1. Introduction:

Diabetics have accelerated levels of oxidative stress and this contributes massively to most cardiovascular, neurological, retinal and renal diabetic complications¹. Diabetes mellitus is a heterogeneous metabolic disorder as old as mankind and its incidence is considered to be high all over the world². It is characterized by hyperglycemia. Hyperglycemia significantly diminishes glutathione levels lowering defences against oxidative stress³.

A multitude of herbs, spices and other plant material has been described for the treatment of diabetes throughout the world⁴. Furthermore, after the recommendations made by WHO (World Health Organization) on diabetes mellitus, investigations on hypoglycemic agents from medicinal plants have become more important. The levels of serum lipids are usually elevated in diabetes mellitus and such an elevation represents the risk factor for coronary heart disease. Moreover, diabetic patients experience a two to three fold increase in cardiovascular morbidity and mortality in comparison to non diabetics⁵.

Amritarishta is a polyherbal hydroalcoholic Ayurvedic preparation and is used as antioxidant and advised as a choice of remedy in mostly all types of fevers⁶. The chief ingredient of Amritarishta is guduchi, dried stem of Tinospora cordifolia. The chemical constituents reported from stems of Tinospora cordifolia belong to different classes such as alkaloids as tinosporin^7-8, glycosides as cordifoliosides-A and cordifolioside-B^9-10, steroids as β- sitosterol¹¹, sesquiterpenoid as tinocordifolin¹² and a large amount of phenolic compounds as gallic aciod, ellagic acid, catechin and epicatechin¹³. These compounds have many notable medicinal properties as antidiabetic¹⁴, hepatoprotective¹⁵, antioxidant¹⁶, antimalarial¹⁷, immunomodulatory¹⁸ and antineoplastic properties¹⁹.

Therefore we undertook the present investigation to evaluate the antidiabetic effect of Amritarishta-T and Amritarishta-M prepared by traditional and modern methods respectively and also marketed Amritarishta in alloxan induced hyperglycemic rats. Effect of these preparations was also evaluated on serum lipid profile of alloxan induced diabetic rats.

2. Materials and Methods:

2.1 Preparation of Amritarishta-T:

This was prepared by the method as given in The Ayurvedic Formulary of India, Part-I^6. All the ingredients of Amritarishta were procured from local market, Jamnagar while jaggery was procured from local market, Mehsana. Authentication of all the ingredients of Amritarishta was done by Dr. G. D. Bagchi, Scientist, Department of Taxonomy and Pharmacognosy, Central Institute of Medicinal and Aromatic Plants, Lucknow. Prepared herbarium has been deposited in the Central Institute of Medicinal and Aromatic Plants, Lucknow for future reference. Identification of all the individual plant material was done as per The Ayurvedic Pharmacopoeia of India. Quantity of ingredients taken for the preparation of batch size 3.072 l of Amritarishta has been calculated according to the formula as given in The Ayurvedic Formulary of India, Part-I, 2000.

According to this method, coarsely powdered stems of guduchi (Tinospora cordifolia) with prescribed ingredients as Aegle marmelos (stem bark), Oroxylum indicum (roots), Gmelina arborea (stem bark), Stereospermum suaveolns (stem bark), Premna integrifolia (stem bark), Hedysarum gangeticum (entire plant), whole plant of Paederia foetida, entire plant of Solanum indicum, entire plant of Solanum xanthocarpum and Tribulus terrestris were placed in polished vessel of brass along with prescribed quantity of water (12.288l) and allowed to steep. After 12 h of steeping, this material was warmed at medium flame until the water for decoction reduced to one fourth of the prescribed quantity(3.072 l) , then the heating was stopped and it was filtered in cleaned vessel and after that jaggery was added and mixed properly. Then, prakshepa dravyas as svet jiraka, raktapuspaka, saptaparni, sunthi, marica, pippali, nagakesara, mustaka, katuka, ativisa and indravaruni in fine powdered form were added and this sweet filtered material was placed for fermentation in incubator for fifteen days at 33±1°C. After 15 days completion of fermentation was confirmed by standard tests²⁰. The fermented preparation was filtered with cotton cloth and kept in clean covered vessel for further next seven days. Then, when the fine suspended particles settled down, it is strained again and poured in amber colored glass bottles previously rinsed with ethyl alcohol, packed and properly labelled.

2.2 Preparation of Amritarishta-M:

Method of preparation of Amritarishta-M was same as followed with Amritarishta-T only in addition to jaggery, yeast was also added for inducing fermentation²¹.

2.3 Animals

Adult Wistar albino rats, weighing between 200-220g of either sex were acclimatized to normal environmental conditions in the animal house for one week. The animals were housed in standard polypropylene cages and maintained under controlled room temperature (22^oC±2^oC) and humidity (55±5%) with 12:12 hour light and dark cycle. All the animals were given a standard chow diet (Hindustan Lever Limited), and water ad libitum. The guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) of the Government of India were followed and prior permission was granted from the Institutional Animal Ethics Committee (CPCSEA No. 07/09).

2.4 Induction of Diabetes

The animals were fasted for 18 h and made diabetic by injecting Alloxan monohydrate (150mg/kg, i.p.) dissolved in sterile normal saline. In order to stave off the hypoglycaemia during the first day, 5% w/v glucose solution was given orally to diabetic rats after four to six hours of alloxan administration. The diabetic state was confirmed when the blood sugar level was greater than 180 mg/dl²².

2.5 Treatment Protocol

All the animals were randomly divided into the six groups with six animals in each group. All the three types of Amritarishta as Amritarishta-T, Amritarishta-M and marketed Amritarishta were given at a dose of 2 ml/kg body weight, orally daily for a period of 21 days to different groups of diabetic animals.

Group I : Normal animals received normal saline as vehicle (2 ml/kg, p.o.)

Group II: Diabetic animals received normal saline as vehicle (2 ml/kg, p.o.)

Group III: Diabetic animals received glibenclamide (10 mg/kg, p.o.)²³

Group IV: Diabetic animals received Amritarishta-T (2 ml/kg, p.o.)

Group V: Diabetic animals received Amritarishta-M (2 ml/kg, p.o.)

Group VI: Diabetic animals received marketed Amritarishta (2 ml/kg, p.o.)

Albino rats were made diabetic by a single intra-peritonial injection of Alloxan monohydrate (Loba Chemie, Mumbai). Alloxan was first weighed individually for each animal according to the weight and then solubilized with 0.2ml saline just prior to injection. Two days after Alloxan monohydrate injection rats with blood glucose level of greater than 180mg/dl were selected for the present study. Treatment with Amritarishta-T, Amritarishta-M and marketed Amritarishta was started 48h after Alloxan injection. Blood samples were drawn at weekly interval until the end of study i.e three weeks. Fasting blood glucose (FBG), blood glutathione (GSH) estimation and body weight measurement were done on day 1^st, 7^th, 14^th and 21^st of the study. On 21^st day blood was collected by retro

Table 1. Antihyperglycaemic effect of Amritarishta-T, M and marketed Amritarishta on fasting blood sugar (FBS) and blood glutathione (GSH) level of diabetic rats

Groups

Dose

(ml/kg bw)

Fasting blood sugar (mg/dl) and

Blood glutathione level (mg/dl)

Day1

Day7

Day14

Day21

FBS

GSH

FBS

GSH

FBS

GSH

FBS

GSH

Normal

2.0

66.8±

2.1

27.71±

0.7

72.01±

2.2

24.25±

0.67

73.5±

2.8

25.0±

0.49

75.6±

2.3

24.5±

0..39

Diabetic control

2.0

205.15±

3.15^a

14.17±

0.71^a

267.72±

2.95^a

14.01±

2.1^a

285.79±4.85^a

13.7±

2.7^a

291.70±

6.5^a

12.79±

2.03^a

Diabetic+ Glibenclamide

10mg/kg

202.15±

3.0

17.8±

1.0

180.63±

6.1^b

20.48±

1.8

136.16±2.3^b

22.73±

1.2

116.72±

4.2^b

25.63±

2.6^b

Diabetic+

Amritarishta-T

2.0

204.15±

2.47

16.18±

0.92

198.48±

1.48^b

18.43±

0.46

183.72±2.43^b

19.86±

0.92

175.36±

2.79^b

21.18±

0.72^b

Diabetic+

Amritarishta-M

2.0

202.98±

1.63

15.94±

0.88

200.17±

2.19^b

18.12±

0.58

185.19±1.78^b

19.27±

0.49

178.94±

1.38^b

20.89±

0.63^b

Diabetic+ marketed Amritarishta

2.0

203.72±

1.89

15.76±

0.69

198.93±

2.73^b

17.95±

0.75

184.74±1.43^b

19.14±

0.67

176.82±

2.87^b

20.56±

0.82^b

All values are expressed as mean ± SD (n = 6);

a P<0.001 significant as compared to normal group

b P<0.001 significant as compared to diabetic control group

Table 2. Effect of Amritarishta-T, M and marketed Amritarishta on the body weight of alloxan induced diabetic rats

Group	Dose (ml/kg bw)	Average body weight ( g)
Group	Dose (ml/kg bw)	Day 1	Day 7	Day 14	Day 21
Normal	2.0	200.2±3.4	203.4±2.6	204.12±4.2	206.19±4.5
Diabetic control	2.0	200.5±4.6	169.43±3.4^a	160.47±4.2^a	142.7±2.4^a
Diabetic+ Glibenclamide	10 mg/kg	208.4±2.4	194.27±1.6^b	190.8±2.7^b	187.37±3.4^b
Diabetic+ Amritarishta-T	2.0	210.26±2.4	195.18±1.6^b	182.41±3.8^b	180.6±2.1^b
Diabetic+ Amritarishta-M	2.0	208.42±1.8	194.72±2.8^b	180.68±1.4^b	179.6±2.7^b
Diabetic+ marketed Amritarishta	2.0	212.81±3.2	196.43±2.3^b	181.42±1.2^b	180.2±1.9^b

All values are expressed as mean ± SD (n = 6);

a P<0.001 significant as compared to normal group

b P<0.001 significant as compared to diabetic control group

Table 3. Effect of Amritarishta–T, M and marketed Amritarishta on serum lipid profile in alloxan induced diabetic rats after three weeks of treatment

Group

Dose

(ml/kg

b.w./d p.o.)

Serum cholesterol (mg/dl)

Serum triglyceride

( mg/dl)

Serum HDL (mg/dl)

Serum LDL

(mg/dl)

Serum creatinine ( mg/dl)

Serum urea (mg/dl)

ALP

(IU/L)

Normal

2.0

155.0±

6.21

84.65±

5.22

47.46±

1.34

91.5±

4.10

0.49±

0.079

25.7±

1.23

118.72±

2.20

Diabetic

control

2.0

275.35±

17.5^a

200.0±

11.72^a

27.17±

0.31^a

195.47±

10.51^a

1.72±

0.032^a

64.85±

1.70^a

318.42±

5.94^a

Diabetic+

Glibenclamide

10 mg/kg

146.13±

6.04^b

109.1±

4.83^b

39.17±

1.71^b

84.1±

3.21^b

0.52±

0.021^b

29.06±

2.05^b

122.63±

3.92^b

Diabetic+

Amritarishta-T

2.0

170.14±

1.29^b

119.62±

2.18^b

36.18±

1.48^b

106.86±2.83^b

0.66±

0.014^b

36.43±

1.46^b

154.68±

3.45^b

Diabetic+

Amritarishta-M

2.0

169.24±

2.14^b

120.47±

1.79^b

35.84±

1.43^b

104.79±3.14^b

0.64±

0.026^b

38.69±

1.21^b

156.48±

1.73^b

Diabetic+

marketed

Amritarishta

2.0

167.48±

3.76^b

118.62±

1.82^b

35.73±

2.79^b

107.63±1.97^b

0.62±

0.037^b

37.94±

2.56^b

157.94±

1.92^b

All values are expressed as mean ± SD (n = 6)

a P<0.001 significant as compared to normal group

b P<0.001 significant as compared to diabetic control group

4. Discussion:

Administration of alloxan caused rapid destruction of pancreatic ß cells in rats, which led to impaired glucose stimulated insulin release and insulin resistance, both of which are marked feature of type II diabetes³². This is because administration of alloxan (150mg/kg i.p.) led to more than 1.5 fold elevation of fasting blood glucose level, which was maintained over a period of three weeks. Three week of daily treatment of Amritarishta-T, Amritarishta-M and marketed Amritarishta(2ml/kg p.o.) caused significant fall in blood glucose level.

GSH, being the most important bio-molecule against chemically induced toxicity can participate in the elimination of reactive intermediates by reduction of hydro-per-oxidase in the presence of glutathione per-oxidase. The most important mechanism implicated in the diabetogenic action of alloxan is by increased generation of oxygen free radicals which cause a decrease in plasma GSH concentration. Hence, drugs that could prevent the generation of these oxygen free radicals or increase the free radical scavenging enzymes may be effective in alloxan induced diabetes³³.

In the present study, the observed significant increase in blood glucose level and a decrease in blood glutathione levels in diabetic rats could be due to destruction of ß –cells by alloxan reinforcing the view that alloxan induced diabetes probably through the generation of oxygen free radicals³⁴.

The standard anti-diabetic drug glibenclamide and the test preparations as Amritarishta-T, Amritarishta-M and marketed Amritarishta showed that they could prevent the development of Dibetes mellitus in albino rats, due to their antioxidant property since they showed a significant decrease in fasting blood glucose level (FBG) along with significant increase in blood glutathione level (GSH) after treatment.

Vehicle control animals were found to be stable in their body weight but diabetic rats showed significant reduction in body weight during 21 days (Table 2). Alloxan caused significant reduction in body weight, which was improved by standard drug (glibenclamide) and test preparations (Amritarishta-T, Amritarishta-M and marketed Amritarishta) nearly equal to the normal.

In general, an increase in blood glucose level is usually accompanied by an increase in plasma cholesterol, triglyceride, LDL levels and a decrease in HDL levels as observed in diabetic patients³⁵. The marked hyperlipidemia that characterizes the diabetic state may be the consequence of the uninhibited actions of lipolytic hormones on fat depots³⁶. Amritarishta-T, Amritarishta-M, marketed Amritarishta and standard anti-diabetic glibenclamide produced significant reduction in fasting blood glucose level. These Amritarishta preparations and glibenclamide also produced significant reduction in serum cholesterol, LDL and triglyceride along with significant rise in HDL level in alloxan induced diabetic rats. The improvement in the lipid profile in diabetic animals after treatment with Amritarishta-T, Amritarishta-M, marketed Amritarishta and standard anti-diabetic glibenclamide could be beneficial in preventing diabetic complications, as well as improving lipid metabolism in diabetic patients³⁷.

A significant decrease in the FBG level, improvement in the lipid profile along with significant rise in the blood glutathione level (GSH) by Amritarishta-T, Amritarishta-M and marketed Amritarishta in alloxan induced diabetic rats suggests that these preparations could be useful as an anti-diabetic agent with cardio-protective activity. The obtained result suggests that presence of alcohol could be beneficial in the faster absorption of poly-phenolic compounds found present in Amritarishta which are responsible for showing scavenging of alloxan induced reactive oxygen species.

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Received on 14.05.2014 Modified on 28.05.2014

Res. J. Pharmacology & P’dynamics. 6(3): July- Sept. 2014; Page 129-134