Research On Chemistry Of Phyllanthus Emblica L. And Catalytic Capability Of GbUGT717L

Phyllanthus is the dried fruit of Phyllanthus emblica L,which belongs to the genus Phyllanthus in family Euphorbiaceae,and Phyllanthus is first recorded in "New Materia Medica" and is a Tibetan medicinal herb.The extracts of Phyllanthus display the activities of resolving phlegm and cough,strengthening stomach and digesting food,clearing away heat and promoting body fluid,digesting and accumulating food,etc.It can also be used to treat cold,fever,cough,sore throat,diphtheria,hot mouth,dyspepsia,chronic hepatitis,hypertension,and obesity,etc.Phyllanthus has a long history of application in traditional Chinese medicine,Tibetan medicine,Mongolian medicine,Egypt,Persia,North America and other traditional medicine systems in more than 30 countries or ethnic groups.At the same time,Phyllanthus has been listed as a dual-purpose list of medicine and food.There are more than 300 species of Phyllanthus,and Phyllanthus is the only wild and cultivated edible plant of Phynanthus genera,which contain more than 300 species.These unique characteristics are possibly related to their chemical composition.Previous phytochemical studies have shown that Phyllanthus is rich in polyphenols,flavonoids,tannins,polysaccharides,organic acids,vitamins,amino acids and trace elements such as zinc,manganese,iron,calcium and selenium.Therefore,it has the effect of anti-oxidation,anti-bacterial,anti-inflammatory,anti-virus,anti-aging,lipid-lowering,blood pressure-lowering,HIV suppression,liver protection and other functions.The purpose of this study is to further study the chemical constituents of Phyllanthus,in order to explore the other characteristic chemical constituents of Phyllanthus and its anti-atherosclerosis mechanism.Twenty-two compounds were obtained by silica gel column chromatography,ODS column chromatography,Sephadex LH-20 column chromatography,semi-preparative high-performance liquid chromatography and recrystallization method.Through their spectra data,physical and chemical properties analysis.19 compounds were identified as dibutyl phthalate(Y1),isovanillic acid(Y2).trans-cinnamic acid(Y3),p-hydroxybenzaldehyde(Y4),pine aldehyde(Y5).quercetin(Y6),kaempferol-3-O-alpha-L-rhamnose(Y7),phthalate-bis(2'-ethylheptyl)ester(Y8),naringenin(Y9),2-hydroxy-3-phenylpropylpropyl methyl ester(Y10),1-(4'-methoxy-4'-oxobutyl)-1 H-pyrrole-2,5-dicardicarboxylic acid(Yll),hydroquinone(Y12),myricetin(Y13),2,5-dicarboxypyrrole-2,5-dicarboxypyrr(Y14),furoic acid(Y15),methyl gallate(Y16),di(2-ethylhexyl)phthalate(Y17),protocatechuic acid(Y18),gallic acid(Y19).Among the above compounds,there are 4 flavonoids,7 phenolic acids,5 aromatic esters,1 furoic acid and 2 alkaloids.Notably,Yll and Y14 are the first pyrrole alkaloids discovered from Phyllanthus and compound Y11 is a new compound,and compounds Y1,Y2,Y4,Y5,Y8,Y10,Y12,Y13,Y14,Y15 and Y17 were isolated from Phyllanthus for the first time.In this paper,the anti-inflammatory activity of extracts of Phyllanthus(EO)and selected representative compounds were also evaluated,including new compounds(Y11),2,5-dicarboxypyrrole(Y14),quercetin,gallic acid,methyl gallate,dibutyl phthalate.Firstly,The anti-inflammatory effects of EO and the above compounds were evaluated.The concentration of inflammatory factors IL-6,TNF-a and MCP-1 in cell supernatant was determined by ELISA,and the concentration of NO in supernatant was determined by Griess.The results showed that the above compounds and EO had different inhibitory effects on the production of inflammatory factors IL-6,TNF-a,MCP-1 and NO.The anti-atherosclerosis activities of pyrrole alkaloids Y11 and its analogues 2,5-dicarboxypyrrole(Y14)and EO were further studied.The same method was used to investigate the effects of different concentrations of Y11 and Y14 on the production of inflammatory cytokines NO,IL-6 and TNF-a.The results showed that Y11 and Y14 inhibited release of inflammatory factors in macrophages in a concentration-dependent manner.Meanwhile,the pathways of Y11,Y14 and EO inhibiting the release of inflammatory factors were investigated by Western blotting method.The results showed that EO,Y11 and Y14 inhibited the release of inflammatory factors through the MAPKs pathway.The oil red staining was used to detect the effect of EO,Y11 and Y14 on cell foam.Under the microscope,we could see that EO,Y11 and Y14 could inhibit the production of red lipid droplets.Meanwhile,the effects of different concentrations of Y11 on ox-LDL induced foam cells were investigated.Under microscope,we found that Y11 could inhibit the production of red lipid droplets in a concentration dependent manner.The results showed that the total extract of Phyllanthus and its two pyrrole alkaloid compounds could reduce the formation of foam cells,and the new compound Y11 decreased foam cell formation in a concentration dependent manner.Inflammation and macrophage foam play an important role in the formation and development of atherosclerosis.These results suggest that extracts of Phyllanthus and its pyrrole alkaloids have antiatherosclerosis effort to some extent.Compound GK-1 is an alkaloid compound with remarkable pharmacodynamics,which isolated from Ginkgo.But it is difficult to isolated from Ginkgo.The chemical synthesis step of GK-1 is long,so that the economy of chemical synthesis is not high In previous studies,N-glycosyltransferase GbUGT717L was expressed in Ginkgo,which can catalyze the reaction of N-indole-3-acetyl-L-aspartic acid(IAA-Asp)with uridine diphosphate glucose(UDPG)to produce glucoside.43 compounds as substrates were carried out to investigate the selectivity of the enzyme to the compounds.The results showed that GbUGT717L enzyme specifically catalyzed the amidation of indoleacetic acid and amino acid(IAA-AA)with UDPG to glucoside,and catalyzed the reaction of 3-methylindole and ortho-,meta-alkyl aniline with UDPG to new product.