Studies On Isolation, Purification And Bioactivity Of Polyphenols In Ilex Paraguariensis

Polyphenols are secondary metabolites in plants, which possess special chemicaland physiological activities. The intake of appropriate amount of polyphenols canreduce the risk of diseases. Therefore, polyphenols have been known as "the seventhnutrient" for human health and are applied to cure and prevent diseases as naturalhealthy food and herbal drug. Ilex paraguariensis (IP) infusion is a popular beverage inLatin America countries for long time. It is proved that IP possessed a variety ofpharmacological activities. IP was found to be a rich source of phenolic compounds, inwhich the content of Caffeoyl derivatives is up to 10.71%. And the IP polyphenols areconsidered to contribute to some of pharmacological activities. However, there arefewer reports on the extraction, isolation, purification and functional evaluation of IPpolyphenols, which badly impeded the utilization of IP. With the frequent exchangebetween China and foreign countries, IP was imported in domestic market. This projectaim at extraction, isolation, purification of IP polyphenols, as well as identification thestructure of the monomer by modem technologies, And evaluation the function of IPpolyphenols, such as anti-oxidant, anti-obesity and anti-hyperlipemia activities and so on.The objective of present study is to offer a reliable theory basis for people to make fulluse of IP polyphenols.1 Extraction, isolation, purification and structure identification of IP polyphenols(1) A method of extraction and purification IP polyphenols were established byorthogonal experiment. The optimum condition was 70% ethanol in 60min at 80℃andsolid to liquid ratio 1:16. The content of polyphenols was 34.53% when this technologywas used. EtoAc (ethyl acetate) was the best reagent for purification, and the content ofpolyphenols reached 80.04 % after purification.(2) HPD500 was selected as the medium for initial isolation of IP polyphenols bycomparing 8 kinds of macroporous adsorption resin's adsorption and separationperformance, adsorption capacity of HPD500 is 35.2mg/g. The method to initiallyseparate IP polyphenols was established using column chromatography. Total IPpolyphenols (TIPP) was separated into three fractions which were named TIPPA,TIPPB and TIPPC after gradient elution with aqueous alcohol. The recovery rate wasup to 75.17%.(2) Sephadex LH-20, HPD500 or polyamide column chromatography were chosen to separate fraction TIPPA, TIPPB, TIPPC respectively, Three separationprocedures were optimized and six phenolic compounds were obtained. The structureidentification showed these six phenolic compounds were 5-O-caffeoylquinic acid(TIPPA1), 3-O-caffeoylquinic acid (TIPPA2), 1-O-caffeoylquinic acid (TIPPA3), rutin(TIPPB4), 3,5-di-O- caffeoylquinic acid (TIPPC5) and 4,5-di-O-caffeoylquinic acid(TIPPC6) respectively. Among them, 1-O-caffeoylquinic acid (TIPPA3) was firstlyisolated from IP.2 Studying the anti-hyperlipemia and anti-obesity activity of IP polyphenois byHigh Throughput Screening (HTS) MethodHTS models of PPARs, FXR, 3T3-L1 were used to evaluate the pharmacologicalactivity of IP ployphenols. The results were as follows.(1) IP ployphenols showed a dual PPARγ/βactivity. Fraction TIPPA showed thehighest PPARγactivity (activity times 1.86). The compound 3,5-di-O- caffeoylquinicacid (TIPPC5) showed the highest PPARβ/δactivity. Its activity times was 1.46, whichwas equal to that of tea ployphenols.(2) TIPPA, TIPPB fractions and TIPPA1, TIPPA2, TIPPA3 compounds hadinhibition on FXR, in which TIPPA exhibited the highest inhibitory activity.(3) The effects of IP ployphenols on PPARαand 3T3-L1 were insignificant.(4) The activation and inhibition effects of IP ployphenols on various modelsindicated that IP polyphenols were involved in anti-hyperlipidemia and lipid & energymetabolism regulation through various pathways. These findings suggested that IPployphenols were active ingredient for anti-obesity in IP.3 Study of in vitro antioxidant properties of IP ployphenolsThe scavenging capacities of IP ployphenols on 1,1-diphenyl-2-picrylhydrazyl,superoxide and hydroxyl free radicals were investigated. Meanwhile, the antioxidantactivity on linoleic acid was studied by thiobarbituric acid (TBA) method. The resultsshowed that IP ployphenols had good effects on scavenging free radicals and onpreventing linoleic acid peroxidation.(1) Except the fraction TIPPB, the hydroxyl scavenging capacities of total andfractional polyphenols were superior to tea ployphenols and L-ascorbic acid. The DPPHscavenging capacities of total and fractional polyphenols was inferior to teapolypheanols, but superior to L-ascorbic acid. In superoxide anion radicals system, theTIPPC fraction had the highest effect and the others were between tea polyphenols andL-ascorbic acid. The capacity of preventing linoleic acid peroxidation of total andfractional polyphenols in IP was lower than tea polyphenols. (2) From the studying of antioxidant properties of the phenolic compounds in IP, inhydroxyl free radicals system, the compounds TIPPC5, TIPPC6, TIPPA1, TIPPA2were more effective in scavenging free radicals than those of tea polyphenols andL-ascorbic acid, but 1-O-caffeoylquinic acid (TIPPA3) was similar to tea ployphenols.Rutin (TIPPB4) was less effective than L-ascorbic acid. The DPPH scavengingactivities of all phenolic compounds were higher than L-ascorbic acid but lower than teapolyphenols, with an exception of rutin (TIPPB4) which was close to L-ascorbic acid.In superoxide anion radicals system, the scavenging activity of a dicaffeoylquinic acidwas higher than tea polyphenolsand, however monocaffeoylquinic acid was lower thantea polyphenols. The preventing Linoleic acid peroxidization capacities of all thephenolic compounds were lower than tea polyphenols.(3) The antioxididant activities of total and fractional polyphenols showed highertendency as compared to the phenolic compounds in IP. The scavenging free radical andpreventing linoleic acid peroxidization capacities of dicaffeoylquinic acids weresuperior to monocaffeoylquinic acids, with the exception of compound5-di-O-caffeoylquinic acid which was more effective in preventing linoleic acidperoxidization than 3,5-di-O-caffeoylquinic acid.4 The inhibitory effects of IP ployphenols on amylase and bacterial activities.(1) The effects of IP ployphenols on amylase were investigated with the percentinhibition ofα- amylase and ptyalin as index. IP ployphenols showed inhibitory activityon amylase. And its inhibitory activity on ptyalin was similar to a-amylase. TIPP,TIPPB and TIPPC exhibited inhibitory activity on amylase, TIPPA had no inhibitoryactivity on amylase, and TIPPB had the highest inhibitory activity on amylase amongtotal and fractional IP ployphenols. Rutin possessed the highest inhibitory amylaseactivity, and the inhibitory rate ofα-amylase and ptyalin was 38.5% and 33.1% at theconcentration of 2.5mg/ml, and 4,5-di-O- caffeoylquinic acid (TIPPC6) had goodinhibitory effects on amylase among phenolic compounds in IP(2) The inhibitory activities on Escherichio coli, solmonella paratyhpi andstreptococcu sp were investigated using antibacterial assay in vitro. IP ployphenolsshowed considerable antibacterial effects, The highest antibacterial effect was againstsolmonella paratyhpi, followed anti-Escherichio coli activity, and the anti-streptococcusp activity was relatively weak. IP ployphenols were more effective against solmonellaparatyhpi than tea ployphenols. Fraction TIPPB, TIPPC and TIPP had greater activitiesagainst Escherichio coli and solmonella paratyhpi than TIPPA, but TIPPA had thehighest anti-streptococcu sp activity.