Studies On Triterpenoid Constituents And Metabolism In Vivo Of Ilicis Rotundae Cortex

Ilex rotunda Thunb.,one of the species in the Ilex genus of the Aquifoliaceae family,is widely distributed in southern China.The bark of this plant is recorded as "Ilicis Rotundae Cortex" in the Pharmacopoeia of the P.R.China(2015).Ilicis Rotundae Cortex has remarkable therapeutic effects on the treatment of swelling pain,rheumatic arthralgia,fever,peptic ulcer and cardiovascular diseases.Phytochemical investigations have indicated the presence of triterpenes,hemiterpene glycosides,phenylpropanoids and phenolics.The recent pharmacological studies showed that this plant had lots of bioactivities,such as anti-arrhythmia,anti-myocardial ischemia,anti-inflammatory and annti-(?).In order to seek the bioactive constituents from I.rotunda,the systematic research on the constituents was carried out.By means of column chromatograph(silica gel,RP C18,sephadex LH-20,and pre-HPLC),a total of twenty-five compounds were isolated from the barks of I.rotunda.On the basis of spectroscopic data,including IR,1H and 13C-NMR,1H-1H COSY,HSQC,HMBC,NOESY,and MS,and their physicochemical properties,twenty-four triterpenes were identified.The structures were elucidated as 23-hydroxy-3,11-dioxodea-12-en-28-oic acid(1),/3,23-dihydroxy-19-oxo-18,19-seco-urs-11,13(18)-dien-28-O-β-D-glucopyrano-side(A)(2),3β,23-dihydroxy-19-oxo-18,19-seco-urs-11,13(18)-dien-28-O-β-D-glucopy-ranoside(B)(3),3β,19a,23-trihydroxy-urs-12-en-28-O-β-D-glucopyranosyl-23-acetyl ester(4),3-O-α-D-glucopyranosyl-rotundic acidic-28-O-β-D-glucopyranoside(5),3-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosy1-(1→2)-β-D-arabinopyranosyl]-3β,19a-dihydroxy-12-en-28-oic acid(6),3-0-[a-L-rhamnopyranosyl-(1→2)-β-D-glucopyrano-sy1-(1→2)-β-D-xylopyranosyl]-3β,19a-dihydroxy-12-en-28-oic acid(7),(20β)-3β,23-dihydroxyurs-12,18-dien-28-oic acid(8),3β-O-[β-D-glucuronopyranosyl-6-O-methyl ester]ilexosapogenin A(9),rotundioic acid(10),mateside(11),19a,23-dihydroxyurs-12-en-28-oic acid 3β-O-β-D-glucuronopyranoside-6-O-methyl ester(12),ilexside Ⅱ(13),pomolic acid(14),28-O-β-D-glucopyranosyl pomolic acid(15),19-anhydro-4-epirotungenic acid(16),3β,23-dihydroxyursa-12,18(19)-dien-28-oic acid 28-β-D-glucopyranosyl ester(17),ilexosapogenin B(18),ilemaminoside A(19),rotungenic acid(20),hedergenin 3-O-β-D-glucuronopyranoside-6’-O-methyl ester(21),oleanolin acid 3-O-β-D-glucuronopyranoside-6’-O-methyl ester(22),gardeniside B(23),3β-[(-L-arabinopyranosyl)oxy]-19α-hydroxyolean-12-en-28-oic acid(24)。Among them,nine compounds(1-9)were new compounds,named rotundinosides E-M.Compound 1 contains an α,β-unsaturated ketone in C ring and one carbonyl resonance at the position of C-3,which is isolated from Ilex genus for the first time;Compounds 2 and 3 are 19-oxo-18,19-seco-ur-sane-type triterpenoids,which are rare from Ilex family;Compound 5 is biogenetically interesting secondary metabolite with an α-linked glucopyranosyl residue,as we knowed that glucosides of secondary metabolites found in plants normally have a β-configuration.Eight compounds(11-12,17,19,21-24)were isolated from this plant for the first time.The results enriched our knowledge about constituents of I.rotunda and could provide basic material for the further activity screening.Although numerous aspects of pharmacology of triterpenoids,in particular its activities as anti-tumor and anti-inflammatory,have been studied,the metabolism in vivo has few been characterized.Pedunculoside is one of the representative triterpenoids in quality standard of Ilicis Rotundae Cortex and embodied in the Pharmacopoeia of the P.R.China(2015).It showed the activity in hypolipidemic according to the pharmacological studies.Therefore,pedunculoside is one of the major bioactive components of I.rotunda.In order to characterize its biotransformation in rats,metabolism study of pedunculoside in vivo was investigated in this paper.In the present study,a simple and rapid method using ultra high performance liquid chromatography/quadrupole-time-of flight mass spectrometry(UPLC-QTOF-MS/MS)was developed for the determination of the pedunculoside and its metabolites.Based on the proposed strategies of dynamic background subtraction(DBS),information dependent acquisition(IDA)and multiple mass defect filter(MMDF),thirteen metabolites(10 phase I and 3 phase Ⅱ metabolites)were identified in rats after oral administration.Among these metabolites,12 metabolites were characterized in the rat feces(M1-M8,M10-M13),4 metabolites were characterized in the rat urine(M6,M8,M10,M12)and 2 metabolites were characterized in the rat plasma(M9,M12).The results indicated that deglycosylation,oxidization,deoxidation,dehydrogenation,sulfation,and glucuronidation were the major metabolic transformation of pedunculoside in vivo.Through the characteristic fragments of pedunculoside and its metabolites,a practical strategy for rapidly screening metabolites was taken out.The ions at loss of 162 Da and 176 Da from the precursor ion corresponded to the characteristic loss of glycoside and glucuronide.The ions at loss of 16 Da and 32 Da from the precursor ion corresponded to the characteristic of oxidation and di-oxidation.The ion at loss of 80 Da from the precursor ion corresponded to the characteristic of sulfation.This study has improved our understanding of the biotransformations and the pharmaceutical applications of pedunculoside in vivo,and provided a practical strategy for rapidly screening and identifying metabolites,hence,this methodology can be widely applied for structural characterization of the metabolites of other compounds with similar structures.