RESEARCH ARTICLE

Anti-diabetic and anti-oxidative effect of composite extractof leaves of some Indian plants on alloxan induced diabeticwistar rats

Brahm Kumar Tiwari • Kanti Bhooshan Pandey •

Nidhi Jaiswal • A. B. Abidi • Syed Ibrahim Rizvi

Received: 21 November 2013 / Accepted: 1 January 2014

� The Korean Society of Pharmaceutical Sciences and Technology 2014

Abstract Use of plant based remedies for the prevention

and treatment of diabetic complications over conventional

therapies have received much emphasis in recent years.

Present study was performed to evaluate the protective

effect of composite extract (CE) of leaves of Aegle

marmelos, Ocimum sanctum, Murraya koenigii and Aza-

dirachta indica on biochemical alterations in alloxan

(ALX) induced diabetic wistar rats. Diabetes was induced

in rats by a single administration of ALX (150 mg/kg

intraperitoneal (i.p) and CE at three different doses (25, 50

and 100 mg/kg/bw/day) were administrated orally in three

group of diabetic rats for 35 days. Another group of same

number of diabetic rats were administrated by insulin

(6 unit/kg/bw/day) by subcutaneous injection for 35 days and

used as standard. Results showed that oral administration of

CE significantly protected the biochemical impairments in

diabetic rats as evidenced by restoration of glutathione

depletion, ascorbic acid level, plasma antioxidant values

and inhibition of lipid peroxidation. The outcome of the

study suggests that CE of A. marmelos, O. sanctum, M.

koenigii and A. indica leaves mimics insulin and passes

capacity to ameliorate the hyperglycemia induced cellular

complications thus may control the development and pro-

gression of diabetes.

Keywords Diabetes � MDA � GSH antioxidant �Composite extract

Introduction

Diabetes mellitus is one of the most prevalent metabolic

diseases in the world affecting nearly 25 % of the popu-

lation (Maiti et al. 2004). It is characterized by condition of

hyperglycaemia due to impairment in insulin secretion or

insulin action. Uncontrolled and long term hyperglycaemic

state mediates biochemical alterations including glycation

of proteins, inactivation of enzymes, and alterations in

structural functions of collagen basement membranes

(Aronson and Rayfield 2002) which ultimately results into

dysfunction of various organs especially the eyes, kidneys,

nerves, heart and blood vessels (Paneni et al. 2013).

Hyperglycaemia induced micro vascular complications are

the leading cause of blindness, renal failure, nerve damage

during diabetes (Cade 2008).

Oxidative stress due to imbalance between reactive

oxygen species (ROS) generation and their neutralization

has been reported to play a very important role in the

causation and progression of diabetes and related conse-

quences (Sajeeth et al. 2011). Hyperlipidemia and oxida-

tive stress frequently co-exist with diabetes mellitus

(Kumar et al. 2012). Oxidative stress critically affects the

glucose transport proteins (GLUT) and insulin receptors

(Abolfathi et al. 2012). Diabetes in experimental animal

models exhibits high oxidative stress due to persistent and

chronic hyperglycaemia, which depletes the activity of

antioxidative enzymes and thus promotes free radical

generation (Bagri et al. 2009). There are reports on per-

turbed plasma antioxidant levels in patients with diabetes

(Chandra et al. 2007).

K. B. Pandey � N. Jaiswal � S. I. Rizvi (&)

Department of Biochemistry, University of Allahabad,

Allahabad 211002, U.P., India

e-mail: sirizvi@gmail.com

B. K. Tiwari � A. B. Abidi

Department of Biochemistry & Biochemical Engineering,

S.H.I.A.T.S. (Deemed University), Allahabad 211007, U.P.,

India

123

Journal of Pharmaceutical Investigation

DOI 10.1007/s40005-014-0116-5

Plant based natural therapies have gained much attention

in recent years to control and limit hyperglycaemia and

thus decrease the chances of diabetic complications (Tag

et al. 2012; Sabu and Kuttan 2002). According to the

World Health Organization (WHO), more than 70 % of the

world’s population use traditional medicine to satisfy their

principal health needs. A number of medicinal plants are

used for the control of the diabetes mellitus (Rizvi and

Mishra 2013; Tiwari et al. 2013).

Aegle marmelos commonly known as the ‘bel’ fruit tree

is frequently used as an alternative medicine in the treat-

ment of diabetic condition (Sankeshi et al. 2013). Other

than this, the plant also finds use as antidiarrheal and

haemostatic agent (Rizvi and Mishra 2013). Ocimum

sanctum or ‘tulsi, is an herbaceous plant found throughout

the south Asian region. Its use as an antihyperglycemic

agent has been reported (Vats et al. 2002), it is also used in

conditions of chronic fever and haemorrhage (Vats et al.

2004). Azadirachta indica is predominantly found in the

Asian sub-region but is also grown in Nigeria and other

parts of Africa; it is functionally used as an anti-diabetic

(Ali Hussain 2002) and to treat gastrointestinal upset,

diarrhoea and skin ulcers (Anyaehie 2009). Murraya

koenigii is a small deciduous tree with very pungent aro-

matic leaves (curry leaf), its use an as antidiabetic is doc-

umented (Arulselvan et al. 2006) besides finding

application as a stimulant and antidysenteric (Lawal et al.

2008).

The anti-diabetic effects of A. marmelos, O. sanctum, M.

koenigii and A. indica, have also been reported in other

studies (Kumar et al. 2013; Bhatti et al. 2013; Murali-

krishnan et al. 2012; Saha and Mazumder 2013; Shailey and

Basir 2012). It is pertinent to emphasize that previous

studies focussed on evaluating the anti diabetic effect of A.

marmelos, O. sanctum, A. indica, and M. koenigii have

yielded moderate results (Sankeshi et al. 2013; Murali-

krishnan et al. 2012; Saha and Mazumder 2013; Arulselvan

et al. 2006), however the studies on their composite extract

(CE) are limited or a few. Keeping this rationale in mind,

the present study was conducted to investigate the effect of

the CE of the leaves of A. marmelos, O. sanctum, M.

koenigii and A. indica at different concentrations on reliable

biomarkers of oxidative stress (Pandey and Rizvi 2011) in

alloxan induced diabetic rats. The results have been com-

pared with the anti diabetic effect observed with insulin.

Materials and methods

Collections of leaves and preparation of CE

The leaves of A. indica, A. marmelos, O. sanctum, M.

koenigii were collected from Sam Higginbottom Institute

of Agriculture, Technology & Sciences, Allahabad, India

orchard and dried at room temperature. After that leaves

were powdered in warring grinder and passed through 40

mesh sieve and after keeping in the filter were transferred

in the soxhelt apparatus at 60 �C for 10 h where 80 %

methanol solvent was used for extracting the extract. After

that obtained extracts were evaporated to dryness on a

boiling water bath and stored in tight dark bottle separately

in the refrigerator.

The aqueous extract of these plants and fruits were

diluted on the day of experimentation and administration

by oral gavage at the dose of 25, 50 and 100 mg/kg body

weight in a fixed volume of 1 mL. CE was prepared by

mixing all four individual plant leaf extracts in same pro-

portion i.e. 25 % weight was contributed by each four plant

extract in 100 % CE).

Animal model and study protocol

All protocols for experiments were approved by the Ani-

mal Care and Ethics Committee of University of Allaha-

bad, Allahabad, India. The experimental male wistar strain

rats (4 ± 0.5 months old) with body weight between

150 ± 15 g were housed in the animal house under tem-

perature varied between 25 ± 5 �C with 12-h light-dark

cycle. The animals were kept in plastic cages and clean

drinking water provided ad libitum, while they were fed

with standard commercial pelleted feed. The adequate

ventilation was maintained in the animal house. Rats were

allowed to acclimatize for 7 days before the start of the

experiment.

The rats were divided in to fallowing 6 groups each

containing of six animals (n = 6). Group I—Control:

received no treatment/supplementation; Group II—Induced

diabetes: rats were injected single dose intra peritonealy

alloxan Group III—Insulin treated diabetic: rats were

treated twice a day by subcutaneous injection of 3 units of

NPH insulin each time; 6 units of insulin/day (NPH hu-

miinsulin, Lilly Egypt), Groups IV, V and VI—CE

administered diabetic rats at concentrations 25, 50 and

100 mg/kg body weight respectively.

Experimental induction of diabetes in rats

Diabetes was induced in experimental rats by single in-

traperitonial injection of alloxan monohydrate (2,4,5,6-

tetraoxyprimidine) dissolved in 0.9 % saline at dose of

150 mg/kg body weight as described elsewhere (Burade

and Kuchekar 2011; Manonmani et al. 2005). Since alloxan

is capable of producing fatal hypoglycaemia as a result of

massive pancreatic insulin release, the rats were then kept

for the next 24 h on 5 % glucose solution bottles in their

cage to prevent hypoglycaemia. After 1 week diabetes was

B. K. Tiwari et al.

123

confirmed by the determination of fasting blood glucose

concentration 241–275 mg/dL with the help of a glucom-

eter (Gluco careTM ultima). Different doses of the metha-

nolic CE were administered for 35 days via gavage

technique (oral route) in diabetic rats caused by alloxon

injection.

Collection of blood, and isolation of erythrocytes

and plasma

After the end of the treatment period, rats were sacrificed

under light anaesthesia. Blood were collected by cardiac

puncture into 10 units/mL heparin rinsed anticoagulant

syringes and then red blood cells were pelleted by centri-

fugation at 8009g for 10 min at 4 �C. Packed red cells

were washed two times by phosphate buffered solution and

proceed for the experiments. Separated plasma was

immediately frozen at -20 �C until use for biochemical

assays.

Determination of erythrocyte reduced glutathione

(GSH)

Erythrocyte GSH was measured following the method of

Beutler (1984) based on the ability of the –SH group to

reduce 5,50-dithiobis, 2-nitrobenzoic acid (DTNB) and

form a yellow colored anionic product whose optical

density is measured at 412 nm on Shimadzu-UV-1800 UV-

VIS spectrophotometer, Japan. Concentration of GSH is

expressed in mg/mL packed RBCs and was determined

from standard plot.

Determination of erythrocyte malondialdehyde (MDA)

content

Erythrocyte MDA was measured according to previous

published method (Pandey and Rizvi 2009). Packed

erythrocytes (0.2 mL) were suspended in 3 mL PBS con-

taining 0.5 mM glucose, pH 7.4. The lysate (0.2 mL) was

added to 1 mL of 10 % trichloroacetic acid (TCA) and

2 mL of 0.67 % thiobarbituric acid (TBA) boiled for

20 min at 90–100 �C, cooled the mixture was centrifuged

at 1,0009g for 5 min and the absorbance of supernatant

was read at 532 nm on Shimadzu-UV-1800 UV-VIS

spectrophotometer, Japan.. The concentration of MDA in

erythrocytes was calculated using extinction coefficient

(e = 1.56 9 105 M-1 cm-1) and is expressed as nmol/mL

of packed erythrocytes.

Determination of plasma ascorbic acid content

Ascorbic acid content was estimated in the plasma by using

the calorimetric method (Jagota and Dani 1982). 2 mL of

plasma was quickly transferred in to a centrifuge tube and

1 mL of 50 % TCA was added to it and mixed well. The

mixture was placed in an ice-bath for 30 min. for complete

deproteinization and then centrifuged at 3009g for 10 min.

In a tube, 1.5 mL supernatant was taken and triple-distilled

water was added to make the final volume 3 mL. Then

300 lL of diluted (1:10 dilution in the distilled water)

Folin and ciocalteu’s phenol reagent was added to the

mixture and tube were incubated at 37 �C for 15 min. The

absorbance of the blue colour that developed was mea-

sured using a spectrophotometer (Shimadzu-UV-1800

UV-VIS spectrophotometer, Japan at 760 nm. A solution

of L-ascorbic acid at concentrations of 0, 2.5, 5, 7.5 and

10 lg/mL was used as standard.

Measurement of plasma antioxidant activity by FRAP

assay

The total antioxidant potential of the plasma samples was

determined by the ferric reducing ability of plasma (FRAP)

assay by the method of Benzie and Strain (Benzie and

Strain 1996). FRAP reagent was prepared from 300 mmol/L

acetate buffer, pH 3.6, 20 mmol/L ferric chloride and

10 mmol/L 2,4,6-tripyridyl-s-triazine made up in 40 mmol/L

hydrochloric acid. All three solutions were mixed together

in the ratio 10:1:1 (v:v:v) respectively, 3 mL of FRAP

reagent was mixed with 100 lL of plasma and the contents

were mixed thoroughly. The absorbance was read at

593 nm at 30 s intervals for 4 min. Aqueous solution of

known Fe2? concentration in the range of 100–1,000 lmol/L

was used for calibration. The FRAP values (lmol Fe(II)/L)

of the plasma was calculated using the regression equation.

Statistical analysis

Statistical analyses were carried out using Graph Pad

PRISM software, US (version 5).The data were expres-

sed as mean ± SD. The data were analyzed by one

way ANOVA followed by Tukey’s Multiple Compari-

son Test. Values P \ 0.05 was considered statistically

significant.

Results

Blood glucose level

Blood glucose levels were measured randomly on 0, 7th,

14th, 21st, 28th, 35th day of study. The raised levels of

blood glucose decline sharply after oral administration of

CE in a dose dependent manner (Fig. 1).

Anti-diabetic and anti-oxidative effect of CE

123

Erythrocyte GSH status

Diabetic rats showed significant decrease in erythrocyte

GSH content in comparison to the control (P \ 0.01). CE

treatment to diabetic animals showed protection against

alloxan induced reduction in GSH content in dose dependent

manner. 100 mg/kg body weight dose of CE was more

effective in protection of GSH depletion than 25 and 50 mg/kg

body weight CE at the concentration 100 mg/kg body weight

mitigated the effect of alloxan as evidenced by the level of

GSH in the treated rats and in the control (Fig. 2).

MDA level in erythrocytes

Alloxan induced diabetic rat showed increased erythrocyte

MDA in comparison to normal rats (P \ 0.01). Diabetic rats

treated with CE and insulin showed significant decrease

in erythrocyte MDA content. CE at dose 25 mg/kg was

sufficient to provide complete protection against lipid

peroxidation in diabetic rat; the effect was comparable to

insulin (3 units /twice a day). Same protective effect on

lipid peroxidation was observed at the higher dose of CE

50 and 100 mg/kg body weight treated rats (Fig. 3).

Ascorbic acid content of plasma

A significant decrease in plasma ascorbic acid content was

observed in alloxan induced diabetic rats when compared

with normal untreated rats (P \ 0.001). Diabetic rats

administrated with CE showed higher ascorbic acid content

than the diabetic rats treated with alloxan only. The effect

shown by CE was similar to the insulin (3 units/twice a

day), however CE at all the concentrations used in the

study was unable to provide complete protection against

diabetes induced depletion of ascorbic acid (Fig. 4).

Antioxidant capacity of plasma

Diabetic rats showed significant decrease in antioxidant

potential as observed by reduced FRAP values compared to

normal control (P \ 0.005). CE administrated diabetic rats

showed higher FRAP values and the effect was concen-

tration dependent. The diabetic rats administrated with

100 mg/kg body weight CE, exhibited higher FRAP val-

ues, CE not only prevented the antioxidant depletion but

also enhanced its level (Fig. 5).

Discussion

Enhanced oxidative stress and diminished plasma antioxi-

dants have been implicated as a risk factor for the

Fig. 1 Time and dose dependent blood glucose level of rats subjected

to alloxan-induced diabetes and CE supplementation during the

experimental period of 35 days. Each value represents the

mean ± SD of six rats in each group, n = 6

Fig. 2 Erythrocyte GSH level in methanolic CE treated alloxan

induced diabetes rats. The level of GSH was significantly lower in

diabetic rats in comparison the normal control rats (#P \ 0.01). CE in

dose dependent manner protected depletion of GSH in alloxan

induced diabetic rats (*P \ 0.01, **P \ 0.05; compared with

diabetic rats). Concentration of GSH is expressed in mg/mL packed

erythrocytes

Fig. 3 Erythrocyte MDA content in methanolic CE treated alloxan

induced diabetes rats. The content of MDA was significantly higher in

diabetic rats in comparison to the normal control rats (#P \ 0.01). CE

in dose dependent manner significantly protected the lipid peroxida-

tion in diabetic rats (*P \ 0.01, **P \ 0.005; compared with diabetic

rats). The concentration of MDA is expressed as nmol/L of packed

erythrocytes

B. K. Tiwari et al.

123

development of diabetic complications (McCune and Johns

2002). GSH is among the most important non-protein thiol

in mammalian cells. GSH has many biological functions,

such as maintaining membrane protein sulfhydryl groups in

the reduced form, acting as a substrate for GSH peroxidase

and detoxification of xenobiotics. Therefore, the mainte-

nance of the GSH level is crucial for cellular defence

against oxidative injury and cellular integrity (Pandey and

Rizvi 2011). We observed decreased levels of GSH in

diabetic rats. Significant protection of GSH depletion in CE

administered rat signifies the anti-diabetic effect of CE

which is comparable with insulin (Fig. 2).

Lipid peroxidation measured in terms of MDA is known

as the potent marker of oxidative stress (Pandey and Rizvi

2010). Lipid peroxidation is a free radical-induced process

leading to oxidative deterioration of polyunsaturated fatty

acid. Increased erythrocyte lipid peroxidation is known to

cause a decrease in the fluidity of the membrane lipid

bilayer, alteration of integrity, permeability, and functional

loss. Elevated level of MDA is one of the characteristics

features of chronic diabetes (Karpen et al. 1982; Pandey

et al. 2009). In agreement with other studies (Aydin et al.

2001; Cho et al. 2005), we have observed a significant

increase MDA levels in erythrocyte and plasma of diabetic

rats when compared to control rats. The CE at doses 25, 50

and 100 mg/kg body weight significant decreased the

peroxidation level of lipids in diabetic rats, the effect quit

similar to the insulin. Interestingly unlike the effect exerted

by CE against GSH depletion which is dose dependent, the

effect of CE on protecting against lipid peroxidation was

the same for all the three doses (Fig. 3).

Ascorbic acid is primary antioxidant in the plasma. In

addition to its antioxidant property, this vitamin serves as a

cofactor in several important enzyme reactions, including

those involved in the synthesis of catecholamines carnitine,

cholesterol, amino acids and certain peptide hormones

(Rizvi et al. 2009). Ascorbic acid supplementation in STZ-

diabetic rats improves both hyperlipidemia and cardiac

function (Dai and McNeill 1995). The importance of

ascorbic acid regeneration involving the concerted action

of plasma membrane redox system in diabetes has been

reported (Rizvi and Srivastava 2010). Our result on the

protection of loss of ascorbic acid in diabetic rats by CE

highlights the use of CE supplementation for the mainte-

nance of intracellular and extracellular redox balance.

Antioxidant capacity of plasma is the primary measure

and marker to evaluate the state and potential of oxidative

stress The FRAP assay offers a putative index of antioxidant,

or reducing, potential of biological fluids within the tech-

nological reach of laboratory and researcher interested in

oxidative stress and its effects (Benzie and Strain 1996). A

decrease in the reducing power/antioxidant capacity in terms

of FRAP in plasma is predictive of alloxan-induced oxidative

stress in experimental rats. CE supplementation to experi-

mental rats augmented antioxidant capacity. The restoration

of plasma antioxidant potential in diabetic rats by CE is an

important observation and adds to the novelty of the results.

Conclusion

Diabetes is a fast growing chronic disease all over the word

affecting social values and economy. Conventional drugs

used for treating diabetes come with high cost and several

side effects. The plant extracts used in the study are from

periannual, ever green and easily available plants. Their

effects on diabetic rats as observed in the study are

Fig. 4 Effect of methanolic CE on alloxan induced diabetic rats on

ascorbic acid level in wistar strain rat. Treatment with alloxan caused

a decreases in ascorbic acid level (#P \ 0.001) compared with

control. Dose dependent treatment with CE showed significant

protection against ascorbic acid depletion (*P \ 0.01, **P \ 0.05,

compared with diabetic rats). Concentration of ascorbic acid is

expressed as lg/mL plasma

Fig. 5 Effect of methanolic CE on alloxon induced diabetes rats on

plasma antioxidant potential of wistar strain rats. Treatment with

alloxan caused a decrease in FRAP values (#P \ 0.005) compared

with control. Does dependent treatment of CE significantly restored

the reduction in antioxidant power of plasma in diabetic rats

(*P \ 0.005, **P \ 0.001; compared with diabetic rats). FRAP

value is expressed in lmol Fe(II)/L plasma

Anti-diabetic and anti-oxidative effect of CE

123

exciting. Taken together, our results contribute towards

validation of enhanced antidiabetic efficacy of A. marme-

los, O. sanctum, M. koenigii and A. indica leaf extracts

when combined. Future studies concerning pharmacologi-

cal aspects may provide further information necessary for

establishing CE as potent anti-diabetic phytochemical.

Acknowledgments The authors are thankful to the University of

Allahabad, Allahabad, India for providing the facilities. Department

of Biochemistry, University of Allahabad is recipient of FIST grant

from Department of Science and Technology, Govt. of India.

Conflict of interest None.

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