Journal of Basic and Clinical Pharmacy
Antioxidant, Antihyperlipidaemic and Antidiabetic Activity of
Eugenia Floccosa Bedd Leaves in Alloxan Induced Diabetic Rats
Mary Jelastin Kala S
, Tresina P.S.
and Mohan V.R.
Department of Chemistry, St. Xavier’s College, Palayamkottai, Tamil Nadu, India.
Ethnopharmacology Unit, Research Department of Botany, V.O.Chidambaram College, Tuticorin-628008, Tamil Nadu, India.
iabetes mellitus is a major and growing public health problem through-
people in 2000, which is expected to increase to 320 million by 2025 .
Besides hyperglycemic, several other factors including dislipidemia or hyperlipi-
demia are involved in the development of micro and macro vascular complications
of diabetes which are the major causes of morbidity and death . Although nu-
merous oral hypoglycemic drugs exist alongside insulin, still there is no promising
therapy to cure diabetes . India has a rich emporium of various potent herbs and
herbal components for treating various diseases including diabetes. In recent years,
numerous traditional medicinal plants were tested for their antidiabetic potential
in the experimental animals [4,5,6].
Eugenia ﬂ occosa Bedd is one of the medicinally important plants belongs to
Myrtaceae family. Th
e leaf paste of E. ﬂ occosa is given to treat rheumatic pain by
Kanikkar tribe of Agasthiarmalai Biosphere Reserve, Tamil Nadu. Th
extract of E. ﬂ occosa has been reported for its anti-tumour activity . Th
investigation is an attempt to study the antidiabetic, antihyperlidaemic and antioxi-
dant activities of ethanol extract of E. ﬂ occosa leaf in alloxan induced diabetic rats.
MATERIALS AND METHODS
e leaves of Eugenia ﬂ occosa Bedd were freshly collected from the well grown
sphere Reserve, Western Ghats, Tamilnadu. Th
e plant were identiﬁ ed and authen-
ticated in Botanical Survey of India, Southern Circle, Coimbatore, Tamilnadu,
India. A voucher specimen was deposited in Ethnopharmacology Unit, Research
Department of Botany, V.O.Chidambaram College, Tuticorin, Tamilnadu.
e E. ﬂ occosa leaves were shade dried at room temperature and the dried leaves
packed in a Soxhlet apparatus and extracted with ethanol Th
e extract were subjected
to qualitative test for the identiﬁ cation of various phytochemical constituents as per
the standard procedures [8,9,10].Th
e ethanol extracts were concentrated in a rotary
e concentrated ethanol extract were used for antidiabetic studies.
Normal healthy male Wistar albino rats (180- 240g) were housed under standard
environmental conditions at temperature (25±2º C) and light and dark (12: 12 h).
Rats were fed with standard pellet diet (Goldmohur brand, MS Hindustan lever
Ltd., Mumbai, India) and water ad libitum.
Acute Toxicity Study
Acute oral toxicity study was performed as per OECD – 423 guidelines (acute
toxic class method), albino rats (n=6) of either sex selected by random sampling
were used for acute toxicity study . Th
e animals were kept fasting for overnight
and provided only with water, after which the extracts were administered orally at
5mg/kg body weight by gastric intubations and observed for 14 days. If mortality
was observed in two out of three animals, then the dose administered was assigned
as toxic dose. If mortality was observed in one animal, then the same dose was
repeated again to conﬁ rm the toxic dose. If mortality was not observed, the proce-
dure was repeated for higher doses such as 50,100, and 2000 mg/kg body weight.
Induction of Experimental Diabetes
Rats were induced diabetes by the administration of simple intraperitioneal dose
of alloxan monohydrate (150 mg/kg) . Two days after alloxan injection, rats
*Corresponding Author E-mail: email@example.com
The ethanol extract of Eugenia floccosa Bedd (Family: Myrtaceae) leaf was investigated for its antioxidant, antihyperli-
pidaemic and antidiabetic effect in Wistar Albino rats. Diabetes was induced in Albino rats by administration of alloxan
monohydrate (150mg/kg, i.p). The ethanol extracts of E. floccosa at a dose of 150 and 300mg/kg of body weight were
administered at single dose per day to diabetes induced rats for a period of 14 days. The effect of ethanol extract of
E. floccosa leaf extract on blood glucose, plasma insulin, creatinine, glycosylated haemoglobin, urea serum lipid pro-
file [total cholesterol (TR), triglycerides (TG), low density lipoprotein – cholesterol (LDL-C), very low density lipoprotein
– cholesterol (VLDL-C), high density lipoprotein – cholesterol (HDL-C) and phospholipid (PL)] serum protein, albumin,
globulin, serum enzymes [serum glutamate pyruvate transaminases (SGPT) and serum glutamate oxaloacetate transam-
inases (SGOT), and alkaline phosphatase (ALP)], lipoprotein peroxidation (LPO) antioxidant enzymes (catalase (CAT),
superoxide dismutase (SOD), reduced glutathione (GSH) and glutathione peroxidase (GPx) were measured in the dia-
betic rats. The ethanol extract of Eugenia floccosa leaf elicited significant reductions of blood glucose (P<0.05), lipid
parameters except HDL-C, serum enzymes and significantly increased HDL-C and antioxidant enzymes. The extracts
also caused significant increase in plasma insulin (P<0.05) in the diabetic rats. From the above results, it is concluded
that ethanol extract of Eugenia floccosa possesses significant antidiabetic, antihyperlipidaemic and antioxidant effects
in alloxan induced diabetic rats.
received on 16-12-2011
accepted on 02-08-2012
available online 15-02-2012
Antioxidant, antihyperlipidaemic, antidiabetic,
E. floccosa, alloxan.
Mary Jelastin Kala S et al.
Vol-003 Issue-001 February
of 200-260 mg/100 ml were taken for the study. All animals were allowed free ac-
cess to water and pellet diet and maintained at room temperature in plastic cages.
In the present investigation, a total of 30 rats (24 diabetic surviving rats and 6
normal rats) were taken and divided into ﬁ ve groups of 6 rats each.
Group I: Normal untreated rats
Group II: Diabetic control rats
Group III: Diabetic rats given ethanol extract of
E. ﬂ occosa leaf (150mg/kg
Group IV: Diabetic rats given ethanol extract of
E. ﬂ occosa leaf (300mg/kg
Group V: Diabetic rats given standard drug glibenclamide (600mg/kg body
e animals were sacriﬁ ced at the end of experimental period of 14 days by decapi-
utes. Serum glucose was measured by the O-toluidine method . Insulin level
was assayed by Enzyme Linked Immunosorbant Assay (ELISA) kit . Urea
estimation was carried out by the method of Varley ; serum creatinine was
estimated by the method of Owen et al . Glycosylated haemoglobin (HBA
Chandrasekharan . Serum total cholesterol (TC) , total triglycerides (TG)
, low density lipoprotein cholesterol (LDL-C), very low density lipoprotein
cholesterol (VLDL- C) , high density lipoprotein cholesterol (HDL-C) 
and phospholipids  were analyzed. Serum protein  and serum albumins
were determined by quantitative colorimetrically method by using bromocresol
e total protein minus the albumin gives the globulin, serum glutamate
pyruvate transaminase (SGPT) and serum glutamate oxaloacetate transaminase
(SGOT) were measured spectrophotometrically by utilizing the method of Reit-
man and Frankel . Serum alkaline phosphatase (ALP) was measured by the
method of King and Armstrong . Catalase (CAT) , superoxide dismutase
(SOD) , lipid peroxidation (LPO) , reduced glutathione (GSH)  and
glutathione peroxidase (GPx)  were analyzed in the normal, diabetic induced
and drug treated rats.
e data were analyzed using student’s t-test statistical methods. For the statistical
e phytochemical screening of ethanol extract of E. ﬂ occosa leaf revealed the pres-
sugar, glycoside, xanthoprotein and ﬁ xed oil. Acute toxicity study revealed the
non-toxic nature of the ethanol extract of E. ﬂ occosa leaf. Table 1 shows the levels
of blood glucose, plasma insulin, urea, creatinine and glycosylated haemoglobin
of normal, diabetic rats and drug treated rats. Th
e alloxan induced diabetic rats
elicited signiﬁ cant rise in blood glucose from 69.50 to 201.00mg/dl (p<0.05) and
a signiﬁ cant decrease in plasma insulin level from 24.50 to 5.40 (p<0.01). On the
contrary, diabetic rats treated with ethanol extract of E. ﬂ occosa leaf exhibited de-
crease in blood glucose and increase in plasma insulin signiﬁ cantly at a dose of
150 mg/kg and 300 mg/kg body weight. It was observed that ethanol extract of
E. ﬂ occosa reversed these eﬀ ects in diabetic animals. Th
e possible mechanism by
which ethanol extract brings about its hyperglycemic action may be by induction
of pancreatic insulin secretion from β cells of islets of langerhans or due to en-
hanced transport of blood glucose to peripheral tissue . Earlier many plants
have been studied for their hypoglycemic and insulin release stimulatory eﬀ ects
A signiﬁ cant elevation in serum constituents, urea and creatinine were ob-
served in alloxan induced diabetic rats (Group II), when compared to control
e ethanol extracts of E. ﬂ occosa leaves were administered orally (150 mg/
kg body weight- Group III and 300mg/kg body weight- Group IV) to rats for 14
days, reversed the urea and creatinine level to near normal. Th
e administration of
glibenclamide (Group V) also decreased the levels of urea and creatinine to some
extent. Alloxan is taken as indications of an abnormal glomerular fraction where
a simple injection of cisplation at a dose of 5mg/kg body weight in rabbits caused
a marked reduction in the glomerular ﬁ ltration rates, which was accompanied by
an increase in the creatinine level, indicating the induction of acute renal failure.
It is conﬁ rmed that there is a signiﬁ cant increase in serum creatinine in albino rats
14 days after alloxan administration. Th
e present results show that, the treatment
with ethanol extract of E. ﬂ occosa leaf was eﬀ ective in preventing alloxan induced
increase in serum creatinine level when compared with the control. Alloxan in-
duced diabetic rats showed signiﬁ cant increase (p<0.05) glycosylated haemoglobin
C) level compared with normal rats. Th
treated rats showed a signiﬁ cant decrease (p<0.05) in the content of glycosylated
haemoglobin. Glycosylated haemoglobin determinations are self monitoring of
blood glucose therefore play an important complementary roles for the manage-
ment of diabetes mellitus .
e levels of serum protein, albumin and globulin of control, alloxan induced
duction in serum protein, albumin and globulin were observed in alloxan induced
Eugenia Floccosa Bedd Leaves
Journal of Basic and Clinical Pharmacy
diabetic rats (Group II) when compared to control (Group I) and glibenclamide
treated rats (Group V). On administration of ethanol extract of E. ﬂ occosa to the
diabetic rats, the levels of protein, albumin and globulin were found to be restored
in normal. Th
ese results were in accordance with the eﬀ ects of Wattakaka volubilis
, Senna auriculata  and Pterocarpus marsupium  in diabetic rats.
Table 2 summarized the eﬀ ect of alloxan on the activity of the hepatic marker
enzymes in serum. In the present study, the levels of SGPT and SGOT in alloxan
induced diabetic rats were elevated. It may be due to leaking out of enzymes from
the tissues and migrating into the circulation by the adverse eﬀ ect of alloxan .
Aspartate amino transaminases and Alanine transaminase were used as markers
to assess the extent of liver damage in streptozotocin induced diabetic rats .
In this study, the ethanol extract of E. ﬂ occosa regulated the activity of SGPT and
SGOT in liver of rats intoxicated with alloxan. Th
e eﬀ ect of glibenclamide on the
recovery of hepatic enzyme activity in serum was very similar to that of the earlier
e restorations of SGPT and SGOT to their respective normal levels af-
strengthen the antidiabegenic eﬀ ect of this extract. Moreover, SGPT and SGOT
levels also act as indicators of liver function and restoration of normal levels of
these parameters indicate normal functioning of liver. Since the alloxan can also
aﬀ ect the liver by free radical mechanism.
In addition to the assessment of SGPT and SGOT levels during diabetes the
measurement of enzymatic activities of phosphatases such as acid phosphatase
(ACP) and alkaline phosphatase (ALP) is of clinical and toxicological importance
as changes in their activities are indicative of tissue damage by toxicants. In the
present study, serum ALP increased in alloxan induced diabetic rats (Table 2).
Elevated level of this enzyme in diabetes may be due to extensive damage to liver in
the experimental animal by alloxan. Treatment with ethanol extract of E. ﬂ occosa
in alloxan induced diabetic rats produces a decline in ALP level.
e levels of serum lipid proﬁ le, total cholesterol (TC), triglycerides (TG),
were presented in Table 3. Alloxan induced rats showed signiﬁ cant increase in se-
rum lipid proﬁ les except HDL-C when compared with normal rats. Th
mide (Group V) and ethanol extract of E. ﬂ occosa (Group III and IV) treated rats
Effect of ethanol extract of Eugenia floccosa leaves on the serum protein, albumin, globulin, SGOT, SGPT and ALP
level of normal, diabetic induced and drug treated adult albino rats.
diabetic induced rats. Similarly HDL-C level decreased in alloxan induced diabetic
rats when compared to normal rats. On administration of ethanol extract of E. ﬂ oc-
cosa and glibenclamide to the diabetic rats, HDL-C level was found to be restored
to normal. Th
e level of serum lipid proﬁ les are usually raised in diabetic rats in the
present study and such elevation represents risk factor for coronary heart diseases
. Lowering of the serum lipid level through dietary or drug therapy seems to
be associated with a decrease in the risk of vascular disease . During diabetes,
enhanced activity of the enzyme, increased lipolysis and releases more fatty ac-
ids into the circulation . Th
e increased fatty acid concentration also increases
the β-oxidation of fatty acids, producing more acetyl Co-A and cholesterol dur-
ing diabetes. In normal condition, insulin increases receptor-mediator removal of
LDL-cholesterol and decreased activity of insulin, during diabetes causes hyperc-
holesterolemia. Hypercholesterolemia and hypertriglycedemia have been reported
to occur in diabetic rats . Th
e increased concentration of free fatty acid may
be due to lipid break-down and this may cause increased generation of NADPH-
dependent microsomal lipid peroxidation. Phospholipids were increased in al-
loxan induced diabetic rats. Phospholipids are present in cell membrane and make
up vast majority of the surface lipoprotein forming a lipid bilayer that acts as an
interface with both polar plasma environment and non-polar lipoprotein of li-
poprotein core . Increased phospholipids levels in tissues were reported by
Venkateswaran et al. ; Pari and Satheesh,  in streptozotocin diabetic rats.
Administration of ethanolic extract of E. ﬂ occosa leaf and glibenclamide decreased
the levels of phospholipids.
e results (Table 4) showed increased lipid peroxidation (LPO) of alloxan
pid peroxidation in liver, kidney and brain of diabetic rats [47,48]. Th
is may be be-
cause the tissues contain relatively high concentration of early peroxidizable fatty
acids. In the present study, an increase in the levels of LPO was found and these
levels were signiﬁ cantly reduced after the supplementation of the ethanol extract of
E. ﬂ ocossa leaf and glibenclamide (Table 4). Th
is indicate that plant extract inhibit
oxidative damage due to the antiperoxidative eﬀ ect of ingredients present in etha-
nol extract of E. ﬂ ocossa. Th
is could be correlated with previous study reported that
Cassia auriculata ﬂ ower  Syzigium cuminii [49,50] Tinospora cardifolia  and
Scoparia dulcis  has antiperoxidative and antihyperlipidemic eﬀ ect of diabetic
animals. Apart from the regulation of carbohydrate metabolism, insulin also plays
an important role in the lipid metabolism. Insulin is a potent inhibitor of lipolysis,
since it inhibits the activity of hormone sensitive lipase in adipose tissue and sup-
presses the release of free fatty acids .
e levels of superoxide dismutase (SOD), catalase (CAT), glutathione
(p<0.05) reduced in alloxan induced rats. Th
ese adverse changes were reversed
to near normal values in ethanol extract of E. ﬂ occosa leaf treated. It is well known
that CAT, SOD and GPx play an important role as protective enzymes against
free radical formation in tissues . It has been shown that the antioxidants sta-
tus of tissues is an important factor in the development of diabetic complications
. Reduced glutathione plays an important role mainly in the detoxiﬁ cation and
metabolism as a cofactor or a substrate for some enzymes and in this way it is an
antioxidant agent protecting tissues from oxidative stress and thus measured as
a common marker of free radical damage. Enzymatic antioxidant such as SOD
and CAT are considered primary enzymes since they are involved in the direct
elimination of ROS  SOD is an important defense enzyme and scavengers O
and hence diminishes the toxic eﬀ ects due to this radical or other
free radicals derived from secondary reaction . CAT is a haemoprotein, which
catalyzes the reduction of hydrogen peroxides . Th
e antioxidant enzymes such
as SOD and CAT are known to be inhibited in diabetes mellitus as a result of
non- enzymatic glycosylation and oxidation . In the present study, the activi-
ties of SOD and CAT decreased in diabetic rats as reported earlier, which could be
due to inactivation caused by alloxan generated ROS . Th
e ethanol extract of
E. ﬂ occosa had reversed the activities of these enzymatic antioxidants, which might
be due to decreased oxidative stress as evidenced by decreased LPO.
In conclusion, the present study has shown that the ethanol extract of E. ﬂ occosa
leaf have antidiabetic, antihyperlipidaemic and antioxidant eﬀ ects. Th
e possible an-
tidiabetic activity of the extracts might be due to stimulation of residual pancreatic
insulin or by increasing peripheral utilization of glucose. Glycosides, ﬂ avonoids,
tannins, organic sulphur compounds, catechol and alkaloids are active ingredients
of hypoglycemic plant . Flavonoids are reported to regenerate the damaged
pancreatic beta cells . Phenols have found to be eﬀ ective antihyperglycemic
agents . In the present study, the phytochemical analysis of ethanol extract of
E. ﬂ occosa clearly pointed out the presence of above said active phytochemicals. It
denotes that the antidiabetic eﬀ ect of ethanol extract of E. ﬂ occosa may be due to
the presence of more than one antihyperglycemic principle and their synergistic
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4.91 ± 0.33a
0.45 ± 3.9a
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Each value is SEM of 6 animals. Comparison made between normal control to diabetic control and drug treated groups. * p < 0.05; **p<0.01 and comparison made between diabetic
control to drug treated groups a p<0.05. x-One unit of SOD is defined as the enzyme concentration which gives 50% inhibition of NBT reduction in one minute. y-One unit of CAT
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