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Der Pharma Chemica, 2014, 6(3):256-260
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ISSN 0975-413X
CODEN (USA): PCHHAX
256
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Chemical constituents of Syzygium samarangense
Consolacion Y. Ragasa
1,2*
, Francisco C. Franco Jr.
2
, Dennis D. Raga
3
and Chien-Chang Shen
4
1
Chemistry Department, De La Salle University Science & Technology Complex Leandro V. Locsin Campus, Biñan
City, Laguna, Philippines
2
Chemistry Department and Center for Natural Sciences and Ecological Research, De La Salle University, 2401
Taft Avenue, Manila, Philippines
3
Biology Department and Center for Natural Sciences and Ecological Research, De La Salle University-Manila
4
National Research Institute of Chinese Medicine, 155-1, Li-Nong St., Sec 2, Taipei, Taiwan
_____________________________________________________________________________________________
ABSTRACT
The dichloromethane extract of the leaves of Syzygium samarangense (Blume) Merr. & Perry afforded
2
′
,4
′
-
dihydroxy-6
′
-methoxy-3
′
-methylchalcone
(1),
2
′
,4
′
-dihydroxy-6
′
-methoxy-3
′
,5
′
-dimethylchalcone
(2),
2
′
-hydroxy-
4
′
,6
′
-dimethoxy-3
′
-methyl chalcone
(3), squalene (4), betulin (5), lupeol (6), sitosterol (7), and a mixture of
cycloartenyl stearate (8a), lupenyl stearate (8b), β-sitosteryl stearate (8c), and 24-
methylenecycloartenyl
stearate
(8d). The structures of 1-3, 5, 7, and 8a-8d were elucidated by 1D and 2D NMR spectroscopy. Sample 8 was tested
for hypoglycemic and antimicrobial potentials. It showed negative hypoglycemic potential and
exhibited moderate
antifungal activity against C. albicans, low activity against T. mentagrophytes and low antibacterial activity against
E. coli, S. aureus and P. aeruginosa. It was inactive against B. subtilis and A. niger.
Keywords: Syzygium samarangense,
Myrtaceae,
cycloartenyl stearate, lupenyl stearate, β -sitosteryl stearate, 24-
methylenecycloartenyl
stearate
_____________________________________________________________________________________________
INTRODUCTION
Syzygium samarangense (syn. Eugenia javanica Linn.) commonly known as ‘makopa’ is grown throughout the
Philippines for its fruits. The tree is used as an antipyretic and a diuretic [1]
. Four flavonoids isolated from the
hexane extract of S. samarangense showed dose-dependent spasmolytic activity [2]. Another study reported that
2
′
,4
′
-dihydroxy-6
′
-methoxy-3
′
,5
′
-dimethylchalcone
from S. samarangense exhibited significant differential
cytotoxicity against the MCF-7 cell line and significant selective cytotoxicity against RAD 52 yeast mutant strain
[3]. Compounds isolated from the hexane extract of the leaves of S.
samarangense:
2
′
,4
′
-dihydroxy-6
′
-methoxy-3
′
-
methylchalcone
,
2
′
,4
′
-dihydroxy-6
′
-methoxy-3
′
-methyl
dihydrochalcone,
2
′
-hydroxy-4
′
,6
′
-dimethoxy-3
′
-
methylchalcone,
α
- and
β
-carotene, lupeol, betulin,
epi-betulinic
acid, and
β
-D-sitosterylglucoside exhibited
significant and selective inhibition against prolyl endopeptidase [4]. An earlier study reported that the methanol
extract of makopa leaves exhibited high antidiabetic activity [5], while a recent study reported that
2
′
,4
′
-di hydroxy-
6
′
-methoxy-3
′
,5
′
-dimethylchalcone
and 5-
O-
methyl-
4
′
-desmethoxy matteucinol from S. samarangense significantly
lowered the blood glucose levels in hyperglycaemic mice when administered 15 minutes after glucose load, while
2
′
,4
′
-dihydroxy-6
′
-methoxy-3
′
,5
′
-dimethylchalcone significantly lowered the blood glucose levels of alloxan
diabetic mice [6]. Recently, we reported the potent analgesic and anti-inflammatory activities and the negligible
toxicity on zebrafish embryonic tissues of a mixture of
cycloartenyl stearate (8a), lupenyl stearate (8b), sitosteryl
stearate (8c), and 24-
methylenecycloartenyl
stearate (8d) from the dichloromethane extract of the air-dried leaves of
S. samarangense [7].
Consolacion Y. Ragasa
et al
Der Pharma Chemica, 2014, 6 (3):256-260
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257
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We report herein the isolation and identification of 2
′
,4
′
-dihydroxy-6
′
-methoxy-3
′
-methylchalcone
(1),
2
′
,4
′
-
dihydroxy-6
′
-methoxy-3
′
,5
′
-dimethylchalcone
(2),
2
′
-hydroxy-4
′
,6
′
-dimethoxy-3
′
-methylchalcone
( 3), squalene ( 4),
betulin (5), lupeol (6), sitosterol (7), and a mixture of cycloartenyl stearate (8a), lupenyl stearate (8b), β-sitosteryl
stearate (8c), and 24-
methylenecycloartenyl
stearate (8d) (Fig.1) from the air-dried leaves of S. samarangense. To
the best of our knowledge this is the first report on the isolation of 8a-8d from the tree. Results of the hypoglycemic
and antimicrobial tests on a mixture of 8a-8d are likewise reported.
R
R
1
4
6
1 0
1 1
1 4
1 7
1 8
1 9
2 0
2 1
2 4
2 6
2 8
3 0
2 8
2 4
2 9
3 0
1 9
1 8
2 0
2 1
3 1
2 6
2 8
3 0
2 7
2 5
2 2
2 0
1 9
1 7
1 4
1 1
6
1
6 R = O H
8 b R = C H
3
(
C H
2
)
1 6
C O O
H O
C H
2
O H
R
R
1
6
1 1
1 4
2 0
2 5
2 6
2 1
1 9
1 8
2 9
1 R = O H , R ' = H
2 R = O H , R ' = C H
3
3 R = O C H
3
, R ' = H
5 '
3 '
1 '
α
β
4
1
O C H
3
O
O H
R
H
3
C
R '
7 R = O H
8 c R = C H
3
(
C H
2
)
1 6
C O O
5
8 a R = C H
3
(
C H
2
)
1 6
C O O
8 d R = C H
3
(
C H
2
)
1 6
C O O
4
Figure 1: The compounds from S. samarangense: 2
′
,4
′
-dihydroxy-6
′
-methoxy-3
′
-methylchalcone
(1),
2
′
,4
′
-dihydroxy-6
′
-methoxy-3
′
,5
′
-
dimethylchalcone
(2),
2
′
-hydroxy-4
′
,6
′
-dimethoxy-3
′
-methylchalcone
(3), squalene (4), betulin (5), lupeol (6), sitosterol (7), cycloartenyl
stearate (8a), lupenyl stearate (8b), β-sitosteryl stearate (8c), and 24-
methylenecycloartenyl
stearate (8d)
MATERIALS AND METHODS
General Experimental Procedures
NMR spectra were recorded on a Varian VNMRS spectrometer in CDCl
3
at 600 MHz for
1
H NMR and 150 MHz
for
13
C NMR spectra. 2D NMR (COSY, HSQC, HMBC) spectra were recorded on a Varian VNMRS spectrometer.
MS was obtained on a Finnigan MAT LCQ mass spectrometer. Column chromatography was performed with silica
gel 60 (70-230 mesh); TLC was performed with plastic backed plates coated with silica gel F
254
; plates were
visualized by spraying with vanillin sulfuric acid, followed by warming.
Sample Collection
Fresh leaves of Syzygium samarangense (5 kg) were collected from Antipolo City in December 2008. Specimens of
the sample were authenticated at the Institute of Biology, University of the Philippines, Diliman, Quezon City. A
voucher specimen # 140 was deposited at the Chemistry Department, De La Salle University-Manila.
Consolacion Y. Ragasa
et al
Der Pharma Chemica, 2014, 6 (3):256-260
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Isolation
The air-dried leaves of Syzygium samarangense (1 kg) were ground in an osterizer, soaked in CH
2
Cl
2
for three days
and then filtered. The filtrate was concentrated under vacuum to afford a crude extract (45.86 g) which was
chromatographed in increasing volumes of acetone in CH
2
Cl
2
at 10 % increment. The CH
2
Cl
2
and 10 % acetone in
CH
2
Cl
2
fractions were combined and rechromatographed in petroleum ether. The less polar fractions afforded 4 (55
mg). The more polar fractions were rechromatographed in increasing percentage of EtOAc in petroleum ether
(0.5%, 1%, 2.5% and 5% by volume) to afford sample 8 which is a mixture of 8a-8d (1g). The 20% and 30%
acetone in CH
2
Cl
2
fractions were combined and rechromatographed (5 ×) in CH
2
Cl
2
to afford 6 (25 mg) and 7 (35
mg). The 40% and 50% acetone in CH
2
Cl
2
fractions were combined and rechromatographed in
CH
3
CN:Et
2
O:CH
2
Cl
2
(1:1:8) by volume ratio. The less polar fractions were rechromatographed (4 ×) in
CH
3
CN:Et
2
O:CH
2
Cl
2
(0.5:0.5:9) by volume ratio to afford 3 (18 mg) and 5 (8 mg). The more polar fractions were
rechromatographed (6 ×) in CH
3
CN:Et
2
O:CH
2
Cl
2
(1:1:8) by volume ratio to afford 1 (24 mg) and 2 (32 mg).
Antimicrobial Test
The microorganisms used were obtained from the University of the Philippines Culture Collection (UPCC). These
are Pseudomonas aeruginosa (UPCC 1244), Bacillus subtilis (UPCC 1149), Escherichia coli (UPCC 1195),
Staphylococcus aureus (UPCC 1143), Candida albicans (UPCC 2168), Trichophyton mentagrophytes (UPCC 4193)
and Aspergillus niger (UPCC 3701). Sample 8 was tested for antimicrobial activity against these microorganisms
using the procedure reported in the literature [8].
Experimental Animals
A total of 50 male albino mice (Mus musculus L.) of an inbred ICR strain (7 weeks old) weighing 19.0 ±2.0 g were
acclimatized for 7 days prior to conducting the bioassay. The animals (n = 9) were procured from the Food and
Drugs Authority, Muntinlupa City, Philippines and housed at the animal containment unit of DLSU-Manila with
12h daylight and 12h darkness with free access to food pellets and water. A 16h fasting period was carried out prior
to each treatment procedure [9]. All animal handling procedures were in accordance with the existing policies and
guidelines of the Philippine Association of Laboratory Animal Science (PALAS) for care and use of laboratory
animals and with Administrative Order 40 of the Bureau of Animal Industry relative to the Rep. Act. No.8485.
Hypoglycemic Test
The anti-diabetes assay was performed modified from the procedure [9]. Oral Glucose Tolerance Test (5 g/kg BW)
was performed on normoglycemic mice, followed by measurement of blood glucose level (mg/dL) using OneTouch
Horizon Glucometer (Lifescan, Johnson & Johnson, USA). Polysorbate 80 (25 mg/kg BW, Tween-80, AJAX,
Finechem Pty. Ltd., Australia) as the negative control for sample 8 (8a-8d). Solosa (16.7
µ
g/kg BW, Glimepiride
solosa, Aventis, Italy) dissolved in distilled H
2
O was orally administered as the positive control, while sample 8
(100 mg/kg BW, 50 mg/kg BW, and 25 mg/kg BW) dissolved in Polysorbate 80 were given as the test compounds.
Blood glucose was measured within a 3h period at 30 minutes intervals. Blood glucose reduction was computed as
percent reduction ([initial blood glucose – final blood glucose] / initial x 100) and was used in the statistical
analysis.
RESULTS AND DISCUSSION
Silica gel chromatography of the dichloromethane extract of the air-dried leaves of S. samarangense afforded 2
′
,4
′
-
dihydroxy-6
′
-methoxy-3
′
-methylchalcone
(1),
2
′
,4
′
-dihydroxy-6
′
-methoxy-3
′
,5
′
-dimethylchalcone
(2),
2
′
-hydroxy-
4
′
,6
′
-dimethoxy-3
′
-methylchalcone
( 3), squalene ( 4), betulin ( 5), lupeol ( 6), sitosterol ( 7), and a mixture of
cycloartenyl stearate (8a), lupenyl stearate (8b), β-sitosteryl stearate (8c), and 24-
methylenecycloartenyl
stearate
(8d).
The structures of 1, 2, 3, 5 and 7 were elucidated by extensive 1D and 2D NMR analyses and confirmed by
comparison of their
1
H and
13
C NMR data with those of 2
′
,4
′
-dihydroxy-6
′
-methoxy-3
′
-methylchalcone [10], 2
′
,4
′
-
dihydroxy-6
′
-methoxy-3
′
,5
′
-dimethylchalcone [11], 2
′
-hydroxy-4
′
,6
′
-dimethoxy-3
′
-methylchalcone (aurentiacin)
[12], betulin [13] and sitosterol [14], respectively. Compound 4 was identified by comparison of its
1
H NMR data
with those of squalene [15]. The structure of 6 was deduced by comparison of its
13
C
NMR data with those of
lupeol [13].
The structures of 8a-8d were elucidated by extensive 1D and 2D NMR spectroscopy. The resonances attributed to
the major compound, 8a suggested a cycloartenol esterified to a fatty acid.
Confirmatory evidences are the
13
C
NMR data of
8a and
cycloartenyl acetate [16] for the triterpene part and the fatty acid ester of 16-
hydroxycycloartenyl palmitate [17] for the fatty acid part, which match in all essential respects. The fatty acid chain
length was determined by the mass spectrum of sample 8 which gave a molecular ion at m/z = 694.2 corresponding
to the molecular formula of C
48
H
86
O
2
and an [M
+
-C
18
H
35
O
2
] of m/z 409 which resulted from the loss of stearic acid
Consolacion Y. Ragasa
et al
Der Pharma Chemica, 2014, 6 (3):256-260
_____________________________________________________________________________
259
www.scholarsresearchlibrary.com
from the molecular ion peak. The resonances assigned to 8b indicated that it is lupenyl stearate [18], while
8c is β-
sitosteryl stearate [19].
On the other hand, 8d is
24-
methylenecycloratenyl stearate as confirmed by similar
13
C
NMR data with
24-methylenecycloartenyl acetate [20] for the triterpene part and the fatty acid ester of 16-
hydroxycycloartenyl palmitate [17] for the fatty acid part.
As part of our continuing search for antimicrobial compounds from Philippine medicinal plants, sample 8 was tested
for possible antimicrobial activities by the agar well method. Results of the study (Table 1) indicated that sample 8
is moderately active against the fungus, C. albicans with an activity index (AI = 0.3), slightly active against the
fungus, T. mentagrophytes (AI = 0.3), slightly active against the bacteria: E. coli (AI = 0.1), P. aeruginosa (0.3) and
S. aureus (AI = 0.1). It was inactive against B. subtilis and A. niger.
Table 1. Antimicrobial Test Results on Sample 8
Organism
Sample (30
µ
g)
Clearing Zone
(mm)
a
Activity Index (AI)
E. coli
Sample 8
11
0.1
Chloramphenicol
b
23
2.8
P. aeruginosa
Sample 8
12
0.2
Chloramphenicol
b
14
1.3
S. aureus
Sample 8
12
0.2
Chloramphenicol
b
25
3.2
B. subtilis
Sample 8
-
d
0
Chloramphenicol
b
20
2.3
C. albicans
Sample 8
13
0.3
Canesten, 0.2g
c
18
0.8
T. mentagrophytes
Sample 8
12
0.2
Canesten, 0.2g
c
55
4.5
A. niger
Sample 8
-
0
Canesten, 0.2g
c
23
1.3
a
Average of three trials;
b
Chloramphenicol disc -
6 mm diameter;
c
Contains 1% chlotrimazile;
d
No clearing zone
An earlier study reported that the methanol extract of the leaves of makopa exhibited high antidiabetes activity [5].
Another study reported that the chalcones ( 1- 3) isolated from S. samarangense have been tested for hypoglycemic
activity where 2 significantly lowered the blood glucose levels of alloxan diabetic mice [6]
. Since 8a-8d were
obtained for the first time from S. samarangense and the leaves are known to have antidiabetes property, sample 8
was tested for hypoglycemic potential.
Glucose challenged mice were given three doses of sample 8 (25 mg/kg BW, 50 mg/kg BW, 100 mg/kg BW),
Solosa® or P80 as experimental, positive and negative controls, respectively. Blood glucose was measured 30
minutes after oral gavage and after every 30 minutes for 3h. Percent blood glucose reduction was found highest in
mice administered with 50 mg/kg BW sample 8 at 0.5h (Table 2). This observed reduction however is statistically
similar with the negative control and 100 mg/kg BW. This implies that there was minimal blood glucose reduction
as affected by the treatment. The effect however was very minimal that it is not possible to statistically identify it
from the effects of the negative control. Glimipiride Solosa on the other hand was found to have its effects at 1.0h
similar to our previous reports [21]. Although the observed blood glucose reduction at 0.5
h
revealed significant
differences (P<0.05) between means, such percent reduction cannot be accounted to the effect of sample 8 in all
dosages of the treatment groups but rather to the net effects of insulin. The results however revealed no
hypoglycemic potential of sample 8.
Table 2.
Percent blood glucose reduction in mice administered with sample 8 across a 3h observation period
Group
0.5 h
1.0 h
1.5 h
2.0 h
2.5 h
Control (P80)
62.07±2.91
ab
27.78±5.94
b
17.40±5.36
-4.91±4.99
4.22±2.37
Glymipiride Solosa
32.96±3.72
c
59.24±3.40
a
19.81±3.44
-11.28±4.7
0.15±4.57
25 mg/Kg BW
sample 8
53.60±4.54
b
26.01±4.10
b
15.30±2.54
0.31±4.83
-0.15±4.39
50 mg/Kg BW
sample 8
64.67±2.76
a
33.63±4.39
b
10.13±2.86
-16.89±3.91
10.16±3.24
100 mg/Kg BW
sample 8
63.05±2.81
ab
21.80±6.09
b
6.87±4.38
-7.93±2.47
6.57±2.62
*means followed by the same letter are not significantly different at α=0.05 DMRT
.
Statistical Analysis
The results were analyzed using SPSS ver. 10.5 for windows. One way Analysis of Variance was performed to
determine the significant effects on anti-diabetic potentials of sample 8. Significant differences within group
Consolacion Y. Ragasa
et al
Der Pharma Chemica, 2014, 6 (3):256-260
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variables were determined by post hoc analysis at 95% DMRT. Results were considered significant at
α
= 0.05.
The data was presented as Mean±SD.
Acknowledgment
The antimicrobial tests were conducted at the University of the Philippines-Natural Sciences Research Institute (UP-
NSRI). A research grant from the De La Salle University Science Foundation through the University Research
Coordination Office is gratefully acknowledged.
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