INTRODUCTION:

Diabetic have significant accelerated levels of oxidative stress and this contributes massively to most neurological, cardiovascular, retinal, renal diabetic complications ¹. A panoply of defenses against oxidative stress has evolved and operate at distinct levels. They are reduced generation of reactive oxygen species, enhancement of antioxidant enzymes like- Superoxide dismutase (SOD), catalase, glutathione peroxidase (GPX), and glutathione reductase (GSH) and repair system at the level of DNA. Hyperglycemia significantly diminishes glutathione levels lowering defenses against oxidative stress. N-acetyl cysteine a precursor of GSH inhibited the development of functional and structural abnormalities of peripheral nerves in experimental diabetes².

Thuja occidentalis, family- cupressaceae, commonly known as Arbor vitae or white cedar, is indigenous to North America and is grown in Europe as an ornamental tree. In folk medicine, Thuja occidentalis has been used to treat bronchial catarrh, enuresis, cystitis, psoriasis, uterine carcinomas, amenorrhea and rheumatism³. Extract of this plant has shown anti oxidant, anti viral, anti diarrhoeal activity^4,5. It has been reported to increase the proliferation of spleen cells as well as increase in TNF-α, IL-6 and IL-1 activity in serum and also have protective effect against radiation-induced toxicity⁶. Today it is mainly used in homeopathy as mother tincture or dilution.

MATERIALS AND METHODS:

Plant material:

Fresh aerial part (twigs) of Thuja occidentalis were collected from Jaipur, Rajasthan, India, in October-2007 and were authenticated by experts of Deptt. Of Botany University of Rajasthan, Jaipur. The voucher specimen is preserved for further research in our laboratory.

Preparation of extract:

Shade dried and powdered twigs (40-mesh size, 1kg) were soxhlet extracted with (EtOH) Ethyl alcohol (90%), the solvent was removed and the residue was triturated with hot (65⁰C) petroleum ether (60-80⁰C). Solvent was evaporated from the petroleum ether soluble portion and the residue dissolved in ethanol. On removal of the ethanol by evaporation, a semi solid reddish brown mass (12.76g) was obtained. Phytochemical investigations showed the presence of flavonoids (quercetin, kaempherol), tannic acids, polysaccharides and proteins.

Animals used:

Wistar albino rats of either sex, weighing 175-220 g, provided by the Sanjeevan college of Pharmacy, Dausa, Rajasthan, India, were used. Animals were maintained under standard environmental conditions and had free access to standard pellet food (Hindustan lever, India) and water. The animals were maintained as per the norms of IAEC and the experiments were cleared by IAEC and the local institutional ethical committee.

Induction of Diabetes:

The animals were fasted for 18hr and made diabetic by injecting Alloxan monohydrate (150mg/kg, i.p) dissolved in sterile normal saline⁷. Diabetic state was confirmed when the blood sugar value was greater than 180mg/dl.

Table-1.The anti-hyperglycemic effect of EFTO (ethanolic fraction of Thuja occidentalis) on the Fasting Blood Sugar Level (mg/dl) and Blood Glutathione Level (mg/dl) of Diabetic rats.

Treatment	Dose (mg/kg p.o)	Blood Glucose (mg/dl)and Blood Glutathione level (mg/dl)
		Day1		Day7		Day14		Day21
		FBS	GSH	FBS	GSH	FBS	GSH	FBS	GSH
Vehicle control	0.2ml	65.74±2.1	26.74±0.68	71.04±2.3	23.25±0.58	76.45±3.0	24.23±0.45	79.15±2.1	25.43±0.25
Diabetic control	0.2ml ^b	203.15±3.0	13.06±0.65	265.15±2.7	13.15±1.1	281.67±4.9	13.70±2.1	280.76±6.3	13.89±1.9
Glibenc lamide control	10	201.15±3.0	16.8±1.0*	179.63±6.1*	19.48±1.8*	135.16± 2.3**	21.73± 1.2**	115.72± 4.2 ***	24.63± 2.6 ***
EFTO control	200	202.49±1.9	14.7±1.1*	185.60±6.5*	17.7±1.5*	155.15±1.5**	19.89± 3.4**	122.15± 7.1 ***	21.07± 1.0**

^avehicle (0.5% Tween 80 solution in normal saline) ^b Alloxan single dose of 150mg/kg i.p. in normal saline on day 0. *P<0.05, **P<0.01, ***P<0.001 as compared to vehicle control on corresponding days

Experimental Design:

All the animals were randomly divided into the four groups with six animals in each group. Group A, B, and C were served as vehicle, diabetic and standard drug (Glibenclamide, 10 mg/kg per day p.o⁸) control, respectively. Preliminary oral LD₅₀doses of EFTO in rats was found to be more than 2000mg/kg,. Groups D was treated with EFTO in one-tenth of LD₅₀ doses of the EFTO (200mg/kg per day p.o)

Table-2. Effect of 21 days treatment with EFTO (ethanolic fraction of Thuja occidentalis) on body weight (g) after Alloxan (150mg/kg i.p.) induced diabetes in rats.

Treatment

Dose

Average Body Weight (g) ± SEM

Day1

Day7

Day14

Day21

Vehicle

control

0.2 ml

200.50±

2.15

201.45

±1.06

202.00

±2.00

206.14

±1.65

Diabetic

control

0.2 ml

200.00±

4.68

170.33

±2.49*

159.25

±2.48*

143.80

±1.60*

Glibencl

amide

Control

10(mg

/kg p.o)

207.35±

2.13

195.16

±1.50*

190.00

±2.15*

188.25

±3.15*

EFTO

200(mg

/kg p.o)

209.55±

2.46

190.27

±3.14*

183.16

±3.62*

181.30

±4.10*

Values are given in average body weight (g) ± SEM for groups of six animals each ^avehicle (0.5% Tween 80 solution in normal saline) ^b Alloxan single dose of 150mg/kg i.p. in normal saline on day 0. *P < 0.05 as compared to vehicle control on corresponding day.

Diabetic Animal Models:

Rats were made diabetic by a single intraperitoneal injection of Alloxan monohydrate (Loba Chemie, Bombay; 150 mg/kg). Alloxan was first weighed individually for each animal according to the weight and then solubilized with 0.2 ml saline (154 mM NaCl) just prior to injection. Two days after alloxan injection, rats with plasma glucose levels of >180 mg/dl were included in the study. Treatment with EFTO was started 48 h after alloxan injection. Blood samples were drawn at weekly

intervals until the end of study (i.e. 3 weeks). Fasting blood glucose, blood glutathione estimation and body weight measurement were done on day 1, 7, 14 and 21 of the study. On day 21, blood was collected by cardiac puncture under mild ether anesthesia from overnight fasted rats and fasting blood sugar was estimated⁹. Serum was separated and analyzed for serum cholesterol¹⁰ , serum triglycerides by enzymatic DHBS colorimetric method¹¹, serum HDL ¹², serum LDL ¹³,serum creatinine¹⁴, serum urea¹⁵ , serum alkaline phosphatase hydrolyzed phenol amino antipyrine method ¹⁶were estimated.

Determination of Blood Glutathione:

Blood glutathione was estimated by the method of Beutler et.al¹⁷.

Statistical analysis:

All the values of body weight, fasting blood sugar, and biochemical estimations were expressed as mean ± standard error of mean (S.E.M.) and analyzed for ANOVA.

RESULTS AND DISCUSSION:

The anti hyperglycemic effect of EFTO on the Fasting Blood Glucose level and Blood Glutathione level of diabetic rats is shown in Table-1. Administration of alloxan (150mg/kg i.p) led to more than 1.5 fold elevation of fasting glucose level which was maintained over a period of 3 weeks. Three weeks of daily treatment of EFTO (200 mg/kg p.o) make fall in blood sugar level by 50%. Effect seems to reach maximum after 14 days of treatment and remained constant in third week.

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. GSH also functions as free radical scavenger and in the repair of free radical caused biological damage .The most important mechanism implicated in the diabetogenic action of alloxan is by increased generation of oxygen free radical, which causes a decrease in plasma GSH

Table-3. Effect of EFTO (ethanolic fraction of Thuja occidentalis) on serum profile in Alloxan (150mg/kg i.p.) induced diabetic albino rats after 21 days of treatment.

Treatment	Dose	Serum cholesterol	Serum triglycerides	Serum H.D.L cholesterol	Serum LDL cholesterol	Serum creatinine	Serum urea	Serum alkaline phosphates
Vehicle control	0.2ML^a	149.00 ±5.1	84.65±5.2	36.46 ±1.4	94.06±5.2	0.51±0.1	24.00 ±1.3	116.16±2.6
Diabetic control	0.2ML^b	270.44 ±16.7	201.45±12.3	29.41 ±1.3	198.99±15.6	1.40±0.1	62.15 ±2.0	313.50±6.3
Glibenc Lamide control	10 (mg/kg p.o)	145.72 ±5.7*	107.00±5.2*	52.75 ±1.6*	72.97±4.9*	0.61±0.1*	31.45 ±2.1*	131.17±4.8*
EFTO	200 (mg/kg p.o)	158.50 ±6.5*	131.66±6.2*	44.70 ±1.9*	94.46±4.2*	0.69±0.3*	33.15 ±3.1*	163.83±16.3*

Values are given as mean± SEM for groups of six animals each ^avehicle (0.5% Tween 80 solution in normal saline)

^b Alloxan single dose of 150mg/kg i.p. in normal saline on day 0. *P < 0.05 as compared to vehicle control on corresponding day.

concentration. Hence, drug that could prevent the generation of these oxygen free radicals or increase the free radical scavenging enzymes may be effective in alloxan-induced diabetese¹⁸. In our study, alloxan treated diabetic rats the GSH (blood glutathione level) decreased significantly (P<0.05). Glibenclamide (10m/kg p.o) treated rats showed increase in GSH levels on both 14^th and 21^st day of treatment. Similarly, EFTO (200mg/kg p.o) treated rats also showed significant increase in GSH levels (P<0.05). 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 diabetes probably through the generation of oxygen free radicals¹⁹. The standard anti diabetic drug taken was Glibenclamide and test drug was EFTO, this shown that they could prevent the development of diabetes mellitus in albino rats, due to anti oxidant property since they shown a significant decrease in fasting blood sugar level (FBS) and increase in GSH (Blood glutathione level) 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 body weight reduction, which is reversed by standard drug (Glibenclamide) and test drug (EFTO).

Serum cholesterol, serum triglycerides, serum LDL, serum creatinine, serum urea and serum alkaline phosphatase levels were decreased significantly, and serum HDL levels were increased significantly in both standard and test drug (Table-3). In the light of results, our study indicates that EFTO (ethanolic fraction of Thuja occidentalis) have good anti diabetic activity. EFTO exhibited significant anti-hyperglycemic activities in alloxan induced hyperglycemic rats without significant change in body weight. EFTO can also improve the condition of diabetes as indicated by parameters like body weight and lipid profile along with serum creatinine, serum urea and serum alkaline phosphatase.

CONCLUSION:

Ethanolic fraction of Thuja occidentalis (EFTO) exhibited significant anti-hyperglycemic activities in alloxan induced diabetic rats. This extract has showed improvement in parameters like body weight and lipid profile by enhancing effect on cellular antioxidant defenses to protect against oxidative damage. Present efforts are directed to isolate the active constituents from this fraction and confirmation of mechanism of action.

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