Reverse Ayurvedic Pharmacology of Ashwagandha as an Adaptogenic Anti-Diabetic Plant: A Pilot Study

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1 Send Orders for Reprints to Current Traditional Medicine, 2015, 1, Reverse Ayurvedic Pharmacology of Ashwagandha as an Adaptogenic Anti-Diabetic Plant: A Pilot Study Ajit K. Thakur 1, Amitabha Dey 1, Shyam S. Chatterjee 2 and Vikas Kumar 1* 1 Neuropharmacology Research Laboratory, Department of Pharmaceutics, Indian Institute of Technology (Banaras Hindu University), Varanasi , India; 2 Stettiner Str. 1, D Karlsruhe, Germany Abstract: Aim of this study was to define oral dose range and treatment regimen of a pharmaceutically well-standardized Withania somnifera root extract (WS) potentially useful for treatments of diabetes-associated comorbidities. Normal and diabetic male mice were treated daily with graded oral doses of WS for 10 consecutive days. On the 1 st, 5 th, 7 th, and 10 th days of treatments, the animals were subjected to a foot shock stress-triggered hyperthermia tests. Changes in body weights and basal core temperatures of experimental animals during the course of the experiments were also recorded. In addition, plasma glucose, insulin, and cortisol levels and weights of adrenal glands and spleen of diabetic animals were quantified after 10 daily WS doses. No statistically significant effects of a single oral dose of WS on any of the quantified parameters were observed even after its highest oral dose (400 mg/kg) tested. However, dose and duration of treatment dependant protective effects of WS against body weight losses and elevated core temperatures caused by daily handling and intermittent foot shock stress, as well as against stress-triggered transient hyperthermia were observed in both normal and diabetic mice after its repeated daily doses. Dose dependant efficacy of the extract against elevated plasma glucose, insulin, and cortisol levels, and alterations in adrenal gland and spleen weights of diabetic animals were apparent after its 10 daily doses. Reported observations add further experimental evidence justifying traditionally known medicinal uses of Withania somnifera for treatment of mental health problems commonly associated with numerous lifestyle-related chronic diseases. Keywords: Ashwagandha, adaptogens, diabetes, foot-shock, hyperthermia, stress, Withania somnifera. 1. INTRODUCTION Ashwagandha is the Sanskrit name of a plant of the Solanacea family (botanical name: Withania somnifera), the roots of which are often used for medicinal purposes in Ayurvedic and other traditionally known systems of medicine in India and several other countries [1]. In Ayurvedic system of medicine and health care, Ashwagandha is considered to be a rejuvenative herb, which in *Address correspondence to this author at the Neuropharmacology Research Laboratory, Department of Pharmaceutics, Indian Institute of Technology (Banaras Hindu University), Varanasi , India; Tel: ; Fax: ; vikas.phe@iitbhu.ac.in Retired Head of Pharmacology Research Laboratories, Dr. Willmar Schwabe GmbH & Co KG, Karlsruhe, Germany. combination with other herbs and natural products is often used for prevention and cure of diverse spectrums of mental health problems commonly associated with chronic diseases. Information now available on medicinal phytochemistry and pharmacology of diverse types of Withania somnifera extracts not only justify such traditionally known medicinal uses of its roots, but also reveal their broad spectra of stress response regulation and other therapeutically interesting bioactivities [2-5]. A few recent reports have also suggested that such extracts could also be used for prevention and cure of hyperglycemia and other diabetes-associated pathologies [6-9]. However, our current understanding of bioactive constituents of such extracts still remains to be far from satisfactory, or at the best speculative only, and as /15 $ Bentham Science Publishers

2 52 Current Traditional Medicine, 2015, Vol. 1, No. 1 Thakur et al. yet only little concentrated efforts have been made to obtain a pharmacologically and analytically well standardized extract from the roots that could be further developed for such purposes. Diabetes is a slowly progressing metabolic disorder spreading like epidemic in all countries irrespective of their socio-economic and cultural background [10]. It has recently been estimated that more than 85% of type-2 diabetic patients live in India, China and other developing countries, and that the global prevalence of diabetes will continue to increase during the next two decades [11]. Comorbidities of anxiety, depression, and other mental health problems are more often encountered in diabetic patients than in normal population [12, 13], and it is now well recognized that serious psychological distress has a significant negative impact on diabetes process and outcome [14, 15]. Since available antidiabetic and other drugs do not properly meet the therapeutic demands of diabetic patients, efforts are now being made in our laboratories to identify novel therapeutic leads from traditionally known medicinal herbs potentially useful for prevention and cure of psychopathologies accompanying diabetes and other metabolic disorders. For such purposes, we have now standardized and pharmacologically validated a mouse bioassay for estimating the pharmacologically interesting dose ranges and treatment regimen of adaptogenic herbal extracts and their bioactive constituents [16-22]. In principle, this mouse bioassay is based on an earlier reported observations made in foot shock stressed rats with an extracts of Withania somnifera [23], and on the well known effects of other adaptogenic herbal extracts on thermoregulatory processes involved in physiological stress responses [24-26]. It has since long been known that thermoregulation is impaired in diabetic patients [27-29], and that diabetic mice also suffer from such impairment [30]. Results of the very first set of experiments conducted to compare the efficacy of a commercially available and pharmaceutically well-characterized Withania somnifera extract in the bioassay using nondiabetic and type-2 diabetic mice are reported and discussed in this communication. 2. MATERIALS AND METHODS 2.1. Animals Adult male albino mice (25±5 g) were obtained from Central Animal House of Institute of Medical Sciences, Banaras Hindu University, Varanasi (Registration number: 542/AB/CPCSEA). All animals were acclimatized to constant laboratory conditions at least for one week before starting the experiment. They were randomly selected and group-housed (six animals per cage) in polypropylene cages (28x19x12.5 cm) at an ambient temperature (25±1 C) and relative humidity (50±10%) with a 12:12 h light/dark cycle (light on at 06:00 and off at 18:00 h). Cages were provided with sawdust and free access to standard rodent diet and water, and were cleaned routinely. Principles of laboratory animal care (NIH publication 85-23, revised in 1985) guidelines were always followed. All the experimental groups in a given set of experiment were tested in parallel (i.e. on the same days of the experiments), and handled, weighed, and observed by single blinded observer and using the same lab equipments. Prior approval from the Central Animal Ethical Committee of Banaras Hindu University, Varanasi, India was obtained for the experimental protocols (Dean/11-12/CAEC/325, dated ) Plant Material The pharmaceutically well-standardized 90% methanolic extract of Withania somnifera roots (WS), analytically characterized to contain withanolides aglycones and glycosides (2.6 %, w/w), and withaferin-a (0.7 %, w/w) by HPLC (Fig. 1), was generously supplied by Natural Remedies Pvt. Ltd., Bangalore, India Animal Grouping and Drug Treatments In the first dose finding experiment, nondiabetic male mice were randomly allotted to six experimental groups of 6 animals each. Five of them were orally treated with 25, 50, 100, 200, or 400 mg/kg daily doses of WS (suspended in 0.3% sodium carboxy-methyl-cellulose) for 10 consecutive days. The sixth group was similarly treated with the vehicle only. All animals were closely observed for apparent behavioral abnormalities during the course of the experiments.

3 Reverse Ayurvedic Pharmacology of Ashwagandha Current Traditional Medicine, 2015, Vol. 1, No Fig. (1). HPLC fingerprint of Withania somnifera extract. In the second analogous experiment, the effects of three graded oral WS doses (25, 50 and 100 mg/ kg/day) in type-2 diabetic mice were tested. Type-2 diabetes was induced in mice by intraperitoneally administered streptozotocin (65 mg/kg) and nicotinamide (120 mg/kg, administered 15 min after streptozotocin treatment) as described elsewhere [31]. Animals with blood glucose levels more than 240 mg/dl (on seventh day after streptozotocin injection) were considered as diabetic and randomly allotted to three WS treatment groups, and to a diabetic control group treated daily with the vehicle only. For the sake of comparison, a control nondiabetic group treated daily with the vehicle was tested in parallel. Each experimental group consisted of 6 animals, and choice of the daily WS doses of this experiment was based on the observation made during the first dose finding experiment Foot Shock Stress-Triggered Hyperthermia Test Body weights and rectal temperatures of the animals were recorded on each experimental day during the oral administrations. 1 hr after the treatments on 1 st, 5 th, 7 th, and 10 th days of the experiments each individual animal of a test group was placed in a black box (24 x 29 x 40 cm) with a grid floor for 1 min. During this period, electric foot shocks were delivered through the grid floor (2 ma, 50 Hz of 2 ms duration) for inducing stress-triggered transient hyperthermia [32]. Five consecutive foot shocks of 2 ma at 10 s interval were given 10 s after placing the animals in the test box. At the end of 1 min, the animals were placed back in their home cages, and 10 min thereafter their rectal temperatures were recorded again. Numerical differences between the rectal temperature of a given animal before oral administrations and 10 min after the foot shock session were calculated and used as an index for stress-triggered transient hyperthermia. Rectal temperatures were measured using a calibrated rectal probe and electronic thermometer (Digital Thermometer; Dr. Gene, India) Plasma Glucose, Insulin, and Cortisol Level and Organs Weights Immediately after the last temperature measurements on the 10 th day of the experiment, diabetic mice were sacrificed by decapitation and blood was collected in EDTA coated tubes kept in ice and centrifuged at 1000 g for 20 min at 4 C to separate plasma (Compufuge CPR-30 Plus, with Rotor No. 8; REMI, India). Plasma was separated and aliquots were stored at -70 C for biochemical estimations. Plasma glucose level was estimated by using biochemical enzyme test kit (Span

4 54 Current Traditional Medicine, 2015, Vol. 1, No. 1 Thakur et al. Diagnostic Ltd., India). Plasma insulin level was estimated using Enzyme-Linked Immunosorbent Assay (ELISA) test kit (DRG Instruements GmbH, Germany). Plasma cortisol was estimated using ELISA kit (Assay Design; Ann Arbor, MI, USA). All biochemical estimations were done by using absorbance microplate reader (imark TM - Bio-Rad Laboratories, California, USA) according to instruction manuals of respective enzyme test kit. Immediately after blood collections, adrenal gland and spleen of the animals were removed and weighed Statistical Analysis Data are expressed as mean values ± standard error of mean (SEM). Statistical analysis was performed by one way analysis of variance (ANOVA) followed by Student-Newman-Keuls multiple comparison test. GraphPad Prism-5 (GraphPad Software Inc., San Diego, CA) software was used for statistical analysis. P value less than 0.05 was considered as statistically significant. 3. RESULTS 3.1. Body Weights Mean body weights of different experimental groups recorded on the 1 st, 5 th, 7 th, and 10 th day of the experiments are summarized in (Figs. 2A and B). It is apparent for the figures that body weights of both nondiabetic and diabetic control groups decreased continuously during the course of the experiments. Such intermittent foot shock stresstriggered body weight losses were dose dependently antagonized by WS treatments in both diabetic and nondiabetic animals. Maximally effective daily WS dose for this stress response parameter in nondiabetic mice was 100 mg/kg, whereas in diabetic animals its efficacy observed after its 50 mg/kg daily doses was almost identical to those observed after its 100 mg/kg daily doses. All tested daily doses of WS were well tolerated and no apparent behavioral abnormalities in nondiabetic animals were observed even after its 10 daily highest oral dose (400 mg/kg) tested Basal Rectal Temperatures These results are summarized in the (Figs. 3A and B). Mean basal rectal temperatures of the vehicle treated nondiabetic on the first day of both the experiments were somewhat higher than the diabetic ones. On this day, no statistically significant (P > 0.05) differences between the mean basal rectal temperatures of vehicle and WS treated nondiabetic experimental groups were observed and such was the case in diabetic animals as well. From the 5 th day onwards, mean basal rectal temperatures of diabetic as well as nondiabetic control groups were significantly (P < 0.05) higher than those recorded for them on the 1 st day of the Fig. (2). Effects of daily treatments with WS on body weights of (A) nondiabetic and (B) diabetic mice. ND= Nondiabetic, D= Diabetic, WS= Withania somnifera root extract. *= p<0.05 vs. ND control values on the same day; = p<0.05 vs. D control values on the same day; a = p<0.05 vs. day 1 values of the same group.

5 Reverse Ayurvedic Pharmacology of Ashwagandha Current Traditional Medicine, 2015, Vol. 1, No Fig. (3). Effects of daily treatments with WS on basal core temperatures of (A) nondiabetic and (B) diabetic mice. ND= Nondiabetic, D= Diabetic, WS= Withania somnifera root extract. *= p<0.05 vs. ND control values on the same day; = p<0.05 vs. D control values on the same day; a = p<0.05 vs. day 1 values of the same group. experiments. Such episodic foot shock triggered slight elevation in rectal temperatures observed on the 10 th experimental day in both nondiabetic and diabetic mice were completely absent in all WS treated groups. However, such statistically significant effects of its 25, 50 and 100 mg/kg daily doses in nondiabetic mice were first observed on the 7 th observational day (Fig. 3A), whereas mean basal rectal temperatures of all the WS treated groups in diabetic animals remained almost constant during the course of the experiment (Fig. 3B). These observations reveal that the number of daily doses of WS necessary for antagonizing episodic foot shock stress triggered slight elevation in core temperatures of diabetic mice is less than those necessary for inducing similar effects in nondiabetic ones, and that its minimally effective stress response suppressing daily dose could be 25 mg/kg or lower Foot Shock Stress-Triggered Transient Hyper-thermia No statistically significant effects of WS treatments in non diabetic mice were observed on the 1 st and 5 th observational days. However, its dose dependent inhibitory effects in nondiabetic mice were observed on the 7 th and 10 th day of the experiment (Fig. 4A). Efficacy of WS treatments in nondiabetic mice for inhibiting foot shock stress triggered transient hyperthermia observed after its 10 daily oral doses were somewhat higher than that observed after its 7 daily doses. Since this efficacy of 10 daily 200 mg/kg WS doses in nondiabetic mice were almost equal to that of its highest dose tested (400 mg/kg/day), this seems to be its maximally effective doses for inhibiting foot shock stress-triggered transient hyperthermia in normal laboratory mice. Magnitude of transient hyperthermia induced by one minute duration of foot shock stress in vehicle treated diabetic and nondiabetic control groups observed on day 1 of the experiments remained almost constant on the 5 th, 7 th and 10 th experimental days. Dose dependant inhibitory effects of WS against foot shock stress-triggered hyperthermia in diabetic mice became also apparent only after its repeated daily doses (Fig. 4B). However, unlike in nondiabetic mice such effects of 25 mg/kg/day WS were also observed on the 7 th and 10 th treatment days, and that of its 50 mg/kg daily doses on the fifth treatment day onwards. Quantitatively though, the efficacy of ten 100 mg/kg daily WS doses in diabetic mice was not as high as that observed after this daily dose in nondiabetic ones.

6 56 Current Traditional Medicine, 2015, Vol. 1, No. 1 Thakur et al. Fig. (4). Log dose response relationships of WS on foot shock stress-triggered hyperthermia in (A) nondiabetic and (B) diabetic mice observed after 1, 5, 7 and 10 daily oral administrations. WS= Withania somnifera root extract. Fig. (5). Effects of 10 daily oral doses of WS on plasma glucose, insulin and cortisol levels of diabetic mice. ND= Nondiabetic, D= Diabetic, WS= Withania somnifera extract. *=p< 0.05 vs. ND control; =p< 0.05 vs. D control.

7 Reverse Ayurvedic Pharmacology of Ashwagandha Current Traditional Medicine, 2015, Vol. 1, No Plasma Glucose, Insulin, and Cortisol Levels in Diabetic Mice As compared to vehicle treated non diabetic group, mean higher levels of glucose and cortisol and lower levels of insulin in the blood plasma of vehicle treated diabetic group were observed on the 10 th day of the experiment (Fig. 5). It is apparent from the figure that all WS daily doses tested significantly suppressed plasma glucose and cortisol levels in diabetic animals, and that such effects of the extract increased with its increasing daily oral doses tested. Mean blood glucose levels of the 100 mg/kg/day WS treated diabetic group was comparable to that observed in vehicle treated nondiabetic one, but such was not the case for the mean plasma cortisol level of the group. Although statistically significant suppressing effect of 25 mg/kg/day doses of WS on plasma glucose level of diabetic mice was observed, this dose of the extract had no significant effects on plasma cortisol levels. Plasma insulin level of 25 mg/ kg/day WS treated diabetic group was also statistically not significantly (P > 0.05) different from that of the vehicle treated diabetic group. Although statistically significant (P < 0.05) and dose dependant beneficial effects of WS on plasma insulin levels in diabetic mice were observed after its 50 and 100 mg/kg/day doses, this mean level of the 100 mg/kg/day WS treated diabetic groups was not as high as that estimated for the vehicle treated nondiabetic group Organ Weights of Diabetic Mice The absolute and relative wet weights of adrenal glands and spleen of different groups are given in Table 1. Relative organ weights are expressed as organ weights in mg/g body weight of an animal. Both absolute and relative weights of both these organs were significantly (P < 0.05) changed in vehicle treated diabetic group. Adrenal gland hypertrophy observed in the vehicle treated diabetic mice was completely absent in the 25 mg/ kg/day WS treated diabetic group. Both absolute and relative mean adrenal gland weights of 50 or 100 mg/kg/day WS treated diabetic groups were even lower than that of the vehicle treated nondiabetic one. Although absolute spleen weights of vehicle treated diabetic mice were lower than those of nondiabetic ones, the relative weights of the organ of diabetic animals were higher than those of nondiabetic ones. Mean absolute spleen weights of WS treated diabetic groups increased with its increasing doses, whereas relative spleen weights of diabetic WS treated groups decreased with its increasing doses. 4. DISCUSSION Observations reported in this communication reaffirm that the mouse bioassay system used in the two described experiments is well suited not only for identifying and pharmacologically characterizing the bioactive constituents of Withania somnifera extracts potentially involved in their stress response suppressing and antidiabetic activities, but also for estimating their pharmacologically interesting dose ranges and treatment regimen. They also add further experimental evidences to the conviction that modulation of homeostatic processes involved in thermoregulation and glucose metabolism are involved in modes of actions of the tested extract, and reveal that its oral efficacy to suppress stress triggered response increases with increasing numbers of treatment days. In addition, they also reveal that therapeutic index or safety margin of the tested extract is fairly high. Although ten daily oral WS dose as high as 400 mg/kg/day was well tolerated by nondiabetic mice, its estimated minimally effective doses for affording significant protections against all metabolic and foot shock stress triggered responses quantified was 25 mg/ kg/day. However, efficacy of WS in protecting diverse foot shock stress triggered responses in nondiabetic and type-2 diabetic mice varies considerably. Its maximally effective daily oral dose in counteracting body weight losses in stressed diabetic mice was 50 mg/kg, whereas that in nondiabetic ones was 100 mg/kg. Such higher efficacy of the extract in diabetic mice was also observed for its protective effects against slight elevation of basal core temperature induced by episodic foot shock stress. Although on the 10 th treatment day this response was almost completely absent in both diabetic and nondiabetic animals treated with 25 mg/kg/day dose of the extract, such were not the cases on the 5 th or 7 th treatment days. Although no statistically significant effects of daily oral doses of WS up to 100 mg/kg/day against this response in nondiabetic mice were observed on the 5 th day of the

8 58 Current Traditional Medicine, 2015, Vol. 1, No. 1 Thakur et al. Table 1. Effects of 10 daily oral doses of WS on absolute and relative weights on adrenal gland and spleen weights of diabetic mice. Treatment groups Absolute organ weight (mg) Relative organ weight (mg/g body weight) Adrenal gland Spleen Adrenal gland Spleen ND Control (0.3% CMC) 5.33± ± ± ±0.14 D Control (0.3% CMC) 6.33±0.33* 72.00±1.84* 0.46±0.01* 5.29±0.20* D + WS 25 mg/kg 4.50± ± ± ±0.27* D + WS 50 mg/kg 4.17±0.31*, 93.67±1.80*, 0.20±0.01*, 4.55±0.15*, D + WS 100 mg/kg 3.67±0.33*, ±2.12*, 0.17±0.02*, 4.98±0.15* ND= Nondiabetic, D= Diabetic, WS= Withania somnifera extract. *=p< 0.05 vs. ND control; =p< 0.05 vs. D control. experiment (Fig. 3A), such responses in diabetic mice observed on that day were almost completely absent in the 25 mg/kg/day WS treated diabetic animals (Fig. 3B). These observations strongly suggest that pharmacological observations made with Withania somnifera extracts in normal animals after their acute oral doses are not very reliable indicators of their adaptogenic potentials, and that their therapeutically interesting pharmacological activity profiles in mice depend not only on the duration of treatments, but also on preexisting metabolic status of the animals. Analogous observations that pharmacological activity profiles of adaptogenic herbal extracts observed after their acute doses can be different, or even opposite, to those after their chronic doses have also been reported by others [33], and it is also known and that numerous phytochemicals could well have inverted U or J shaped dose response curves [34]. Moreover, it has often been pointed out that repeated dose studies are necessary for proper pharmacological characterization of numerous psychoactive and other drugs [35-37], and that psychopharmacological activity profiles of drugs observed in normal animals are not reliable predictors of their efficacy in diabetic and other patients suffering from lifestyle-associated chronic diseases [38, 39]. However, yet little concentrated efforts have been made to identify a pharmacological screening procedure that could be used for better understanding of complexities arising from such facts, or for translating existing knowledge on traditionally known medicinal plants according to the evidence based concepts of modern medicine. In principle, the stress induces hyperthermia paradigm used in the bioassay in this pilot study is analogous to those now often used for identifying anxiolytic activity of test agents in rodents [32, 40]. Hereupon, the foot shock stress procedure used in the bioassay is a shorter version of the one used earlier for studying the effects of prolonged treatments with W. somnifera root extract against prolonged chronic foot shock stress-triggered pathologies [23]. Diabetes is a chronic metabolic stressor [41], and numerous observations in our laboratories and elsewhere have consistently revealed that anxiety, and depressive and cognitive states of diabetic animals are not identical those of nondiabetic ones [42-48]. Therefore, the same bioassay procedure was used in the second experiment to quantify the efficacy of three lower (but effective in nondiabetic mice) oral WS doses in type-2 diabetic mice. Results of this experiment revealed somewhat higher efficacy of WS in diabetic mice, and suggested that this could as well be due to its additional beneficial effects on glucose metabolism, which in turn could be due its modulating actions on biological processes and mechanisms regulating endocrine, immune, and other bodily functions. Observed effects of WS on plasma glucose, insulin, and cortisol levels (Fig. 5), and the weights adrenal gland and spleen (Table 1) of stressed diabetic mice are in agreement with this inference. However, it must be noted that although the mean blood glucose level of the 100 mg/kg/day WS treated diabetic group was within the normal values observed in the vehicle treated nondiabetic group, such was not the case for its effects on blood insulin level. These observations

9 Reverse Ayurvedic Pharmacology of Ashwagandha Current Traditional Medicine, 2015, Vol. 1, No could indicate that beneficial effect of WS on blood glucose levels, body weight gains and core temperatures observed in diabetic animals are not entirely due to its effects on insulin homeostasis. That such could indeed be the case is indicated also by the observed effects of WS on the other three parameters quantified in diabetic animals. Elevated plasma cortisol level observed in diabetic mice was completely reversed after 10 daily 50 mg/kg WS doses, and mean blood cortisol level of the 100 mg/kg/day WS treated group was significantly lower than that of the stressed nondiabetic control group. Moreover, WS treatments dose dependently decreased adrenal gland weights and increased spleen weights of diabetic mice, whereupon the mean adrenal gland weights of the 25 mg/kg/day WS treated diabetic group was higher, and the mean spleen weight of of the group lower, than that of the corresponding values of the nondiabetic control group. These observations made in type-2 diabetic mice could indicate that its observed antihyperglycemic and other beneficial effects is most probably due its modulating effects on biological processes regulating stress responses and diverse other functions of the hypothalameicpituitary-adrenal axis. Although such suggestions for the modes of action of Withania somnifera extracts has often been made [49], and reports on clinical efficacy of such extracts against stress triggered psychopathologies have continued to appear during more recent years [50, 51], as yet no very definitive statements on bioactive constituents of such extracts involved in their stress response modulating efficacy can be made. Since the observations made with WS in this study are quite analogous to those observed in one such clinical study conducted with another type of Withania somnifera extract [51], it seems reasonable to suggest that the mouse bioassay system used in the experiments reported in this communication could be a useful one for comparing pharmacological quality and relative potencies of such therapeutically used extracts. Since the bioassay enables quantitative assessments of beneficial effects of a Withania somnifera extract on 8 different parameters in the same animals, it should be also useful not only for identifying their diverse bioactive constituents, but also for clarifying possible pharmacological interactions between them (by appropriate uses of activity guided fractionation procedures, and recombination experiments etc.). Moreover, this bioassay could also be used for proper selection of starting plant materials and their processing and extraction procedures necessary for obtaining sustainable and reproducible therapeutic benefits offered by this wildly growing and easily cultivable ayurvedic medicinal plant. Encouraged by the observations made during this pilot study, efforts are now being made in our laboratories to identify the bioactive secondary metabolites of Withania somnifera potentially involved in the therapeutically interesting activity profile of the tested extract. Although using the same mouse bioassay we have already identified a few bioactive secondary metabolites of Withania somnifera, further efforts will still be necessary to clarify possible interactions between them and numerous other already known bioactive secondary metabolites of the plant. Ultimate goal of these efforts is to obtain an analytically as well as pharmacologically better characterized and more efficacious Withania somnifera extract that could be further developed as a phyto-pharmaceutical or nutraceutical according to evidence based concepts of modern medicine. 5. CONCLUSIONS Withania somnifera extracts are desensitisors of stress-triggered biological responses involving thermoregulatory mechanisms, and have therapeutic potentials for prevention and cure of diabetes associated mental health problems. Repeated dose studies in rodents are essential prerequesites for morer realistic assesments of their pharmacologically interesting dose ranges. CONFLICT OF INTEREST The author(s) confirm that this article content has no conflict of interest. ACKNOWLEDGEMENTS Thanks are due to Natural Remedies Pvt. Ltd., Bangalore, India for generous supply of the pharmaceutically standardised Withania somnifera extract along with its HPLC finger print.

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