Study On Chemical Constituents Of N-butyl Alcohol Fraction Of Litsea Coreana L. And Anti-inflammatory And Immunomodulatory Activity In Vitro

Laoying tea is leaf of Litsea Coreana level which has been used as a tea in Guizhou,Sichuan and south of Anhui province in China. Modern pharmacological and clinicalstudies have shown that he extract from the leaves of Litsea Coreana L. exerts variousbiological actions, such as antioxidant, hepatoprotective hypolipidaemic,anti-inflammatory and immunomodulatory activity. And the mechanism of the liverprotection and anti-inflammatory has been clarified by experiments. It contains manychemical compositions, such as flavonoid glycoside, saponin, organic acid, cumoric,anhydride and tannin et al. Total flavonoids is the effective component in the extractfrom leaves of Litsea Coreana L. We had studied on the chemical constituents ofLitsea Coreana L. and obtained Quercetin, Kaempferol,（+）-Catechin, β-Sitosterol,Daucosterin,quercetin-3-O-β-D-galactopyr-anoside, kaempferol-3-O-β-D-glucopyran-oside, quercetin-3-O-β-D-glucopyranoside and kaempferol-3-O-β-D-galactopyran-oside. The research was aimed to accumulate and purify the total compounds, separateand elucidate individual compounds from the acetoacetate extract, and then screen theanti-inflammatory and immunomodulatory activity of isolated monomers in vitro. First Section：To accumulate and purify the chemical constituents and identifythe components from the extracts of Litsea Coreana L．Litsea coreana L．（10kg）were was refluxing extracted with10times the amountof volume of70%ethanol reflux extraction for three times, respectively for2h,1h,1h. The extract was filtered and mergered. The solution were combined concentratedunder reduced pressure. Then the extract was suspended in H2O and successivelypartitioned with petroleum ether, chloroform, EtOAc and n-BuOH in turn. Theconstituents were isolated from the n-BuOH fraction and purified by D101macroporous column, Silica column, Sephadex LH-20column chromatography andhigh performance liquid chromatography.20compounds were isolated from then-butanol extract of Lietsea coreana L.. The compounds were identified by means ofphysical and chemical properties, HPLC, TLC and spectroscopic analysis such as UV,1H-NMR,¹³C-NMR and MS．14compounds were identified as n-Butyl phthalate,Flavanone-7-O-β-D-glucopyranoside, Kaempferol-3-O-β-D-（6-O-trans-p-coumaroyl）mannopyranoside, Kaempferol-3-O-β-D-（6-O-trans-p-coumaroyl） glucopyranoside,trans-p-Coumaric acid, Salicylic acid, Protocatechuic acid, Iso-epiphyllocoumarin,Phyllocoumarin, Epiphyllocoumarin, Isophyllocoumarin, Kaempferol-3-O-α-L-rhamnose,5-（2-phenylethyl）-3-hydroxyphenol-β-D-glucopyranoside. Among thosecompounds, Isoepiphyllocoumarin, Isophyllocoumarin and5-（2-phenylethyl）-3-hydroxyphenol-β-D-glucopyranoside are three new compounds. The other elevencompounds are obtained from the plant for the first time．Second section: The activity screening of the monomers of compounds onanti-inflammatory and immunomodulatory in vitroThe experiment meaned to study the structure-function relationship of the monomersof compounds on immune function of rats. Ten Sprague-Dawiey rats were fed for10¹5days. Then the rats were killed by bleeding of the Femoral artery. Immunecells（peritoneal macrophages） were obtained，then cultured in vitro with the sixcompounds we had obtained from the n-butanol fraction. Some immune indexes were detected respectively such as the productions of IL-1, prostaglandin E2（PGE2） andtumor necrosis factor-α （TNF-α） were measured by the method of enzyme linkedimmunosorbent assay, making a primary screening to the compounds onanti-inflammatory and immunomodulatory activity. We found that all the sixcompounds have activity on immune regulation, but the activity of anti-inflammatoryand immunomodulatory of Iso-epiphyllocoumarin, Phyllocoumarin, Epiphyllocou-marin, Isophyllocoumarin and5-（2-phenylethyl）-3-hydroxyphenol-β-D-glucopyran-oside are strong. Because of the differences of structure-function relationship betweencompounds, the regulation of IL-1and TNF-а by the configuration ofIsoepiphyllocoumarin and Isophyllocoumarin are stronger than the configuration ofPhyllocoumarin and Epiphyllocoumarin, but the regulation of PGE2by theconfiguration of Phyllocoumarin and Epiphyllocoumarin are stronger than theconfiguration of Isoepiphyllocoumarin and Isophyllocoumarin.