ANTIDIABETIC EFFECT OF CONYZA AEGYPTIACA (L.) DRYAND EX. AITON IS IMPROVED BY SWIMMING IN RAT.

Conyza aegyptiaca (C. aegyptiaca) is one medicinal plant used for the treatment of diabetes by African populations. We showed previously that C. aegyptiaca is not toxic in rats, prevents hepatic glucose release and has anti-hyperglycemic properties. In addition to medical


ISSN: 2320-5407
Int. J. Adv. Res. 7 (7), 220-230 221 Introduction:-Diabetes mellitus (DM) is a metabolic disorder characterized by hyperglycemia due to defection of insulin secretion, insulin action or both (Goldenberg and Punthakee, 2013;Hussain et al., 2007). The prevalence of DM is increasing across the world (World Health Organisation, 2016). Type 2 diabetes (T2D) is one of the major public health challenges of the 21st century. Overweight, sedentary lifestyle and dietary components are risk factors but also central themes for prevention (Hussain et al., 2007). Regular physical activity reduces the risk of T2D in adults by 20-60% in a dose response manner (Hussain et al., 2007;Vuori, 2001).
Because of the high cost of conventional anti-diabetic drugs (Gning et al., 2007), traditional treatment based on plants becomes an alternative option for financially poor populations who may have problems of accessibility to modern drugs. To overcome diabetes, physical activity practice is recommended (World Health Organisation, 2016). For these reasons, the development of new therapies from plants that are able to control diabetes mellitus is of great interest.
Several medicinal plants have been found to be successful in diabetes treatment and some have been tested and their active compounds identified (Grover et al., 2002). This has led researchers to intensify research of suitable treatment from plant origin for diabetes care. Conyza aegyptiaca (L.) Dryand ex. Aiton, belonging to the Asteraceae family, is a well-known plant used by the population of Togo and Benin (West Africa). Also called EGBE VEVE in Mina (local dialect), Conyza aegyptiaca (C. aegyptiaca) is widely used to overcome malaria, sickle cell disease, and sore throat (Akpagana et al., 1996). Recently, an ethnobotanic study found that this plant is also used for obesity and diabetes treatment by West African populations (Gbekley et al., 2015).
The anti-diabetic effect attributed to medicinal plants is generally reflected in their efficacy on inhibition of intestinal absorption, stimulation of insulin secretion by the pancreas, inhibition of glucose production by the liver or stimulation of the storage of blood glucose by the liver and muscles (Hanan and Mona, 2014). More ever, physical exercise reduced body weight, visceral fat accumulation (Irwin et al., 2003;Ross et al., 2000), insulin resistance (Kelley and Goodpaster, 1999;Rice et al., 1999), improved glucose tolerance (Dengel et al., 1998;Kelley and Goodpaster, 1999) and normalized lipid profile (Kraus et al., 2002;Rice et al., 1999).
In previous study, we showed that C. aegyptiaca extracts prevent liver glucose liberation in vitro (Akakpo et al., 2016), thus promoting glucose storage in liver. We also showed that C. aegyptiaca aerial parts extracts is safe and has antihyperglycemia potential as it normalized induced high blood glucose level without causing hypoglycemia (Akakpo et al., 2017). Here, we tested the antidiabetic effect of C. aegyptiaca alone and in association with forced swimming on streptozotocin-induced diabetes rats.

Materials and Methods:-Plant material:
The plant material was composed of the aerial parts of C. aegyptiaca. The leaves and stem of the plant were shadedried and ground into a fine powder. The powder was then extracted with water and ethanol.

Extraction:
For the aqueous extraction, decoction was done on a heating plate during 30 minutes by addition of 1000 ml distilled water to 100 g of powder. The mixture was filtered with Whitman paper and evaporated under rota vapor at 65-70°C. The ethanolic extraction was done by macerating 100 g of powder in ethanol diluted at 50° with distilled water, during 24 hours mixed by homogenizer. After filtration with Whitman paper, the product was evaporated with the rota vapor at 40-45°C. Extracts were conserved at 4°C.

Animal material:
Male and female albinos Wistar rats weighing 200 to 250 g at the age of eight to ten weeks were used for the study. Animals were housed in polypropylene cages and maintained under standard conditions with an alternated cycle of twelve hours light and twelve hours dark. They had free access to food and water. Room temperature was maintained at 25°C with a relative humidity of 35-60%.

222
Antidiabetic activity: To induce diabetes, the rats were administered nicotinamide (NAD) 230 mg/kg (ip) 15 min before streptozotocin (STZ) 65 mg/kg (iv) after 16 h fasting (Masiello et al., 1998;Srinivasan and Ramarao, 2007). Seventy two hours later, rats were classified diabetics if glucose level ≥175 mg/dl. Animals were divided into 5 groups of 2 females and 3 males each. Group 1 received Glibenclamide 10 mg/kg bw, groups 2 and 3 received aqueous and ethanolic extract at 500 mg/kg bw of C. aegyptiaca respectively, group 4 (diabetics without treatment) and group 5 (non-diabetic) received only distilled water at 10 ml/kg bw, once a day during 14 days.
Antidiabetic activity associated with sport and physical activity: Rats were submitted to swimming during 28 days (Table 1) (Association Canadienne du Diabète, 2008; Snowling NJ, 2006). The swimming was performed in a glass jar (90cm x 50cm x 50cm) filled with water up to 35 cm (Kamakura et al., 2001;Matsumoto et al., 1996). Five groups of five rats each were used in this study and biochemical parameters were measured at D7, D14, D21 and D28.

Statistical analysis:
Data are expressed as mean ± standard error of the mean (SEM) and evaluated by Student's t-test using SigmaPlot statistical analysis software 2014 (Systat Software, Inc. San Jose, CA, USA). A level of p <0.05 was set as significant.
Results:-Antidiabetic activity: Figure 1 showed variation of glucose level in diabetic rats. The first day of treatment (Day 1), glucose level significantly increased compared to D0 (p=0.0009). In diabetic rats without treatment, glucose level remained significantly high during the experience (p=0.0460) compared to treated groups. At D7, glucose level significantly decreased in ethanolic extract group (p=0.0450), in aqueous extract group (p=0.0031) and in Glibenclamide group (p=0.0046). The decrease remained until D14 (Fig.1).
Creatinine level did not change in all groups. In contrast, the level of the enzyme AST significantly decreased in group treated with C. aegyptiaca aqueous extracts (p=0.0007), Glibenclamide treated group (p=0.0009) and ethanolic treated group (p=0.0047) compared to D0. The level of the enzyme ALT also significantly decreased respectively in diabetic without treatment group (p=0.0091) and in Glibenclamide treated group (p=0.0046) ( Table  2). Aqueous and ethanolic extracts had no significant effect on TC and TG. However, LDL-C level significantly decreased in aqueous extract group (p=0.0472) whereas HDL-C level significantly increased (p=0.0481) at D14 compared to D0 (Table 4). In the group of rats taken Glibenclamide 10 mg/kg, triglycerides (p=0.0471) and LDL-C (p=0.0009) levels significantly decreased whereas HDL-C (p=0.0009) level significantly increased (Table 3). Rats treated with aqueous extract showed a significant increased level of potassium (p=0.0471) and sodium (p=0.0092). Only potassium level significantly increased in group of ethanolic extract (p=0.0046). Calcium, chloride and magnesium levels did not change in any group (Table 5). No variation in electrolytes levels was recorded in normal rats. In contrast, diabetic rats without treatment showed significant potassium and sodium levels increase (p=0.0451) ( Table 4).
Antidiabetic effect of C. aegyptiaca combined with sport and physical activity: Aqueous extract 500 mg/kg bw (p=0.0091) and Glibenclamide 10 mg/kg (p=0.0046) significantly decreased glucose level at D7 (Fig. 2A). The association of Sport and Physical Activity (SPA) to C. aegyptiaca aqueous extract and Glibenclamide did not induce additional glucose level variation. In ethanolic extract group however, SPA caused further glucose level decrease (p=0.0450) ( Fig. 2A). Glucose level was unchanged in normal rats submitted to SPA 223 or not (Fig. 2B). Glucose level was significantly decreased in diabetic rats submitted to SPA alone at D7 (p=0.0080) and D14 (p=0.0470) compared to diabetic rats without SPA at D7 and D14 (Fig. 2B). Table 5 showed results of the combined effect of C. aegyptiaca and SPA on liver and kidney function. The practice of swimming showed an additive decreasing effect on creatinine level in the aqueous extract taken groups of rats (p=0.0092) and in diabetic rats without treatment (p=0.0461).
Discussion:-C. aegyptiaca (L.) Dryand ex. Aiton is a widely used plant to treat malaria, gonorrhoea (Goldenberg and Punthakee, 2013), and cardiovascular diseases (Hussain et al., 2007). It was recently reported that this plant treats diabetes (World Health Organisation, 2016). However, the use of C. aegyptiaca to treat diseases is based on empirical knowledge, with no scientific basis (Vuori, 2001), as is the case for most of medicinal plants in Africa. World Health Organization (Gning et al., 2007) promotes ethnobotanic studies and pharmaceutical research to improve disease treatment based on medicinal plants.
In our previous work, we showed that C. aegyptiaca displayed inhibitory properties of hepatic release of glucose (World Health Organisation, 2016) and possesses anti-hyperglycemic effects (Grover et al., 2002) in rats. In this study, we analyzed the antidiabetic properties of C. aegyptiaca alone and when combined with SPA. Our results showed that C. aegyptiaca extracts induce a significant decrease in blood glucose levels in diabetic rats compared to untreated diabetic rats at a level comparable to Glibenclamide, an antidiabetic molecule used as a reference in this study. This finding confirms the anti-hyperglycemic properties we showed earlier (Grover et al., 2002) and supports the empirical knowledge that C. aegyptiaca treats diabetes (World Health Organisation, 2016). C. aegyptiaca contains alkaloids and flavonoids (Akakpo et al., 2016) that are involved in stimulating insulin secretion (Sharma et al., 2008). SPA alone significantly lowered blood glucose levels in diabetic rats at a level similar to ethanolic extract but less than aqueous extracts. A further decrease in blood glucose was observed when SPA was associated with the ethanolic extract. The combination of SPA with the aqueous extract did not induce any further drop in blood glucose. These results suggest that SPA produces an additive effect on the C. aegyptiaca-induced blood glucose lowering.
Regular physical activity lowers blood glucose, decreases body fat, improves lipid balance, and generally helps control T2D risk factors and prevent its complications ( Our results showed a significant decrease in blood creatinine levels in diabetic rats subjected to SPA. During effort, blood drained by organs like kidneys decreases in favor of muscles and induces decreases in creatinine level (Rice et al., 1999;Ross et al., 2000). Decrease in blood creatinine level and other parameters such as glomerular filtration rate at the end of exercise is transient and reversed approximately 24 hours later (Ross et al., 2000). SPA did not produce an additive effect on transaminases levels reached with C. aegyptiaca extracts and Glibenclamide. These results show that C. aegyptiaca has hepatoprotective properties (World Health Organisation, 2016) that SPA obviously does not influence. Altered lipid parameters may lead to atherosclerosis and coronary heart disease (Kelley and Goodpaster, 1999). CT, TG and LDL-C levels are reported higher in diabetic patients compared to nondiabetic controls (Kraus et al., 2002;Akpovi et al., 2015). As in the case of blood glucose, we observed an additive effect of C. aegyptiaca and SPA in the normalization of disturbed lipid profile in diabetic rats. This result supports the idea that regular SPA reduces fat mass and improves lipid balance (Akpagana et al., 1996). SPA also promotes muscle use of free fatty acids by increasing the number of mitochondria and mitochondria enzymes involved in beta-224 oxidation (Akakpo et al., 2016). Our study is the first to show that C. aegyptiaca reverses disturbed lipid profile in streptozotocin-induced diabetic rat.
Numerous studies have reported a wide variety of electrolyte disorders in diabetics (Akakpo et al., 2017;Yessoufou et al., 2013;Srinivasan and Ramarao, 2007;Prashanth et al., 2001). In this study, we measured a significant increase in K + and Na + in diabetic rats compared to control, while Ca 2+ , Cland Mg 2+ remained unchanged. High levels of K + are reported in diabetic rats (Prashanth et al., 2001). Chronic hyperkalemia in diabetes was reported to be associated with hypoaldosteronism syndrome and the reduction of tubular K + secretion (Liamis et al., 2012). In this syndrome, patients generally have asymptomatic hyperkalemia characterized by mild to moderate renal impairment (Prashanth et al., 2001). Since glucose is an osmotically active substance, hyperglycemia increases serum osmolarity, draining excess fluid out of the cells in favors of blood circulation. This leads to a reduction in serum Na + levels by hemodilution and hyponatremia (Masiello et al., 1998).
In this study, we showed that SPA significantly improves the electrolytes profile in both diabetic rats and nondiabetic control rats. This effect of SPA is independent of C. aegyptiaca extracts. Physical exercise and nutrition are the cornerstones of the diabetes prevention mechanism. In healthy individuals and animals, physical activity alone can delay or even prevent the development of T2D later in life. Results showed here suggest that the antidiabetic effect of C. aegyptiaca extracts are mainly due to their antioxidant properties (Akakpo et al., 2016). SPA enhances the activity of antioxidant enzymes in diabetic rats subjected to swimming (Vogiatzi et al., 2009). In doing so, SPA potentiates the antidiabetic effect of C. aegyptiaca extracts by strengthening the body's defense against very harmful oxidative stress in diabetics (Rakieten et al., 1963).
In conclusion, we showed that streptozotocin-induced diabetes in albino Wistar rats is accompanied by hyperglycemia, an imbalance in blood electrolyte levels, and dyslipidemia characterized by high levels of CT and TG and impaired kidneys and liver function. All these disturbances were substantially corrected by C. aegyptiaca extracts. SPA alone reduced blood glucose levels and lipid parameters CT and TG without reaching normal levels. However, the combination of SPA with C. aegyptiaca extracts further reduced levels of disturbed parameters in diabetic rats. Together, our results showed that SPA potentiates the antidiabetic effect of C. aegyptiaca extracts.

Authors' contributions:
B. Huguette Akakpo is a PhD student who conducts the experiences, treats data and writes the manuscript. She also co-finances the study.

Conflicts of interest:
No potential conflict of interest was reported by the authors.