trolox equivalent antioxidant capacity assay for evaluation of antioxidant capacity measurements of mixtures [J]. Food Chemistry, 1999, (66): 511-517.
Silvana Tommasini DR, Rita Ficarra, Maria Luisa Calabrò, Rosanna Stancanelli, & Paola Ficarra, Improvement in solubility and dissolution rate of flavonoids by complexation withβ-cyclodextrin. Journal of Pharmaceutical and Biomedical Analysis, 2004, (35): 379-387.
Singleton VL, & Rossi JA, Colorimetry of total phenolics with phosphor-molybdic-phosphotungstic acid reagent. American Journal of Enology and Viticulture, 1965, 16(3): 144-158.
Tamirs S, Eizenberg M, & Somjen D, et al., Estrogen-like activity of glabrene and other constituents isolated from licorice root. Journal of Steroid Biochemistry &Molecular Biology, 2001, (78): 291-298.
Taso R, & Deng ZY, Seperation procedures for naturally occurring antioxidant phytochemicals [J]. Journal Chromatography B, 2004, (812): 85-99.
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