| 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 leavesThe 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, O2-·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 leavesThe result of anti-fatigue evaluation test showed that the 20% ethanol eluted product (EU1â… ) and 60% ethanol eluted product (EU1â…¡) could both prolong the weight-loaded swimming time, reduce the content of blood LA and SUN, and increase the content of hepatic glycogen. However, EU1â… showed more obvious influence on these anti-fatigue evaluation parameters than EU1â…¡, which meant that EU1â… had more significant anti-fatigue effect than EU1â…¡. Meanwile, only EU1â… could obviously enhance the lactate dehydrogenase (LDH) activity, which meant that EU1â… could reduce the content of LA by enhancing LDH activity.The result of in vivo anti-oxidation test showed that only EU1â… could reduce the content of malondialchehyche (MDA), enhance superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) activity, but EU1â…¡had not obvious effect on the three parameters, which was identical to the fact that the anti-fatigue effect of EU1â… was better than EU1â…¡, 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 EU1â… and EU1â…¡did not have the obvious effect of enhancing the mics's hypoxia tolerance ability, therefore, the function that EU1â… and EU1â…¡exhixbited the anti-fatigue effect by improving hypoxia tolerance ability was not significant.The result of in vitro anti-oxidation test showed that EU1â… and EU1â…¡had the function of scavenging·OH, O2-·and DPPH·in different degree. However, the scavenging effect on each free radicals of EU1â… was better than EU1â…¡, which was identical to the fact that the anti-fatigue of EU1â… was better than EU1â…¡, 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 EU1â… and EU1â…¡had 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 leavesThe result of anti-fatigue evaluation test showed that ethyl acetate extract (EU2â… ) and butyl alcohol extract (EU2â…¡) had some influence on mice body weight. EU2â… and EU2â…¡could both prolong the weight-loaded swimming time, reduce the content of blood LA and increase the content of hepatic glycogen, but only EU2â…¡could reduce the content of SUN, and EU2â…¡showed more obvious influence on these anti-fatigue evaluation parameters than EU2â… , which meant that EU2â…¡had more significant anti-fatigue effect than EU2â… . Meanwile, only EU2â…¡could obviously enhance the LDH activity, which meant that EU2â…¡could reduce the content of LA by enhancing LDH activity.The result of in vivo anti-oxidation test showed that both EU2â… and EU2â…¡could reduce the content of MDA, but only EU2â…¡could obviously enhance SOD and GSH-PX activity, which was identical to the fact that the anti-fatigue of EU2â…¡was better than EU2â… , 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 EU2â… and EU2â…¡did not have the obvious effect of enhancing the mics's hypoxia tolerance ability, therefore, the function that EU2â… and EU2â…¡exhixbited the anti-fatigue effect by improving hypoxia tolerance ability was not significant.The result of in vitro anti-oxidation test showed that EU2â… and EU2â…¡had the function of scavenging·OH, O2-·and DPPH·in different degree. However, EU2â…¡showed more obvious scavenging effect on each free radicals, which was identical to the fact that the anti-fatigue of EU2â…¡was 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 EU2â… and EU2â…¡had 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 leavesThe result of anti-fatigue evaluation test showed that flavonoids purified product (EU3â… ) and chlorogenic acid purified product (EU3â…¡) could both prolong the weight-loaded swimming time, but only EU3â… could reduce the content of blood LA and SUN, increase the content of hepatic glycogen, so EU3â… showed more obvious influence on these anti-fatigue evaluation parameters than EU3â…¡, which meant that EU3â… had more significant anti-fatigue effect than EU3â…¡. Meanwile, only EU3â… could obviously enhance the LDH activity, which meant that EU3â… could reduce the content of LA by enhancing LDH activity.The result of in vivo anti-oxidation test showed both EU3â… and EU3â…¡could reduce the content of MDA, but only EU3â…¡could obviously enhance SOD and GSH-PX activity, which proved that EU3â…¡had more obvious capacity in vivo anti-oxidation.The result of hypoxia tolerance test showed that EU3â… could enhance the mics's hypoxia tolerance ability, but EU3â…¡could not. Therefore, EU3â… might exhixbit the anti-fatigue effect by improving hypoxia tolerance ability, but EU3â…¡might not.The result of in vitro anti-oxidation test showed that EU3â… and EU3â…¡had the function of scavenging·OH, O2-·and DPPH·in different degree. However, the scavenging effect on each free radicals of EU3â…¡was better than EU3â… , which meant that EU3â…¡had better capacity in vitro anti-oxidation. The result of dose-respones relationship test showed that the in vitro anti-oxidation capacity of EU3â… and EU3â…¡had good dose-respones relationship.The test result showed that EU3â… (flavonoids) might enhance the anti-fatigue capacity by improving the body's energy metabolization, but EU3â…¡(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 leavesSilica 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.
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