Pharmacology, Biochemistry and Behavior 136 (2015) 13–20

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Protective effect of berberine, an isoquinoline alkaloid ameliorates ethanol-induced oxidative stress and memory dysfunction in rats Shaktipal Patil a,⁎, Santosh Tawari a, Dharmendra Mundhada a, Sayyed Nadeem b a b

Agnihotri College of Pharmacy, Pharmacology Division, Bapuji Wadi, Sindhi (Meghe), Wardha 442 001, Maharashtra, India Technocarts Institute of Technology Pharmacy, Anand Nagar, Bhopal 462 021, Madhya Pradesh, India

a r t i c l e

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Article history: Received 6 April 2015 Received in revised form 30 June 2015 Accepted 1 July 2015 Available online 6 July 2015 Keywords: Berberine Ethanol induced memory Memory impairment Oxidative stress Morris water maze Vitamin C

a b s t r a c t Memory impairment induced by ethanol in rats is a consequence of changes in the CNS that are secondary to impaired oxidative stress and cholinergic dysfunction. Treatment with antioxidants and cholinergic agonists are reported to produce beneficial effects in this model. Berberine, an isoquinoline alkaloid is reported to exhibit antioxidant effect and cholinesterase (ChE) inhibitor activity. However, no report is available on the influence of berberine on ethanol-induced memory impairment. Therefore, we tested its influence against cognitive dysfunction in ethanol-induced rats using Morris water maze paradigm. Lipid peroxidation and glutathione levels as parameter of oxidative stress and cholinesterase (ChE) activity as a marker of cholinergic function were assessed in the cerebral cortex and hippocampus. Forty five days after ethanol treated rats showed a severe deficit in learning and memory associated with increased lipid peroxidation, decreased glutathione, and elevated ChE activity. In contrast, chronic treatment with berberine (25–100 mg/kg, p.o., once a day for 45 days) improved cognitive performance, and lowered oxidative stress and ChE activity in ethanol treated rats. In another set of experiments, berberine (100 mg/kg) treatment during training trials also improved learning and memory, and lowered oxidative stress and ChE activity. Chronic treatment (45 days) with vitamin C, and donepezil during training trials also improved ethanol-induced memory impairment and reduced oxidative stress and/or cholinesterase activity. In conclusion, the present study demonstrates that treatment with berberine prevents the changes in oxidative stress and ChE activity, and consequently memory impairment in ethanol treated rats. © 2015 Elsevier Inc. All rights reserved.

1. Introduction Alzheimer's disease (AD) is a frequent and predominant cause of dementia in the elderly, neurological disturbance characterized by the presence of senile plaques and decrease in cholinergic neurotransmission (Parihar and Hemnani, 2004; Scarpini et al., 2003). Thus, most pharmacological research has focused on acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors to alleviate the cholinergic deficit associated with AD (Jung et al., 2009; Rao et al., 2007). Ethanol consumption is the most common cause of peripheral as well as CNS toxicity. Ethanol induced brain damage produces some of the most insidious effects of alcoholism, including cognitive deficits such as learning and memory impairment (Pfefferbaum et al., 1998; White, 2003). Many studies have demonstrated that chronic ethanol consumption is accompanied by oxidative damage to cellular proteins, lipids and DNA (Mansouri et al., 2001; McDonough, 2003) as well as reduced levels of the endogenous antioxidants like glutathione and superoxide dismutase (Reddy et al., 1999; Thirunavukkarasu et al., 2003). ⁎ Corresponding author at: Agnihotri College of Pharmacy, Department of Pharmacology, Wardha 442 001, Maharashtra, India. E-mail address: [email protected] (S. Patil).

http://dx.doi.org/10.1016/j.pbb.2015.07.001 0091-3057/© 2015 Elsevier Inc. All rights reserved.

Ethanol enhances oxidative stress through the generation of oxygen free radicals and lipid peroxidation (Nordmann et al., 1990) and depletion of endogenous antioxidants such as alpha-tocopherol, glutathione, ascorbate and vitamin E. Ethanol is oxidized to acetaldehyde by cytochrome P450, which increases reactive oxygen species, with concomitant changes in redox balance (Mantle and Preedy, 1999; Zima et al., 2001). In addition, increased acetylcholinesterase activity (which causes degradation of acetylcholine (ACh), a neurotransmitter associated with learning and memory) in the brain has been correlated with memory in disruption ethanol-treated rats (Iliev et al., 1999; Rico et al., 2007). Importantly, galantamine, an acetylcholinesterase inhibitor improves the rate of learning, short-term memory and spatial orientation, in a prolonged alcohol intake model of ACh deficit in male Wistar rats (Iliev et al., 1999). Hippocampal neurogenesis is involved in hippocampal mediated learning and memory formation. It has been proposed that changes in neurogenesis in the hippocampus may be involved in some of the alterations of cognitive function observe during aging (Drapeau et al., 2003). Berberine is an isoquinoline alkaloid reported to show anxiolytic, analgesic, anti-inflammatory, antipsychotic, antidepressant and antiamnesic effects (Imanshahidi and Hosseinzadeh, 2008; Kulkarni and

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Dhir, 2010). Berberine has multiple pharmacological effects. It is a acetylcholinesterase inhibitor similar to galantamine (Ingkaninan et al., 2006), a drug treating AD, and might be a low-molecular-weight neurotrophic drug to neurodegenerative disorder such as AD by potentiating the nerve growth factor (NGF) induced differentiation in neural cells (Shigeta et al., 2002). It is suggested that berberine can block transient outward potassium current (IA) and delayed rectifier potassium current (IK) in a concentration-dependent manner in acutely isolated CA1 pyramidal neurons of rat hippocampus (Wang et al., 2004). A recent study demonstrated that mutations in voltage-gated potassium channel KCNC3 caused adult-onset ataxia (Waters et al., 2006) and potassium channels are regarded to play a key role in neurodegeneration including AD (Nelson, 2006). So berberine might be useful for the treatment of neurodegenerative disorders including AD. Berberine might decrease the level of ROS via inhibiting NMDA-receptor. Because, ROS participate in both early and late steps of apoptosis (Tabakman et al., 2002) and reducing ROS production might be responsible for the neuroprotective effects of berberine. Long-term administration of berberine attenuates scopolamine-induced amnesia (Weng-Huang Peng et al., 1997). Therefore, the present study was designed to investigate the protective role of berberine on cognitive dysfunction in ethanol-induced rats. 2. Experimental procedures 2.1. Subjects Adult male Wistar rats born and reared in the Animal House of the Agnihotri College of Pharmacy, Wardha, from a stock originally purchased from Shree Farms, Bhandara, India were used in the present study. Young, healthy male rats (200–250 g) were group housed (three per cage) and maintained at 23 ± 2 °C under 12:12 h light (08:00–20:00 h)/dark cycle with free access to rodent chow and tap water. Animals were naive to drug treatments and experimentation at the beginning of all studies. All tests were conducted between 08:00 and 13:00 h. All experimental protocols were approved by the Institutional Animal Ethics Committee and carried out under strict compliance with ethical principles and guidelines of Committee for Purpose of Control and Supervision of Experimental Animals, Ministry of Environment and Forests, Government of India, New Delhi, India. 2.2. Drugs and solutions Berberine hydrochloride (purity: 97%) (Sami Labs, Bangalore, India), ethanol (Changshu Yangyuan Chemical, China), vitamin C (Sisco Research Laboratories, Mumbai, India), and donepezil hydrochloride (Eisai, Mumbai, India) were used in the present study. All the drugs were dissolved in double distilled water. Drug solutions were prepared fresh and their doses are expressed in terms of their free bases. 2.3. Experimental induction of alcoholic dementia Alcoholic dementia was induced by 15% w/v ethanol (2 g/kg) in double distilled water once a day, administered intraperitoneally, over a period of 30 s for 45 days. The rats serving as controls were given 1 ml/kg double distilled water (DDW) per oral (p.o.) (Baydas et al., 2005). 2.4. Treatment schedule 2.4.1. Treatment 1 (chronic treatment) Separate groups of rats (n = 6) were orally administered berberine (25, 50 and 100 mg/kg), vitamin C (100 mg/kg), and vehicle (1 ml/kg) 1 h before ethanol dosing was started from the first day of experiment once a day (20:00 h) for next 45 days (days 1–45), and at the end of 45 days rats were subjected to Morris water maze test & elevated plus maze test. Similar treatments were given to DDW control rats. The

learning and memory were evaluated (days 46–51 for Morris water maze test and days 46–47 for elevated plus maze test) (Table 1).

2.4.2. Treatment 2 (Acute treatment) In another set of experiment, 45 days after confirmation of dementia, rats (n = 6) were administered orally berberine (25, 50 and 100 mg/kg), vitamin C (100 mg/kg), donepezil (3 mg/kg) or vehicle (1 ml/kg) 1 h before ethanol dosing once a day (20:00 h) during training trials for next 5 days (days 46–50). Similar treatments were given to DDW control rats. After these treatments, rats were subjected to Morris water maze test. The learning and memory were evaluated during days 46–51 (Table 1).

2.5. Assessment of cognitive function 2.5.1. Morris water maze test Cognitive function of rats was assessed by using Morris water maze test (MWM) as described earlier (Morris et al., 1982; Tiwari et al., 2009; Tuzcu and Baydas, 2006). The test apparatus was a circular water tank (180 cm in diameter and 60 cm high) made up of dark gray plastic that was partially filled with water (24 ± 1 °C). Full cream milk (1.5 l) was used to render the water opaque. The pool was divided virtually into four equal quadrants, labeled A–B–C–D. A platform (12.5 cm in diameter and 38 cm high) was placed in one of the four maze quadrants (the target quadrant) and submerged 2.0 cm below the water surface. The platform remained in the same quadrant during the entire experiment. The rats were required to find the platform using only distal spatial extra-maze cues existing in the testing room. The cues were maintained stable throughout the testing. The rats received four consecutive daily training trials for 5 days (days 46–50 after alcoholic dementia 45 days), with each trial having a ceiling time of 60 s and a trial interval of approximately 30 s. The rat had to swim until it climbed onto the platform submerged underneath the water. After climbing onto the platform, the animal remained there for 20 s previous to the commencement of the next trial. The escape platform was reserved in the same position relative to the distal cues. If the rat was unsuccessful to reach the escape platform within the maximally allowed time of 60 s, it was gently located on the platform and allowed to remain there for the same amount of time. The time to reach the platform (latency in seconds) was measured. To test possible deficits in sensorimotor processes, rats were experienced in the water maze with a visible platform on a new location on the final day of training (Kamal et al., 2000). The test with the visual platform does not require special orientation (McNamara and Skelton, 1993) and was used to show possible deficits in sensorimotor processes. For the test, target platform was placed inside the pool 1 cm above the water line. Rats were allowed to swim for 60 s. Time to reach the platform was recorded as escape latency. After completion of the last trial, rats were gently dried with a towel, kept warm for an hour and returned to their home cages.

2.5.2. Memory consolidation test A probe trial was performed wherein the extent of memory consolidation was assessed (Kamal et al., 2000; Tuzcu and Baydas, 2006). The time spent in the target quadrant indicates the degree of memory consolidation that has taken place after learning. The probe trial was conducted on day 51, wherein the individual rat was placed into the pool as in the training trial, but that the hidden platform was removed from the pool. The time spent in the target quadrant was considered for 60 s. In probe trial, each rat was placed in a starting position directly opposite to where the platform was located. Further, the number of times crossing over the platform site of each rat was also measured and calculated.

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Table 1 Treatment schedule.

Treatment 1 Treatment 2

Days 1–45: chronic exposure

Days 46–51: Morris water maze

Days 46–47: elevated plus maze

Ethanol or DDW, with berberine, vitamin C, or DDW Ethanol or DDW

Ethanol or DDW, with berberine, vitamin C, or DDW Ethanol or DDW, with berberine, vitamin C, donepezil, or DDW

Not applicable No testing occurred

2.6. Elevated plus maze test Memory acquisition and retention were tested using the elevated plus maze test (EPM) at the 46 day. The apparatus consisted of two closed, two crossed arms and the other, open. Each rat was placed on the open arm, facing outwards. The time taken by the rat to enter the closed arm in the first trial (acquisition trial) was noted and was called as initial transfer latency. Cutoff time was set at 90 s and in case a rat could not find the closed arm within this period, it was quietly pushed into one of the closed arms and allowed to explore the maze for 30 s. Second trial (retention trial) was performed 24 h after the acquisition trial and a retention transfer latency was noted (Sharma and Gupta, 2002).

2.7.4. Estimation of glutathione Glutathione (GSH) estimation was done according to the method of Ellman (1959). Briefly, 160 μl of supernatant was added to 2 ml of Ellman's reagent (5′5 dithiobis [2-nitrobenzoic acid] 10 mM, NaHCO3 15 mM) and the mixture was incubated at room temperature for 5 min and absorbance was read at 412 nm (Ellman, 1959).

2.8. Statistical analysis Results were expressed as mean ± S.E.M. The data were analyzed by two-way or one-way analysis of variance (ANOVA) followed by Bonferroni and Tukey's multiple comparison tests, respectively. Statistical significance was considered at P b 0.05 in all the cases.

2.7. Biochemical estimation

3. Results

2.7.1. Post-mitochondrial supernatant preparation After behavioral tests, the animals were submitted to euthanasia being previously anesthetized with ethyl ether and brain structures were removed and separated into the cerebral cortex and hippocampus. Cerebral cortex and hippocampus were rinsed with ice cold saline (0.9% sodium chloride) and homogenized in chilled phosphate buffer (pH 7.4). The homogenates were centrifuged at 800 ×g for 5 min at 4 °C to separate the nuclear debris. The supernatant thus obtained was centrifuged at 10,500 ×g for 20 min at 4 °C to get the post-mitochondrial supernatant, which was used to assay cholinesterase activity.

3.1. Effect of berberine on body weight

2.7.2. Cholinesterase activity (ChE) Cholinergic dysfunction was assessed by measuring ChE levels in the cerebral cortex and hippocampus according to the method described previously by Ellman et al. (1961) with slight modifications. The assay mixture contained 0.05 ml of supernatant, 3 ml of 0.01 M sodium phosphate buffer (pH 8), 0.10 ml of 0.75 mM acetylthiocholine iodide (AcSCh) and 0.10 ml Ellman reagent (5′5 dithiobis [2-nitrobenzoic acid] 10 mM, NaHCO3 15 mM). The change in absorbance was measured at 412 nm for 5 min. The results were calculated using molar extinction coefficient of chromophore (1.36 × 104 M−1 cm−1). All samples were run in duplicate or triplicate and the enzyme activity was expressed in μmol AcSCh/min/g of protein (Ellman et al., 1961).

2.7.3. Estimation of lipid peroxidation (LPO) The malondialdehyde (MDA) content, a measure of lipid peroxidation, was assayed in the form of thiobarbituric acid reactive substances (TBARSs) by the method of Wills (1965). Briefly, 0.5 ml of post-mitochondrial supernatant and 0.5 ml of Tris HCl were incubated at 37 °C for 2 h. After incubation 1 ml of 10% trichloroacetic acid was added and centrifuged at 1000 ×g for 10 min. To 1 ml of supernatant, 1 ml of 0.67% thiobarbituric acid was added and the tubes were kept in boiling water for 10 min. After cooling 1 ml double distilled water was added and absorbance was measured at 532 nm. Thiobarbituric acid reactive substances were quantified using an extinction coefficient of 1.56 × 105 M− 1 cm− 1 and expressed as nmol of malondialdehyde per mg protein. Tissue protein was estimated using the Biuret method and the brain malondialdehyde content expressed as nmol of malondialdehyde per milligram of protein (Wills, 1965).

As shown in Fig. 1, fifty one days after the start of ethanol administration, there was a marked decline in the body weights of ethanol control rats as compared to age-matched control rats. Chronic berberine (25, 50 and 100 mg/kg) treatment significantly and dose dependently increased the body weight of ethanol control rats [F(6, 40) = 16.75, P b 0.0001], whereas chronic treatment with vitamin C (100 mg/kg) did not influence the same (P N 0.05) compared to ethanol control rats. Treatment with berberine (100 mg/kg) in DDW control animals had no significant influence on body weight (P N 0.05) compared to DDW control rats (Fig. 1). As depicted in Fig. 1, one-way ANOVA revealed that berberine (25 and 50 mg/kg) treatment during trials (days 46–50) significantly did not influence the body weight of ethanol control rats (P N 0.05). During trial, treatment with berberine (25 mg/kg), vitamin C (100 mg/kg) and donepezil (3 mg/kg) did not influence the body weight (P N 0.05) compared to ethanol control rats during trails. Treatment with berberine (100 mg/kg) in DDW control rats had no significant influence on body weight (P N 0.05) compared to DDW control rats (Fig. 1).

Fig. 1. Effect of chronic treatment with berberine (45 days) and acute treatment with berberine during training trails (5 days) on body weights. Each value represents mean ± S.E.M. of 5–6 observations. *P b 0.001 vs. DDW control group, @P b 0.001, #P N 0.05 vs. ethanol control group (One-way ANOVA followed by Tukey's post hoc test). DDW control: Double Distilled Water control; DDW + Ber 100: Double Distilled Water berberine (100 mg/kg) treated; E control: ethanol control; E + Ber 25: ethanol berberine (25 mg/kg) treated; E + Ber 50: ethanol berberine (50 mg/kg) treated; E + Ber 100: ethanol berberine (100 mg/kg) treated; E + Vit C 100: ethanol vitamin C (100 mg/kg) treated.; E + Donep 3: ethanol donepezil (3 mg/kg) treated.

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3.2. Effects of berberine on ethanol-induced cognitive dysfunction 3.2.1. Effects of berberine treatment (treatment schedule-1) on performance of Morris water maze test The cognitive function was assessed in the Morris water maze test. The mean escape latency for the trained rats decreased over the course of the 20 learning trials in all groups. Ethanol control rats exhibited significantly higher escape latency on days 2, 3, 4, and 5 during training trials compared to DDW control rats (P b 0.05). Two way repeat measure ANOVA revealed that chronic berberine treatment significantly influenced escape latency in ethanol control rats; treatment effect [F(6, 170) = 17.02, P b 0.0001] and time effect [F(4, 170) = 251.5, P b 0.0001]. Further, post hoc test revealed that berberine treatment (25, 50 and 100 mg/kg) significantly decreased escape latency compared to ethanol control rats on days 2, 3, 4 and 5 (P b 0.05). Chronic treatment with vitamin C (100 mg/kg) in ethanol treated rats showed similar results. Treatment with berberine (100 mg/kg) in vehicle treated rats had no significant influence on escape latency (P N 0.05) compared to DDW control rats (Fig. 2). The probe trial measures how well the rats had learned and consolidated the platform location during the 5 days of training. One-way ANOVA revealed that ethanol control rats spent less time in the target quadrant as compared to the DDW control rats, and treatment with berberine, and vitamin C significantly influenced the same [F(6, 40) = 23.28, P b 0.0001]. Further, post hoc test revealed that berberine (25, 50 and 100 mg/kg), and vitamin C (100 mg/kg) significantly increased time spent in the target quadrant compared to ethanol control rats (P b 0.05). Treatment with berberine (100 mg/kg) in DDW control rats had no significant influence on time spent in the target quadrant (P N 0.05) compared to DDW control rats (Fig. 3).

3.2.2. Effects of berberine treatment during trials (treatment schedule 2) on performance of Morris water maze test Two-way repeat measure ANOVA revealed that treatment with berberine and donepezil during training trials, significantly influenced the transfer latency; treatment effect [F(7, 200) = 19.03, P b 0.0001] and time effect [F(4, 200) = 243.4, P b 0.0001] (Fig. 4). Further, post hoc test revealed that berberine (100 mg/kg) and donepezil (3 mg/kg) during training trials, significantly reduced escape latency on day 5 (P b 0.05), whereas vitamin C (100 mg/kg), and berberine per se had no effect on escape latency (Fig. 4).

Fig. 2. Effect of chronic treatment with berberine (45 days) on the performance of spatial memory acquisition phase in Morris water maze test. Each value represents mean ± S.E.M. of 5–6 observations. **P b 0.01 and ***P b 0.001 vs. DDW control group. #P b 0.05, &P b 0.01 and @P b 0.001 vs. ethanol control group (Two-way repeat measure ANOVA followed by Bonferroni multiple comparison test). DDW control: Double Distilled Water control; DDW + Ber 100: Double Distilled Water berberine (100 mg/kg) treated; E control: ethanol control; E + Ber 25: ethanol berberine (25 mg/kg) treated; E + Ber 50: ethanol berberine (50 mg/kg) treated; E + Ber 100: ethanol berberine (100 mg/kg) treated; E + Vit C 100: ethanol vitamin C (100 mg/kg) treated.

Fig. 4. Effect of acute treatment with berberine during training trials (5 days) on the performance of spatial memory acquisition phase in Morris water maze test. Each value represents mean ± S.E.M. of 5–6 observations. *P b 0.05 and ***P b 0.001 vs. DDW control group. #P b 0.05, &P b 0.01 and @P b 0.001 vs. ethanol control group (two-way repeat measure ANOVA followed by Bonferroni multiple comparison test). DDW control: Double Distilled Water control; DDW + Ber 100: Double Distilled Water berberine (100 mg/kg) treated; E control: ethanol control; E + Ber 25: ethanol berberine (25 mg/kg) treated; E + Ber 50: ethanol berberine (50 mg/kg) treated; E + Ber 100: ethanol berberine (100 mg/kg) treated; E + Vit C 100: ethanol vitamin C (100 mg/kg) treated; E + Donep 3: ethanol donepezil (3 mg/kg) treated.

Fig. 3. Effect of chronic treatment with berberine (51 days) on time spent in target quadrant during probe trial in Morris water maze test. Each bar represents mean ± S.E.M. of 5–6 observations. @P b 0.001 vs. DDW control group, *P b 0.05, **P b 0.01 and ***P b 0.001 vs. ethanol control group (One-way ANOVA followed by Tukey's post hoc test). DDW control: Double Distilled Water control; DDW + Ber 100: Double Distilled Water berberine (100 mg/kg) treated; E control: ethanol control; E + Ber 25: ethanol berberine (25 mg/kg) treated; E + Ber 50: ethanol berberine (50 mg/kg) treated; E + Ber 100: ethanol berberine (100 mg/kg) treated; E + Vit C 100: ethanol vitamin C (100 mg/kg) treated.

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In the probe trial, one-way ANOVA revealed that ethanol control rats spent less time in the target quadrant as compared to the DDW control rats, and rats treated with berberine and donepezil significantly influenced the same [F(7, 47) = 9.012, P = 0.0001] (Fig. 5). Further, post hoc test revealed that berberine (100 mg/kg) and donepezil (3 mg/kg) treatment significantly increased time spent in the target quadrant compared to ethanol control rats (P b 0.05), whereas vitamin C (100 mg/kg) ethanol treated rats also had a significant influence on this parameter. However, berberine (25 and 50 mg/kg) had no significant influence on time spent in the target quadrant (P N 0.05). 3.2.3. Effects of berberine on performance of elevated plus maze test (EPM) The effects of various chronic treatments in ethanol control rats and DDW control rats on mean transfer latencies in the EPM test are presented in Fig. 6. One-way ANOVA revealed that chronic treatment with berberine (25, 50 and 100 mg/kg) and vitamin C (100 mg/kg), had no significant effect on the transfer latencies of the first day (acquisition) compared to that of the vehicle treated ethanol control rats (P N 0.05). Student-t test revealed that transfer latency on day 2 (retention) significantly reduced when compared with transfer latency on day 1 in vehicle treated DDW control rats (P = 0.0002). Further, one-way ANOVA revealed that berberine (25, 50 and 100 mg/kg) and vitamin C (100 mg/kg) treatment had significant influence on transfer latency tested on day 2 (retention) [F(6, 41) = 18.30, P b 0.0001]. Post hoc test revealed that berberine (50 and 100 mg/kg) and vitamin C (100 mg/kg) significantly decreased transfer latency as compared to vehicle treated ethanol control rats on day 2 (P b 0.05). Treatment with berberine (100 mg/kg) in vehicle treated rats had no significant influence on transfer latency (P N 0.05) compared to vehicle treatment in DDW control rats in both sessions (Fig. 6). 3.3. Effect of berberine on ethanol-induced changes in cholinesterase activity Cholinesterase (ChE) activity was expressed as AcSCh formed. The changes in ChE activity in cerebral cortex and hippocampus after chronic administration of berberine are presented in Fig. 7. As can be observed, ChE activity was significantly increased in the cortex [F(6, 41) = 6.296, P = 0.0001] and hippocampus [F(6, 41) = 9.737, P b 0.0001] of ethanol control group compared to the DDW control group. Chronic treatment with berberine (25, 50 and 100 mg/kg) significantly decreased the ChE activity in cortex compared to ethanol control rats (P b 0.05, 0.01, and 0.001). Similarly, chronic treatment with berberine (50 and 100 mg/kg) significantly decreased the ChE activity in hippocampus compared to ethanol control rats (P b 0.01 and 0.001). These effects were comparable to that of vitamin C. Chronic berberine treatment

Fig. 5. Effect of acute treatment with berberine during training trials (5 days) on time spent in target quadrant during probe trial in Morris water maze test. Each bar represents mean ± S.E.M. of 5–6 observations. @P b 0.001 vs. DDW control group, *P b 0.05, **P b 0.01 and ***P b 0.001 vs. ethanol control group (One-way ANOVA followed by Tukey's post hoc test). DDW control: Double Distilled Water control; DDW + Ber 100: Double Distilled Water berberine (100 mg/kg) treated; E control: ethanol control; E + Ber 25: ethanol berberine (25 mg/kg) treated; E + Ber 50: ethanol berberine (50 mg/kg) treated; E + Ber 100: ethanol berberine (100 mg/kg) treated; E + Vit C 100: ethanol vitamin C (100 mg/kg) treated; E + Donep 3: ethanol donepezil (3 mg/kg) treated.

Fig. 6. Effect of chronic treatment with berberine (45 days) on the performance of special memory acquisition and retention phase in elevated plus maze test. Each value represents mean ± S.E.M. of 5–6 observations. @P b 0.001 vs. DDW control group during respective sessions (one-way ANOVA followed by Tukey's post hoc test); #P = 0.0002 vs. DDW control group during acquisition (t-test). *P b 0.05, **P b 0.01 and ***P b 0.001 vs. ethanol control group during retention (One-way ANOVA followed by Tukey's post hoc test). DDW control: Double Distilled Water control; DDW + Ber 100: Double Distilled Water berberine (100 mg/kg) treated; E control: ethanol control; E + Ber 25: ethanol berberine (25 mg/kg) treated; E + Ber 50: ethanol berberine (50 mg/kg) treated; E + Ber 100: ethanol berberine (100 mg/kg) treated; E + Vit C 100: ethanol vitamin C (100 mg/kg) treated.

in vehicle treated rats did not influence the ChE activity as compared to DDW control rats (P N 0.05) (Fig. 7). In another set of experiment, one-way ANOVA indicated that treatment with berberine during training trials significantly influenced the cortical [F(7, 47) = 7.368, P b 0.0001] and hippocampal [F(7, 47) = 8.284, P b 0.0001] ChE activity (Fig. 8). Treatment with berberine (100 mg/kg) significantly decreased the ChE activity in the cortex and hippocampus as compared to ethanol control rats (P b 0.01). Similarly, treatment with donepezil (3 mg/kg) significantly decreased the ChE activity in the cortex and hippocampus as compared to ethanol control rats (P b 0.01), whereas, vitamin C did not influence the same (P N 0.05). Berberine per se did not influence the ChE activity (Fig. 8).

3.4. Effect of berberine on parameters of oxidative stress in brain 3.4.1. Effect of berberine on ethanol-induced changes in lipid peroxidation Effects of chronic administration of berberine on lipid peroxidation (LPO) are depicted in Fig. 9. There was a significant rise in MDA levels in cortical [F(6, 41) = 11.71, P b 0.0001] and hippocampal [F(6, 41) = 28.32, P b 0.0001] tissue of rat brain in ethanol control rats as compared to DDW control rats. Berberine (50 and 100 mg/kg), and vitamin C (100 mg/kg) significantly reduced MDA levels as compared to ethanol

Fig. 7. Effect of chronic treatment with berberine (45 days) on cholinesterase activity in cerebral cortex and hippocampus of rat brain. Cholinesterase activity was expressed as AcSCh formed. Each value represents mean ± S.E.M. of 5–6 observations. @P b 0.001 vs. DDW control group, *P b 0.05, **P b 0.01 and ***P b 0.001 vs. ethanol control group (One-way ANOVA followed by Tukey's post hoc test). DDW control: Double Distilled Water control; DDW + Ber 100: Double Distilled Water berberine (100 mg/kg) treated; E control: ethanol control; E + Ber 25: ethanol berberine (25 mg/kg) treated; E + Ber 50: ethanol berberine (50 mg/kg) treated; E + Ber 100: ethanol berberine (100 mg/kg) treated; E + Vit C 100: ethanol vitamin C (100 mg/kg) treated.

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Fig. 8. Effect of acute treatment with berberine during training trails (5 days) on cholinesterase activity in cerebral cortex and hippocampus of rat brain. Cholinesterase activity was expressed as AcSCh formed. Each value represents mean ± S.E.M. of 5–6 observations. @P b 0.001 vs. DDW control group, *P b 0.05, **P b 0.01 and ***P b 0.001 vs. ethanol control group (One-way ANOVA followed by Tukey's post hoc test). DDW control: Double Distilled Water control; DDW + Ber 100: Double Distilled Water berberine (100 mg/kg) treated; E control: ethanol control; E + Ber 25: ethanol berberine (25 mg/kg) treated; E + Ber 50: ethanol berberine (50 mg/kg) treated; E + Ber 100: ethanol berberine (100 mg/kg) treated; E + Vit C 100: ethanol vitamin C (100 mg/kg) treated; E + Donep 3: ethanol donepezil (3 mg/kg) treated.

control rats in the cortex and hippocampus (P b 0.05). Berberine per se did not influence the MDA levels (Fig. 9). In addition, effects of berberine treatment during training trials on lipid peroxidation are depicted in Fig. 10. Similarly, there was a significant rise in MDA levels in hippocampal and cortical tissue of rat brain in ethanol control rats as compared to DDW control rats. One-way ANOVA indicated that ethanol control and berberine treatment significantly influenced the MDA levels [cortex: F(7, 47) = 22.38, P b 0.0001; hippocampus: F(7, 47) = 13.77, P b 0.0001]. Further, post hoc test revealed that berberine (100 mg/kg) significantly reduced MDA levels as compared to ethanol control rats (P b 0.05). However, treatment with vitamin C decreased the LPO but failed to attain statistical significance. Berberine per se and donepezil did not influence the MDA levels (Fig. 10).

Fig. 10. Effect of acute treatment with berberine during training trails (5 days) on lipid peroxidation (LPO) levels in cerebral cortex and hippocampus of rat brain. Each value represents mean ± S.E.M. of 5–6 observations. @P b 0.001 vs. DDW control group, *P b 0.05, **P b 0.01 and ***P b 0.001 vs. ethanol control group (One-way ANOVA followed by Tukey's post hoc test). DDW control: Double Distilled Water control; DDW + Ber 100: Double Distilled Water berberine (100 mg/kg) treated; E control: ethanol control; E + Ber 25: ethanol berberine (25 mg/kg) treated; E + Ber 50: ethanol berberine (50 mg/kg) treated; E + Ber 100: ethanol berberine (100 mg/kg) treated; E + Vit C 100: ethanol vitamin C (100 mg/kg) treated; E + Donep 3: ethanol donepezil (3 mg/kg) treated.

control rats (P b 0.05). Berberine per se did not influence the GSH levels (Fig. 11). In addition, effects of berberine treatment during training trials on GSH are depicted in Fig. 12. Similarly, there was a significant fall in GSH levels in hippocampal and cortical tissue of rat brain in ethanol control rats as compared to DDW control rats. One way ANOVA indicated that ethanol control and treatment with berberine significantly influenced the GSH levels [cortex: F(7, 47) = 20.95, P b 0.0001; hippocampus: F(7, 47) = 18.96, P b 0.0001]. Further, post hoc test revealed that berberine (100 mg/kg) significantly increased GSH levels as compared ethanol control rats (P b 0.01). However, treatment with vitamin C increased the GSH levels but failed to attain statistical significance. Berberine per se and donepezil did not influence the GSH levels (Fig. 12). 4. Discussion

3.4.2. Effect of berberine on ethanol-induced changes in glutathione levels Effects of chronic administration of berberine on GSH levels are depicted in Fig. 11. There was a significant fall in GSH levels in cortical [F(6, 41) = 16.97, P b 0.0001] and hippocampal [F(6, 41) = 27.0, P b 0.0001] tissue of rat brain in ethanol control rats as compared to DDW control rats. Berberine (50 and 100 mg/kg) and vitamin C (100 mg/kg) treatment significantly increased GSH levels as compared to ethanol

Fig. 9. Effect of chronic treatment with berberine (45 days) on lipid peroxidation (LPO) levels in cerebral cortex and hippocampus of rat brain. Each value represents mean ± S.E.M. of 5–6 observations. @P b 0.001 vs. DDW control group, **P b 0.01 and ***P b 0.001 vs. ethanol control group (One-way ANOVA followed by Tukey's post hoc test). DDW control: Double Distilled Water control; DDW + Ber 100: Double Distilled Water berberine (100 mg/kg) treated; E control: ethanol control; E + Ber 25: ethanol berberine (25 mg/kg) treated; E + Ber 50: ethanol berberine (50 mg/kg) treated; E + Ber 100: ethanol berberine (100 mg/kg) treated; E + Vit C 100: ethanol vitamin C (100 mg/kg) treated.

This study demonstrated the role of berberine, an isoquinoline alkaloid on the behavioral and biochemical function of ethanol treated rats. Ethanol-induced alcohol dependent rats exhibited marked impairment in cognitive function which was associated with marked increase in cholinesterase activity and lipid peroxidation (LPO), and reduced glutathione (GSH) level in the brain. Chronic treatment with berberine significantly and dose dependently ameliorated cognitive deficits,

Fig. 11. Effect of chronic treatment with berberine (45 days) on glutathione (GSH) levels in cerebral cortex and hippocampus of rat brain. Each value represents mean ± S.E.M. of 5–6 observations. @P b 0.001 vs. DDW control group, **P b 0.01 and ***P b 0.001 vs. ethanol control group (One-way ANOVA followed by Tukey's post hoc test). DDW control: Double Distilled Water control; DDW + Ber 100: Double Distilled Water berberine (100 mg/kg) treated; E control: ethanol control; E + Ber 25: ethanol berberine (25 mg/kg) treated; E + Ber 50: ethanol berberine (50 mg/kg) treated; E + Ber 100: ethanol berberine (100 mg/kg) treated; E + Vit C 100: ethanol vitamin C (100 mg/kg) treated.

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Fig. 12. Effect of acute treatment with berberine during training trails (5 days) on glutathione (GSH) levels in cerebral cortex and hippocampus of rat brain. Each value represents mean ± S.E.M. of 5–6 observations. @P b 0.001 vs. DDW control group, *P b 0.05, **Pb 0.01 and ***P b 0.001 vs. ethanol control group (One-way ANOVA followed by Tukey's post hoc test). DDW control: Double Distilled Water control; DDW + Ber 100: Double Distilled Water berberine (100 mg/kg) treated; E control: ethanol control; E + Ber 25: ethanol berberine (25 mg/kg) treated; E + Ber 50: ethanol berberine (50 mg/kg) treated; E + Ber 100: ethanol berberine (100 mg/kg) treated; E + Vit C 100: ethanol vitamin C (100 mg/kg) treated; E + Donep 3: ethanol donepezil (3 mg/kg) treated.

reduced cholinesterase activity and lipid peroxidation, and increased glutathione level in alcohol dependent rats. Further, short-term treatment with berberine also significantly reversed ethanol induced behavioral and biochemical changes. Chronic alcohol administration is known to cause memory deficits associated with enhanced oxidative stress and is well supported by numerous studies. Khalil et al. found that ethanol administration significantly increased the time to find the platform (latency period), indicating that ethanol induces deficit in spatial reference memory which was associated with increased levels of β-EN in the cerebral cortex and hippocampus of ethanol treated rats (Khalil et al., 2005). Some studies also suggested that galantamine improves the speed of learning, short-term memory and spatial orientation of rats in conditions of prolonged alcohol intake indicating the deficits in cholinergic neurotransmission in chronic ethanol administered rats (Iliev et al., 1999). Studies reporting ethanol induced decreases in endogenous antioxidant levels in brain further support ethanol-induced oxidative stress in the brain (Reddy et al., 1999). The present study revealed that the Morris water maze test and elevated plus maze test were used for the assessment of memory functions. A decreased escape latency in Morris water maze task in repeated trials demonstrates intact learning and memory function. Chronic ethanol treated rats did not show a significant decline in the escape latency as compared to control and per se group, whereas berberine treatment of ethanol-treated rats decreased the time to reach the hidden platform. In probe trial also, the time spent in target quadrant is significantly decreased in ethanol treated rats as compared to control and per se group, which was significantly reversed dose dependently on treatment with both the berberine (25, 50 and 100 mg/kg) and vitamin C (100 mg/kg). EPM test is suggested to be a simple method for the evaluation of learning and memory processes. Since the animals are able to remember the configuration of the open and enclosed arms, they escape from the unsafe open arm more rapidly on the second trial. It is possible to evaluate the fear motivated learning, which underlies the transfer latency procedure in this test. Similarly, EPM performance in ethanol treated rats was also severely impaired. This test also revealed that alcoholic dementia reduced learning and memory performance. The results from EPM further substantiated the findings with the MWM as both the treatments reduced the initial transfer latencies after 24 h in ethanol-treated rats. However, in both the memory assessment paradigms, elevated plus maze is a widely used animal model for the determination of anti anxiety activity. But people have widely used it as a memory assessment paradigm also. Decrease in transfer latency after 24 h of training indicates improvement in memory function (Kumar and Gupta, 2002; Sharma and Gupta, 2002). This excludes the possibility that the activity per se may have contributed to the changes

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in MWM and EPM in vehicle treated alcohol dependent rats and the berberine and vitamin C treated alcohol dependent rats. The biochemical estimations indicated a significant increase in cholinesterase activity and lipid peroxidation levels and marked decrease in the activity of glutathione levels in the cerebral cortex and hippocampus of ethanol treated rats. Treatment with berberine returned the levels of lipid peroxidation, and reduced glutathione towards their control values. The effect was more pronounced with donepezil and vitamin C treatment. Bhutada et al., 2010 showed that chronic treatment with vitamin C protected the cognitive dysfunction in diabetic rats. The clinically used antidementic drug donepezil ameliorated scopolamine induced memory impairment by reducing ChE activity and oxidative stress and restoring cerebral circulation (Agrawal et al., 2008). Chronic treatment with both the isoforms of vitamin E decreases nitric oxide levels in both brain regions significantly in a dose-dependent manner, this observation is supported by the findings from Osakada et al., 2004 We have also observed increased cholinesterase levels in both brain regions (cortex and hippocampus) of ethanol treated rats suggesting the involvement of enhanced cholinesterase activity in chronic ethanol-induced cognitive dysfunction. The berberine (25, 50 and 100 mg/kg) treatment showed a significant decrease in elevated cholinesterase activity in cortex and hippocampus of ethanol treated rats in a dose-dependent manner. Similarly, donepezil (3 mg/kg) treatment showed a significant decrease in cholinesterase activity in cortex and hippocampus of ethanol treated rats. An increase in reactive species production due to oxidative stress (Bonnefont-Rousselot, 2002; Evans et al., 2002) seems to play a key role in neuronal damage (Arvanitakis et al., 2004). Oxidative damage to the rat synapse in the cerebral cortex and hippocampus has been previously reported to contribute to the deficit of cognitive functions (Biessels et al., 1996; Fukui et al., 2001; Gispen and Biessels, 2000). The results of the present study revealed that treatment with berberine dose dependently prevented the learning and memory deficits in ethanol-induced rats and these effects were similar to vitamin C treatment. Interestingly, Liu et al., 2008 reported that berberine restored the malondialdehyde, superoxide anion and superoxide dismutase. It is also shown that berberine inhibited reactive oxygen species (ROS) generation (Zhou et al., 2008a,b). Therefore, in the present study, berberine might have protected ethanol-associated memory dysfunction by reducing oxidative stress in ethanol-induced rats. In conclusion, the findings of the present investigation suggest that berberine exerts its beneficial effects on ethanol-induced memory dysfunction and it may be attributed to its antioxidant and ChE inhibitory activity. Thus berberine may be projected in the treatment of cognitive and neural dysfunction associated with chronic alcoholism. References Agrawal, R., Tyagi, E., Shukla, R., Nath, C., 2008. Effect of insulin and melatonin on acetylcholinesterase activity in the brain of amnesic mice. Behav. Brain Res. 189, 381–386. Arvanitakis, Z., Wilson, R.S., Bienias, J.L., Evans, D.A., Bennett, D.A., 2004. Diabetes mellitus and risk of Alzheimer disease and decline in cognitive function. Arch. Neurol. 61, 661–666. Baydas, Giyasettin, Yasar, Abdullah, Tuzcu, Mehmet, 2005. Comparison of the impact of melatonin on chronic ethanol-induced learning and memory impairment between young and aged rats. J. Pineal Res. 39, 346–352. Bhutada, P., Mundhada, Y., Bansod, K., Bhutada, C., Tawari, S., Dixit, P., Mundhada, D., 2010. Ameliorative effect of quercetin on memory dysfunction in streptozotocininduced diabetic rats. Neurobiol. Learn. Mem. 94, 293–302. Biessels, G.J., Kamal, A., Ramakers, G.M., Urban, I.J., Spruijt, B.M., Erkelens, D.W., Gispen, W.H., 1996. Place learning and hippocampal synaptic plasticity in streptozotocininduced diabetic rats. Diabetes 45, 1259–1266. Bonnefont-Rousselot, D., 2002. Glucose and reactive oxygen species. Curr. Opin. Clin. Nutr. Metab. Care 5, 561–568. Drapeau, E., Mayo, W., Aurousseau, C., 2003. Spatial memory performances of aged rats in the water maze predicts levels of hippocampal neurogenesis. Proc. Natl. Acad. Sci. U. S. A. 100, 14385–14390. Ellman, G., Courtney, K., Andres, V., Featherstone, R., 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88–95 (1961). Ellman, G., 1959. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82 (1), 70–77.

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