Int J Pharm Pharm Sci, Vol 7, Issue 9, 90-94Original Article


AMELIORATIVE EFFECTS OF ARTEMISIA JUDAICA L. EXTRACT AGAINST ALLOXAN-INDUCED BIOCHEMICAL ALTERATIONS IN MALE WISTAR RATS

EMAN G.E. HELALa, NOURAN ABOU-AOUFa, HANY NADY YOUSEFb, FATIMAH M YOUSEFc, AL SAYEDA MOHAMMAD KHATTABa

aDepartment of Zoology, Faculty of Science, Al-Azhar University (Girls), Nasr City, Cairo, Egypt, bDepartment of Biological and Geological Sciences, Faculty of Education, Ain Shams University, Roxy, Cairo, Egypt, cDepartment of Food and Nutrition, Faculty of Home Economics, King Abdulaziz, University, Jeddah, Saudi Arabia.
Email: hany_barsoum@edu.asu.edu.eg

Received: 24 May 2015 Revised and Accepted: 06 Jul 2015

ABSTRACT

Objective: The current study aims to evaluate the potential of crude leaf extract of Artemisia judaica L. (AJE) in reducing the biochemical abnormalities accompanied to alloxan-induced diabetes in male Wistar rats.

Methods: Thirty male albino rats (100-110 g) were divided equally into three groups including control, diabetic and diabetic+AJE. Diabetes was induced by using a single dose of alloxan (120 mg/kg of body weight). Serum biochemical parameters, including insulin, glucose, triglycerides, total cholesterol (TC), high-density lipoprotein cholesterol (HDL), low-density lipoprotein cholesterol (LDL), very low density lipoprotein cholesterol (VLDL), total proteins, albumin, globulin, renal markers (creatinine, urea, uric acid), activities of aspartate transaminase (AST), alanine transaminase (ALT) and gamma-glutamyltransferase (ɤGT) were measured in all groups. Also, values of homeostasis model assessment of insulin resistance (HOMA-IR) and ratios of albumin: globulin (A: G), TC/HDL (risk factor-1), LDL/HDL (risk factor-2) were calculated for each group.

Results: Diabetic rats showed reduction in body weight and marked decline in the values of serum insulin, protein profile indices and HDL accompanied with marked elevation in values of glucose, HOMA_IR, triglycerides, TC, LDL, VLDL, TC/HDL, LDL/HDL, renal markers and activities of the estimated enzymes. Supplementation of diabetic rats with AJE, twice daily for 30 days, significantly ameliorated most of the estimated biochemical parameters.

Conclusion: The current results demonstrate that AJE possesses a hypoglycemic effect and acts as a protective factor against metabolic abnormalities induced by diabetes mellitus.

Keywords: Artemisia judaica, Alloxan, Diabetes, Lipid profile, Renal markers, Hyperglycemia.

INTRODUCTION

Diabetes mellitus (DM) is a chronic disease characterized by hyperglycemia resulting from deficiency of insulin production, insulin action, or both [1, 2]. The chronic hyperglycemia of diabetes is associated with disturbances in carbohydrate, protein, and fat metabolisms, in addition to long-term complications affecting the eyes, kidneys, nerves, heart and blood vessels [3-6]. According to WHO, about 143 million people worldwide suffering from diabetes and the number may likely to double by the year 2030 [7]. The use of plant materials for medicinal purposes is an ancient practice which has become even more relevant in modern perspective for general health and for specific diseases [8]. Recently there is a growing interest in herbal remedies due to the side effects associated with the available oral hypoglycemic agents for the treatment of diabetes mellitus [9]. Also the search for improved, safe and natural antidiabetic agents has been recommended by World Health Organization [10].

Artemisia judaica (family Asteraceae), also known as “ Shih kharasani” in Arabic, is a perennial fragrant shrub which grows widely in Sinai Peninsula of Egypt. It is widely used in folk medicine and is recommended as a healer plant by Bedouins there [11]. Al-Mustafa and Al-Thunibat [12] reported that A. judaica is one of medicinal plants which has potential of antioxidant activity and used as a traditional anti-diabetic agent. The current study was designed to evaluate the efficacy of AJE in reducing the metabolic abnormalities accompanied to alloxan-induced diabetes in male albino rats.

MATERIALS AND METHODS

Plant materials

Samples of mature fresh green leaves of A. judaica L. were collected from the Southern Sinai, Egypt. The plant was identified and authenticated by a botanist at the Department of Biological and Geological Sciences, Faculty of Education, Ain Shams University, Cairo, Egypt.

Preparation of aqueous extract of A. judaica leaves

After being thoroughly rinsed with sterile distilled water, leaves of shih were ground dried in the shade, and then powdered with a blender. Crude hot water extract of the plant leaves was prepared by boiling 2g of the plant powder with 200 ml distilled water for 15 min. The obtained extract was allowed to cool at room temperature then filtered through Whatman No.2 filter Paper. The resultant extract was stored in a glass container in refrigerator. This extract was freshly prepared each two days.

Experimental animals

Thirty adult male albino rats of the Wistar strain weighing 100-110g and of similar age (8-10 weeks) were obtained from the animal house of Theodor Bilharz Research Institute (TBRI), El-Giza, Egypt. They were housed in clear plastic cages (2 animals/cage) with wood chips as bedding and given a standard pellet rodent diet, in addition of water ad libitum. The rats were maintained under standard laboratory conditions at 25±2 °C, relative humidity 55±5% and normal photoperiod (12h light/dark cycle). All animal experiments were performed under protocols approved by the local Institutional Animal Ethics Committee of Ain Shams University.

Induction of diabetes

Diabetes mellitus was induced in animals by a single intraperitoneal injection of alloxan (120 mg/kg body weight) dissolved in freshly prepared physiological saline. After three days of alloxan injection, rats were deprived of food overnight and they were then given glucose (3 g/kg body weight) by gastric intubation. After 2h of oral glucose administration, blood samples were taken from tail vein and the fasting blood glucose (FBG) concentration was determined by means of one touch ultra glucometer and compatible blood glucose strips. Animals with fasting blood glucose levels ≥ 300 mg/dl were considered as mild diabetic animals and included in the experiment. The control rats were injected with physiological saline alone as placebo.

Experimental protocol

Experimental animals were divided into three groups (ten for each) as follows:

Group I: (Control group): Untreated non-diabetic rats.

Group II: (Diabetic group): Rats were injected intraperitoneally with a single dose of alloxan (120 mg/kg dissolved in saline solution).

Group III: Diabetic rats treated orally with AJE (28.5 mg/kg twice/day) for 30 days.

Blood collection

At the end of the experiment and under diethyl ether anaethesia, blood samples were taken from the retro-orbital plexus of the overnight fasted animals. Blood samples were centrifuged at 4000 rpm for 10 min at 4 °C. The clear supernatant sera were quickly removed and immediately stored at-80 °C till used for further analysis of biochemical parameters.

Biochemical estimations

Serum samples were analyzed to estimate the levels of glucose according to Tietz [13] and insulin according to Reeves [14]. Insulin resistance was estimated using homeostasis model assessment (HOMA-IR) from fasting serum glucose and insulin using the following equation [15]:

HOMA-IR = fasting serum glucose (mg/dl) × fasting serum insulin (μU/L)/405

Serum creatinine, urea and uric acid were estimated by colorimetric methods according to Tietz [13], while levels of aspartate transaminase (AST), alanine transaminase (ALT) and glutamyltransferase (γGT) in sera were determined colorimetrically following Schumann and Klauke [16]. Serum albumin and total proteins were measured according to the method of Burtis et al.[17]. Globulin was calculated by subtracting albumin from total proteins [13]. Serum total lipids, total cholesterol [18]; triglycerides[19] and HDL[20] were estimated colorimetrically using high quality kits according to manufacturer's protocol; while VLDL was calculated as triglyceride/5 and LDL was calculated applying the Friedwald's equation [21].

Friedewald's equation: LDL (mg/dl) = TC-HDL-[TG/5].

Risk factor 1 = TC/HDL

Risk factor 2 = LDL/HDL

Statistical analysis

The results were expressed as mean±SEM of 10 rats per group and the statistical significance was evaluated by one way analysis of variance (ANOVA) followed by Duncan post Hoc test using the SPSS/17.0 software. Values were considered statistically significant at P<0.05.

RESULTS

Fig. 1. Depicts that diabetic rats showed marked decline (P<0.05) in their body weight (-4.55%) when compared to the control group. Supplementation of AJE to the diabetic rats for 30 days returned the body weight towards normalcy.

Table 1 shows that levels of insulin were significantly (P<0.05) decreased (-38.50%) and levels of glucose were significantly (P<0.05) increased (300.50 %) in sera of diabetic rats relative to the corresponding control rats. Oral administration of AJE to the diabetic rats for one month improved significantly (P<0.05) the levels insulin and glucose in sera relative to the control group.

In addition, HOMA_IR values were significantly higher (P<0.05) in diabetic rats (147.19%) when compared to the corresponding controls, while treatment of diabetic rats with AJE returned HOMA_IR values to normalcy.

Marked elevations (P<0.05) in the activities of AST (43.81%), ALT (49.24%) and γ GT (323.68%) were recorded in sera of the diabetic rats when compared to the values of the corresponding controls. On the other hand, treatment of diabetic rats with AJE significantly (P<0.05) abolished the disturbances occurred in the activities of these enzymes.

Fig. 1: Body weight changes in the control and experimental male albino rats
Values are expressed as mean±SEM for 10 rats in each group. *P<0.05, AJE: Artemisia judaica extract

Parameters of serum protein profile (total proteins, albumin, globulin and A/G ratio) in control and experimental groups are presented in table 3. Diabetic animals showed marked decline (P<0.05) in serum total proteins (-28.38%), albumin (-26.41%) and globulin (-31.01%) relative to the corresponding controls. Treatment of diabetic rats with AJE resulted in significant (P<0.05) modulation of the measured serum protein profile parameters. The values of A/G ratio showed non-significant changes between control and experimental groups.

Table 4 shows the indices of lipid profile in sera of control and experimental groups of rats. Diabetic animals showed marked elevation (P<0.05) in total lipids (201.69%), total cholesterol (64.11%), triglycerides (113.34%), LDL (104.93%), VLDL (113.37%) and ratios of TC/HDL (102.66%) and LDL/HDL (159.42) accompanied with marked decline in HDL (-20.47 %) relative to the corresponding controls. Treatment of diabetic rats with AJE improved the sera lipid profile indices.

Biochemical parameters of renal function elevated markedly (P<0.05) in sera of diabetic rats with percentage of change 82.42%, 95.44% and 248.08% higher than those of control rats for creatinine, urea and uric acid, respectively (table 5). Treatment of diabetic rats with AJE returned these parameters towards normalcy.

Table 1: Levels of serum insulin and glucose and values of HOMA_IR in control and experimental groups

Parameters

Groups

Control

Diabetic

Diabetic+AJE

Insulin (µU/l)

4.13±0.45

2.54±0.37*

3.80±0.68*

Glucose (mg/dL)

87.44±0.78

350.20±0.85*

94.04±0.62*

HOMA_IR

0.89±0.23

2.20±0.36*

0.88±0.08

Values are expressed as mean±SEM for 10 rats in each group. * P<0.05, AJE: Artemisia judaica extract, HOMA_IR: homeostasis model assessment of insulin resistance.


Table 2: Activities (U/l) of serum AST, ALT and ɤGT in sera of control and experimental groups

Parameters

Groups

Control

Diabetic

Diabetic+AJE

AST

37.02±0.59

53.24±1.08*

34.36±0.70*

ALT

22.46±0.56

33.52±0.60*

28.06±0.55*

ɤGT

2.28±0.12

9.66±0.08*

4.50±0.07*

Values are expressed as mean±SEM for 10 rats in each group. * P<0.05, AJE: Artemisia judaica extract, AST: aspartate transferase, ALT: alanine transferase, ɤGT: gamma-glutamyltransferase.


Table 3: Levels of serum proteins (g/dl) and A/G ratio in control and experimental groups

Parameters

Groups

Control

Diabetic

Diabetic+AJE

Total proteins

7.40±0.08

5.30±0.11*

8.20±0.09*

Albumin

4.24±0.07

3.12±0.06*

4.46±0.05

Globulin

3.16±0.05

2.18±0.07*

3.72±0.09*

A/G ratio

1.34±0.05

1.42±0.04

1.22±0.05*

Values are expressed as mean±SEM for 10 rats in each group. * P<0.05, AJE: Artemisia judaica extract.


Table 4: Levels of serum lipid profile indices (mg/dl) and values of TC/HDL and LDL/HDL ratios in control and experimental groups

Parameters

Groups

Control

Diabetic

Diabetic+AJE

Total lipids

474.00±8.12

1430.00±11.40*

463.60±5.00

Total cholesterol

141.08±0.37

231.52±0.53*

184.62±0.59*

Triglycerides

133.10±0.77

283.96±0.86*

147.72±0.45*

HDL

47.88±0.49

38.08±0.39*

43.70±0.54*

LDL

66.56±048

136.40±0.59*

111.48±0.61*

VLDL

26.62±0.16

56.80±0.17*

29.52±0.10*

TC/HDL

3.00±0.00

6.08±0.04*

4.22±0.06*

LDL/HDL

1.38±0.02

3.58±0.04*

2.54±0.05*

Values are expressed as mean±SEM for 10 rats in each group. * P<0.05, AJE: Artemisia judaica extract. HDL: high-density lipoprotein cholesterol, LDL: low-density lipoprotein cholesterol, VLDL: very low density lipoprotein cholesterol.


Table 5: levels (mg/dl) of serum creatinine, urea and uric acid in control and experimental groups

parameters

Groups

Control

Diabetic

Diabetic+AJE

Creatinine

0.91±0.01

1.66±0.08*

0.92±0.01

Urea

32.46±0.55

63.44±0.59*

37.02±0.62*

Uric acid

2.08±0.06

7.24±0.09*

3.46±0.07*

Values are expressed as mean±SEM for 10 rats in each group. * P<0.05, AJE: Artemisia judaica extract.

DISCUSSION

Diabetes mellitus is one of the major diseases affecting many people on the globe. Traditional plant based remedies are still the first choice in the developing countries because of their cost effectiveness, easy availability and minimum or no side effects [22, 23]. More than 800 plant species have been identified throughout the world to have antidiabetic properties [24]. From these medicinal plants is Artemisia judaica which is commonly used in folk medicine in Egypt. The present investigation was carried out to examine the efficacy of AJE in reducing the metabolic abnormalities accompanied to alloxan-induced diabetes in male albino rats.

Induction of diabetes by alloxan resulted in loss of body weight reflecting the catabolic effect of diabetes on protein metabolism by retarding protein synthesis and stimulating protein degradation [25]. In addtion, diabetic rats showed significant decrease (p<0.05) in levels of serum insulin accompanied with marked elevation (p<0.05) in levels of blood glucose when compared to the control rats. This may be due to damages caused by alloxan in beta cells which lead to decrease in endogenous insulin secretion. In the same context, Szkudelski [26] showed that Alloxan selectively destroys the pancreatic insulin secreting β-cells and induces hyperglycemia. Treatment of diabetic rats with AJE returned the body weight, insulin and glucose levels towards normalcy. These results indicated that the AJE possesses significant hypoglycemic and anti-hypoinsulinemia effects that may be attributed to its content of flavonoids, essential oil, saponins, terpenes, and tannins. The hypoglycemic action of AJE may be through increase glucose uptake and glycogen synthesis, inhibiting for α-glucosidase and α-amylase, reduction of insulin resistance, reduction of oxidative stress and protecting against tissue damage, generation of beta cells in pancreas. These results are in agreement with a previous study suggested that A. judaica significantly reduced the blood glucose level in diabetic rats [27].

Furthermore, a new hormone called Betatrophin found to be secreted by liver and adipose tissues. This hormone prompts beta cells in the pancreas to multiply and produce more insulin [28]. AJE has a variety of chemical constituents such as essential oils (artemisyl-oil, apiperitone-oil, piperitone and trans-ethyl cinnamate), which have antioxidant, anti-inflammation and anti-hypo insulinemic activity [29]. Also, these essential oils might be stimulating normal beta cells for insulin production enhanced peripheral uptake of glucose where insulin increased significantly and caused regeneration of hepatocytes [30]. These hepatocytes produced more β-trophin enhancing insulin production by β-cells of pancreas and enhancing body weight.

HOMA-IR has considered as a robust tool for the surrogate assessment of insulin resistance [31, 32]. In the current study, HOMA_IR values were significantly higher (P<0.05) in diabetic rats when compared to the corresponding controls. This may be attributed to high glucose concentrations leading to the development of insulin resistance in peripheral tissues owing to impairment of both insulin secretion and insulin sensitivity [33]. After AJE administration, the level of HOMA_IR returned to the normal values. These findings indicated that AJE exhibited antihyperglycemic properties, enhanced insulin release and peripheral uptake of glucose in alloxan-induced diabetic rats.

Activities of ALT, AST and γGT reflect the state of hepatocyte injury [34, 35]. In our study, there is an elevation in the activities of these enzymes in diabetic rats when compared with the control group indicating a state of hepatocyte injury. This may be attributed to hyperglycemia which promotes reactive oxygen species (ROS) accumulation, accelerates cellular damage and significantly contributes to the diabetic complications development and progression [36]. Furthermore, an overall significant reduction in serum total protein, albumin and globulin in diabetic animal’s consequents with slight non-significant elevation in A/G ratio were observed in the present study. These observations are in analogy to the results obtained[37] and Chandramohan et al. [38]. Hyperglucagonemia during insulin deficiency accelerates protein catabolism [39]. This may explain the reduction in serum total protein, albumin and globulin in diabetic animals as hypoinsulinemia increases the rate of protein degradation and may have a direct adverse effect on the synthesis and secretion of albumin and globulin.

Results of the present work showed that, treatment of alloxan-induced diabetic rats with AJE significantly reduced the activities of ALT, AST and γGT and serum protein profile parameters were returned back to the normal levels. These results indicated that AJE has a hepato protective effect and improving liver function. This may be due to the presence of flavonoids (apigenin, cirsimaritin, flavonoid glycosides) which have a hypoglycemic action in addition to a potent antioxidant action attenuating the oxidative stress induced by free radicals, so they can ameliorate the functions of the liver by protecting the hepatocytes and inhibiting the production proinflammatory mediators like TNF-α, IL-12, IL-2 cytokines which has been associated with inflammatory diseases[40].

Diabetic animals showed marked elevation (P<0.05) in the values of total lipids, total cholesterol, triglycerides, LDL,VLDL and ratios of TC/HDL and LDL/HDL accompanied with marked decline in HDL relative to the corresponding controls. Insulin has a potent inhibitory effect on lipolysis in adipocytes. Therefore, hypoinsulinemia is associated with excess lipolysis and increased influx of free fatty acids to the liver [41, 42]. This stimulates over production of triglycerides, LDL and VLDL by the hepatocytes [41]. According to Kinosian et al. [43], the changes in TC/HDL and LDL/HDL ratios are better predictors of coronary heart disease than the changes in LDL alone. In the present study, diabetic animals were subjected to the risk of coronary heart disease as evidenced by their high ratios of TC/HDL and LDL/HDL. These observations are consistent to earlier results obtained by many investigators [44-46]. Results of the current investigation showed that, AJE significantly ameliorated all the estimated sera lipid profile indices indicating that AJE has potential role of antioxidant activity and it can be used as anti-diabetic agent to decrease the risk of atherosclerosis. This may be attributed to the flavonoid compounds (apigenin, cirsimaritin) which were found in water extracts of A. judaica. These compounds act as antioxidants and reduce the levels of cholesterol and triglycerides significantly through their protective role against free radicals by scavenging activity [30, 47].

Elevated levels of blood urea, creatinine and uric acid are likely evidence of impaired kidney functions. The obtained results from the current study showed high levels of renal markers in sera of diabetic rats relative to the corresponding control ones. Renal dysfunction indicated by elevation of renal markers in diabetic rats has been proved by many investigators [38, 44, 48]. Diabetes mellitus is characterized by hyper glycaemia that is strongly linked to nephropathy mediated via oxidative stress. This may be due to high activities of xanthine oxidase, lipid peroxidation as well as impairment of the urea cycle enzyme activities [49]. Treatment of diabetic rats with AJE reversed the renal markers towards the normal values indicating that A. judaica can ameliorate renal function abnormalities and provides protection against the oxidative renal damage through the antioxidant capacity of its constituents. These findings are in consistence with the results obtained [46] on treatment of diabetic rats with aqueous extract of Olea europaea plant. Also Chandramohan et al. [38]. Obtained similar results after treatment of diabetic rats with 3-hydroxymethyl xylitol for 45 days.

In conclusion, The present results suggest that AJE has a hypoglycemic acts as a beneficial agent against metabolic abnormalities induced by diabetes.

CONFLICT OF INTERESTS

Declared None

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