Study On The Chemical Constituents And Anti-inflammatory Activity Of Piper Puberulum

Piper puberulum,which is mainly distributed in Guangxi,Guangdong,Guizhou,and southern coastal islands in China,belongs to the genus Piper of the family Piperaceae.The whole plant of this species,commonly known as “Diu-Bing-Duan” in Chinese,has been used as a traditional Yao medicine to treat rheumatism,abdominal pain,headache,dysmenorrhea and other diseases.According to the studies shown that Piper puberulum has a variety of constituents,such as alkaloids,terpenoids,lignans and other compounds,which have analgesic,anti-inflammatory and anti-platelet aggregation properties.The aerial parts of P.puberulum were investigated on their chemical constituents systematically by Diaion HP20 macroporous resin chromatography,positive-phase silica gel chromatography,MCI,reversed-phase ODS,Sephadex LH-20 and semi-preparative HPLC.Seventy-three compounds were obtained from Et OAc fraction,including thirty-one alkaloids(1-11,19-38),nine lignans(12-15,39-43),twenty terpenoids(16-17,44-61),and thirteen other components(18,62-73).Compounds 1-11 were new alkaloids,compounds 12-15 were new lignans,compound 16-17 was a new terpenoids,compound 18 was a new other component.These compounds were identified by spectroscopic methods as follows: puberulumines A-K(1-11),puberulin E(12a/12b),piperneolignan E(13a/13b),puberulins F-G(14-15),piperterpenes A-B(16-17),2-Methoxybenzoic acid-7-O-β-D-(6′-acetyl)-glucoside(18),semiimmersumine A(19),semiimmersumine B(20),liriodenine(21),norcepharadione B(22),piperolactam A(23),piperolactam B(24),cepharanone B(25),piperolactam C(26),aristololactam II(27),aduncamide(28),houttuynamide A(29),N-cis-feruloyl tyramine(30),N-trans-feruloyl tyramine(31),N-cis-glucosyl tyramide(32),N-trans-glucosyl tyramide(33),paprazine(34),N-trans-feruloil-3′-O-metoxidopamina(35),1,2-dihydro-6,8-dimethoxy-7-hydroxy-1-(3,5-dimethoxy-4-hydroxyphenyl)-Nl,N2-bis-[2-(4-hydroxyphenyl)ethyl]-2,3-naphthal ene dicarboxamide(36),1-(m-methoxycinnamoyl)pyrrolidine(37),N-isobutyl-2E,4Edecadienamide(38),neotaiwanensol B(39),nudibaccatumin A(40),piperneolignan D(41),diallylcatechol(42),neotaiwanensol A(43),6α-etoxieudesm-4(15)-eno-1β-ol(44),litseagermacrane(45),10α-hydroxycadin-4-en-15-al(46),10β,15-Hydroxy-α-cadinol(47),1β-hydroxy-4(15),5E,10(14)-germacratriene(48),4α-hydroxy-10β-ethoxy-1β,5α(H)-guai-6(7)-ene(49),(+)-aphanamol I(50),1α-hydroxyisodauc-4-en-15-al(51),isodauc-6-ene-10β,14-diol(52),4-epi-isodauc-6-ene-10β,14-diol(53),(3R*,3a S*,8S*,8a S*)-1,2,3,3a,6,7,8,8aoctahydro-8-hydroxy-8a-methyl-3-(1-methylethyl)-5-azulenemethanol(54),5-epi-oposit-4(15)-eno-1β,7-diol(55),(7R*)-opposit-4(15)-ene-1β,7-diol(56),4-megastigmen-3,9-dione(57),(3R,6R,7E)-3-hydroxy-4,7-megastigmadien-9-one(58),(3S,5R,6S,7E)-3,5,6-trihydroxy-7-megastigmen-9-one(59),pressafonin A(60),pressafonin B(61),3,4-dihydroxy-1-propenylbenzene(62),3,4-(1-propenyl)-phthalic acid diethyl ester(63),5-methoxy-3,4-(1-propenyl)-phthalic acid diethyl ester(64),cinnamic acid(65),nudibaccatumin B(66),(+)-5-hydroxy-3,4-dimeth-yl-5-pentylfuran-2(5H)-one(67),indole aldehyde(68),caffealdehyde(69),8-(5′-oxo-2′,5′-dihydrofuran-2′-yl)-octanoic acid(70),12-oxo-octadecadienoic acid methyl ester(71),methyl-(+)-(3R,4E,6Z,15E)-3-methoxyoctadecatrienoate(72),methyl linolenate(73).Rotamers 7-11 were determined the relative configuration of the new compounds by combining coupling constants and NOESY spectra,and their absolute configuration were further determined by comparing single crystal X-ray diffraction data and experimental ECD curves.Racemic or scalemic mixtures of 12-13 was analyzed and separated by Daicel Chiralpak AD-H column,and their absolute configurations were further established by using single crystal X-ray diffraction data,experimental and calculated ECD curve to their absolute configuration.Compounds 16-18 were confirmed their relative configurations according to coupling constants and NOESY spectra,and compound 18 was further comfirmed by acid hydrolysis and its optical rotation value to determine the configuration of its glucose fragment.Compounds 1-11 and 19-38 were assessed for their antineuroinflammatory activities by the Griess reaction in LPS-stimulated BV-2 cells,the results showed that compounds 3,5,10-11,20-23,26-31,34 and 38 displayed moderate inhibitory effects with IC50 values of 0.93-45.00 μM(Positive control MINO 15.00 ± 1.80 μM).In particular,the IC50 values of compounds 3,5,10,20,21,23,27,and 34 were lower than those of the positive control,particulary,the IC50 value of compound 27 was 0.93 μM.Compounds 12-17 and 39-61 were assessed for their antineuroinflammatory activities by the Griess reaction in LPS-stimulated BV-2 cells,the results showed that compounds 12 a,16-17,40,42,44-48,50-51,53-54 and 56-61 displayed moderate inhibitory effects with IC50 values of 2.16-34.90 μM(Positive control MINO 27.25 ± 7.06 μM).In particular,the IC50 values of compounds 12 a,17,40,42,46-48,50,54 and 58-61 were lower than those of the positivecontrol,particulary,the IC50 value of compound 60 was 2.16 μM.Among the compounds were isolated from P.puberulum,most are amide alkaloids,some are terpenoids,and a few are lignans and others.According to the anti-inflammatory activity studies,the methylenedioxy group may be an effective group to inhibit the release of NO from BV-2 cells,because the apophine or aristolochic acid lactams alkaloids containing the methylenedioxy group have good biological activity,and their IC50 values are lower than the positive control.