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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm
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
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
588
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.
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.
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.
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.
590
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.
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.
591
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