杜仲叶抗疲劳功效分子机制与抗氧化作用关联性研究
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摘要
杜仲(Eucommia ulmoides)在我国有着悠久的药用历史,其具有降血压、补肾、益肝、强筋骨及安胎等功效。杜仲作为中药材的使用部位是杜仲皮,然而现代研究表明,杜仲叶与杜仲皮的成分含量相近,甚至某些成分含量较杜仲皮高。因此,杜仲叶可以代皮入药,这为解决杜仲皮资源匮乏、杜仲资源综合利用提供了很好的途径。
研究表明,杜仲叶具有抗疲劳功效,但是目前所见的相关报道大多是以杜仲叶粗提物为研究对象,并且抗疲劳功效成分尚不明确,抗疲劳作用机理也不清楚。所以,目前以杜仲叶为原料开发的抗疲劳功能食品仍属于第二代保健食品,这类保健食品存在作用机理不清楚、功效成分不明确的问题。
针对上述问题,本课题以杜仲叶为研究对象,对杜仲叶功效成分逐步进行分离纯化,采用动物试验评价各步产物的抗疲劳功效,以筛选出成分更加明确的杜仲叶抗疲劳功效成分。同时,分析各分离纯化产物体内和体外抗氧化作用,进一步研究杜仲叶抗疲劳功效与抗氧化作用之间的关联性,探索杜仲叶抗疲劳作用机理。最后,将最终得到的具有抗疲劳功效的杜仲叶纯化产物进行细致的分离,得到单体化合物,采用红外光谱和紫外光谱测定各单体的结构式,初步推测其分子结构,从分子水平上阐释杜仲叶抗疲劳作用机理。
本论文完成的研究内容如下:
1.杜仲叶粗提物试验
抗疲劳功效评价结果显示,杜仲叶水提物(EUH)和醇提物(EUE)均能显著延长小鼠负重游泳时间、降低血乳酸(LA)和血清尿素氮(SUN)含量及提高肝糖原含量,但EUE对各抗疲劳功效评价指标的影响比EUH显著,表明EUE的抗疲劳功效强于EUH。
常压耐缺氧试验结果显示,EUH和EUE不能明显地提高小鼠常压耐缺氧能力,因此,EUH和EUE并不能通过改善常压耐缺氧能力表现抗疲劳功效。
体外抗氧化试验结果显示,EUH和EUE均具有不同程度的清除羟自由基(·OH)、超氧阴离子(O_2~-·)及1,1-二苯基-2-苦苯肼自由基(DPPH·)的作用,但是EUE对各自由基的清除效果均优于EUH,这与EUE的抗疲劳功效强于EUH的结论相一致,证明杜仲叶粗提物体外抗氧化作用与抗疲劳功效正相关。
试验结果还表明,杜仲叶粗提物中的总黄酮与总多酚可能为杜仲叶粗提物表现抗疲劳功效和体外抗氧化作用的功效成分。
2.杜仲叶一次分离纯化产物试验
抗疲劳功效评价结果显示,20%乙醇洗脱液(EU1Ⅰ)和60%乙醇洗脱液(EU1Ⅱ)均能延长小鼠负重游泳时间、降低血LA和SUN含量及提高肝糖原含量,但EU1Ⅰ对各抗疲劳功效评价指标的影响比EU1Ⅱ显著,表明EU1Ⅰ具有强于EU1Ⅱ的抗疲劳功效。同时,EU1Ⅰ可以显著提高乳酸脱氢酶(LDH)的活性,表明EU1Ⅰ可以通过提高机体LDH活性降低血LA含量。
体内抗氧化作用试验结果显示,只有EU1Ⅰ可以降低丙二醛(MDA)含量、提高超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-PX)活性,而EU1Ⅱ对这三项指标的影响并不明显,表明EU1Ⅰ具有比EU1Ⅱ更显著的体内抗氧化作用,这与EU1Ⅰ的抗疲劳功效强于EU1Ⅱ的结论相一致,证明杜仲叶一次分离纯化产物体内抗氧化作用与抗疲劳功效正相关。
常压耐缺氧试验结果显示, EU1Ⅰ和EU1Ⅱ不能明显地提高小鼠常压耐缺氧能力,因此,EU1Ⅰ和EU1Ⅱ不能通过改善常压耐缺氧能力表现抗疲劳功效。
体外抗氧化试验结果显示,EU1Ⅰ和EU1Ⅱ均具有不同程度的清除·OH、O_2~-·及DPPH·的作用,但是EU1Ⅰ对各自由基的清除效果均优于EU1Ⅱ,表明EU1Ⅰ具有比EU1Ⅱ更显著的体外抗氧化作用,这与EU1Ⅰ的抗疲劳功效强于EU1Ⅱ的结论相一致,证明杜仲叶一次分离纯化产物体外抗氧化作用与抗疲劳功效正相关。量效关系试验结果表明,EU1Ⅰ和EU1Ⅱ的体外抗氧作用具有很好的量效关系。
试验结果还表明,多酚类物质可能为杜仲叶一次分离纯化产物表现抗疲劳功效和抗氧化作用的主要功效成分。
3.杜仲叶二次分离纯化产物试验
抗疲劳功效评价结果显示,乙酸乙酯萃取物(EU2Ⅰ)和正丁醇萃取物(EU2Ⅱ)对小鼠体重有一定的影响。EU2Ⅰ和EU2Ⅱ均能延长小鼠负重游泳时间、降低血LA含量及提高肝糖原含量,但只有EU2Ⅱ可以显著降低SUN含量,EU2Ⅱ对各抗疲劳功效评价指标的影响比EUⅠ显著,表明EU2Ⅱ具有强于EU2Ⅰ的抗疲劳功效。同时,只有EU2Ⅱ可以显著提高LDH活性,表明EU2Ⅱ可以通过提高机体LDH活性降低血LA含量。
体内抗氧化作用试验结果显示,EU2Ⅰ和EU2Ⅱ均可以降低MDA含量,但是,只有EU2Ⅱ可以显著提高SOD和GSH-PX活性,表明EU2Ⅱ具有比EU2Ⅰ更显著的体内抗氧化作用,这与EU2Ⅱ的抗疲劳功效强于EUⅠ的结论相一致,证明杜仲叶二次分离纯化产物体内抗氧化作用与抗疲劳功效正相关。
常压耐缺氧试验结果显示,EU2Ⅰ和EU2Ⅱ不能明显地提高小鼠常压耐缺氧能力,因此,EU2Ⅰ和EU2Ⅱ不能通过改善常压耐缺氧能力表现抗疲劳功效。
体外抗氧化试验结果显示,EU2Ⅰ和EU2Ⅱ均具有不同程度的清除·OH、O_2~-·及DPPH·的作用,但是EU2Ⅱ对各自由基的清除效果尤其显著,表明EU2Ⅱ具有更为显著的体外抗氧化作用,这与EU2Ⅱ具有更明显的抗疲劳功效的结论相一致,证明杜仲叶二次分离纯化产物体外抗氧化作用与抗疲劳功效也正相关。量效关系试验表明,EU2Ⅰ和EU2Ⅱ的体外抗氧作用也具有很好的量效关系。
试验结果还表明,多酚类物质可能为杜仲叶二次分离纯化产物表现抗疲劳功效和抗氧化作用的主要功效成分。
4.杜仲叶三次分离纯化产物试验抗疲劳功效评价结果显示,黄酮纯化产物(EU3Ⅰ)和绿原酸纯化产物(EU3Ⅱ)均能延长小鼠负重游泳时间,但是只有EU3Ⅰ可以显著降低血LA和SUN含量及提高肝糖原含量,EU3Ⅰ对各抗疲劳功效评价指标的影响比EU3Ⅱ显著,表明EU3Ⅰ具有明显的抗疲劳功效。同时,只有EU3Ⅰ可以显著提高LDH活性,表明EU3Ⅰ可以通过提高机体LDH活性降低由血LA含量。
体内抗氧化作用实验结果显示,EU3Ⅰ和EU3Ⅱ均可以降低MDA含量,但是,只有EU3Ⅱ可以显著提高SOD和GSH-PX活性,证明EU3Ⅱ具有更为明显的体内抗氧化作用。
常压耐缺氧试验结果显示,EU3Ⅰ可以提高小鼠常压耐缺氧能力,而EU3Ⅱ不能明显地提高小鼠常压耐缺氧能力。因此,EU3Ⅰ可能是通过提高小鼠常压耐缺氧能力表现抗疲劳功效,而EU3Ⅱ不能通过改善常压耐缺氧能力表现抗疲劳功效。
体外抗氧化试验结果显示,EU2Ⅰ和EU2Ⅱ均具有不同程度的清除·OH、O_2~-·及DPPH·的作用,但是EU3Ⅱ对各自由基的清除效果尤其显著,表明EU3Ⅱ具有更强的体外抗氧化能力。量效关系实验表明,EU3Ⅰ和EU3Ⅱ的体外抗氧能力同样具有很好的量效关系。
试验结果表明,EU3Ⅰ(黄酮类物质)可能是通过改善机体能量代谢提高机体抗疲劳能力;而EU3Ⅱ(绿原酸)可能是通过对中枢神经系统影响或者是与杜仲叶中多种成分协同作用的方式提高机体运动耐受力.
5.杜仲叶抗疲劳功效成分鉴定
采用硅胶柱层析和薄层层析的方法对杜仲叶正丁醇萃取纯化产物进行分离、纯化和分析,得到6种单体化合物,经红外光谱和紫外光谱初步鉴定,其中4种可能为槲皮素、表儿茶素、绿原酸和芦丁,另外2种初步推测为黄酮苷和黄酮类化合物。
从杜仲叶正丁醇萃取物中分离出的化合物多为黄酮、多酚以及黄酮苷类物质,这些成分具有显著的抗氧化活性。由此可见,抗氧化功效成分的存在使杜仲叶表现出了抗氧化作用,并且使杜仲叶显示了抗疲劳功效。
杜仲叶中抗氧化功效成分可以清除体内产生的自由基,使机体免受自由基引起的损伤,由此延缓疲劳的产生,提高机体抗疲劳能力,这就是杜仲叶抗疲劳作用机理。该机理符合抗疲劳产生机理中的自由基学说,本研究为该理论学说提供了支持,也进一步表明了杜仲叶抗疲劳功效与抗氧化作用之间的关联性。
然而,本研究没有深入探讨杜仲叶绿原酸提高机体运动耐受力的机制,这也是探索杜仲叶抗疲劳作用机理的另一个切入点,有待于进一步研究。
Eucommia ulmoides is used as a herbin China for a long history, and it has the function of antihypertensive, tonifying kidney, reinforcing liver, sinewing bone, anti-abortion, etc. Although the part of Eucommia ulmoides plant used as a Chinese herb is the barks, modern research shows that Eucommia ulmoides bark and leaves have the similar components, and even the contents of some components in leaves are higher than these in bark. Therefore, the Eucommia ulmoides leaves can also be used as an herb instead of bark, and this provide a very good chance to resolve the lack of the resources of Eucommia ulmoides bark and to take the comprehensive utilization of Eucommia ulmoides resources.
Researches have shown that Eucommia ulmoides leaves have anti-fatigue effect, but the study objects of relative researches are usually the crude extracts of Eucommia ulmoides leaves, so the functional components of anti-fatigue effect are not clear, and also the mechanism of anti-fatigue effect is not known. Therefore, the functional foods which are developed from raw material of Eucommia ulmoides leaves are still the 2nd gerneration of functional foods, which have the problem of unclear mechanism and unclear functional components.
In order to solve this problem, in this research, Eucommia ulmoides leaves were studied as the subject, and the functional components were separated and purified step by step.The purified products from every step were evaluated on the anti-fatigue effect by animal experiments in order to screen out more specific anti-fatigue components from Eucommia ulmoides leaves. Meanwhile, the separated and purified products were taken in the in vivo and in vitro antioxidant tests in order to study the relationship between the anti-fatigue effect of Eucommia ulmoides leaves and anti-oxidation function of Eucommia ulmoides leaves, and furthermore to explore the mechanism of anti-fatigue effect of Eucommia ulmoides leaves. Finally, the last separated and purified anti-fatigue product of Eucommia ulmoides leaves were separated and purified more detailedly, and monomers were gotten. And then IR and UV spectroscopy were used to analyze the monomers structure. The molecular structure of each monomer was identified primarily, and the mechanism of anti-fatigue of Eucommia ulmoides leaves could be interpreted on the molecular level.
The contents of this research were shown as follows:
1. The tests on crude extract of Eucommia ulmoides leaves
The result of anti-fatigue evaluation test showed that the water extract of Eucommia ulmoides leaves (EUH) and ethanol extract of Eucommia ulmoids leaves (EUE) could both prolong the weight-loaded swimming time, reduce the content of blood lactic acid(LA)and serum urea nitrogen (SUN), and increase the content of hepatic glycogen. However, EUE showed more obvious influence on these anti-fatigue evaluation parameters than EUH, which meant that EUE had more significant anti-fatigue effect than EUH.
The result of hypoxia tolerance test showed that EUH and EUE did not have the obvious effect of enhancing the mics’s hypoxia tolerance ability, therefore, the function that EUH and EUE exhixbited the anti-fatigue effect by improving hypoxia tolerance ability was not significant.
The result of in vitro anti-oxidation test showed that EUH and EUE had the function of scavenging·OH, O_2~-·and DPPH·in different degree. However, the scavenging effect on each free radicals of EUE was better than EUH, which was identical to the fact that the anti-fatigue of EUE was better than EUH, and this proved that the in vitro anti-oxidation and anti-fatigue effect of Eucommia ulmoids leaves crude extract had the positive correlation.
The result of test also showed that flavonoids and polyphenols might be the functional components of exhibiting capacity of anti-fatigue and in vitro anti-oxidation in crude extract of Eucommia ulmoids leaves.
2. The tests on 1st seperated product of Eucommia ulmoides leaves
The result of anti-fatigue evaluation test showed that the 20% ethanol eluted product (EU1Ⅰ) and 60% ethanol eluted product (EU1Ⅱ) could both prolong the weight-loaded swimming time, reduce the content of blood LA and SUN, and increase the content of hepatic glycogen. However, EU1Ⅰshowed more obvious influence on these anti-fatigue evaluation parameters than EU1Ⅱ, which meant that EU1Ⅰhad more significant anti-fatigue effect than EU1Ⅱ. Meanwile, only EU1Ⅰcould obviously enhance the lactate dehydrogenase (LDH) activity, which meant that EU1Ⅰcould reduce the content of LA by enhancing LDH activity.
The result of in vivo anti-oxidation test showed that only EU1Ⅰcould reduce the content of malondialchehyche (MDA), enhance superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) activity, but EU1Ⅱhad not obvious effect on the three parameters, which was identical to the fact that the anti-fatigue effect of EU1Ⅰwas better than EU1Ⅱ, and this proved that the in vivo anti-oxidation and anti-fatigue effect of 1st seperated product of Eucommia ulmoides leaves had the positive correlation.
The result of hypoxia tolerance test showed that EU1Ⅰand EU1Ⅱdid not have the obvious effect of enhancing the mics’s hypoxia tolerance ability, therefore, the function that EU1Ⅰand EU1Ⅱexhixbited the anti-fatigue effect by improving hypoxia tolerance ability was not significant.
The result of in vitro anti-oxidation test showed that EU1Ⅰand EU1Ⅱhad the function of scavenging·OH, O_2~-·and DPPH·in different degree. However, the scavenging effect on each free radicals of EU1Ⅰwas better than EU1Ⅱ, which was identical to the fact that the anti-fatigue of EU1Ⅰwas better than EU1Ⅱ, and this proved that the in vitro anti-oxidation and anti-fatigue effect of 1st seperated product of Eucommia ulmoides leaves had the positive correlation. The result of dose-respones relationship test showed that the in vitro anti-oxidation capacity of EU1Ⅰand EU1Ⅱhad good dose-respones relationship.
The result of test also showed that polyphenols might be the leading components of exhibiting capacity of anti-fatigue and anti-oxidation in 1st seperated product of Eucommia ulmoides leaves.
3. The tests on 2nd seperated product of Eucommia ulmoides leaves
The result of anti-fatigue evaluation test showed that ethyl acetate extract (EU2Ⅰ) and butyl alcohol extract (EU2Ⅱ) had some influence on mice body weight. EU2Ⅰand EU2Ⅱcould both prolong the weight-loaded swimming time, reduce the content of blood LA and increase the content of hepatic glycogen, but only EU2Ⅱcould reduce the content of SUN, and EU2Ⅱshowed more obvious influence on these anti-fatigue evaluation parameters than EU2Ⅰ, which meant that EU2Ⅱhad more significant anti-fatigue effect than EU2Ⅰ. Meanwile, only EU2Ⅱcould obviously enhance the LDH activity, which meant that EU2Ⅱcould reduce the content of LA by enhancing LDH activity.
The result of in vivo anti-oxidation test showed that both EU2Ⅰand EU2Ⅱcould reduce the content of MDA, but only EU2Ⅱcould obviously enhance SOD and GSH-PX activity, which was identical to the fact that the anti-fatigue of EU2Ⅱwas better than EU2Ⅰ, and this proved that the in vivo anti-oxidation and anti-fatigue effect of 2nd seperated product of Eucommia ulmoides leaves had the positive correlation.
The result of hypoxia tolerance test showed that EU2Ⅰand EU2Ⅱdid not have the obvious effect of enhancing the mics’s hypoxia tolerance ability, therefore, the function that EU2Ⅰand EU2Ⅱexhixbited the anti-fatigue effect by improving hypoxia tolerance ability was not significant.
The result of in vitro anti-oxidation test showed that EU2Ⅰand EU2Ⅱhad the function of scavenging·OH, O_2~-·and DPPH·in different degree. However, EU2Ⅱshowed more obvious scavenging effect on each free radicals, which was identical to the fact that the anti-fatigue of EU2Ⅱwas more significant, and this proved that the in vitro anti-oxidation and anti-fatigue effect of 2nd seperated product of Eucommia ulmoides leaves had the positive correlation. The result of dose-respones relationship test showed that the in vitro anti-oxidation capacity of EU2Ⅰand EU2Ⅱhad good dose-respones relationship.
The result of test also showed that polyphenols might be the leading components of exhibiting capacity of anti-fatigue and anti-oxidation in 2nd seperated product of Eucommia ulmoides leaves.
4. The tests on 3rd seperated product of Eucommia ulmoides leaves
The result of anti-fatigue evaluation test showed that flavonoids purified product (EU3Ⅰ) and chlorogenic acid purified product (EU3Ⅱ) could both prolong the weight-loaded swimming time, but only EU3Ⅰcould reduce the content of blood LA and SUN, increase the content of hepatic glycogen, so EU3Ⅰshowed more obvious influence on these anti-fatigue evaluation parameters than EU3Ⅱ, which meant that EU3Ⅰhad more significant anti-fatigue effect than EU3Ⅱ. Meanwile, only EU3Ⅰcould obviously enhance the LDH activity, which meant that EU3Ⅰcould reduce the content of LA by enhancing LDH activity.
The result of in vivo anti-oxidation test showed both EU3Ⅰand EU3Ⅱcould reduce the content of MDA, but only EU3Ⅱcould obviously enhance SOD and GSH-PX activity, which proved that EU3Ⅱhad more obvious capacity in vivo anti-oxidation.
The result of hypoxia tolerance test showed that EU3Ⅰcould enhance the mics’s hypoxia tolerance ability, but EU3Ⅱcould not. Therefore, EU3Ⅰmight exhixbit the anti-fatigue effect by improving hypoxia tolerance ability, but EU3Ⅱmight not.
The result of in vitro anti-oxidation test showed that EU3Ⅰand EU3Ⅱhad the function of scavenging·OH, O_2~-·and DPPH·in different degree. However, the scavenging effect on each free radicals of EU3Ⅱwas better than EU3Ⅰ, which meant that EU3Ⅱhad better capacity in vitro anti-oxidation. The result of dose-respones relationship test showed that the in vitro anti-oxidation capacity of EU3Ⅰand EU3Ⅱhad good dose-respones relationship.
The test result showed that EU3Ⅰ(flavonoids) might enhance the anti-fatigue capacity by improving the body’s energy metabolization, but EU3Ⅱ(chlorogenic acid) might enhance exercise capacity by influencing on the central nervous system or cooperative effect with other component in Eucommia ulmoides leaves.
5. Identification for functional anti-fatigue components from Eucommia ulmoides leaves
Silica gel column and thin-layer chromatography methods were used to separate, purify and analyze the butyl alcohol extract of Eucommia ulmoides leaves, and 6 compounds were gotten. By identification from IR and UV spectroscopy, 4 of them might be primarily identified as quercetin, L-epicatechin, chlorogenic acid and rutin, and antother 2 might be primarily identified as flavonoid glycosides and flavonoids.
The components separated from the butyl alcohol extract of Eucommia ulmoides leaves were flavonoids, polyphenols and flavonoids glycosides, which showed obvious anti-oxidation activity. Therefore, the presence of anti-oxidant made Eucommia ulmoides leaves exhibit anti-oxidation capacity, and made Eucommia ulmoides leaves show anti-fatigue effect.
The anti-oxidation functional components in Eucommia ulmoides leaves could scanvenging free radicals produced in body, and protect body from damage caused by free radicals, so the fatigue was delayed, and the anti-fatigue capacity was enhanced, and this was the mechanism of anti-fatigue effect from Eucommia ulmoides leaves. This mechanism met the free radicals theory for interpreting anti-fatigue mechanism. This research provided the support for this theory, and also showed the relationship between anti-fatigue effect and anti-oxidation fuction of Eucommia ulmoides leaves.
Howver, this research did not deeply probe into the mechanism of enhancing exercise capacity from chlorogenic acid in Eucommia ulmoides leaves, and this was another entry point to explore the anti-fatigue mechanism of Eucommia ulmoides leaves, which should be researched further.
研究表明,杜仲叶具有抗疲劳功效,但是目前所见的相关报道大多是以杜仲叶粗提物为研究对象,并且抗疲劳功效成分尚不明确,抗疲劳作用机理也不清楚。所以,目前以杜仲叶为原料开发的抗疲劳功能食品仍属于第二代保健食品,这类保健食品存在作用机理不清楚、功效成分不明确的问题。
针对上述问题,本课题以杜仲叶为研究对象,对杜仲叶功效成分逐步进行分离纯化,采用动物试验评价各步产物的抗疲劳功效,以筛选出成分更加明确的杜仲叶抗疲劳功效成分。同时,分析各分离纯化产物体内和体外抗氧化作用,进一步研究杜仲叶抗疲劳功效与抗氧化作用之间的关联性,探索杜仲叶抗疲劳作用机理。最后,将最终得到的具有抗疲劳功效的杜仲叶纯化产物进行细致的分离,得到单体化合物,采用红外光谱和紫外光谱测定各单体的结构式,初步推测其分子结构,从分子水平上阐释杜仲叶抗疲劳作用机理。
本论文完成的研究内容如下:
1.杜仲叶粗提物试验
抗疲劳功效评价结果显示,杜仲叶水提物(EUH)和醇提物(EUE)均能显著延长小鼠负重游泳时间、降低血乳酸(LA)和血清尿素氮(SUN)含量及提高肝糖原含量,但EUE对各抗疲劳功效评价指标的影响比EUH显著,表明EUE的抗疲劳功效强于EUH。
常压耐缺氧试验结果显示,EUH和EUE不能明显地提高小鼠常压耐缺氧能力,因此,EUH和EUE并不能通过改善常压耐缺氧能力表现抗疲劳功效。
体外抗氧化试验结果显示,EUH和EUE均具有不同程度的清除羟自由基(·OH)、超氧阴离子(O_2~-·)及1,1-二苯基-2-苦苯肼自由基(DPPH·)的作用,但是EUE对各自由基的清除效果均优于EUH,这与EUE的抗疲劳功效强于EUH的结论相一致,证明杜仲叶粗提物体外抗氧化作用与抗疲劳功效正相关。
试验结果还表明,杜仲叶粗提物中的总黄酮与总多酚可能为杜仲叶粗提物表现抗疲劳功效和体外抗氧化作用的功效成分。
2.杜仲叶一次分离纯化产物试验
抗疲劳功效评价结果显示,20%乙醇洗脱液(EU1Ⅰ)和60%乙醇洗脱液(EU1Ⅱ)均能延长小鼠负重游泳时间、降低血LA和SUN含量及提高肝糖原含量,但EU1Ⅰ对各抗疲劳功效评价指标的影响比EU1Ⅱ显著,表明EU1Ⅰ具有强于EU1Ⅱ的抗疲劳功效。同时,EU1Ⅰ可以显著提高乳酸脱氢酶(LDH)的活性,表明EU1Ⅰ可以通过提高机体LDH活性降低血LA含量。
体内抗氧化作用试验结果显示,只有EU1Ⅰ可以降低丙二醛(MDA)含量、提高超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-PX)活性,而EU1Ⅱ对这三项指标的影响并不明显,表明EU1Ⅰ具有比EU1Ⅱ更显著的体内抗氧化作用,这与EU1Ⅰ的抗疲劳功效强于EU1Ⅱ的结论相一致,证明杜仲叶一次分离纯化产物体内抗氧化作用与抗疲劳功效正相关。
常压耐缺氧试验结果显示, EU1Ⅰ和EU1Ⅱ不能明显地提高小鼠常压耐缺氧能力,因此,EU1Ⅰ和EU1Ⅱ不能通过改善常压耐缺氧能力表现抗疲劳功效。
体外抗氧化试验结果显示,EU1Ⅰ和EU1Ⅱ均具有不同程度的清除·OH、O_2~-·及DPPH·的作用,但是EU1Ⅰ对各自由基的清除效果均优于EU1Ⅱ,表明EU1Ⅰ具有比EU1Ⅱ更显著的体外抗氧化作用,这与EU1Ⅰ的抗疲劳功效强于EU1Ⅱ的结论相一致,证明杜仲叶一次分离纯化产物体外抗氧化作用与抗疲劳功效正相关。量效关系试验结果表明,EU1Ⅰ和EU1Ⅱ的体外抗氧作用具有很好的量效关系。
试验结果还表明,多酚类物质可能为杜仲叶一次分离纯化产物表现抗疲劳功效和抗氧化作用的主要功效成分。
3.杜仲叶二次分离纯化产物试验
抗疲劳功效评价结果显示,乙酸乙酯萃取物(EU2Ⅰ)和正丁醇萃取物(EU2Ⅱ)对小鼠体重有一定的影响。EU2Ⅰ和EU2Ⅱ均能延长小鼠负重游泳时间、降低血LA含量及提高肝糖原含量,但只有EU2Ⅱ可以显著降低SUN含量,EU2Ⅱ对各抗疲劳功效评价指标的影响比EUⅠ显著,表明EU2Ⅱ具有强于EU2Ⅰ的抗疲劳功效。同时,只有EU2Ⅱ可以显著提高LDH活性,表明EU2Ⅱ可以通过提高机体LDH活性降低血LA含量。
体内抗氧化作用试验结果显示,EU2Ⅰ和EU2Ⅱ均可以降低MDA含量,但是,只有EU2Ⅱ可以显著提高SOD和GSH-PX活性,表明EU2Ⅱ具有比EU2Ⅰ更显著的体内抗氧化作用,这与EU2Ⅱ的抗疲劳功效强于EUⅠ的结论相一致,证明杜仲叶二次分离纯化产物体内抗氧化作用与抗疲劳功效正相关。
常压耐缺氧试验结果显示,EU2Ⅰ和EU2Ⅱ不能明显地提高小鼠常压耐缺氧能力,因此,EU2Ⅰ和EU2Ⅱ不能通过改善常压耐缺氧能力表现抗疲劳功效。
体外抗氧化试验结果显示,EU2Ⅰ和EU2Ⅱ均具有不同程度的清除·OH、O_2~-·及DPPH·的作用,但是EU2Ⅱ对各自由基的清除效果尤其显著,表明EU2Ⅱ具有更为显著的体外抗氧化作用,这与EU2Ⅱ具有更明显的抗疲劳功效的结论相一致,证明杜仲叶二次分离纯化产物体外抗氧化作用与抗疲劳功效也正相关。量效关系试验表明,EU2Ⅰ和EU2Ⅱ的体外抗氧作用也具有很好的量效关系。
试验结果还表明,多酚类物质可能为杜仲叶二次分离纯化产物表现抗疲劳功效和抗氧化作用的主要功效成分。
4.杜仲叶三次分离纯化产物试验抗疲劳功效评价结果显示,黄酮纯化产物(EU3Ⅰ)和绿原酸纯化产物(EU3Ⅱ)均能延长小鼠负重游泳时间,但是只有EU3Ⅰ可以显著降低血LA和SUN含量及提高肝糖原含量,EU3Ⅰ对各抗疲劳功效评价指标的影响比EU3Ⅱ显著,表明EU3Ⅰ具有明显的抗疲劳功效。同时,只有EU3Ⅰ可以显著提高LDH活性,表明EU3Ⅰ可以通过提高机体LDH活性降低由血LA含量。
体内抗氧化作用实验结果显示,EU3Ⅰ和EU3Ⅱ均可以降低MDA含量,但是,只有EU3Ⅱ可以显著提高SOD和GSH-PX活性,证明EU3Ⅱ具有更为明显的体内抗氧化作用。
常压耐缺氧试验结果显示,EU3Ⅰ可以提高小鼠常压耐缺氧能力,而EU3Ⅱ不能明显地提高小鼠常压耐缺氧能力。因此,EU3Ⅰ可能是通过提高小鼠常压耐缺氧能力表现抗疲劳功效,而EU3Ⅱ不能通过改善常压耐缺氧能力表现抗疲劳功效。
体外抗氧化试验结果显示,EU2Ⅰ和EU2Ⅱ均具有不同程度的清除·OH、O_2~-·及DPPH·的作用,但是EU3Ⅱ对各自由基的清除效果尤其显著,表明EU3Ⅱ具有更强的体外抗氧化能力。量效关系实验表明,EU3Ⅰ和EU3Ⅱ的体外抗氧能力同样具有很好的量效关系。
试验结果表明,EU3Ⅰ(黄酮类物质)可能是通过改善机体能量代谢提高机体抗疲劳能力;而EU3Ⅱ(绿原酸)可能是通过对中枢神经系统影响或者是与杜仲叶中多种成分协同作用的方式提高机体运动耐受力.
5.杜仲叶抗疲劳功效成分鉴定
采用硅胶柱层析和薄层层析的方法对杜仲叶正丁醇萃取纯化产物进行分离、纯化和分析,得到6种单体化合物,经红外光谱和紫外光谱初步鉴定,其中4种可能为槲皮素、表儿茶素、绿原酸和芦丁,另外2种初步推测为黄酮苷和黄酮类化合物。
从杜仲叶正丁醇萃取物中分离出的化合物多为黄酮、多酚以及黄酮苷类物质,这些成分具有显著的抗氧化活性。由此可见,抗氧化功效成分的存在使杜仲叶表现出了抗氧化作用,并且使杜仲叶显示了抗疲劳功效。
杜仲叶中抗氧化功效成分可以清除体内产生的自由基,使机体免受自由基引起的损伤,由此延缓疲劳的产生,提高机体抗疲劳能力,这就是杜仲叶抗疲劳作用机理。该机理符合抗疲劳产生机理中的自由基学说,本研究为该理论学说提供了支持,也进一步表明了杜仲叶抗疲劳功效与抗氧化作用之间的关联性。
然而,本研究没有深入探讨杜仲叶绿原酸提高机体运动耐受力的机制,这也是探索杜仲叶抗疲劳作用机理的另一个切入点,有待于进一步研究。
Eucommia ulmoides is used as a herbin China for a long history, and it has the function of antihypertensive, tonifying kidney, reinforcing liver, sinewing bone, anti-abortion, etc. Although the part of Eucommia ulmoides plant used as a Chinese herb is the barks, modern research shows that Eucommia ulmoides bark and leaves have the similar components, and even the contents of some components in leaves are higher than these in bark. Therefore, the Eucommia ulmoides leaves can also be used as an herb instead of bark, and this provide a very good chance to resolve the lack of the resources of Eucommia ulmoides bark and to take the comprehensive utilization of Eucommia ulmoides resources.
Researches have shown that Eucommia ulmoides leaves have anti-fatigue effect, but the study objects of relative researches are usually the crude extracts of Eucommia ulmoides leaves, so the functional components of anti-fatigue effect are not clear, and also the mechanism of anti-fatigue effect is not known. Therefore, the functional foods which are developed from raw material of Eucommia ulmoides leaves are still the 2nd gerneration of functional foods, which have the problem of unclear mechanism and unclear functional components.
In order to solve this problem, in this research, Eucommia ulmoides leaves were studied as the subject, and the functional components were separated and purified step by step.The purified products from every step were evaluated on the anti-fatigue effect by animal experiments in order to screen out more specific anti-fatigue components from Eucommia ulmoides leaves. Meanwhile, the separated and purified products were taken in the in vivo and in vitro antioxidant tests in order to study the relationship between the anti-fatigue effect of Eucommia ulmoides leaves and anti-oxidation function of Eucommia ulmoides leaves, and furthermore to explore the mechanism of anti-fatigue effect of Eucommia ulmoides leaves. Finally, the last separated and purified anti-fatigue product of Eucommia ulmoides leaves were separated and purified more detailedly, and monomers were gotten. And then IR and UV spectroscopy were used to analyze the monomers structure. The molecular structure of each monomer was identified primarily, and the mechanism of anti-fatigue of Eucommia ulmoides leaves could be interpreted on the molecular level.
The contents of this research were shown as follows:
1. The tests on crude extract of Eucommia ulmoides leaves
The result of anti-fatigue evaluation test showed that the water extract of Eucommia ulmoides leaves (EUH) and ethanol extract of Eucommia ulmoids leaves (EUE) could both prolong the weight-loaded swimming time, reduce the content of blood lactic acid(LA)and serum urea nitrogen (SUN), and increase the content of hepatic glycogen. However, EUE showed more obvious influence on these anti-fatigue evaluation parameters than EUH, which meant that EUE had more significant anti-fatigue effect than EUH.
The result of hypoxia tolerance test showed that EUH and EUE did not have the obvious effect of enhancing the mics’s hypoxia tolerance ability, therefore, the function that EUH and EUE exhixbited the anti-fatigue effect by improving hypoxia tolerance ability was not significant.
The result of in vitro anti-oxidation test showed that EUH and EUE had the function of scavenging·OH, O_2~-·and DPPH·in different degree. However, the scavenging effect on each free radicals of EUE was better than EUH, which was identical to the fact that the anti-fatigue of EUE was better than EUH, and this proved that the in vitro anti-oxidation and anti-fatigue effect of Eucommia ulmoids leaves crude extract had the positive correlation.
The result of test also showed that flavonoids and polyphenols might be the functional components of exhibiting capacity of anti-fatigue and in vitro anti-oxidation in crude extract of Eucommia ulmoids leaves.
2. The tests on 1st seperated product of Eucommia ulmoides leaves
The result of anti-fatigue evaluation test showed that the 20% ethanol eluted product (EU1Ⅰ) and 60% ethanol eluted product (EU1Ⅱ) could both prolong the weight-loaded swimming time, reduce the content of blood LA and SUN, and increase the content of hepatic glycogen. However, EU1Ⅰshowed more obvious influence on these anti-fatigue evaluation parameters than EU1Ⅱ, which meant that EU1Ⅰhad more significant anti-fatigue effect than EU1Ⅱ. Meanwile, only EU1Ⅰcould obviously enhance the lactate dehydrogenase (LDH) activity, which meant that EU1Ⅰcould reduce the content of LA by enhancing LDH activity.
The result of in vivo anti-oxidation test showed that only EU1Ⅰcould reduce the content of malondialchehyche (MDA), enhance superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) activity, but EU1Ⅱhad not obvious effect on the three parameters, which was identical to the fact that the anti-fatigue effect of EU1Ⅰwas better than EU1Ⅱ, and this proved that the in vivo anti-oxidation and anti-fatigue effect of 1st seperated product of Eucommia ulmoides leaves had the positive correlation.
The result of hypoxia tolerance test showed that EU1Ⅰand EU1Ⅱdid not have the obvious effect of enhancing the mics’s hypoxia tolerance ability, therefore, the function that EU1Ⅰand EU1Ⅱexhixbited the anti-fatigue effect by improving hypoxia tolerance ability was not significant.
The result of in vitro anti-oxidation test showed that EU1Ⅰand EU1Ⅱhad the function of scavenging·OH, O_2~-·and DPPH·in different degree. However, the scavenging effect on each free radicals of EU1Ⅰwas better than EU1Ⅱ, which was identical to the fact that the anti-fatigue of EU1Ⅰwas better than EU1Ⅱ, and this proved that the in vitro anti-oxidation and anti-fatigue effect of 1st seperated product of Eucommia ulmoides leaves had the positive correlation. The result of dose-respones relationship test showed that the in vitro anti-oxidation capacity of EU1Ⅰand EU1Ⅱhad good dose-respones relationship.
The result of test also showed that polyphenols might be the leading components of exhibiting capacity of anti-fatigue and anti-oxidation in 1st seperated product of Eucommia ulmoides leaves.
3. The tests on 2nd seperated product of Eucommia ulmoides leaves
The result of anti-fatigue evaluation test showed that ethyl acetate extract (EU2Ⅰ) and butyl alcohol extract (EU2Ⅱ) had some influence on mice body weight. EU2Ⅰand EU2Ⅱcould both prolong the weight-loaded swimming time, reduce the content of blood LA and increase the content of hepatic glycogen, but only EU2Ⅱcould reduce the content of SUN, and EU2Ⅱshowed more obvious influence on these anti-fatigue evaluation parameters than EU2Ⅰ, which meant that EU2Ⅱhad more significant anti-fatigue effect than EU2Ⅰ. Meanwile, only EU2Ⅱcould obviously enhance the LDH activity, which meant that EU2Ⅱcould reduce the content of LA by enhancing LDH activity.
The result of in vivo anti-oxidation test showed that both EU2Ⅰand EU2Ⅱcould reduce the content of MDA, but only EU2Ⅱcould obviously enhance SOD and GSH-PX activity, which was identical to the fact that the anti-fatigue of EU2Ⅱwas better than EU2Ⅰ, and this proved that the in vivo anti-oxidation and anti-fatigue effect of 2nd seperated product of Eucommia ulmoides leaves had the positive correlation.
The result of hypoxia tolerance test showed that EU2Ⅰand EU2Ⅱdid not have the obvious effect of enhancing the mics’s hypoxia tolerance ability, therefore, the function that EU2Ⅰand EU2Ⅱexhixbited the anti-fatigue effect by improving hypoxia tolerance ability was not significant.
The result of in vitro anti-oxidation test showed that EU2Ⅰand EU2Ⅱhad the function of scavenging·OH, O_2~-·and DPPH·in different degree. However, EU2Ⅱshowed more obvious scavenging effect on each free radicals, which was identical to the fact that the anti-fatigue of EU2Ⅱwas more significant, and this proved that the in vitro anti-oxidation and anti-fatigue effect of 2nd seperated product of Eucommia ulmoides leaves had the positive correlation. The result of dose-respones relationship test showed that the in vitro anti-oxidation capacity of EU2Ⅰand EU2Ⅱhad good dose-respones relationship.
The result of test also showed that polyphenols might be the leading components of exhibiting capacity of anti-fatigue and anti-oxidation in 2nd seperated product of Eucommia ulmoides leaves.
4. The tests on 3rd seperated product of Eucommia ulmoides leaves
The result of anti-fatigue evaluation test showed that flavonoids purified product (EU3Ⅰ) and chlorogenic acid purified product (EU3Ⅱ) could both prolong the weight-loaded swimming time, but only EU3Ⅰcould reduce the content of blood LA and SUN, increase the content of hepatic glycogen, so EU3Ⅰshowed more obvious influence on these anti-fatigue evaluation parameters than EU3Ⅱ, which meant that EU3Ⅰhad more significant anti-fatigue effect than EU3Ⅱ. Meanwile, only EU3Ⅰcould obviously enhance the LDH activity, which meant that EU3Ⅰcould reduce the content of LA by enhancing LDH activity.
The result of in vivo anti-oxidation test showed both EU3Ⅰand EU3Ⅱcould reduce the content of MDA, but only EU3Ⅱcould obviously enhance SOD and GSH-PX activity, which proved that EU3Ⅱhad more obvious capacity in vivo anti-oxidation.
The result of hypoxia tolerance test showed that EU3Ⅰcould enhance the mics’s hypoxia tolerance ability, but EU3Ⅱcould not. Therefore, EU3Ⅰmight exhixbit the anti-fatigue effect by improving hypoxia tolerance ability, but EU3Ⅱmight not.
The result of in vitro anti-oxidation test showed that EU3Ⅰand EU3Ⅱhad the function of scavenging·OH, O_2~-·and DPPH·in different degree. However, the scavenging effect on each free radicals of EU3Ⅱwas better than EU3Ⅰ, which meant that EU3Ⅱhad better capacity in vitro anti-oxidation. The result of dose-respones relationship test showed that the in vitro anti-oxidation capacity of EU3Ⅰand EU3Ⅱhad good dose-respones relationship.
The test result showed that EU3Ⅰ(flavonoids) might enhance the anti-fatigue capacity by improving the body’s energy metabolization, but EU3Ⅱ(chlorogenic acid) might enhance exercise capacity by influencing on the central nervous system or cooperative effect with other component in Eucommia ulmoides leaves.
5. Identification for functional anti-fatigue components from Eucommia ulmoides leaves
Silica gel column and thin-layer chromatography methods were used to separate, purify and analyze the butyl alcohol extract of Eucommia ulmoides leaves, and 6 compounds were gotten. By identification from IR and UV spectroscopy, 4 of them might be primarily identified as quercetin, L-epicatechin, chlorogenic acid and rutin, and antother 2 might be primarily identified as flavonoid glycosides and flavonoids.
The components separated from the butyl alcohol extract of Eucommia ulmoides leaves were flavonoids, polyphenols and flavonoids glycosides, which showed obvious anti-oxidation activity. Therefore, the presence of anti-oxidant made Eucommia ulmoides leaves exhibit anti-oxidation capacity, and made Eucommia ulmoides leaves show anti-fatigue effect.
The anti-oxidation functional components in Eucommia ulmoides leaves could scanvenging free radicals produced in body, and protect body from damage caused by free radicals, so the fatigue was delayed, and the anti-fatigue capacity was enhanced, and this was the mechanism of anti-fatigue effect from Eucommia ulmoides leaves. This mechanism met the free radicals theory for interpreting anti-fatigue mechanism. This research provided the support for this theory, and also showed the relationship between anti-fatigue effect and anti-oxidation fuction of Eucommia ulmoides leaves.
Howver, this research did not deeply probe into the mechanism of enhancing exercise capacity from chlorogenic acid in Eucommia ulmoides leaves, and this was another entry point to explore the anti-fatigue mechanism of Eucommia ulmoides leaves, which should be researched further.
引文
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