International Journal o f Molecular Sciences Article Antiallergic Phorbol Ester from the Seeds of Aquilaria malaccensis MichalKorinek1,2,†,VitthalD.Wagh1,†,I-WenLo1,Yu-MingHsu1,Hsue-YinHsu3, Tsong-LongHwang4,5,6,Yang-ChangWu1,7,8,9,Yuan-BinCheng1,10,Bing-HungChen2,11,*and Fang-RongChang1,10,12,13,* 1 GraduateInstituteofNaturalProducts,CollegeofPharmacy,KaohsiungMedicalUniversity,Kaohsiung807, Taiwan;[email protected](M.K.);[email protected](V.D.W.);[email protected](I.-W.L.); [email protected](Y.-M.H.);[email protected](Y.-C.W.);[email protected](Y.-B.C.) 2 DepartmentofBiotechnology,CollegeofLifeScience,KaohsiungMedicalUniversity,Kaohsiung807,Taiwan 3 DepartmentofLifeSciences,TzuChiUniversity,Hualien970,Taiwan;[email protected] 4 GraduateInstituteofNaturalProducts,CollegeofMedicine,ChangGungUniversity,Taoyuan333,Taiwan; [email protected] 5 ResearchCenterforIndustryofHumanEcologyandGraduateInstituteofHealthIndustryTechnology, ChangGungUniversityofScienceandTechnology,Taoyuan333,Taiwan 6 DepartmentofAnesthesiology,ChangGungMemorialHospital,Taoyuan333,Taiwan 7 SchoolofPharmacy,CollegeofPharmacy,ChinaMedicalUniversity,Taichung404,Taiwan 8 ChineseMedicineResearchandDevelopmentCenter,ChinaMedicalUniversityHospital, Taichung404,Taiwan 9 CenterforMolecularMedicine,ChinaMedicalUniversityHospital,Taichung404,Taiwan 10 CenterforInfectiousDiseaseandCancerResearch,KaohsiungMedicalUniversity,Kaohsiung807,Taiwan 11 TheInstituteofBiomedicalSciences,NationalSunYat-SenUniversity,Kaohsiung804,Taiwan 12 CancerCenter,KaohsiungMedicalUniversityHospital,Kaohsiung807,Taiwan 13 DepartmentofMarineBiotechnologyandResources,NationalSunYat-senUniversity, Kaohsiung804,Taiwan * Correspondence:[email protected](B.-H.C.);[email protected](F.-R.C.); Tel.:+886-7-312-1101(ext.2676)(B.-H.C.);+886-7-312-1101(ext.2162)(F.-R.C.); Fax:+886-7-3125-339(B.-H.C.);+886-7-311-4773(F.-R.C.) † Theseauthorscontributedequallytothiswork. AcademicEditor:GopinadhanPaliyath Received:21January2016;Accepted:10March2016;Published:21March2016 Abstract: The Aquilaria malaccensis (Thymelaeaceae) tree is a source of precious fragrant resin, calledagarwood,whichiswidelyusedintraditionalmedicinesinEastAsiaagainstdiseasessuch as asthma. In our continuous search for active natural products, A. malaccensis seeds ethanolic extract demonstrated antiallergic effect with an IC value less than 1 µg/mL. Therefore, the 50 present research aimed to purify and identify the antiallergic principle of A. malaccensis through a bioactivity-guided fractionation approach. We found that phorbol ester-rich fraction was responsible for the antiallergic activity of A. malaccensis seeds. One new active phorbol ester, 12-O-(2Z,4E,6E)-tetradeca-2,4,6-trienoylphorbol-13-acetate, aquimavitalin (1) was isolated. The structureof1wasassignedbymeansof1Dand2DNMRdataandhigh-resolutionmassspectrometry (HR-MS). Aquimavitalin (1) showed strong inhibitory activity in A23187- and antigen-induced degranulationassaywithIC valuesof1.7and11nM,respectively,withatherapeuticindexup 50 to71,000. TheantiallergicactivitiesofA.malaccensisseedsandaquimavitalin(1)haveneverbeen revealedbefore. TheresultsindicatedthatA.malaccensisseedsandthepurecompoundhavethe potentialforuseinthetreatmentofallergy. Keywords: Aquilaria malaccensis seeds; antiallergic; degranulation; phorbol ester; bioactivity-guidedfractionation Int.J.Mol.Sci.2016,17,398;doi:10.3390/ijms17030398 www.mdpi.com/journal/ijms Int.J.Mol.Sci.2016,17,398 2of13 1. Introduction Imunoglobulin E (IgE)-mediated allergy is a common immune system disorder affecting approximately235millionpeopleworldwide,particularlythepopulationindevelopedcountries[1]. Althoughtodayweareabletotreatthesymptomsofallergy,availablemedicationshaveundesirable effects,especiallywithaprolongeduse. Therefore,thereisaneedtosearchforalternativetreatment. In general, some natural sources are considered as safe and easily available. Mast cells and their degranulationplayacrucialroleinIgE-mediatedallergicinflammatoryresponses,suchasallergic rhinitis,acuteasthma,andatopiceczema[2]. β-Hexosaminidaseisanenzymereleasedalongwith histaminefrommastcells(ratbasophilicleukemiacells,RBL-2H3cells)uponactivationandservesas awell-acceptedinvitromodelinallergy[3]. AgarwoodisapricelessfragrantresinouswoodfromtheAquilariaspecies(Thymelaeaceae),which is formed as a defense mechanism to fend off pathogens. Agarwood is widely used in religious, aromatic,andmedicinalpreparations[4,5]. AquilariaspecieshasbeentraditionallyusedinThai[6]and Korean[7]medicine,intheAyurvedicpractice,aswellastraditionalChinesemedicinetotreatvarious diseases,particularlythediseasesassociatedwithinflammation[8]. AgarwoodfromtheAquillaria specieshasbeenusedascardiotonic,carminative,antiasthmatic,aphrodisiac,astringentremedy,and hasbeenfoundeffectiveagainstdiarrhea,dysentery,gout,rheumatism,paralysis,andparasites,and it has been beneficial for skin diseases [9]. The Aquilaria species was previously found to possess antidepressant [10,11], antineuroinflammatory [12], analgesic, antiinflammatory [13], antioxidant, antibacterial[6],antihyperglycemicinvivo[14],andlaxativeactivityinvivo[15]. Aquilaria malaccensis Lam. (syn. Aquilaria agallocha Roxb.) (Thymelaeaceae) is a tropical tree nativetoMalaysia,locallyknownas“Karas”. ItisdistributedintherainforestsofIndonesia,Thailand, Cambodia,Laos,Malaysia,Philippines,andIndia[16].ThealcoholicextractofA.malaccensisstemsand barkexhibitedcardiotonicactivity[17],andcytotoxicityagainstEagle’scarcinomaofthenasopharynx andP-388lymphocyticleukemiacellsinvitro[18]. Theaqueousextractshowedantitrypanosomal[19], antibacterial [20], and antiallergic activity invitro and invivo [7]. The study on the composition of agarwoodfromA.malaccensisutilizinggaschromatography-massspectrometry(GC-MS)revealed thepresenceofchromones,aromaticcompounds,sesquiterpenes,monoterpenes,steroidsandfatty acids[21]. Inapreviousphytochemicalinvestigation,ferurylglycerideandphorbolesterwereisolated fromA.malaccensisbark[18]. However,therewasnoinvestigationreportingoncompositionandbioactivityofA.malaccensis seeds(AMS). Inthecurrentstudy,weinvestigatedantiallergic,antiinflammatory,andcytotoxicactivitiesof AMSextractanditsfractions. Withinaprojectofcontinuousscreeningforactivenaturalproducts, AMSshowedstrongantiallergiceffectwithanIC valuelessthan1µg/mLindegranulationassay. 50 Therefore, a phytochemical investigation of AMS was undertaken through a bioactivity-guided fractionation approach. The active components of the most active fraction were further defined asamixtureofphorbolesters,and,moreover,thenewactivephorbolesterpossessingpolyunsaturated fattyacid(1)wasisolated. 2. ResultsandDiscussion 2.1. Antiallergic,Antiinflammatory,CytotoxicEffectsofA.malaccensisSeeds(AMS) The preliminary bioactivity screening of AMS ethanolic extract (A-EtOH) showed potent antiallergic (IC 0.92 and 3.9 µg/mL in A23187 and antigen-induced β-hexosaminidase assay, 50 respectively) (Table 1), and antiinflammatory activities (90.1% and 85.3% inhibition of superoxide generationandelastasereleaseat10µg/mL,respectively)(Table2). Allpartitionedfractionsexcept water layer displayed significant antiallergic and antiinflammatory activities (A-BuOH, A-EtOAc, A-Hexane,A-MeOH). Int.J.Mol.Sci.2016,17,398 3of13 Table1.AntiallergicactivityofAquilariamalaccensisseedsextracts,fractionsandaquimavitalin(1). InhibitoryEffecton Viability,RBL-2H3 Inhibitionofβ-HexosaminidaseRelease,DegranulationAssay,RBL-2H3Cellsa Enzyme Sample IC50(µg/mL)b A23187-Induced TherapeuticalIndexc Antigen-Induced TherapeuticalIndexc β-Hexosaminidase(%)d (%Viabilityat100µg/mL) IC50(µg/mL)b IC50(µg/mL)b A-EtOH >100(86.0%) 0.92 >109.0 3.9 >25.7 12.7˘4.2(100µg/mL) A-BuOH >100(93.3%) 1.1 >92.1 6.0 >16.7 7.3˘5.5(100µg/mL) A-Water >100(94.0%) – – – – N/Ae A-EtOAc >100(90.3%) 0.56 >177.9 0.86 >116.8 13.3˘2.1(100µg/mL) A-Hexane >100(95.3%) 0.83 >120.1 5.1 >19.5 13.7˘2.5(100µg/mL) A-MeOH 96.8 0.0089 10,910.9 0.069 1405.2 5.3˘3.2(10µg/mL) AM4 98.0 0.0034 28,677.6 0.0065 15,098.4 4.7˘4.0(10µg/mL) AM4-4 70.6 4.8ˆ10´5 1,477,328.2 6.8ˆ10´4 103,776.5 N/Ae AM4-4-7 73.8 7.4ˆ10´4 99,680.2 0.0065 11,309.9 N/Ae AM4-4-8 73.4 7.6ˆ10´6 9,645,374.3 8.0ˆ10´5 917,440.9 N/Ae Aquimavitalin(1) 71.5 0.0010(0.0017µM) 71,538.5 0.0068(0.011µM) 10,550.2 4.3˘4.5(10µg/mL) a Dexamethasone (10 nM) inhibited 54.0% ˘ 4.0% of A23187-induced β-hexosaminidase release and 54.3% ˘ 7.2% of antigen-induced β-hexosaminidase release; b IC50 values express the concentration of the sample required to inhibit cell growth or degranulation by 50%; c Therapeutic index was calculated by dividing IC50 value from3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide(MTT)viabilityassaywithcorrespondingIC50 valuefromdegranulationassay;d Resultsarepresentedas mean˘SD(n=3);eN/A,notapplicable;A-EtOH:crudeethanolicextractofAquilariamalaccensisseeds;A-BuOH:n-butanollayerfromAquilariamalaccensisseeds;A-Water:water layerfromAquilariamalaccensisseeds;A-EtOAc:ethylacetatelayerfromAquilariamalaccensisseeds;A-Hexane:n-hexanelayerfromAquilariamalaccensisseeds;A-MeOH:methanol layerfromAquilariamalaccensisseeds,AM:subfractionsofmethanollayerfromAquilariamalaccensisseeds. Int.J.Mol.Sci.2016,17,398 4of13 Table2.AntiinflammatoryeffectsofA.malaccensisseedsextractsonsuperoxideaniongenerationand elastasereleaseinfMLP/CB-inducedhumanneutrophilsa. Sample SuperoxideAnionGeneration(Inh%) ElastaseRelease(Inh%) A-EtOH 90.1˘5.3 ** 85.3˘0.8 ** A-BuOH 93.9˘8.3 ** 77.6˘2.4 ** A-Water 11.4˘1.6 * 2.7˘4.1 – A-EtOAc 94.8˘5.6 ** 85.4˘1.8 ** A-Hexane 103.4˘1.8 ** 80.2˘4.0 ** A-MeOH 96.5˘8.0 ** 90.4˘6.0 ** AM1 54.5˘5.7 ** 99.2˘2.3 ** AM2 68.7˘5.0 ** 47.5˘5.3 ** AM3 105.9˘3.4 ** 86.8˘2.0 ** AM4 100.7˘8.1 ** 70.9˘1.0 ** AM5 102.6˘1.5 ** 93.5˘3.7 ** AM6 102.4˘2.0 ** 99.3˘2.3 ** aPercentageofinhibition(Inh%)at10µg/mLconcentration;resultsarepresentedasmean˘SEM(n=3–4); *p<0.05,**p<0.001comparedwiththecontrolvalue(formyl-methionyl-leucyl-phenylalanine/cytochalasinB, fMLP/CB). The effects of the AMS samples on degranulation in both A23187- and antigen-induced β-hexosaminidaseassaysweredose-dependent(TablesS1andS2). Toclarifythatantiallergicactivity ofthesampleswasduetoinhibitionofβ-hexosaminidaserelease,andnotfalsepositiveasaresultof directinhibitionofβ-hexosaminidaseenzymaticactivity[22],theenzymewasextractedandtested withtheactivesamples. Noneofthesamplesinhibitedtheenzymaticactivityofβ-hexosaminidase (Table1). As the methanol layer proved the best antiallergic activity (IC 0.0089 and 0.069 µg/mL in 50 A23187 and antigen-induced degranulation assay, respectively), it was further separated using silica gel column chromatography to yield six fractions, AM1–AM6 (subfractions of methanol layer from Aquilaria malaccensis seeds). Among them, fraction AM4 showed the most remarkable antiallergicactivityinhibitingβ-hexosaminidasereleasefrommastcellsinducedbyeitherA23187 (IC 0.0034µg/mL)orantigen(IC value0.0065µg/mL). 50 50 Incytotoxicityassayagainstapanelofthreecancercelllines(humanhepatocellularcarcinoma cells HepG2, human breast adenocarcinoma cells A549, and human lung adenocarcinoma cells MDA-MB231), only some of the AMS fractions showed cytotoxic activities at a 20-µg/mL level (Table3)(A-BuOH57.1%againstA549,AM456.5%againstMDA-MB231and79.3%againstA549, AM656.0%againstMDA-MB231cellline). Moreover,consideringweakcytotoxicityofAMStowards RBL-2H3cells,theantiallergicactivefractionAM4exertedtherapeuticindexupto28,000. Tofurther ruleoutthepossibilitythatAM4causesdirectmastcellactivation,weexaminedthecapacityofAM4 toelicitdegranulationbyitself. ResultsshowedthattheAM4treatmentsdidnotcausesignificant degranulationascomparedwithuntreatedcontrol(Figure1). ThesedataimpliedthatAM4isthebest targetforfurtherphytochemicalanalysis. 2.2. ChemicalAnalysisandBioactivity-GuidedFractionation Followingbioactivity-guidedfractionationoftheactivefractions,theAM4wasfurtherseparated, yieldingseveralactivefractions,AM4-3,AM4-4,andAM4-5(TablesS1andS2). AM4-4 (IC 4.8 ˆ 10´5 µg/mL, therapeutic index 1477328, A23187-induced; and 50 IC 6.8ˆ10´4µg/mL,therapeuticindex103776,antigen-inducedβ-hexosaminidaseassay)afforded 50 themostactivefractionAM4-4-8(IC 7.6ˆ10´6µg/mL,therapeuticindex9645374,A23187-induced; 50 andIC 8.0ˆ10´5µg/mL,therapeuticindex917440,antigen-induceddegranulationassay),anda 50 newcompound,aquimavitalin(1)(IC valuesof0.0017µM,therapeuticindex71,538,A23187-induced; 50 andIC 0.011µM,therapeuticindex10,550,antigen-induceddegranulationassay)(Figure2). 50 Int. J. Mol. Sci. 2016, 17, 398 5 of 13 Int.J.Mol.Sci.2016,17,398 5of13 AM2 2.4 11.5 19.8 AM3 25.5 46.3 39.5 Table3.CytotoAxiMcs4c reeningof2A3..m4 alaccensissee5d6s.e5x tractsonca7n9c.e3r celllinesa. AM5 7.9 39.9 29.2 Sample HepG2b MDA-MB231c A549d AM6 5.3 56.0 39.5 A-EtOHdoxorubicin e 16.091.3 9377.7.2 98.0 29.7 A-BuOH 4.2 34.4 57.1 a Percentage of inhibition (%) at 20 µg/mL concentration (n = 1); b Hep-G2: human hepatocellular A-Water ´9.3 6.8 13.3 carcinoma cells; c MDA-MB231: human breast adenocarcinoma cells; d A549: human lung A-EtOAc 1.5 41.2 23.5 Int. J. Mol. Sci. 2016, 17, 398 5 of 13 adenocarcinAo-mHae xcealnlse; e Positive contr2o5l. 1(2 µg/mL). 42.5 16.8 A-MeOH ´0.8 30.3 32.7 AM2 2.4 11.5 19.8 AM1 8.1 1.7 ´12.6 AM3 25.5 46.3 39.5 AM2 2.4 11.5 19.8 AM3 AM4 25.523.4 5466.5.3 79.3 39.5 AM4 AM5 23.47.9 3596.9.5 29.2 79.3 AM5 7.9 39.9 29.2 AM6 5.3 56.0 39.5 AM6 5.3 56.0 39.5 doxorubicidnoexorubicin e 91.391.3 9977.7.7 98.0 98.0 a Percentage of inhibition (%) at 20 µg/mL concentration (n = 1); b Hep-G2: human hepatocellular aPercentageofinhibition(%)at20µg/mLconcentration(n=1);bHep-G2:humanhepatocellularcarcinoma cacreclilsn;ocmMaD Ac-eMllBs;2 31c :hMumDaAn-bMreBa2st3a1d: enhoucmarcainno mbarecaesllts ;dadAe5n49o:chaurcminanomlunag acdelelnso; cadr ciAno5m49a: cehllus;mePaons itlivueng adceonntorcoalr(2ciµngo/mmaL c).ells; e Positive control (2 µg/mL). Figure 1. Activity of phorbol ester-rich fraction (AM4) and aquimavitalin (1) on stimulant-free degranulation in RBL-2H3 cells. The RBL-2H3 cells were treated with AM4 (10 µg/mL) and aquimavitalin (10 µg/mL) for 10 h. Tyrode’s buffer supplemented with glucose, bovine serum albumin (BSA) and glutamine was used as a medium. A23187 (1 µM) was used as a positive control. Data are expressed as mean ± SD (n = 3). ** p < 0.001 compared with the control value. 2.2. Chemical Analysis and Bioactivity-Guided Fractionation Following bioactivity-guided fractionation of the active fractions, the AM4 was further separated, yielding several active fractions, AM4-3, AM4-4, and AM4-5 (Tables S1 and S2). FAFiiMgguu4rr-ee4 11(.I. CAA50c c4tti.iv8vi it×tyy 1 0oo−ff5 pµphhgoo/mrrbbLool,l teehssetterearr--prrieiccuhht ifcfrr iaanccdttiioeoxnn 1((4AA7MM73442)) 8aa, nnAdd2 3aa1qq8uu7ii-mminaadvvuiittcaaelliidnn; a((11n))d oo InnC s5s0tt ii6mm.8uu ×llaa 1nn0tt-−-4ff rrµeeege /mL, theradpeeguratincu liantdioenx in10R3B7L7-62,H a3nctiegllesn.-iTnhdeucReBdL -β2H-h3excoelslasmwienriedatrseea teadsswayit)h aAffMor4de(1d0 µthge/ mmLo)satn dactive degranulation in RBL-2H3 cells. The RBL-2H3 cells were treated with AM4 (10 µg/mL) and fractiaaoqqnuu iiAmmMaavv4iitt-aa4lli-in8n ((1(I10C0µ5 0gµ /7gm./6mL ×L) )f1 o0fro−16r 0 µ1hg0./ Tmhy.rL oT,d ytehr’osedbreau’pfsf eeburustifucfpe ripn lsedumepxepn 9ltee6md4e5wn3it7teh4d,g Alwu2cito3hs1 e8g,7lbu-oicvnoidsneue, csbeeorduv;m iannea dlbs euIrCmu5im0n 8.0 × 10−5 µ(aBglbS/umAm)Lai,nn t d(hBegSrlAuapt)a aemnuidtni ceg lwiuntadasmeuxisn e9ed1 w7a4sa4sa 0um,s aeenddt iaiugsm ean. mA-ien2d3di1uu8mc7e.(d 1A µd2M3e1g)8rw7a n(a1su µulaMstei)do wnasa asas uspsaoeysd)it ,ai vase nadc po oans tnirtoeivlw.eD ccaootnmatraporole. und, aquimeDxaaptvraei tasasrleeid nex a(ps1r)me (seIsCaend50 ˘ avsaS mlDuee(ansn =o ±f 3 S0)D..0* *0(n1p 7=< µ30)M..0 *0*, 1 tphc <oe mr0a.0pp0ae1ru ecdtoimcw ipintahdretehdxe w7c1oit,nh5t3 rt8ohl,e A vcao2lnu3te1r.8o7l v-ianldueu. ced; and IC50 0.011 µM, therapeutic index 10,550, antigen-induced degranulation assay) (Figure 2). 2.2. Chemical Analysis and Bioactivity-Guided Fractionation Following bioactivity-guided fractionation of the active fractions, the AM4 was further separated, yielding several active fractions, AM4-3, AM4-4, and AM4-5 (Tables S1 and S2). AM4-4 (IC50 4.8 × 10−5 µg/mL, therapeutic index 1477328, A23187-induced; and IC50 6.8 × 10−4 µg/mL, therapeutic index 103776, antigen-induced β-hexosaminidase assay) afforded the most active fraction AM4-4-8 (IC50 7.6 × 10−6 µg/mL, therapeutic index 9645374, A23187-induced; and IC50 8.0 × 10−5 µg/mL, therapeutic index 917440, antigen-induced degranulation assay), and a new compound, aquimavitalin (1) (IC50 values of 0.0017 µM, therapeutic index 71,538, A23187-induced; and IC50 0.011 µM, therapeutic index 10,550, antigen-induced degranulation assay) (Figure 2). Figure 2. Structure of aquimavitalin (1). Figure2.Structureofaquimavitalin(1). Figure 2. Structure of aquimavitalin (1). Int.J.Mol.Sci.2016,17,398 6of13 According to 1H NMR of the crude (A-EtOH), methanolic (A-MeOH) and the subsequent active fractions (AM4 and AM4-4) (Figure S1), we found the proportional relationship of the antiallergicactivitywiththeincreaseinsignalstypicalforphorbolditerpenes(δ 7.5,H-1;δ 5.6,H-7; H H δ 4.0,H-20). H 2.3. StructureElucidationofAquimavitalin(1) Compound 1 (Figures S2–S8) was isolated as a colorless oil. It was assigned the molecular formula C H O Na, according to high-resolution electrospray ionization mass spectrometry 36 50 8 (HR-ESIMS) (m/z 633.33980 [M + Na]+, calcd. 633.33979), indicating 12 degrees of unsaturation. Its IR spectrum revealed the presence of hydroxyl (3413 cm´1), carbonyl (1710 cm´1) and olefinic (1615cm´1)functionalities. The NMR data of compound 1 (1H, 13C and heteronuclear multiple quantum coherence, HMQC, Table 4) confirmed the presence of α, β-unsaturated carbonyl (δ 7.57, s, H-1, δ 160.8, H C C-1; δ 132.8, C-2; δ 209.3, C-3), trisubstituted double bond (δ 5.68, brs, H-7, δ 129.1, C-7; C C H C δ 140.6, C-6), oxygenated methylene (δ 3.95, d, J = 12.8 Hz, 4.02, d, J = 12.8 Hz, H-20, δ C H C 67.9, C-20), oxygenated methane (δ 5.43, d, J = 10.4 Hz, H-12, δ 75.9, C-12), four methyls, a H C methylene and four methines. Furthermore, signals for acetyl group (δ 2.10, s, H-22, δ 21.1, H C C-22; δ 173.9, C-21) and fatty acid moiety including six olefinic protons, six methylenes and C terminalmethylgroupweredetected. The1HNMRdatawascloselyrelatedtoknowncompound 12-O-(2Z,4E,6E)-deca-2,4,6-trienoylphorbol-13-acetate[18]exceptofthelengthofthefattyacidmoiety (TableS3). Table4.1Dand2DNMRdataofaquimavitalin(1)inCDCl a. 3 Position δH,Multiplicity(JinHz) δC,Type COSY(1H–1H) HMBC(1H–13C) NOESY(1H–1H) 1 7.57(s) 160.8CH 10,19 4,10 18 2 – 132.8C – – – 3 – 209.3C – – – 4 – 73.6C – – – 5α 2.48(d,J=18.8) 38.3CH2 7 4,6,7 5,20 5β 2.58(d,J=18.8) – – – – 6 – 140.6C – – – 7 5.68(brs) 129.1CH 5,8 14,20 14,20 8 3.26(t,J=5.2) 38.8CH 7,14 6,14,15 11,17 9 – 78.4C – – – 10 3.22(brs) 55.9CH 1,19 – – 11 2.13(m) 43.0CH 12,18 – 17,18 12 5.43(d,J=10.4) 75.9CH 11 11,13,15,18,11 18 13 – 65.7C – – – 14 1.08(d,J=5.2) 36.1CH 8 7,13,15,16 – 15 – 25.6C – – – 16 1.19(s) 23.8CH3 – 13,14,15,17 – 17 1.24(s) 16.7CH3 – 13,14,15,16 – 18 0.88(d,overlap) 14.0CH3 11 9,11,12 – 19 1.73(brs) 10.0CH3 1,10 1,2,3 – 20a 4.02(d,J=12.8) 67.9CH2 – 5,6,7 – 20b 3.95(d,J=12.8) – – – – 21 – 173.9C – 22 – 22 2.10(s) 21.1CH3 – – – 11 – 166.3C – – – 21 5.57(d,J=11.2) 115.6CH 31 – 31 31 6.59(t,J=11.6) 145.6CH 21,41 11,51 – 41 7.39(dd,J=15.2and11.6) 126.5CH 31,51 – 61 51 6.46(dd,J=14.8and10.4) 142.4CH 41,61 – 71 61 6.20(dd,J=15.2and10.8) 130.1CH 51,71 – – 71 5.92(dt,J=15.2and7.2) 141.0CH 61,81 – – 81 2.13(m) 33.0CH2 71,91 61,71,91 91 91 1.38(m) 28.9CH2 81 101 – 101 1.26–1.28(m,overlap) 29.1CH2 – – – 111 1.26–1.28(m,overlap) 29.1CH2 – – – 121 1.26–1.28(m,overlap) 31.7CH2 – – – 131 1.26–1.28(m,overlap) 22.6CH2 – – – 141 0.86(t,J=7.2) 14.4CH3 – 121,131 – a 1Hand13CNMRdata(δ)weremeasuredat400and100MHz,respectively;chemicalshiftsareinppm; COSY: Correlation spectroscopy; HMBC: Heteronuclear multiple bond correlation spectroscopy; NOESY:NuclearOverhausereffectspectroscopy. IInntt.. JJ.. MMooll.. SSccii.. 22001166,, 1177,, 339988 77 ooff 1133 1100′′ 11..2266––11..2288 ((mm,, oovveerrllaapp)) 2299..11 CCHH22 –– –– –– 1111′′ 11..2266––11..2288 ((mm,, oovveerrllaapp)) 2299..11 CCHH22 –– –– –– 1122′′ 11..2266––11..2288 ((mm,, oovveerrllaapp)) 3311..77 CCHH22 –– –– –– 1133′′ 11..2266––11..2288 ((mm,, oovveerrllaapp)) 2222..66 CCHH22 –– –– –– 1144′′ 00..8866 ((tt,, JJ == 77..22)) 1144..44 CCHH33 –– 1122′′,, 1133′′ –– aa 11HH aanndd 1133CC NNMMRR ddaattaa ((δδ)) wweerree mmeeaassuurreedd aatt 440000 aanndd 110000 MMHHzz,, rreessppeeccttiivveellyy;; cchheemmiiccaall sshhiiffttss aarree iinn Int.J.pMpppomlm.S;; ci.CC2O0O1SS6YY,1:: 7 ,3CC9oo8rrrreellaattiioonn ssppeeccttrroossccooppyy;; HHMMBBCC:: HHeetteerroonnuucclleeaarr mmuullttiippllee bboonndd ccoorrrreellaattiioonn7 of13 ssppeeccttrroossccooppyy;; NNOOEESSYY:: NNuucclleeaarr OOvveerrhhaauusseerr eeffffeecctt ssppeeccttrroossccooppyy.. TTThhheee 11HH1H––11–HH1 Hccoorrrrceeollraartteiioolannt iossppneeccttsrrpooessccctoorppoyysc (o(CCpOOySSYY(C)) OccooSrrYrree)llaattciiooonrnrsse l((aFFtiiiggouunrrsee 33())F iiignnuddriicecaattee3dd) tthhineed ppicrraeetsseeednncceet hooeff CC--1100//CC--11//CC--1199,, CC--55//CC--77//CC--88//CC--1144,, aanndd CC--1122//CC--1111//CC--1188 mmooiieettiieess ffoorr bbaacckkbboonnee,, presence of C-10/C-1/C-19, C-5/C-7/C-8/C-14, and C-12/C-11/C-18 moieties for backbone, CC--22’’//CC--33’’//CC--44’’//CC--55’’//CC--66’’//CC--77’’//CC--88’’//CC--99’’ ffoorr ffaattttyy aacciidd mmooiieettyy.. TThhee CCOOSSYY ccoorrrreellaattiioonnss ttooggeetthheerr wwiitthh C-2’/C-3’/C-4’/C-5’/C-6’/C-7’/C-8’/C-9’forfattyacidmoiety. TheCOSYcorrelationstogetherwith lloonngg--rraannggee hheetteerroonnuucclleeaarr mmuullttiippllee bboonndd ccoorrrreellaattiioonn ssppeeccttrroossccooppyy ((HHMMBBCC)) ccoorrrreellaattiioonnss ((FFiigguurree 33)) long-range heteronuclear multiple bond correlation spectroscopy (HMBC) correlations (Figure 3) ffrroomm HH--1199//CC--11,, CC--22,, CC--33;; HH--11//CC--44;; HH--55//CC--44;; HH--2200//CC--55,, CC--66,, CC--77;; HH--88//CC--66,, CC--1144,, CC--1155;; HH--1122 ttoo CC--1111,, fromH-19/C-1,C-2,C-3;H-1/C-4;H-5/C-4;H-20/C-5,C-6,C-7;H-8/C-6,C-14,C-15;H-12toC-11, CC--1133,, CC--1155,, CC--1188;; HH--1188//CC--99;; HH--1166 aanndd HH--1177// CC--1133,, CC--1144,, CC--1155 eessttaabblliisshheedd tthhee ttiigglliiaannee ((pphhoorrbbooll)) ttyyppee C-13,C-15,C-18;H-18/C-9;H-16andH-17/C-13,C-14,C-15establishedthetigliane(phorbol)type ddiitteerrppeennee bbaacckkbboonnee ooff ccoommppoouunndd 11 [[2233,,2244]].. diterpenebackboneofcompound1[23,24]. FFFiiiggguuurrreee 333... KKKeeeyyy 111HHH–––111HHH cccooorrrrrreeellalaattitiooionnn ssspppeeeccctttrrrooossscccooopppyyy (((CCCOOOSSSYYY))) (((bbbooolllddd))) aaannnddd HHHMMMBBBCCC (((aaarrrrrrooowww))) cccooorrrrrreeelllaaatttiiiooonnnsss ooofff aaaqqquuuiiimmmaaavvviiitttaaallliiinnn (((111)))... TTThhheee rrreeelllaaatttiiivvveee cccooonnnfffiiiggguuurrraaatttiiiooonnn wwwaaasss aaassssssiiigggnnneeeddd bbbyyy mmmeeeaaannnsss ooofff nnnuuucccllleeeaaarrr OOOvvveeerrrhhhaaauuussseeerrr eeeffffffeeecccttt ssspppeeeccctttrrrooossscccooopppyyy (((NNNOOOEEESSSYYY))) ccocoorrrrerrleealltaaittoiiononsnsso foo1ff( F11i g((uFFriiegg4uu)rr.eeT h44e)).. c rTTohhsese- pccerraooksssss--oppfeeHaakk-8ss/ oHoff- 1HH1,--H88//-HH11--11/11H,, -H1H7--11a11n//dHHH--11-771 7/aannHdd- 8 HHin--d11i77c//aHHte--d88 iitnnhddatiicctaahtteeeydd a ttrhheaaattl ltthhβee-yyo r aaierrnee t eaadllll. ββM--ooorrriieeeonnvtteeedrd,..t hMMeoocrroeeroorvevleearrt,,i otthnheeb ecctoowrrrreeeellnaattHiioo-nn1 /bbHeett1ww8e/eeeHnn- 1HH2--s11u//HHgg11e88s//tHHed--11t22h asstuutgghggeeefssattetetddy ttahhcaaidtt ttmhheeo iffeaatttyttyyi s aaaccliisddo mmβ-oooiiereitteyyn iitsse daall[ss2oo3 ]ββ.--Aoorrdiieednnittteieoddn a[[22ll33y]],.. 1AAsddhddoiiwttiiooendnaanlllleyyg,, a11t isvshheooswwpeeecddi finnceeggoaaptttiiivvceea lssrppoeetccaiitffiiioccn oo(pp´ttii3cc.aa8ll) rrsooimttaaittliiaoornnt o((−−1332..88-O)) ss-ii(mm2Ziill,aa4rrE t,t6ooE 11)22-d--OOec--(a(22-2ZZ,,,444,EE6-,,66trEEie))--nddoeeycclaap--h22,o,44r,,b66-o-ttlrr-ii1ee3nn-ooayycellpptahhtooerr(bb´oo1ll--511.333-)-aa[cc1ee8tt]aa.ttee ((−−1155..33)) [[1188]].. FFFiiiggguuurrreee 444... KKKeeeyyy NNNOOOEEESSSYYY (((dddooouuubbbllleee---hhheeeaaadddeeeddd aaarrrrrrooowww))) cccooorrrrrreeelllaaatttiiiooonnnsss ooofff aaaqqquuuiiimmmaaavvviiitttaaallliiinnn (((111)))... GGGrrreeeyyy cccooolllooorrr rrreeeppprrreeessseeennntttsss cccaaarrrbbbooonnn,,, rrreeeddd cccooolllooorrr oooxxxyyygggeeennn aaannnddd wwwhhhiiittteee cccooolllooorrr ppprrroootttooonnn... The fatty acid was identified as (2Z,4E,6E)-tetradeca-2,4,6-trienoic acid according to 1D NMR and COSY correlations supported by following HMBC correlations, H-31/C-11 (δ 166.3), C C-51 (δ 142.4); H-81/C-61 (δ 130.1), C-71 (δ 141.0), C-91 (δ 28.9); H-91/C-101 (δ 29.1) and C C C C C H-141/C-121 (δ 31.7), C-131 (δ 22.6). The geometry of the double bonds was assigned by C C coupling constants in 1H NMR. The NMR data were in agreement with those of (2Z,4E,6E)-ethyl Int.J.Mol.Sci.2016,17,398 8of13 tetradeca-2,4,6-trienoate [25]. The fatty acid moiety was attached to phorbol backbone at C-12 by virtueofHMBCcorrelationfromH-12toC-11 (δ 166.3). Therefore,compound1wasidentifiedas C 12-O-(2Z,4E,6E)-tetradeca-2,4,6-trienoylphorbol-13-acetateandnamedasaquimavitalin. 2.4. AntiallergicActivityofAquimavitalin(1) Indegranulationassay,aquimavitalin(1)showedsignificantβ-hexosaminidaserelease-inhibitory activity with IC values of 0.0017 µM (therapeutic index 71,538) using A23187 as an inducer and 50 0.011µM(therapeuticindex10,550)usingantigenasaninducer. Aquimavitalin(1)didnotinhibit β-hexosaminidase enzymatic activity (Table 1), neither trigger the degranulation of unstimulated mastcells(Figure1). Accordingtoourresults,phorbolester-richfractions(AM4-4,AM4-4-8)showed strongeractivity(uptopg/mLlevel)thanapurecompound. Thisphenomenonmaybearesultof synergisticeffectsofphorbolestersinthemixture. Ingeneral,phorbolesters,particularlyphorbol-12-myristate-13-acetate(PMA),arewell-knownas irritant,proinflammatoryandcocarcinogenic. Nevertheless,phorbolesterswerepreviouslyreported toexertantiinflammatory,anti-HIV,antiparasiticandanticanceractivities[26]. BothfreeC-20hydroxy, andC-12and/orC-13estermoietieswereimportantfortheactivitiesofphorbolesters[26].Importantly, it was suggested that unsaturation of ester functionality may play a crucial role in bioactivity of phorbols[26,27]. Previously,12-O-(2Z,4E,6E)-deca-2,4,6-trienoylphorbol-13-acetate,aphorbolester possessingsimilarconjugatedfattyacidmoietyas1,wasisolatedfromA.malaccensisbarkandexerted cytotoxicactivityinP-388lymphocyticleukemiacellsinvitro[18]. Instructure-activityrelationship studyonphorbolesterscontainingfattyacidswithdifferentlevelofunsaturationandcarbonchain length,phorbolesterscarryingconjugatedunsaturatedfattyacidasacylgroupshowedirritantbut veryweaktumor-promotingactivities.[27]. Thisisthefirststudytoreportontheantiallergicpotential ofpurephorbolesterwiththetherapeuticindexupto71,000. TheantiallergicactivityofAMStogether withidentificationofitsactivecomponentprovidesscientificsupportforthefolkuseofA.malaccensis againstasthma. 3. MaterialsandMethods 3.1. GeneralProcedures SephadexLH-20(MerckKGaA,Darmstadt,Germany),silicagel60(MerckKGaA)andGeduran Si 60 (Merck KGaA) were used for column chromatography. TLC plates (Silica Kiesel 60 F254) were from Merck KGaA. Jasco V-530 ultraviolet spectrophotometer (Jasco International Co., Ltd, Tokyo, Japan) was used to measure UV spectra. IR spectra were obtained on an FT-IR-4100 Jasco spectrophotometer (Jasco). Optical rotations were achieved by a Jasco P-2000 digital polarimeter (Jasco). NMR spectra were obtained by JEOL JNM ECS 400 MHz. Electrospray ionization mass spectrometry (ESIMS) data were collected on a Waters micromass ZQ mass spectrometer (Waters Corporation,Milford,MA,USA).High-resolutionESIMSdatawasaccomplishedbyaBrukerAPEXII spectrometer(FT-ICR/MS,FTMS)(BrukerDaltonicsInc.,Billerica,MA,USA).Dulbecco’smodified Eagle’smedium(highglucose)powder(DMEM),3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), p-nitrophenyl-N-acetyl-D-glucosaminide (p-NAG), penicillin and streptomycin, dexamethasone,calciumionophoreA23187,anddimethylsulfoxide(DMSO)werepurchasedfrom Sigma-Aldrich(St. Louis,MO,USA).Fetalbovineserum(FBS)wasobtainedfromHyclone(Logan, UT,USA).Mouseanti-DNPIgEantibodywasagenerousgiftfromDr. DanielH.Conrad(Virginia CommonwealthUniversity,Richmond,VA,USA). 3.2. PlantMaterial The seeds of A. malaccensis were obtained from Hsue-Yin Hsu, Tzu Chi University, Hualien, Taiwan,inNovember2014. TheplantmaterialwasidentifiedbyHsue-YinHsu,DepartmentofLife Sciences, TzuChiUniversity, Hualien, Taiwan. Avoucherspecimen(codeno. KMU-AMS1)was Int.J.Mol.Sci.2016,17,398 9of13 depositedintheGraduateInstituteofNaturalProducts,CollegeofPharmacy,KaohsiungMedical University,Kaohsiung,Taiwan. 3.3. ExtractionandIsolation Air-driedandpowderedseedsofA.malaccensis(462g)wereextractedwith90%EtOHatroom temperature(3ˆ5L)andthenconcentratedunderreducedpressure. Thecombinedextractswere concentrated and obtained crude ethanolic extract (A-EtOH, 27.7 g) was suspended in water and partitioned with ethyl acetate (3 ˆ 1 L). The water layer was partitioned with n-butanol (3 ˆ 1 L) to yield water layer (A-Water, 1654.0 g) and n-butanol layer (A-BuOH, 398.2 g). The EtOAc layer (A-EtOAc,25.6g)wasfurtherpartitionedwithn-hexaneand90%aqueousMeOHtoobtainn-hexane layer(A-Hexane,7.1g)andMeOHlayer(A-MeOH,16.2g). TheMeOHlayer(A-MeOH)wassubjected toacolumnchromatographyoversilicagel(23cmˆ4cm,silicagel60,0.063–0.200mm,Merck)undera gradientelutionofn-hexane/CH Cl /MeOHtoyieldsixfractions(AM1,6:3:1;AM2,6:4:1;AM3,6:6:1; 2 2 AM4,6:8:1;AM5,6:10:1andAM6,6:10:2). Followingbioactivitydata,fractionAM4(3212.0g)was furtherfractionatedoveraSephadexLH-20column(CH Cl /MeOH,1:1)toobtaineightsub-fractions 2 2 (AM4-1toAM4-8). FractionAM4-3(762.0mg)wassubjectedtocolumnchramtography(17cmˆ4cm, GeduranSi60,0.040-0.063mm,Merck)undergradientelutionofEtOAc/n-hexane(from1:10to4:1) yielding15fractions. FractionAM4-4(173.7mg)wasfurtherseparatedbycolumnchromatography on silica gel (30 cm ˆ 1.5 cm, Geduran Si 60, 0.040–0.063 mm, Merck) under gradient elution of EtOAc/n-hexane(from1:15to4:1)toobtainfractionAM4-4-7(37.6mg)andAM4-4-8(6.8mg)and aquimavitalin(1)(43.9mg)togetherwithother8subfractions. Theyieldofaquimavitalin(1)was 0.0095%fromdryplantmaterial,0.16%fromcrudeEtOHextract. 3.4. ExperimentalDataofAquimavitalin(1) Aquimavitalin(1): Colourlessoil;rαs25´3.75(c0.067,CHCl );UV(MeOH)λ (logε)303(2.78), D 3 max 233 (2.75) nm; IR (neat) v 3413, 2965, 2922, 1710, 1615, 1377, 1258, 1092, 802; 1H NMR (CDCl , max 3 400MHz)and13CNMR(CDCl ,100MHz): seeTable4;ESIMSfoundm/z611.3[M+H]+and633.3 3 [M+Na]+;HR-ESIMSfound(m/z633.33980[M+Na]+,(calcd. forC H O Na: 633.33979). 36 50 8 3.5. CellCulture Themucosalmastcell-derivedratbasophilicleukemia(RBL-2H3)celllinewaspurchasedfrom theBioresourceCollectionandResearchCenter(Hsin-Chu, Taiwan). CellsweregrowninDMEM mediumsupplementedwith10%FBSand100U/mLpenicillinplus100µg/mLstreptomycin. Cells wereculturedin10cmcellculturedishesat37˝Cinahumidifiedchamberwith5%CO inair. 2 3.6. CellViabilityAssay Amethylthiazoltetrazolium(MTT)assaywasusedtomeasurethepotentialtoxiceffectsofthe samplesonRBL-2H3cells[28].Briefly,RBL-2H3cells(2ˆ104cells/well)wereseededina96-wellplate overnightandtreatedwithvariousconcentrationsofsamples(10–100µg/mL)for24h. MTTsolution (0.5mg/mL)wasaddedtothewells(80µLperwell)andincubatedfor1h. Theformedformazan crystalsweredissolvedinDMSO(80µL).Theabsorbanceat595nmwasmeasuredusingmicroplate reader(MultiskanAscent,ThermoScientific,Waltham,MA,USA).Thedegreeofcellviabilityofeach samplewascalculatedasthepercentageofcontrolvalue(untreatedcells). Themaximaltolerateddose ofDMSOwas0.5%. Allexperimentswererepeatedatleasttwotimes. 3.7. Degranulationβ-HexosaminidaseAssayInducedbyA23187orAntigen ThedegreeofA23187-andantigen-induceddegranulationinRBL-2H3cellswasdeterminedbya β-hexosaminidasereleaseassayasdescribedpreviously[28,29]withfollowingmodifications.RBL-2H3 cells were seeded in a 96-well plate (2 ˆ 104 cells/well) for A23187-induced and in 48-well plate Int.J.Mol.Sci.2016,17,398 10of13 (3ˆ104cells/well)forantigen-inducedexperiment. Cellsweretreatedwithvariousconcentrations of the samples for 20 h. Dexamethasone (10 nM) was used as a positive control. The cells for theantigen-inducedexperimentwerefirstsensitizedwithanti-DNPIgE(5µg/mL)foratleast2h. Afterthoroughwashingbypre-warmedTyrode’sbuffer(135mMNaCl,5mMKCl,1.8mMCaCl , 2 1.0mMMgCl ,5.6mMglucose,20mMHEPESatpH7.4),thecellswerestimulatedbyeithercalcium 2 ionophoreA23187(1µM)orantigenDNP-BSA(100ng/mL)inTyrode’sbufferfor1h. Unstimulated cells were either lysed with 0.5% Triton X-100 solution for the total amount of β-hexosaminidase releaseorleftuntreatedforspontaneousreleaseofβ-hexosaminidase. Thenaliquotsofsupernatants (50µL)wereincubatedwithequalvolumeof1µMofp-NAG(50µL)preparedin0.1Mcitratebuffer (pH4.5)servingasasubstrateforthereleasedβ-hexosaminidase. After1hofincubationat37˝C, thereactionwasquenchedbytheadditionof100µLofstopbuffer(0.1MNa /NaHCO ,pH10.0). 2 3 Absorbancewasmeasuredat405nmonamicroplatereader(MultiskanAscent,ThermoScientific). Theinhibitionpercentageofβ-hexosaminidasereleasewascalculatedasthepercentageofcontrol value(untreatedstimulatedcells). ThemaximaltolerateddoseofDMSOwas0.5%. Allexperiments wererepeatedthreetimes. 3.8. EffectonEnzymaticActivityofβ-Hexosaminidase Totestthepossibleeffectofthesampleonenzymaticactivity,followingassaywasperformed. Thecellsuspension(2ˆ106cells)in2mLofTyrode’sbufferwassonicatedfor5min. Thesolutionwas thencentrifuged,andthesupernatantwasdilutedwith8mLofTyrode’sbuffer. Theenzymesolution (45µL)andtestsamplesolution(5µL)weretransferredintoa96-wellmicroplateandenzymeactivity wasexaminedasdescribedabove(Section3.7). Allexperimentswererepeatedthreetimes. 3.9. DirectDegranulationβ-HexosaminidaseAssayInducedbytheSample Thedegreeofβ-hexosaminidasereleasetriggeredbythesampleinRBL-2H3cellswasdetermined byamodifiedβ-hexosaminidasereleaseassay. Briefly,RBL-2H3cells(4ˆ104cells/well)wereseeded ina48-wellplateandtreatedwiththesamplesfor10h. Tyrode’sbuffersupplementedwith5.6mM glucose,2mg/mLBSAand2mMglutaminewasusedtopreparethesamplesandtreatthecells. Then, 50µLofsupernatantsweretransferredintoa96-wellmicroplateandexaminedasdescribedabove (Section3.7). A23187(1µM)wasusedasapositivecontrol. Allexperimentswererepeatedthreetimes. 3.10. PreparationofHumanNeutrophils Human neutrophils from venous blood of healthy, adult volunteers (20–30 years old) were isolatedusingastandardmethodofdextransedimentationpriortocentrifugationinaFicoll-Hypaque gradientandhypotoniclysisoferythrocytes[30]. Purifiedneutrophilscontaining>98%viablecells, asdeterminedbythetrypan-blueexclusionmethod[31], wereresuspendedinaCa2+-freeHank’s bufferedsaltsolution(HBSS)atpH7.4andweremaintainedat4˝Cpriortouse. 3.11. SuperoxideAnionGenerationAssayandElastaseReleaseInhibitionAssay Neutrophil superoxide anion generation was determined using superoxide dismutase (SOD)-inhibitorycytochromereductionaccordingtodescribedprocedures[32,33]. Degranulationof azurophilicgranuleswasdeterminedbymeasuringtheelastasereleaseasdescribedpreviously[33]. Allexperimentswererepeatedatleastthreetimes. 3.12. CytotoxicAssay MTTassaywasusedaccordingtothemethoddescribedbefore[34]. Briefly,HepG2(1ˆ104cells), A549(5ˆ103 cells),andMDA-MB-231(1ˆ104 cells)wereseededinto96-wellplates,followedby treatmentwiththeAMSsamplesatconcentrationof20µg/mL.After72h,themediumwasremoved and100µLofMTTsolution(0.5mg/mL)wasaddedtoeachwell. Theplateswerethenincubatedat