Vitamin C as antioxidat supplement in Type 2 Diabetes Mellitus along with Oral Hypogylemic agents...

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Sushanta et al. World Journal of Pharmacy and Pharmaceutical Sciences

ANTIOXIDANT EFFECT OF VITAMIN C ON TYPE 2 DIABETES

MELLITUS PATIENTS ALONG WITH TWO DIFFERENT ORAL

HYPOGLYCEMIC AGENTS FOR SMOOTH GLYCEMIC CONTROL

*Sushanta Kr. Das1, P.R. Anand Vijayakumar2, R. Senthil2, Jayesh Kumar Bhatt2, S.

Gupta2 1Department of Pharmacy Practice, RVS College of Pharmaceutical Sciences, 242B, Trichy

Road, Sulur, Coimbatore- 641402, Tamilnadu. India. 2Department of Pharmacy Practice & Pharmacology, JSS College of Pharmacy, Off Campus

JSS University, Post Box- 20, Rocklands, Ootacamund – 643001, Tamilnadu, India.

ABSTRACT

Background: Type 2 diabetes mellitus (T2DM) is a global epidemic

with major challenge to healthcare systems. T2DM patients have

increase the level of vitamin C’s primary oxidation product,

dehydroascorbate may be due to inadequate intracellular delivery of

vitamin C because it’s entry into cells depends on insulin. Chronic

vitamin C deficiency in T2DM patients can lead to a range of

complications. Supplementation with antioxidants as promising

complementary treatment can exert beneficial effects.

Objective: To investigate if antioxidant effect of vitamin C in

combination with two different oral hypoglycemic agents in T2DM

patients may produce smooth glycemic control or not.

Subject/Methods: 93 T2DM patients participated in this randomized

open label controlled study at out-patient department centre of

government headquarters hospital, Ootacamund, The Nilgiris, Tamil

Nadu, India. Enrolled patients were randomised in to six groups.

Control groups received glibanclamide, metformin and combination of these two drugs,

whereas in intervention groups viz; glibanclamide, metformin and combination of two drugs,

received vitamin C tablet (500mg/day) for a period of three months. Random blood sugar

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Article Received on 01 September 2012,

Revised on 14 September2 012, Accepted on 25 September2012

*Correspondence for

Author:

* Mr. Sushanta Kr. Das.

Assistant Professor

RVS College of

Pharmaceutical Sciences

242B, Trichy Road

Sulur, Coimbatore- 641402

Tamilnadu. India.

[email protected]

[email protected]

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were measured and simultaneously fasting blood samples were analysed for level of HbA1C,

ascorbic acid, TBAR, SOD, catalase, glutathione estimation by using specific assay methods.

Results: 93 T2DM patients participated in this randomized open label controlled study, were

randomized into two groups; control (n=47) and intervention (n=46). Control group received

glibenclamide/metformin/glibenclamide-metformin combination. Intervention group received

glibenclamide/metformin/glibenclamide-metformin combination along with vitamin C

(500mg/day) for three months. Blood samples were analyzed by using specific assay

methods. Mean serum levels of superoxide dismutase and ascorbic acid were significantly

increased with significant decrease of thiobarbituric acid reactive substances and HbA1C in

intervention group when compared to control group.

Conclusion: Our result shows that vitamin C supplementation with oral hypoglycemic agents

decreases oxidative stress & improves glycemic control, also has potential implications for

the prevention of further complications in T2DM patients.

Key words: T2DM, vitamin C, glibenclamide, metformin.

INTRODUCTION

Diabetes mellitus is a chronic and progressive disease state of worldwide significance. It can

affect children, young, and adults of all ages. Type 2 diabetes mellitus (T2DM) is a global

epidemic with an estimated worldwide prevalence is 285 million people in 2010, and set to be

increases to 438 million by 20301. The health, social, and economic burden is great

consequently, T2DM presents a major challenge to healthcare systems around the world 2, 3, 4.

Diabetes mellitus is primarily a metabolic disorder arising from a lack of or resistance to

insulin, which results in the impairment of uptake and storage of glucose and reduced glucose

utilization for energy purposes, that lead to the condition called hyperglycemia. Prolonged

exposure to elevated glucose induces repeated acute changes in intracellular metabolism as

well as cumulative long-term changes in the structure and function of macromolecules5.

The environment, stress factor and chain reaction - our own body metabolism generates free

radicals that are very reactive chemical species, can cause oxidative injury to the living

beings by attacking the macromolecules. There is a critical balance in the generation of

oxygen free radicals and antioxidant defense systems used by organisms to deactivate and

protect themselves against free radical toxicity under normal physiological conditions6, 7, 8.

Upset in the oxidant/antioxidant equilibrium lead to a condition called oxidative stress, which

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is known to be a component of molecular and cellular tissue damage mechanisms in a wide

range of human diseases including diabetes.

Patients with type 2 diabetes mellitus have 2 to 4 fold increase in the prevalence of

cardiovascular disease compared to non-diabetes mellitus patients9. It has been suggested that

free radical activity is increased in diabetes that contribute to increased oxidative stress

through, not only increased non-enzymatic glycosylation and auto oxidative glycosylation but

also metabolic stress resulting from changes in energy metabolism, alterations in sorbitol

pathway, changes in the level of inflammatory mediators. The status of antioxidant defense

systems and localized tissue damage results from hypoxia and ischemic reperfusion injury10.

People with type 2 diabetes have a relative vitamin C deficiency despite adequate dietary

intake, and increase the level of its primary oxidation product, dehydroascorbate that could be

due to inadequate intracellular delivery of vitamin C because vitamin C entry into cells

depends on insulin11. Chronic vitamin C deficiency in people with type 2 diabetes can lead to

a range of complications. Supplementation with antioxidants as a promising complementary

treatment can exert beneficial effects in diabetes. Although antioxidant has a beneficial effect

in type 2 diabetes mellitus, only limited studies have dealt with antioxidant supplement in

type 2 diabetes mellitus with Indian population. Hence, therefore, the present study was

designed to evaluate the role of antioxidant supplement (vitamin C) in patients with type 2

diabetes mellitus along with two different oral hypoglycemic drugs and their combination for

smooth glycemic control in south Indian population.

ETHICS

Written consent was obtained from all participants after debriefing session. The project

protocol was approved by Institutional Ethics Committee (IEC), JSS College of pharmacy,

Ootacamund, The Nilgiris, Tamil Nadu, India. All procedures followed were in accordance

with the ethical standards of the responsible committee on human experimentation.

MATERIALS & METHODS

A randomized open label controlled trial include 93 study patients divided into two groups

control (n=47) and intervention (n=46). Each group were subdivided in to three groups viz.

control-metformin CM (n=16), control-glibenclamide CG (n=16), control-combination CC

(n=15) and intervention-metformin IM (n=16), intervention-glibenclamide IG (n=15),

intervention-combination IC (n=15). The study was carried out at outpatient department

centre of Government headquarters hospital, Ootacamund, The Nilgiris, Tamil Nadu, India

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during the period of April 2005 to February 2006. Demographic data and general health

characteristics include height, body weight, body mass index, social habit, smoking status and

food habits were collected on standard structured data collection form by face to face

interview. Patients with type 2 diabetes mellitus aged between 35 to 65 years, duration of

disease ≥ 2 years, either sex with or without co-morbidities and on oral hypoglycemic agents

were included in the study, whereas exclusion criteria includes, type 1 diabetes, juvenile

diabetes, pregnant women and lactating mothers, voluntary withdrawal and significant

hepatic & renal dysfunction patients.

Patients were randomized by using computer assisted randomization procedure and assigned

to different treatment groups, control group (n=47) received, metformin (CM, n=16),

glibenclamide (CG, n=16), combination (CC, n=15), whereas in intervention group (n=46),

metformin (IM, n=16), glibenclamide (IG, n=15), combination (IC, n=15) received additional

vitamin C (500mg OD) tablet for a period of three months.

Random blood glucose level were measured at all visits including the day of enrolment and 5

ml of blood samples were collected from each patients at the day of enrolment and at the end

of 3 months trial.

Blood samples were collected in heparinized sample collection tubes and the plasma was

separated by centrifugation at 4000 rpm for 20 min. The isolated plasma was stored at -700 C

until biochemical analysis. Random blood glucose was measured by using Accu-chek Active

Blood Glucose Monitor® by Roche, Germany. HbA1C were determined by using semi-auto

analyzer (Micro lab 200 Merck). Ascorbic acid was estimated by the method of Roe and

Kuether12: this involves oxidation of ascorbic acid by copper followed by treatment with 2, 4-

dinitrophenylhydrazine that undergoes rearrangement to form a product with absorption

maximum at 520 nm. Superoxide dismutase (SOD) was determined using spectrophotometric

method by Kakkar13: based on inhibition of the formation of nicotine amide adenine

dinucleotide, phenazine methosulfate and amino blue tetrazolium formazan. Lipid peroxides

were estimated by measurement of thiobarbituric acid reactive substances (TBARs) in plasma

by the method of Yagi14: the pink chromogen produced by the reaction of thiobarbituric acid

with malondialdehyde, a secondary product of lipid peroxidation was estimated. The

absorbance of clear supernatant was measured against reference blank at 535 nm. Catalase

was assayed colorimetrically at 620 nm as described by Sinha15: the reaction mixture (1.5 ml,

vol) contained 1.0 ml of 0.01 M phosphate buffer (pH 7.0), 0.1 ml of erythrocyte lysate and

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0.4 ml of 2 M H2 O2 . The reaction was stopped by the addition of 2.0 ml of

dichromateacetic acid reagent (5% potassium dichromate and glacial acetic acid were mixed

in 1:3).

STATISTICS

The data obtained from the study were analyses by using one way of analysis of variance

(ANOVA) using the Graph Pad “Instat” and the “p” value of ≤ 0.05 were considered as

statistically significant. All the obtained data were plotted in the graph pad column and

comparison were done with different control group Vs different intervention group. Standard

deviation (SD), Mean, standard error mean (SEM) and p values were obtained from the graph

pad itself.

RESULTS

Table 1 shows the Demographic characteristic of the investigated study populations. The

mean age limit was 49.27±7.25 years in control diabetic patients and 48.07±6.25 years in

intervention group. In control group 35 patients had alone diabetes and 22 patients had co

morbidity with diabetes whereas in intervention group 21 patients had alone diabetes and 25

patients had co-morbidity along with diabetes mellitus. Body mass Index was slightly

elevated in both control and intervention patients with duration of disease 5.83±1.99 years in

control group and 6.56±1.91 years in intervention group.

Table 2 shows the circulatory lipid peroxide, oxidative stress parameter, antioxidant status

and HbA1C level of control group and intervention group. The parameters include TBAR,

superoxide dismutase, Catalase, ascorbic acid and HbA1C. Mean serum levels of superoxide

dismutase and ascorbic acid were significantly increased with significant decrease of TBAR

and HbA1C level (vitamin C 500mg/day p≤0.05) in intervention group when compared to

control diabetic patients after three months of vitamin C supplementation. There were no

significant changes in the Catalase level in both the groups. Moreover in intervention group

there were no significant changes with respect to different drug regimen and their

combination.

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Table.1. Demographic comparison of control and Intervention group

Variables Control Group Intervention group

No. of Patients 47 46

Male/Female 12/35 17/29

Age (years) 49.27±7.25 48.07 ±6.25 NS

Smoker 3 4

Alcoholic 4 2

Non Vegetarian 32 33

Duration of disease 5.83±1.99 6.56±1.91 NS

Co-morbidty 22 25

Body Mass Index (kg/m2) 24.75 ±3.09 24.52±2.89 NS

Data expressed in mean ± S.D (NS-Not Significant)

DISCUSSION

In this study most of population was non-vegetarian and overweight, since the Nilgiris is

situated above 2400 meter, the climatic condition with the low atmospheric temperature

affects the life style of the residents. This is the important factor which may affects the basal

metabolic rate of the populations, forced to have the carbohydrate -rich non- vegetarian diet

and one of the epidemic cause for more diabetes7,8.

The extent of lipid peroxidation (TBAR) was decreased and superoxide dismutase (SOD) was

increased in intervention group when compared to control group. This clearly indicates the

antioxidant supplement really useful to minimize the oxidative stress and thereby reduce

related risks. The increased TBARs level observed in study patients as most of them were

coming under the age group of 41- 50 and above 50 years. This might be due to the

pathological condition, worsens with the age due to altered defense system and elevated free

radical production16.

In fast urban lifestyle, fast foods, changing diet pattern, lack of exercise, obesity, smoking

etc. are responsible for the diabetic epidemic17, these factors are greatly undermine the body’s

ability to remove toxic “free radicals “which are constantly being produced in the body”.

The role of oxidative stress in the causation of chronic tissue damage is being increasingly

recognized if antioxidant deficiency occurs. The low antioxidant level in diabetes may have

resulted from lower intake of antioxidant or increased oxidative stress. So, in this condition

the supplementation of antioxidants expected to be beneficial8.

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Table 2 Comparison of lipid peroxide, oxidative stress, antioxidant status & HbA1C between control and intervention groups

TBAR- thiobarbituric acid reactive substances, SOD- superoxide dismutase, HbA1C- glycosylated hemoglobin.

Data expressed in mean ± S.D (**p≤0.05, NS-Not Significant)

Parameter

Control group (n=47) Intervention group (n=46)

Metformin

(n=16)

glibenclamide

(n=16)

combination

(n=15)

metformin

(n=16)

glibenclamide

(n=15)

Combination

(n=15)

TBAR

Mmol/mg

protein

Before 168.47±16.83 147.27±13.47 154.50±13.54 163.25±17.75 162.58±16.62 160.09±15.12

After 186.13±16.74 167.53±13.14 175.20±9.22 130.93±19.67** 136.00±12.24** 128.14±7.88**

SOD

IU/mg

protein

Before 7.25±1.73 7.18±0.86 8.35±1.40 7.99±1.54 9.22±2.49 8.61±2.11

After 5.15±1.85 5.39±1.01 6.30±1.447 14.58±1.45** 15.78±2.35** 14.76±1.73**

Catalase

IU/mg

protein

Before 8.89±0.95 8.22±1.33 8.60±0.76 12.93±1.38 13.79±1.44 13.39±0.98

After 5.34±1.10 5.50±1.23 5.47±0.87 19.74±1.90 NS 18.81±1.14 NS 18.78±1.36 NS

Ascorbic

Acid

µg /mg

protein

Before 106.13±13.99 109.13±16.37 108.40±17.75 103.58±10.29 109.25±13.55 104.91±12.30

After 97.43±12.19 98.80±16.04 98.26±16.04 126.53±12.59** 120.88±7.50** 121.53±7.62**

HbA1C% Before 9.83±1.03 9.11±1.70 9.71±1.63 9.78±1.03 9.83±1.83 9.88±1.85

After 9.65±1.01 9.92±1.92 9.80±1.99 9.10±1.05** 9.11±1.65** 9.08±1.76**

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In our study most of the enrolled patients coming under economically deprived background.

Thus lower consumption of fruits and vegetables may have contributed to reduce the

consumption of vitamin C and carotenes among the patients with diabetes which may lead to

increase oxidative stress. These data are supported by the Enstrom study where consumption

of vitamin C decreases the risk of premature death18. There were no significant changes with

respect to different drug regimen and their combination.

Limitations of this study are as it was conducted at government hospital so turn out of the

patients were very irregular with a higher drop out, thus forced us to limit the number of

subject of the study. Different dose of vitamic C with more number of subjects will give a

much better result for clinical application. Use of different antioxidant other than vitamin C,

like vitamin E and alpha lipoic acid would have given a better comparison. More over a

placebo group would have been best used to measure the actual effect of vitamin C.

CONCLUSIONS

Although our results may have some clinical implications, measurement of antioxidants is not

routinely performed in clinical practice, reviewing the intake of food rich in antioxidants

particularly citrus fruits and green leafy vegetables and the appropriate antioxidant

supplements among patients with the metabolic syndrome may be instructive for the smooth

control of diabetes. If their dietary intake of vitamins A, C and E fails to meet the

recommended daily allowance, health care professionals should encourage people with the

metabolic syndrome to increase their intake of vitamins, preferably through the consumption

of healthy food sources rich in these vitamins or otherwise through the use of appropriate

vitamin supplements. The future studies may direct towards extended duration of treatment

and large number of patients.

ACKNOWLEDGEMENTS

The authors would like to acknowledge the support and co-operation received from the

medical and supporting staff of government headquarters hospital, Ootacamund, The Nilgiris,

Tamil Nadu, India and also all the participants who willingly enrolled themselves in the

study.

REFERENCES

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