RevistaBrasileiradeFarmacognosia25(2015)177–179
w w w . s b f g n o s i a . o r g . b r / r e v i s t a
Short
communication
Propolins
and
glyasperin
A
from
stingless
bee
nests
Consolacion
Y.
Ragasa
a,b,∗,
Richard
Anthony
F.
Galian
b,
Virgilio
D.
Ebajo
Jr.
b,
Remil
M.
Aguda
c,
Cleofas
R.
Cervancia
d,
Chien-Chang
Shen
eaChemistryDepartment,DeLaSalleUniversityScience&TechnologyComplex,LeandroV.LocsinCampus,Bi˜nanCity,Laguna4024,Philippines bChemistryDepartment,DeLaSalleUniversity,2401TaftAvenue,Manila1004,Philippines
cInstituteofChemistry,UniversityofthePhilippinesLosBa˜nos,College,Laguna4031,Philippines
dInstituteofBiologicalSciences,UniversityofthePhilippinesLosBa˜nos,College,Laguna4031,Philippines
eNationalResearchInstituteofChineseMedicine,155-1,Li-NongSt.,Sec.2,Taipei112,Taiwan
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received26December2014 Accepted9March2015 Availableonline30March2015
Keywords:
Philippinepropolis
Tetragonulabiroi
PropolinA PropolinE PropolinH GlyasperinA
a
b
s
t
r
a
c
t
Chemicalinvestigationofthedichloromethaneextractsofthepropoliscollectedfrombee(Tetragonula biroiFriese)hivesinSanRoque,Sorsogon,PhilippinesaffordedpropolinA(1),propolinE(2),propolin H(3),glyasperinA(4),squalene,amixtureoflupeol,␣-amyrinand-amyrin,andanothermixture ofurs-12-en-3-one,olean-12-en-3-oneandlup-12-en-3-one.Thestructuresof2–4wereelucidatedby extensive1Dand2D(COSY,HSQC,andHMBC)NMRspectroscopy,while1wasidentifiedbycomparison ofitsNMRdatawith2.
©2015SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Allrightsreserved.
Introduction
Propolishasbeenreportedtocontainflavonoidsand pheno-liccompoundsthathaveshownpharmacologicalactivities(Toreti etal.,2013).Propolisisknowntoexhibitnumerousmedicinal prop-erties, includingcytotoxic(Matsuno et al., 1997; Kimotoet al., 2001),freeradicalscavenging(Basnetetal.,1997),anti-HIV(Ito et al., 2001), antimicrobial (Park et al., 1998), and antiherpes (Vynogradetal.,2000).Reviewsonthechemicalconstituentsand biologicalactivitiesofpropolishavebeenprovided(Toretietal., 2013;Huangetal.,2007;Bankovaetal.,2000).
A recent study on Philippine propolis identified by LC–MS artepillinC and pinobanksin-3-O-hexanoate aspossible pheno-lic compounds in propolis of stingless bees, Tetragonula biroi
and exudates from Persea americana Mill, Artocarpus
hetero-phyllus Lam, Mangifera indica L., Canarium ovatum Engl, and
Nephelium lappaceum L.that are utilized by T.biroi as propolis
∗ Correspondingauthor.
E-mail:consolacion.ragasa@dlsu.edu.ph(C.Y.Ragasa).
source (Belina-Aldamita et al., 2013). In another study, the flavonoids and phenolic compounds found in propolis and M. indica, A. heterophyllus, C. ovatum, Chrysophyllum cainito and
Achrassapotawereidentifiedaspinobanksin-5,7-dimethylether,
artepillin C, luteolin-5-methylether, pinobanksin-3-O-(butyrate orisobutyrate)andkaempferidebymatchingtheretentiontime and the positive and negative ion chromatograms of the sam-ples to the literature data of known flavonoid and phenolic compounds from the study of Gardana et al. (2007) (Alvarez et al., 2013). The ethanol extract of Philippine propolis was also reported as a potential inhibitor of QS-mediated viola-ceinproduction in Chromobacteriumviolaceum (Lamberte etal., 2011).
We report herein the isolation of propolin A (1), propolin E (2), propolin H (3), glyasperinA (4), and squalene from the dichloromethane extractsofthepropolis collectedfrombee (T. biroiFriese)hivesinSanRoque,Sorsogon,Philippines.Amixture oflupeol,␣-amyrin and-amyrin,and anothermixtureof urs-12-en-3-one,olean-12-en-3-one andlup-12-en-3-onewerealso obtainedfromthedichloromethane extractsofthepropolis.To thebestofourknowledgethisisthefirstreportontheisolationof
http://dx.doi.org/10.1016/j.bjp.2015.03.006
178 C.Y.Ragasaetal./RevistaBrasileiradeFarmacognosia25(2015)177–179
thesecompoundsfromPhilippinepropolisandthefirstreporton theisolationof4frompropolis.
Materialsandmethods
NMRspectrawererecordedonaVarianVNMRSspectrometer inCDCl3at600MHzfor1HNMRand150MHzfor13CNMRspectra. The2DNMRexperiments(COSY,HSQC,andHMBC)forpropolin E,propolinH,andglyasperinAwereperformedbyusingVarian gCOSY,gHSQCADandgHMBCADpulseprograms.CDspectrawere recordedbyaJASCOJ-715spectropolarimeter.Column chromatog-raphywasperformedwithsilicagel60(70–230mesh).Thinlayer chromatographywasperformedwithplasticbackedplatescoated withsilicagelF254andtheplateswerevisualizedbysprayingwith vanillin/H2SO4solutionfollowedbywarming.
The propolis used in this study was collected from bee (TetragonulabiroiFriese)hivesinSanRoque,Sorsogon,Philippines inMay2012.
Inthefirst isolation,propolis (50g) waspulverizedby mor-tar and pestle, soaked in 200ml of CH2Cl2 for three days and then filtered. The filtrate was concentrated under vacuum to afford the crude extract (27.5g) which was chromatographed usingincreasingproportionsofacetoneinCH2Cl2 at10% incre-mentaseluents. TheCH2Cl2 fractionfrom thechromatography ofthecrudeextract wasrechromatographed (4×)inpetroleum
ethertoaffordsqualene(10mg).The60%acetoneinCH2Cl2 frac-tionfromthechromatographyofthecrudeextractwascombined andrechromatographedinCH3CN:Et2O:CH2Cl2(0.5:0.5:9by vol-ume).Theless polarfractionswererechromatographed (3×)in
CH3CN:Et2O:CH2Cl2(0.5:0.5:9byvolume)toafford2(8mg)after washingandtriturationwithpetroleumether.Themorepolar frac-tionswererechromatographed(4×)inCH3CN:Et2O:CH2Cl2(1:1:8 byvolume)toafford1(4mg)afterwashingandtriturationwith petroleumether.
Inthesecondisolation,driedpropolis(100g)waspulverized withmortarandpestle,soakedin400mlCH2Cl2 forthree days andthenfiltered.Thefiltratewasconcentratedundervacuumto affordthe crude extract (71g). Thecrude extract was fraction-atedby silica gelchromatography using increasingproportions ofacetone inCH2Cl2 (10%increment) aseluents. The10% ace-tonein CH2Cl2 fractionwas rechromatographed(2×)using 5%
EtOAcinpetroleumetheraseluenttoaffordamixtureof urs-12-en-3-one,olean-12-en-3-one,andlup-12-en-3-one(18mg)after washingwithpetroleumether.The20%acetoneinCH2Cl2fraction wasrechromatographed(3×)using10%EtOAcinpetroleumether
aseluenttoaffordamixtureoflupeol,␣-amyrin,and-amyrin (22mg)afterwashingwithpetroleumether.The50%acetonein CH2Cl2fractionwastrituratedinpetroleumetherandthen rechro-matographedusingCH2Cl2,followedbyEt2O:CH3CN:CH2Cl2(1:1:8 by volume) as eluents. The fractions eluted with CH2Cl2 were combinedandrechromatographed(5×)usingEt2O:CH3CN:CH2Cl2 (1:1:8byvolume)aseluenttoafford3(15mg)aftertriturationwith petroleumether. The fractions eluted with Et2O:CH3CN:CH2Cl2 (1:1:8byvolume)fromtherechromatographyofthe50%acetonein
CH2Cl2fractionwererechromatographed(6×)inthesamesolvent
toafford4(12mg)aftertriturationwithpetroleumether. Com-pounds1,2andsqualenewerealsodetectedinthesecondisolation, butwerenolongerpurified.
Resultsanddiscussion
The structuresof 2–4 were elucidatedby extensive 1D and 2DNMRspectroscopy,while 1wasidentified bycomparisonof its 1H NMR data withthose of 2. Propolin A (1) (Chen et al., 2003, 2008), propolin E (2) (Chen et al., 2008; Weng et al., 2007)andpropolin H(3)(Weng etal.,2007)werereportedas constituentsofTaiwanese propolis.GlyasperinA(4)was previ-ouslyisolated fromtheroots of Glycyrrhiza aspera(Zenget al., 1992).
Propolinswithdiversebiologicalactivitieshavebeenisolated from propolis. Propolins A–F isolated from Taiwanese propolis couldeffectivelyinducehumanmelanomacellapoptosisandwere strongantioxidantagents(Chenet al.,2004).PropolinA exhib-ited cytotoxic properties against humanmelanoma, C6glioma, andHL-60withIC50valuesof6.0,3.5,and7.5g/ml,respectively (Chenetal.,2003).PropolinsAandBinducedcytotoxicityeffect in humanmelanomaA2058cells and were strongantioxidants (Chen et al., 2007a). The anti-methicillin resistant
Staphylococ-cus aureus (MRSA) activity of propolin D (MIC 8–16mg/l) and
propolin C (MIC 8–32mg/l) was reported (Raghukumar et al., 2010).On theotherhand,propolinGwasreportedasapotent caspase-dependentinducerofapoptosisinbraincancercells.With Taiwanesepropolisextract,propolinGdemonstratedaprotective effectagainstoxidativestressinratcorticalneurons(Huangetal., 2007).AnotherstudyreportedthatpropolinHinhibitedthe pro-liferationofhumanlungcarcinomacelllinesinMTTassay(Weng etal.,2007).
Authors’contributions
CYRconductedtheexperiments,elucidatedthestructuresof thecompoundsandwrotethemanuscript.C-CShenobtainedthe NMR,CDandHR-MSdataandreviewedthemanuscript.RFGand VEconductedtheexperiments.RAandCCcollectedthepropolis andcontributedtotheintroduction.Alltheauthorsapprovedthe finalmanuscript.
Conflictsofinterest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgment
AresearchgrantfromtheDeLaSalleUniversityScience Foun-dation through the University Research Coordination Office is gratefullyacknowledged.
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