Study On Isolation And Identification Of Ilex Latifolia Thunb And Its Bioactivities

Ilex latifolia Thunb (I. latifolia) is a kind of traditional Chinese beverage, which isderived from the leaves of Folium llicis latifoliae and mainly distributed in the region ofSouth China. I. latifolia contains more than200kinds of chemical ingredients, such assaponins, amino acids, polyphenols, flavonoids, vitamin C, caffeine, protein, etc. The tea hasa bitter taste, followed by cool and sweet. It has various pharmacological activities withantihypertensive, lose weight, antitumor, anti-aging, and quicken the blood vessels. In recentyears, an increasing research on I. latifolia was mainly concentrated in the health protection oftea and beverage, but the study of functional components in I. latifolia remains to be furtherin-depth discussion, which is in order to promote the development of tea resource.The total extract was obtained by the extraction of heating reflux with95%ethanol andthen portioned with petroleum ether, ethyl acetate, n-butanol and water, which yieldedpetroleum ether fraction, ethyl acetate fraction, n-butanol fraction, and water fraction,respectively. Different models, antioxidant (ferric reducing/antioxidant power assay, DPPHradical scavenging assay and superoxide anion scavenging assay), anticomplement, antitumorand anti-inflammatory properties were use to evaluate the activity of the total extract (95%ethanol) and four fractions extracted from I. latifolia. Results showed that95%ethanol extractand ethyl acetate fraction were found to have significant DPPH radical scavenging activity,reducing power activity, obviously inhibitory effect on the complementary system with thehemolysis inhibition rate, cytotoxicity against human cervix carcinoma Hela cells andinhibition effects on NO production in macrophage RAW264.7cells. Consequently, ethylacetate fraction was selected for further separation and purification.In order to analyze the effective component, four chemical constituents (flavonoids,saponins, polysaccharides and alkaloids) were obtained from I. latifolia by different extractionmethods. Their contents were determined by standard curves of four reference substances, be21.58%,28.75%,24.55%, and14.67%, respectively. In addition, the antioxidant,anticomplementary, antitumor and anti-inflammatory activities of the four chemicalconstituents were investigated. Results showed that polysaccharides exhibited a stronginhibitory effect on the complementary system. Flavonoids caused a significant scavengingactivity of DPPH and inhibition effect on NO production in LPS-induced RAW264.7macrophages. Saponins show a better effect of antiproliferative against Hela cells andalkaloids exhibited a high reducing power of antioxidantion. Four constituents all displayed lower activity of superoxide anion scavenging assay.Ethyl acetate fraction was separated and purified by various column chromatography(including silica gel column, Sephadex LH-20gel column and ODS column) and the preparedHPLC. Five compounds were obtained and their structure was identified by massspectrometry and nuclear magnetic resonance as ethyl caffeate, ursolic acid, chlorogenic acid,3,4-di-O-caffeoylquinic acid methyl eater,3,5-di-O-caffeoylquinic acid methyl eater,respectively.Antioxidant, antitumor and anti-inflammatory activities of the five compounds isolatedfrom ethyl acetate fraction was investigated. Results showed that compound2and3showedstrong antioxidant activity both DPPH radical scavenging and reducing power activity. Allcompounds displayed lower activity of superoxide anion scavenging assay. In the evaluationof cytotoxicity against tumor cell lines, compound4and5exhibited obviously action. Inaddition, compound1and2has cytotoxicity against RAW264.7cells, which is unsuitable forthe study of anti-inflammatory activity. Other three compounds showed a certain extentinhibition effect on NO production in LPS-induced RAW264.7macrophages. Among them,compound3showed the strongest anti-inflammatory activity than others.