Purification Of Caffeoylquinic Acid From ILEX Kudingcha C. J.Tseng And The Study Of Their Anti-Obesity Efficacy

The development of our society and the change of the environment lead to great changes in people’s life pace, eating habits and their dietary structure. People become more worried about the following health problems which brought by the changes, and they are paying more attention to the research in order to solve these health problems. Early in the year 1948, WHO had defined obesity as a chromic metabolic disease, which has been a threaten to more than 500 million patients. While the pharmaceutical researchers are striving to find anti-obesity compound drugs, people never stop using natural products to keep healthy and control their weight. As a natural alternative beverage for tea, Kuding tea has a long history of almost 2000 years. It has been confirmed that kuding tea have many functions like antiovesity, hypolipidemic, antibacterial, anti-inflammatory and the alexipharmic effects. The study of this article is based on Ilex Kudingcha C. J. Tseng. We established an HPLC method to analysis 6 main active components, Caffeoylquinic acids (CQA), which are separated and purified from kudingcha polyphenols. We made some efforts to investigate the inhibitory effects of CQAs on the a-amylase, a-glucosidase and porcine pancreatic lipase, and also the effects on the proliferation and differentiation of 3T3-L1 cells. These experiments showed the anti-obesity properties of CQA compounds at the level of enzyme and cell. Finally, we had a preliminary study on the intestinal microecology regulation by the extracts of kudingcha.1. The separation and purification of CQAs. Six caffeoylquinic acid (CQA) derivatives were separated by the column and semi-preparative high performance liquid chromatography (HPLC) from the crude extract of Ilex kudingcha C.J. Tseng. A HPLC method with DAD as detector and TSKgel ODS-80 TsQA as column was developed for the simultaneous determination of CQAs in kudingcha made from Ilex kudingcha C.J. Tseng. Our results demonstrated that six CQA derivatives with 95%purity,3-CQA,4-CQA, 5-CQA,3,4-diCQA,3,5-diCQA and 4,5-diCQA, could be obtained through the purification of Ilex kudingcha C.J. Tseng by semi-preparative high performance liquid chromatography (HPLC). Chromatographic conditions:TSKgel ODS-80TsQA column, oven temperature 40℃. gradient elution of methanol and formic acid (5%o). flow rate 0.5 mL/min, detector at 326 nm. As results, the correlation coefficients were 0.9992-0.9999 in the linear range, the average recovery was 96.35%, and the relative standard deviations were all less than 3%.2. The enzyme inhibition activity of CQAs. In this study, we took the 6 CQAs from kudingcha polyphenols as samples, and explored the mechanism of anti-obesity and lipid lowering in the level of enzyme. To investigate the inhibition effect of CQAs on a-amylase, the starch azure was used as substrate. The results showed obvious effects on enzyme inhibition by all the six CQAs. Among the six compounds,3,5-diCQA showed the strongest inhibition effect, with the IC50 to 0.2842 mg/mL. Using pNPG as the substrate, a system for determination CQAs inhibition of a-glucosidase was established. A comparison of inhibition effect can be concluded as 4,5-diCQA> 5-CQA> 3,4-diCQA> 3,5-diCQA> 4-CQA> 3-CQA. Moreover, through the Lineweave-Burk curve we drew, we deduced that the inhibition effect of CQAs on a-glucosidase were all noncompetitive inhibition. In addition, with the substrate of pNP laurate, the inhibition effect and type of CQAs inhibitor for porcine pancreatic lipase were determined. There was a comparison of inhibition effect of six CQAs which was:3,5-diCQA> 4,5-diCQA> 3,4-diCQA> 3-CQA> 4-CQA> 5-CQA. All the di-CQAs showed stronger inhibition effect than nomo-CQAs in this experiment. Finally, through the Lineweave-Burk curve we drew, we included that the inhibition effect of CQAs on lipases were all noncompetitive inhibition.3. The inhibition effect of CQAs on the proliferation and differentiation of 3T3-L1 cell. Taking the six CQAs isolated from Ilex kudingcha as samples, the effect of kudingcha polyphenols on 3T3-L1 preadipocyte was studied. At first, MTT assay was used to measure the survival rate of 3T3-L1 cell proliferation in 24,48 and 72 h after the treatment of six CQAs. Insulin, IBMX and DEX were added into DMEM to induce the differentiation of 3T3-L1. As compared with the control group, CQAs showed significant inhibition effect on the conversion of 3T3-L1 preadipocytes to adipocytes. Meanwhile, the inhibition is does dependent.4. Intestinal microecology regulation of kudingcha polyphenols. The water extracts of Ilex Kudingcha, the extracts of Ilex Kudingcha polyphenols and 3,5-diCQA were used as samples in the experiment. The effects of these samples on the intestinal microbiota in vitro were evaluate by monitoring the bacterial populations and analyzing the concentrations of short-chain fatty acids (SCFA) at different fermentation times. It was found that the samples exhibited proliferate effects on the growth of Bifidobacterium groups within 36 h, while the population of total bacteria was not affected. Moreover, by HPLC method, we found that the concentration of formic acid, lactic acid, acetic acid and propionic acid in culture with the 3 samples were relative higher than that of the control. The results suggested that Ilex kudingcha might affect the intestinal microecology by modifing intestinal flora and generating SCFA that might be due to its polyphenols, and Ilex kudingcha might be able to improve thebalance of human intestinal micro-ecology. However, there still need more experiments to figure out the mechanism.