Antiallergic Phorbol Ester from the Seeds of Aquilaria malaccensis

13 Pages·2016·1.01 MB·English

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.:[phone](ext.2676)(B.-H.C.);[phone](ext.2162)(F.-R.C.);
Fax:[phone](B.-H.C.);[phone](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