The Antihyperglycemic, Hepatoprotective and Renoprotective Potentials of the Aqueous Extract of Costus lucanusianus on Streptozotocin-induced Diabetic Rats

Aim: This study sought to evaluate the effect of Costus lucanusianus on streptozotocin-induced diabetic rats. Methodology: The effect of daily oral administration of aqueous extract of this plant on streptozotocin-induced diabetic rats was monitored for four weeks. Fasting blood glucose, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) activities were determined. In addition, total protein, creatinine and urea levels were also measured. there administration of aqueous extract of Costus lucanusianus decreased the activities of these marker enzymes. Conclusion: These findings suggest the antihyperglycemic, hepatoprotective and renoprotective potentials of this plant which can make it suitable candidate in the management of diabetes mellitus.


INTRODUCTION
Diabetes mellitus (DM) has been distinguished with persistently elevated blood glucose leading to acute or long term complications [1]. Globally, DM presents increased public health issue.The prevalence of DM in all age groups rate is expected to rise 8% to (170 million) in 2000 and the 4.4% (366 million) in 2030 [2]. The therapeutic agents currently used for treatment of diabetes have undesirable effects and fail to significantly alter diabetic complications [3]. Attention is now being shifted to traditional practices and herbal remedies with more efficacious and minimal side effects than the currently used oral hypoglycemic agents for the treatment of diabetes [4,5]. Costus lucanusianus is a common species in the forest zone of tropical Africa. It is widely used as a medicinal plant in tropical Africa but little is known about the constituents and their pharmacological activities [6]. This merits further research. In fact, saponins as the key ingredients in Chinese medicines responsible for both antiglycation and antioxidant activities had also been addressed in few studies [7][8][9].

Materials
Costus lucanusianus leaves were obtained from an open forest in Akungba Akoko, Ondo state and were identified by Dr. Obembe O.A, a taxonomist from the department of Plant Science and Biotechnology, Adekunle Ajasin University Akungba Akoko. Assay kits for AST, ALT, ALP, LDH, creatinine, urea and total protein were products of Randox Laboratory Ltd, Ardmore, Diamond Road, Crumlin, Co. Anrtim, United Kingdom. Streptozotocin (Sigma, Brazil) and other analytical grade chemicals were used for this study.

Plant Extract Preparation
C. lucanusianus leaves were collected from a farm in Akungba-Akoko, Ondo State, Nigeria. A modification of [10] method was used to prepare the extract. Briefly, the shade dried leaves were crushed and then soaked in distilled water for 72 hours in a plastic container and covered with cheesecloth. The contents were stirred several times a day and at the end of the third day the contents were filtered through two layers of cheesecloth. The extract was quantified by drying 1 mL of the homogeneous filtrate (by controlled heating i.e. in an oven kept at 40°C) in a pre-weighed watch glass; this was done in triplicates and the average determined. The extract was kept in the freezer until use, when it was allowed to thaw at room temperature.

Experimental Animals and Protocols
Thirty-five (35) adult male and Wistar rats (average weight 228 g) were obtained from the Animal Unit of the University of Ibadan Teaching Hospital (UCH), Ibadan, Nigeria. The rats were kept in a well-ventilated room, with 12 h light and 12 h dark cycles. They were given free access to food (standard pelleted feed) and water and allowed to acclimatize for three weeks before the commencement of the study. Treatment of the animals conformed to the guidelines in the Principles of Laboratory Animal Care (NIH Publication 85-23, revised 1985) and was approved by the local Institutional Review Board (IRB).
Seven groups of five rats each were used for this study, namely: Group 1: normal control rats given distilled water; Group 2: diabetic control rats given distilled water; Group 3: Metformin treated diabetic rats, orally given 200 mg/kg; Group 4: C. lucanusianus treated diabetic rats orally given 100 mg/kg; Group 5: C. lucanusianus treated diabetic rats orally given 200 mg/kg body weight of C. lucanusianus aqueous leaf extract; Group 6: normal control rats orally given 100 mg/kg body weight of C. lucanusianus aqueous leaf extract; Group 7: normal rats orally given 200 mg/kg body weight of C. lucanusianus aqueous extract.

Biochemical Assays
Serum glucose (Reddy's laboratories, Hyderabad, India), Assay kits for AST, ALT, ALP, LDH, creatinine, urea and total protein were products of Randox Laboratory Ltd, Ardmore, Diamond Road, Crumlin, Co. Anrtim, United Kingdom. They were estimated using a commercial diagnostic kit. Serum protein and urea were estimated by the method described by [11,12].

Statistical Analysis
All the grouped data was statistically evaluated via the statistical package for social sciences version 15. Hypothesis testing method included one-way analysis of various (ANOVA) followed by least significant difference test. P-values of less than 0.05 were considered to indicate statistical significance. All the results were expressed as mean ± SD for 5 animals in each group.

Effect of Costus lucanusianus Leaves on Fasting Blood Sugar of Diabetic Rats
The result obtained in Figure 1 showed that there was a significant increase (P<0.05) in the fasting blood sugar of the diabetic rats when compared to the normal rats. However, the treatment with aqueous extract of Costus lucanusianus leaf on the diabetic rats showed a significant reduction of fasting blood sugar (P<0.05) in the treatment groups when compared to the diabetic rats. It is worthy of note that the reduction in the fasting blood sugar is also concentration dependent.

Effect of Costus Lucanusianus on Body Weight of the Diabetic Rats
It was observed that there was a significant decrease in the diabetic control when compared to the normal rats. However, on administration of metformin and Costus lucanusinus, there was an increase in body weight of metformin treated group and Costus lucanusianus treated group respectively throughout the period of monitoring.
The general increase in body weight observed in Costus lucanusianus treated rats as compared with water control rats indicates that Costus lucanusianus has an increasing effect on body weight.

Effect of Costus lucanusianus Leaf on Aspartate Aminotransferase (AST) Activity
The result obtained above showed that there was a significant increase (P<0.05) in the serum AST activity of the diabetic control when compared to the normal control rats. In contrast, the cardiac and hepatic AST activities were significantly reduced in the diabetic control when compared to the normal control. Furthermore, there was a significant reduction (P<0.05) in the serum AST activities of the Costus lucanusianus treated groups when compared to the diabetic control.

Effect of Costus lucanusianus Leaf on Alanine Aminotransferase (ALT) Activity
The result in Figure 4 showed that there was a significant increase (P<0.05) in the serum ALT activity of the diabetic control when compared to the normal control rats. In contrast, the cardiac and hepatic ALT activities were significantly reduced in the diabetic control when compared to the normal control. Furthermore, there was a significant reduction (P<0.05) in the serum ALT activities of the Costus lucanusianus treated groups when compared to the diabetic control.

Effect of Costus lucanusianus Leaf on Alkaline Phosphatase (ALP) Activity
The result obtained in Figure 5 revealed that there was a significant increase in the activity of ALP in diabetic rats when compared to the normal control. However, in the liver ALP activity there was a significant reduction in diabetic control when compared to the normal control.

Effect of Costus lucanusianus Leaf on Lactate Dehydrogenase (LDH) Activity
The result obtained in Figure 6 revealed that there was a significant increase in the activity of LDH in diabetic rats when compared to the normal control. However, in the liver LDH activity there was a significant reduction in diabetic control when compared to the normal control.

Effect of Costus lucanusianus Leaf on Urea Level
The result obtained in Figure 7 revealed that there was a significant increase in the level of urea in diabetic rats when compared to the normal control. However, in the kidney urea level there was a significant reduction in diabetic control when compared to the normal control.

Effect of Costus lucanusianus Leaf on Creatinine Level
The result obtained in Figure 8 revealed that there was a significant increase in the level of creatinine in diabetic rats when compared to the normal control. However, in the kidney creatinine level there was a significant reduction in diabetic control when compared to the normal control.

Effect of Costus lucanusianus Leaf on Total Protein Level
The result obtained in Figure 9 revealed that there was a significant decrease in the level of total protein in diabetic rats when compared to the normal control. However, in the kidney total protein level there was a significant reduction in diabetic control when compared to the normal control.

DISCUSSION
The reduction in the fasting blood glucose observed in the Costus lucanusianus treated diabetic groups agrees with the report of [13] which showed that some medicinal plants exhibited antidiabetic activity against the streptozotocin induced diabetic rats. The possible mechanism by which Costus lucanusianus extract bring about its hypoglycaemic action in diabetic rats may be improving glycemic control mechanism and insulin secretion from remnant pancreatic beta cells in diabetic rats [14]. Previous work on the phytochemical screening of this plant revealed that it contains saponins which have been known to possess antidiabetic property and are promising compounds with potential to be developed into new drugs for antidiabetes [5].
Aminotransferases (AST and ALT) mediate the catalysis of amino-transfer reactions and they are markers for clinical diagnosis of liver injury [15] while ALP is responsible for removing phosphate group from nucleotides and proteins. This enzyme is primarily produced in the liver and brain [16], and also used as marker of hepatic functions [17]. As revealed in this present study, the significant increase in the activities of serum AST, ALT and ALP observed in diabetic control group as compared to normal control group was mainly due to the leakage of these enzymes from liver cytosol into the blood stream which is an indication of hepatoxic effect of STZ [15] and consistent with previous studies which described increased liver oxidative stress in this model of experimental diabetes mellitus [18]. Moreover, it was suggested that the elevation in serum AST and ALT activities in diabetic rats could be related to excessive accumulation of glutamate and alanine in the serum of diabetic animals, as a result of amino acids mobilization from protein stores [19]. However, Costus lucanusianus treated groups exhibited significant decrease in serum AST, ALT and ALP activities which could be attributed to their ability to protect/repair liver tissue damage. Plant foods are rich in saponins and antioxidant polyphenol phytochemicals with hepatoprotective properties [20].
Indeed, elevated LDH levels observed in the experimental diabetic animals are associated with impaired glucose-stimulated insulin secretion [21]. Thus, increased activity of LDH interferes with normal glucose metabolism and insulin secretion in the β-cells of pancreas and it may therefore be directly responsible for insulin secretory defects in diabetes. However, treatment with Costus lucanusianus to diabetic rats reversed the LDH activity to near normalcy. Similarly treatment with resveratrol to diabetic rats decreased the activity of LDH [22] most probably by regulating the proportion of pyruvate and NADH thereby promoting the mitochondrial oxidation of (pyruvate) glucose. The protective effects due to treatment with Costus lucanusianus strongly indicate the possibility of the extract being able to prevent any leakages of marker enzymes. There are some reports on reversal of LDH in diabetic rats with treatment with Murray koenigii, Ocimum sanctum [23].
Renal disease is one of the most common and severe complications of diabetes. Insulin is a physiological factor, which plays an important role in the maintenance of protein balance, since it not only stimulates the uptake of amino acids and protein synthesis, but also inhibits protein degradation [24]. In addition, significant elevations in serum creatinine and urea levels indicate impaired renal function of diabetic animals. Aqueous extract of Costus lucanusianus leaf increased the total protein and lowered the serum urea and creatinine levels by enhancing the renal function that is generally impaired in diabetic rats. This result is in agreement with a previous study [25].
Reduction in serum protein level observed in diabetic control group may be due to microproteinuria which are clinical important markers of diabetic nephropathy and may be due to increased protein catabolism [26]. Also formation of protein-MDA adducts may have contributed to the decrease in serum protein level due to increased lipid peroxidation [27]. However, the marked increase in the serum total protein of Costus lucanusianus treated group may be due to the hepatoprotective properties of their phytochemical constituents and also availability of amino acids. Myricetin is the active principle detected in this plant [28].

CONCLUSION
In the present study, the findings may suggest the antihyperglycemic, hepatoprotective and renoprotective potentials of this plant which can make it suitable candidate in the management of diabetes mellitus.