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Singapore Med J 2002 Vol 43(12) : 617-621
Original Article
Effect of Cassia Auriculata Flowers on
Blood Sugar Levels, Serum and Tissue
Lipids in Streptozotocin Diabetic Rats
L Pari, M Latha
Department of
Biochemistry
Faculty of Science
Annamalai University
Annamalai Nagar
Tamil Nadu-608 002
India
L Pari, MSc, MPhil,
PhD
Reader
M Latha, MSc, MPhil,
PhD
Scholar
Correspondence to:
Dr L Pari
Tel: +914144 38343
Fax: +914144 22265
Email: Paribala@
sancharnet.in
ABSTRACT
Aim of the study: The main aim was to
demonstrate the effects of Cassia auriculata
flowers on blood glucose and lipid levels in
experimental diabetic rats.
Methodology: Aqueous extract of Cassia
auriculata flowers was administered orally and
different doses of the extract on blood glucose,
haemoglobin, glycosylated haemoglobin, serum
and tissue lipids, hexokinase and glucose-
6-phosphatase in streptozotocin-induced diabetic
rats were studied. Glibenclamide was used as
standard reference drug.
Results: Cassia auriculata flower extract (CFEt),
at doses of 0.15, 0.30 and 0.45 g/kg body weight
for 30 days, suppressed the elevated blood
glucose and lipid levels in diabetic rats. Cassia
auriculata at 0.45 g/kg was found to be comparable
to glibenclamide.
Conclusion: Our findings indicate that the Cassia
auriculata flowers possess antihyperlipidaemic
effect in addition to antidiabetic activity.
Keywords: Blood glucose, Cassia auriculata,
Carbohydrate enzymes, Insulin, Lipids
Singapore Med J 2002 Vol 43(12):617-621
INTRODUCTION
Diabetes mellitus is characterised by hyperglycaemia
together with biochemical alterations of glucose
and lipid metabolism(1). Liver is an insulin dependent
tissue, which plays a pivotal role in glucose and lipid
homeostasis and is severely affected during
diabetes(2). Liver participates in the uptake, oxidation
and metabolic conversion of free fatty acids, synthesis
of cholesterol, phospholipids and triglycerides.
During diabetes a profound alteration in the
concentration and composition of lipid occurs(3).
Decreased glycolysis, impeded glycogenesis and
increased gluconeogenesis are some of the changes
of glucose metabolism in the diabetic liver(4).
Many traditional plant treatments for diabetes
mellitus are used throughout the world(5). Few of
the traditional plant treatments for diabetes have
received scientific scrutiny, and the World Health
Organisation has recommended that this area
warrants attention(6).
This paper describes the study of Cassia auriculata
L. (Cesalpinaceae, common name: Tanner’s Cassia)
a common plant in Asia, has been widely used in
traditional medicine as a cure for rheumatism,
conjunctivitis and diabetes(7). In addition, Cassia
auriculata has been widely used in Ayurvedic
medicine as ‘Avarai Panchaga Choornam’ and the main
constituent of Kalpa herbal tea, has come under
extensive study in the light of its antidiabetic effects.
We have recently reported the antiperoxidative effect
of Cassia auriculata flowers in streptozotocin diabetic
rats(8). This study was thus initiated with the aim of
evaluating the effects of an aqueous extract of
Cassia auriculata flowers on the blood glucose level,
serum and tissue lipids in streptozotocin diabetic rats.
MATERIALS AND METHODS
Animals
All the experiments were carried out with male Wistar
rats aged seven to eight weeks (180-200 g), obtained
from the Central Animal House, Rajah Muthiah
Medical College, Annamalai University, India. The
animals were housed in polypropylene cages and
provided with water and standard pellet diet
(Karnataka Agro Food Corporation Limited,
Bangalore, India) ad libitum. The animals used in the
present study were approved by the ethical committee,
Annamalai University.
Chemicals
Streptozotocin was obtained from Himedia Laboratory
Limited, Mumbai, India. All other reagents used were
of analytical grade.
Plant Material
Cassia auriculata flowers were collected freshly from
Neyveli, Cuddalore District, Tamil Nadu, India. The
618 : 2002 Vol 43(12) Singapore Med J
plant was identified and authenticated at the
Herbarium of Botany Directorate in Annamalai
University. A voucher specimen (No.231) was deposited
in the Botany Department of Annamalai University.
Preparation of plant extract
Five hundred g of Cassia auriculata flowers were
extracted with 1,500 ml of water by the method of
continuous hot extraction at 60ºC for six hours and
evaporated. The residual extract was dissolved in
water and used in the study(9).
Induction of experimental diabetes
A freshly prepared solution of streptozotocin
(45 mg/kg i.p) in 0.1 M citrate buffer, pH 4.5 was
injected intraperitoneally in a volume of 1 ml/kg.
After 48 hours of streptozotocin administration,
rats with moderate diabetes having glycosuria and
hyperglycaemia (i.e. with a blood glucose of 200-
300 mg/dl) were taken for the experiment(10).
Experimental procedure
In the experiment, a total of 36 rats (30 diabetic
surviving rats, six normal rats) were used. The rats
were divided into six groups of six rats each.
Group 1: Normal untreated rats.
Group 2: Diabetic control rats given 1 ml of aqueous
solution daily using an intragastric tube
for 30 days.
Group 3: Diabetic rats given CFEt (0.15 g/kg body
weight) in 1ml of aqueous solution daily
using an intragastric tube for 30 days.
Group 4: Diabetic rats given CFEt (0.30 g/kg body
weight) in 1 ml of aqueous solution daily
using an intragastric tube for 30 days.
Group 5: Diabetic rats given CFEt (0.45 g/kg body
weight) in 1 ml of aqueous solution daily
using an intragastric tube for 30 days.
Group 6: Diabetic rats given glibenclamide (600 µg/
kg body weight)(11) in 1 ml of aqueous solution
daily using an intragastric tube for 30 days.
At the end of 30 days, the animals were deprived
of food overnight and sacrificed by decapitation.
Blood was collected in two different tubes (i.e.,)
one with anticoagulant- potassium oxalate and
sodium fluoride for plasma and another without
Table I. Blood glucose, plasma insulin, total haemoglobin, glycosylated haemoglobin, changes in body weight and urine sugar of
normal and experimental animals.
Groups Body weight (g) Fasting Blood Plasma Haemoglobin Glycosylated Urine
Initial Final Glucose insulin (g/dl) haemoglobin sugarA
(mg/dl) (µU/ml) (mg/gHb)
Normal 196 ± 10.40 208 ± 9.80 97.50 ± 8.04a16.03 ± 1.04a12.85 ± 0.72a0.22 ± 0.01aNil
Diabetic control 201 ± 15.70 151 ± 13.66••• 232.00 ± 15.40b4.35 ± 0.95b5.60 ± 0.45b0.81 ± 0.07b+ + +
Diabetic + Cassia auriculata (0.15g/kg) 193 ± 17.70 198 ± 15.33*** 216.66 ± 20.80b4.90 ± 0.41b6.91 ± 0.61c0.68 ± 0.03c+ +
Diabetic + Cassia auriculata (0.30g/kg) 198 ± 18.30 208 ± 10.32*** 158.60 ± 14.20c7.05 ± 0.64c9.54 ± 0.93d0.48 ± 0.04d+
Diabetic + Cassia auriculata (0.45g/kg) 202 ± 19.68 214 ± 12.72*** 113.3 ± 10.30ad 14.16 ± 0.67d11.5 ± 0.91e0.37 ± 0.04eNIL
Diabetic + Glibenclamide (600 µg/kg) 195 ± 11.80 206 ± 13.43*** 124.6 ± 10.32d12.70 ± 0.65e10.36 ±1.01d0.47 ± 0.04dTRACE
Values are given as mean ± S.D for six rats in each group.
Values not sharing a common superscript letter differ significantly at p<0.05 (DMRT).
Duncan procedure, Range for the level 2.89, 3.03, 3.13, 3.20, 3.25.
Diabetic control was compared with normal, ••• p<0.001.
Experimental groups were compared with diabetic control, *** p<0.001.
A - Indicates 0.25% sugar and (+ + +) indicates more than 1% sugar.
Table II. Changes in levels of cholesterol, free fatty acids, triglycerides and phospholipids in serum of normal and experimental
animals.
Groups Cholesterol Free fatty acids Triglycerides Phospholipids
(mg/100 ml) (mg/100 ml) (mg/100 ml) (mg/100 ml)
Normal 74.00 ± 1.49a69.43 ± 4.06a44.53 ± 3.36a80.25 ± 1.57a
Diabetic control 98.66 ± 4.03b83.86 ± 6.67b62.83 ± 1.50b98.75 ± 4.28b
Diabetic + Cassia auriculata (0.45 g/kg) 83.46 ± 2.18c75.06 ± 1.55c53.93 ± 2.70c85.50 ± 2.86c
Diabetic + Glibenclamide (600 µg/kg) 90.26 ± 1.37d78.51 ± 0.87d58.46 ± 1.70d90.00 ± 2.12d
Values are given as mean ± S.D for six rats in each group.
Values not sharing a common superscript letter differ significantly at p<0.05 (DMRT).
Duncan procedure, Range for the level 2.95, 3.09, 3.20.
Singapore Med J 2002 Vol 43(12) : 619
anticoagulant for serum separation. Plasma and
serum were separated by centrifugation.
Liver was immediately dissected out, washed in
ice cold saline, patted dry and weighed.
Analytical Procedure
Fasting blood glucose was estimated by O-toluidine
method (Sasaki et al)(12). Plasma insulin level was
assayed by Enzyme Linked Immunosorbent Assay
(ELISA) kit, using human insulin as standard.
Haemoglobin was estimated by the method of
Drabkin and Austin(13) and glycosylated haemoglobin
by the method of Sudhakar Nayak and Pattabiraman(14).
Lipids were extracted from serum and tissues by
the method of Folch et al(15). Total cholesterol and
triglycerides were estimated by the method of
Zlatkis et al(16) and Foster and Dunn(17) respectively.
Free fatty acids and phospholipids were analysed by
the method of Falholt et al(18) and Zilversmit et al(19).
Hexokinase and glucose-6-phosphatase were
assayed by the method of Brandstrup et al(20) and
Koida and Oda(21).
Statistical analysis
All values were expressed as the mean obtained from
a number of experiment (n). Data from all the tables of
normal animals, diabetic control animals, reference drug
treated and CFEt treated animals were compared
by ANOVA followed by Duncan’s Multiple Range
Test (DMRT)(22).
RESULTS
Blood glucose and Plasma insulin
Table I shows the levels of blood glucose, plasma
insulin, total haemoglobin, glycosylated haemoglobin,
changes in body weight and urine sugar of normal and
experimental rats. There was a significant elevation in
blood glucose and glycosylated haemoglobin levels,
while the plasma insulin and total haemoglobin levels
decreased significantly in streptozotocin diabetic
rats when compared with normal rats. Administration
of CFEt and glibenclamide tends to bring the
parameters significantly towards the normal. The
effect of CFEt at a dose of 0.45 g/kg body weight was
more highly significant than 0.15 and 0.30 g/kg body
weight and therefore the dose was used for further
biochemical studies.
In diabetic rats, the urine sugar was (+++) but in
the case of CFEt treated rats at a dose of 0.15 and
0.30 g/kg body weight showed decreased urine sugar
(++) and (+) respectively. CFEt at a dose of 0.45 g/kg
body weight, showed urine sugar as seen in normal
rats. These effects were compared with glibenclamide.
Serum and tissue lipids
The effect of CFEt on serum and tissue lipids of
Table III. Changes in levels of cholesterol, free fatty acids, triglycerides and phospholipids in liver of normal and experimental
animals.
Groups Cholesterol Free fatty acids Triglycerides Phospholipids
(mg/100 g wet tissue) (mg/100 g wet tissue) (mg/100 g wet tissue) (g/100 g wet tissue)
Normal 329.04 ± 2.88a607.70 ± 30.68a347.88 ± 13.04a1.66 ± 0.11a
Diabetic control 512.70 ± 5.88b915.22 ± 50.27b621.35 ± 8.40b2.54 ± 0.08b
Diabetic + Cassia auriculata (0.45 g/kg) 420.14 ± 4.40c774.09 ± 46.86c442.98 ± 13.05c2.02 ± 0.05c
Diabetic + Glibenclamide (600 µg/kg) 441.98 ± 5.36d806.67 ± 25.30c530.19 ± 11.70d2.29 ± 0.10d
Values are given as mean ± S.D for six rats in each group.
Values not sharing a common superscript letter differ significantly at p<0.05 (DMRT).
Duncan procedure, Range for the level 2.95, 3.09, 3.20.
Table IV. Changes in activities of hexokinase and glucose-6-phosphatase in liver of normal and experimental animals.
Groups Hexokinase Glucose- 6-phosphatase
(unitsA/g protein) (unitsB/mg protein)
Normal 146.66 ± 6.09a0.168 ± 0.013a
Diabetic control 107.48 ± 5.74b0.242 ± 0.023b
Diabetic + Cassia auriculata (0.45 g/kg) 128.70 ± 9.44c0.186 ± 0.011ac
Diabetic + Glibenclamide (600 µg/kg) 123.20 ± 5.40c0.200 ± 0.008c
Values are given as mean ± S.D for six rats in each group.
Values not sharing a common superscript letter differ significantly at p<0.05 (DMRT).
Duncan procedure, Range for the level 2.95, 3.09, 3.20.
A - µ moles of glucose phosphosylated/min.
B - µ moles of Pi liberated/min.
620 : 2002 Vol 43(12) Singapore Med J
normal and experimental rats is summarised in
Table II and III respectively. A marked increase in
the frequency of cholesterol, free fatty acids,
triglycerides and phospholipids were observed
in diabetic control rats. Treatment with CFEt
significantly reduced the lipid levels.
Hepatic hexokinase and glucose-6-phosphatase
The activities of carbohydrate enzymes are represented
in Table IV. Activity of hexokinase in liver decreased
markedly while the glucose-6-phosphatase activity
increased significantly in diabetic control rats.
Treatment with CFEt in diabetic rats increased the
hexokinase activity and decreased the glucose-
6-phosphatase activity.
DISCUSSION
Streptozotocin is well known for its selective
pancreatic islet β-cell cytotoxicity and has been
extensively used to induce diabetes mellitus in
animals. It interferes with cellular metabolic oxidative
mechanisms(23). Intraperitoneal administration of
streptozotocin (45 mg/kg) effectively induced
diabetes in normal rats as reflected by glycosuria,
hyperglycaemia, polyphagia, polydipsia and body
weight loss when compared with normal rats(24). In
our present study we have observed that an aqueous
extract of Cassia auriculata flower can reverse these
effects. The possible mechanism by which CFEt
brings about its antihyperglycemic action may be
by potentiation of pancreatic secretion of insulin
from β-cell of islets or due to enhanced transport
of blood glucose to peripheral tissue. This was
clearly evidenced by the increased level of insulin
in diabetic rats treated with CFEt. In this context
a number of other plants have also been reported
to have antihyperglycemic and insulin-release
stimulatory effect(25,26).
We have observed a decrease in total haemoglobin
during diabetes and this may be due to the formation
of glycosylated haemoglobin. Increase in the level of
haemoglobin in animals given CFEt may be due to
decreased level of blood glucose and glycosylated
haemoglobin.
CFEt administration to streptozotocin dosed
animals reversed the weight loss. The ability of CFEt
to recover body weight loss seems to be due to its
antihyperglycemic effect.
Excess of fatty acids in serum produced by
the streptozotocin-induced diabetes promotes
conversion of excess fatty acids into phospholipids
and cholesterol in liver. These two substances along
with excess triglycerides formed at the same time in
liver may be discharged into blood in the form of
lipoproteins(27). The abnormal high concentration of
serum lipids in the diabetic subject is due, mainly to
increase in the mobilisation of free fatty acids from
the peripheral fat depots, since insulin inhibits
the hormone sensitive lipase. Hypercholesterolemia
and hypertriglyceridemia have been reported to
occur in streptozotocin diabetic rats(28,29) and
significant increase observed in our experiment was
in accordance to these studies. The marked
hyperlipidaemia that characterise the diabetic state
may therefore, be regarded as a consequence of the
uninhibited actions of lipolytic hormones on the
fat depots(30).
The antihyperlipidaemic effect of CFEt may be
due to the down regulation of NADPH and NADH,
a cofactor in the fat metabolism. Higher activity
of glucose-6-phosphatase provides H+ which binds
with NADP+ in the form of NADPH and is helpful
in the synthesis of fats from carbohydrates. When
glycolysis slows down because of cellular activity,
the pentose phosphate pathway still remain active
in liver to breakdown glucose that continuously
provides NADPH which converts acetyl radicals
into long fatty acid chains. CFEt may be capable
of oxidising NADPH. Enhanced hexokinase activity
in CFEt treated rats suggests greater uptake of
glucose from blood by the liver cells.
Activities of enzymes suggest that enhanced
lipid metabolism during diabetes is shifted towards
carbohydrate metabolism and it enhances the
utilisation of glucose at the peripheral sites. One of
the possible actions of CFEt may be due to its
inhibition of endogenous synthesis of lipids.
Metabolic aberration in streptozotocin diabetic
rats suggest a high turnover of triglycerides and
phospholipids. CFEt may antagonise the metabolic
aberration and thereby restore the normal metabolism
by tilting the balance from high lipids to high
carbohydrate turnover. Alteration of fatty acid
composition by increased lipid levels contribute to
lowering the resistance of tissues and higher rate
of oxidative stress. Decreased activity of glucose-
6-phosphatase through pentose phosphate shunt
results in high reduced glutathione to oxidised
glutathione ratio (GSH/GSSG)(27), which is coupled
with conversion of NADPH to NADP. CFEt may
produce high NADP+ which results in down
regulation of lipogenesis and lower risk of the tissues
for oxidative stress and high resistance for diabetes.
It can be concluded from the data that CFEt
significantly reduces the levels of serum and tissue
lipids, which are actively raised in streptozotocin
diabetes rats. CFEt has beneficial effect on
plasma insulin and hexokinase activity. Moreover
Singapore Med J 2002 Vol 43(12) : 621
its antihyperlipidaemic effect could represent
a protective mechanism against the development
of atherosclerosis.
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