Food and Chemical Toxicology 43 (2005) 1433–1439
www.elsevier.com/locate/foodchemtox
Antihyperlipidemic effect of Eugenia jambolana seed kernel
on streptozotocin-induced diabetes in rats
Kasiappan Ravi, Subbaih Rajasekaran, Sorimuthu Subramanian
*
Department of Biochemistry and Molecular Biology, University of Madras, Guindy Campus, Chennai 600 025, India
Received 11 April 2005; accepted 11 April 2005
Abstract
Abnormalities in lipid profile are one of the most common complications in diabetes mellitus, which is found in about 40% of
diabetics. In the present study, anti-hyperlipidemic efficacy of Eugenia jambolana seed kernel (EJs-kernel) was evaluated in streptozotocin (STZ)-induced diabetic rats and the efficacy was compared with standard hypoglycemic drug, glibenclamide. The effect of
oral administration of ethanolic extract of EJs-kernel (100 mg/kg body weight) was examined on the levels of cholesterol, phospholipids, triglycerides and free fatty acids in the plasma, liver and kidney tissues of STZ (55 mg/kg body weight)-induced diabetic rats.
The plasma lipoproteins and tissues fatty acid composition were also monitored. STZ-induced diabetic rats, showed significant
increase in the levels of cholesterol, phospholipids, triglycerides and free fatty acids which were considerably restored to near normal
in EJs-kernel or glibenclamide treated animals. The plasma lipoproteins (HDL, LDL, VLDL-cholesterol) and fatty acid composition were altered in STZ-induced diabetic rats and these levels were also reverted back to near normalcy by EJs-kernel or glibenclamide treatment. It may be concluded that, EJs-kernel possesses hypolipidemic effect, which may be due to the presence of
flavonoids, saponins, glycosides and triterpenoids in the extract. The hypolipidemic effect mediated by EJs-kernel may also be anticipated to have biological significance and provide a scientific rationale for the use of EJs-kernel as an anti-diabetic plant.
Ó 2005 Elsevier Ltd. All rights reserved.
Keywords: Antihyperlipidemic; Eugenia jambolana; Diabetes; Streptozotocin; Fatty acids
1. Introduction
Diabetes mellitus, an endocrine disorder, is a major
source of morbidity in developed countries. The metabolism of all fuels including carbohydrates, fats and proteins are altered in diabetes and patients with diabetes
have lipid disorders and an increased risk of coronary
heart disease, peripheral vascular disease and cerebrovascular disease (Brown, 1994; Stamler et al., 1993). Diabe-
Abbreviations: Eugenia jambolana seed kernel-EJs-kernel; HDL,
High density lipoprotein; LDL, Low density lipoprotein; VLDL, Very
low density lipoprotein; STZ, Streptozotocin.
*
Corresponding author. Tel.: +91 44 22351269; fax: +91 44
22300488.
E-mail address: [email protected] (S. Subramanian).
0278-6915/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2005.04.004
tes is associated with profound alterations in the plasma
lipid and lipoprotein profile and with an increased risk of
premature atherosclerosis, coronary insufficiency and
myocardial infarction (Betteridge, 1997).
Accumulation of lipids in diabetes is mediated
through a variety of derangements in metabolic and regulatory processes, especially insulin deficiency, thereby
rendering the diabetic patient more prone to hypercholesterolemia and hypertriglyceridemia (Jaiprakash
et al., 1993). One of the major pathogenesis of lipid
metabolism disturbances in diabetes is the increased
mobilization of fatty acids from adipose tissue and secondary elevation of free fatty acid level in the blood
(Singh et al., 1987). Excessive lipolysis has been found
to occur during diabetes. One of the consequences of
excessive mobilization of fatty acid is the production
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K. Ravi et al. / Food and Chemical Toxicology 43 (2005) 1433–1439
of ketone bodies in the liver. The excessive lipolysis in
diabetic adipose tissue leads to increase free fatty acids
in circulation. They enter the liver and are esterified to
form triglycerides (Hem, 1977). Fatty acids are required
for both the structure and function of every cell in the
body and they form an important component of cell
membranes. Several authors have reported that, the
fatty acid compositions of various tissues are altered in
both experimental and human diabetes (Faas et al.,
1988; Tilvis and Miettinen, 1985).
Many indigenous Indian medicinal plants have been
found to be useful to successfully manage diabetes.
Despite the introduction of hypoglycemic agents from
natural and synthetic sources, diabetes and its secondary
complications continue to be a major medical problem
in the world population. Eugenia jambolana Lam
(Fam: Myrtaceae) commonly called as Jamun, Black
plum or Indian Black berry. Eugenia jambolana seeds
have hypoglycemic, anti-inflammatory, neuropsychopharmacological, anti-bacterial, anti-HIV and antidiarrheal effects (De Lima et al., 1998). Bhatia and
Bajaj, 1975) have reported that the Eugenia jambolana
seed contains several biologically active constituents
such as flavonoids, gallic acid, ellagic acid, glycosides,
triteripenoids and saponins. Recently, we have reported
the anti-diabetic and anti-oxidant property of Eugenia
jambolana seed kernels on streptozotocin-induced diabetic rats (Ravi et al., 2003, 2004a,b,c). The present
study was aimed to investigate the effect of Eugenia jambolana seed kernel on lipid constituents and fatty acid
composition in plasma and tissues of streptozotocininduced diabetic rats. The efficacy was compared with
a standard hypoglycemic drug, glibenclamide.
2. Materials and methods
2.1. Chemicals
Streptozotocin was procured from Sigma Chemical
Co., St. Louis, MO, USA. All other chemicals used were
of analytical grade.
water to remove the traces of pulp from the seeds and
were dried at room temperature. The kernel of the seeds
was selectively separated from the seed coat. The kernel
was powdered in an electrical grinder and stored at 5 °C
until further use. 100 g of kernel powder was extracted
with petroleum ether (60–80 °C) to remove lipids. It
was then filtered and the residue was extracted with
95% ethanol by soxhalation. Ethanol was evaporated
in a rotary evaporator at 40–50 °C under reduced pressure. The yield of kernel was—3.2 g/100 g.
2.4. Animals
Male albino Wistar rats, weighing about 150–180 g
obtained from Tamil Nadu Veterinary and Animal Sciences University, Chennai, India were used for the present investigations. The animals were maintained on
standard rat feed supplied by Hindustan Lever Ltd.,
Bangalore, India. The experiments were conducted
according to the ethical norms approved by Ministry
of Social Justices and Empowerment, Government of
India and Institutional Animal Ethics Committee
Guidelines.
2.5. Experimental induction of diabetes
The animals were fasted overnight and diabetes was
induced by a single intraperitoenal injection of a
freshly prepared solution of streptozotocin (STZ)
(55 mg/kg body weight) in 0.1 M cold citrate buffer
(pH 4.5) (Rakieten et al., 1963). The animals were
allowed to drink 5% glucose solution overnight to
overcome the drug- induced hypoglycemia. Control
rats were injected with citrate buffer alone. The animals
were considered as diabetic, if their blood glucose values were above 250 mg/dl on the third day after STZ
injection. The treatment was started on the fourth
day after STZ injection and this was considered as
first day of treatment. The treatment was continued
for 30 days.
2.6. Experimental design
2.2. Plant material
Fresh, mature Eugenia jambolana fruits were collected from a tree in Kolli Hills, Tamil Nadu, India.
The plant was identified at the Herbarium of Botany,
CAS in Botany, University of Madras. An exemplar
voucher specimen (no. 1283) was deposited in the
department herbarium.
2.3. Preparation of plant extract
The fruits of jambolana were washed well and pulp
was removed. The seeds were washed with distilled
The rats were divided into four groups comprising of
six animals in each group as follows:
Group I. Control rats receiving 0.1 M citrate buffer
(pH 4.5).
Group II. Diabetic control rats.
Group III. Diabetic rats treated with EJs-kernel
extract (100 mg/kg b.w/day) in aqueous solution
orally for 30 days.
Group IV. Diabetic rats treated with glibenclamide
(600 lg/kg b.w/day) in aqueous solution orally for
30 days (Pari and Uma Maheswari, 2000).
K. Ravi et al. / Food and Chemical Toxicology 43 (2005) 1433–1439
At the end of the experimental period, the rats were
anaesthetized and killed by cervical dislocation. Blood
was collected with anti-coagulant and the serum was
used for the estimation of total cholesterol, triglycerides,
free fatty acids and phospholipids. The liver and kidney
were excised, rinsed in ice-cold saline.
Total lipid was extracted from the liver and kidney
tissues according to the method of Folch et al. (1957).
Total cholesterol was estimated by the method of Parekh and Jung (1970). Triglycerides were estimated by
the method of Rice (1970). Free fatty acid content was
estimated by the method of Hron and Menahan
(1981). Phospholipid was estimated by the method of
Bartlette (1959) by digestion with perchloric acid and
the phosphorus liberated was estimated by the method
of Fiske and Subbarow (1925). Serum lipoproteins were
fractionated by dual precipitation techniques (Burstein
and Scholnick, 1972). Analysis of fatty acid composition
in lipid extract was performed in gas chromatography
according to the method of Morrison and Smith (1964).
2.7. Statistical analysis
All the grouped data were statistically evaluated with
SPSS/10 software. Hypothesis testing methods included
one way analysis of variance (ANOVA) followed by
least significant difference (LSD) test. P-values of less
1435
than 0.05 were considered to indicate statistical significance. All the results were expressed as mean ± S.D.
for six animals in each group.
3. Results
Table 1 depicts the levels of serum total cholesterol,
triglycerides, free fatty acids and phospholipids in control and experimental groups of rats. A significant
(p < 0.05) increase in the levels of plasma lipids was
observed in diabetic rats (Group II), which were restored, to near normal by the treatment of EJs-kernel
(Group III) or glibenclamide (Group IV).
Fig. 1 shows the levels of serum HDL, LDL and
VLDL-cholesterol in control and experimental groups
of rats. The levels of LDL and VLDL-cholesterol were
significantly (p < 0.05) increased, whereas the HDLcholesterol was markedly (p < 0.05) decreased in rats
induced with STZ (Group II) when compared with control rats (Group I). The diabetic rats treated with EJskernel (Group III) or glibenclamide (Group IV) for 30
days, LDL and VLDL-cholesterol were significantly
reduced, whereas the HDL-cholesterol was significantly
increased.
Comparison of liver lipid contents in control and
experimental groups of rats are shown in Table 2. A
Table 1
Levels of serum total cholesterol, phospholipids, triglycerides, free fatty acids in control and experimental groups of rats
Groups
Total cholesterol (mg/dl)
Phospholipids (mg/dl)
Triglycerides (mg/dl)
Free fatty acids (mg/dl)
Control
Diabetic control
Diabetic + EJs-kernel
Diabetic + glibenclamide
82.8 ± 5.6
198.7 ± 9.7*a
85.3 ± 4.5*b
90.1 ± 3.7*c
81.2 ± 7.9
153.5 ± 13.4*a
88.5 ± 6.4*b
91.7 ± 8.5*c
75.3 ± 6.3
140.8 ± 12.4*a
89.2 ± 7.2*b
93.4 ± 8.2*c
80.3 ± 7.3
158.2 ± 11.2*a
87.3 ± 6.9*b
86.8 ± 8.4*c
Values are given as mean ± SD for groups of six animals each. Values are statistically significant at *p < 0.05.
a
Diabetic rats were compared with control rats.
b
EJs-kernel treated diabetic rats were compared with diabetic rats.
c
Glibenclamide treated diabetic rats were compared with diabetic rats.
Fig. 1. Levels of HDL, LDL, VLDL-cholesterol in the serum of control and experimental groups of rats. Values are given as mean ± SD for groups
of six animals each. Values are statistically significant at *p < 0.05; aDiabetic rats were compared with control rats; bEJs-kernel treated diabetic rats
were compared with diabetic rats; cGlibenclamide treated diabetic rats were compared with diabetic rats.
1436
K. Ravi et al. / Food and Chemical Toxicology 43 (2005) 1433–1439
Table 2
Levels of liver total cholesterol, phospholipids, triglycerides, free fatty acids in control and experimental groups of rats
Groups
Total cholesterol (mg/g)
Phospholipids (mg/g)
Triglycerides (mg/g)
Free fatty acids (mg/g)
Control
Diabetic control
Diabetic + EJs-kernel
Diabetic + glibenclamide
6.8 ± 0.91
13.7 ± 1.4*a
7.4 ± 0.82*b
7.9 ± 0.45*c
20.5 ± 1.8
31.2 ± 2.9*a
22.6 ± 1.8*b
24.3 ± 2.1*c
3.5 ± 0.52
6.2 ± 0.65*a
3.9 ± 0.53*b
4.2 ± 0.78*c
6.5 ± 0.81
11.0 ± 1.4*a
7.1 ± 0.75*b
6.9 ± 1.01*c
Values are given as mean ± SD for groups of six animals each. Values are statistically significant at *p < 0.05.
a
Diabetic rats were compared with control rats.
b
EJs-kernel treated diabetic rats were compared with diabetic rats.
c
Glibenclamide treated diabetic rats were compared with diabetic rats.
compared to control rats (Group I). Administration of
EJs-kernel (Group III) or glibenclamide (Group IV) to
diabetic rats tends to bring the levels of kidney lipid contents to near normal level.
Figs. 2 and 3 show the alterations in the fatty acid
composition of total lipids in liver and kidney of control
and experimental groups of rats. There was a significant
elevation in palmitic acid (16:0), stearic acid (18:0) and
oleic acid (18:1) observed in tissues of diabetic rats
(Group II). In contrast, there was a significant decrease
in linolenic acid (18:3) and arachidonic acid (20:4)
observed in tissues of diabetic rats. These altered fatty
significant increase (p < 0.05) in the levels of total cholesterol, phospholipids, triglycerides and free fatty acids
were observed in diabetic (Group II) rats when compared with control rats (Group I). Administration of
EJs-kernel (Group III) or glibenclamide (Group IV) to
diabetic rats tends to bring the levels of hepatic lipids
to near normal level.
The levels of total cholesterol, triglycerides, phospholipids and free fatty acids in kidney of control and experimental groups of rats are shown in Table 3. A
significant increase (p < 0.05) in the kidney lipid contents was observed in diabetic rats (Group II) when
Table 3
Levels of kidney total cholesterol, phospholipids, triglycerides, free fatty acids in control and experimental groups of rats
Groups
Total cholesterol (mg/g)
Phospholipids (mg/g)
Triglycerides (mg/g)
Free fatty acids (mg/g)
Control
Diabetic control
Diabetic + EJs-kernel
Diabetic + glibenclamide
5.2 ± 0.43
8.1 ± 0.73*a
6.1 ± 0.75*b
6.6 ± 1.3*c
13.3 ± 1.8
19.5 ± 1.7*a
14.1 ± 1.3*b
13.5 ± 0.98*c
2.8 ± 0.35
4.7 ± 0.53*a
2.9 ± 0.27*b
3.1 ± 0.53*c
5.9 ± 0.72
10.4 ± 2.1*a
6.2 ± 0.68*b
7.1 ± 1.2*c
Percentage of fatty acid /100mg
tissue
Values are given as mean ± SD for groups of six animals each. Values are statistically significant at *p < 0.05.
a
Diabetic rats were compared with control rats.
b
EJs-kernel treated diabetic rats were compared with diabetic rats.
c
Glibenclamide treated diabetic rats were compared with diabetic rats.
35
30
25
*a
*b
Control
Diabetic control
*c
*a
Diabetic+EJs-kernel
Diabetic+Glibenclamide
20
*b
15
*c
*a
*b
*c
*a
*b
*c
10
*b
5
*c
*a
0
Palmitic acid(16:0)
Stearic acid(18:0)
Oleic acid(18:1)
Linolenic acid(18:3)
Arachidonic
acid(20:4)
Fatty acid composition
Fig. 2. Fatty acid composition in liver of total lipids in control and experimental groups of rats. Values are given as mean ± SD for groups of six
animals each. Values are statistically significant at *p < 0.05; aDiabetic rats were compared with control rats; bEJs-kernel treated diabetic rats were
compared with diabetic rats; cGlibenclamide treated diabetic rats were compared with diabetic rats.
Percentage of fatty acid / 100 mg tissue
K. Ravi et al. / Food and Chemical Toxicology 43 (2005) 1433–1439
1437
40
35
30
*a
Control
*b
Diabetic control
*c
Diabetic+EJs-kernel
*a
25
Diabetic+Glibenclamide
20
*b
*c
*c
*a
15
*b
*b
10
*c
*a
*a
5
*b
*c
0
Palmitic acid(16:0)
Stearic acid(18:0)
Oleic acid(18:1)
Linolenic acid(18:3) Arachidonic acid(20:4)
Fatty acid composition
Fig. 3. Fatty acid composition in kidney of total lipids in control and experimental groups of rats. Values are given as mean ± SD for groups of six
animals each. Values are statistically significant at *p < 0.05; aDiabetic rats were compared with control rats; bEJs-kernel treated diabetic rats were
compared with diabetic rats; cGlibenclamide treated diabetic rats were compared with diabetic rats.
acid composition were restored to near normal by EJskernel (Group III) or glibenclamide (Group IV) treated
diabetic rats.
4. Discussion
In the present study, a marked increase in the lipid
content of serum, liver and kidney were found in STZinduced diabetic rats. The increase in the serum lipids
on the diabetic subject is mainly due to the increased
mobilization of free fatty acids from peripheral deposits,
since insulin inhibits the hormone sensitive lipase (AlShamaony et al., 1994). On the other hand, glucagon,
catecholamines and other hormones enhance lipolysis.
The marked hyperlipemia that characterizes the diabetic
state may therefore be regarded as a consequence of the
uninhibited actions of lipolytic hormones on the fat depots (Goodman and Gilman, 1985).
Diabetes is also known to be associated with an increase in the synthesis of cholesterol, which may be
due to the increased activity of HMG CoA reductase.
A number of observations indicate that plasma HDL
cholesterol is low in untreated insulin-deficient diabetics
(Goodman et al., 1982; Glasgow et al., 1981), which was
associated with a decline in HDL-turnover rate. Further
the HDL-cholesterol levels correlate with lipoprotein lipase (LPL) levels in IDDM patients (Nikkila et al.,
1977). It is shown that VLDL and chylomicrons contribute surface apoproteins and lipids to HDL during
hydrolysis by LPL. Increased LDL-cholesterol may
arise from glycosylation of the lysyl residues of apoprotein B as well as from decreasing affinity for the LDL
receptor and hence, decreased metabolism (Golay
et al., 1986). The ability of LDL-cholesterol to form lipid peroxides was found specifically responsible for the
atherogenesis in diabetic patients (Kondo et al., 2001).
Oral administration of EJs-kernel extract normalized
these effects, possibly by controlling the hydrolysis of
certain lipoproteins and their selective uptake and
metabolism by different tissues.
Hypertriglyceridemia is a common finding in patients
with diabetes mellitus and is responsible for vascular
complications (Kudchodkar et al., 1988). Bruan and
Severson (1992) has reported that deficiency of lipoprotein lipase (LPL) activity may contribute significantly to
the elevation of triglycerides in diabetes. Lopes-Virella
et al. (1983) reported that treatment of diabetes with
insulin served to lower plasma triglyceride levels by
returning lipoprotein lipase levels to normal. The restoration of triglycerides levels following EJs-kernel treatment is supported by above reports. Thus, it may be
concluded that EJs-kernel extract may have a stimulatory effect on insulin.
The elevated serum phospholipid levels are a consequence of elevated lipoproteins. Jain et al. (2000) suggested that the levels of glycemic control and elevated
levels of HDL cholesterol and decreased levels of triglycerides in the blood are significantly correlated with the
phospholipid levels. The serum cholesterol/phospholipid
ratio is one of the important markers of development of
atherosclerosis. The restoration of phospholipids by
EJs-kernel extract may be controlled mobilization of
serum triglycerides; controlling the tissue metabolism
and improving the level of insulin secretion and action
presumably mediate cholesterol and phospholipids.
An increase in the total lipids of liver and kidney in
STZ-induced diabetic rats may indicate an increased
synthesis of lipids and storage capacity, which may have
caused an increase in serum triglycerides and phospholipids. Rajalingam et al. (1993) reported that variety of
derangements in metabolic and regulatory mechanisms
due to insulin deficiency is responsible for the observed
accumulation of lipids. The increase in total lipids
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K. Ravi et al. / Food and Chemical Toxicology 43 (2005) 1433–1439
observed in diabetic rats was due to the impairment of
insulin secretion, which resulted in enhanced mobilization of lipid from the adipose tissue to the plasma.
In alloxan-induced diabetic rats the tissue cholesterol,
phospholipids and triglycerides were significantly increased whereas insulin treatment has an appreciable effect on the lipids of these tissues. Changes in the fatty
acids during diabetes are closely associated with the
activity of Na+/K+-ATPase in the kidney. Accumulation
of fatty acids results in higher levels of their metabolites
such as acyl-carnitine and long chain acyl-CoA and this
may be a cause for diabetic nephropathy (Parving and
Hommel, 1989). Thus, the diabetic complications associated with renal tissue may be partly due to abnormalities
in lipid metabolism.
Fatty acids undergo changes during the process of injury, repair and cell growth (Cameron and Cotter,
1997). Seigneur et al. (1994) have reported that there is
a significant alteration in the fatty acid composition of
serum and variety of tissues in experimental diabetes.
In the present study, the increased concentrations of palmitic acid, oleic acid and stearic acid in rats induced
with STZ were correlated with other reports that may
have an preferential synthesis of stearic acid and other
total saturated fatty acids in type 1 diabetic patients (Tilvis and Miettinen, 1985). Seigneur et al. (1994) have reported that the increased concentration of oleic acid was
observed in the membranes of both type 1 and type 2
diabetic patients. This alteration has drawn special
attention because of the possible causal relationship
with platelet aggregation and prostaglandin production
in diabetic patients.
Linolenic acid and arachidonic acid are rich in polyunsaturated fatty acids, which is the major target for
reactive oxygen species (ROS) mediated damage. D6
desaturase a key enzyme of fatty acid desaturation,
has been shown to be suppressed in diabetes (Eck
et al., 1979). In the present study, the decreased levels
of linolenic acid and arachidonic acid were observed in
diabetic rats, which might be due to the decreased activity of D6 desaturase enzyme. The reduced availability of
essential fatty acid intermediates in diabetes is further
exacerbated by increased destruction due to elevated
levels of reactive oxygen species (Cameron and Cotter,
1997). Shin et al. (1995) have reported that, insulin therapy restores fatty acid composition in tissues of rats
induced with streptozotocin. EJS-kernel treatment in
rats induced with STZ restored the fatty acid composition to near normal level and this may be due to the
decreased toxicity of reactive oxygen species by its
anti-oxidant property. Thus the anti-oxidant effect of
EJs-kernel has to be proved in STZ-induced diabetic
rats (Ravi et al., 2004b).
In conclusion, the alterations in lipid profiles during
experimental diabetes were restored to near normal
levels by EJs-kernel treatment. Several authors reported
that, saponins, flavonoids, phenolic compounds, glycosides and triterpenoids have hypolipidemic and
hypocholesterolemic effects (Rupasinghe et al., 2003;
Leontowicz et al., 2002). Hence it may be concluded that
the hypolipidemic effect produced by the extract may be
due to the presence of flavonoids, saponins, glycosides,
and triterpenoids. The hypolipidemic effect mediated
by EJs-kernel may be anticipated to have biological significance and provide a scientific rationale for the use of
EJs-kernel as an anti-diabetic plant.
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