INTRODUCTION:

Diabetes Mellitus is an endocrine disorder characterized by altered glucose homeostasis leading to derangements in the carbohydrate, protein and lipid metabolism, resulting from partial or complete deficiency in insulin synthesis or due to peripheral resistance to insulin action. Diabetes is rapidly emerging at an alarming rate and is considered to be one of the biggest health catastrophes in the world¹, causing significant health and economic burdens on patients and communities². The successful therapy depends greatly on the patient's lifestyle and the physicians experience in identifying the right combination of pharmacological interventions and proper lifestyle³.

Oral hypoglycemic drugs used for the treatment of diabetes such as sulfonylureas, biguanides, a- glucosidase inhibitors, thiazolidenediones and insulin are often associated with undesirable side-effects or diminution in response after prolonged use⁴. Moreover, providing modern medical healthcare across the world is still a far-off goal due to economic constraints. Thus, it is necessary that we continue to look for new and, if possible, more efﬁcacious drugs, and the vast reserves of phytotherapy may be an ideal target.

Plants have played a signiﬁcant role in maintaining human health and improving quality of life for thousands of years. In particular, herbs have been used as food and for medicinal purposes for centuries. In herbal medicine, the term herb refers not only to seed-producing plants but also bark, roots, leaves, seeds, ﬂowers, and the fruit of trees. According to the World Health Organization, about three-quarters of the world’s population relies on traditional medicine for primary healthcare needs and most of this treatment involves use of plant extracts or their active components⁵. However, the mechanism of action of most herbal medicines has not been fully understood, and experience obtained from their traditional use over the years should not be ignored⁶. Therefore, it is prudent to look for options in herbal medicine for diabetes.

Tridax procumbens Linn. (Family-Asteraceae; common name-Dhaman grass) is common herb found in India. The whole plant and seeds are reported to be used to treat various aliments, such as bronchial catarrh, dysentery, diarrhea, preventing hair loss, and to check hemorrhage from cuts^{7, 8}. Pharmacological studies have shown that T. procumbens possess properties like-anti inflammatory, hepatoprotective, wound healing, Immunomodulatory, antimicrobial, antiseptic, and hypotensive, bradycardiac effects^{9, 10, 11,
12, 13}.

In the absence of systematic studies, the present study was aimed to evaluate the antidiabetic, hypolipidemic and antioxidant nature of Tridax Procumbens in alloxan induced diabetic rats.

MATERIALS AND METHODS:

Plant Material:

Fresh, mature leaves of Tridax procumbens Linn. were collected from University of Madras, Guindy, Chennai. The plants were identified and authenticated and a voucher specimen was deposited at the Department of Botany, University of Madras, Chennai.

Preparation of Plant extract:

The Tridax procumbens leaves were dried at room temperature and powdered in an electrical grinder, which was then stored in an airtight container at 5° C until further use. The powdered leaf was delipidated with petroleum ether (60 - 80° C) for overnight. It was then filtered and soxhalation was performed with 95% Ethanol. Ethanol was evaporated in a rotary evaporator at 40 – 50° C under reduced pressure.

Preliminary Phytochemical screening:

The ethanolic extract of Tridax procumbens leaves were subjected to preliminary phytochemical screening of various plant constituents^14,15.

Experimental animals:

Male albino Wistar rats (150-180 g) were purchased from TANUVAS, Madavaram, Chennai. The rats were housed in polypropylene cages lined with husk and kept in animal house, Department of Biochemistry. It was renewed every 24 hours. The rats were fed with commercial pelleted rats chow (VRK Nutritional Solutions, Maharashtra, India) and had free access to water. The experimental rats were maintained in a controlled environment (12:12 hours light/dark cycle) and temperature (30 ± 2° C). The experiments were designed and conducted in accordance with the ethical norms approved by Ministry of Social Justices and Empowerment, Government of India and Institutional Animal Ethics Committee Guidelines for the investigation of experimental pain in conscious rats. The rats were acclimatized for one week before starting the experiments.

Induction of Diabetes Mellitus:

Rats were induced diabetes by single intraperitonial injection of alloxan monohydrate dissolved in sterile normal saline at a dose 120 mg/Kg, after 18 hours fasting to induce hyperglycemia. After 1 hour alloxan administration, the animals were fed on standard pellets and water ad libitum. Rats were supplied with 5% Glucose solution for 48 hours after alloxan injection in order to prevent severe hypoglycaemia. After 1 week time for the development and aggravation of diabetes, the rats with moderate Diabetes having persistant glycosuria and hyperglycemia (Blood glucose range of above 250 mg/dL) were considered as diabetic rats and used for the experiment. The treatment was started on the eighth day after alloxan injection and this was considered as first day of treatment.

Experimental Design:

The rats were grouped into 4 groups, comprising minimum of 6 rats in each group as follows:

Group I : Control Rats (Water and food ad libitum)

Group II : Alloxan induced diabetic rats

Group III : Diabetic Rats treated with Tridax procumbens leaf extract (400 mg/Kg body weight/day) in aqueous solution orally for 30 days.

Group IV : Diabetic Rats treated with gliclazide (5mg/Kg body weight/day) in aqueous solution orally for 30 days.

During the experimental period, body weight and blood glucose levels of all the rats were determined at regular intervals. At the end of the experimental period, the rats were fasted over night, anaesthetized, and sacrificed by cervical decapitation. The blood was collected with and without anticoagulant for plasma and serum separation respectively.

Whole blood was used for glucose ¹⁶and urea¹⁷ estimation. Plasma was separated and used for insulin assay using ELISA kit for rats. Levels of hemoglobin and glycosylated hemoglobin were estimated according to methods of Drabkin and Austin¹⁸ and Nayak and Pattabiraman¹⁹, respectively. Plasma was used for protein assay by Lowry’s method ²⁰ and serum for determination of creatinine ²¹ and uric acid ²² .

Pancreatic tissue was selectively excised, washed in ice-cold saline and then homogenized in Tris Hel buffer (pH 7.4) using a Teflon homogenizer. The pancreatic tissue homogenate was then centrifuged at 5000g to remove cellular debris and supernatant was used for the determination of lipid peroxidation and enzymatic antioxidants. Lipid peroxidation was determined using thiobarbituric acid reactive substances by the method of Ohkawa et al.²³. Levels of vitamin C, vitamin E, ceruloplasmin and glutathione (GSH) were determined by the methods of Omaye et al.²⁴, Desai ²⁵, Ravin ²⁶, and Sedlak and Lindsay²⁷, respectively. Enzymatic antioxidants such as superoxide dismutase²⁸, catalase²⁹ and glutathione peroxidase³⁰ in pancreatic supernatant.

Oral Glucose Tolerance Test (OGTT):

At the end of the experimental period, a fasting blood sample was collected from all the groups of rats to perform oral glucose tolerance test, rats were fasted for 12 h before the test and 2 g/kg glucose solution was administered orally. Blood samples were taken by severing the tip of the tail 1 h before and at 0.5, 1, 1.5 and 2 h after glucose administration. Blood glucose was determined using ortho toluidine reagent.

Lipid profile:

Plasma was used for the estimation of lipid profile. Cholesterol content was estimated by the method of Parekh and Jung³¹. Triglyceride was estimated by the method of Rice³². HDL Cholesterol fraction was separated by the precipitation techniques of Burstein and Scholnick³³ and the cholesterol content was determined by method of Parekh and Jung³¹.

Statistical analysis:

All the grouped data were statistically evaluated with SPSS 16.00 software. Hypothesis testing methods included one-way analysis of variance followed by least signiﬁcant difference (LSD) test. p<0.05 was considered to indicate statistical signiﬁcance. All results are expressed as mean ± standard deviation (SD) for six rats in each group.

RESULTS:

Table 1 shows the qualitative analysis of phytochemicals in the ethanolic extract of Tridax procumbens leaves. Phytochemical evaluation revealed the presence of alkaloids, flavonoids, proteins, carbohydrates, saponins, tannins, glycosides and phenols.

Table 1: Preliminary phytochemical screening of Tridax procumbens leaves extract

PHYTOCONSTITUENTS	INFERENCE
Alkaloids	+
Flavonoids	+
Carbohydrates	+
Glycosides	+
Saponins	+
Tannins	+
Phytosterol	+
Triterpenoids	+
Proteins	-
Aminoacids	-
Anthraquinones	-
Phenols	+

Fig 1 shows the changes in the levels of blood glucose, after oral administration of glucose (3g/Kg) in normal control and experimental group of rats. The data of OGTT revealed that the blood glucose value in normal control rat reach peak at 60 minutes after the oral glucose load and gradually reverted back to near normal levels after 120 minutes. In diabetic control rats, the peak increase in blood glucose concentration was observed after 60 minutes and remained high over the next 60 minutes. Treated group showed definite lower peak blood glucose values, 60 minutes after glucose load also gives lower values almost at the end of 120 minutes.

Fig 1: Effect of T .Procumbens extract on the blood glucose level (mg/dl) in the experimental groups of rats receiving an oral glucose load.

The effect of oral administration of T. procumbens leaf extract on the levels of blood glucose, plasma insulin, hemoglobin, glycosylated hemoglobin and urine sugar in the control and experimental groups of rats were depicted in Table 2. The elevated levels of blood glucose, glycosylated hemoglobin in the diabetic group of rats were reverted to near normal level by the administration of T. procumbens leaves extract. Conversely, the decreased levels of plasma insulin, hemoglobin in diabetic group of rats were elevated by the administration of T. procumbens leaves extract to diabetic rats for 30 days. Urine sugar which is present in the diabetic group of rats was absent in T. procumbens leaves extract as well as gliclazide treated diabetic group of rats. The results are comparable with gliclazide, an oral hypoglycemic drug. Urine sugar present in the diabetic rats was found to be absent in the rats treated with leaves extract.

The effect of oral administration of T. procumbens leaf extract on the levels of total protein, urea, uric acid and creatinine are presented in Table 3. The altered levels of these parameters were reverted back to near normalcy upon the treatment with the leaf extract.

Table 4 represents the effect of T. procumbens leaf extract on the levels of lipid peroxides in the plasma and pancreas of control and experimental groups of rats. The levels of lipid peroxides were significantly (p<0.05) elevated in the diabetic group of rats. Upon oral administration of T. procumbens leaf extract as well as gliclazide to diabetic group of rats were significantly (p<0.05) reverted to normal levels when compared to control group of rats.

Table 2. Effect of T .Procumbens extract on the levels of biochemical parameters in the experimental groups of rats.

Groups	Glucose (mg/dl)	Insulin (µU/ml)	Hemoglobin (g/dl)	Glycosylated hemoglobin (%)	Urine sugar
Control	85.46 ± 9.24	16.29 ± 2.30	15.85 ± 2.66	6.21 ± 1.32	Nil
Diabetic	286.62 ± 19.18*	5.49 ± 1.24*	11.85 ± 1.89*	14.05 ± 2.96*	+++
Diabetic + T. procumbens extract	150.31 ± 15.01^@	11.55 ± 2.51^@	12.96 ± 2.45^@	8.21 ± 1.90^@	Nil
Diabetic + gliclazide	132.27 ± 12.79^@	12.99 ± 2.04^@	13.61 ± 2.06^@	7.96 ± 2.12^@	Nil

Values are given as mean ± SD for groups of six rats in each. Values are statistically significant at p < 0.05. Statistical significance was compared within the groups as follows: *compared with control, ^@compared with diabetic rats.

Table 3. Effect of T .Procumbens extract on the levels of protein, urea, creatinine and uric acid in experimental groups of rats.

Groups	Protein (g/dl)	Urea (mg/dl)	Creatinine (mg/dl)	Uric acid (mg/dl)
Control	8.05 ± 1.25	20.28 ± 2.14	0.90 ± 0.10	2.39 ± 0.75
Diabetic	5.60 ± 0.92*	46.80 ± 3.85*	1.98 ± 0.18*	4.95 ± 1.06*
Diabetic + T. procumbens extract	6.91 ± 0.96^@	31.02 ± 3.14^@	1.30 ± 0.12^@	3.20 ± 0.89^@
Diabetic + gliclazide	7.06 ± 0.89^@	28.95 ± 2.64^@	1.19 ± 0.10^@	2.94 ±0.90^@

Table 4 . Effect of T .Procumbens extract on the level of TBARS in plasma and pancreas of experimental groups of rats.

Groups	TBARS
Groups	Plasma	Pancreas
Control	4.61 ± 0.74	48.27 ± 4.65
Diabetic	8.45 ± 1.50*	80.12 ± 9.59*
Diabetic + T. procumbens extract	5.21 ± 1.13^@	60.72 ± 6.74^@
Diabetic + gliclazide	5.36 ± 1.06^@	59.94 ± 7.21^@

Units: mM/100 g in tissues; nM/ml in plasma. Values are given as mean ± SD for groups of six rats in each. Values are statistically significant at p < 0.05. Statistical significance was compared within the groups as follows: *compared with control, ^@compared with diabetic rats.

The effect of T. procumbens leaf extract on the plasma levels of non-enzymatic antioxidants such as vitamin C, vitamin E, reduced glutathione and ceruloplasmin in control and experimental groups of rats are shown in table 5. The diminished levels of non-enzymic antioxidants in the diabetic group of rats were significantly (p<0.05) improved to near normal values by the oral administration of T. procumbens leaf extract as well as glycIazide, after 30 days of treatment.

Table 5. Effect of T .Procumbens extract on the levels of vitamin C, vitamin E, ceruloplasmin and GSH in plasma of experimental groups of rats.

Groups	Vitamin C	Vitamin E	Ceruloplasmin	GSH
Control	1.52 ± 0.16	0.76 ± 0.11	12.86 ± 1.95	32.98 ± 4.10
Diabetic	0.49 ± 0.07*	0.35 ± 0.05*	5.21 ± 0.96*	16.52 ± 2.97*
Diabetic + T. procumbens extract	0.95 ± 0.10^@	0.62 ± 0.09^@	10.71 ± 1.69^@	26.59 ± 3.10^@
Diabetic + gliclazide	0.89 ± 0.12^@	0.59 ± 0.07^@	11.30 ± 1.91^@	29.34 ± 3.41^@

Units: mg/dl. Values are given as mean ± SD for groups of six rats in each. Values are statistically significant at p < 0.05. Statistical significance was compared within the groups as follows: *compared with control, ^@compared with diabetic rats.

Table 6 depicts the activities of pancreatic enzymatic antioxidants such as superoxide dismutase, catalase and glutathione peroxidase in the control and experimental groups of rats. The decreased activity of enzymic antioxidants observed in the diabetic group of rats were significantly (p<0.05) elevated to near normal levels after treatment with T. procumbens leaf extract as well as gliclazide.

Table 6. Effect of T .Procumbens extract on the activity of SOD, Catalase and GPx, in pancreas of experimental groups of rats.

Groups	SOD	Catalase	GPx
Control	5.12 ± 1.06	16.24 ± 2.18	6.94 ± 1.06
Diabetic	1.48 ± 0.55*	5.92 ± 1.24*	3.18 ± 0.39*
Diabetic + T. procumbens extract	3.84 ± 0.82^@	12.65 ± 1.96^@	4.96 ± 0.68^@
Diabetic + gliclazide	3.69 ± 0.94^@	14.29 ± 1.48^@	5.29 ± 0.81^@

Activity is expressed as: 50% of inhibition of epinephrine autoxidation/min/mg of protein for SOD; µM of hydrogen peroxide decomposed/min/mg of protein for catalase; µM of glutathione oxidized/min/mg of protein for GPx, mg/100 g tissue for GSH. Values are given as mean ± SD for groups of six rats in each. Values are statistically significant at p < 0.05. Statistical significance was compared within the groups as follows: *compared with control, ^@compared with diabetic rats.

The levels of cholesterol, triglycerides, HDL and LDL in control and experimental groups of rats are shown in Table 7. The levels of cholesterol, triglycerides, LDL and were significantly increased whereas the HDL-cholesterol was significantly decreased in alloxan-induced diabetic rats. Treatment with extract as well as gliclazide significantly ameliorated these levels to near normal levels.

DISCUSSION:

Alloxan is widely employed to induce diabetes mellitus in experimental animals due to the fact that it causes a massive reduction of the insulin secreting β cells of islets of langerhans, resulting in a decrease in endogenous insulin release, which paves the ways for the decreased utilization of glucose by the tissue³⁴. Alloxan also increases the oxidative stress which is the possible mechanism of its diabetogenic action³⁵.

Oral administration of Tridax procumbens leaves extract to alloxan induced diabetic rats resulted in significant reduction in blood glucose level indicating that the activation of β cells and granulation returns to normal, which shows an insulinogenic effect. Similarly, it was reported that the aqueous as well as alcoholic extracts from Tridax procumbens leaves (200mg/Kg body weight) administered orally for seven days produced a significant decrease in the blood glucose level of alloxan induced diabetic rats³⁶. The results of our study fall in line with earlier reports.

Beside this Tridax procumbens might involve in extrapancreatic action in alloxan induced diabetic rats, might be by stimulating the peripheral glucose utilization or by enhancing glycogenic and glycolytic pathways with concominant decrease in glycogenolysis and gluconeogensis. In the above context a similar hypoglycemic mechanism was shown by whole plant extract Tridax procumbens ³⁷. The elevated blood glucose level observed in the diabetic rats was significantly decreased in Tridax procumbens treated rats suggesting insulin secretory effect of Tridax procumbens from the remnant b cells.

Glycosylated hemoglobin is a standard biochemical marker in assessment of diabetes. Glycated Haemoglobin is a unique form of haemoglobin used primarily to identify the average plasma glucose concentration over prolonged periods of time. It is formed in non-enzymatic pathway by hemoglobin’s normal exposure to persistent high plasma levels of glucose³⁸. Diabetic rats showed higher levels of glycated hemoglobin indicating their poor glycemic control. Oral administration of the leaf extract to diabetic rats decreased the level of glycosylated hemoglobin and increased the total hemoglobin concentration.

The accelerated proteolysis of uncontrolled diabetes occurs as a result of dearranged glucagon mediated regulation of cAMP formation in insulin deficiency. This readily accounts for the observed decrease in the protein content in diabetes mellitus. Thus, in alloxan induced diabetic rats, insulin deficiencies account for the observed decrease in the total protein content. The formation of glycated protein also decreases plasma proteins in diabetes. Administration of Tridax procumbens leaves extract to alloxan induced diabetic rats significantly inhibits proteolysis caused by insulin deficiency and thus increases the levels of total protein to near normal. It was reported that the diabetic rat treated with ethanolic extract of Tridax procumbens leaves have shown a significant changes in protein content of liver and kidney³⁹.

The biochemical parameters such as Urea, Creatinine and Uric acid are considered as significant markers for renal dysfunction. Renal dysfunction is one of the pathophysiological condition occurs in diabetic condition. Urea is the end product of protein catabolism. The diabetic animals manifest a negative nitrogen balance related to proteolysis in muscles and other tissues which is coupled with lowered protein synthesis⁴⁰ and increased protein catabolism accelerates urea synthesis thereby resulting in hyperuremia. The accelerated proteolysis of uncontrolled diabetes occurs as a result of deranged glucagonmediated regulation of cyclic AMP formation in insulin deficiency⁴¹. This readily accounts for the observed decrease in the total protein content in diabetes mellitus.

The serum creatinine concentration is the variable used not only to assess impairment of kidney function but also to detect the toxic effects of certain compounds derived from medicinal plants on kidney, in order to determine its efficacy in the treatment of diabetic rats. Serum uric acid is significantly associated with the risk of diabetes. Serum uric acid has been shown to be associated with oxidative stress and production of tumour necrosis factor-α⁴² In addition, a recent study in rats showed that fructose-induced hyperuricemia plays a pathogenic role in metabolic syndrome⁴³. Thus, lowering uric acid may be a novel treatment target for preventing diabetes. The levels of urea, serum creatinine and uric acid were restored to near normalcy by treatment with Tridax procumbens leaves extract as well as gliclazide in alloxan induced diabetic rats.

Oxidative stress definitely refers to the situation of an imbalance between the production of Reactive Oxygen Species (ROS) and antioxidant defense. There is emerging evidence that the formation of ROS is a direct consequence of hyperglycemia⁴⁴. Antioxidant treatment could be a beneficial therapeutic strategy for diabetic complications⁴⁵. Induction of diabetes in rats with alloxan uniformly results in an increase in thiobarbituricacid reactive substances (TBARS), an indirect evidence of intensified free-radical production. Due to increased level of free radicals, levels of TBARS elevate in liver and pancreas. The accumulation of TBARS during progression of diabetes may play a role in pancreatic and hepatocytic damage associated with diabetes.

The prevention of the formation of hydroxyl radicals would be an efficient means to reduce oxidative stress induced damage; the leaves extract which possesses a potent antioxidant system has the ability to prevent it. Thus, increase in TBARS associated with diabetes was prevented by treatment with Tridax procumbens leaves extract.

The enzymatic antioxidants such as SOD, catalase, glutathione peroxidase and glutathione S-transferase are involved in the scavenging of reactive oxygen metabolites that are produced as result of chronic hyperglycemia. The enzyme SOD is involved in the dismutation of superoxide anion into hydrogen peroxide thereby diminishes the toxic effects due to superoxide radicals and other free radicals derived from secondary reactions.⁴⁶. The increase in superoxide radical in diabetes may inhibit the activity of catalase and glutathione peroxidase.^47,48 . However, glutathione peroxidase is very sensitive even at lower concentrations of hydrogen peroxide. The activity of glutathione-S-transferase is essential for the regeneration of reduced glutathione from oxidized glutathione, because reduced glutathione is involved in the direct scavenging of reactive oxygen species as well as act as a co-enzyme for the activity of several detoxifying enzymes including glutathione peroxidase. In diabetic milieu, the activities of all these enzymatic antioxidants are declined notably because of the detonated production of free radicals arise out of chronic hyperglycemia.

The decrease in the levels of antioxidants in diabetic state represents the increased utilization of these antioxidants due to hyperglycemia-mediated oxidative stress. Oral administration Tridax procumbens leaf extract caused a significant increase in the levels of non-enzymatic antioxidants such as vitamin C, vitamin E, reduced glutathione and ceruloplasmin and enzymatic antioxidants such as SOD, catalase, glutathione peroxidase and glutathione-S-transferase suggesting that the Tridax procumbens leaf extract possess free radical scavenging and antioxidant activity. The presence of biologically active phytochemicals present in the leaves extract may be responsible for the observed antioxidant properties.

Lipids play an important role in maintaining the integrity of biomembrane structure and functions. In recent years, considerable interest has been directed towards the investigation of plasma lipids and lipoproteins. Pattern in Diabetes Mellitus is due to the fact that abnormal lipid level leads to the development of coronary artery disease as a complication in diabetic patients⁴⁹.

Hypercholesterolemia and hypertriglyceridemia have been reported to exist in alloxan induced diabetic rats. Accumulation of cholesterol in liver due to elevated plasma free fattyacids has been reported in diabetic rats⁵⁰. The higher concentration of plasma total cholesterol observed in diabetic rats is probably due to mobilization of free fatty acids from the peripheral fat depots⁵¹.

Alterations in the erythrocyte membranes lipid composition may be a reflection of alterations in the plasma lipid profile. HDL removes cholesterol from non-hepatic tissues to liver through the process known as reverse cholesterol transport. Several studies have documented reduction in plasma HDL cholesterol in diabetic rats and diabetic patients due to defect in reverse cholesterol transport⁵².

The result of the present study reveal that the ora; administration of Tridax procumbens leaves extract as well as gliclazide treated significantly decreased the levels of cholesterol, triglycerides, LDL with concomitant increase in HDL cholesterol are presumably mediated by controlling the lipid metabolism through improving the glycemic status.

CONCLUSION:

The results of the present study, supports the use of Tridax procumbens leaves extract for the treatment of diabetes mellitus. In conclusion, Tridax procumbens leaves extract possess potent antidiabetic, hypolipidemic and antioxidant properties. The extract exhibited anti-hyperglycemic activity comparable to that of a standard anti-diabetic drug, gliclazide. It can be concluded from the present study that the phytochemicals present in the extract may account for the observed pharmacological action. The present study also warrants further studies to isolate and characterize the active principle responsible for its pharmacological action.

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Received on 16.09.2011 Modified on 02.10.2011

Asian J. Research Chem. 4(11): Nov., 2011; Page 1732-1738

Groups	Total cholesterol	Triglycerides	LDL	HDL
Control	90.24 ± 10.56	59.74 ± 6.21	45.24 ± 6.49	29.85 ± 2.36
Diabetic	186.28 ± 16.30*	148.36 ± 12.34*	138.39 ± 10.24*	16.42 ± 1.91*
Diabetic + T. procumbens extract	119.74 ± 12.94^@	85.51 ± 9.36^@	76.14 ± 7.26^@	24.13 ± 2.19^@
Diabetic + gliclazide	105.28 ± 9.45^@	80.14 ± 6.98^@	68.97 ± 6.69^@	22. 18 ± 1.05^@