chapter - 3 - shodhganga : a reservoir of indian theses...

Chapter - 3

In Vivo Antidiabetic Activity Studies

Education is what survives when what

has been learnt has been forgotten.

- B. E. Skinner

Chapter – 3 : In vivo antidiabetic activity studies Introduction

75

Introduction

Diabetes mellitus is a leading metabolic disorder worldwide, caused by

inherited and / or acquired deficiency in production of insulin in β cells of pancreas,

or by the desensitization of insulin receptors for insulin. Such a deficiency results in

the increased concentration of glucose in the blood which results in secondary

complications affecting eyes, kidneys, nerves and arteries (Ismail, 2009).

Experimental evidences suggest the involvement of free radicals in the pathogenesis

of diabetes (Matteucci and Giampietro, 2000) and more importantly in the

development of diabetic complications (Lipinski, 2001).

The number of people suffering from diabetes all over the world has increased

and the disease now kills more people than AIDS. Based on a report by Shaw et al.

(2010), diabetes among adults is expected to increase from 285 million in the year

2010 to 439 million or more by the year 2030. Several reports indicate that annual rate

of DM will increase in future worldwide, especially in India. It has been proposed that

approximately 87 million Indians will be affected by DM by the year 2030 (Shaw

et al., 2010). Out of the two types of diabetes, the incidence of non-insulin dependent

diabetes mellitus (NIDDM) is much higher than the insulin dependent diabetes

mellitus (IDDM) (Hussain, 2002).

In conventional therapy, IDDM is treated with exogenous insulin and NIDDM

with oral hypoglycemic agents (Jarald et al., 2008). Though, different types of oral

agents are available in the treatment of NIDDM, in the last few years there has been

an exponential growth in the use of herbal medicines for diabetes and these drugs are

gaining popularity both in developed and developing countries because of their

natural origin and less side effects (Grover et al., 2002b).

A systematic review of literature on the research work on A. paniculata,

T. cordifolia and T. foenum-graecum, have revealed that very few studies have been

carried out to show their in vivo antidiabetic activity. Majority of the studies on A.

paniculata have been carried out using the aerial parts. Antidiabetic activity studies

on T. cordifolia have been carried out using only root and stem. Earlier in vivo studies

Chapter – 3 : In vivo antidiabetic activity studies Introduction

76

on T. foenum-graecum have shown only the use of seeds. Hence, we have carried out

in vivo antidiabetic activity studies on these plants by taking only the leaves. In vitro

screening of different solvent leaf extracts for antioxidant activities played a strategic

role in the selection of the suitable solvent extract for the antidiabetic study. The

ethanol leaf extract of these plants exhibited highest in vitro antioxidant activity. This

provides deep insight into the use of extracts for therapeutic purpose and totalitarian

approach emphasized in traditional medicine. Hence, ethanol extract was selected for

carrying out the in vivo antidiabetic activity studies. The ethanol leaf extract of all the

three plants were investigated for their efficacy and safety in rats. The extracts were

tested for an array of biochemical parameters in rats like blood glucose levels, serum

urea levels, serum and urine creatinine levels and antioxidant enzyme activities in

liver. Histopathological studies of islet cells of pancreas in normal and streptozotocin

induced diabetic rats were also carried out. Details of the work carried out are

presented in this chapter.

Chapter – 3 : In vivo antidiabetic activity studies Material and Methods

77

Material and Methods

Plant material

Fresh and healthy leaves of A. paniculata and T. cordifolia were obtained from

local growers of Mysore. Trigonella foenum-graecum leaves were obtained from the

local market, Mysore. The sample specimen was identified based on the taxonomical

characteristics. The leaves were washed thoroughly in distilled water and the surface

water was removed by air drying under shade. The leaves were subsequently dried in

a hot air oven at 40°C for 48 h, powdered to 100-120 mesh in an apex grinder [Apex

Constructions, London] and used for extraction.

Chemicals

Ethanol was of AR grade from Merck (Mumbai, India). Streptozotocin was

from Sisco Research Laboratories (Mumbai, India). Sodium carbonate, copper

sulphate, phenol reagent, sodium potassium tartarate, formaldehyde, sodium

phosphate and hydrogen peroxide were from SD Fine Chemicals (Mumbai, India).

Creatinine and urea estimation kits were from Anamol Laboratories (Tarapur, India).

Superoxide dismutase and glutathione peroxidase estimation kits were from

RANDOX Laboratories (UK).

Preparation of solvent extract

Extraction was carried out according to the method of Okigbo et al. (2005).

Fifty grams each of the powdered material was extracted initially with 300 ml of

chloroform, hexane, methanol and ethanol separately for 24 h at 23 ± 2°C. The extract

was filtered with sterile Whatman No. 1 filter paper into a clean conical flask. Second

extraction was carried out with same amount of solvent for another 24 h at 23 ± 2°C

and filtered. The extracts were later pooled and transferred into the sample holder of

the rotary flash evaporator [Buchi Rotavapor R-124, Switzerland] for the evaporation

of the solvents. The evaporated extracts so obtained were preserved at 4°C in airtight

bottles until further use. The suspensions of the extracts were prepared by dissolving

the weighed amounts in freshly prepared 1% gum arabic solution.


78

Animals

Adult healthy Albino rats of Wistar strain of either sex weighing 120-160 g

with no prior drug treatment were used in the present study. Animals were maintained

at 22 ± 2°C with 12 h light and dark cycle. The animals were fed on a standard pellet

diet and had free access to water throughout the experiment. Animals that are

described as fasting were deprived of food for at least 16 h but were allowed free

access to drinking water. Animal study was performed in Central Animal Facility, JSS

Medical College, Mysore, with due permission from Institutional Animal Ethics

Committee (R.No. JSS/MC/IEC/5064/CPCSEA).

Experimental design

The animals were randomly divided into the 15 groups with six animals in

each group.

Group A saline or normal control

Group B and Group C normal controls fed with 250 mg/kg b.w.

and 500 mg/kg b.w. of A. paniculata leaf

extract respectively

Group B1 and Group C1 normal controls fed with 250 mg/kg b.w.

and 500 mg/kg b.w. of T.cordifolia leaf


Group B2 and Group C2 normal controls fed with 250 mg/kg b.w.

and 500 mg/kg b.w. of T. foenum-graecum

leaf extract respectively

Group D diabetic control

Group E diabetic rats treated with glibenclamide

(0.5 mg/kg b.w.)

Group F and Group G diabetic rats treated with 250 mg/kg b.w.

and 500 mg/kg b.w. of A. paniculata leaf


Group F1 and Group G1 diabetic rats treated with 250 mg/kg b.w.

and 500 mg/kg b.w. of T. cordofolia leaf


Group F2 and Group G2 diabetic rats treated with 250 mg/kg b.w.

and 500 mg/kg b.w. of T. foenum-graecum

leaf extract respectively


79

Saline, leaf extract and the standard drug were administered orally using a

mouth gauge. During the study period, body weight, food and water intake of rats

were monitored regularly.

Induction of diabetes

After initial determination of 16 h fasting blood glucose levels (blood drawn

through the tail vein puncture) animals were given single intraperitoneal injection of

streptozotocin at a dose of 45 mg/kg b.w. freshly dissolved in cold 0.9% saline

(Venkateswaran and Pari, 2003). Following injection, animals were carefully

observed for first 24 h for evidence of allergic reaction, behavioural changes and

convulsions. Fasting blood glucose levels were recorded after 5 days. Animals that

developed stable hyperglycemia with fasting blood glucose levels more than 200 mg/dl

were selected for the study.

Blood collection

Rats were fasted overnight; blood was drawn from the tail by tail vein

puncture during the experimental period and from cardiac puncture at the end of the

experimental period (after 28 days) under anaesthesia. Serum was separated by

centrifuging the collected blood samples at 10,000 rpm for 10 min at 4°C and used for

the estimation of creatinine and urea.

Urine collection

Urine from normal control rats, diabetic and diabetic treated rats were

collected under a layer of toluene by keeping the rats in metabolic cages for 24 h.

Collected urine samples were filtered through filter paper, centrifuged and stored at

4°C until further analysis.

Preparation of liver tissue homogenate

After 28 days, rats were dissected under anaesthesia. Liver was rinsed in ice

cold distilled water followed by chilled 0.9% saline. About 1 g of liver tissue was

homogenized in 10 ml of 10 mM phosphate buffered saline (pH 7.4) using REMI

homogenizer fitted with a teflon plunger (REMI Laboratory Instruments, Mumbai,

India). The homogenate was centrifuged at 10,000 rpm at 4°C for 15 min and the

supernatant was used for the determination of antioxidant liver enzymes viz.,

superoxide dismutase, catalase and glutathione peroxidase.


80

Analytical Methods

Blood glucose estimation

Blood samples were collected by tail vein puncture at weekly intervals for a

period of 28 days. Fasting blood glucose was measured by glucose oxidase-peroxidase

(GOD-POD) method (Trinder, 1969) in mg/dl using a digital glucometer (Braun

OmnitestR

EZ, Germany).

Protein estimation

Protein estimation was carried to the method of Lowry et al. (1951). The blue

colour developed by the reduction of phosphomolybdic-phosphotungstic components

in the Folin-Ciocalteau reagent by the aminoacids tyrosine and tryptophan present in

the protein is measured at 660 nm.

Procedure

Reagent A – 2% sodium carbonate in 0.1 N sodium hydroxide

Reagent B – 0.5% copper sulphate in 1% potassium sodium tartarate

Reagent C – 50 ml of reagent A and 1 ml of reagent B mixed prior to use

Reagent D – 1 ml of Folin-Ciocalteau phenol reagent diluted with 2.5 ml of distilled water

To 1 ml of the sample containing 50-200 µg of protein, 5 ml of reagent C was

added and mixed thoroughly and allowed to stand at room temperature for 10 min. To

this, 0.5 ml of reagent D was added, vortexed and allowed to stand at room

temperature for 30 min and absorbance was measured at 660 nm in a

spectrophotometer. Standard BSA (0-200 µg/ml) was treated similarly and a standard

graph was plotted.

Creatinine estimation

Creatinine estimation was carried out according to the method of Folin and

Wu (1919) in both serum and urine samples using creatinine estimation kit according

to manufacturer’s instruction. Creatinine when treated with alkaline picrate solution

yields a bright orange red complex. Intensity of the colour at 520 nm corresponds to

the concentration of creatinine in the sample.


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Procedure

For urine: To 10 µl of the urine sample, 90 µl of distilled water was added. To this,

600 µl of picric acid reagent provided in the kit was added and mixed thoroughly

followed by the addition of 2 ml of 0.2 N sodium hydroxide. The mixture was

allowed to stand at room temperature for 15 min. The colour intensity developed was

measured at 520 nm. Standard creatinine (10 mg/dl concentration) provided in the kit

was also treated similarly. Creatinine levels were expressed as mg/day and calculated

according to manufacturer’s instruction as below:

Urine creatinine = Absorbance of sample / Absorbance of standard × Dilution factor

For serum: To 200 µl of serum sample, 1.2 ml of picric acid reagent was added,

vortexed and centrifuged at 2000 rpm for 10 min. To 700 µl of supernatant, 400 µl of

0.2 N sodium hydroxide was added and allowed to stand at room temperature for 15 min.

Colour intensity developed was measured at 520 nm. Standard creatinine (10 mg/dl

concentration) was also treated similarly. Creatinine levels were expressed as mg/dl

and calculated according to manufacturer’s instructions as below:

Serum creatinine = Absorbance of sample / Absorbance of standard × 2

Serum urea estimation

Serum urea was estimated according to enzymatic UV-kinetic method of

Talke and Schubert (1965). In the presence of urease, urea is hydrolyzed to ammonia

ion and carbon dioxide. In the presence of glutamate dehydrogenase (GLDH) the

formed ammonium ion reacts with α-ketoglutarate and NADH to form glutamate and

NAD+. An equimolar quantity of NADH undergoes oxidation during the reaction and

therefore the decrease in absorbance at 340 nm due to NADH oxidation is directly

proportional to the urea concentration in the sample. Urea was estimated according to

manufacturer’s instruction.

Procedure

Reagent R1 – 50 mM/l of tris buffer (pH 7.7) containing 10 KU/l of urease and 900

IU/l of glutamate dehydrogenase

Reagent R2 – 50 mM/l of tris buffer (pH 7.7) containing 0.25 mM/l of NADH and 10

mM/l of α-ketoglutarate


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Reagent R1 and reagent R2 were mixed in the ratio of 4:1 to prepare the

desired volume of working reagent. To 10 µl of serum sample 1 ml of working

reagent was added and allowed to react for 30 sec. The absorbance at 340 nm was

read at 30th

and 60th

second. Standard urea (42.8 mg/dl) provided in the kit was also

treated similarly. Urea concentrations were expressed as mg/dl and calculated

according to the manufacturer’s instructions as below:

Urea (mg/dl) = Absorbance of sample / Absorbance of standard × 42.8

Liver antioxidant enzyme activity assay

Estimation of superoxide dismutase (SOD)

The activity of SOD was assayed according to the method of Woolliams et al.

(1983) in a Daytona analyser (RANDOX Laboratories Ltd., United Kingdom) using

RANDOX SOD kit. This method employs xanthine and xanthine oxidase to generate

superoxide radicals which reacts with 2-(4-iodophenyl)-3-(4-nitrophenol)-5-

phenyltetrazolium chloride (I.N.T.) to form a red formazan dye. The SOD activity is

then measured by the degree of inhibition of this reaction. One unit of SOD is that

which causes a 50% inhibition of the rate of reduction of I.N.T. under the conditions

of the assay.

Procedure

Reagent R1 – 0.05 mM/l of xanthine and 0.025 mM/l of I.N.T. in 20 ml of buffer

[CAPS (40 mM/l, pH 10.2) + EDTA (0.94 mM/l)]

Reagent R2 – 80 U/l of xanthine oxidase in 10 ml of redistilled water

Subsequent dilutions of the standard (4.48 U/ml) were prepared with 0.01

mM/l phosphate buffer (pH 7) to produce a standard curve. To 5.6 µl of diluted

standard, 189 µl of reagent R1 and 28 µl of reagent R2 were added. The initial

absorbance after 30 sec and final absorbance after 3 min was taken at 505 nm.

Suitably diluted samples were treated similarly. SOD units/mg protein was calculated

according to manufacturer’s instruction as below:

SOD units/ml = SOD units/ml from standard curve × dilution factor

SOD units/mg protein = SOD units/ml mg protein/ml


83

Estimation of catalase

The activity of catalase was assayed according to the method of Luck (1971).

The UV absorption of hydrogen peroxide (H2O2) solution can be easily measured

between 230 and 250 nm. On decomposition of H2O2 by catalase, the absorption

decreases with time. The enzyme activity can be arrived at from this decrease.

Procedure

Before the assay, the standard H2O2 (30% w/v) dilution was set with

phosphate buffer (10 mM, pH 7.0) so that at 240 nm for 2 min, the absorbance is 0.50.

To the reaction mixture consisting of 1 ml H2O2 in phosphate buffer, 50 µl of sample

was added and change in absorbance at 240 nm for 2 min was monitored. Results

were expressed as units/min/mg protein.

Specific activity = ΔA / min / ε of H2O2 × mg of protein

Estimation of glutathione peroxidase (GSH-Px)

The activity of glutathione peroxidase was assayed according to the method of

Paglia and Valentine (1967) in a Daytona analyser using glutathione peroxidase kit.

Glutathione peroxidase catalyses the oxidation of glutathione by cumene

hydroperoxide. In the presence of glutathione reductase and NADPH the oxidised

glutathione is immediately converted to the reduced form with a concomitant

oxidation of NADPH to NADP+. The decrease in absorbance at 340 nm is measured.

Procedure

Reagent R1 – 4 nM/l of glutathione, 0.5 U/l gluthione reductase and 0.34 mM/l

NADPH in 6.5 ml of buffer (0.05 M/l phosphate buffer of pH 7.2 +

4.23 mM/l of EDTA)

Reagent R2 – 10 µl of 0.18 mM/l cumene hydroperoxide in 10 ml of saline

In this assay, 20 µl of sample was mixed with 1 ml of reagent R1 and 40 µl of

reagent R2 and mixed well. The initial absorbance of sample and reagent blank was

read after 1 min and again after 1 and 2 min at a wavelength of 340 nm. The reagent

blank value was subtracted from that of the sample. Glutathione peroxidase units/mg

protein was calculated according to the manufacturer’s instruction as below:

GSH-Px Units / l of homogenate = 8412 × ΔA 340 nm / minute

GSH-Px Units / mg protein = U / l homogenate / mg protein


84

Histopathological studies

At the end of the study, the rats were sacrificed, whole pancreas from each

animal was removed, washed in normal saline and fixed in 10% formalin, embedded

in paraffin and sections of 3-5 µm thickness were cut and routinely stained with basic

dye haematoxylin and acidic dye eosin to differentiate the nucleus and cytoplasm

(Kiernan, 2008). The sections were studied at 10× and 40× magnifications for the islet

cell characteristics using a binocular compound microscope.

Statistical analysis

All the values of fasting blood sugar, biochemical estimations, body weight,

and food and water intake were expressed as mean ± standard error of mean (SEM).

Statistical difference was evaluated by using one way analysis of variance (ANOVA)

followed by Turkey’s test. Data were considered statistically significant at P value ≤ 0.05.

Statistical analysis was performed using Graph Pad statistical software.

Chapter – 3: In vivo antidiabetic activity studies Results and Discussion

85

Results and discussion

Effect of leaf extracts on blood glucose level

Administration of streptozotocin produced 68.7% increase in fasting blood

glucose levels of diabetic control rats compared to the normal control rats and the

increased glucose levels in group D rats was maintained over a period of four weeks.

Administration of A. paniculata ethanol leaf extract to streptozotocin induced diabetic

rats for four weeks produced a significant blood glucose reduction (Table 3.1).

Reduction in blood glucose was observed from the first week by both extract and

glibenclamide. At the end of 4th

week, 500 mg/kg b.w. of extract produced 29.2%

blood glucose reduction in group G rats. Similar to group G rats, there was a lowering

of 25.2% blood glucose in the rats treated with 250 mg/kg b.w. of the extract. Among

the two doses of the extracts used, 500 mg/kg b.w. of the extract showed greater

reduction in blood glucose level which was comparable to glibenclamide.

Table 3.1 : Effect of ethanol extract of Andrographis paniculata leaves on glucose

levels in streptozotocin induced diabetic rats (n=6)

Groups Fasting blood glucose levels (mg/dl)

Day 0 Day 7 Day 14 Day 21 Day 28

Group A

Normal control rats

(0.9% NaCl)

77.0 ± 1.82a

78.25 ± 0.95a

77.75± 2.21a

79.50 ± 2.64a

79.25 ± 1.70a

Group D

STZ induced

diabetic control

246.25 ± 9.53b

284.0 ± 5.47c

309.50 ± 8.73d

319.75 ± 7.80c

319.50 ± 8.66c

Group E

STZ+ glibenclamide

(0.5 mg/kg b.w.)

248.50 ± 13.52b

222.2 ± 7.27b

190.0 ± 4.54b

178.25 ± 6.80b

157.75 ± 8.95b

Group F

STZ+extract

(250 mg/kg b.w.)

235.75 ± 10.65b

219.50 ± 7.41b 205.50 ± 6.24

c 189.75 ± 10.24

b 176.25 ± 9.06

b

Group G

STZ+extract

(500 mg/kg b.w.)

233.0 ± 12.03b

216.75 ± 8.65b

198.50 ± 8.18bc

179.75 ± 10.07b 163.25 ± 12.33

b

Values are expressed as mean ± S.E.M.

Mean values with different superscripts are significantly different from each other as indicated by Turkey’s HSD (P ≤ 0.05)


86

The effect of two different doses of ethanol leaf extracts of T. cordifolia on the

fasting blood glucose levels of diabetic rats is given in Table 3.2. In the diabetic rats,

the ethanol extract at a dose of 500 mg/kg b.w. produced a 20.7% fall in the blood

glucose levels at the end of the experimental period. The glibenclamide treated

diabetic rats showed greater reduction in the blood glucose level than the extract

treated rats. Eventhough, there was a reduction in blood glucose levels with 250

mg/kg b.w. of the extract, the decrease was much lower when compared to group G1

and group E animals.

Table 3.2 : Effect of ethanol extract of Tinospora cordifolia leaves on glucose

levels in streptozotocin induced diabetic rats (n=6)



Group A

Normal control rats

(0.9% NaCl)

77.0 ± 1.82a

78.25 ± 0.95a

77.75± 2.21a

79.50 ± 2.64a

79.25 ± 1.70a

Group D

STZ induced diabetic

control

246.25 ± 9.53b

284.0 ± 5.47d

309.50 ± 8.73e

319.75 ± 7.80e

319.50 ± 8.66d

Group E

STZ+ glibenclamide

(0.5 mg/kg b.w.)

248.50 ± 13.52b

222.2 ± 7.27b

190.0 ± 4.54b

178.25 ± 6.80b

157.75 ± 8.95b

Group F1

STZ+extract

(250 mg/kg b.w.)

249.75 ± 13.74b

241.0 ± 9.05c

231.75 ± 6.80d

224.25 ± 8.88d

207.50 ± 6.65c

Group G1

STZ+extract

(500 mg/kg b.w.)

244.0 ± 13.63b

226.50 ± 4.65b

215.0 ± 5.71c

205.50 ± 5.68c

193.25 ± 6.44c



The antihyperglycemic effect of T. foenum-graecum ethanol leaf extract on the

fasting blood sugar levels of diabetic rats is shown in Table 3.3. Daily treatment of

ethanol extract of T. foenum-graecum leaves at a concentration of 250 mg/kg b.w. and

500 mg/kg b.w. for a period of 28 days led to a dose dependent fall in blood glucose

levels (23.5% and 31.2% respectively). The glucose lowering effect of the extract

seems to reach a maximum after 25 days of treatment and remains almost constant


87

during the next 4-5 days. The effect was more pronounced with animals receiving

500 mg/kg b.w. of extract which can be compared to that of glibenclamide. The

fasting blood glucose levels in normal control animals were found to be stable all

throughout the experimental period.

Table 3.3 : Effect of ethanol extract of Trigonella foenum-graecum leaves on

glucose levels in streptozotocin induced diabetic rats (n=6)



Group A

Normal control rats

(0.9% NaCl)

77.0 ± 1.82a

78.25 ± 0.95a

77.75± 2.21a

79.50 ± 2.64a

79.25 ± 1.70a

Group D


control

246.25 ± 9.53b

284.0 ± 5.47c

309.50 ± 8.73d

319.75 ± 7.80d

319.50 ± 8.66d

Group E

STZ+ glibenclamide

(0.5 mg/kg b.w.)

248.50 ± 13.52b

222.2 ± 7.27b

190.0 ± 4.54b

178.25 ± 6.80b

157.75 ± 8.95b

Group F2

STZ+extract

(250 mg/kg b.w.)

248.25 ± 10.65b

240.0 ± 12.27b

226.50 ± 8.58c

208.5 ± 7.41c

189.75 ± 6.39c

Group G2

STZ+extract

(500 mg/kg b.w.)

243.25 ± 12.03b

229.0 ± 11.40b

204.0 ± 6.21b

184.50 ± 5.44b

167.25 ± 7.80b



Diabetes mellitus, characterized by hyperglycemia, is the most common and

serious metabolic disorder that is considered to be one of the five leading causes of

death in the world (Rahimi et al., 2005). Streptozotocin induced diabetes produced a

significant increase in blood glucose levels in the diabetic rats. It has been postulated

but, is still debated that the fasting hyperglycemia in NIDDM arises from the hepatic

overproduction of glucose (De-Fronzo, 1992). However, studies by Wi et al. (1998)

suggested that the post absorptive hyperglycemia in STZ induced diabetic rats is

mainly due to the decreased peripheral glucose clearance, while increased hepatic

glucose output might also be a contributing factor at a very high STZ dose.


88

In case of A. paniculata leaf extract, there was a greater reduction in the blood

glucose at a concentration of 500 mg/kg b.w. when compared to 250 mg/kg b.w. This

shows that the effect of ethanol extract was dose dependent. Earlier investigations on

the antihyperglycemic activity of the organic extracts of A. paniculata whole plant by

various authors also substantiate the results of the present study (Zhang and Tan,

2000a; Zhang and Tan, 2000b; Husen et al., 2004). In the previous studies on

A. paniculata, aqueous extract has been shown to reduce the blood glucose level only

at higher doses (Hossain et al., 2007; Fasola et al., 2010). Moreover, our results on

leaf extract also indicate a prolonged duration of antidiabetic action in a chronic study

which could be due to multiple sites of action possessed by the active principle of

A. paniculata. The antidiabetic effect of the extract may be due to the presence of one

or more than one antihyperglycemic principle and their synergistic properties

(Mukherjee et al., 2006) as A. paniculata contains polyphenols, flavonoids and

diterpenoids as the major bioactive components (Rao et al., 2004; Xu et al., 2010). In

this study also we have shown the presence of polyphenols, flavonoids, tannins,

terpenoids and glycosides in the ethanol leaf extract. The possible mode of action may

be the extra-pancreatic action such as increased glucose uptake (Yu et al., 2003; Rao,

2006) or α-glucosidase inhibition (Subramanian et al., 2008) or mediation of

β-endomorphin (Yu et al., 2008).

Tinospora cordifolia is a widely used plant in folk and Ayurvedic systems of

medicine. Many researchers have shown the antidiabetic activity of its stem and root

extracts (Wadood et al., 1992; Grover et al., 2001; Rathi et al., 2002; Prince and

Menon, 2003). Due to this reason the leaf extract of the plant was evaluated and the

data also confirmed the traditional indications. Earlier investigations on the

antidiabetic activity of T. cordifolia root aqueous extract has shown a significant

reduction in blood glucose in alloxan diabetic rats at a concentration of 2.5 g/kg b.w.

(Prince and Menon, 2000). Various workers have shown potent antidiabetic activity

of T. cordifolia stem extract in reducing the blood sugar level in STZ induced diabetic

rats (Rajalakshmi et al., 2009; Puranik et al., 2010). There are reports on the

antihyperglycemic effects of Tinospora sinensis (Pimpriker et al., 2009) and

Tinospora crispa (Noor and Ashcroft, 1989). The results of the present study on the

leaf extract of T. cordifolia substantiate the earlier work on the antidiabetic activity of

stem and root extracts. The ethanol extract of T. cordifolia also produced significant


89

but less antihyperglycemic activity in comparison with that of ethanol extracts of

A. paniculata and T. foenum-graecum at a concentration of 500 mg/kg b.w. which

may be attributed to the presence of lower concentration of active principle in the

extract or may be due to the lower dose used. The antidiabetic effect of T. cordifolia

leaf extract may be due to the presence of diterpene lactones, steroids, phenols and

aliphatic compounds (Singh et al., 2003) or other constituents present in the extract

which could act synergistically or independently to show the antidiabetic action. The

possible mechanism by which the ethanol extract brings about its hypoglycemic

action is by potentiating the insulin effect of plasma by increasing either the

pancreatic secretion of insulin from β cells of islets of langerhans or its release from

bound insulin (Prince and Menon, 2000).

Treatment with ethanol leaf extract of T. foenum-graecum showed significant

decrease in blood glucose levels which was comparable to glibenclamide. Decrease in

blood glucose levels was found to be more effective with 500 mg/kg b.w. which

shows its dose dependent effects. Glibenclamide showed rapid normalization of blood

glucose due to its insulin releasing effects (Nima and Jagruti, 2010). Earlier studies by

various workers have shown the antihyperglycemic activity of T. foenum-graecum

seeds in a dose dependent manner in experimentally induced diabetic rats (Ali et al.,

1995; Vats et al., 2002; Xue et al., 2007). A study by Abdel-Barry et al. (1997)

showed that the intraperitoneal administration of ethanol leaf extract of T. foenum-

graecum at a concentration of 0.8 g/kg b.w. caused a reduction in blood glucose

concentration in alloxan diabetic rats. From the results of the present study it is

evident that the antihyperglycemic activity of the ethanol extract can be achieved at a

lower concentration (0.5 g/kg b.w.) when given orally. The dietary fibre fraction of

fenugreek seeds has been shown to reduce the elevated blood glucose level of type 2

diabetic rats (Hannan et al., 2003). As the leaves also contain the dietary fibre the

possible constituent for the antihyperglycemic effect may be in part the dietary fibre

or compounds other than that. From the phytochemical analysis, it was shown that the

ethanol leaf extract contains phenols, polysterols, saponins, cardiac glycosides and

flavonoids. So, the antidiabetic activity of the plant extract may involve one or more

compounds which decrease blood glucose suggesting that the natural constituents

could act synergistically to induce a hypoglycemic effect (Marles and Farnsworth,

1995; Roy et al., 2005). The hypoglycemic effect of fenugreek seed has been


90

evidenced with polar solvents in diabetic animals (Zia et al., 2001; Moorthy et al.,

2010). Similarly, our study has shown the hypoglycemic effect of fenugreek leaves in

a polar solvent like ethanol. The probable mechanism, by which the plant extract

lowered the blood glucose levels in diabetic rats, may be by increasing glycogenesis,

inhibiting gluconeogenesis in the liver, inhibiting the absorption of glucose from the

intestine or stimulation of insulin secretion (Mowla et al., 2009).

Effect of leaf extract on body weight

Prior to STZ administration, there were no significant differences in the

average body weights of all the 15 groups of experimental animals. By the end of the

first week after DM was experimentally induced, the weights of groups D, E, F, and G

were significantly reduced as shown in Table 3.4. This weight loss continued for four

weeks in diabetic control animals (135.50 g to 86.25 g). Administration of

A. paniculata ethanol leaf extract (250 mg/kg b.w. and 500 mg/kg b.w.) and

glibenclamide significantly increased the body weights in diabetic rats as compared to

diabetic control rats. The effect of 500 mg/kg b.w. of extract on the body weight of

diabetic rats was better than glibenclamide.

Table 3.4 : Effect of treatment of Andrographis paniculata leaf extract on body

weight of streptozotocin induced diabetic rats (n=6)

Groups Average body weight (g)


Group A

Normal control rats

(0.9% NaCl)

142.50 ± 5.0d

146.25 ± 2.50b

152.50 ± 5.0c

160.0 ± 4.08c

166.25±4.78c

Group D


control

135.50 ± 5.77bc

115.0 ± 5.77a

105.0 ± 5.77a

95.0 ± 4.08a

86.25 ± 4.78a

Group E

STZ+ glibenclamide

(0.5 mg/kg b.w.)

132.50 ± 5.0bc

120.0 ± 8.16a

125.0 ± 5.77b

123.75 ± 4.78b

126.25 ± 2.50b

Group F

STZ+extract

(250 mg/kg b.w.)

120.0 ± 0.00a

117.0 ± 2.44a

118.75 ± 2.50b

122.50 ± 2.88b

123.75 ± 4.78b

Group G

STZ+extract

(500 mg/kg b.w.)

125.0 ± 5.77ab

118.75 ± 2.50a

121.25 ± 2.50b

125.0 ± 4.08b

125.50 ± 5.25b




91

Change in the body weight of diabetic rats after administration of ethanol leaf

extract of T. cordifolia is summarised in Table 3.5. There was a decrease in the body

weight of diabetic rats as compared to normal rats. Administration of the extract

(250 mg/kg b.w. and 500 mg/kg b.w.) did not significantly increase the body weights

of groups F1 and G1 rats as compared to glibenclamide treated rats. As compared to

the steady decrease in the body weights of diabetic control rats, the effect of

500 mg/kg b.w. of the extract on body weight was significant.

Table 3.5 : Effect of treatment of Tinospora cordifolia leaf extract on body




Group A

Normal control rats

(0.9% NaCl)

142.50 ± 5.0b

146.25 ± 2.50b

152.50 ± 5.0c

160.0 ± 4.08c

166.25±4.78d

Group D


control

135.50 ± 5.77ab

115.0 ± 5.77a

105.0 ± 5.77a

95.0 ± 4.08a

86.25 ± 4.78a

Group E

STZ+ glibenclamide

(0.5 mg/kg b.w.)

132.5 ± 5.0ab

120.0 ± 8.16a

125.0 ± 5.77b

123.75 ± 4.78b

126.25 ± 2.50c

Group F1

STZ+extract

(250 mg/kg b.w.)

125.0 ± 5.77a

117.50 ± 15.0a

112.50 ± 12.58ab

110.0 ± 9.12b

103.75 ± 9.46ab

Group G1

STZ+extract

(500 mg/kg b.w.)

125.0 ± 5.77a

117.50 ± 12.50a

115.0 ± 12.90ab

112.50 ± 9.57b

110.0 ± 14.10bc



The effect of administration of T. foenum-graecum ethanol leaf extract on

body weights of diabetic rats is summarised in Table 3.6. The body weight of animals

in groups F2 and G2 increased on treatment with the extract of T. foenum-graecum leaf

over a period of 28 days which can be compared to that of group E animals. At the

end of the experimental period there was a significant difference in the weights of

groups F2 and G2 as compared to group D. Animals in group A showed a steady

increase in their body weights during the experimental period.


92

Table 3.6 : Effect of treatment of Trigonella foenum-graecum leaf extract on body




Group A

Normal control rats

(0.9% NaCl)

142.50 ± 5.0b

146.25 ± 2.50b

152.50 ± 5.0c

160.0 ± 4.08c

166.25±4.78c

Group D

STZ induced

diabetic control

135.50 ± 5.77ab

115.0 ± 5.77a

105.0 ± 5.77a

95.0 ± 4.08a

86.25 ± 4.78a

Group E

STZ+ glibenclamide

(0.5 mg/kg b.w.)

132.50 ± 5.0ab

120.0 ± 8.16a

125.0 ± 5.77b

123.75 ± 4.78b

126.25 ± 2.50b

Group F2

STZ+extract

(250 mg/kg b.w.)

127.50 ± 5.0a

117.50 ± 2.88a

116.25 ± 4.78b

120.0 ± 4.08b

122.5 ± 2.88b

Group G2

STZ+extract

(500 mg/kg b.w.)

133.75 ± 4.78ab

121.25 ± 2.50a

123.75 ± 2.50b

124.50 ± 4.20b

125.0 ± 4.08b



Streptozotocin induced diabetes is characterized by a severe loss in body

weight (Al-Shamaony et al., 1994; Kalaiarasi and Pugalendi, 2009). The decrease in

the body weight of diabetic rats in our study was due to the loss or degradation of

structural proteins (since structural proteins are known to contribute to the body

weight) to provide amino acids for gluconeogenesis during insulin deficiency

resulting in muscle wasting and weight loss (Swanston-Flatt et al., 1990; Rajkumar

et al., 1991). Due to insulin deficiency, protein content is decreased in muscular tissue

by proteolysis (Babu et al., 2007). Murray et al. (2003) have shown that protein

synthesis is decreased in all tissues due to decreased production of ATP and absolute

or relative deficiency of insulin. In the present study, diabetic control rats showed

marked reduction in their body weights when compared to normal rats. The weight

loss was reverted by administration of A. paniculata, T. cordifolia and T. foenum-

graecum extracts to the diabetic rats for a period of 28 days. The ability of the extracts

to protect body weight loss in diabetic rats seems to be the result of their ability to

reduce hyperglycemia (Whitton and Hems, 1975). Zhang and Tan (2000a) in their


93

study on antihyperglycemic activity of ethanol extract of aerial parts of A. paniculata

have shown a significant increase in the body weight of STZ diabetic rats receiving

the extract. Prince and Menon (2000) have shown the ability of T. cordifolia root

extract in preventing loss of body weight in alloxan induced diabetic rats. The results

of the present study substantiate the earlier work done on different parts of these

plants.

Effect of leaf extracts on food and water intake

The mean food and water intake of rats receiving A. paniculata ethanol leaf

extract are represented in Tables 3.7 and 3.8. The untreated diabetic rats had severe

polyphagia and polydipsia by the end of the experimental period with respective

increase in food and fluid intake. The mean food and water intake were significantly

lower in the extract treated group (groups F and G) than the glibenclamide treated

group and the diabetic control group. Extract of 500 mg/kg b.w. had more pronounced

effect on food and water intake than 250 mg/mg b.w. of extract. The normal control

animals showed a gradual increase in their food and water intake which can be

considered normal.

Table 3.7 : Effect of treatment of Andrographis paniculata leaf extract on food

intake in streptozotocin induced diabetic rats (n=6)

Groups Treatment Food intake (g/rat/week)

Day 7 Day 14 Day 21 Day 28

A Normal control rats

(0.9% NaCl) 57.75 ± 1.25

b 63.25 ± 1.70

c 65.50 ± 1.29

c 65.75 ± 0.95

d

D STZ induced diabetic

control 56.0 ± 1.41

ab 59.0 ± 0.81

b 59.50 ± 1.29

b 59.75 ± 2.21

c

E STZ+ glibenclamide

(0.5 mg/kg b.w.) 54.0 ± 1.41

a 49.50 ± 2.38

a 44.50 ± 1.29

a 44.0 ± 1.15

b

F

STZ+extract

(250 mg/kg b.w.) 54.50 ± 1.29

a 49.0 ± 0.81

a 42.50 ± 2.08

a 42.0 ± 2.16

ab

G

STZ+extract

(500 mg/kg b.w.) 53.25 ± 0.95

a 47.75 ± 1.70

a 42.0 ± 1.41

a 39.75 ± 1.70

a




94

Table 3.8 : Effect of treatment of Andrographis paniculata leaf extract on water


Groups Treatment Water intake (ml/rat/week)



(0.9% NaCl) 93.50 ± 2.38

a 97.50 ± 1.29

a 99.25 ± 1.25

b 101.75 ± 1.50

b


control 104.50 ± 1.29

c 111.0 ± 3.16

b 116.75 ± 2.21

c 122.25 ± 2.21

c


(0.5 mg/kg b.w.) 99.50 ± 1.73

b 94.75 ± 2.75

a 91.0 ± 0.81

a 90.50 ± 1.29

a

F

STZ+extract

(250 mg/kg b.w.) 96.25 ± 1.25

ab 93.75 ± 1.70

a 88.50 ± 1.29

a 87.50 ± 1.29

a

G

STZ+extract

(500 mg/kg b.w.) 96.0 ± 2.58

ab 94.75 ± 2.87

a 89.25 ± 4.19

a 87.25 ± 0.95

a



The changes in the food and water intake of rats treated with T. cordifolia

ethanol leaf extract are represented in Tables 3.9 and 3.10. At the end of the 28th

day

period, there was a decrease in both food and water intake in the extract treated

groups (groups F1 and G1) when compared to the diabetic control group. But, in the

glibenclamide treated group, the reduction of food and water intake was much lower

than the extract treated groups.

Table 3.9 : Effect of treatment of Tinospora cordifolia leaf extract on food intake

in streptozotocin induced diabetic rats (n=6)




(0.9% NaCl) 57.75 ± 1.25

b 63.25 ± 1.70

d 65.50 ± 1.29

d 65.75 ± 0.95

d


control 56.0 ± 1.41

ab 59.0 ± 0.81

c 59.50 ± 1.29

c 59.75 ± 2.21

c


(0.5 mg/kg b.w.) 54.0 ± 1.41

a 49.50 ± 2.38

a 44.50 ± 1.29

a 44.0 ± 1.15

a

F1

STZ+extract

(250 mg/kg b.w.) 54.75 ± 1.25

ab 53.75 ± 0.95

b 52.25 ± 1.89

b 49.50 ± 1.29

b

G1

STZ+extract

(500 mg/kg b.w.) 54.75 ± 1.70

ab 51.75 ± 0.95

ab 49.50 ± 1.29

b 47.25 ± 1.70

ab




95

Table 3.10 : Effect of treatment of Tinospora cordifolia leaf extract on water





(0.9% NaCl) 93.50 ± 2.38

a 97.50 ± 1.29

a 99.25 ± 1.25

c 101.75 ± 1.50

c


control 104.50 ± 1.29

c 111.0 ± 3.16

b 116.75 ± 2.21

d 122.25 ± 2.21

d


(0.5 mg/kg b.w.) 99.50 ± 1.73

b 94.75 ± 2.75

a 91.0 ± 0.81

a 90.50 ± 1.29

a

F1

STZ+extract

(250 mg/kg b.w.) 100.25 ± 2.75

b 99.50 ± 1.73

a 97.75 ± 1.50

bc 97.0 ± 1.82

b

G1

STZ+extract

(500 mg/kg b.w.) 99.75 ± 1.70

b 97.25 ± 1.70

a 95.0 ± 0.81

b 92.75 ± 1.70

a



The food and water intake of experimental animals treated with T. foenum-

graecum ethanol leaf extract are shown in Tables 3.11 and 3.12. Treatment with the

extract lowered the food and water intake of the groups F2 and G2. The result was

more evident with respect to rats treated with 500 mg/kg b.w. of extract than

250 mg/kg b.w. which was comparable to group E animals.

Table 3.11 : Effect of treatment of Trigonella foenum-graecum leaf extract on

food intake in streptozotocin induced diabetic rats (n=6)




(0.9% NaCl) 57.75 ± 1.25

b 63.25 ± 1.70

c 65.50 ± 1.29

d 65.75 ± 0.95

d


control 56.0 ± 1.41

ab 59.0 ± 0.81

b 59.50 ± 1.29

c 59.75 ± 2.21

c


(0.5 mg/kg b.w.) 54.0 ± 1.41

a 49.50 ± 2.38

a 44.50 ± 1.29

ab 44.0 ± 1.15

b

F2

STZ+extract

(250 mg/kg b.w.) 54.25 ± 0.95

a 51.25 ± 1.25

a 46.0 ± 1.41

b 41.50 ± 1.29

ab

G2

STZ+extract

(500 mg/kg b.w.) 54.25 ± 1.50

a 48.0 ± 2.16

a 42.50 ± 2.08

a 39.50 ± 1.29

a




96

Table 3.12 : Effect of treatment of Trigonella foenum-graecum leaf extract on water





(0.9% NaCl) 93.50 ± 2.38

a 97.50 ± 1.29

b 99.25 ± 1.25

b 101.75 ± 1.50

c


control 104.50 ± 1.29

c 111.0 ± 3.16

c 116.75 ± 2.21

c 122.25 ± 2.21

d


(0.5 mg/kg b.w.) 99.50 ± 1.73

b 94.75 ± 2.75

ab 91.0 ± 0.81

a 90.50 ± 1.29

b

F2

STZ+extract

(250 mg/kg b.w.) 96.75 ± 1.25

ab 93.75 ± 0.95

ab 89.25 ± 0.95

a 88.5 0± 1.29

ab

G2

STZ+extract

(500 mg/kg b.w.) 96.5 ± 2.08

ab 91.0 ± 1.82

a 87.75 ± 2.06

a 85.0 ± 3.36

a



The increase in food and water intake in diabetic rats is a classical symptom of

DM which was observed in all diabetic rats. The increase which was seen in the initial

period was decreased after the administration of the plant extracts for a period of

28 days. The metabolic disorders were corrected as shown by the reduction in

polyphagia, polyuria and polydipsia in diabetic rats treated with plant extracts. This

could be the result of improved glycemic control produced by ethanol extracts of

A. paniculata, T. cordifolia and T. foenum-graecum which was better seen with the

higher doses. The results obtained in our study supports the earlier study by Zhang and

Tan (2000a) on crude extract of A. paniculata aerial parts at a dose of 400 mg/kg b.w.

Effect on kidney parameters

Serum and urinary creatinine levels and serum urea levels of normal and

diabetic rats treated with A. paniculata ethanol leaf extract is shown in Table 3.13.

Diabetic control rats exhibited higher serum creatinine, urinary creatinine and serum

urea levels compared to those of normal rats. The creatinine and urea levels were

significantly decreased by glibenclamide and the extract due to 28 days of treatment. In

groups F and G animals there was a reduction in serum creatinine (74.1% and 71.9%),

urinary creatinine (63.4% and 60.6%) and serum urea (50.5% and 49.3%) levels as

compared to diabetic untreated animals. In the normal control rats fed with the extract

(groups B and C) the creatinine and urea levels were similar to the normal control rats.


97

Table 3.13 : Effect of treatment of Andrographis paniculata leaf extract on serum

creatinine, urinary creatinine and serum urea levels in normal and

streptozotocin induced diabetic rats (n=6)

Groups Treatment

Serum

creatinine

(mg/dl)

Urinary

creatinine

(mg/day)

Serum urea

(mg/dl)


(0.9% NaCl) 0.47 ± 0.04

a 18.72 ± 1.83

a 23.74 ± 0.73

a

B

Normal rats with extract

(250 mg/kg b.w.) 0.44 ± 0.04

a 18.42 ± 0.75

a 23.79 ± 0.26

a

C


(500 mg/kg b.w.) 0.43 ± 0.08

a 18.25 ± 0.34

a 23.72 ± 0.43

a

D STZ induced

diabetic control 1.82 ± 0.17

b 51.52 ± 3.76

c 61.94 ± 1.54

d


(0.5 mg/kg b.w.) 0.60 ± 0.07

a 23.61 ± 2.38

b 29.73 ± 0.59

b

F

STZ+extract

(250 mg/kg b.w.) 0.51 ± 0.01

a 20.25 ± 0.91

ab 31.40 ± 0.62

c

G

STZ+extract

(500 mg/kg b.w.) 0.47 ± 0.04

a 18.85 ± 0.71

a 30.63 ± 0.46

bc



Table 3.14 represents the creatinine and urea levels in normal and diabetic rats

treated with T. cordifolia ethanol leaf extract. There was a reduction in the creatinine

and urea levels in the extract fed diabetic rats. Extract of 500 mg/kg b.w. showed

greater reduction in the serum creatinine (65.3%), urinary creatinine (47.9%) and

serum urea (47.0%) levels. In the diabetic control rats there was a significant increase

in the creatinine and urea levels. In the normal rats fed with the extract (groups B1 and

C1) the creatinine and urea levels were normal.


98

Table 3.14 : Effect of treatment of Tinospora cordifolia leaf extract on serum

creatinine, urinary creatinine and serum urea levels in normal and

streptozotocin induced diabetic rats (n=6)

Groups Treatment

Serum

creatinine

(mg/dl)

Urinary

creatinine

(mg/day)

Serum urea

(mg/dl)


(0.9% NaCl) 0.47 ± 0.04

ab 18.72 ± 1.83

a 23.74 ± 0.73

a

B1


(250 mg/kg b.w.) 0.48 ± 0.02

ab 18.45 ±1.04

a 23.62 ± 0.43

a

C1


(500 mg/kg b.w.) 0.43 ± 0.04

a 18.45 ± 1.86

a 23.65 ± 0.34

a

D STZ induced


d 51.52 ± 3.76

d 61.94 ± 1.54

e


(0.5 mg/kg b.w.) 0.60 ± 0.07

abc 23.61 ± 2.38

b 29.73 ± 0.59

b

F1

STZ+extract

(250 mg/kg b.w.) 0.68 ± 0.07

c 28.45 ± 1.92

c 35.53 ± 0.56

d

G1

STZ+extract

(500 mg/kg b.w.) 0.63 ± 0.03

bc 26.80 ± 2.64

bc 32.78 ± 0.68

c



The effect of T. foenum-graecum ethanol leaf extract on serum and urinary

creatinine and serum urea levels is shown in Table 3.15. The extract at a concentration

of 500 mg/kg b.w. showed a greater decrease in serum creatinine (73.6%), urinary

creatinine (62.7%) and serum urea (50.6%) levels when compared to diabetic

untreated rats. There were no changes in the creatinine and urea levels in normal

extract treated controls (groups B2 and C2) as compared to the normal controls.


99

Table 3.15 : Effect of treatment of Trigonella foenum-graecum leaf extract on

serum creatinine, urinary creatinine and serum urea levels in

normal and streptozotocin induced diabetic rats (n=6)

Groups Treatment

Serum

creatinine

(mg/dl)

Urinary

creatinine

(mg/day)

Serum urea

(mg/dl)


(0.9% NaCl) 0.47 ± 0.04

ab 18.72 ± 1.83

a 23.74 ± 0.73

a

B2


(250 mg/kg b.w.) 0.41 ± 0.08

a 18.72 ± 0.62

a 23.62 ± 0.79

a

C2


(500 mg/kg b.w.) 0.36 ± 0.05

a 18.32 ± 0.36

a 23.42 ± 0.74

a

D STZ induced


c 51.52 ± 3.76

c 61.94 ± 1.54

d


(0.5 mg/kg b.w.) 0.60 ± 0.07

b 23.61 ± 2.38

b 29.73 ± 0.59

b

F2

STZ+extract

(250 mg/kg b.w.) 0.50 ± 0.03

ab 22.22 ± 2.30

ab 32.63 ± 0.29

c

G2

STZ+extract

(500 mg/kg b.w.) 0.48 ± 0.01

ab 19.17 ± 0.84

a 30.55 ± 0.44

b



Diabetic nephropathy is the leading cause of DM related morbidity and

mortality. The pathogenesis of diabetic nephropathy is related to chronic

hyperglycemia and hemodynamic alterations in renal microcirculation and structural

changes in glomerulus as evident by the significant elevation in creatinine and urea

levels (Cryer, 2001). Measurement of creatinine and urea levels reflects the function

of kidneys (Smith et al., 2006). Most of the reports on toxic effects of herbal

medicines and dietary supplements are associated with hepatoxicity. But, reports of

other toxic effects including kidney, nervous system, blood, cardiovascular,

dermatologic effects, mutagenecity and carcinogenicity have also been published in

medical literature (Niggermann and Gruber, 2003; Pak et al., 2004). Streptozotocin

induced diabetes is associated with generation of free radicals and oxidant tissue

damage, increasing the risk of renal complications (Elena et al., 2001). The ROS

produced affect the renal functions by promoting varieties of vasoactive mediators


100

such as thromboxane, which in turn causes renal vasoconstriction and alter the

glomerular filtration rate (Craven et al., 1992). Hence measurement of these two

parameters enables the study of toxic effects of the drug on the kidney.

The increased creatinine and urea levels in the diabetic rats indicates renal

damage due to abnormal glucose regulation, including elevated glucose and

glycosylated protein tissue levels, hemodynamic changes within the kidney tissue and

increased oxidative stress (Veeramani et al., 2008). Treatment of ethanol leaf extracts

of A. paniculata, T. cordifolia and T. foenum-graecum caused a reduction in the

creatinine and urea levels thereby enhancing the renal function that is generally

impaired in diabetic rats. The results of the present study are in agreement with other

previous studies on the herbal formulation D-400 (Dubey et al., 1994), mesocarp

extract of Balanites aegyptiaca (Mansour and Newairy, 2000), fruit extract of

Terminalia catappa (Nagappa et al., 2003), chloroform root extract of A. paniculata

(Rao, 2006), ginger and clove oils (Atef et al., 2007) and Annona squamosa extract

(Kaleem et al., 2008) which showed lowering of creatinine and urea levels on

supplementation with various plant extracts.

The normal levels of creatinine and urea in the normal healthy rats fed with

the extracts of A. paniculata, T. cordifolia and T. foenum-graecum at a concentration

of 250 mg/kg b.w. and 500 mg/kg b.w. for a period of 28 days revealed the non-toxic

nature of the ethanol extract and also showed the normal kidney function. No

mortality was observed in the extract treated rats and behaviour of the treated rats also

appeared normal. Rajalakshmi et al. (2009) in their study on stem extract of

T. cordifolia have shown the non-toxic nature of the methanol extract. Similar results

were observed in the ethanol leaf extract of T. cordifolia.

Effect on liver antioxidant enzymes

As shown in Table 3.16, STZ induced diabetic control rats showed a

significant decrease in SOD, catalase and GSH-Px levels as compared to the normal

rats. Treatment with A. paniculata ethanol leaf extract for 28 days produced a

significant increase in the activities of the antioxidant enzymes comparable to that of

glibenclamide treated group. Treatment with 500 mg/kg b.w. of extract produced

more pronounced effect with an increase in the activities of SOD (10.29 U/mg


101

protein), catalase (8.57 U/min/mg protein) and GSH-Px (5.31 U/mg protein). The

activities of the enzymes in the non diabetic rats treated with extracts (groups B and

C) were similar to the normal control rats.

Table 3.16 : Effect of ethanol extract of Andrographis paniculata leaves on liver

antioxidant enzymes in normal and streptozotocin induced diabetic

rats (n=6)

Groups Treatment

Superoxide

dismutase

(units/mg

protein)

Catalase

(units/min/mg

protein)

Glutathione

peroxidase

(units/mg protein)


(0.9% NaCl) 10.50 ± 0.54

b 10.70 ± 1.13

c 5.73 ± 0.28

c

B


(250 mg/kg b.w.) 10.00 ± 0.49

b 10.60 ± 0.66

c 5.70 ± 0.14

c

C


(500 mg/kg b.w.) 10.32 ± 0.44

b 10.97 ± 0.81

c 5.82 ± 0.16

c

D STZ induced


a 6.37 ± 0.48

a 3.45 ± 0.36

a


(0.5 mg/kg b.w.) 10.02 ± 0.33

b 8.82 ± 0.63

b 5.26 ± 0.27

bc

F

STZ+extract

(250 mg/kg b.w.) 9.78 ± 0.43

b 8.05 ± 0.20

b 4.82 ± 0.21

b

G

STZ+extract

(500 mg/kg b.w.) 10.29 ± 0.40

b 8.57 ± 0.29

b 5.31 ± 0.26

bc



The activities of the liver antioxidant enzymes in normal and diabetic rats

treated with T. cordifolia ethanol leaf extract is summarised in Table 3.17. Treatment

with the extract increased the enzyme activities but, the increase was lower when

compared to the glibenclamide treated rats. The effect of 500 mg/kg b.w. of extract

was more than 250 mg/kg b.w. of extract. There was an increase in the activity of

SOD in the non diabetic rats treated with 250 mg/kg b.w and 500 mg/kg b.w. of the

extract. Marginal increase in the activities of catalase and GSH-Px were found with

group C1 rats.


102

Table 3.17 : Effect of ethanol extract of Tinospora cordifolia leaves on liver

antioxidant enzymes in normal and streptozotocin induced diabetic

rats (n=6)

Groups Treatment

Superoxide

dismutase

(units/mg

protein)

Catalase

(units/min/mg

protein)

Glutathione

peroxidase

(units/mg

protein)


(0.9% NaCl) 10.50 ± 0.54

cd 10.70 ± 1.13

d 5.73 ± 0.28

cd

B1


(250 mg/kg b.w.) 10.89 ± 0.14

cd 10.57 ± 0.48

d 5.75 ± 0.26

cd

C1


(500 mg/kg b.w.) 11.41 ± 0.82

d 11.10 ± 0.31

d 5.93 ± 0.19

d

D STZ induced


a 6.37 ± 0.48

a 3.45 ± 0.36

a


(0.5 mg/kg b.w.) 10.02 ± 0.33

c 8.82 ± 0.63

c 5.26 ± 0.27

bc

F1

STZ+extract

(250 mg/kg b.w.) 7.88 ± 0.47

b 7.10 ± 0.54

ab 4.84 ± 0.26

b

G1

STZ+extract

(500 mg/kg b.w.) 8.63 ± 0.53

b 7.95 ± 0.26

bc 4.98 ± 0.10

b



Table 3.18 shows the level of antioxidant enzyme activities in liver of normal

and diabetic rats treated with T. foenum-graecum ethanol leaf extract. Administration

of the leaf extract for 28 days produced a marked increase in the enzyme activities.

There was a significant increase in the activity of SOD (11.50 U/mg protein), catalase

(8.32 U/min/mg protein) and GSH-Px (5.48 U/mg protein) in diabetic rats treated with

500 mg/kg b.w. of extract which was greater than the glibenclamide treated rats.

There was an increase in the enzyme activities in group B2 and C2 animals.


103

Table 3.18 : Effect of ethanol extract of Trigonella foenum-graecum leaves on

liver antioxidant enzymes in normal and streptozotocin induced

diabetic rats (n=6)

Groups Treatment

Superoxide

dismutase

(units/mg

protein)

Catalase

(units/min/mg

protein)

Glutathione

peroxidase

(units/mg protein)


(0.9% NaCl) 10.50 ± 0.54

bc 10.70 ± 1.13

d 5.73 ± 0.28

cd

B2


(250 mg/kg b.w.) 12.39 ± 0.75

d 11.12 ± 0.40

d 6.08 ± 0.18

de

C2


(500 mg/kg b.w.) 14.15 ± 0.26

e 11.35 ± 0.42

d 6.30 ± 0.24

e

D STZ induced


a 6.37 ± 0.48

a 3.45 ± 0.36

a


(0.5 mg/kg b.w.) 10.02 ± 0.33

b 8.82 ± 0.63

c 5.26 ± 0.27

bc

F2

STZ+extract

(250 mg/kg b.w.) 10.08 ± 0.43

b 7.52 ± 0.61

ab 4.93 ± 0.16

b

G2

STZ+extract

(500 mg/kg b.w.) 11.50 ± 1.26

cd 8.32 ± 0.40

bc 5.48 ± 0.17

c



There is an increasing evidence in both experimental and clinical studies

which suggests that diabetes is associated with oxidative stress, leading to an

increased production of ROS, including superoxide radical, hydrogen peroxide and

hydroxyl radical (Young et al., 1995; Baynes and Thorpe, 1999; Arulselvan and

Subramanian, 2007; Gokce and Haznedaroglu, 2008; Bagri et al., 2009). Recently,

much attention has been focussed on the role of oxidative stress and it has been

suggested that oxidative stress may constitute the key and common events in the

pathogenesis of different diabetic complications (Maritim et al., 2003; Sepici-Dincel

et al., 2007). Several lines of evidences indicate that free radicals may play an

essential role in the mechanism of β cell damage and diabetogenic effect of STZ

(Ohkuwa et al., 1995). Streptozotocin induced hyperglycemia induces free radical

generation which thereby leads to DNA damage, protein degradation, lipid

peroxidation and finally culminating into damage to various organs of the body like

liver, kidney, brain and eyes (Feldman, 1988; Saxena et al., 1993; Garg and Bansal,

2000; Yazdanparast et al., 2007).


104

Diabetics and experimental animal models exhibit high oxidative stress due to

persistent and chronic hyperglycemia, which thereby depletes the activity of

antioxidative defence system and thus promote de novo free radical generation

(Nazirogilu and Butterworth, 2005; Kamalakannan and Prince, 2006). Free radical

production caused by hyperglycemia may occur via at least three different routes: non

enzymatic glycation (Cariello et al., 1992), auto-oxidation of glucose (Jiang et al.,

1990) and intracellular activation of the polyol pathway (Cariello, 2000). An

imbalance of oxidant/antioxidant defence systems result in alterations in the activity

of antioxidant enzymes such as SOD, catalase and GSH-Px (Maritim et al., 2003).

Antioxidant enzymes have been shown to play an important role in maintaining

physiological levels of oxygen and hydrogen peroxide by hastening the dismutation of

oxygen radicals and eliminating organic peroxides and hydroperoxides generated

from inadvertent exposure to STZ (Pari and Latha, 2004).

Enzymatic antioxidants such as SOD and catalase are considered primary

enzymes since they are involved in the direct elimination of ROS (Arulselvan and

Subramanian, 2007). Superoxide dismutase is the first line of defence against free

radical attacks. Its function is to catalyse the conversion of superoxide radicals to

hydrogen peroxide and hence diminishes the toxic effects due to this radical or other

free radicals derived from secondary reaction (Manonmani et al., 2005). The

hydrogen peroxide produced by SOD is excreted as water, based on the activity of

GSH-Px and catalase, thereby protecting the body from oxygen toxicity (Punitha

et al., 2005). Hence in the present study activities of SOD, catalase and GSH-Px in

non diabetic and diabetic rats treated with extracts were evaluated.

In the present study, the activity of SOD, catalase and GSH-Px were decreased

in liver homogenate of diabetic control rats compared to normal rats, indicating

dysfunction in antioxidant defensive system which could be due to free radical

induced inactivation or glycation of the enzyme in diabetic state (Al-Azzawie and

Alhamdani, 2006). Schettler et al. (1994) suggested that the reduced antioxidant

production is due to increased oxygen metabolites causing a decrease in the activity of

the antioxidant defence system. Various studies in the past reported conflicting results

regarding the status of antioxidant enzymes in DM. Majority of the authors reported

the decreased enzymatic antioxidant activities in diabetic rats (Pari and Latha, 2004;

Pavana et al., 2007; Sarkhail et al., 2007; Kapoor et al., 2009). A few authors have


105

shown an increased activity of catalase in diabetic erythrocytes (Terekhina et al.,

1998) or tissues (Cekic et al., 1999). Hazem et al. (2007) reported elevated levels of

enzymatic antioxidants SOD and reduced catalase in diabetic rats.

Treatment with A. paniculata, T. cordifolia and T. foenum-graecum ethanol

leaf extracts for a period of 28 days reversed the activities of these enzymatic

antioxidants, which could be due to decreased oxidative stress. Zhang and Tan

(2000b) reported reduced activities of hepatic SOD, catalase and GSH-Px in untreated

diabetic rats. Oral administration of ethanol extract of aerial parts of A. paniculata for

a period of 14 days resulted in a significant elevation of activities of SOD and

catalase, but the extract had no significant effect on GSH-Px activity (Zhang and Tan,

2000b). But, the present study with A. paniculata ethanol leaf extract has shown the

increased activities of all the three antioxidant enzymes and shows agreement with the

earlier study of Trivedi and Rawal (2001). Alcoholic root extract of T. cordifolia

administered orally for six weeks normalized the antioxidant status of SOD, catalase

and GSH-Px (Prince and Menon, 2001; Prince et al., 2004a; Prince et al., 2004b). The

results from the present study on T. cordifolia ethanol leaf extract support the earlier

studies. Trigonella foenum-graecum seeds have been shown to normalise the

antioxidant functions of liver enzymes in diabetic rats (Ravikumar and Anuradha,

1999; Anuradha and Ravikumar, 2001; Genet et al., 2002). Similar results were

observed in diabetic rats treated with T. foenum-graecum leaf extract in the present

study. Various other plant extracts, Punica granatum (Bagri et al., 2009), Rosmarinus

officinalis (Bakirel et al., 2008) and Phlomis anisodonta (Sarkhail et al., 2007) have

shown significant increase in the activities of SOD, catalase and GSH-Px on

administration to diabetic animals. Normal antioxidant enzyme levels in non diabetic

control rats treated with A. paniculata and T. cordifolia extracts indicates the normal

functioning of the liver and also the non-toxic nature of the extracts. The

improvement in the enzyme activities in non diabetic control rats treated with

T. foenum-graecum extracts is indicative of better liver function. The result of our study

is in agreement with the earlier study on fenugreek seeds (Muralidhara et al., 1999).

Theoretically, hypoglycemic plants act through a variety of mechanisms such

as improving insulin sensitivity, augmenting glucose dependent insulin secretion and

stimulating the regeneration of islets of langerhans in pancreas of STZ induced

diabetic rats (Sezik et al., 2005). Moreover, the role of antioxidant compounds in both

protection and therapy of diabetics have been considered in various scientific


106

researches. Treatment of STZ injected diabetic animals with N-acetly-L-Cysteine, a

well known antioxidant, prevented hyperglycemia through reduced oxidative stress

and restoring β cell function (Takatori et al., 2004). In this regard, it is anticipated that

the ethanol leaf extracts of A. paniculata, T. cordifolia and T. foenum-graecum acts by

decreasing the oxidative damage to pancreatic tissue. However, by this speculation,

we do not exclude the possibilities of other mechanisms by which the ethanol leaf

extracts exerts their effects.

Histopathological studies

Figures 3.1 and 3.2 are the photomicrographs of the pancreas of a normal rat

showing normal architecture and normal islets of langerhans. Figures 3.3 and 3.4 are the

photomicrographs of the pancreas of diabetic untreated rat. There were lymphocytic

infiltrations. Atrophy and destruction of β cells were marked. Islet cells were small and

shrunken. Figures 3.5 and 3.6 are photomicrographs of the pancreas of diabetic rat

treated with glibenclamide. There was an increase in the number of islet cells.

Hematoxylin and eosin sections of the pancreas of diabetic rats treated with

ethanol leaf extract of A. paniculata are shown in Figures 3.7 to 3.10. In animals

treated with 250 mg/kg b.w. and 500 mg/kg b.w. of extract there were regenerative

changes in tissue architecture of islet cells of pancreas. In animals treated with 500

mg/kg b.w. of extract, there was a marked increase in the number and size of islet

cells especially in the β cell region compared to animals treated with 250 mg/kg b.w.

of extract. Figures 3.11 to 3.14 are the photomicrographs of the pancreas of the

diabetic rats treated with 250 mg/kg b.w. and 500 mg/kg b.w. of T. cordifolia ethanol

leaf extract. In animals treated with 250 mg/kg b.w. of extract, there was a slight

improvement in the architecture of the islet cells as compared to diabetic control rats.

But in animals treated with 500 mg/kg b.w. of extract, there was a further

improvement in the architecture of islet cells with many cells showing normal shape

and size. Figures 3.15 to 3.18 are the photomicrographs of the pancreas of diabetic

rats treated with 250 mg/kg b.w. and 500 mg/kg b.w. of T. foenum-graecum ethanol

leaf extract. In diabetic rats treated with 250 mg/kg b.w. of the extract, the restoration

of the normal cellular population and size of islets were noted especially in the central

β cell region. In animals treated with 500 mg/kg b.w. of the extract, there was not

only an increase in the cellular population and size of the islets but also an increase in

the number of islets. The regeneration noted in the β cell regions was comparable to

that noted with glibenclamide.


107

Fig. 3.1: Normal rat Fig. 3.2: Normal rat

pancreas (10×) pancreas (40×)

Fig. 3.3: Diabetic untreated Fig. 3.4: Diabetic untreated

rat pancreas (10×) rat pancreas (40×)

Fig. 3.5: Glibenclamide treated Fig. 3.6: Glibenclamide treated



108

Fig. 3.7: A. paniculata treated Fig. 3.8: A. paniculata treated

(250 mg/kg b.w.) diabetic (250 mg/kg b.w.) diabetic


Fig. 3.9: A. paniculata treated Fig. 3.10: A. paniculata treated

(500 mg/kg b.w.) diabetic rat (500 mg/kg b.w.) diabetic

pancreas (10×) rat pancreas (40×)


109

Fig. 3.11: T. cordifolia treated Fig. 3.12: T. cordifolia treated



Fig. 3.13: T. cordifolia treated Fig. 3.14: T. cordifolia treated




110

Fig 3.15: T. foenum-graecum treated Fig 3.16: T. foenum-graecum treated



Fig. 3.17: T. foenum-graecum treated Fig. 3.18: T. foenum-graecum treated




111

Pancreas is a compound tubular, alveolar, partly exocrine and partly endocrine

gland. The exocrine part of the pancreas is in the form of serous acini, secreting the

secretions into intralobular duct. The endocrine part of the pancreas is in the form of

numerous rounded collections of cells known as islet of langerhans, embedded within

the exocrine part. Each islet is separated by the surrounding alveoli by a thin layer of

reticular tissue. The average islet in rats is 150 µm in diameter and contains about 45 ng

of insulin. There are four major endocrine cell types in mammalian islets; the insulin

producing β cells, the glucagon producing α cells, the somatostatin producing δ cells and

pancreatic polypeptide producing pp cells. The β cells are polyhedral, being truncated

pyramids and are usually well granulated with secretory granules 250-300 nm in

diameter. It has been estimated that each rat β cell contains about 10,000 granules

(Bonner-Weir and Smith, 1994).

Streptozotocin (N-[methylnitrocarbamoyl]-D-glucosamine) is well known for

its selective pancreatic islet β cell cytotoxicity and in many animal species STZ

induces diabetes that resembles human hyperglycemic non-ketotic DM (Weir et al.,

1981; Papaccio et al., 2000). Further, rats treated with STZ displays many of the

features seen in human subjects with uncontrolled DM and are invaluable when

studying the mechanism by which hyperglycemia may contribute to complications

such as nephropathy, retinopathy and neuropathy (Obrosova et al., 2005). So, in the

present investigation, the histological changes in the tissues of pancreas of diabetic

rats and effect of the extracts of A. paniculata, T. cordifolia and T. foenum-graecum

on the islet cells of pancreas were studied.

Microscopic examination shows abundant patches of β cells in the pancreas of

normal rats which are absent in diabetic pancreas (Anil et al., 1996). The decrease in

the cellularity, small and shrunken islets and destruction of β cells within islets of

langerhans observed in diabetic rats in the present study reflects the cytotoxicity of

STZ (Mitra et al., 1996; Szudelski, 2001; Xiu et al., 2001; Selvan et al., 2008). The

β cells in some islets of diabetic pancreas were found to be fusiform. The change in

shape of cells can be attributed to the partial damage by STZ. Aybar et al. (2001) have

reported that the use of lower dose of STZ produces an incomplete destruction of

pancreatic β cells eventhough rats become permanently diabetic. Streptozotocin

destroys β cells selectively and a single adequate dose produces lasting hyperglycemia


112

and insulin deficiency (Szaleczky et al., 1999). It has been reported that STZ enters

β cells via a glucose transporter (GLUT2) and causes alkylation of DNA. Other

studies indicated that cytotoxic effects of STZ are dependent upon DNA alkylation by

site specific action with DNA bases (Benneth and Pegg, 1981) and by free radical

generation during STZ metabolism (Bolzan and Bianchi, 2002).

Andrographis paniculata ethanol extract significantly increased the number

and size of islet cells especially in the β cell region. A recent study has shown that

andrographolide-lipoic acid conjugate (andrographolide analogue) had both

hypoglycemic and β cell protective effects (Zhang et al., 2009). The protective effect

of A. paniculata ethanol leaf extract may be due to the andrographolide or other

diterpenoids, flavonoids or polyphenols present in the plant (Matsuda et al., 1994;

Cheung et al., 2001; Pholphana et al., 2004; Rao et al., 2004).

A study by Rajalakshmi et al. (2009) on T. cordifolia stem have shown that

the alterations in the islet cells caused due to STZ was corrected to normal by

methanol extract treatment at a dose of 250 mg/kg b.w. for a period of 100 days. The

result obtained after treatment with T. cordifolia leaf extract to diabetic rats in the

present study is consistent with the earlier results and has shown the reversal of tissue

architecture of number of islet cells in diabetic treated rats but not to the extent of

becoming normal. It may be due to the shorter experimental period of 28 days as

compared to 100 days. A study by Puranik et al. (2010) on T. cordifolia stem extract

did not show any change in the altered architecture of the islet cells of pancreas even

after the treatment and the results of our study are contrary to this study. The

antihyperglycemic activity of T. cordifolia leaf extract may be through the increased

insulin secretion by pancreatic β cells or due to increased entry of glucose into the

peripheral tissues and organs (Prince and Menon, 2000). The antidiabetic activity of

T. cordifolia leaves may be due to the presence of diterpene lactones, phenols and

aliphatic compounds (Singh et al., 2003).

There was regeneration of islet cells especially in the β cell region in the

pancreatic tissues of rats treated with T. foenum-graecum ethanol leaf extract.

Nagappa et al. (2003) in their study had shown the regeneration of β cell due to the

presence of β carotene in Terminalia catappa fruits. The activity of T. foenum-


113

graecum ethanol leaf extract in the present study might be due to the presence of

β carotene (Srinivasan, 2006) or other phytochemicals present in its leaves.

Photomicrographical data in our studies reinforce the healing of pancreas, by

T. foenum-graecum leaf extract as a plausible mechanism of their antidiabetic activity.

Various other studies have reported the regeneration islet cells of pancreas.

Shanmugasundaram et al. (1990) have reported the activity of Gymnema sylvestre leaf

extract in regenerating the damaged endocrine tissue and increasing β cell numbers

partially. Sharma et al. (2003) have reported that oral administration of Eugenia

jambolana seed extract reversed the abnormalities in the islet of langerhans of alloxan

induced diabetic rabbits.

chapter - 3 - shodhganga : a reservoir of indian theses...

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