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Journal of Ethnopharmacology 124 (2009) 586591

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Hypoglycemic effect of quassinoids from Brucea javanica (L.) Merr


(Simaroubaceae) seeds
Ajmi NoorShahida a,b , Tin Wui Wong b , Chee Yan Choo a,
a
b

MedChem Herbal Research Group, Faculty of Pharmacy, Universiti Teknologi MARA, 40450 Shah Alam, Selangor, Malaysia
Non-Destructive Biomedical and Pharmaceutical Research Centre, Universiti Teknologi MARA, 40450 Shah Alam, Selangor, Malaysia

a r t i c l e

i n f o

Article history:
Received 28 November 2008
Received in revised form 20 April 2009
Accepted 30 April 2009
Available online 9 May 2009
Keywords:
Brucea javanica (L.) Merr
Simaroubaceae
Quassinoids
Diabetes
STZ induced diabetic rats
Acute toxicity

a b s t r a c t
Ethnopharmacological relevance: The seeds of Brucea javanica (L.) Merr (Simaroubaceae) are recommended
by traditional practitioners for the treatment of diabetes mellitus.
Aim of the study: To identify the compounds responsible for blood glucose lowering effect and evaluate
the acute toxicity of the compounds.
Materials and methods: Extracts, fractions and subfractions were administered to normoglycemic mice
and the blood glucose concentration was monitored for 8 h. Bioactive compounds isolated through column chromatography were administered to normoglycemic mice and streptozotocin (STZ) rats with
monitoring of blood glucose concentration at 08 h. The acute toxicity was evaluated in mice.
Results: Bioactivity-guided fractionation led to the isolation of bruceines E (1) and D (2). Normoglycemic
mice administered with 1 mg/kg of 1 and 2 exhibited signicant blood glucose concentration reduction of 40.07 11.45% and 48.82 13.34%, respectively. STZ induced diabetic rats administered with 1
and 2 exhibited signicant blood glucose concentration reduction of 73.57 13.64% and 87.99 2.91%,
respectively.
Conclusion: The reduction of blood glucose concentration by both bruceines was comparable to glibenclamide and they might act as an insulin secretagogue. The presence of a hydroxyl moiety at C2 in 1
reduced the toxic effect by 9-fold compared to 2.
2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
Brucea javanica (L.) Merr (Simaroubaceae) is a shrub of about 3 m
height. It grows in tropical areas distributing from Southeast Asia to
Northern Australia (Kamperdick et al., 1995; Kim et al., 2003). Compounds from Brucea javanica exhibited various biological activities
such as antimalarial (ONeill et al., 1987), amoebicidal (Wright et al.,
1988) cytotoxic and antileukemic (Lee et al., 1979, 1984; Cassady
and Suffness, 1980; Sakaki et al., 1986; Anderson et al., 1991;
Fukamiya et al., 1992; Luyengi et al., 1996; Su et al., 2002; Kim et
al., 2004), anti-protozoan (Sawangjaroen and Sawangjaroen, 2005),
anti-HIV (Okano et al., 1996), anti-inammatory (Hall et al., 1983)
and antibabesial (Subeki et al., 2007) effects. Chemical constituents
reported from Brucea javanica included quassinoids (Polonsky et
al., 1980; Kim et al., 2003, 2004), quassinoid glycosides (Lee et al.,
1979; Sakaki et al., 1986; Fukamiya et al., 1992), apotirucallane-type
triterpenoids, lignan (Luyengi et al., 1996), alkaloids (Liu et al., 1990)
and alkaloid glycoside (Kitagawa et al., 1994). Locally, this plant is
known as lada pahit due to its bitter taste. In Malaysia, the seeds

Corresponding author. Tel.: +60 3 55442877; fax: +60 3 55442725.


E-mail address: choo715@salam.uitm.edu.my (C.Y. Choo).
0378-8741/$ see front matter 2009 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2009.04.058

of Brucea javanica are used by the traditional practitioners for the


treatment of diabetes (Shamsul et al., 2003). Diabetes patients are
recommended to take 510 seeds a day. Nonetheless, there is no
known research to investigate the blood glucose lowering property
of Brucea javanica seeds. The objective of this study is to identify
the compounds responsible for blood glucose lowering effect.
2. Materials and methods
2.1. Plant material
The dried seeds of Brucea javanica were purchased from the
traditional medicine market in Malacca, Malaysia. The identity of
Brucea javanica was authenticated by Emeritus Professor Dato Dr.
Abdul Latiff Mohamed from Universiti Kebangsaan Malaysia, Bangi,
Malaysia. The voucher specimen (FP/UiTM/BJ/01/05) was deposited
at the Faculty of Pharmacy, Universiti Teknologi MARA, Malaysia.
2.2. Preparation of extracts
The seeds (1 kg) of Brucea javanica were oven dried at 40 1 C,
grounded and soaked in 80% (v/v) aqueous methanol for 48 h at
room temperature. Later, the methanolic extract was ltered and

A. NoorShahida et al. / Journal of Ethnopharmacology 124 (2009) 586591

dried under reduced pressure at 40 1 C using the rotary evaporator. The dried methanolic extract was suspended in deionised
water and sequentially partitioned with n-hexane (3 1 L), chloroform (3 1 L) and n-butanol (3 1 L). The solvent of each partitioned
extract was evaporated to dryness with the rotary evaporator to
afford the dried hexane (3.5 g), chloroform (6.3 g) and butanol
(21.8 g) soluble extracts. The aqueous soluble residual left after
sequential partitioning was also dried under reduced pressure with
the rotary evaporator at 40 1 C to afford the residue extract. All
the dried extracts were stored at 4 C until further evaluation.
2.3. Bioactivity-guided fractionation of compounds
The bioactive butanol extract (5 g) was subjected to open
silica gel (0.0630.200 mm, Merck) column chromatography (5 cm 50 cm) eluting sequentially with 300 mL each of
CHCl3 :MeOH in ratios of 9:1, 7:3, 1:1 and 0:10 to afford four pooled
fractions, namely, BJA (0.09 g), BJB (2.09 g), BJC (0.98 g), and BJD
(1.17 g). The fractions were pooled together based on the similar
Rf values on thin layer chromatography (TLC) developed with the
mobile phase system of CHCl3 :MeOH (7:3), sprayed with 10% (v/v)
sulfuric acid and heated to 110 C for 10 min.
The bioactive BJB fraction (1.0 g) was further subjected to
ash silica gel (0.0400.063 mm, Merck) column chromatography
(3 cm 50 cm) eluting with CHCl3 :MeOH (15:1, 10:1, 9:1, 8:2, 7.2
and 0:10). Six fractions, namely, BJ1 (10.8 mg), BJ2 (278.0 mg), BJ3
(206.7 mg), BJ4 (195.8 mg), BJ5 (86.6 mg) and BJ6 (159.2 mg) were
pooled together based on the similar Rf values on TLC. Recrystallization of BJ1 and BJ2 with methanol afforded crystals bruceine
E (1) (88.9%) and bruceine D (2) (88.4%), respectively (Fig. 1). The
crystals were structurally elucidated using NMR, IR, MS, MS and UV
spectrometry. The purity of both 1 and 2 were evaluated on an analytical column 250 mm 4.6 mm (Inertsil, RP18, 3 m, GL Sciences
Inc., Japan) monitored at 200500 nm with a diode-array detector
(Agilent 1200, Germany) and ow rate of 1 mL/min with 25% MeOH
in deionised water.
Bruceine E (1) was obtained as colorless needles, m.p.
261264 C. UV max (MeOH) nm (log ): 204 (3.17); []D 20 + 30
(c 0.1; MeOH); IR max (KBr) cm1 : 3542, 1710. MS m/z: 395
[MH2 O+H]+ . 1 H NMR (500 MHz, pyridined5 ): 1.58 (3H, overlapped, CH3 -10), 1.58 (3H, overlapped, CH3 -4), 1.62 (1H, ddd, J = 13.8,
13.4, 2.6 Hz, H-6), 2.08 (3H, s, CH3 -13), 2.22 (1H, dt, J = 13.4, 2.6 Hz),
2.71 (1H, d, J = 4.7 Hz, H-9), 2.80 (1H, br d, J = 13.8 Hz, H-5), 4.10 (1H,
dd, J = 7.1, 3.5 Hz, H-1), 4.30 (1H, d, J = 6.9 Hz, H-20), 4.58 (1H, br s,
H-2), 4.63 (1H, d, J = 5.0 Hz, H-12), 5.15 (1H, d, J = 6.9 Hz, H-20), 5.23
(1H, d, J = 5.7 Hz, 14-OH), 5.49 (1H, d, J = 4.7 Hz, H-11), 5.51 (1H, overlapped, H-7), 5.75 (1H, d, J = 1.3 Hz, H-3), 6.00 (1H, d, J = 4.9 Hz, H-15),
6.30 (1H, d, J = 6.3 Hz, 2-OH), 7.11 (1H, s, 11-OH), 7.31 (1H, d, J = 5.0 Hz,
12-OH), 7.57 (1H, d, J = 3.5 Hz, 1-OH), 7.65 (1H, d, J = 4.9 Hz); 13 C NMR

587

(125 MHz, pyridined5 ): 12.5 (CH3 -4), 19.5 (CH3 -13), 21.0 (CH3 10), 28.2 (C-6), 43.2 (C-5), 45.0 (C-6), 46.5 (C-7), 50.6 (C-8), 70.4
(C-9), 70.5 (C-10), 73.4 (C-11), 76.2 (C-12), 80.5 (C-13), 81.5 (C-14),
82.6 (C-14), 82.7 (C-1), 84.5 (C-13), 126.3 (C-3), 135.8 (C-4), 175.2
(C-16).
Bruceine D (2) was obtained as colorless needles, m.p.
294296 C. UV max (MeOH) nm (log ): 240 (3.99); []D 20 + 29
(c 0.1; MeOH); IR max (KBr) cm1 : 3457, 1708. MS m/z: 411 [M+H]+ .
1 H NMR (500 MHz, pyridined ): 1.46 (3H, s, CH -10), 1.66 (1H,
5
3
ddd, J = 2.9, 14.6, 12.3 Hz, H-6), 1.71 (3H, s, CH3 -4), 2.09 (3H, s, CH3 13), 2.30 (1H, dt, J = 2.9, 2.9, 14.6 Hz, H-6), 2.90 (1H, d, J = 5.5 Hz,
H-9), 3.07 (1H, br d, J = 12.3 Hz, H-5), 4.27 (1H, d, J = 6.9 Hz, H-20),
4.33 (1H, d, J = 2.5 Hz, H-1), 4.60 (1H, d, J = 3.5 Hz, H-12), 4.90 (1H, d,
J = 5.5 Hz, 11-OH), 4.96 (1H, overlapped, H-20), 5.43 (1H, t, J = 5.5,
5.5 Hz, H-11), 5.45 (1H, br s, H-7), 6.12 (1H, overlapped, H-3), 6.12
(1H, overlapped, H-15), 6.66 (1H, d, J = 2.5 Hz, 1-OH), 7.29 (1H, s, 14OH), 7.45 (1H, d, J = 3.5 Hz, 12-OH), 8.01 (1H, d, J = 5.0 Hz, 15-OH);
13 C NMR (125 MHz, pyridined ): 11.5 (CH -4), 19.5 (CH -13), 22.2
5
3
3
(CH3 -10), 28.0 (C-6),43.5 (C-5), 45.8 (C-9), 48.7 (C-10), 50.3 (C-8),
69.9 (C-20), 70.5 (C-15), 75.6 (C-11), 79.5 (C-7), 81.6 (C-12), 82.5
(C-14), 83.2 (C-1), 84.8 (C-13), 124.9 (C-3), 163.5 (C-4), 175.2 (C-16),
198.5 (C-2).
2.4. Experimental animals
Normoglycemic male Mus musculus mice (Kebayan Enterprise,
Malaysia), weighed between 25 and 30 g and Sprague Dawley male
rats (Kebayan Enterprise, Malaysia) weighed between 200 and
250 g were used in the evaluation of blood glucose lowering effect
of Brucea javanica extracts and pure compounds. Both the mice and
rats were housed in a standard environment at an ambient temperature of 25 1 C with a relative humidity of 65 5% and 12 h
light/dark cycle. They were given free access to standard pelletized
food (GoldCoin Enterprise, Malaysia) and water ad libitium. They
were acclimatized for at least 1 week and subjected to 12 h of fasting prior to experiment unless otherwise stated. All experiments
were conducted after approval was obtained from the Local Animal
Ethical Committee.
2.5. STZ induced diabetic rats
The STZ solution was freshly prepared using 0.1 M citrate
buffer with pH 4.5. The STZ solution was administered by a single intraperitoneal (i.p.) injection to the fasted rats at a dose of
60 mg/kg body weight (BW). The rats were then provided with
5% glucose solution to prevent death owing to oxidative stress
as it was envisaged that binding of STZ on glucose receptors of
-cells can be effected at a lower propensity in the presence of
glucose (Kamtchouing et al., 1998). The rats with blood glucose

Fig. 1. Quassinoids from Brucea javanica.

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Fig. 2. Proles of blood glucose concentration of mice (n = 5) administered (i.p.) with 1 mg/kg BW of butanol fractions. *p < 0.05, in relation to control.

concentration above 13.3 mmol/L or 250 mg/dL were subjected to


further experiment (Sekar et al., 1990).
2.6. Crude extracts
The mice were divided into groups of 10 each. Methanol, butanol
and aqueous residue extracts were dissolved using 0.2% (w/v) of
polyethylene glycol 3000 (PEG 3000) solution to prepare a dose
of 32 mg extract/kg BW of mouse, respectively. Similar dose of
32 mg extract/kg were prepared for both the hexane and chloroform
extracts dissolved using 2% (w/v) dimethylsulfoxide (DMSO) solution. The blood was withdrawn from the vein of the tail immediately
prior to the administration of extracts (0 h) and throughout 8 h after
the oral administration of extracts. The blood glucose concentration
of each mouse was examined using the glucometer (Ascensia Elite,
Bayer Corporation, USA). Mice administered orally with either 0.2%
(w/v) of PEG 3000 solution or 2% (w/v) DMSO solution served as
the negative control. Glibenclamide at a dose of 3 mg/kg was used
as a positive control.
2.7. Butanol fractions and subfractions
The butanol fractions and subfractions were administered to
mice via the intraperitoneal route (i.p.) rather than oral route due
to the small quantity of semi-puried fractions and subfractions
collected after column chromatography. A dose of 1 mg/kg BW was
used owing to blood glucose lowering property was indicated at low

doses in preliminary study. A dose of 1 mg/kg BW of BJA, BJB, BJC and


BJD was administered intraperitoneally to the mice in groups of 5
each. BJA was dissolved in 2% (w/v) DMSO solution, while the other
fractions BJB, BJC and BJD were dissolved in 0.2% (w/v) PEG 3000
solution. The solubilizers of either 2% w/v DMSO or 0.2% (w/v) PEG
3000 solution were similarly administered to mice as the negative
control. Glibenclamide, as positive control was dissolved in 0.2%
(w/v) PEG 3000 solution and was administered intraperitoneally at
a dose of 0.3 mg/kg BW of mouse.
All the butanol subfractions, BJ16, were dissolved in 0.2% (w/v)
PEG 3000 solution and were administered intraperitoneally to mice
in groups of 10 each at a dose of 1 mg/kg BW of mouse. The blood
glucose lowering effects of the fractions and subfractions were
examined.
2.8. Dose-blood glucose lowering response of bruceine E (1) and
bruceine D (2) in normoglycemic mice
Both 1 and 2 were dissolved individually in 0.2% (w/v) PEG
3000 solution. They were administered intraperitoneally to mice
in groups of 10 each at doses of 0.25, 0.5 and 1 mg/kg BW of mouse.
The blood was withdrawn from the vein of the tail immediately
prior to the administration of the pure compounds (0 h) and every
2 h after the intraperitoneal administration for 8 h (2, 4, 6 and 8 h).
Glibenclamide, as positive control was dissolved in 0.2% (w/v) PEG
3000 solution and was administered intraperitoneally at a dose of
0.3 mg/kg BW of mouse.

Fig. 3. Proles of blood glucose concentration of mice (n = 10) administered (i.p.) with 1 mg/kg BW of butanol subfractions. *p < 0.05, in relation to control.

A. NoorShahida et al. / Journal of Ethnopharmacology 124 (2009) 586591

589

Fig. 4. Proles of blood glucose concentration of mice (n = 10) administered (i.p.) with various doses of bruceine E (1). *p < 0.05, in relation to control.

2.9. Dose-blood glucose lowering response of bruceine E (1) and


bruceine D (2) in STZ induced diabetic rats
Forty STZ induced diabetic rats were divided into eight groups
of 5 each with group 1 receiving 0.2% (w/v) PEG 3000 solution as
the vehicle while the other six groups receiving 1, 2 and 3 mg/kg
BW of STZ induced diabetic rat with either 1 or 2 through intraperitoneal administration. The blood glucose lowering effects of 1 and 2
in STZ induced diabetic rats were determined from the blood withdrawn from the tail vein at 0 h and every 2 h for the duration of 8 h.
Glibenclamide, as positive control was dissolved in 0.2% (w/v) PEG
3000 solution and was administered intraperitoneally at a dose of
3 mg/kg BW of STZ induced diabetic rat.
2.10. Acute toxicity
The pure compound 1 was prepared in doses of 10, 25, 35, 50
and 75 mg/kg BW of mouse and 2 was prepared in doses of 2,
3, 4 and 5 mg/kg BW of mouse and administered via intraperitoneal route to mice in groups of 10 each. The mice were given
standard pelletized food and water ad libitum following the administration of isolated pure compounds. The mice were kept under
observation for 24 h after receiving the pure compounds. The acute
toxicity of pure compounds was expressed by LD50 value of which
denotes the dose which resulted in 50% mortality of mice in
24 h. The LD50 values were calculated from the log doseprobit
relationship.

2.11. Statistical analysis


Two-way ANOVA was used to evaluate all data expressed in
mean and standard deviation by SPSS statistical package version
14. The level of signicance for all statistical evaluation was set at
p < 0.05.
3. Results and discussion
Preliminary study with various doses of crude methanolic
extract indicated the lowest dose of 32 mg/kg exhibited signicant reduction of blood glucose level by 41.14 14.57% against that
of control. The blood glucose concentration reduction was comparable to glibenclamide (47.22 6.43%). Following partitioning of
methanol extract, the bioactive constituents were identied in the
polar butanol extract (39.14 13.50%) while the hexane, chloroform or the aqueous extracts exhibited no signicant blood glucose
concentration reduction.
3.1. Blood glucose lowering response of butanol fractions BJAD in
normoglycemic mice
Further fractionation of the butanol extract afforded four
fractions, namely, BJAD. The fraction, BJB, exhibited signicant (p < 0.05) blood glucose concentration reduction after 6 h
(42.63 10.53%) and 8 h (46.25 14.15%) administration (Fig. 2).
Further fractionation of BJB afforded six subfractions, BJ16. The

Fig. 5. Proles of blood glucose concentration of mice (n = 10) administered (i.p.) with various doses of bruceine D (2). *p < 0.05, in relation to control.

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Fig. 6. Proles of blood glucose concentration of STZ induced diabetic rats (n = 5) administered (i.p.) with various doses of bruceine E (1). *p < 0.05, in relation to control.

blood glucose concentration reduction was signicant (p < 0.05) in


mice fed with both BJ1 and BJ2 with reductions of 40.07 11.45%
and 48.82 13.34%, respectively, following 8 h of administration.
Both the bruceines E (1) and D (2) isolated as the major constituents
of BJ1 and BJ2, respectively were further subjected to blood glucose
concentration evaluation (Fig. 3).
3.2. Dose-blood glucose lowering response of bruceine E (1) and
D (2) in normoglycemic mice
Following 8 h of bruceine E (1) or D (2) administration in normoglycemic mice, a doseresponse relationship was exhibited at
all the administered doses (Figs. 4 and 5). Normoglycemic mice
administered with 1 mg/kg of bruceine D (2) exhibited signicant
(p < 0.05) blood glucose concentration reduction (53.04 17.05%)
after 6 h while the effect of blood glucose concentration reduction of 40.07 11.45% was only exhibited after 8 h of bruceine E
(1) administration. The blood glucose concentration reduction prole of bruceine E (1) and D (2) were similar to the positive control,
glibenclamide.
3.3. Determination of LD50 value of bruceine E (1) and bruceine D
(2)
Both, bruceine E (1) and bruceine D (2) exhibited LD50 values of
31.86 and 3.52 mg/kg BW of mouse, respectively. Bruceine E (1) was
9-fold less toxic than bruceine D (2). The LD50 value of 1 was 32-

fold higher than its effective dose at 1 mg/kg BW of normoglycemia


mouse. While, the LD50 value of 2 was 3.5-fold higher than its effective blood glucose lowering dose at 1 mg/kg BW of normoglycemic
mouse. The chemical structure of 1 differed from 2 only at the C2
position with the presence of a hydroxyl moiety instead of ketone
moiety. Presence of the ketone moiety in 2 may possess the toxicity
effects but does not affect its potential in lowering the blood glucose
concentration in mice. Nonetheless, 2 is 145 times less toxic than
bruceantin (Anderson et al., 1991), a quassinoid suspended from
human clinical trial due to lack of efcacy (Cragg and Newman,
2005). Recently, 2 was reported to be a potent anti-proliferative
action on pancreatic cancer cell lines but was also less cytotoxic on
the non-tumorigenic Hs-68 broblasts (Lau et al., 2009).

3.4. Dose-blood glucose lowering response of bruceine E (1) and


D (2) in STZ induced diabetic rats
STZ induced diabetic rats administered through the intraperitoneal route with 2 mg/kg of bruceine E (1) exhibited signicant
blood glucose concentration reduction of 73.57 13.64% (Fig. 6).
Administration of 1 mg/kg of bruceine D (2) exhibited similar
blood glucose concentration reduction of 87.99 2.91% (Fig. 7). The
prole of blood glucose concentration reduction for both the bruceines were similar to STZ induced diabetic rats administered with
3 mg/kg of the positive control, glibenclamide. Though bruceine E
(1) or D (2) have antitumor (Lee et al., 1979), Epstein Barr virus early
antigen inhibitory (Okano et al., 1995), antiplasmodial (ONeill et al.,

Fig. 7. Proles of blood glucose concentration of STZ induced diabetic rats (n = 5) administered (i.p.) with various doses of bruceine D (2). *p < 0.05, in relation to control.

A. NoorShahida et al. / Journal of Ethnopharmacology 124 (2009) 586591

1987), antiamoebic (Wright et al., 1988), antibabesial (Subeki et al.,


2007), anti-inammatory (Hall et al., 1983) properties, this is the
rst report on its antihyperglycemic effect.
4. Conclusion
In the present study, we demonstrated for the rst time that
the two quassinoids, namely bruceine E (1) and D (2) exhibited
potential blood glucose lowering effect in both the normoglycemic
mice and STZ induced diabetic rats. Both the bruceines exhibited
a doseresponse relationship. Presence of the hydroxyl moiety in
the C2 position reduced the toxicity effect. The blood glucose concentration reduction proles of quassinoids in STZ induced diabetic
rats and normoglycemic mice were comparable to glibenclamide.
Both 1 and 2 might act as an insulin secretagogue on surviving cells of islets of Langerhans to release more insulin to reduce the
concentration of blood glucose. It can be concluded that the present
study may support the use of Brucea javanica seeds at low doses as
recommended by the traditional practitioners in taking 510 seeds
per day for the treatment of diabetes.
Acknowledgements
The authors are grateful for the nancial support from the Fundamental Research Grant Scheme (FRGS) and Pasca scholarship
from the Ministry of Science, Technology and Innovation (MOSTI),
and facility support from Ministry of Higher Education. The authors
also wished to thank Prof. Dr. K. Takeya and Dr. Y. Hitotsuyanagi from
the Tokyo University of Pharmacy and Life Science, Japan for NMR
and MS measurements.
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