| Purposes:Portulaca oleracea L.is an annual herb of the family Portulacaceae,produced in the north and south of the country and widespread in temperate and tropical regions of the world.It prefers fertile soil,is drought-and water-resistant,and lives well in sunny places such as vegetable gardens,farmland,roadsides,and ruins.P.oleracea has long been used as a dualpurpose plant for medicinal purposes.It could clear heat and dampness,detoxify swellings,reduce inflammation,quench thirst and diuretic properties.Modern pharmacological studies have shown that P.oleracea has anti-inflammatory,antioxidant,anti-ageing,anti-bacterial,antitumor,and neuroprotective properties,which are inextricably linked to its intrinsic chemical composition of alkaloids,organic acids,flavonoids and lignans,etc.The aim of this project is to extract and isolate the monomeric chemical constituents of P.oleracea,screen them and evaluate their anti-inflammatory,anti-cholinesterase,and antioxidant activities,and then select the most active monomeric constituent for metabolic studies in rats,to provide a technical route and theoretical basis for basic research,clinical applications,and the development of new drugs.Materials and Methods:1.250 kg of P.oleracea was extracted with 50% ethanol at reflux to obtain the alcoholic extract,which was separated and purified by a series of column chromatography methods including silica gel,polyamide,ODS and Sephadex LH-20,and finally the monomeric compounds were prepared by UHPLC,and the monomeric compounds were structurally identified using UV,IR,NMR and UHPLC-ESI-Q-TOF/MS.2.The cytotoxicity of oleraceacid A,oleraceacid B and oleraisoquinoline,5-hydroxymethylfuran-2-carboxylic acid and 1H-pyrrole-2,5-dicarboxylic acid was firstly tested by CCK-8 and MTT assay respectively,followed by the establishment of LPS-induced RAW264.7 cells inflammation model,the anti-inflammatory activities of the above five monomeric compounds were assessed by ELISA.In addition,the anti-acetylcholinesterase activities of the five monomers were assessed by modified Ellman assay,and the antioxidant activities of oleraisoquinoline and oleraceacid B were assessed by DPPH radical scavenging assay.3.Metabolism studies of oleraisoquinoline in rats were carried out by tail vein administration.The plasma,urine,and feces samples were collected from rats after administration and the metabolites were identified using UHPLC-ESI-Q-TOF/MS as well as to deduce its possible metabolic pathways.Results:1.In this study,27 monomeric compounds were isolated from P.oleracea by efficient and non-polluting isolation methods,among which three new compounds,named oleraisoquinoline,oleraceacid A and oleraceacid B;12 first-subdivision compounds,named(7E,10E)-octadeca-7,10-dienoic acid,(10E,13E)-octadeca-10,13-dienoic acid,(7E,10E)-hexadeca-7,10-dienoic acid,methyl tridecanoate,5-(hydroxymethyl)furan-2-carboxylic acid,5-(hydroxymethyl)furan-2-ol,furan-2,5-dione,4-hydroxybenzamide,4-amino-3-hydroxybenzoic acid,2-methoxy-5-hydroxybenzoic acid,p-aminobenzamide and phenylglycine;and 12 known compounds,namely methyl(9E,12E)-octadeca-9,12-dienoate,1H-pyrrole-2,5-dicarboxylic acid,1H-indole-3-carboxaldehyde,6,7-dihydroxy-3,4-dihydroisoquinolin-1-one,esculetin,gallic acid,p-hydroxybenzaldehyde,mesalazine,2,5-dihydroxybenzoic acid,salicylic acid,p-aminobenzoic acid and p-hydroxy-cinnamic acid.2.The results of the cytotoxicity assay showed that oleraceacid A,oleraceacid B and olerisoquinoline,5-hydroxymethylfuran-2-carboxylic acid and 1H-pyrrole-2,5-dicarboxylic acid were not cytotoxic at concentrations below 25 μM;the results of the anti-inflammatory assay showed that all the five monomers had anti-inflammatory effects;the results of the antiacetylcholine activity assay showed that the above five monomeric compounds had different degrees of acetylcholinesterase inhibition;the results of the antioxidant activity assay showed that the monomeric compounds oleraisoquinoline and oleraceacid B both had strong antioxidant effects.Notably,oleraisoquinoline exhibited strong anti-inflammatory,antiacetylcholinesterase,and antioxidant activities in the above three experiments.3.After tail intravenous administration of oleraisoquinoline in rats,4,17 and 2metabolites were found in rat’s plasma,urine and feces,respectively,and their metabolic pathways were obtained including oxidation,hydrolyzation,hydroxylation,sulfonation,acetylation,methylation and glucuronidation,and sulfonation was the main mode of metabolism.Conclusions:1.Three new monomeric compounds,the alkaloid oleraisoquinoline,the organic acid oleraceacid A and the lignan oleraceacid B,as well as 12 first-component monomeric compounds and 12 known monomeric compounds,were isolated from Portulaca oleracea L..2.The anti-inflammatory and anti-acetylcholinesterase activities of oleraisoquinoline,oleraceacid A,oleraceacid B,5-(hydroxymethyl)furan-2-carboxylic acid and 1H-pyrrole-2,5-dicarboxylic acid were evaluated in this study,and the antioxidant activities of oleraisoquinoline and oleraceacid B were also selectively evaluated based on their structures.The results showed that all the above five monomeric compounds exhibited varying degrees of anti-inflammatory and anti-acetylcholinesterase activities,and that both oleraisoquinoline and oleraceacid B had strong antioxidant activity.3.The UHPLC-ESI-Q-TOF/MS method was applied to the metabolism of the new alkaloid oleraisoquinoline in P.oleracea,and a total of 21 metabolites were found and the possible metabolic pathways were inferred as oxidation,hydrolyzation,hydroxylation,sulfonation,acetylation,methylation and glucuronidation. |
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