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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-

out the world, with an estimated worldwide prevalence of 150 million 

people in 2000, which is expected to increase to 320 million by 2025 [1]. 

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 [2]. Although nu-

merous oral hypoglycemic drugs exist alongside insulin, still there is no promising 

therapy to cure diabetes [3]. 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

 e ethanol 

extract of E. fl occosa has been reported for its anti-tumour activity [7]. Th

 e current 

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.


Plant Material


  e leaves of Eugenia fl occosa Bedd were freshly collected from the well grown 

healthy plants inhabiting the natural forests of Kothiayar, Agasthiarmalai Bio-

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.


Preparation of plant extract for phytochemical screening 

and antidiabetic studies


 e E. fl occosa leaves were shade dried at room temperature and the dried leaves 

were powdered in a Wiley mill. Hundred grams of powdered E. fl occosa leaves was 

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 

evaporator. Th

  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 [11]. 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) [12].  Two days after alloxan injection, rats 

*Corresponding Author E-mail: vrmohan_2005@yahoo.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.

Journal of Basic and Clinical Pharmacy

Mary Jelastin Kala S et al.


Vol-003 Issue-001 February 



screened for diabetes having glycosuria and hypoglycemia with blood glucose level 

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.

Experimental Design

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 

body weight)

Group IV: Diabetic rats given ethanol extract of 


E. fl occosa leaf (300mg/kg 

body weight)

Group V: Diabetic rats given standard drug glibenclamide (600mg/kg body



Biochemical analysis


  e animals were sacrifi ced at the end of experimental period of 14 days by decapi-

tation. Blood was collected, sera separated by centrifugation at 3000g for 10 min-

utes. Serum glucose was measured by the O-toluidine method [13]. Insulin level 

was assayed by Enzyme Linked Immunosorbant Assay (ELISA) kit [14]. Urea 

estimation was carried out by the method of Varley [15]; serum creatinine was 

estimated by the method of Owen et al [16]. Glycosylated haemoglobin (HBA



estimation was carried out by a modifi ed colorimetric method of Karunanayake and 

Chandrasekharan [17]. Serum total cholesterol (TC) [18], total triglycerides (TG) 

[19], low density lipoprotein cholesterol (LDL-C), very low density lipoprotein 

cholesterol (VLDL- C) [20], high density lipoprotein cholesterol (HDL-C) [21] 

and phospholipids [22] were analyzed. Serum protein [23] and serum albumins 

were determined by quantitative colorimetrically method by using bromocresol 

green. Th

  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 [24]. Serum alkaline phosphatase (ALP) was measured by the 

method of King and Armstrong [25]. Catalase (CAT) [26], superoxide dismutase 

(SOD) [27], lipid peroxidation (LPO) [28], reduced glutathione (GSH) [29] and 

glutathione peroxidase (GPx) [30] 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 

tests a p values of less than 0.01 and 0.05 was taken as signifi cant.



  e phytochemical screening of ethanol extract of E. fl occosa leaf revealed the pres-

ence of alkaloid, catechin, coumarin, tannin, saponin, steroid, fl avonoid, phenol, 

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 [31]. 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 

rats. Th

  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

  e ethanol extract of E. fl occosa leaf 

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 [37].


  e levels of serum protein, albumin and globulin of control, alloxan induced 

diabetic rats and drug treated rats were presented in Table 2. A signifi cant re-

duction in serum protein, albumin and globulin were observed in alloxan induced 

Table 1: 

Effect of ethanol extracts of Eugenia floccosa leaves on the serum insulin, glucose, urea, creatinine and HBA



level of normal, diabetic induced and drug treated adult albino rats.


Insulin (MIu/ml)

Glucose (mg/dl)

Urea (mg/dl)

Creatinine (mg/dl)

Glycosylated Hb 


Group I






Group II






Group III






Group IV

17.80±1.3** a





Group V






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; aa p<0.01

 Antioxidant, Antihyperlipidaemic and Antidiabetic Activity of 

Eugenia Floccosa Bedd Leaves

Journal of Basic and Clinical Pharmacy

Vol-003 Issue-001 February 




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 

[6], Senna auriculata [38] and Pterocarpus marsupium [36] 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 [34]. 

Aspartate amino transaminases and Alanine transaminase were used as markers 

to assess the extent of liver damage in streptozotocin induced diabetic rats [39]. 

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 

study [40].


  e restorations of SGPT and SGOT to their respective normal levels af-

ter treatment with both glibenclamide and ethanol extract of E. fl occosa, further 

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), 

LDL-C, VLDL-C and HDL-C in control, diabetic induced and drug treated rats 

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

 e glibencla-

mide (Group V) and ethanol extract of E. fl occosa (Group III and IV) treated rats 

Table 2: 

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.


Protein (g/dl)

Albumin (g/dl)


SGPT (u/l)

SGOT (u/l)

ALP (u/l)

Group I







Group II







Group III







Group IV







Group V







Each value is SEM of 6 animals.  Comparison made between normal control to diabetic control and drug treated groups * p < 0.05

Table 3: 

Effect of ethanol extract of Eugenia floccosa leaves on the serum lipid profile of normal, diabetic induced and drug 

treated adult albino rats.


TC (mg/dl)


LDL-C (mg/dl)

VLDL (mg/dl)

HDL (mg/dl)

PL (mg/dl)

Group I







Group II







Group III







Group IV







Group V





      29.31 ±4.23


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; *** p<0.001 and comparison  made 

 between diabetic control to drug treated groups a p<0.05; aa p<0.01  

Journal of Basic and Clinical Pharmacy

Mary Jelastin Kala S et al.


Vol-003 Issue-001 February 



showed a signifi cant decrease in the content of lipid profi le when compared with 

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 

[41]. Lowering of the serum lipid level through dietary or drug therapy seems to 

be associated with a decrease in the risk of vascular disease [42]. During diabetes

enhanced activity of the enzyme, increased lipolysis and releases more fatty ac-

ids into the circulation [43]. 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 [41]. 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 [44]. Increased phospholipids levels in tissues were reported by 

Venkateswaran et al. [45]; Pari and Satheesh, [46] 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 

induced diabetic rats.  Earlier studies have reported that there was an increased li-

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 [35] Syzigium cuminii [49,50] Tinospora cardifolia [51] and 

Scoparia dulcis [52] 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 [53].


 e levels of superoxide dismutase (SOD), catalase (CAT), glutathione 

peroxidase (GPx) and reduced glutathione (GSH) (Table 4) were signifi cantly 

(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 [54]. It has been shown that the antioxidants sta-

tus of tissues is an important factor in the development of diabetic complications 

[55]. 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 [56] SOD is an important defense enzyme and scavengers O



anion from H




 and hence diminishes the toxic eff ects due to this radical or other 

free radicals derived from secondary reaction [57]. CAT is a haemoprotein, which 

catalyzes the reduction of hydrogen peroxides [58]. 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 [59]. 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 [60]. 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 [61]. Flavonoids are reported to regenerate the damaged

pancreatic beta cells [62]. Phenols have found to be eff ective antihyperglycemic

agents [63]. 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

eff ects.



  anks to Dr. Sampathraj, Honorary Advisor, Samsun Clinical Research Labora-

tory, Tirupur for their assistance in animal studies.


Sicree R, Shaw J, Zimmet P. Diabetes and impaired glucose tolerance In: 


Gan. D (Ed.) Diabetes Atlas. International Diabetes Federation, 3



International diabetes Federation. Brussels, Belgium 2006; pp15-103.

Table 4: 

Effect of ethanol extracts of Eugenia floccosa leaves on the LPO, SOD, CAT, GPX and GSH enzymes  in the plasma 

of normal, diabetic induced, and drug treated adult albino rats.


LPO (mmole/dl)

SOD (unit x/mg 


CAT (unit y/mg 


GPX(unit z/mg 


GSH (mg/dl)

Group I

0.83± 0.34

6.93± 0.74

0.49±  0.31

7.36± 0.81

91.54± 3.05

Group II

3.11± 0.98*

14.56± 0.54**

2.96± 0.88*

13.23± 0.92**

27.31± 2.86**

Group III

1.15 ±0.88

9.39± 0.65

0.57± 0.77a

8.59 ±1.65


Group IV

0.74 ±  0.41a 

4.91 ± 0.33a

0.45 ± 3.9a

5.81 ± 1.64a


Group V

0.89± 0.14a

7.99± 0.33

0.84±  0.14

8.03± 0.65a

87.4± 5.18a

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 

is defined as the μ mole of hydrogen peroxide consumed per minute. z- One unit of GPx is defined as the μ g of glutathione consumed per minute.

 Antioxidant, Antihyperlipidaemic and Antidiabetic Activity of 

Eugenia Floccosa Bedd Leaves

Journal of Basic and Clinical Pharmacy

Vol-003 Issue-001 February 




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