Withaferin A attenuates Alcohol Abstinence Signs in Rats

Kotagale, Kedia, Gite, Rahmatkar, Gawande, Umekar, and Taksande: Withaferin A attenuates Alcohol Abstinence Signs in Rats

Authors

https://s3-us-west-2.amazonaws.com/jourdata/pj/PharmacognJ-10-6-1190_g000.jpg

INTRODUCTION

Alcohol withdrawal syndrome is potentially life-threatening in addicted people and associated maladies constitute a serious health and social issues.1 Abstinence from chronic ethanol consumption leads to the manifestation of a variety of somatic and affective symptoms attributed to central nervous system hyperexcitability, like irritability, anxiety, restlessness and dysphoria.2 Despite the tremendous advances made in the treatment of alcoholism and/or its abstinence, remarkably, the majority of these agents, including naltrexone and benzodiazepines etc. have unpleasant side effect.3,4

Withaferin A is a steroidal lactone, an active compound isolated from Withania somnifera (WS) (Family- Solanaceae). WS, known as ashwagandha in Ayurveda or its active principles, including withaferin A has been used as an antioxidant, adaptogen, antistress,5,6 anti-inflammatory, neuroprotective, anxiolytic, antidepressant, immunomodulatory, memory enhancer, anti-ulcer and anti-carcinogenic agents.7-12 In addition, WS extract has been inhibited the morphine-induced acquisition and expression in conditioned place preference,13 ethanol conditioned place preference14 and self-administration,15 ethanol withdrawal-induced anxiety in rats.16 In the present work, we have assessed the effect of withaferin A on somatic and affective symptoms of ethanol withdrawal syndrome in rats.

MATERIALS AND METHODS

Subjects

Adult healthy Sprague Dawley rats weighing 200-220 g (3-4 months old) were group housed (four per cage) under controlled temperature (25±2°C) and light (12 h light/dark cycle, light on at 07.00 am) environment with free access to food and water. Experimental protocols were approved by the Institutional Animal Ethical Committee and executed in strict accordance with the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals, Govt. of India. The behavioral assessments were conducted during the light cycle.

Drugs

Withaferin A was purchased from Natural Remedies Private Limited, Bangalore, India and administered intraperitoneally (ip) as a solution (1 ml/kg) in dimethylsulphoxide (DMSO) prepared just before the experiments. Ethanol (99% w/v) (Merck, India) was given through liquid diet. Doses and timing of the drug injections with respect to the behavioral testing employed in the protocols were selected on the basis of previous experiments carried in our laboratory and available literature.17,18

Induction of ethanol dependence and withdrawal

All different groups of rats (n = 16 - 20) housed individually were given free access to nutritionally balanced liquid diet (Novartis India Ltd., Mumbai) without ethanol for 7 days to allow adaptation to the novel food. Water was made freely available. From 8th day onwards, half of the animals (n = 6) had given free access to ethanol uninterrupted for 21 days through liquid diet.19 Initially, 2.4% v/v ethanol was introduced into the liquid diet for 3 days, which was increased to 4.8% v/v for the following 4 days and finally to 7.2% v/v for 14 days (ethanol-fed group). The remaining rats kept on the control liquid diet (nutritionally balanced) (pair-fed group). Every day fresh ethanol and control liquid diet (100 ml/rat) was introduced into the cage each morning at 0900 h. The weight of each rat was recorded everyday and daily ethanol intake was monitored and expressed as g/kg/day. From day 22 (0900 h), pair-fed groups were continued on the same diet, whereas to ethanol-fed groups, ethanol diet was stopped and animals were fed with isocaloric liquid diet (without ethanol) until the end experiment. Control rats (n = 6) were pair-fed with an isocaloric liquid diet containing sucrose as a caloric substitute to ethanol.

This protocol had two segments: In the first design, withaferin A (5, 10 and 20 mg/kg) was injected (ip) during the development of ethanol dependence phase (days 15 - 21). In the second design,withaferin A (5, 10 and 20 mg/kg) was administered to rats 30 min before ethanol withdrawal assessment. Separate groups (n = 6) of rats were assigned to different doses. Appropriate vehicle-treated controls were also maintained simultaneously.

Behavioral changes during ethanol withdrawal

At 24 h post ethanol withdrawal, the pair-fed groups, as well as ethanol-fed groups, were subjected to evaluation of ethanol withdrawal syndrome. This time point was selected since peak withdrawal somatic and affective signs were apparent at this time point.17,18, 20,21 Withdrawal signs characterized by changes in somatic signs were measured in open field for 15 min.19 This was followed by evaluation of anxiety parameters in elevated plus maze (EPM),18 locomotion (actophotometer) and depressive behavior (Porsolt’s forced swim test, FST) each for 5 min duration.17 All the animal experiments were performed during the light cycle between 0900 and 1200 h.

Somatic signs

At 24 h of ethanol withdrawal, animals were observed individually for 15 min and the frequency of various stereotyped behavioral signs that included grooming, sniffing, head weaving, gnawing, chewing, scratching, genital licking and body shake were recorded. Ethanol abstinence score (EAS) consisting of the sum of all the stereotyped behaviors was compiled and expressed as mean ± SEM.22 Wet dog shakes, tremors, tail stiffness, abnormal gait and posture, abdominal constriction and teeth chattering were also assessed for the incident.

Anxiety related behavior

The anxiety in animals was evaluated in elevated plus maze (EPM), the earlier reported experimental protocol was followed.18 Briefly, after 24 h of withdrawal rats were placed on the central platform of EPM with open arm facing. In EPM following variables were recorded during 5 min, frequencies and time spent in closed and open arms respectively. After each test, the platform of the maze was wiped and cleaned with damp cotton. All subjects were experimentally naive at the beginning of each study and used only once to avoid ‘one trial tolerance’ to EPM test.

Locomotor activity

Locomotor activities of the rats were recorded by an automatic actophotometer (38 × 38 × 14 cm) (VJ instruments, Karanja, India). Any movement of the rat that interrupted photo beams was recorded as a total of horizontal, vertical and ambulatory activities and expressed as mean motor counts ± SEM. Locomotor counts were not recorded for first 5 min after placement and the spontaneous locomotor count of each animal was recorded for following 5 min. After each animal’s recording, the grid floor of actophotometer was carefully cleaned. The data are expressed as a mean number of counts for 5 min.

Depressive behavior

Porsolt’s forced swim test (FST) was used to determine the depressive behavior in ethanol-withdrawal rats.23-24 Animals were individually allowed for forced to swim in a cylindrical glass tank (46 cm × 20 cm) containing 30 cm of water and the time of immobility was recorded. Two swimming sessions were carried out with an initial 15 min ‘pre-test’ followed by a 6 min test after 24 h. Reduction in the duration of immobility was considered as attenuation of the depressive behavior of ethanol withdrawal. A trained observer blind to the treatments monitored the immobility time, which is the measure of depression.

Determination of blood ethanol, corticosterone and ACTH levels

Blood samples were collected from a separate group of animals at 1 h before (0800 h) and at 24 h after (0900 h) ethanol withdrawal from a rat tail vein in heparinized tubes and centrifuged at 13000 × g for 15 min at 4°C to separate plasma and stored at -20°C. The separated plasma samples were then used to determine ethanol, corticosterone and adreno-corticotropic hormone (ACTH).Plasma ethanol concentrations were determined by nicotinamide adenine dinucleotide-alcohol dehydrogenase enzymatic assay (Sigma-Aldrich, St. Louis, MO, USA).25 Plasma corticosterone concentrations were measured using a quaternary gradient HPLC system equipped with a Crestpak C18T-5 column and PDA detector (MD2010 plus) (Jasco, Japan) as per the earlier described procedure.26,27ACTH concentrations in plasma were measured by two-site ELISA assay using a commercial kit (Sigma-Aldrich, St. Louis, MO, USA).28

Data Analysis

The results are presented as mean ± SEM. The data obtained from ethanol-withdrawal and pair-fed rats were compared by unpaired t-test. The effects of different acute drug treatments were statistically analyzed by one-way analysis of variance (ANOVA) followed by post hoc Dunnett’s test or Bonferroni multiple comparison tests. A value of P<0.05 was considered significant.

RESULTS

Ethanol consumption, body weight, and ethanol levels

Daily ethanol consumption of the rats in ethanol-fed control and withaferin A treated groups ranged from 13.6 ± 1.3 to 15.8 ± 2.1 g/kg during exposure to ethanol (7.2 % w/v) of last 2 weeks. However, this difference was not statistically significant. Average liquid food consumption of the above two groups was 42.5 ± 4.2 and 44.3 ± 6.1 ml/rat from day 1 – 21 and was not different from the pair-fed control animals.

On the 21st day of the experiment, the mean body weight of pair-fed and ethanol-fed animals were 207.2 ± 4.9 g (against the average body weight of 204.3 ± 3.5 g on day 1) and 208.5 ± 3.8 g (against the average body weight of 211.6 ± 6.8 g on day 1) respectively and these body weight changes in pair-fed and ethanol-fed animals were statistically insignificant.

Blood ethanol concentration was found to be 173.79 ± 7.68 mg/dl and 6.31 ± 0.99 mg/ml, 1 h before (0800 h) and at 24 h after (0900 h) ethanol withdrawal respectively (Table 1). The decreased blood ethanol level 24 h after withdrawal was found significantly correlated with the abstinence signs.

Table 1

Blood ethanol concentration in withaferin A treated ethanol withdrawn rats. Separate groups of rats received ethanol in the liquid modified diet for 21 days and treated with withaferin A (5 – 20 mg/kg, ip) from day 15 - 21 of ethanol presentation. Blood ethanol concentration was measured 24 h before and after withdrawal. Data expressed as mean ethanol concentration (mg/dl) ± SEM (n=6).

Treatment groupsBlood ethanol concentration (mg/dl)
Before withdrawal24 h after withdrawal
Vehicle (1 ml/kg, ip)173.79 ± 7.686.31 ± 0.99
Withaferin A (5 mg/kg, ip)169.32 ± 6.735.44 ± 0.71
Withaferin A (10 mg/kg, ip)174.44 ± 8.157.86 ± 0.84
Withaferin A (20 mg/kg, ip)170.79 ± 7.086.76 ± 0.81

Behavioral changes during ethanol withdrawal

The significant stereotyped behaviors (somatic behaviors) such as grooming (t = 3.055, df = 2, P = 0.0185), scratching (t = 4.085, df = 2, P = 0.0047), head shake (t = 3.705, df = 2, P = 0.0076), genital licking (t = 3.831, df = 2, P = 0.0064), chewing (t = 3.055, df = 2, P = 0.0185), body shakes (t = 3.020, df = 2, P = 0.0194) etc. were evident in ethanol dependent rats when compared with control pair fed animals at 24 h post withdrawal. The sum of all the stereotyped behaviors expressed as EAS was significantly higher in ethanol withdrawn rats as compared to pair fed animals (t = 3.49, df = 10, P < 0.01; unpaired t test) (Figure 1). However, tail stiffness, abnormal gait, abnormal posture, abdominal constriction, wet dog shakes, tremor or teeth chattering were not observed in the withdrawn, pair fed as well as treated animals.

Figure 1

Effect of withaferin A on ethanol withdrawal-induced somatic behaviors. Rats were given free access to ethanol through liquid modified diet for of 21 days and from the 22ndday onwards they were deprived of ethanol. Animals were treated with withaferin A (5 – 20 mg/kg, ip) once daily from day 15 -21 of ethanol dependence. Somatic behaviors (grooming, jumping, genital licking, leg licking, head shakes etc.) were determined for 15 min at 24 h following ethanol withdrawal. Each bar indicates mean EAS ± SEM (n = 6). @P < 0.001 vs vehicle-treatedpair-fed rats; *P < 0.05, **P < 0.01vs vehicle-treatedethanol-withdrawn animals (One way ANOVA post hoc Bonferroni test).

https://s3-us-west-2.amazonaws.com/jourdata/pj/PharmacognJ-10-6-1190_g003.jpg

A significant locomotor hyperactivity was observed in ethanol-withdrawal animals at the 24 h of post-withdrawal testing as compared to control animals (t = 3.86, df = 10, P < 0.01; unpaired t-test) (Figure 2). Similarly, when tested in FST, immobility time was increased as compared to pair-fed animals (t = 3.93, df = 10, P < 0.01; unpaired t-test) (Figure 3).

Figure 2

Effect of withaferin A on ethanol withdrawal-induced locomotor hyperactivity. Rats were given free access to ethanol through liquid modified diet for 21 days and from the 22nd day onwards they were deprived of ethanol. Animals were treated with withaferin A (5 - 20 mg/kg, ip) once daily from day 15 -21 of ethanol dependance. Locomotor activity was determined for 5 min in actophotometer at 24 h following ethanol withdrawal. Each bar indicates mean locomotor counts ± SEM (n = 6). @P < 0.01 vs vehicle-treated pair-fed rats; *P < 0.05, **P < 0.01 vs vehicle-treated ethanol-withdrawn animals (One way ANOVA post hoc Bonferroni test).

https://s3-us-west-2.amazonaws.com/jourdata/pj/PharmacognJ-10-6-1190_g001.jpg
Figure 3

Effect of withaferin A on ethanol withdrawal-induceddepressive behaviors. Rats were given free access to ethanol through liquid modified diet for of 21 days and from the 22nd day onwards they were deprived of ethanol. Animals were treated with withaferin A (5 - 20 mg/kg, ip) once daily from day 15 -21 of ethanol dependence. Immobility duration was determined for 5 min in FST at 24 h following ethanol withdrawal. Each bar indicates mean immobility time (s) ± SEM (n = 6). @P < 0.01 vs vehicle-treated pair-fed rats; *P < 0.05, **P < 0.01 vs vehicle-treated ethanol-withdrawn animals (One way ANOVA post hoc Bonferroni test).

https://s3-us-west-2.amazonaws.com/jourdata/pj/PharmacognJ-10-6-1190_g002.jpg

As depicted in Figure 4, animals withdrawn from the chronic ethanol exhibited the anxiogenic effect indicated by animal behavior in the maze. Significant decrease in % time spent in open arm (t = 5.49, df = 10, P < 0.001; unpaired t-test) and % open arm entries (t = 3.84, df = 10, P < 0.01; unpaired t-test) was observed in ethanol-withdrawn rats when compared with pair-fed group.

Figure 4

Effect of withaferin A on ethanol withdrawal-inducedanxiety-related behaviors. Rats were given free access to ethanol through liquid modified diet for of 21 days and from the 22nd day onwards they were deprived of ethanol. Animals were treated with withaferin A (5 – 20 mg/kg, ip) once daily from day 15 -21 of ethanol dependance. The number of entries (A)and time spent (B) were determined for 5 min in EPM at 24 h following ethanol withdrawal. Each bar indicates mean % entries/time spent in open arm ± SEM (n = 6). @P < 0.001 vs vehicle-treatedpair-fed rats; *P < 0.05, **P < 0.01, ***P < 0.001 vs vehicle-treatedethanol-withdrawn animals (One way ANOVA post hoc Bonferroni test).

https://s3-us-west-2.amazonaws.com/jourdata/pj/PharmacognJ-10-6-1190_g004.jpg

Withaferin A attenuates ethanol withdrawal-induced behaviors

Withaferin A (5 - 20 mg/kg, ip) treatment 30 min before 24 h post-ethanol withdrawal assessment did not alter the scores of somatic behavioral signs in ethanol abstinence animals. Moreover, a single administration of withaferin A (5 - 20 mg/kg, ip) before withdrawal assessment did not influence the locomotion, immobility time as well as EPM indices as compared to ethanol-withdrawn rats (Data not shown).

Chronic treatment with withaferin A (10 and 20 mg/kg, ip) from day 15-21 significantly prevented the ethanol withdrawal induced elevated scores of somatic behaviors [F (4, 29) = 7.34; P < 0.001] (Figure 1) and hyperlocomotion [F (4, 29) = 9.46; P < 0.001] (Figure 2) as well as depressive behavior [F (4, 29) = 7.83; P < 0.001] (Figure 3). The posthoc Dunnett mean comparisons demonstrated that withaferin A (10 and 20 mg/kg) treatment for 7 days in ethanol dependent rats significantly attenuated the elevated somatic scores (P < 0.05 and P < 0.01), locomotor activity (P < 0.05 and P < 0.01 respectively) and immobility time (P< 0.05 and P < 0.01 respectively) and when compared against vehicle treated ethanol withdrawn rats.

Similarly, as shown in Figure 4, ethanol-withdrawn rats treated with withaferin A (10 - 20 mg/kg, ip) from day 15-21 along with ethanol exposure displayed a significant reduction in the withdrawal-induced anxiety as evident from decreased percent entries into the open arms [F (4, 29) = 12.84; P < 0.001] (Figure 4A) and percent time spent [F (4, 29) = 9.17; P < 0.001] (Figure 4B) as compared to ethanol-withdrawn rats administered with vehicle for 7 days. Post-hoc Dunnett analysis demonstrated a significant reversal of ethanol withdrawal-inducedanxiety behaviors [% entries in open arm (P < 0.01 and P < 0.01) and % time spent in open arm (P < 0.05 and P < 0.01) in animals treated with withaferin A (10 and 20 mg/kg, respectively) in ethanol-dependentrats compared to vehicle-treated controls. Administration of withaferin A in pair-fed animals did not alter basal somatic behaviors, immobility time, locomotor activity and plus-maze behaviors.

Effect of withaferin A on ethanol withdrawal-induced elevated plasma ACTH and corticosterone levels

As shown in Table 1, blood ethanol levels in withaferin A (5-20 mg/kg, ip) administered ethanol-withdrawn rats were not significantly different from vehicle-treated abstinence animals. However, as depicted in Table 2, ethanol withdrawal results in a significant elevation in plasma corticosterone levels were as compared to their concentration before withdrawal (t = 6.44, df = 10, P < 0.001; unpaired t-test). In order with the behavioral observations, Withaferin-A [10 (P < 0.001) and 20 mg/kg (P < 0.001)] administration significantly attenuated the elevated plasma corticosterone levels in ethanol-withdrawn rats [F (4, 29) = 16.20; P < 0.001].

Table 2

Plasma corticosterone concentration in withaferin A treated ethanol withdrawn rats. Separate groups of rats received ethanol in the liquid modified diet for 21 days and treated with withaferin A (5 – 20 mg/kg, ip) from day 15 – 21 of ethanol presentation. Plasma corticosterone concentration was measured 30 min before and 24 h after withdrawal. Data expressed as mean corticosterone level (ng/ml) ± SEM (n=6). #P < 0.001 vs vehicle treated ethanol fed rats 30 min before ethanol withdrawal, *P < 0.001 vs vehicle treated ethanol fed rats 24 h after ethanol withdrawal.

Treatment groupsPlasma corticosterone concentration (ng/ml)
Before withdrawal24 h after withdrawal
Vehicle (1 ml/kg, ip)61.82 ± 3.63166.8 ± 15.91#
Withaferin A (5 mg/kg, ip)76.59 ± 4.50140.4 ± 8.35
Withaferin A (10 mg/kg, ip)74.93 ± 6.31102.9 ± 8.07*
Withaferin A (20 mg/kg, ip)75.02 ± 7.8989.81 ± 11.49*

In addition, ELISA analysis revealed a significant rise in the levels of ACTH follows ethanol withdrawal (t = 4.72, df = 10, P < 0.001; unpaired t test). Treatment withaferin A [10 (P < 0.05) and 20 mg/kg (P < 0.05)] dose dependently reversed the elevated plasma ACTH levels as compare to control rats [F (4, 29) = 5.11; P < 0.01] (Table 3). Administration of withferin A (5 – 20 mg/kg, ip), however, did not alter the basal corticosterone as well as ACTH levels in pair-fed animals (data not shown).

Table 3

Plasma ACTH concentration in Withaferin A treated ethanol withdrawn rats. Separate groups of rats received ethanol in the liquid modified diet for 21 days and treated with withaferin A (5 – 20 mg/kg, ip) from day 15 – 21 of ethanol presentation. Plasma ACTH concentration was measured 24 h before and after withdrawal. Data expressed as mean ACTH level (pg/ml) ± SEM (n=6). #P < 0.001 vs vehicle treated ethanol fed rats 30 min before ethanol withdrawal, *P < 0.05 vs vehicle treated ethanol fed rats 24 h after ethanol withdrawal.

Treatment groupsPlasma ACTH concentration (pg/ml)
Before withdrawal24 h after withdrawal
Vehicle (1 ml/kg, ip)122.2 ± 13.82252.4 ± 23.91#
Withaferin A (5 mg/kg, ip)148.4 ± 16.50205.2 ± 35.78
Withaferin A (10 mg/kg, ip)140.7 ± 15.31167.9 ± 8.66*
Withaferin A (20 mg/kg, ip)145.2 ± 10.89164.3 ± 15.07*

DISCUSSION

The main observations of this study are that withaferin A, an active constituent of WS, has an inhibitory effect on alcohol abstinence signs in rats. In line with the earlier reports, in current findings, we have observed the approximately 9-15 g/kg/day up to 15 days, which further produces the physical dependence in rats.29,30 In the present study, very high blood ethanol levels (174.44 ± 8.15 mg/dl) observed just before ethanol withdrawal indicates an adequate quantity of ethanol consumed (13.6 ± 1.3 to 15.8 ± 2.1 g/kg) for the induction of abstinence signs. Pilot studies from our laboratory as well as earlier published studies have demonstrated that ethanol withdrawal displays its peak behavioral and biochemical signs following 24 h of ethanol withdrawal. As depicted in Table 1, ethanol withdrawal in animal resulted insignificant decline in the blood ethanol concentration. In our study, ethanol abstinence was characterized by increased scores of stereotyped behaviors, motor hyperactivity, behavioral despair and anxiety-like behavior in EPM test. Our previous findings that ethanol withdrawal anxiety18 and depression17 in rodents support the development of psychological dependence. Negative reinforcement in ethanol withdrawal is one of the potential reason for ethanol dependence and reinstatement.31,32 Similarly, dysphoria like the state is very common following the withdrawal of substances of abuse like ethanol, nicotine, and morphine and has been demonstrated in several animal models of depression.33,35 This negative emotional state of ethanol withdrawal has been suggested a key factor in the maintenance of ethanol addiction (i.e., alcohol self-administration to prevent withdrawal).36

WS has been widely investigated for their diverse pharmacological activities, phytochemically it contains steroidal lactones, alkaloids, flavonoids, tannin etc. Withaferin A, predominantly found in WS known as withanolide. Several publish report acclaimed its different pharmacological activities viz. anti-inflammatory, anti-stress,12,37 immunomodulatory,10 inhibition of cognitive deficits in Alzheimer’s disease38 and anxiolytic–antidepressant action in rats7 without inducing any significant toxicity. In the present study, we evaluated the effects of acute and chronic injections of withaferin A on the somatic manifestations, anxiety, and depression-like effect of ethanol withdrawal. Our results demonstrate that subchronic withaferin A administration (day 15- 21) during ethanol exposure prevented the somatic expression of ethanol withdrawal as revealed by a significant decrease in the most prominent somatic signs of ethanol withdrawal and also prevented the anxiety and depression-like behavior in ethanol-withdrawn rats. However, acute injection of withaferin A 30 min prior withdrawal testing did not influence any of the ethanol withdrawal signs. It is of interest to note that withaferin A (glyco with anolide) has been reported to have both anxiolytic and antidepressant activity in rats, supporting the contention that this bioactive component of WS is the effective mood regulator.7

Activation of the hypothalamo-pituitary-adrenocortical axis (HPA) during stress is a well-known phenomenon, an increase in plasma corticosterone in rodents and cortisol in humans have been utilized as markers of withdrawal associated stress and its intensity.39-40 Single ethanol withdrawal induces the maladaptive drinking symptoms like anxiety, hyperactivity etc. which have been correlated with the elevated corticotrophin-releasing factor levels.41 Interestingly, these maladaptive symptoms of alcohol withdrawal have been reversed.42 Therefore the CRF has been proposed as a key mediator in drug and ethanol dependence.43 Current reports suggest the neuroadaptative alterations activated by chronic alcohol exposure leads to up-regulation of CRF system.44 Importantly, ethanol-withdrawn animals demonstrated a marked elevation in ACTH and corticosterone levels, which was reversed by withaferin A treatment. It is likely that withaferin A by its interaction with the central stress regulating system might have attenuated ethanol withdrawal syndrome in rats.45 However, the role of other proposed biological targets of WS like GABA and opioids receptors needs further investigation. Thus, withaferinA-induced anti-stress effect and stabilization of HPA axis activity could have facilitated the inhibitory effect of withaferin A on ethanol withdrawal syndrome.

In conclusion, this study demonstrated that prolong administration of withaferin A during exposure to ethanol attenuate the ethanol withdrawal-induced somatic hyperexcitability, anxiety, and dysphoria like state in chronically ethanol-exposed animals. These findings strongly support further investigation of withaferin A and other bioactive components of WS in alcohol addiction. The data clearly projects withaferin A as a new potential therapeutic intervention in alcohol abuse associated complications.

CONFLICT OF INTEREST

The authors have no conflict of interest.

ABBREVIATIONS

WS

Withania somnifera

HPA

Hypothalamic Pituitary adrenal

EPM

Elevated Plus Maze

FST

Forced Swim Test

EAS

Ethanol Abstinence Score

ACTH

Adreno-Corticotropic Hormone.

ACKNOWLEDGEMENT

The work was funded under the institutional research grant.

REFERENCES

1 

Campos J, Roca L, Gude F, Gonzalez-Quintela A. , authors. Long-term mortality of patients admitted to the hospital with alcohol withdrawal syndrome. Alcohol Clin Exp. Res. 2011; 35 (6): 1180 –6

2 

Uzbay IT, Kayir H, Celik T, Yuksel N , authors. Acute and chronic tianeptine treatments attenuate ethanol withdrawal syndrome in rats. Prog. Neuropsychopharmacol. Biol Psychiatry. 2006; 30 (3): 478 –85

3 

Malcolm RJ. , author. GABA systems, benzodiazepines, and substance dependence. J Clin Psychiatry. 2003; 64 (3): 36 –40

4 

Leggio L, Kenna GA, Swift RM , authors. New developments for the pharmacological treatment of alcohol withdrawal syndrome. A focus on non-benzodiazepine GABAergic medications. Prog Neuropsychopharmacol Biol Psychiatry. 2008; 32 (5): 1106 –17

5 

Bhattacharya A, Ghosal S, Bhattacharya SK , authors. Antioxidant effect of Withania somnifera glycowithanolides in chronic footshock stress-induced perturbations of oxidative free radical scavenging enzymes and lipid peroxidation in rat frontal cortex and striatum. J Ethnopharmacol. 2001; 74 (1): 1 –6

6 

Bhattacharya SK, Muruganandam AV , authors. Adaptogenic activity of Withania somnifera: an experimental study using a rat model of chronic stress. Pharmacol Biochem Behav. 2003; 75 (3): 547 –55

7 

Bhattacharya SK, Bhattacharya A, Sairam K, Ghosal S , authors. Anxiolytic-antidepressant activity of Withania somniferaglycowithanolides: an experimental study. Phytomedicine. 2000; 7 (6): 463 –9

8 

Ahmad M, Saleem S, Ahmad AS, Ansari MA, Yousuf S, Hoda MN, Islam F , authors. Neuroprotective effects of Withania somnifera on 6-hydroxydopamine induced Parkinsonism in rats. Hum Exp Toxicol. 2005; 24 (3): 137 –47

9 

Owais M, Sharad KS, Shehbaz A, Saleemuddin M , authors. Antibacterial efficacy of Withania somnifera (ashwagandha) an indigenous medicinal plant against experimental murine salmonellosis. Phytomedicine. 2005; 12 (3): 229 –35

10 

Rasool M, Varalakshmi P , authors. Immunomodulatory role of Withania somnifera root powder on experimental induced inflammation: An in vivo and in vitro study. Vascul Pharmacol. 2006; 44 (6): 406 –10

11 

Yang Z, Garcia A, Xu S, Powell DR, Vertino PM, Singh S, Marcus AI , authors. Withania somnifera root extract inhibits mammary cancer metastasis and epithelial to mesenchymal transition. PLoS One. 2013; 8 (9): e75069

12 

Gupta A, Singh S , authors. Evaluation of anti-inflammatory effect of Withania somnifera root on collagen-induced arthritis in rats. Pharm Biol. 2014; 52 (3): 308 –20

13 

Ruiu S, Longoni R, Spina L, Orrù A, Cottiglia F, Collu M, Kasture S, Acquas E. , authors. Withania somnifera prevents acquisition and expression of morphine-elicited conditioned place preference. Behav Pharmacol. 2013; 24 (2): 133 –43

14 

Spina L, Longoni R, Rosas M, Collu M, Peana AT, Espa E, Kasture S, Cotti E, Acquas E. , authors. Withania somnifera Dunal (Indian ginseng) impairs acquisition and expression of ethanol-elicited conditioned place preference and conditioned place aversion. J Psychopharmacol. 2015; 29 (11): 1191 –9

15 

Peana AT, Muggironi G, Spina L, Rosas M, Kasture SB, Cotti E, Acquas E , authors. Effects of Withania somnifera on oral ethanol self-administration in rats. Behav Pharmacol. 2014; 25 (7): 618 –28

16 

Gupta GL, Rana AC , authors. Effect of Withania somnifera Dunal in ethanol-induced anxiolysis and withdrawal anxiety in rats. Indian J Exp Biol. 2008; 46 (6): 470 –5

17 

Kokare DM, Singru PS, Dandekar MP, Chopde CT, Subhedar NK , authors. Involvement of alpha-melanocyte stimulating hormone (α-MSH) in differential ethanol exposure and withdrawal related depression in rat: neuroanatomical-behavioral correlates. Brain Res. 2008; 1216: 53 –67

18 

Taksande BG, Kotagale NR, Patel MR, Shelkar GP, Ugale RR, Chopde CT , authors. Agmatine, an endogenous imidazoline receptor ligand modulates ethanol anxiolysis and withdrawal anxiety in rats. Eur J Pharmacol. 2010; 637 (1-3): 89 –101

19 

Uzbay IT, Erden BF , authors. Attenuation of ethanol withdrawal signs by high doses of L-arginine in rats. Alcohol Alcohol. 2003; 38 (3): 213 –8

20 

Macey DJ, Schulteis G, Heinrichs SC, Koob GF , authors. Time-dependent quantifiable withdrawal from ethanol in the rat: effect of method of dependence induction. Alcohol. 1996; 13 (2): 163 –70

21 

Kokare DM, Chopde CT, Subhedar NK , authors. Participation of alpha-melanocyte stimulating hormone in ethanol-induced anxiolysis and withdrawal anxiety in rats. Neuropharmacology. 2006; 51 (3): 536 –45

22 

Uzbay IT, Erden BF, Tapanyigit EE, Kayaalp SO , authors. Nitric oxide synthase inhibition attenuates signs of ethanol withdrawal in rats. Life Sci. 1997; 61 (22): 2197 –209

23 

Porsolt RD, Bertin A, Jalfre M , authors. Behavioural despair in mice: a primary screening test for antidepressants. Arch Int Pharmacodynamic Theory. 1977; 229 (2): 327 –36

24 

Goyal SN, Kokare DM, Chopde CT, Subhedar NK , authors. Alpha-melanocyte stimulating hormone antagonizes antidepressant-like effect of neuropeptide Y in Porsolt’s test in rats. Pharmacol Biochem Behav. 2006; 85 (2): 369 –77

25 

Zapata A, Gonzales RA, Shippenberg TS , authors. Repeated ethanol intoxication induces behavioural sensitization in the absence of a sensitized accumbens dopamine response in C57BL/6J and DBA/2J Mice. Neuropsychopharmacol. 2006; 31 (2): 396 –405

26 

Sheikh N, Ahmad A, Siripurapu K, Kuchibhotla V, Singh S, Palit G. , authors. Effect of Bacopa monniera on stress induced changes in plasma corticosterone and brain monoamines in rats. J Ethnopharmacol. 2007; 111 (3): 671 –2

27 

Taksande BG, Faldu DS, Dixit MP, Sakaria JN, Aglawe MM, Umekar MJ, Kotagale NR , authors. Agmatine attenuates chronic unpredictable mild stress inducedbehavioral alteration in mice. Eur J Pharmacol. 2013; 720 (1-3): 115 –20

28 

Makrigiannakis A, Semmler M, Briese V, Eckerle H, Minas V, Mylonas I, Friese K, Jeschke U , authors. Maternal serum corticotropin-releasing hormone and ACTH levels as predictive markers of premature labor. Int J Gynaecol Obstet. 2007; 97 (2): 115 –9

29 

Majchrowicz E. , author. Induction of physical dependence upon ethanol and the associated behavioral changes in rats. Psychopharmacologia. 1975; 43 (3): 245 –54

30 

Uzbay IT, Yesilyurt O, Celik T, Ergün H, Isimer A. , authors. Effects of agmatine on ethanol withdrawal syndrome in rats. Behav Brain Res. 2000; 107 (1-2): 153 –9

31 

Roelofs SM. , author. Hyperventilation, anxiety, craving for alcohol: a subacute alcohol withdrawal syndrome. Alcohol. 1985; 2 (3): 501 –5

32 

Zhang Z, Morse AC, Koob GF, Schulteis G , authors. Dose- and time-dependent expression of anxiety-like behavior in the elevated plus-maze during withdrawal from acute and repeated intermittent ethanol intoxication in rats. Alcohol Clin Exp Res. 2007; 31 (11): 1811 –19

33 

Cryan JF, Hoyer D, Markou A , authors. Withdrawal from chronic amphetamine induces depressive-like behavioral effects in rodents. Biol Psychiatry. 2003; 54 (1): 49 –58

34 

Rothwell PE, Thomas MJ, Gewirtz JC , authors. Distinct profiles of anxiety and dysphoria during spontaneous withdrawal from acute morphine exposure. Neuropsychopharmacology. 2009; 34 (10): 2285 –95

35 

Frankowska M, Golda A, Wydra K, Gruca P, Papp M, Filip M , authors. Effects of imipramine or GABA(B) receptor ligands on the immobility, swimming and climbing in the forced swim test in rats following discontinuation of cocaine self-administration. Eur J Pharmacol. 2010; 627 (1-3): 142 –9

36 

Koob GF , author. Corticotropin-releasing factor, neuroplasticity (sensitization), and alcoholism. Proc Natl Acad Sci USA. 2008; 105 (26): 8809 –10

37 

Bhattacharya SK, Muruganandam AV , authors. Adaptogenic activity of Withania somnifera: an experimental study using a rat model of chronic stress. Pharmacol Biochem Behav. 2003; 75 (3): 547 –55

38 

Sehgal N, Gupta A, Valli RK, Joshi SD, Mills JT, Hamel E, Khanna P, Jain SC, Thakur SS, Ravindranath V. , authors. Withania somnifera reverses Alzheimer’s disease pathology by enhancing low-density lipoprotein receptor-related protein in liver. Proc Natl Acad Sci USA. 2012; 109 (9): 3510 –5

39 

Munro CA, Oswald LM, Weerts EM, McCaul ME, Wand GS , authors. Hormone responses to social stress in abstinent alcohol-dependent subjects and social drinkers with no history of alcohol dependence. Alcohol Clin Exp Res. 2005; 29 (7): 1133 –8

40 

Borlikova GG, Le Merrer J, Stephens DN , authors. Previous experience of ethanol withdrawal increases withdrawal-induced c-fos expression in limbic areas, but not withdrawal-induced anxiety and prevents withdrawal-induced elevations in plasma corticosterone. Psychopharmacology (Berl). 2006; 185 (2): 188 –200

41 

Becker HC. , author. Effects of alcohol dependence and withdrawal on stress responsiveness and alcohol consumption. Alcohol Res. 2012; 34 (4): 448 –58

42 

Funk CK, Zorrilla EP, Lee MJ, Rice KC, Koob GF , authors. Corticotropin-releasing factor 1 antagonists selectively reduce ethanol self-administration in ethanol-dependent rats. Biol Psychiatry. 2007; 61 (1): 78 –86

43 

Heilig M, Koob GF , authors. A key role for corticotropin-releasing factor in alcohol dependence. Trends Neurosci. 2007; 30 (8): 399 –06

44 

Sommer WH, Rimondini R, Hansson AC, Hipskind PA, Gehlert DR, Barr CS, Heilig MA , authors. Upregulation of voluntary alcohol intake, behavioral sensitivity to stress, and amygdala crhr1 expression following a history of dependence. Biol Psychiatry. 2008; 63 (2): 139 –45

45 

Rasmussen DD, Boldt BM, Bryant CA, Mitton DR, Larsen SA, Wilkinson CW , authors. Chronic daily ethanol and withdrawal: Long-term changes in the hypothalamo-pituitary-adrenal axis. Alcohol Clin Exp Res. 2000; 24 (12): 1836 –49

GRAPHICAL ABSTRACT

https://s3-us-west-2.amazonaws.com/jourdata/pj/PharmacognJ-10-6-1190_g005.jpg

SUMMARY

  • Alcoholism and withdrawal of its chronic intake are serious social and medical problems. There is no appropriate drug therapy which helps the patients to overcoming the withdrawal symptoms. Present study investigated the beneficial effects of withaferin A during ethanol dependence and withdrawal, hence projected new therapeutic drug molecule for the effective management of alcoholism.