Anti-tumor And Anti-inflammatory Constituents From Physalis Pubescens And The Pharmacokinetic Study Of Physapubescin In Rats

The genus Physalis(family Solanaceae)includes approximately 120 species worldwide,which are mainly distributed in the tropical and temperate regions of America and in the temperate regions of Europe and Asia.Five species and two varieties are found in China.Recently,increasing attention has been drawn to the Physalis genus especially on their phytochemistry and pharmacology.Physalis pubescens L.is also known as Deng-Long-Cao in China,and it has been used as a traditional folk medicine to treat sore throat,cough,urethritis,hematuria,and orchitis.The fruits of P.pubescens L.,with a golden yellow skin and which tastes both sweet and sour,is a popular fruit in the north-east region of China.Earlier phytochemical investigations on this species have led to the characterization of a series of withanolides which were reported to have anti-inflammatory and anti-tumor activities.Our recent findings also suggest that withanolides could be potentially useful in the treatment of human tumors.1.Anti-tumor and anti-inflammatory constituents from Physalis pubescens L..To fully understand the anti-tumor and anti-inflammatory constituents in this plant,further investigation on the extracts of the fruits of P.pubescens L.and the whole herb of P.pubescens L.were carried out which led to the isolation of 68 compounds including 20 new ones(1～20).On the basis of chemical evidences and spectral analyses(UV、ECD、IR、1D and 2D-NMR、HRMS、X-ray single crystal diffraction),the structures of these compounds were elucidated as follows:physapubefruside A(1),(1S,3S,20S,22R,24S,25S)1,3,24,25-tetrahydroxywitha-5-en-22,26-olide(2),(1S,3S,20S,22R,24S,25S)-1,3,24,25tetrahydroxywitha-5-en-22,26-olide 3-O-β-D-glucopyranoside(3),(1S,3S,20S,22R,24S,25S)1,3,24,25-tetrahydroxywitha-5-en-22,26-olide 3-O-[β-D-glucopyranosyl(1→6)]-β-Dglucopyranoside(4),(20S,22R,24R,2 5S,26R)-22,26-epoxy-1α,3β,24,25,26pentahydroxyergost-5-ene(5),(22R,24R,25S,26Φ)-22,26-epoxy-1α,3β,24,25,26(α/β)pentahydroxyergost-5-ene 3-O-β-D-glucopyranoside(6),(20S,22R,24R,25S,26R)-22,26epoxy-24,26-dimethoxy-1α,3β,25-trihydroxyergost-5-ene(7),(20S,22R,24R,25S,26R)-22,26epoxy-24,26-dimethoxy-1α,3β,25-trihydroxyergost-5-ene 3-O-[β-D-glucopyranosyl(1→6)]-βD-glucopyranoside(8),(20S,22R,24R,25S,26Φ)-15α-acetoxy-5,6β:22,26-diepoxy-3β,4β,24,25,26(α/β)-pentahydroxyergost-1-one(9),(20S,22R,24R,25S,26Φ)-15α-acetoxy-5,6β:22,26diepoxy-3-O-methyl-3β,4β,24,25,26(α/β)-pentahydroxyergost-1-one(10),(20S,22R,24R,25S,26Φ)-15α-acetoxy-5,6β:22,26-diepoxy-3-O-n-butyl-3β,4β,24,25,26(α/β)-pentahydroxyergost1-one(11),(20S,22R,24R,25S,26Φ)-15α,16α-diacetoxy-5,6β:22,26-diepoxy-3β,4β,24,25,26(α/β)-pentahydroxyergost-2-en-1-one(12),(20S,22R,24R,25S,26Φ)-15α,16α-diacetoxy5,6β:22,26-diepoxy-3-O-methyl-3β,4β,24,25,26(α/β)-pentahydroxyergost-2-en-1-one(13),(20S,22R,24R,25S,26Φ)-15α,16α-diacetoxy-5,6β:22,26-diepoxy-3-O-n-butyl-3β,4β,24,25,26(α/β)-pentahydroxyergost-2-en-1-one(14),withapubeside A(15),(1S,3S,20S,22R,24S,25R)22,25-epoxy-25-O-n-butyl-1α,3β,25-trihydroxyergost-5ene-26-one 3-O-[β-D-glucopyranosyl(1→6)]-β-D-glucopyranoside(16),(20S,22R,24S,25S,26R)-15α-acetoxy-5,6β:22,26:24,25-triepoxy-3-O-methyl-3β,4β,26β-trihydroxyergost-1-one(17),(20S,22R,24S,25S,26R)-15αacetoxy-5,6β:22,26:24,25-triepoxy-26-methoxy-4β-hydroxyergost-2-en-1-one(18),(20S,22R,24S,25S,26Φ)-15α,16α-diacetoxy-5,6β:22,26:diepoxy-24-methoxy-4β,25,26(α/β)trihydroxyergost-2-en-1-one(19),1α,3α-dihydroxy-7α-eremophila-9,11-dien-3-O-[α-Larabinopyranosyl-(1→6)]-β-D-glucopyranoside(20),(22R,24R,25S,26R/S)22,26-epoxy-1α,3β,24,25,26(α/β)-pentahydroxyergost-5-ene 3-O-[β-D-glucopyranosyl(1→6)]-β-D-glucopyranoside(21),(22R,24S,25S)24,25;22,26-diepoxy-1α,3β,dihydroxy-witha-5enolide 3-O-[β-D-glucopyranosyl(1→6)]-β-D-glucopyranoside(22),(22R,24R,25S,26R/S)-22,26-epoxy-24-O-methyl-1α,3β,24,25α,26(α/β)-pentahydroxyergost-5ene 3-O-[β-D-glucopyranosyI(1→6)]-β-D-glucopyranoside(23),physapubescin(24),(20S,22R,24S,25S,26R)-15α-acetoxy-5,6β:22,26:24,25-triepoxy-26-methoxy-4β-hydroxyergo st-2-en-1-one(25),physapubescin I(26),(20S,22R,24R,25S,26Φ)-15α-acetoxy5,6β:22,26-diepoxy-4β,24,25,26(α/β)-tetrahydroxyergost-2-en-1-one(27),(20S,22R,24S,25S,26Φ)-15α-acetoxy-5,6β;22,26:diepoxy-24-methoxy-4β,25,26(α/β)-trihydro xyergost-2-en-1-one(28),(20S,22R,24R,25S,26Φ)-15α,16α-diacetoxy-5,6β:22,26-diepoxy4β,24,25,26(α/β)-tetrahydroxyergost-2-en-1-one(29),physapubescin D(30),2α,3βdihydroxy-5α-pregn-16-en-20-one 3-O-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside(31),(22E,24S)-5α,8α-epidioxy-24-methyl-cholesta-6,22-dien-3β-ol(32),(22E,24S)-5α,8α-epidioxy-24-methyl-cholesta-6,9(11),22-trien-3β-ol(33),alkesterol B(34),(1’S,6’R)-8’-hydroxyabscisic acid β-D-glucoside(35),(6S,9R)-roseoside(36),(6S,9S)-roseoside(37),staphylionoside D(38),p-menth-4(8)-ene-12-diol 1-O-α-L-arabinopyranosyl-(1→6)-β-D-glucopyranoside(39),loliolide(40),bergapten(41),imperatorin(42),osthol(43),xanthotoxin(44),isopimpinellin(45),auraptenol(46),kaempferol(47),kaempferol 3-O-β-D-glucopyranoside(48),kaempferol 3-O-β-D-(2-O-β-D-glucopyranosyl)-glucopyranoside(49),luteolin(50),quercetin 3-O-β-D-(2-O-β-D-glucopyranosyl)glucopyranoside(51),diosmetin(52),eriodictyol(53),pyrocatechol 1-O-β-D-glucopyranoside(54),benzylβ-D-glucopyranosyl(1→6)-β-D-glucopyranoside(55),2-phenylethyl-O-β-D-glucopyranoside(56),p-hydroxybenzene propanoic acid(57),3,4-dihydroxybenzenepropionic acid(58),p-coumaric acid(59),(1-O-p-coumaroyl)-(6-O-β-glucosyl)-β-glucoside(60),1-O-transcinnamoyl-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside(61),1-(β-D-ribofuranosyl)-1,2,4-triazole(62),1-(4-hydroxy-3,5-dimethoxyphenyI)ethanone(63),n-butyl-β-D-fructopyranoside(64),icariside E5(65)and ambinine(66).There are 29 withanolides,6 other type steroids,7 terpenes,6 coumarins,7 flavonoids,8 phenolic profiles,and 5 others.Compounds 31～33,35,38,46,52～58,60,62,64 and 66 were isolated from the genus Physalis for the first time.Withanolides,the representative bioactive constituents of this plant,were reported to have significant bioactivities.It is important to explore this class of compounds from P.pubescens L.as comprehensive as possible.Therefore,the fragmentation pathways and corresponding fragmentation rules of withanolides in positive MS/MS mode were thoroughly investigated in this study by analysing 14 isolated withanolides.As a result,diagnostic ions for the rapid screening of withanolides and classification of different types of withanolides were determined based on their MS/MS fragmentation patterns.Furthermore,an integrated approach using UPLC-Q/TOF-MS/MS together with a novel three-step data mining strategy was developed for the systematic analysis of withanolides in complex samples.Consequently,40 withanolides were efficiently discovered and identified from the crude extracts of P.pubescens L..Compounds 18,19,and 24-29 exhibited significant cytotoxicity against human prostate cancer cells(C4-2B and 22Rvl),human renal carcinoma cells(786-O,A-498,Caki-2,and ACHN),human melanoma cells(A375 and A375-S2),human hepatoma carcinoma cells(HepG2),and human normal hepatic cell line(L02)with IC50 values in the range of 0.19～9.70μM.Compounds 18,19,24,25,and 29 showed potent inhibitory activities against LPS-induced nitric oxide production in RAW 264.7 macrophages with IC50 values in the range of 0.32～5.56μM.Physapubescin decreased the expression of HIF-2a and increased the expression of CCAAT/enhancer-binding protein homologus protein(CHOP),which led to up-regulation of death receptor 5(DR5),activation of caspase-8 and-3,cleavage of poly(ADP-Ribose)polymerase(PARP)and apoptosis.2.Pharmacokinetic study of physapubescin in rats.Physapubescin,a representative withanolide from P.pubescens L.,shows potent antitumor and anti-inflammatory activity.To clarify the real effective materials and the intracorporal process of physapubescin,a strategy was established and introduced to identify the metabolites in vivo based on UPLC-Q/TOF-MS and MetabolynxTM data processing software,and a quantitative determination method was developed to study the pharmacokinetic of physapubescin and its major metabolite M19.A total of 28 metabolites were identified in rat biological samples after oral administration of physapubescin and a possible metabolic pathway and transformation process were summarized.The pharmacokinetic study clarified the in vivo pharmacokinetic characteristics of physapubescin and its major metabolite M19 in rats,so as to provide a basis for revealing the material foundation and guiding rational clinical application of P.pubescens L..From the above results,a series of compounds with anti-tumor and anti-inflamatory activities were discovered,which indicated the material foundation of efficacy of P.pubescens L.both in vitro and in vivo.The metabolism and pharmacokinetic studies of physapubescin threw light on the bioactive material basis and the intracorporal process of physapubescin.Additionally,it also provides basis to develop and utilize the traditional Chiness medicine P.pubescens L.