Chemical investigation of the dichloromethane extracts of the propolis collected from bee (Tetragonulabiroi Friese) hives in San Roque, Sorsogon, Philippines afforded propolin A (1), propolin E (2), propolinH (3), glyasperin A (4), squalene, a mixture of lupeol, α-amyrin and β-amyrin, and another mixtureof urs-12-en-3-one, olean-12-en-3-one and lup-12-en-3-one. The structures of 2–4 were elucidated byextensive 1D and 2D (COSY, HSQC, and HMBC) NMR spectroscopy, while 1 was identified by comparisonof its NMR data with 2. © 2015 2015 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editora Ltda. All rights reserved

Aguda, Remil M.

Cervancia, Cleofas R.

Ebajo, Virgilio D.

Galian, Richard Anthony F.

Ragasa, Consolacion Y.

Shen, Chien Chang

Revista Brasileira de Farmacognosia

AbstractChemical investigation of the dichloromethane extracts of the propolis collected from bee (Tetragonula biroi Friese) hives in San Roque, Sorsogon, Philippines afforded propolin A (1), propolin E (2), propolin H (3), glyasperin A (4), squalene, a mixture of lupeol, α-amyrin and β-amyrin, and another mixture of urs-12-en-3-one, olean-12-en-3-one and lup-12-en-3-one. The structures of 2–4 were elucidated by extensive 1D and 2D (COSY, HSQC, and HMBC) NMR spectroscopy, while 1 was identified by comparison of its NMR data with 2

Shen, Chien-Chang

Elsevier - Publisher Connector 

Propolins and glyasperin A from stingless bee nests 

Animo Repository - De La Salle University Research

Propolins and glyasperin a from stingless bee nests

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0Revista Brasileira de Farmacognosia 25 (2015) 177–179
www.sbfgnos ia .org .br / rev is ta
hort  communication
ropolins  and  glyasperin  A  from  stingless  bee  nests
onsolacion  Y.  Ragasaa,b,∗, Richard  Anthony  F.  Galianb, Virgilio  D.  Ebajo  Jr. b,
emil  M.  Agudac,  Cleofas  R.  Cervanciad, Chien-Chang  Shene
Chemistry Department, De La Salle University Science & Technology Complex, Leandro V. Locsin Campus, Bin˜an City, Laguna 4024, Philippines
Chemistry Department, De La Salle University, 2401 Taft Avenue, Manila 1004, Philippines
Institute of Chemistry, University of the Philippines Los Ban˜os, College, Laguna 4031, Philippines
Institute of Biological Sciences, University of the Philippines Los Ban˜os, College, Laguna 4031, Philippines
National Research Institute of Chinese Medicine, 155-1, Li-Nong St., Sec. 2, Taipei 112, Taiwan
 r  t i  c  l  e  i  n  f  o
rticle history:
eceived 26 December 2014
ccepted 9 March 2015
vailable online 30 March 2015
a  b  s  t  r  a  c  t
Chemical  investigation  of the  dichloromethane  extracts  of  the propolis  collected  from  bee  (Tetragonula
biroi  Friese)  hives  in  San  Roque,  Sorsogon,  Philippines  afforded  propolin  A (1),  propolin  E  (2),  propolin
H  (3),  glyasperin  A  (4), squalene,  a mixture  of lupeol,  -amyrin  and  -amyrin,  and  another  mixture
of  urs-12-en-3-one,  olean-12-en-3-one  and  lup-12-en-3-one.  The  structures  of 2–4  were  elucidated  byeywords:
hilippine propolis
etragonula biroi
ropolin A
ropolin E
ropolin H
lyasperin A
extensive  1D  and  2D  (COSY,  HSQC,  and  HMBC)  NMR  spectroscopy,  while  1 was identiﬁed  by  comparison
of  its NMR  data  with  2.
© 2015  Sociedade  Brasileira  de  Farmacognosia.  Published  by Elsevier  Editora  Ltda.  All  rights  reserved.ntroduction
Propolis has been reported to contain ﬂavonoids and pheno-
ic compounds that have shown pharmacological activities (Toreti
t al., 2013). Propolis is known to exhibit numerous medicinal prop-
rties, including cytotoxic (Matsuno et al., 1997; Kimoto et al.,
001), free radical scavenging (Basnet et al., 1997), anti-HIV (Ito
t al., 2001), antimicrobial (Park et al., 1998), and antiherpes
Vynograd et al., 2000). Reviews on the chemical constituents and
iological activities of propolis have been provided (Toreti et al.,
013; Huang et al., 2007; Bankova et al., 2000).
A recent study on Philippine propolis identiﬁed by LC–MS
rtepillin C and pinobanksin-3-O-hexanoate as possible pheno-
ic compounds in propolis of stingless bees, Tetragonula biroi
nd exudates from Persea americana Mill, Artocarpus hetero-
hyllus Lam, Mangifera indica L., Canarium ovatum Engl, and
ephelium lappaceum L. that are utilized by T. biroi as propolis
∗ Corresponding author.
E-mail: consolacion.ragasa@dlsu.edu.ph (C.Y. Ragasa).
http://dx.doi.org/10.1016/j.bjp.2015.03.006
102-695X/© 2015 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editorasource (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
Achras sapota were identiﬁed as pinobanksin-5,7-dimethyl ether,
artepillin C, luteolin-5-methyl ether, pinobanksin-3-O-(butyrate
or isobutyrate) and kaempferide by matching the retention time
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-
cein production in Chromobacterium violaceum (Lamberte et al.,
2011).
We  report herein the isolation of propolin A (1), propolin
E (2), propolin H (3), glyasperin A (4), and squalene from the
dichloromethane extracts of the propolis collected from bee (T.
biroi Friese) hives in San Roque, Sorsogon, Philippines. A mixture
of lupeol, -amyrin and -amyrin, and another mixture of urs-
12-en-3-one, olean-12-en-3-one and lup-12-en-3-one were also
obtained from the dichloromethane extracts of the propolis. To
the best of our knowledge this is the ﬁrst report on the isolation of
 Ltda. All rights reserved.
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(78 C.Y. Ragasa et al. / Revista Brasileir
hese compounds from Philippine propolis and the ﬁrst report on
he isolation of 4 from propolis.
aterials and methods
NMR  spectra were recorded on a Varian VNMRS spectrometer
n CDCl3 at 600 MHz  for 1H NMR  and 150 MHz  for 13C NMR  spectra.
he 2D NMR  experiments (COSY, HSQC, and HMBC) for propolin
, propolin H, and glyasperin A were performed by using Varian
COSY, gHSQCAD and gHMBCAD pulse programs. CD spectra were
ecorded by a JASCO J-715 spectropolarimeter. Column chromatog-
aphy was performed with silica gel 60 (70–230 mesh). Thin layer
hromatography was performed with plastic backed plates coated
ith silica gel F254 and the plates were visualized by spraying with
anillin/H2SO4 solution followed by warming.
The propolis used in this study was collected from bee
Tetragonula biroi Friese) hives in San Roque, Sorsogon, Philippines
n May  2012.
In the ﬁrst isolation, propolis (50 g) was pulverized by mor-
ar and pestle, soaked in 200 ml  of CH2Cl2 for three days and
hen ﬁltered. The ﬁltrate was concentrated under vacuum to
fford the crude extract (27.5 g) which was chromatographed
sing increasing proportions of acetone in CH2Cl2 at 10% incre-
ent as eluents. The CH2Cl2 fraction from the chromatography
f the crude extract was rechromatographed (4×)  in petroleum
ther to afford squalene (10 mg). The 60% acetone in CH2Cl2 frac-
ion from the chromatography of the crude extract was  combined
nd rechromatographed in CH3CN:Et2O:CH2Cl2 (0.5:0.5:9 by vol-
me). The less polar fractions were rechromatographed (3×)  in
H3CN:Et2O:CH2Cl2 (0.5:0.5:9 by volume) to afford 2 (8 mg)  after
ashing and trituration with petroleum ether. The more polar frac-
ions were rechromatographed (4×)  in CH3CN:Et2O:CH2Cl2 (1:1:8
y volume) to afford 1 (4 mg)  after washing and trituration with
etroleum ether.
In the second isolation, dried propolis (100 g) was  pulverized
ith mortar and pestle, soaked in 400 ml  CH2Cl2 for three days
nd then ﬁltered. The ﬁltrate was concentrated under vacuum to
fford the crude extract (71 g). The crude extract was  fraction-
ted by silica gel chromatography using increasing proportions
f acetone in CH2Cl2 (10% increment) as eluents. The 10% ace-
one in CH2Cl2 fraction was rechromatographed (2×)  using 5%
tOAc in petroleum ether as eluent to afford a mixture of urs-12-
n-3-one, olean-12-en-3-one, and lup-12-en-3-one (18 mg)  after
ashing with petroleum ether. The 20% acetone in CH2Cl2 fraction
as rechromatographed (3×)  using 10% EtOAc in petroleum ether
s eluent to afford a mixture of lupeol, -amyrin, and -amyrin
22 mg)  after washing with petroleum ether. The 50% acetone in
H2Cl2 fraction was triturated in petroleum ether and then rechro-
atographed using CH2Cl2, followed by Et2O:CH3CN:CH2Cl2 (1:1:8
y volume) as eluents. The fractions eluted with CH2Cl2 were
ombined and rechromatographed (5×)  using Et2O:CH3CN:CH2Cl2
1:1:8 by volume) as eluent to afford 3 (15 mg)  after trituration with
etroleum ether. The fractions eluted with Et2O:CH3CN:CH2Cl2
1:1:8 by volume) from the rechromatography of the 50% acetone inarmacognosia 25 (2015) 177–179
CH2Cl2 fraction were rechromatographed (6×)  in the same solvent
to afford 4 (12 mg)  after trituration with petroleum ether. Com-
pounds 1, 2 and squalene were also detected in the second isolation,
but were no longer puriﬁed.
Results and discussion
The structures of 2–4 were elucidated by extensive 1D and
2D NMR  spectroscopy, while 1 was  identiﬁed by comparison of
its 1H NMR  data with those of 2. Propolin A (1) (Chen et al.,
2003, 2008), propolin E (2) (Chen et al., 2008; Weng et al.,
2007) and propolin H (3) (Weng et al., 2007) were reported as
constituents of Taiwanese propolis. Glyasperin A (4) was previ-
ously isolated from the roots of Glycyrrhiza aspera (Zeng et al.,
1992).
Propolins with diverse biological activities have been isolated
from propolis. Propolins A–F isolated from Taiwanese propolis
could effectively induce human melanoma cell apoptosis and were
strong antioxidant agents (Chen et al., 2004). Propolin A exhib-
ited cytotoxic properties against human melanoma, C6 glioma,
and HL-60 with IC50 values of 6.0, 3.5, and 7.5 g/ml, respectively
(Chen et al., 2003). Propolins A and B induced cytotoxicity effect
in human melanoma A2058 cells and were strong antioxidants
(Chen et al., 2007a). The anti-methicillin resistant Staphylococ-
cus aureus (MRSA) activity of propolin D (MIC 8–16 mg/l) and
propolin C (MIC 8–32 mg/l) was reported (Raghukumar et al.,
2010). On the other hand, propolin G was reported as a potent
caspase-dependent inducer of apoptosis in brain cancer cells. With
Taiwanese propolis extract, propolin G demonstrated a protective
effect against oxidative stress in rat cortical neurons (Huang et al.,
2007). Another study reported that propolin H inhibited the pro-
liferation of human lung carcinoma cell lines in MTT  assay (Weng
et al., 2007).
Authors’ contributions
CYR conducted the experiments, elucidated the structures of
the compounds and wrote the manuscript. C-C Shen obtained the
NMR, CD and HR-MS data and reviewed the manuscript. RFG and
VE conducted the experiments. RA and CC collected the propolis
and contributed to the introduction. All the authors approved the
ﬁnal manuscript.
Conﬂicts of interest
The authors declare no conﬂicts of interest.
Acknowledgment
A research grant from the De La Salle University Science Foun-
dation through the University Research Coordination Ofﬁce is
gratefully acknowledged.
References
Alvarez, P.L.J., Cruz, M.B., Micor, J.R.L., Fajardo Jr., A.C., Cervancia, C.R., Hizon-Fradejas,
A.B.,  2013. Identiﬁcation of ﬂavonoids and phenolic compounds in propolis from
stingless bee (Tetragonula biroi Friese) nests and extracts from ﬁve tree sources
using tandem liquid chromatography–mass spectrometry. Philipp. Entomol. 20,
91–99.
Bankova, V.S., De Castro, S.L., Marcucci, M.C., 2000. Propolis: recent advances in
chemistry and plant origin. Apidologie 31, 3–15.
Basnet, P., Matsuno, T., Neidlein, R., 1997. Potent free radical scavenging activity of
propol isolated from Brazilian propolis. Z. Naturforsch. C 52, 828–833.
Belina-Aldamita, M.A., Lechiga, J.A., Mateo, J.M.C., Micor, J.R.L., Cervancia, C.R.,
Hizon-Fradejas, A.B., 2013. Detection of phenolic compounds in stingless bee
(Tetragonula biroi Friese) propolis and ﬁve tree sources using tandem liquid
chromatography–mass spectrometry. J. Nat. Stud. 12, 30–38.
a de Fa
C
C
C
C
G
H
I
K
wanese propolis induces G1 arrest in human lung carcinoma cells. J. Agric. FoodC.Y. Ragasa et al. / Revista Brasileir
hen, C.-N., Wu,  C.-L., Shy, H.-S., Lin, J.-K., 2003. Cytotoxic prenylﬂavanones from
Taiwanese propolis. J. Nat. Prod. 66, 503–506.
hen, C.-N., Weng, M.-S., Wu,  C.-L., Lin, J.-K., 2004. Comparison of radical scavenging
activity, cytotoxic effects and apoptosis induction in human melanoma cells by
Taiwanese propolis from different sources. Evid.-Based Complement. Alternat.
Med. 1, 175–185.
hen, C.-N., Wu,  C.-L., Lin, J.-K., 2007a. Apoptosis of human melanoma cells induced
by the novel compounds propolin A and propolin B from Taiwanese propolis.
Cancer Lett. 245, 218–231.
hen, Y.-W., Wu,  S.-W., Ho, K.-K., Lin, S.-B., Huang, C.-Y., Chen, C.-N., 2008. Charac-
terisation of Taiwanese propolis collected from different locations and seasons.
J.  Sci. Food Agric. 88, 412–419.
ardana, C., Scaglianti, M.,  Pietta, P., Simonetti, P., 2007. Analysis of the polyphenolic
fraction of propolis from different sources by liquid chromatography–tandem
mass spectrometry. J. Pharm. Biomed. Anal. 45, 390–399.
uang, W.-J., Huang, C.-H., Wu,  C.-L., Lin, J.-K., Chen, Y.-W., Lin, C.-L., Chuang, S.-
E.,  Huang, C.-Y., Chen, C.-N., 2007. Propolin G, a prenylﬂavanone, isolated from
Taiwanese propolis, induces caspase-dependent apoptosis in brain cancer cells.
J.  Agric. Food Chem. 55, 7366–7376.
to, J., Chang, F.R., Wang, H.K., Park, Y.K., Ikegaki, M.,  Kilgore, N., Lee, K.-H., 2001.
Anti-AIDS agents. 48. Anti-HIV activity of moronic acid derivatives and the new
melliferone-related triterpenoid isolated from Brazilian propolis. J. Nat. Prod.
64,  1278–1281.
imoto, T., Aga, M.,  Hino, K., 2001. Apoptosis of human leukemia cells induced
by Artepillin C, an active ingredient of Brazilian propolis. Anticancer Res. 21,
221–228.rmacognosia 25 (2015) 177–179 179
Lamberte, L.E., Cabrera, E.C., Rivera, W.L., 2011. Activity of the ethanolic extract of
propolis (EEP) as a potential inhibitor of quorum sensing-mediated pigment
production in Chromobacterium violaceum and virulence factor production in
Pseudomonas aeruginosa. Philipp. Agric. Sci. 94, 14–22.
Matsuno, T., Matsumoto, Y., Saito, M.,  Morikawa, J., 1997. Isolation and character-
ization of cytotoxic diterpenoid isomers from propolis. Z. Naturforsch. C 52,
702–704.
Park, Y.K., Koo, M.H., Areu, J.A.S., Ikegaki, M.,  Cury, J.A., Rosalen, P.L., 1998.
Antimicrobial activity of propolis on oral microorganisms. Curr. Microbiol. 36,
24–28.
Raghukumar, R., Vali, L., Watson, D., Fearnley, J., Seidel, V., 2010. Antimethicillin-
resistant Staphylococcus aureus (MRSA) activity of ‘paciﬁc propolis’ and isolated
prenylﬂavanones. Phytother. Res. 24, 1181–1187.
Toreti, V.C., Sato, H.H., Pastore, G.M., Park, Y.K., 2013. Recent progress of propolis for
its  biological and chemical compositions and its botanical origin. Evid.-Based
Complement. Alternat. Med., http://dx.doi.org/10.1155/2013/697390.
Vynograd, N., Vynograd, I., Sosnowski, Z., 2000. A comparative multi-centre study of
the efﬁcacy of propolis, acyclovir and placebo in the treatment of genital herpes
(HSV). Phytomedicine 7, 1–6.
Weng, M.-S., Liao, C.-H., Chen, C.-N., Wu,  C.-L., Lin, J.-K., 2007. Propolin H from Tai-Chem. 55, 5289–5298.
Zeng, L., Fukai, T., Nomura, T., Zhang, R.-Y., Lou, Z.-C., 1992. Four new prenylated
ﬂavonoids, glyasperins A, B, C, and D from the roots of Glycyrrhiza aspera.  Hete-
rocycles 34, 575–587.


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Propolins and glyasperin a from stingless bee nests

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