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Behavioural Brain Research 220 (2011) 30–41

Contents lists available at ScienceDirect

Behavioural Brain Research

journa l homepage: www.e lsev ier .com/ locate /bbr

esearch report

rotection of cholinergic and antioxidant system contributes to the effect oferberine ameliorating memory dysfunction in rat model oftreptozotocin-induced diabetes

ravinkumar Bhutadaa,∗, Yogita Mundhadaa, Kuldeep Bansoda, Santosh Tawaria, Shaktipal Patil a,ankaj Dixitb, Sudhir Umathec, Dharmendra Mundhadaa

Agnihotri College of Pharmacy, Pharmacology Division, Bapuji Wadi, Sindhi (Meghe), Wardha 442 001, Maharashtra, IndiaCollege of Pharmacy, IPS Academy, Hukmakhedi, Knowledge Village, Rajendranagar, AB Road, Indore 452 012, Madhya Pradesh, IndiaDepartment of Pharmaceutical Sciences, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur 440 033, Maharashtra, India

r t i c l e i n f o

rticle history:eceived 12 May 2010eceived in revised form 6 January 2011ccepted 14 January 2011

eywords:erberineiabetesonepezilementiaetforminorris water mazexidative stressitamin C

a b s t r a c t

Memory impairment induced by streptozotocin in rats is a consequence of changes in CNS that aresecondary to chronic hyperglycemia, impaired oxidative stress, cholinergic dysfunction, and changes inglucagon-like peptide (GLP). Treatment with antihyperglycemics, antioxidants, and cholinergic agonistsare reported to produce beneficial effect in this model. Berberine, an isoquinoline alkaloid is reportedto exhibit anti-diabetic and antioxidant effect, acetylcholinesterase (AChE) inhibitor, and increases GLPrelease. However, no report is available on influence of berberine on streptozotocin-induced memoryimpairment. Therefore, we tested its influence against cognitive dysfunction in streptozotocin-induceddiabetic rats using Morris water maze paradigm. Lipid peroxidation and glutathione levels as parametersof oxidative stress and choline esterase (ChE) activity as marker of cholinergic function were assessed inthe cerebral cortex and hippocampus. Thirty days after diabetes induction rats showed a severe deficitin learning and memory associated with increased lipid peroxidation, decreased reduced glutathione,and elevated ChE activity. In contrast, chronic treatment with berberine (25–100 mg/kg, p.o., twice daily,

30 days) improved cognitive performance, lowered hyperglycemia, oxidative stress, and ChE activityin diabetic rats. In another set of experiment, berberine (100 mg/kg) treatment during training trialsalso improved learning and memory, lowered hyperglycemia, oxidative stress, and ChE activity. Chronictreatment (30 days) with vitamin C or metformin, and donepezil during training trials also improveddiabetes-induced memory impairment and reduced oxidative stress and/or choline esterase activity. In

udy dnd co

conclusion, the present ststress and ChE activity, a

. Introduction

Diabetes mellitus (DM) is the most common endocrine disor-er characterized by increased blood glucose levels resulting fromefective insulin secretion, resistance to insulin action or bothhat is associated with long term complications and affects eyes,idneys, blood vessels, heart, and nerves [23,48]. DM is strongly

ssociated with degenerative and functional disorders of the cen-ral nervous system (CNS) [31,49]. These effects of diabetes inhe CNS are a series of neurochemical, neurophysiological, andtructural abnormalities [7,58]. DM is often associated with severe

∗ Corresponding author at: Agnihotri College of Pharmacy, Department of Phar-acology, Wardha 442 001, Maharashtra, India. Tel.: +91 7152 254785;

ax: +91 7152 232548.E-mail address: psbhutada@live.com (P. Bhutada).

166-4328/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.bbr.2011.01.022

emonstrates treatment with berberine prevents the changes in oxidativensequently memory impairment in diabetic rats.

© 2011 Elsevier B.V. All rights reserved.

complications, and there is an increasing appreciation that cogni-tive function declines in DM [13,14,53,60,65,71]. Diabetic childrenand adults exhibit reduced psychomotor efficiency, cognitive flex-ibility, and rapid information-processing [13,16,54]. Furthermore,diabetic patients also seem to double the probability of developingAlzheimer’s disease and other dementias [4,11]. These evidencessuggest the strong association between DM and cognitive dysfunc-tion.

The mechanism causing brain damage in DM appears to bea multi-factorial process. A growing body of literature suggest,diabetes-related cognitive dysfunction is largely a consequenceof changes within the CNS that are secondary to chronic

hyperglycemia [7,15,35,41–43,59]. The cerebrovascular changes[32,40,70], oxidative stress [5,52], increased advanced glycationend products [66,67], and impairments in cerebral insulin sig-naling systems [26] are thought to be the underlying causes fordiabetic dementias. Moreover, anti-oxidants [25,63], antihyper-

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lycemics and insulin sensitizing agents [55] are reported to reduceognitive dysfunction in diabetes. Recent evidences suggest thatlucagon-like peptide-1 (GLP-1) plays imperative role in diabetes38], and cognitive dysfunction, learning, and neuroprotection [17].owever, at present, no specific treatments are available for theanagement and/or prevention of cognitive dysfunction in DM

10].Berberine is an isoquinoline alkaloid reported to exhibit anxi-

lytic, analgesic, anti-inflammatory, antipsychotic, antidepressant,nd anti-amnesic effect [28,34]. A number of clinical and pre-linical investigations have showed beneficial effects of berberinen the diabetes [24,36,37,61,72,74] that are mainly attributed to:nhanced insulin expression, � cell regeneration and potential asn anti-oxidant [61,75]. Recently, berberine is reported to ame-iorate spatial memory impairment by activating microglia andenile plaque clearance [77]. Moreover, berberine is also reported tonhibit acetylcholinesterase enzyme activity and play an importantole in metabolic syndrome [27]. In addition, Peng et al. [51] showednti-amnesic effect of berberine is related to increase in periph-ral and central cholinergic neuronal system activity. Other thanhis, berberine is also reported to reduce diabetic nephropathy andndothelial dysfunctions in diabetic animals [39,68]. Moreover, it islso reported that berberine enhances GLP-1 release and biosynthe-is, and may play important role in pathologies of diabetes-inducedognitive dysfunction through GLP-1 receptor modulation [73].rom these evidences, we hypothesized that berberine may influ-nce diabetes-induced cognitive dysfunction. However, there areo reports concerning this issue. Therefore, the present study wasesigned to investigate the protective role of berberine on cognitiveysfunction in streptozotocin (STZ)-induced diabetic rats.

. Experimental procedures

.1. Subjects

Adult male Wistar rats born and reared in the Animal House of the Agnihotriollege of Pharmacy, Wardha, from a stock originally purchased from Shree Farms,handara, India were used in the present study. Young healthy male rats (200–225 g)ere 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. Thenimal studies were approved by the Institutional Animal Ethics Committee (viderotocol no. 05, dated 19/09/2009), constituted for the purpose of control and super-ision of experimental animals by Ministry of Environment and Forests, Governmentf India, New Delhi, India. Animals were naive to drug treatments and experimen-ation at the beginning of all studies. All tests were conducted between 08:00 and3:00 h.

.2. Drugs and solutions

Berberine hydrochloride (purity: >97%) (Sami Labs, Bangalore, India), strepto-otocin (Sigma–Aldrich Co, St. Louis, MO), Vitamin C (Sisco Research Laboratories,umbai, India), donepezil hydrochloride (Eisai, Mumbai, India), and metformin

ydrochloride (Dr. Reddy’s Laboratories, Hyderabad, India) were used in the presenttudy. All the drugs were dissolved in double distilled water except STZ, which wasissolved in citrate buffer (pH 4.4). Drug solutions were prepared fresh and theiroses are expressed in terms of their free bases.

.3. Experimental induction of diabetes

Diabetes was induced in rats by using an earlier reported method [6,64]. Inrief, STZ was dissolved in 0.1 M sodium citrate buffer, pH 4.4 and administered athe dose of 60 mg/kg through i.p. route. Streptozotocin-treated rats received 5% oflucose solution instead of water for 24 h after injection of STZ in order to reduceeath due to hypoglycemic shock. Blood samples were taken from the tail vein 48 hfter STZ injection to measure blood glucose levels. Only animals with fasting bloodlucose levels over 250 mg/dl were considered diabetic and used for the furthertudy.

.4. Treatments schedule

.4.1. Treatment 1As soon as diabetes was confirmed, separate groups of rats (n = 6) were adminis-

ered orally with berberine (25, 50, or 100 mg/kg), vitamin C (100 mg/kg), metformin500 mg/kg), or vehicle (1 ml/kg) twice daily (07:00–19:00 h) for next 30 days (day

n Research 220 (2011) 30–41 31

1–30), and at the end of this treatment schedule rats were subjected to Morris watermaze test. Similar treatments were given to control (non-diabetic) rats. The learningand memory was evaluated during day 31–36.

2.4.2. Treatment 2In another set of experiment, 30 days after confirmation of diabetes, rats (n = 6)

were administered with berberine (25, 50, or 100 mg/kg), vitamin C (100 mg/kg),metformin (500 mg/kg), donepezil (3 mg/kg) or vehicle (1 ml/kg), twice daily(08:00–20:00 h) during training trials for next 5 days (day 31–35) by p.o. route. Simi-lar treatments were given to control (non-diabetic) rats. After these treatments, ratswere subjected to Morris water maze test. The learning and memory was evaluatedduring day 31–36.

2.5. Assessment of cognitive function

2.5.1. Morris water maze (MWM) testCognitive function of rats was assessed by using MWM test as described ear-

lier [6,46,62,63]. The test apparatus was circular water tank (180 cm in diameterand 60 cm high) made up of dark gray plastic that was partially filled with water(24 ± 1 ◦C). Full cream milk was used to render the water opaque. The pool wasdivided virtually into four equal quadrants, labeled A–B–C–D. A platform (12.5 cmin diameter and 38 cm high) was placed in one of the four maze quadrants (the tar-get quadrant) and submerged 2.0 cm below the water surface (Fig. 1). The platformremained in the same quadrant during the entire experiment. The rats were requiredto find the platform using only distal spatial extra-maze cues available in the testingroom. The cues were maintained constant throughout the testing. The rats receivedfour consecutive daily training trials for 5 days (Day 31–35 after diabetes induction),with each trial having a ceiling time of 90 s and a trial interval of approximately 30 s.The rat had to swim until it climbed onto the platform submerged underneath thewater. After climbing onto the platform, the animal remained there for 20 s beforethe commencement of the next trial. The escape platform was kept in the same posi-tion relative to the distal cues. If the rat failed to reach the escape platform within themaximally allowed time of 90 s, it was gently placed on the platform and allowed toremain there for the same amount of time. The time to reach the platform (latencyin seconds) was measured.

To test possible deficits in sensorimotor processes, rats were tested in the watermaze with a visible platform on a new location on the final day of training [29].The test with the visual platform does not require special orientation [44] and wasused to show possible deficits in sensorimotor processes. For the test, black targetplatform was placed inside the pool 1 cm above the water line. Rats were allowedto swim for 60 s. Time to reach the platform was recorded as escape latency. Aftercompletion of the last trial, rats were gently dried with a towel, kept warm for anhour and returned to their home cages.

2.5.2. Memory consolidation testA probe trial was performed wherein the extent of memory consolidation was

assessed [29,63]. The time spent in the target quadrant indicates the degree of mem-ory consolidation that has taken place after learning. The probe trial was performedon day 36, wherein the individual rat was placed into the pool as in the trainingtrial, except that the hidden platform was removed from the pool. The time spentin target quadrant was measured for 60 s. In probe trial, each rat was placed at astart position directly opposite to platform quadrant. Further, the number of timescrossing over the platform site of each rat was also measured and calculated.

2.6. Open field test

One hour after the probe trial, the rats were transferred to an open-field appa-ratus, measuring 60 cm × 40 cm × 28 cm, with the floor divided into 12 squares. Theopen field session lasted for 5 min and during this time, an observer, recorded thenumber of crossing (horizontal activity) and rearing (vertical activity) responsesmanually. The test was carried out to rule out the possibility of motor disabilities.

2.7. Evaluation of blood glucose levels and body weight

Blood glucose levels were measured with a portable glucometer (Ascensia, BayerUSA). In brief, blood sample was withdrawn from the rats using tail vein rupturemethod, and drop of blood was placed on the glucometer strip loaded in the glu-cometer for blood glucose determination. During the experiment, blood glucoselevels and body weights were verified in the interim (10, 20, 30 and 36 days afterthe beginning of treatment).

2.8. Biochemical estimation

2.8.1. Post mitochondrial supernatant preparation

After behavioral tests rats were sacrificed by decapitation and brain structures

were removed and separated into cerebral cortex and hippocampus for the bio-chemical studies. Cerebral cortex and hippocampus were rinsed with ice cold saline(0.9% sodium chloride) and homogenized in chilled 50 mM phosphate buffer (pH7.4). The homogenates were centrifuged at 4600 rpm for 10 min at 4 ◦C to separatethe nuclear debris. The supernatant thus obtained was centrifuged at 15,000 rpm for

3 l Brain Research 220 (2011) 30–41

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Fig. 1. Schematic representation of Morris water maze (MWM) test apparatus andplacement of platform.

Fig. 2. Effect of chronic treatment with berberine (30 days) on the performanceof spatial memory acquisition phase in Morris water maze. Each value representsmean ± S.E.M. of 5–6 observations. **P < 0.01 and ***P < 0.001 vs. saline treatment innon-diabetic group. #P < 0.05, &P < 0.01 and @P < 0.001 vs. saline treatment in diabeticgroup (Two-way repeat measure ANOVA followed by Bonferroni multiple compari-

2 P. Bhutada et al. / Behavioura

0 min at 4 ◦C to get the post mitochondrial supernatant, which was used to assayholinesterase activity.

.8.2. Cholinesterase activityCholinergic dysfunction was assessed by measuring choline esterase (ChE) levels

n cerebral cortex and hippocampus according to the method described previ-usly by Ellman et al. [19] with slight modifications. The assay mixture contained.05 ml of supernatant, 3 ml of 0.01 M sodium phosphate buffer (pH 8.0), 0.10 ml of.75 mM acetylthiocholine iodide (AcSCh) and 0.10 ml Ellman reagent (5′5 dithio-is [2-nitrobenzoic acid] 10 mM, NaHCO3 15 mM). The change in absorbance waseasured at 412 nm for 5 min. Results were calculated using molar extinction coef-

cient of chromophore (1.36 × 104 M−1 cm−1). All samples were run in duplicate orriplicate and the enzyme activity were expressed in �mol AcSCh/min/g of protein.

.8.3. Estimation of lipid peroxidationMalondialdehyde (MDA), a product of lipid peroxidation was measured by

ethod described earlier [50] with slight modification. Briefly, the sample of 0.1 mlupernatant was taken and mixed with 0.2 ml 8.1% sodium dodecyl sulphate (SDS),.5 ml 20% glacial acetic acid and 1.5 ml of 0.8% thiobarbituric acid (TBA). Followinghese additions, tubes were mixed and heated at 95 ◦C for 1 h on a water bath andooled under tap water before mixing 1 ml of distilled water and 5 ml mixture of-butanol and pyridine (15:1). The mixture was centrifuged at 4000 rpm for 10 min.he amount of MDA formed was measured by absorbance of upper organic layer atwavelength of 532 nm using appropriate controls. A calibration curve was plot-

ed using malondialdehyde bis-(dimethoxy acetyl) as a standard. The values werexpressed as nmol of MDA/mg of protein.

.8.4. Estimation of glutathioneGlutathione (GSH) estimation was done according to the method of Ellman [18].

riefly, 160 �l of supernatant was added to 2 ml of Ellman’s reagent (5′5 dithiobis2-nitrobenzoic acid] 10 mM, NaHCO3 15 mM) and the mixture was incubated atoom temperature for 5 min and absorbance was read at 412 nm.

.9. Statistical analysis

Results were expressed as mean ± S.E.M. The data were analyzed by two-way orne-way analysis of variance (ANOVA) followed by Bonferroni and Tukey’s multipleomparison tests, respectively. Statistical significance was considered at P < 0.05 inll the cases.

. Results

.1. Effect of berberine on blood glucose levels and body weight

As shown in Table 1, thirty-eight days after streptozotocinnjection, plasma glucose levels were elevated in diabetic ratss compared to non-diabetic control rats (P < 0.001). Also, thereas a marked decline in the body weights of streptozotocin-

reated rats as compared to age-matched control rats (P < 0.001).ne-way ANOVA revealed that chronic treatment with berberine

25–100 mg/kg) or metformin (500 mg/kg) significantly (P < 0.001)educed blood glucose levels [F(7, 39) = 57.10, P < 0.0001] andncreased body weights in diabetic rats [F(7, 39) = 28.94, P < 0.0001],

hereas vitamin C (100 mg/kg) did not influence these parametersP > 0.05) when compared with vehicle treatment in diabetic rats.er se berberine had no influence on blood glucose levels and bodyeight (Table 1) (Fig. 2).

Further analysis revealed that berberine (50 and 100 mg/kg) oretformin (500 mg/kg) treatment significantly reduced the blood

lucose levels [berberine: P < 0.01; metformin: P < 0.001] but didot influence the body weights (P > 0.05) of diabetic rats [F(8,5) = 56.52, P < 0.0001]. During trials, treatment with berberine

25 mg/kg), vitamin C (100 mg/kg) and donepezil (3 mg/kg) didot influence the blood glucose levels and/or the body weightsP > 0.05) compared to vehicle treatment during trials in diabeticats. Per se berberine had no influence on blood glucose levels andody weight (Table 2).

son test). ND control: non-diabetic control; ND + Ber 100: non-diabetic berberine(100 mg/kg) treated; D control: diabetic control; D + Ber 25: diabetic berberine(25 mg/kg) treated; D + Ber 50: diabetic berberine (50 mg/kg) treated; D + Ber 100:diabetic berberine (100 mg/kg) treated; D + Vit C: diabetic vitamin C (100 mg/kg)treated; D + Met: diabetic metformin (500 mg/kg) treated.

P. Bhutada et al. / Behavioural Brain Research 220 (2011) 30–41 33

Fig. 3. Effects of chronic treatment with berberine (30 days) on escape latency during visible platform trial in Morris water maze. Each bar represents mean ± S.E.M. of 5–6observations (One-way ANOVA followed by Tukey’s post hoc test). ND control: non-diabetic control; ND + Ber 100: non-diabetic berberine (100 mg/kg) treated; D control:diabetic control; D + Ber 25: diabetic berberine (25 mg/kg) treated; D + Ber 50: diabetic berberine (50 mg/kg) treated; D + Ber 100: diabetic berberine (100 mg/kg) treated;D + Vit C: diabetic vitamin C (100 mg/kg) treated; D + Met: diabetic metformin (500 mg/kg) treated.

Table 1Effect of chronic treatment with berberine (30 days) on body weights and blood glucose levels (mean ± S.E.M. of 5–6 observations) in the different groups of rats at the onsetand at the end of the experiments.

Group Treatment Body weight (g) Blood glucose (mg/dl)

Onset of study End of study Onset of study End of study

ND control Vehicle (1 ml/kg) 212.67 ± 4.02 258.67 ± 3.57 104.00 ± 4.58 106.83 ± 5.73ND + Ber 100 Ber (100 mg/kg) 212.67 ± 3.68 253.33 ± 6.20 101.17 ± 5.06 110.17 ± 4.36D control Vehicle (1 ml/kg) 213.00 ± 3.54 172.83 ± 12.25* 101.00 ± 3.67 392.50 ± 8.60*

D + Ber 25 Ber (25 mg/kg) 213.00 ± 3.93 255.33 ± 5.86@ 99.83 ± 5.40 295.83 ± 24.17@

D + Ber 50 Ber (50 mg/kg) 213.00 ± 3.45 278.00 ± 8.33@ 103.67 ± 4.70 241.67 ± 16.89@

D + Ber 100 Ber (100 mg/kg) 213.00 ± 3.38 269.67 ± 7.25@ 105.83 ± 3.64 195.17 ± 13.92@

D + Vit C Vit C (100 mg/kg) 213.00 ± 3.67 189.40 ± 10.58 103.67 ± 6.22 365.00 ± 25.75D + Met Met (500 mg/kg) 213.00 ± 3.30 288.00 ± 3.48@ 103.67 ± 5.95 154.67 ± 9.89@

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* P < 0.001 vs. non-diabetic control group.@ P < 0.001 vs. diabetic control group (One-way ANOVA followed by Tukey’s p

100 mg/kg) treated; D control: diabetic control; D + Ber 25: diabetic berberine (25erberine (100 mg/kg) treated; D + Vit C: diabetic vitamin C (100 mg/kg) treated; D

.2. Effects of berberine on diabetes-induced cognitiveysfunction

.2.1. Effects of chronic berberine treatment (treatment schedule) on performance of Morris water maze test

The cognitive function was assessed in the MWM test. The meanscape latency for the trained rats was decreased over the coursef the 20 learning trials in all the groups (Fig. 2). Control dia-etic rats (vehicle treated) exhibited significantly higher escape

able 2ffect of berberine treatment during training trail (5 days) on body weights and blood glnset and at the end of the experiments.

Group Treatment Body weight (g)

Onset of study

ND control Vehicle (1 ml/kg) 212.17 ± 3.88ND + Ber 100 Ber (100 mg/kg) 212.33 ± 3.59D control Vehicle (1 ml/kg) 212.83 ± 3.57D + Ber 25 Ber (25 mg/kg) 213.17 ± 4.00D + Ber 50 Ber (50 mg/kg) 213.17 ± 3.51D + Ber 100 Ber (100 mg/kg) 213.17 ± 3.36D + Vit C Vit C (100 mg/kg) 213.00 ± 3.66D + Donep Donep (3 mg/kg) 213.16 ± 3.20D + Met Met (500 mg/kg) 213.33 ± 3.12

* P < 0.001 vs. non-diabetic control group.# P < 0.01.@ P < 0.001 vs. diabetic control group (One-way ANOVA followed by Tukey’s post ho

100 mg/kg) treated; D control: diabetic control; D + Ber 25: diabetic berberine (25 mg/kerberine (100 mg/kg) treated; D + Vit C: diabetic vitamin C (100 mg/kg) treated; D + Donreated.

c test). ND control: non-diabetic control; ND + Ber 100: non-diabetic berberineg) treated; D + Ber 50: diabetic berberine (50 mg/kg) treated; D + Ber 100: diabeticdiabetic metformin (500 mg/kg) treated.

latency on day 3, 4, and 5 during training trials compared to non-diabetic vehicle treated rats (P < 0.01). Two-way repeat measureANOVA revealed that berberine (25, 50, and 100 mg/kg) signifi-cantly decreased escape latency [F(7, 156) = 12.76, P < 0.0001] indiabetic rats compared to vehicle treatment in diabetic rats on day

2, 3, 4, and 5 [F(4, 156) = 273.3, P < 0.0001]. Chronic treatment withvitamin C (100 mg/kg) and metformin (500 mg/kg) in diabetic ratsshowed similar results. However, berberine per se had no effect onescape latency (Fig. 2).

ucose levels (mean ± S.E.M. of 6 observations) in the different groups of rats at the

Blood glucose (mg/dl)

End of study Onset of study End of study

260.83 ± 11.26 107.67 ± 8.03 103.17 ± 5.12266.00 ± 13.40 98.33 ± 5.16 105.17 ± 5.43173.17 ± 6.68* 101.33 ± 4.03 392.00 ± 8.27*

186.00 ± 5.11 102.33 ± 5.84 365.67 ± 12.50179.33 ± 7.97 102.00 ± 6.24 307.67 ± 26.27#

169.00 ± 4.90 104.33 ± 4.08 299.17 ± 16.83#

171.33 ± 6.62 107.00 ± 5.74 387.17 ± 13.23168.00 ± 6.62 104.5 ± 5.76 356.83 ± 26.30173.50 ± 5.01 106.83 ± 5.06 217.33 ± 12.34@

c test). ND control: non-diabetic control; ND + Ber 100: non-diabetic berberineg) treated; D + Ber 50: diabetic berberine (50 mg/kg) treated; D + Ber 100: diabeticep: diabetic donepezil (3 mg/kg) treated; D + Met: diabetic metformin (500 mg/kg)

34 P. Bhutada et al. / Behavioural Brai

Fig. 4. Effect of chronic treatment with berberine (30 days) on time spent in eachquadrant during probe trial in Morris water maze. Each bar represents mean ± S.E.M.of 5–6 observations. (A) Probe trial performance as measured by comparing timespent in the target quadrant with an average of time spent in all three non-targetquadrants, *P < 0.001, #P < 0.01, vs. avg. time spent in non-target quadrant in respec-tive group (two-way ANOVA followed by Bonferroni’s post hoc test); (B) probe trialperformance as measured by comparing the time each rat spent in the target quad-rant with the time it spent in its next most preferred quadrant, *P < 0.001, &P < 0.05,vs. time spent in next preferred quadrant in respective group (two-way ANOVA fol-lowed by Bonferroni’s post hoc test); (C) difference score index of selective search.This measure was obtained by subtracting the time each rat spent in its next mostpreferred quadrant from the time it spent in the target quadrant. Selective search isindicated by the magnitude of the difference score. A score of 0 indicates no selec-tive search. *P < 0.001 vs. non-diabetic control group; #P < 0.01, $P < 0.001, vs. diabeticcontrol group (one-way ANOVA followed by Tukey’s post hoc test).

n Research 220 (2011) 30–41

The performance of all the groups in the trial with the visibleplatform was not significantly different (P > 0.05) (Fig. 3). Theseobservations suggest that there were no changes in sensorimotorfunction due to long-standing STZ-induced diabetes. Further, themean latencies in all the groups were similar in the first trial sug-gest that motor performance (ability to swim) was unaffected bythe hyperglycemia and/or treatments.

The probe trial data of the Morris water maze study was ana-lyzed as per the method reported by Bolding and Rudy [12] andBhutada et al. [6], as this method is capable of expressing memoryretention in terms of selective search behavior, which specifi-cally correlates with learning impairment. The data from probetrial is depicted in Fig. 4A–C, which provides three representa-tions of selective performance on the retention test. Fig. 3A is astandard measure and compares time spent in the target quad-rant against the average time spent in other three quadrants.Two-way ANOVA indicated a significant preference for trainingquadrant [F(1, 78) = 51.86, P < 0.0001] for various treatments [F(7,78) = 6.63, P < 0.0001]. It was further observed that the target quad-rant preference was completely lost in diabetic animals (P > 0.05).The treatment with berberine at all doses significantly preventedthe memory impairment as indicated by the increase in the timespent in target quadrant (P < 0.001). These effects of berberine weresimilar to that shown by vitamin C and metformin treatment.

However, it is reported that this standard measure of selec-tive search underestimates forgetting of place information e.g.,although the time an individual rat spent in the training quadrantmight exceed chance (20 s), it might also have spent as much ormore time in another quadrant, in which case the individual rat didnot display selective search of the training quadrant. To considerthis outcome, the time each rat spent in the target quadrant wascompared with the time it spent in its other most preferred quad-rant (Fig. 4B). Two-way ANOVA indicated a significant differencebetween time spent in training quadrant and the next preferredquadrant [F(1, 78) = 166.6, P < 0.0001]. It was further observed thatthere was a significant difference between time spent in targetquadrant against that of next preferred quadrant in diabetic ani-mals (P < 0.001). The treatment with berberine (50 and 100 mg/kg)significantly prevented the memory impairment as indicated sig-nificant difference in the time spent in target quadrant againstthat of next preferred quadrant (P < 0.001). Interestingly, per seberberine (50 mg/kg) improvised the selective search. Vitamin Cand metformin treatment had similar influence to that of berberinetreatment.

Fig. 4C compares the groups using a difference score derivedfrom the data presented in Fig. 4B. An analysis of variance revealeddifferences among the groups’ retention intervals [F(7, 45) = 13.56,P < 0.0001]. It was further observed that the diabetes significantlyimpaired the memory retention as indicated by a significantly dif-ferent (P < 0.001) lower score as compared to non-diabetic controlgroup. This impairment was significantly prevented by berberinetreatment at all tested doses. Vitamin C and Metformin had similareffects as that of berberine. However, per se berberine (50 mg/kg)was unable to modulate the selective search.

Furthermore, similar results were obtained from the former

platform crossings experiments (Fig. 5). Diabetic rats crossedover the platform less frequently as compared to the non-diabetic control rats, and berberine (50 and 100 mg/kg), metformin(500 mg/kg), and vitamin C (100 mg/kg) significantly increasednumber of crossings over the platform compared to vehicle

ND control: non-diabetic control; ND + Ber 50: non-diabetic berberine (50 mg/kg)treated; D control: diabetic control; D + Ber 12.5: diabetic berberine (12.5 mg/kg)treated; D + Ber 50: diabetic berberine (50 mg/kg) treated; D + Ber 100: diabeticberberine (100 mg/kg) treated; D + Vit C: diabetic vitamin C (100 mg/kg) treated;D + Met: diabetic metformin (500 mg/kg) treated.

P. Bhutada et al. / Behavioural Brain Research 220 (2011) 30–41 35

Fig. 5. Effects of chronic treatment with berberine (30 days) on number of crossings during probe trial in Morris water maze. Each bar represents mean ± S.E.M. of 5–6o 0.001h 00 mgt rberind

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and donepezil (3 mg/kg) significantly increased number of cross-ings over the platform compared to vehicle treated diabetic rats(P < 0.05). However, berberine per se had no effect on number ofcrossings (Fig. 8).

Fig. 6. Effects of berberine treatment during training trials (5 days) on the per-formance of spatial memory acquisition phase in Morris water maze. Each valuerepresents mean ± S.E.M. of 6 observations. *P < 0.05 and ***P < 0.001 vs. non-diabeticcontrol group. #P < 0.05, &P < 0.01 and @P < 0.001 vs. diabetic control group (Two-way repeat measure ANOVA followed by Bonferroni multiple comparison test). ND

bservations. @P < 0.001 vs. saline treatment in non-diabetic rats, *P < 0.05, and ***P <oc test). ND control: non-diabetic control; ND + Ber 100: non-diabetic berberine (1reated; D + Ber 50: diabetic berberine (50 mg/kg) treated; D + Ber 100: diabetic beiabetic metformin (500 mg/kg) treated.

reated diabetic rats (P < 0.05) [F(7, 39) = 6.884, P < 0.0001]. How-ver, berberine per se had no effect on number of crossings (Fig. 5).

.2.2. Effects of berberine treatment during trials (treatmentchedule 2) on performance of Morris water maze test

Two-way repeat measure ANOVA revealed that berberine100 mg/kg), metformin (500 mg/kg), and donepezil (3 mg/kg) dur-ng training trials, significantly reduced escape latency on day 5P < 0.05), whereas vitamin C (100 mg/kg) and berberine per sead no effect on escape latency [F(8, 180) = 9. 794, P < 0.0001; F(4,80) = 236.3, P < 0.0001] (Fig. 6).

In the probe trial, Two-way ANOVA indicated a significant pref-rence for training quadrant [F(1, 90) = 148.1, P < 0.0001] for variousreatments [F(8, 90) = 5.04, P < 0.0001]. It was further observedhat the target quadrant preference was completely lost in dia-etic animals (P > 0.05). The treatment with berberine (100 mg/kg)ignificantly prevented the memory impairment as indicated byhe increase in the time spent in target quadrant (P < 0.001) inoth diabetic and non-diabetic animals. These effects of berberineere similar to that shown by donepezil (3 mg/kg) and metformin

500 mg/kg). However, vitamin C treatment failed to have any effectFig. 7A).

The time spent in target quadrant vs. the time spent in otherost preferred quadrant is shown in Fig. 7B. It was observed

hat there was a significant difference between time spent inarget quadrant against that of next preferred quadrant in dia-etic animals (P < 0.05). The treatment with berberine (100 mg/kg)ignificantly prevented the memory impairment as indicated byhe increase in the time spent in target quadrant (P < 0.001) inoth diabetic and non-diabetic animals. These effects of berberineere similar to that shown by donepezil (3 mg/kg) and metformin

500 mg/kg). However, vitamin C treatment failed to have anyffect. Finally, the differential scores were compared (Fig. 7C), andt was observed that the selective search in diabetic animals wasompletely lost (P < 0.001). This impairment was significantly pre-ented by berberine treatment at 100 mg/kg (P < 0.001). Donepezilnd metformin had similar effects as that of berberine. However,itamin C was unable to modulate the impaired selective searchehavior in diabetic animals.

Moreover, similar results were obtained from the former plat-orm crossings experiments. Diabetic rats crossed over the platformess frequently as compared to the non-diabetic control rats, andreatment with berberine and donepezil significantly influencedhe same [F(8, 45) = 5.826, P < 0.0001] (Fig. 8). Further, post hoc

vs. saline treatment in diabetic group (One-way ANOVA followed by Tukey’s post/kg) treated; D control: diabetic control; D + Ber 25: diabetic berberine (25 mg/kg)e (100 mg/kg) treated; D + Vit C: diabetic vitamin C (100 mg/kg) treated; D + Met:

test revealed that berberine (100 mg/kg), metformin (500 mg/kg)

control: non-diabetic control; ND + Ber 100: non-diabetic berberine (100 mg/kg)treated; D control: diabetic control; D + Ber 25: diabetic berberine (25 mg/kg)treated; D + Ber 50: diabetic berberine (50 mg/kg) treated; D + Ber 100: diabeticberberine (100 mg/kg) treated; D + Vit C: diabetic vitamin C (100 mg/kg) treated;D + Donep: diabetic donepezil (3 mg/kg) treated; D + Met: diabetic metformin(500 mg/kg) treated.

36 P. Bhutada et al. / Behavioural Brai

Fig. 7. Effect of berberine treatment during training trials (5 days) on time spentin each quadrant during probe trial in Morris water maze. Each bar representsmean ± S.E.M. of 6 observations. (A) Probe trial performance as measured by com-paring time spent in the target quadrant with an average of time spent in all threenon-target quadrants, *P < 0.001 vs. avg. time spent in non-target quadrant in respec-tive group (two-way ANOVA followed by Bonferroni’s post hoc test); (B) probe trialperformance as measured by comparing the time each rat spent in the target quad-rant with the time it spent in its next most preferred quadrant, *P < 0.001, &P < 0.05vs. time spent in next preferred quadrant in respective group (Two-way ANOVA fol-lowed by Bonferroni’s post hoc test); (C) difference score index of selective search.This measure was obtained by subtracting the time each rat spent in its next mostpreferred quadrant from the time it spent in the target quadrant. Selective search isindicated by the magnitude of the difference score. A score of 0 indicates no selectivesearch. *P < 0.001 vs. non-diabetic control group; &P < 0.05, #P < 0.01, @P < 0.001 vs.diabetic control group (One-way ANOVA followed by Tukey’s post hoc test).ND control: non-diabetic control; ND + Ber 100: non-diabetic berberine (100 mg/kg)treated; D control: diabetic control; D + Ber 25: diabetic berberine (25 mg/kg)treated; D + Ber 50: diabetic berberine (50 mg/kg) treated; D + Ber 100: diabeticberberine (100 mg/kg) treated; D + Vit C: diabetic vitamin C (100 mg/kg) treated;D + Donep: diabetic donepezil (3 mg/kg) treated; D + Met: diabetic metformin(500 mg/kg) treated.

n Research 220 (2011) 30–41

3.2.3. Effects of berberine on locomotor activity in open field testAn animal model of DM has a great deal of potential to alter

motor activity and anxiety-like behaviors that would alter escapelatency. Thus, the effects of berberine on cognitive dysfunctionin diabetic rats may be related to motor activity and anxiety. Torule out this possibility open field study was carried out. One-wayANOVA revealed that none of the treatment had significant influ-ence on the locomotor activity [F(7, 39) = 0.4588, P = 0.8581] andnumber of rearing [F(6, 39) = 0.0858, P = 0.9988] (Fig. 9).

3.3. Effect of berberine treatment on diabetes-induced changes incholinesterase activity

Cholinesterase (ChE) activity was expressed as AcSCh formed.The changes in ChE activity in cerebral cortex and hippocam-pus after chronic administration of berberine are presented inFig. 10. As can be observed, ChE activity was significantly increasedin the cortex [F(7, 39) = 5.729, P = 0.0001] and hippocampus [F(7,39) = 6.595, P < 0.0001] of diabetic control group compared to thenon-diabetic control group. Chronic treatment with berberine (25,50, and 100 mg/kg) significantly decreased the ChE activity in cor-tex compared to diabetic control rats (P < 0.05, 0.01, and 0.001).Similarly, chronic treatment with berberine (50 and 100 mg/kg) sig-nificantly decreased the ChE activity in hippocampus compared todiabetic control rats (P < 0.01 and 0.001). These effects were com-parable to that of vitamin C and metformin. Chronic berberinetreatment in non-diabetic rats did not influence the ChE activityas compared to non-diabetic control rats (P > 0.05) (Fig. 10).

In another set of experiment, one-way ANOVA indicated thattreatment with berberine during training trials significantly influ-enced the cortical [F(8, 45) = 7.176, P < 0.0001] and hippocampal[F(8, 45) = 7.058, P < 0.0001] ChE activity (Fig. 11). Treatment withberberine (100 mg/kg) significantly decreased the ChE activity incortex and hippocampus as compared to diabetic control rats(P < 0.01). Similarly, treatment with donepezil (3 mg/kg) signifi-cantly decreased the ChE activity in cortex and hippocampus ascompared to diabetic control rats (P < 0.01), whereas, vitamin Cdid not influence the same (P > 0.05). Further, administration ofmetformin in this schedule reduced the ChE activity in cortexas well as hippocampus but, failed to attain statistical signifi-cance levels. Berberine per se did not influence the ChE activity(Fig. 11).

3.4. Effect of berberine on parameters of oxidative stress in brain

3.4.1. Effect of berberine on diabetes-induced changes in lipidperoxidation

Effects of chronic administration of berberine on lipid peroxi-dation (LPO) are depicted in Table 3. There was a significant risein MDA levels in hippocampal [F(7, 39) = 30.41, P < 0.0001] andcortical [F(7, 39) = 12.26, P < 0.0001] tissue of rat brain in diabeticrats as compared to non-diabetic control rats. Berberine (50 and100 mg/kg), metformin (500 mg/kg), and vitamin C (100 mg/kg) sig-nificantly reduced MDA levels as compared to diabetic rats in cortexand hippocampus (P < 0.05). Berberine per se did not influence theMDA levels (Table 3).

In addition, effects of berberine treatment during training trialson lipid peroxidation are depicted in Table 4. Similarly, there was asignificant rise in MDA levels in hippocampal and cortical tissue ofrat brain in diabetic rats as compared to non-diabetic control rats.One-way ANOVA indicated that diabetes and berberine treatment

significantly influenced the MDA levels [cortex: F(8, 45) = 9.597,P < 0.0001; hippocampus: F(8, 45) = 10.92, P < 0.0001]. Further, posthoc test revealed that berberine (100 mg/kg), donepezil, and met-formin significantly reduced MDA levels as compared to diabeticrats (P < 0.05). However, treatment with vitamin C decreased the

P. Bhutada et al. / Behavioural Brain Research 220 (2011) 30–41 37

Fig. 8. Effects of berberine treatment during training trials (5 days) on number of crossings during probe trial in Morris water maze. Each bar represents mean ± S.E.M. of 6observations. @P < 0.001 vs. saline treatment in non-diabetic rats, *P < 0.05 and **P < 0.001 vs. saline treatment in diabetic group (One-way ANOVA followed by Tukey’s posthoc test). ND control: non-diabetic control; ND + Ber 100: non-diabetic berberine (100 mg/kg) treated; D control: diabetic control; D + Ber 25: diabetic berberine (25 mg/kg)treated; D + Ber 50: diabetic berberine (50 mg/kg) treated; D + Ber 100: diabetic berberine (100 mg/kg) treated; D + Vit C: diabetic vitamin C (100 mg/kg) treated; D + Donep:diabetic donepezil (3 mg/kg) treated; D + Met: diabetic metformin (500 mg/kg) treated.

Table 3Effect of chronic treatment with berberine (30 days) on lipid peroxidation (LPO) and glutathione levels.

Group Treatment LPO (nmol/mg protein) Glutathione (�mol/mg protein)

Cortex Hippocampus Cortex Hippocampus

ND control Vehicle (1 ml/kg) 2.28 ± 0.16 1.86 ± 0.09 3.08 ± 0.13 3.25 ± 0.13ND + Ber 100 Ber (100 mg/kg) 2.00 ± 0.12 1.69 ± 0.10 3.27 ± 0.12 3.27 ± 0.10D control Vehicle (1 ml/kg) 4.26 ± 0.30* 4.17 ± 0.17* 1.86 ± 0.08* 1.79 ± 0.09*

D + Ber 25 Ber (25 mg/kg) 3.76 ± 0.30 3.75 ± 0.22 2.01 ± 0.08 2.02 ± 0.08D + Ber 50 Ber (50 mg/kg) 2.95 ± 0.26# 3.26 ± 0.23$ 2.46 ± 0.10# 2.41 ± 0.10$

D + Ber 100 Ber (100 mg/kg) 2.56 ± 0.18@ 2.10 ± 0.10@ 3.00 ± 0.15@ 3.12 ± 0.12@

D + Vit C Vit C (100 mg/kg) 3.27 ± 0.20 3.31 ± 0.23$ 2.46 ± 0.11$ 2.45 ± 0.20#

D + Met Met (500 mg/kg) 2.62 ± 0.17@ 2.23 ± 0.16@ 3.10 ± 0.08# 3.18 ± 0.10@

* P < 0.001 vs. non-diabetic control group.$ P < 0.05.

oc tesc ic cont( iabeti

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TE

c(D

# P < 0.01.@ P < 0.001 vs. diabetic control group (One-way ANOVA followed by Tukey’s post hontrol; ND + Ber 100: non-diabetic berberine (100 mg/kg) treated; D control: diabet50 mg/kg) treated; D + Ber 100: diabetic berberine (100 mg/kg) treated; D + Vit C: d

PO but failed to attain statistical significance. Berberine per se didot influence the MDA levels.

.4.2. Effect of berberine on diabetes-induced changes in

lutathione levels

Effects of chronic administration of berberine on GSH levelsre depicted in Table 3. There was a significant fall in GSH lev-ls in hippocampal [F(7, 39) = 26.26, P < 0.0001] and cortical [F(7,9) = 23.39, P < 0.0001] tissue of rat brain in diabetic rats as com-

able 4ffect of berberine treatment during training trail (5 days) on lipid peroxidation (LPO) an

Group Treatment LPO (nmol/mg protein

Cortex

ND control Vehicle (1 ml/kg) 2.29 ± 0.16ND + Ber 100 Ber (100 mg/kg) 2.25 ± 0.10D control Vehicle (1 ml/kg) 4.26 ± 0.30*

D + Ber 25 Ber (25 mg/kg) 4.16 ± 0.16D + Ber 50 Ber (50 mg/kg) 3.61 ± 0.18D + Ber 100 Ber (100 mg/kg) 2.94 ± 0.20$

D + Vit C Vit C (100 mg/kg) 3.49 ± 0.35D + Donep Donep (3 mg/kg) 2.93 ± 0.27$

D + Met Met (500 mg/kg) 3.18 ± 0.25#

* P < 0.001 vs. non-diabetic control group.# P < 0.05.$ P < 0.01 vs. diabetic control group (One-way ANOVA followed by Tukey’s post hoc tes

ontrol; ND + Ber 100: non-diabetic berberine (100 mg/kg) treated; D control: diabetic cont50 mg/kg) treated; D + Ber 100: diabetic berberine (100 mg/kg) treated; D + Vit C: diabet+ Met: diabetic metformin (500 mg/kg) treated.

t). Each value represents mean ± SEM of 5-6 observations. ND control: non-diabeticrol; D + Ber 25: diabetic berberine (25 mg/kg) treated; D + Ber 50: diabetic berberinec vitamin C (100 mg/kg) treated; D + Met: diabetic metformin (500 mg/kg) treated.

pared to non-diabetic control rats. Berberine (50 and 100 mg/kg),vitamin C (100 mg/kg), and metformin (500 mg/kg) treatment sig-nificantly increased GSH levels as compared to control diabetic rats(P < 0.05). Berberine per se did not influence the GSH levels (Table 3).

In addition, effects of berberine treatment during training trialson GSH are depicted in Table 4. Similarly, there was a significantfall in GSH levels in hippocampal and cortical tissue of rat brainin diabetic rats as compared to non-diabetic control rats. One-way ANOVA indicated that diabetes and treatment with berberine

d glutathione levels.

) Glutathione (�mol/mg protein)

Hippocampus Cortex Hippocampus

1.82 ± 0.14 3.21 ± 0.12 3.10 ± 0.131.77 ± 0.11 3.34 ± 0.12 3.05 ± 0.153.79 ± 0.24* 1.72 ± 0.09* 1.74 ± 0.09*

3.67 ± 0.11 1.76 ± 0.08 1.85 ± 0.083.41 ± 0.32 1.84 ± 0.10 2.02 ± 0.062.69 ± 0.22# 2.55 ± 0.22$ 2.41 ± 0.14#

3.31 ± 0.26 2.09 ± 0.12 2.00 ± 0.082.40 ± 0.29$ 2.42 ± 0.22# 2.62 ± 0.24$

2.54 ± 0.27# 2.41 ± 0.14# 2.38 ± 0.17$

t). Each value represents mean ± SEM of 6 observations. ND control: non-diabeticrol; D + Ber 25: diabetic berberine (25 mg/kg) treated; D + Ber 50: diabetic berberineic vitamin C (100 mg/kg) treated; D + Donep: diabetic donepezil (3 mg/kg) treated;

38 P. Bhutada et al. / Behavioural Brai

Fig. 9. Effects of berberine on locomotor activity and number of rearing in openfield test. Each bar represents mean ± S.E.M. of 5–6 observations (One-way ANOVAfollowed by Tukey’s post hoc test). ND control: non-diabetic control; ND + Ber 100:ndtm

sPhf(ld

4

ldmisicbsc

mlipwr

on-diabetic berberine (100 mg/kg) treated; D control: diabetic control; D + Ber 25:iabetic berberine (25 mg/kg) treated; D + Ber 50: diabetic berberine (50 mg/kg)reated; D + Ber 100: diabetic berberine (100 mg/kg) treated; D + Vit C: diabetic vita-

in C (100 mg/kg) treated; D + Met: diabetic metformin (500 mg/kg) treated.

ignificantly influenced the GSH levels [cortex: F(8, 45) = 17.32,< 0.0001; hippocampus: F(8, 45) = 13.37, P < 0.0001]. Further, postoc test revealed that berberine (100 mg/kg), donepezil, and met-

ormin significantly increased GSH levels as compared diabetic ratsP < 0.05). However, treatment with vitamin C increased the GSHevels but failed to attain statistical significance. Berberine per seid not influence the GSH levels.

. Discussion

This study analyzed the role of berberine, an isoquino-ine alkaloid on the behavioral and biochemical function ofiabetic rats. STZ-induced diabetes produced marked impair-ent in cognitive function which was associated with marked

ncrease in cholinesterase activity and increased oxidativetress in the brain. Chronic treatment with berberine signif-cantly and dose dependently ameliorated cognitive deficits,holinergic dysfunction and oxidative stress markers in dia-etic rats. Further, short-term treatment with berberine alsoignificantly reversed STZ-induced behavioral and biochemicalhanges.

Streptozotocin-induced diabetes in rats is a well-documentedodel of experimental diabetes. STZ produced significant weight

oss and treatment with berberine restored the same, which isn accordance with earlier reports [25,33,39]. Morris-water mazeerformance in diabetic rats was severely impaired as comparedith non-diabetic rats, confirming earlier findings [9,63]. The cur-

ent study explored those findings demonstrating that diabetes

n Research 220 (2011) 30–41

reduced learning and memory performance. Furthermore, thepresent findings indicate that the impaired performance of diabeticrats is related to cognitive impairment rather than to sensorimo-tor deficits [9], since performance of diabetic rats were similar tonon-diabetic rats in the task with the visible platform. In addition,in the first trial the mean escape latencies in all the groups weresimilar, implying that their motor performance (ability to swim)was unaffected by the persistent hyperglycemia and/or berberinetreatment.

The beneficial effects of berberine observed in the present studycould be multivariate. It is reported that most of the diabetescomplications including cognitive impairments are caused dueto protracted hyperglycemia [10,63], and antihyperglycemics andinsulin sensitizing agents are reported to reduce memory deficit indiabetic condition [55]. An exhaustive review by Messier [45] sug-gested that better glycemic control improves cognition and thatthere is a cognitive benefit to restricting hyperglycemic episodesin diabetes. In present study, berberine treatment significantlyreduced blood glucose levels which is well in accordance of earlierstudies [24,36,37,61,74,75]. Therefore, the restoration of cognitivefunction observed in diabetic rats in current study may be due to theability of berberine to reduce hyperglycemia. This is also supportedby observation in present study that metformin, a known anti-hyperglycemics improved performance of diabetic rats in MWMtest.

An increase in reactive species production due to oxidativestress seems to play a key role in neuronal damage [4,12,20].Elevated intracellular glucose oxidation is responsible for glucotox-icity in the neurons [47]. Oxidative damage to the rat synapse in thecerebral cortex and hippocampus has been previously reported tocontribute to the deficit of cognitive functions [8,21,23]. In addition,it is reported that antioxidants prevent the diabetes-induced cog-nitive dysfunction [25,63]. In the present study, lipid peroxidationlevels were significantly increased, whereas reduced glutathioneactivity was markedly decreased in the cerebral cortex and hip-pocampus of diabetic rats, which is well in accordance to earlierreports [63]. Treatment with berberine returned the levels of lipidperoxides and reduced glutathione towards their control valuesin diabetic rats. Treatment with vitamin C also showed berberinelike effects on these parameters. In one of the studies, berberinerestored the malondialdehyde, superoxide anion and superoxidedismutase [39]. It is also showed that berberine inhibits reactiveoxygen species (ROS) generation [76]. Therefore, berberine mightprotect diabetes-associated memory decline by reducing oxidativestress in rats.

Cholinergic neurotransmission is a crucial process underlyingmemory and cognitive function. Cholinergic basal forebrain neu-rons in the nucleus basalis magnocellularis innervate the cerebralcortex, amygdaloid complex, or hippocampus and are essential forlearning and memory formation [2,22]. One of the most impor-tant mechanisms responsible for correct cholinergic function isperformed by enzyme choline esterase (ChE) [3]. Several studieshave found an increased ChE activity in brain, and consequentlybe associated to cognitive impairments in diabetics [33,56,57],which is further supported by observation in the present study thatdonepezil-an AChE inhibitor improved the diabetes-induced cog-nitive dysfunction. In the present study, treatment with berberinerestored the increased levels of ChE in diabetic rats. Interestingly,berberine is reported to non-competitively inhibit ChE activ-ity [27,30,69]. So berberine might ameliorate the pathologies ofdiabetes-induced cognitive dysfunction through inhibiting the ChE

activity.

Recent gathering evidences suggest that glucagon-like peptide-1 (GLP-1) plays important role in diabetes and Alzheimer disease[38]. It was demonstrated that GLP-1 receptor is involved in learn-ing and neuroprotection [17]. More recently, Abbas et al. [1]

P. Bhutada et al. / Behavioural Brain Research 220 (2011) 30–41 39

Fig. 10. Effects of chronic treatment with berberine (30 days) on ChE activity in cerebral cortex and hippocampus. Choline esterase (ChE) activity was expressed as AcSChformed. Each bar represents mean ± S.E.M. of 5–6 observations. **P < 0.01 and ***P < 0.001 vs. saline treatment in non-diabetic rats, #P < 0.05, $P < 0.01, and &P < 0.001 vs. salinetreatment in diabetic group in respective brain area (One-way ANOVA followed by Tukey’s post hoc test). ND control: non-diabetic control; ND + Ber 100: non-diabeticberberine (100 mg/kg) treated; D control: diabetic control; D + Ber 25: diabetic berberine (25 mg/kg) treated; D + Ber 50: diabetic berberine (50 mg/kg) treated; D + Ber 100:diabetic berberine (100 mg/kg) treated; D + Vit C: diabetic vitamin C (100 mg/kg) treated; D + Met: diabetic metformin (500 mg/kg) treated.

Fig. 11. Effects of berberine treatment during training trials (5 days) on ChE activity in cortex and hippocampus. Choline esterase (ChE) activity was expressed as AcSChformed. Each bar represents mean ± S.E.M. of 5–6 observations. **P < 0.01 and ***P < 0.001 vs. saline treatment in non-diabetic rats, #P < 0.05 and &P < 0.001 vs. saline treatmenti post h( mg/kb + Dont

rstiicttitrb

gmiietn

A

h

n diabetic group in respective brain area (One-way ANOVA followed by Tukey’s100 mg/kg) treated; D control: diabetic control; D + Ber 25: diabetic berberine (25erberine (100 mg/kg) treated; D + Vit C: diabetic vitamin C (100 mg/kg) treated; Dreated.

eported that GLP-1 knockout mice exhibited impairment in acqui-ition and recognition during MWM task and clearly demonstratedhat GLP-1 receptor function in the brain affects synaptic plastic-ty and cognitive processes. GLP-1 enhances glucose-dependentnsulin secretion and lowers blood glucose in type 2 diabetes andurrently, exenatide-GLP-1 receptor agonist is approved for thereatment in type 2 diabetics. Interestingly, Yu et al. [73] reportedhat berberine enhances GLP-1 release and biosynthesis. So berber-ne ameliorated pathologies of diabetes-induced cognitive dysfunc-ion may involve GLP-1 receptor modulation. Further studies areequired to confirm the role of GLP-1 modulation in the effects oferberine.

In conclusion, the findings of the present investigation sug-est that berberine exerts its beneficial effects on STZ-inducedemory dysfunction may be attributed to its antidiabetic, antiox-

dant, and ChE inhibition activity, which could find clinical usen treating cognitive and neural dysfunction in diabetics. But tolucidate the exact mechanism of this modulatory effect, ando examine its potential therapeutic effects further studies areecessary.

cknowledgements

This work was supported by the Mr. Shankarprasad Agni-otri, President and Mr. Sachin Agnihotri, Chairman, Jai Mahakali

[

oc test). ND control: non-diabetic control; ND + Ber 100: non-diabetic berberineg) treated; D + Ber 50: diabetic berberine (50 mg/kg) treated; D + Ber 100: diabeticep: diabetic donepezil (3 mg/kg) treated; D + Met: diabetic metformin (500 mg/kg)

Shikshan Sanstha, Wardha. We are grateful to Dr. Reddy’s Lab,Hyderabad, India for Metformin hydrochloride and Sami Labs, Ban-galore, India for Berberine hydrochloride, as generous gift samples.

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