| Acanthopanax sessiliflorus?Rupr.et Maxim?Seem belongs to Acanthopanax genus of the family Araliaceae.It is a shrub distributes mainly in China,Korea and Japan.It has traditionally been used as a tonic and a sedative,as well as in the treatment of rheumatism and diabetes.The chemical constituents,anti-inflammatory activity and pharmacokinetics of main active componentswere investigated in this study.The chemical constituents of 70%ethanol extract of Acanthopanax sessiliflorus fruits were isolated by using multiple column chromatographic techniques,and 44 compounds were obtained.The structures of 42 compounds were fully elucidated by chemical and spectroscopic methods?1D,2D-NMR,MS?.The identified compounds include twelve triterpenes:3-oxo-24-methylenecycloartan?1?,schizandronic acid?2?,isomangiferolic acid?3?,betulin?4?,betulinic acid?5?,eleutheroside K?6?,22-?-hydroxychiisanogenin?7?,chiisanogenin?8?,22?-hydroxychiisanoside?9?,?1R,11??1,4-epoxy-11-hydroxy-3,4-secolupane-20?30?-ene-3,28-dioic acid?10?;one steroids:stigmasterol?11?;four flavonoids:isooirenitn?12?,rutin?13?,isorhamnetin-3-o-rutinoside?14?,catechin-7-O-?-glucopyranoside?15?;three ionones:icariside B1?16?,icariside B2?17?,?6R,7E,9R?-9-hydorxy-4,7-megastigmadien-3-one 9-O-?-D-apiofuranosyl-?1-6?-?-D-glucopyranoside?18?;seven lignans:?+?-l-hydroxypinoresinol-l-O-?-D-glucoside?19?;six benzoic acids:?7S,8R?dihydrodehydrodiconiferyl alcohol 4-O-?-D-glucopyranoside?20?,balanophonin?21?,berchemol-4'-O-?-D-glucoside?22?,lariciresinol-4,4'-di-O-?-D-glucopyranoside?23?,icariside E3?24?,?7S,8R?-erythro-7,9,9'-trihydroxy-3,3'-dimethoxy-8-O-4'-neolignan-4-O-?-D-glucopyranosideerythro?25?;six phenylpropanoids:coniferin?26?,4-O-?2-O-?-D-glucopyranosyl-1-hydroxymethylethly?-dihydroconiferyl alcohol?27?,1-allyl-3-methoxyhenyl-6-O-?-D-apiofuranosyl-?1''-6'?-?-D-glucopyranoside?28?,hovetrichoside G?29?,eugenyl?-rutinoside?30?,5-dimethoxypheny propylenol-4-O-?-D-glucopyranoside?31?;six phenolic acids:?vanillic acid-4-O-?-D-glucopyside??32?,salidroside?33?,4-hydroxybenzyl-?-D-glucopyranoside?34?,4-O-?-D-glucopyranosyl-3,5-dimethoxygallic acid?35?,vanillate glucoside?36?,2-hydroxy-5-?2-hydroxyethyl?phenyl-O-?-D-glucopyranoside?37?;five nitrogens compounds:perlolyrine?38?,flazine?39?,adenosine?40?,1-methyl-1,2,3,4-tetrahydro-?-carboline-3-carboxylic acid?41?,3-hydroxy-L-proline?42?.Compounds2,3,14,18,19,23,24,25,27,28,31,37,41,42were isolated from the Acanthopanax genus for the first time;compounds 6,13,16,17,22,32and 39 were isolated from this plant for the first time.Lipopolysaccharide?LPS?-activated RAW264.7 cells were used to explore the anti-inflammatory effect of Acanthopanax sessiliflorusfruits.First,cytotoxicity of Acanthopanax sessiliflorus fruits methanol extracts of ethyl acetate and the n-butanol layer were determined by MTT assay.The result showed that ethyl acetate or n-butanol layer?50,100,200 mg/L?did not induce significant cytotoxicity.Therefore,these concentrations were used for subsequent studies.To evaluate the inflammatory effect of n-butanol layer and ethyl acetate layer,we examined nitrite levels to investigate their inhibitory effects on NO production in LPS-induced RAW264.7 cells.Both of extracts of ethyl acetate and the n-butanol layer reduced LPS-induced NO production significantly in a dose-dependent manner and n-butanol layer exhibited the strongest inhibition.Then,we selected five triterpenoid?compound 5,6,7,8,10?isolated from n-butanol to study the anti-inflammatory activity of Acanthopanax sessiliflorus in vitro using the method mentioned above.The results showed that compound 5,6,10 have obvious anti-inflammatory activity and the anti-inflammatory activity of compound 10 is potent.Therefore,we further investigated anti-inflammatory mechanism of compound 10 at the molecular level.In this experiment,we selected iNOS and COX-2 as the investigation targets.We evaluated the expression of iNOS and COX-2 at the mRNA and protein levels through the Western Blot and PCR technique.The data demonstrated that LPS increased both the protein and mRNA expression levels of iNOS and COX-2,whereas corynoline significantly reversed the LPS-induced up-regulation in a dose-dependent manner.The results indicated that compound 10 inhibited NO production by down-regulating the levels of iNOS and COX-2.A specific,simple and sensitive UPLC-ESI-MS/MS method was developed and validated for the pharmacokinetic study of four triterpenoid compounds in rat plasma.The four compounds and internal standard were extracted from plasma samples by liquid-liquid extraction.The analysis was carried out on an ACQUITY UPLCTM BEH C18 column?1.7?m,50 mm×2.1 mm,i.d.?with mobile phase of acetonitrile-water containing 5 mmol/L ammonium acetate?90:10,v/v?.The detection was performed on a triple quadrupole tandem mass spectrometer by multiple-reaction monitoring?MRM?mode via electrospray ionization?ESI?source.This validated method was successfully applied to a pharmacokinetic study in rats after oral administrations of four triterpenoid and the extract of Acanthopanax sessiliflorus fruits.22-?-hydroxychiisanogenin and chiisanogenin showed a linear pharmacokinetics in rats in the studied dose range.Compared with the same dose of chiisanogenin,the AUC0-t of extract of Acanthopanax sessiliflorus fruits was decreased.A selective and sensitive UPLC-MS/MS method was developed and validated?for the determination and pharmacokinetic study of protocatechuic acid,scopolin,?-?-pinoresinol-4,4'-di-O-?-D-glucopyranoside,acanthoside D,acanthoside B and hyperin in rat plasma.Sample preparation involved a liquid-liquid extraction of the analysis.LC separation was achieved on a UPLC C18 column with a mobile phase consisting of water containing 0.1%formic acid-methanol.The detection was accomplished by multiple-reaction montiring?MRM?scanning with electrospray ionization?ESI-?source operating in the positive ionization mode.The current UPLC-MS/MS assay was validated for linearity,intra-day and inter-day precisions,accuracy,extraction recovery and stability and was applied to a pharmacokinetic study of the six lignans and a phenolic acid after oral administration of Acanthopanax sessiliflorus extract to rats.The maximum concentration(Cmax)was198.1±60.3ng/mL,734.1±203.4ng/mL,447.1±104.5ng/mL,356.7±145.2ng/mL,409.5±133.2ng/mL,103.7±46.3ng/mL;The time to reach the maximum plasma concentration(Tmax)was 1.5±0.5 h,0.5±0.3 h,1.0±0.4h,0.5±0.3h,0.5±0.2 h,0.25±0.3 h. |
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