Indo American Journal of Pharmaceutical Research, 2014

ALTERATION OF ANTI-OXIDATIVE STATUS

PREVENTION BY HERBAL FORMULATION

INDUCED

BY

ISSN NO: 2231-6876

ALLOXAN

AND

Shiv kumar Jayant, Chandan Singh Sagar, Sapneh Sharma

SOS in Biochemistry, Jiwaji University, Gwalior.
ARTICLE INFO
Article history
Received 13/12/2014
Available online
28/12/2014

Keywords
Type I Diabetes,
Oxidative Stress,
Antioxidant Enzymes,
Natural Herbal Drugs.

ABSTRACT
Diabetes mellitus is a group of metabolic disorder characterised by hyperglycemia and
glycosuria with disturbance in carbohydrate, lipid and protein metabolism resulting from
relative deficiency of insulin secretion or action. Reactive oxygen species are common by
product of many oxidative biochemicals and physiological processes. Antioxidant enzymes
such as SOD, catalase, GPx and GR are the first line of defence against oxidative stress.
Decrease the activities of these enzymes change redox status of the cell. Present studies
clearly showed a dose and exposure frequency dependent decrease in the activities of SOD,
CAT, GPx and GR in brain and liver of diabetic rat tissues. Diabetic rats were treated with
oral administration of Aegle marmelos and Azadirachta Indica plant extracts increase the
activity of antioxidant enzymes and decrease of blood glucose levels as compared to normal
control rats. These herbal plant extracts showed hypoglycemic and hypolipidimic effect on
diabetic rats.

Copy right 2014 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical
Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

www.iajpr.com

Page

Please cite this article in press as Shiv Kumar Jayant et al. Alteration of anti-oxidative status induced by alloxan and prevention
by herbal formulation. Indo American Journal of Pharm Research.2014:4(12).

5838

Corresponding author
Shiv Kumar Jayant
Research Scholar, School of Studies in Biochemistry,
Jiwaji University, Gwalior 474 011, India
09977710704
shivjayant7aug@gmail.com

Vol 4, Issue 12, 2014.

Shiv Kumar Jayant et al.

ISSN NO: 2231-6876

INTRODUCTION
Diabetes mellitus is a group of metabolic diseases in which a person has high blood sugar, either because the body does not
produce enough insulin. Its half life of about 3-5 minutes and is cleared through liver where approximately 50% of insulin delivered is
removed from the circulation [2]. Diabetes mellitus is characterized by increased production of Reactive Oxygen Species (ROS),
cause reduction in antioxidant defence and altered cellular redox status. Free radicals are formed excessively in diabetes by glucose
oxidation, non enzymatic glycation of protein and the subsequent oxidative degradation of glycated protein. The high levels of free
radicals are high and the simultaneous refuse of antioxidant defence mechanism can lead to damage of cellular organelles and
enzymes, increase lipid peroxidation and serum glucose [2]. Alloxan induced diabetes cause the pancreatic beta cell of islets to swell
and degenerate. Diabetes mellitus has increase vascular permeability and lipid peroxidation end product in diabetic rats [3].
There are about 800 plants which have been reported to show anti-diabetic potential [4]. A wide collection of plant-derived
active principles representing numerous bioactive compounds has established their role for possible use in the treatment of diabetes
[4].
The aim of the present study was designed to investigate the potential of an aqueous extract of Aegle marmelos and Azadirachta
Indica in controlling blood glucose and antioxidant enzymes level in alloxan induced diabetic rats, compared to normal control and
diabetic untreated rats.
MATERIALS AND METHOD
Plant material:
The fresh leaves of Aegle marmelos (bael) and Azadirachta Indica (Neem) were collected from botanical garden in school of
studies in Botany, jiwaji University, Gwalior.
Experimental animals:
Male albino rats of Wistar strain (weight 120 20g) was used in the proposed study. Animals were obtained from the animal
facilities of Defence Research and Development Establishment, Gwalior, India, and was maintained under controlled conditions of
temperature (25 20C), relative humidity of (50 15%), and normal photoperiod (light-dark cycle of 12 hrs) in the animal room of
our department on standard pellet diet and tap water ad libitum. Animals was housed throughout the experiment in polypropylene
cages (with each cage housing six animals) containing paddy husk as bedding and allowed to acclimatize to the environment of animal
room for 7 days before the start of experiment.

Page

Figure 1: Effect of oral administration of Aegle marmelos and Azadirachta indica for 14 days on the blood glucose levels of in
the diabetic rat tissues.

5839

Experimental design:
The rats were divided into four groups comprising of six animals in each group as follows1. Normal control rats
2. Diabetic rats
3. Diabetic + plant extract of Aegle marmelos (bael) treated rats
4. Diabetic + plant extract of Azadirachta Indica (Neem) treated rats

www.iajpr.com

Vol 4, Issue 12, 2014.

Shiv Kumar Jayant et al.

ISSN NO: 2231-6876

Figure 2: Effect of oral administration of Aegle marmelos and Azadirachta indica for 14 days on the levels of SOD, CAT, GPx
and GR in the diabetic rat tissues.
Table 1: Effect of oral administration of Aegle marmelos and Azadirachta indica extract on glucose level in alloxan induced
diabetic rats.
Level of glucose in 14 days experimental animals
S No. Groups
0 days
1.
Control
1143
2.
Diabetic
427.333.79ab***
3.
Diabetic + Aegle marmelos
424.334.73ac***bc#
4.
Diabetic + Azadirachta Indica 4194ad***bd#
Glucose concentration is expressed as mg/dl.
Results are expressed as mean S.D. of four set of observation.
Significance is based on #p>0.05, * p<0.05, **p<0.01, ***p<0.001

7 days
118.674.16
431.335.69ab***
337.334.04ac***bc***
3484ad***bd*

14 days
122.674.16
439.334.04ab***
239.3313.06ac***bc***
249.675.13ad***bd***

Control
Diabetic
Diabetic+ Aegle marmelos
Brain 0.530.05 0.270.03ab**
0.380.04ac*bc*
Liver 0.850.06 0.480.06ab**
0.650.07ac*bc*
SOD
Brain 4.730.55 2.030.12ab**
3.50.26ac*bc***
Liver 8.90.4
4.870.35ab*** 7.230.31ac**bc***
CAT
Brain 12.10.36 4.90.66ab***
9.970.15ac***bc***
Liver 20.57.35 10.80.56ab*** 17.60.5ac**bc***
GR
Brain 15.20.4
7.270.55ab*** 110.36ac***bc***
Liver 30.10.4
14.10.4ab***
26.371.1ac**bc***
Antioxidant enzymes concentration is expressed as n mole/gm protein.
Results are expressed as mean S.D. of four set of observation.
Significance is based on #p>0.05, * p<0.05, **p<0.01, ***p<0.001

www.iajpr.com

Diabetic+ Azadirachta Indica

0.40.04ad*bd*
0.690.08ad*bd*
3.970.15ad#bd***
7.60.46ad*bd**
10.50.3ad***bd***
17.970.4ad**bd***
11.430.21ad***bd***
26.970.45ad***bd***

Page

Experiment
GPx

5840

Table 2: Effect of oral administration of Aegle marmelos and Azadirachta Indica for two weeks on the levels of antioxidant
enzymes in diabetic rat tissues.

Vol 4, Issue 12, 2014.

Shiv Kumar Jayant et al.

ISSN NO: 2231-6876

Induction of diabetes:
Type I diabetes was induced by giving single intravenous injection of alloxan monohydrate 65 mg/kg body weight, dissolved
in 0.9% solution of sodium chloride [5]. The animals were checked for blood glucose level 48 h after alloxan injection and blood
sugar level above 200 mg/dl was used for the experiment.
Aegle marmelos (bael) leaves were found from botanical garden of jiwaji university gwalior, cleaned and Aqueous extract of
Aegle marmelos was prepared and 250 mg/kg body weight was given orally to the rats of group 3 with the help of cannula, daily for
two weeks.
Azadirachta Indica (Neem) leavess were purchased from the local herbal market, cleaned, and Aqueous extract of
Azadirachta Indica leaves were prepared and 100 mg/kg body weight was given orally to the rats of group 3 and 6 with the help of
cannula, daily for two weeks.
Rats were humanly killed 24 h after the last treatment by cervical dislocation; different tissues were excised off, washed with
0.9% NaCl and used for different estimations. Animals were handled, ethically treated and humanly killed as per the rules and
instructions of Ethical Committee of Animal Care (Reg. No. 501/01/a/CPCSEA, Ref No. IAEC/JU/2012/01) of Jiwaji University,
Gwalior, India, in accordance with the Indian National law on animal care and use.
Estimations:
Superoxide dismutase (SOD, E.C.No. 1.15.1.1):
SOD activity was assayed by estimating the inhibition of auto oxidation of epinephrine [6]. The 10% tissue homogenates
were prepared in 0.9% NaCl, centrifuged at 15,000 g for 15 min and the corresponding supernatants were used for enzyme assay.
Reaction mixture containing 0.5 ml sodium carbonate buffer (0.3 M, pH 10.2), 0.5 ml EDTA (0.6 mM), 0.5 ml homogenate and 1.0 ml
water, was incubated at room temperature for 10 min. Reaction was initiated by addition of 0.5 ml epinephrine (1.8 mM) and
absorbance change per min was recorded for 5 min at 480 nm. Specific activity is expressed as % inhibition of auto oxidation of
epinephrine by the enzyme min-1 mg-1 protein.
Catalase (CAT, E.C.No. 1.11.1.6):
CAT activity was estimated by the method of Aebi et al. [7]. The 10% tissue homogenates were prepared in 1.15% KCl,
centrifuged at 5000g for 10 min and the corresponding supernatants were used for CAT estimation. Reaction mixture containing 0.8
ml phosphate buffer (K2HPO4/ NaH2PO4, 50 mM, pH 7.0), 0.1 ml homogenate and 0.1 ml triton X-100 (0.02%) was incubated at
room temperature for 10 min. Reaction was initiated by addition of 2.0 ml H 2O2 (0.03 M prepared in potassium phosphate buffer, pH
7.0) and absorbance change per min was recorded for 5 min at 240 nm. Specific activity is expressed as mole H 2O2 decomposed min1
mg-1 protein.
Glutathione peroxidase (GPx, E.C.No. 1.11.1.9):
GPx activity was estimated as described by Paglia and Valentine [8]. The 10% homogenates were prepared in 1.15% KCl,
centrifuged at 5000 g for 10 min and the corresponding supernatants were used for GPx estimation. Reaction mixture containing 0.3
ml sodium phosphate buffer (0.1 M, pH 7.4), 0.1 ml GSH (0.15 M), 0.05 ml sodium azide (2.25 M), 0.05 ml homogenate, 0.1 ml
NADPH (0.84 mM) and 0.05 ml glutathione reductase (2 U/ml) was incubated at room temperature for 10 min. Reaction was initiated
by addition of 0.05 ml H2O2 (0.001 mM) and absorbance change per min was recorded for 5 min at 340 nm. Specific activity is
expressed as nmole NADPH oxidized min-1 mg-1 protein.
Glutathione reductase (GR, E.C.No. 1.6.4.2):
Glutathione reductase activity in tissue homogenate was determined by measuring the rate of conversion of NADPH to
NADP+ by the enzyme [9]. A 10 % (w/v) homogenate was prepared in 1.15% KCl and centrifuged at 5,000 g for 10 min. The
resulting supernatant obtained was used for enzyme assay. A typical reaction mixture containing 0.7 ml phosphate buffer (0.1 M, pH
7.5), 0.1 ml GSSG (0.2mM) and 0.1 ml suitably diluted homogenate was incubated at room temperature for 10 min. Reaction was
initiated by the addition 0.1 ml NADPH (0.12 mM) and absorbance change was recorded for 5 min at 340 nm. Specific activity was
expressed as mole NADPH oxidized min-1mg-1 protein taking molar coefficient of NADPH as 6300 M-1cm-1.
Protein in the tissues samples was estimated by the method of Lowry et al. [10] using bovine serum albumin as standard.

Page

5841

Statistical analysis:
Results are expressed as mean S.D. of four different sets of observation taken on different days. All the statistical analyses
were performed using one-way analysis of variance (ANOVA) with post hoc Dunnetts multiple comparison test applied across
treatment groups for each tissue. Significance level was based on p < 0.05.

www.iajpr.com

Vol 4, Issue 12, 2014.

Shiv Kumar Jayant et al.

ISSN NO: 2231-6876

RESULT AND DISCUSSION

ROS cause lipid peroxidation and damage protein by chemical modifications through cross-linking and fragmentation.
Therefore oxidative stress has been considered to contribute to the pathological processes of diabetic complications. The present
results revealed significant increase in the level of blood glucose and decrease antioxidant enzyme level in diabetic rat tissues.
Treatment of diabetic rats with Aegle marmelos and Azadirachta Indica extract reduced blood glucose, and increase antioxidant
enzyme potential as compared to control rat tissues. This elevation is more notable in long duration of diabetic and is due to increased
peroxidation of lipid in plasma membranes [11].
We observed a significant decrease in reduced Glutathione peroxidise (GPx), Superoxide dismutase (SOD) and Catalases as
compared to the control subjects. The decrease in the GSH level in tissue may be related to decrease activity of GPx because this
enzyme uses for its activity reduced glutathione (GSH) as substrate [12, 13]. The auto oxidation of glucose results in the formation of
hydrogen peroxide (H2O2), which inactivate SOD [14]. The activity of SOD decreases in erythrocytes also due to ageing in diabetic
subjects this could be due to increase in the glycation of SOD [15].
The blood glucose level of all the rats was tested by taking the blood from the tail vein and using electronic glucometer. The
anti diabetic effects of the extracts on the fasting blood sugar levels of diabetes [Table 1] and administration of alloxan (65 mg/kg, i.v)
led to 3-4 fold elevation of fasting blood glucose levels, which was maintained for period of 2 weeks. It was observed that oral
administration of aquous extract of Aegle marmelos and Azadirachta Indica extract significantly decreased the blood glucose levels in
diabetic rats. The present study results showed that oral administration of Aegle marmelos and Azadirachta Indica extract daily for 14
days, to the diabetic rats caused 20.5%, 16.% decrease on 7th day and 44%, 40% decrease in the blood glucose level on day 14 th of the
start of treatmet [Table 2]. The results clearly showed the hypoglycemic potential of Aegle marmelos and Azadirachta Indica plants
extract.
The levels of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR)
were significantly reduced in alloxan induced rats. These adverse changes were reversed to near normal values in extract of Aegle
marmelos and Azadirachta Indica leaf treated. It is well known that SOD, CAT, GPx and GR play an important role as protective
enzymes against free radical formation in tissues. In the present study indicates the reduction in the activity of these antioxidant
enzymes in the liver and the brain, when compared with control. The hepatic activities of SOD, CAT, GPx and GR were decrease by
45.3%, 47.4%, 43.5%, 53.2% and 57%, 51.2%, 49%, 52.2% decrease of brain in alloxan induced rats compared with control rats
(Table 2).
Oral administration of Aegle marmelos and Azadirachta Indica plant extracts for 14 days showed antidiabetic potential
against alloxan diabetes induced alterations in the activities of SOD, CAT, GPx and GR. The activities of SOD, CAT, GPx and GR
were increased by 47.8%, 62.9%, 35.4%, 87% and 56%, 66.4%, 43.7%, 91% in the liver and 72.4%, 68.9%, 28.3%, 51.3% and 95%,
77%, 48.1%, 57.2% in the brain of diabetic rats given Aegle marmelos and Azadirachta Indica plant extracts treatment for 2 weeks
when compared with diabetic control rats (Table 2).

www.iajpr.com

Page

REFERENCE
1. Marques RG, Fontaine MJ, Rogers J, C-peptide: much more than a byproduct of insulin biosynthesis. Pancreas 2004;
29(3):231-8.
2. Amran AA, Zakaria Z, Othman F and Morat O, Effect of Garcinia Atroviridis on Oxidative Stress and Atherosclerotic
Changes in Experimental Guinea Pigs. Am. J. Pharmacol. Toxicol 2010; 5: 65-70.
3. Ceriello A, Acute hyperglycaemia and oxidative stress generation. Diabet Med 1997; 14: S45-S49.
4. Patil R, Patil R, Ahirwar B and Ahirwar D, Current status ofIndian medicinal plants with antidiabetic potential: a
review,Asian Pacific Journal of Tropical Biomedicine, 2011; vol. 1, no. 2, pp.S291S298.
5. Resmi CR, venukumar MR, Latha MS, Antioxidant activity of Albizzia lebbeck (linn) benth in alloxan diabetic rats. Indian J.
Physiol. Pharmacol 2006; 50, 297302.
6. Mishra HP and Fridovich I, The role of superoxide anion in the autooxidation of epinephrine and a simple assay for
superoxide dismutase. J. Biol. Chem 1972; 247, 3170- 3175.
7. Aebi H, Catalase in vitro. Method Enzymol 1984; 105:121-126.
8. Pagalia DE and Valentine WN, Studies on the quantitative and qualitative characterization of erythrocyte glutathione
peroxidise. J Lab Clin Med 1967; 95, 351-358.
9. Tayarani I, Cioez I, Clement M and Bourre JL, Antioxidant enzymes and related trace element in aging brain capillaries and
choroids plexes. J Neurochem 1989; 53, 817-824.
10. Lowry OH, Rosenbrough NJ, Farr AL and Randall R, Protein estimation with folin phenol regent. J. Biol. Chem 1951; 193,
365-370.
11. Kesavulu MM, Rao BK, Giri R, Vijaya J and Subramanyam G, Lipid peroxidation and antioxidant enzyme status in Type 2
diabetics with coronary heart disease. Diabetes Res. Clin. Pract 2001; 53: 33-39.

5842

CONCLUSION
Diabetes mellitus decrease antioxidant enzymes activity and increase serum glucose levels. Oral administration of aqueous
plant extracts of Aegle marmelos and Azadirachta Indica leaves homogenate increase antioxidant enzymes activity and decrease
serum glucose level in diabetic rats. Further human studies are necessary to found the role of these herbal drugs in controlling type I
diabetes and its complications.

Vol 4, Issue 12, 2014.

Shiv Kumar Jayant et al.

ISSN NO: 2231-6876

12. Jos J, Rybak M, Patin PH, Robert JJ, Boitard C, Thevenin R, Etudedes enzymes antioxidants dans le diabete insulinoindependent de I enfant etde I adolescent. Diabete Metab 1990; 16: 493-503.
13. Domingues C, Ruiz E, Gussinye M, Carrascosa A, oxidative stress at onset and in early stages of type I diabetes in children
and adolescents. Diabetes Care 1998; 21: 1736-42.
14. Fajans S, Diabetes mellitus, definition, classification, tests. Endocrinology Degroat L, 3rded, Saunders Co, USA 1995; pp.
1411-22.
15. Arai K, Maguchi S, Fujii S, Ishibashi H, Oikawa K, Taniguchi N, Glycation inactivation of human Cu-Zn superoxide
dismutase. J Biol Chem 1987; 262: 16969-72.

Page

5843

54878478451141238

www.iajpr.com