The Anti-oxidant and the Anti-diabetic Effects of Terminalia chebula and Withania somnifera in Subclinically Diabetic Dogs

Diabetes mellitus (DM) is an emerging metabolic syndrome of greater importance in dogs because of its multi-systemic terminal effects and cachexia. The characteristic features of clinical canine diabetes mellitus are polydipsia, polyuria, polyphagia and weight loss similar to the human syndrome. The prime factors associated with the increased incidence of DM in dogs were obesity, altered food habits, lack of exercise, genetic manipulations and inbreeding Temelkova-Kurktschiev and Stefanov, 2012; Raffan et al., 2015). Earlier studies reported the prevalence of DM among dog populations in the range of 0.0005% to 1.5% (Wilkinson, 1960; Mattheuws et al., 1984) whereas, recent studies from India revealed the existence of clinical/subclinical DM among dog populations in alarming levels, that is 1.8% to 7.2% (Das and Lodh, 2015; Devi et al., 2016). Geriatric female dogs were at high risk than their male counterparts may be due to their hormonal peculiarities leading to insulin resistance (Marmor et al., 1982). In dogs, clinical DM was expressed as elevated blood glucose concentrations (180-220 mg/dl) which is above the renal threshold and the major contributing factor was untreated pancreatitis (Nelson and Reusch, 2014; Rand, 2020).

Prior to clinical manifestation of DM, there is a prediabetic stage often called as subclinical diabetes in humans as well as dogs. According to Buysschaert and Bergman (2011), prediabetes is the presence of Impaired Fasting Glucose (IFG) and/or Impaired Glucose Tolerance (IGT). Experimental studies on pre-diabetic dogs showed high subcutaneous fat deposits, diminished insulin sensitivity and impaired fasting glucose (Ionut et al., 2010).

Insulin therapy is often essential for diabetic dogs since virtually all dogs have Insulin Dependent Diabetes Mellitus (IDDM). Also, oral hypoglycemic drugs such as sulphonylurea, metformin etc. were not effective in dogs suffering from IDDM (Nelson, 2000).

In diabetes mellitus, chronic hyperglycemia lead to multiple biochemical damages. Oxidative stress may be the basis of pathogenesis and clinical manifestations of this multi-systemic syndrome (Giugliano et al., 1996). Free radicals were synthesized in disproportionately higher rate and quantities in DM patients, by means of elevated glucose oxidation, non-enzymatic glycation of proteins followed by their oxidative degradation. Hence, the vital role of antioxidant therapy is significant. Antioxidants cannot be considered as a complete curative for DM. But intake of antioxidants can reduce the oxidative stress by interfering with the synthesis of free radicals or by inactivating them and thereby ameliorate the progression of pathological damages related to the disease (Cheng et al., 2003). Earlier, the role and usage of natural antioxidants in prevention of oxidative damage associated with experimental diabetes in rats has been studied (Coldiron et al., 2002; Vessal, et al., 2003). Strong natural antioxidants of plant origin included carotenoids, limonoids, tocopherols, ascorbates and polyphenols (Bajpai et al., 2005). Also, some plants showed hypoglycemic activity by interfering with insulin release/stimulation (Pari and Latha, 2002).

The development of new drugs and therapeutic strategies with least side effects were of greater interest to the scientific community at every point of time. Scientists were striving to evaluate antidiabetic activities of such natural products and its scientific validation (Florence et al., 2014). The ethanolic extract of T.chebula fruit showed potent hypoglycemic activity in streptozotocin-induced diabetic rats and was found more efficacious than glibenclamide, a standard hypoglycemic drug in commercial use (Kumar et al., 2006). Ethanolic extract of W. somnifera root have potent anti-inflammatory as well as anti-diabetic activity (Udayakumar et al., 2009). Keeping this as background data, the present study was carried out to evaluate the ameliorative role of T. chebula and W. somnifera via redox modulation in subclinically diabetic dogs.

Animal selection
 
The work was carried out at Division of Medicine, ICAR- Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh during 2015-2016. Subclinically diabetic dogs were identified based on preliminary screening of random blood glucose, fasting blood glucose and Benedict’s test for the presence of sugar in urine (Kaneko et al., 2008). Animals selected via preliminary tests were further confirmed through glycated haemoglobin estimation by ion exchange chromatography using commercial kit (Coral clinical systems, India) as per Trivelli et al., (1971), serum fructosamine estimation by nitrobluetetrazolium reduction method (Sahu and Sarkar, 2008) and serum insulin estimation by ELISA (Bioassay technology laboratory, China).

Plant extract preparation
 
T. chebula fruits and W. somnifera roots were procured and later identified by Dr. R.K. Tiwari, Reader and Head, Department of Dravyaguna, SRM Govt. Ayurvedic College, Bareilly, UttarPradesh. T. chebula dried fruit was crushed to separate outer seed husk and the seed was ground into powder. Dried W. somnifera roots were also ground into powder form. Extraction of the coarse powder in 70% ethanol was done using Soxhlet extraction apparatus by running 20 cycles at 60°C. After filtration through Whatman No.1 filterpaper, the extract was then dried using evaporator. The dried extract was then crushed into fine powder and was filled in commercially available gelatin capsule covers at the rate of 700 mg powder in each capsule. Capsules were then preserved in a refrigerator at 4°C till usage.
 
Experimental study design
 
The study is approved by Institutional Animal Ethics Committee (IAEC) under institute project IVRI/MED/12-15/008. Thirty subclinically diabetic dogs were selected based on the diabetic biomarkers (Kaneko et al., 2008) and were divided into five groups of six animals each; Group I was positive control without any treatment, Group II fed with T. chebula extract @ 100 mg/kg body weight, Group III fed with W. somnifera extract @ 100 mg/kg body weight, Group IV fed with both T. chebula extract and W. somnifera extract @ 100 mg/kg body weight each and Group V fed with standard antioxidant N-acetyl cysteine @ 10 mg/kg body weight. The antioxidants were administered for 30 days at the above mentioned daily dosages. Six apparently healthy dogs were kept without any treatment as negative control (Group VI). The animals were evaluated based on various diabetic biomarkers and oxidative stress markers on days 0, 15 and 30 of the study.
 
Blood sample processing
 
Blood samples were collected from the cephalic vein of the dogs in sterile vials with sodium fluoride (2 mg/ml) and heparin (0.2 mg/ml) as anticoagulants for the assessment of blood glucose and oxidative stress indices, respectively. About 2 ml of blood was collected into another clot activator vial and the serum was garnered for the evaluation of other diabetic biomarkers. Heparinised blood samples were centrifuged at 3000 rpm for 10 min to remove plasma and buffy coat and the red blood cells (RBCs) were harvested. The RBCs were washed thrice using normal saline solution and 10% hemolysate was prepared in chilled distilled water. This hemolysate was used for the estimation of oxidative stress indices namely LPO, SOD and Catalase activities. Reduced glutathione (GSH) level was estimated in 50% RBC suspension prepared by mixing with chilled normal saline (1:1).
 
Estimation of diabetic biomarkers
 
Random blood glucose level and fasting glucose level were estimated using portable glucometer (Accu-Chek, Roche Diagnostics) and Glucose oxidase and peroxidase (GOD-POD) method, respectively (Trinder 1969). Glycated haemoglobin (GHb), serum fructosamine and serum insulin concentration were estimated as per the method mentioned above.
 
Estimation of oxidative stress indices
 
Reduced glutathione (GSH) concentration in RBC suspension was estimated by 5, 5-dithiobis-(2-nitrobenzoic acid) (DTNB) method as per Prins and Loos (1969). LPO level in the RBC hemolysate was estimated by the method of Placer (1967) based on the formation of a colored complex between the lipid peroxidation products and thiobarbituric acid (TBA). Superoxide dismutase (SOD) activity was estimated by the method described by Madesh and Balasubramanian (1998). And finally, catalase (CAT) activity in hemolysate was appraised by the method of Bergmayer (1983) using H₂O₂ as a substrate.
 
Statistical analysis
 
The values are expressed as mean ± S.E. Data were analyzed by Analysis of Variance followed by the Post-Hoc Tukey test using statistical software package, SPSS 16.0. A value of P<0.05 was considered to be statistically significant.

Effect of different treatments on diabetic biomarkers and oxidative stress indices of subclinically diabetic dogs are presented on (Table 1). All the treatment groups showed significant (P<0.01) reduction in random blood glucose, fasting blood glucose, glycated hemoglobin, serum fructosamine and serum insulin concentration than the positive control group after treatment. Among treatment groups, group V (N-acetylcysteine) showed better improvement in random blood glucose (15.19%), fasting blood glucose (15.23%), glycated haemoglobin (15.19%), serum fructosamine (33.5%) and Insulin (20%) levels followed by group IV (15.14%, 14.67%, 13%, 31.03%, 19.74%) treated with combination of T. chebula and W. somnifera, group II (14.09%, 13.66%, 6.6%, 26.22%, 16.13%) treated with T. chebula and group III (8.2%, 8.1%, 3.58%, 25.56%, 6.7%) treated with W. somnifera, respectively, on day 30 of the therapy (Fig 1-5). Significant improvement in oxidative biomarkers was noticed in all the four treatment groups on day 30. N-acetylcysteine treated group (group V) showed highest increment in antioxidant systems such as GSH (33.33%), SOD (59%) and CAT (69.57%), followed by group IV (20.45%, 45%, 50%) treated with combination of herbs, group II (8.5%, 36%, 31.96%) treated with T. chebula and group III (6.5%, 29.16%, 27.8%) treated with W. somnifera, respectively, after therapy (Fig. 6-8). However, better reduction of LPO was exhibited by Group V (32.38%) followed by Group IV (29.94%), Group II (22.86%) and Group III (21.91%), respectively (Fig 9). No significant (P>0.01) difference in diabetic as well as oxidative biomarkers were observed between Group IV and Group V animals after treatment, unfolding the possibility of W. somnifera and T. chebula combination as an alternative to the standard antioxidant drug in management of subclinical diabetes mellitus in dogs.





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Varied biological properties of T. chebula is attributed by its tannins, vitamin C, gallic acid, anthraquinones, triterpenoids and other miscellaneous compounds (Juang et al., 2004). The antioxidant constituents are flavonol glycosides, triterpenoids and chebulin. T. chebula extract showed significant antioxidant and antidiabetic effect in alloxan-induced diabetic rats when administered at the dose rate 100 mg/kg body weight (Sabu and Kuttan, 2002). About thirty five pharmacologically important chemical constituents were reported from the root of W. somnifera. It includes various alkaloids, steroidal lactones, withanolides, withaferins, saponins etc. Among them, withaferin A and withanolide D were responsible for most of the pharmacological activity of W. somnifera (Mishra et al., 2000). It is reported to reduce hepatic lipoperoxidation and stimulate thyroidal activity (Verma, 2010). Immunostimulant property of Withania somnifera has also been detected in poultry vaccinated against New Castle’s Disease (Mohanambal et al., 2017). Udayakumar et al. (2009) demonstrated the hypoglycaemic effect of W. somnifera root ethanolic extract on experimentally induced diabetic rats at 100mg/kg body weight.

N-acetyl cysteine has significant therapeutic potential in ameliorating oxidative stress by means of its GSH precursor effect as well as free radical scavenging ability (Gillissen et al., 1997; Messonier, 2001). This antioxidant effect of N-acetylcysteine would have contributed in improving the diabetic biomarkers of the subclinically diabetic dogs by modulating redox mechanisms and preventing further progression of disease. Dried ripe fruit of T. chebula Retz (Combretaceae) is a vital constituent of Triphala, a traditional medicine in Asia (Perry and Metzger 1980). Antidiabetic effect of chebulic acid derived from T. chebula has been reported by Lee et al., (2010). W. somnifera (Dunal), a well-known adaptogen, has proven hypocholesterolemic and antioxidant properties (Singh et al., 2011). Also, it possesses anti-inflammatory, anticancer, antistress, immunomodulatory, rejuvenating properties along with endocrine, cardiopulmonary and central nervous system modulatory properties (Mishra et al., 2000). In vivo and In vitro antioxidant potential of T. chebula and W. somnifera has already been reported (Singh, 2007; Deepa, 2014). Previous reports also suggest W. somnifera as an anti-diabetic agent (Udayakumar, 2009).

Subclinical diabetic/prediabetic animals were apparently healthy with persistently elevated fasting blood glucose concentrations above the reference range, but below the threshold concentration necessary for glycosuria (Rucinsky et al., 2010). The present study reports elevated glycated haemoglobin, serum fructosamine and serum insulin level in prediabetic dogs which is in agreement with Deepa (2014) and Kaneko (2008). Elevated glycated hemoglobin (HbA1c) is important predictive biomarker of chronic hyperglycemia, whereas, the serum fructosamine provide the previous 2-3 weeks’ estimate (Mared et al., 2012). Recently canine HbA1c has shown least correlation with haematological parameters and therefore it can be suggested as a reliable biomarker of canine diabetes (Neumann, 2020). Canine diabetes mellitus is usually compared to latent autoimmune diabetes mellitus in human beings; initial hyperstimulation of pancreatic â cells followed by insulin deficient phase due to â cell exhaustion (Shanik et al., 2008). The present study explains that the antioxidant therapy can curb this pathological mechanism by modulating oxidative metabolisms preventing exhaustive damage to â cells. Similarly, the modulation of host immune mechanisms by the antioxidant N-acetylcysteine in Th1 cytokine dominant disease conditions was previously suggested by Ajith et al., (2017). Also, the human form of IDDM is usually considered as a Th1 dominant condition with increased expression of Th1 cytokines (Eizirik et al., 2009). Hence, early detection and strategic management of subclinical DM using better antioxidants is necessary for preventing its progression to clinical form, improving lifespan and ensuring better quality life.

Subclinical diabetes mellitus in dogs is a disease condition which needs more attention from clinical veterinary practitioners. Significant oxidative impairments were observed in even subclinical stages of diabetes mellitus and may be elaborated in the pathogenesis of clinical diabetes. N- acetylcysteine, T. chebula and W. somnifera ameliorate oxidative stress in subclinical diabetes mellitus and prevent further evolution of the disease. Hence, the current study established the antidiabetic potential of W. somnifera and T. chebula via improved oxidant-antioxidant profile. Further, they prevented damages on the body system resulted from approppriate glucose utilization, which itself is a triggering force for free radical generation and unstable body system. The present study emphasizes the early detection of subclinical form of diabetes and stress the need for antioxidant therapy in preventing progression of the clinical disease.