Food and Chemical Toxicology 50 (2012) 1036–1044

Contents lists available at SciVerse ScienceDirect

Food and Chemical Toxicology

journal homepage: www.elsevier .com/locate/ foodchemtox

Protective effects of total alkaloidal extract from Murraya koenigii leaveson experimentally induced dementia

Vasudevan Mani a,⇑, Kalavathy Ramasamy b, Aliya Ahmad a, Milind Parle c, Syed Adnan Ali Shah d,Abu Bakar Abdul Majeed a

a Brain Research Laboratory, Faculty of Pharmacy, Puncak Alam Campus, Universiti Teknologi MARA (UiTM), 42300 Bandar Puncak Alam, Selangor, Malaysiab Collaborative Drug Discovery Research Group, Faculty of Pharmacy, Puncak Alam Campus, Universiti Teknologi MARA (UiTM), 42300 Bandar Puncak Alam, Selangor, Malaysiac Pharmacology Division, Department of Pharmaceutical Sciences, Guru Jambheshwar University of Science and Technology, Hisar, Indiad Research Institute of Natural Products for Drug Discovery, Faculty of Pharmacy, Puncak Alam Campus, Universiti Teknologi MARA (UiTM), 42300 Bandar Puncak Alam,Selangor, Malaysia

a r t i c l e i n f o

Article history:Received 26 July 2011Accepted 22 November 2011Available online 28 November 2011

Keywords:Murraya koenigiiAmnesiaMemoryAlzheimer’s diseaseAcetylcholinesteraseBACE1

0278-6915/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.fct.2011.11.037

⇑ Corresponding author. Tel.: +60 332584611; fax:E-mail address: vasudevan@puncakalam.uitm.edu

a b s t r a c t

Dementia is a syndrome of gradual onset and continuous decline of higher cognitive functioning. It is acommon disorder in older persons and has become more prevalent today. The fresh leaves of Murrayakoenigii are often added to various dishes in Asian countries due to the delicious taste and flavor that theyimpart. These leaves have also been proven to have health benefits. In the present study, the effect of totalalkaloidal extract from M. koenigii leaves (MKA) on cognitive functions and brain cholinesterase activityin mice were determined. In vitro b-secretase 1 (BACE1) inhibitory activity was also evaluated. The totalalkaloidal extract was administered orally in three doses (10, 20 and 30 mg/kg) for 15 days to differentgroups of young and aged mice. Elevated plus maze and passive avoidance apparatus served as theexteroceptive behavioral models for testing memory. Diazepam-, scopolamine-, and ageing-inducedamnesia served as the interoceptive behavioral models. MKA (20 and 30 mg/kg, p.o.) showed significantimprovement in memory scores of young and aged mice. Furthermore, the same doses of MKA reversedthe amnesia induced by scopolamine (0.4 mg/kg, i.p.) and diazepam (1 mg/kg, i.p.). Interestingly, thebrain cholinesterase activity was also reduced significantly by total alkaloidal extract of M. koenigii leaves.The IC50 value of MKA against BACE1 was 1.7 lg/mL. In conclusion, this study indicates MKA to be a use-ful remedy in the management of Alzheimer’s disease and dementia.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Dementia is characterized by a progressive decline in cognitivefunction depending on neurodegeneration, which particularly af-fects elder population in their daily activities such as memory,speaking, and problem dissolving. The most well-known type ofdementia is Alzheimer’s disease (AD), which is a neurodegenerativedisease related to cognitive and behavioral impairments. Althoughthe primary cause of AD remains unclear, it may be considered thatthe b-amyloid (Ab) and tau protein aggregation, reduced acetylcho-line (ACh), and glutamatergic deficit are regarded as principal path-ogenesis of AD (Kashani et al., 2008). Recent studies have speculatedthat free radicals produced during oxidative stress and/or inflamma-tory processes are also pathologically important in AD (Ferreiraet al., 2006).

The histopathological AD characteristics are shown throughthe accumulation of Ab peptide in plaques and the deposition of

ll rights reserved.

+60 332584602..my (V. Mani).

hyperphosphorylated tau in neurofibrillary tangles. Ab is generatedfrom b-amyloid precursor protein (b-APP) through sequential cleav-age by membrane proteases of b- andc-secretases. The b-secretase 1(BACE 1) first cleaves APP to generate a C-terminus fragment(b-CTF), an immediate substrate for c-secretase, which furthercleaves b-CTF to yield Ab (Willem et al., 2009). Since mutations inb-APP causes the accumulation of Ab1–42 that leads to familial ADforms (Hardy and Selkoe, 2002), Ab accumulation has been sug-gested to play a key role in the etiology of AD.

Murraya koenigii (Linn.) Spreng (Family: Rutaceae), commonlyknown as ‘curry patta’ in Hindi is popular as a spice and condimentamong the Asians. The fresh leaves and its dried powder aretraditionally added to gravy and other vegetables for the distinc-tive flavor and aroma. It is also widely used as a folk medicinefor the treatment of stomachaches, influenza, rheumatism, trau-matic injury, dysentery, and used as an astringent (Joseph andPeter, 1985). The leaves are reported to have hypoglycemic andanti- dia-betic (Krishna and Usha, 2009), hepatoprotective(Sathaye et al., 2010), antibacterial (Ningappa et al., 2010),antioxidant (Tachibana et al., 2001, 2003), wound healing (Gupta

V. Mani et al. / Food and Chemical Toxicology 50 (2012) 1036–1044 1037

et al., 2009), chemomodulatory (Dasgupta et al., 2003), immuno-modulatory (Shah et al., 2008), antidiarrhoeal (Mandal et al.,2010) and anti-inflammatory (Manoj et al., 2009) activities. Severalcarbazole alkaloids namely murrayanine, mahanimbine, girinim-bine, murrayacine, isomurrayazoline, isomahanimbine, koenimbi-dine mahanine, koenine, koenigine, koenidine, koenimbine, and8,80-biskoenigine have been isolated from M. koenigii leaves (Iyerand Devi, 2008). Our previous study demonstrated the antiamnesicpotential of powdered M. koenigii leaves with normal diet fed con-tinuously for 30 days to mice (Vasudevan and Parle, 2009). More-over, a carbazole alkaloid mahanimbine from M. koenigii leaveswas found to inhibit acetylcholinesterase (AChE) activity in vitro(Kumar et al., 2010). In continuation of our research work, thepresent study was extended with evaluation of total alkaloidal ex-tract from M. koenigii leaves on reversal of memory impairmentand acetylcholinesterase activity in experimental animals. In vitroBACE1 inhibitory activity was also studied.

2. Materials and methods

2.1. Plant material and extraction

The fresh leaves of M. koenigii were collected from the local market at PuncakAlam (Malaysia). The plant material was identified, authenticated and depositedin the herbarium (PID 24101011) at the Biodiversity and Environment Division, For-est Research Institute, Malaysia. The collected leaves were dried under shade andcrushed to moderately coarse powder. The dried leaves (2 kg) were extracted with95% methanol (MeOH) using soxhlet apparatus. The MeOH extract was then con-centrated under reduced pressure using rotavapour and acidified with 0.5 MH2SO4. The acidic extract was washed with chloroform to remove neutral compo-nents. The aqueous acidic fraction was then made basic with ammonia (pH 10)and extracted again with chloroform until the aqueous layer was free of alkaloids.The combined chloroform extracts were evaporated in rotary evaporator to yield to-tal alkaloidal extract of M. koenigii leaves as a dark brown residue (0.124% w/w ofthe dry starting material) (Rujjanawate et al., 2003). The presence of alkaloids con-formed with preliminary phytochemical evaluation and NMR analysis.

2.2. Vehicle

Diazepam, scopolamine hydrobromide, piracetam and metrifonate were dilutedseparately in normal saline and injected by intraperitoneal. MKA was suspended in0.5% (w/v) carboxymethylcellulose sodium (CMC) and administered orally toanimals.

2.3. Animals

All the experiments were carried out using male, Swiss Albino mice procuredfrom the animal house at Institute of Medical Research, Kuala Lumpur, Malaysia.Young (3–4 months) and aged (12–15 months) mice weighing about 24 and 35 g,respectively, were used in the present study. The animals had free access to stan-dard laboratory food and water ad libitum, and they were housed in a natural(12 h each) light–dark cycle. The animals were acclimatized to the laboratory con-ditions for at least 5 days before behavioral experiments. Experiments were carriedout between 0900 and 1800 h. The committee on animal research, Universiti Tekno-logi MARA, Malaysia, approved the experimental protocol (600-FF(PT.5/2)) and thecare of laboratory animals was taken as per the guidelines of the Guide for the Careand Use of Laboratory Animals (National Institute of Health Publication).

2.4. Acute toxicity studies

Acute toxicity studies were performed according to the organization for eco-nomic co-operation and development (OECD) guidelines (Ecobichon, 1997). MaleSwiss mice selected by random sampling technique were employed in this study.The animals were fasted for 4 h with free access to water only. MKA was adminis-tered orally at a dose of 5 mg/kg initially and mortality if any was observed for3 days. If mortality was observed in two out of three animals, then the dose admin-istered was considered as toxic dose. However, if the mortality was observed in onlyone out of three animals then the same dose was repeated again to confirm thetoxic effect. If no mortality was observed, then only higher (50, 300 and 2000 mg/kg) doses of MKA were employed for further toxicity studies.

2.5. Drug treatment

In the present investigation, the mice were divided into different groups andvarious interoceptive and exteroceptive memory models were employed. Eachgroup comprised of a minimum of six animals. MKA (10, 20 and 30 mg/kg) was

administered orally for 15 successive days to young and aged mice. On the 15thday, 90 min after the last dose of MKA or piracetam or vehicle the mice were ex-posed to the training session using elevated plus maze and passive avoidance appa-ratus. Retention (memory) was recorded after 24 h (on 16th day). Amnesia wasinduced in separate groups (interoceptive models) of young mice by scopolamine(0.4 mg/kg, i.p.) or diazepam (1 mg/kg, i.p.) on the 15th day 90 min after the lastdose of drug administration. Then the animals were exposed to training session45 min after the scopolamine or diazepam injection. The retention (memory) wasmeasured after 24 h (on 16th day). Piracetam (400 mg/kg, i.p.) was used as anestablished nootropic agent and was injected for 7 days to positive control groups.MKA (10, 20 and 30 mg/kg) was administrated orally for 15 days to separate groupsof young and aged mice for biochemical study. Metrifonate (50 mg/kg, i.p., 60 minbefore dissecting brain) served as the positive control for comparison of brain cho-linesterase activities. All control group animals received the vehicle (0.5% w/v CMC)for 15 consecutive days.

2.6. Elevated plus-maze

The elevated plus-maze was used to evaluate spatial long-term memory, fol-lowing the procedure described previously (Itoh et al., 1990). Briefly, the apparatusconsisted of two open arms (16 cm � 5 cm) and two enclosed arms (16 cm �5 cm � 12 cm). The arms extended from a central platform (5 cm � 5 cm), and themaze was elevated to a height of 25 cm from the floor. On the first day (i.e. 15thday of drug treatment), each mouse was placed at the end of an open arm, facingaway from the central platform. Transfer latency (TL), the time taken by the mouseto move into one of the enclosed arms, was recorded on the first day (training ses-sion) for each animal. If the animal did not enter an enclosed arm within 90 s, it wasgently pushed into one enclosed arm and the TL was assigned as 90 s. The mousewas allowed to explore the maze for another 2 min, and then returned to its homecage. Retention of this learned-task (memory) was examined 24 h after the first daytrial (i.e. 16th day, 24 h after last dose). Significant reduction in TL value of retentionindicated improvement in memory.

2.7. Passive avoidance paradigm

The step-down type of passive avoidance task was used to examine the long-term memory based on negative reinforcement (Maurice et al., 1997). The appara-tus consisted of a box (27 cm � 27 cm � 27 cm) having three walls of wood and onewall of plexiglass, featuring a grid floor (made up of 3 mm stainless steel rods set8 mm apart), with a wooden platform (10 cm � 7 cm � 1.7 cm) in the center ofthe grid floor. Electric shock (20 V, A.C.) was delivered to the grid floor. Training(i.e. 15th day of drug treatment) was carried out in two similar sessions. Eachmouse was gently placed on the wooden platform set in the center of the grid floor.When the mouse stepped-down placing all its paws on the grid floor, shocks weredelivered for 15 s and the step-down latency (SDL) was recorded. SDL was definedas the time (in seconds) taken by the mouse to step down from the wooden plat-form to grid floor with all its paws on the grid floor. Animals showing SDL in therange of 2–15 s during the first test were used for the second session and the reten-tion test. The second-session was carried out 90 min after the first test. During sec-ond session, if the animals stepped down before 60 s, electric shocks were deliveredonce again for 15 s. During the second test, animals were removed from shock freezone, if they did not step down for a period of 60 s and were subjected to retentiontest. Retention (memory) was tested after 24 h (i.e. 16th day, 24 h after last dose) ina similar manner, except that the electric shocks were not applied to the grid floorobserving an upper cut-off time of 300 s.

2.8. Collection of brain samples

The animals were sacrificed by cervical decapitation under light anesthesia onthe 15th day, 60 min after administration of the last dose of MKA. Immediately afterdecapitation, the whole brain was carefully removed from the skull. For preparationof brain homogenate, the fresh whole brain was weighed and transferred to a glasshomogenizer and homogenized in an ice bath after adding 10 volumes of normalsaline solution. The homogenate was centrifuged at 3000 rpm for 10 min and theresultant cloudy supernatant liquid was used for estimation of brain acetylcholin-esterase activity.

2.9. Estimation of brain cholinesterase

The assay of acetylcholinesterase was based on an improved Ellman method in a96 well plate reader using QuantiChrome assay kit (USA) (Kovarik et al., 2003). Theprocedure is based on production of thiocholine by the action of acetylcholinesterasewhich forms yellow color with 5,5-dithiobis-2-nitrobenzoic acid (DTNB). The inten-sity of the product color, measured at 412 nm, is proportionate to the enzyme activ-ity in the sample.

1038 V. Mani et al. / Food and Chemical Toxicology 50 (2012) 1036–1044

2.10. BACE1 inhibitory activity

MKA at concentration ranging from 0.1 to 100 mg/mL were assayed for BACE1inhibition using a fluorescence resonance energy transfer (FRET) assay (Pan VeraCo.), that uses baculovirus-expressed BACE1 and a specific substrate (Rh-EVNLDAEFK-quencher) based on the Swedish mutation of the amyloid precursorprotein (APP). This peptidic substrate becomes highly fluorescent upon enzymaticcleavage. A mixture of 10 ll of BACE1 substrate (Rh-EVNLDAEFK-quencher, in50 nM ammonium bicarbonate), 10 ll of test compound, and 10 ll of BACE1 (b-secretase) enzyme [(50 mM Tris (pH7.5), 10% glycerol) (1.0 U/mL)] were incubatedfor 60 min at room temperature and protected from light. Then 10 ll of BACE1 stopbuffer (2.5 M sodium acetate) was added to the mixture. Fluorescence was readusing a multiwell spectrofluorometer (InfiniteM200, TECAN) under excitation at545 nm and the emitted light at 585 nm. Reaction was measured at a fixed concen-tration of substrate (250 nM) with the above mentioned MKA concentrations andthe IC50 value was determined. IC50 was defined as the concentration of BACE1inhibitor that is required to inhibit 50% of BACE1 activity.

To investigate the inhibition pattern of MKA on BACE1, the inhibition constantKi value was measured. The BACE1 inhibitory activity was measured at differentconcentrations of the substrate (62.5, 125 and 250 nM). A graph of 1/Vo (RFU/min) against the inhibitor concentration was plotted to determine the Ki value.

2.11. Statistical analysis

All the results were expressed as Mean ± Standard Error (SEM). Data was ana-lyzed using one-way ANOVA followed by Tukey–Kramer multiple comparisons test.p-Values <0.05 were considered as statistically significant.

3. Results

3.1. Phytochemical analysis

The MKA extract gave positive test with Dragendroffs, Hagers,Mayers, and Wagners reagents. This indicated the presence of

Fig. 1. 1H NMR spectrum (500 M

alkaloids in MKA extract. Furthermore, the 1H NMR spectrum(500 MHz, CDCl3) of MKA extract also supported the presence ofcarbazole alkaloids (Fig.1) in MKA extract. The 1H NMR spectrumof MKA extract, when compared with the previous literature,showed characteristic signals of carbazole alkaloids (Fig.2) atdownfield region, for instance, the aromatic protons resonated atd 7.91 as doublet for H-5, d 7.80 as broad singlet for N–H, d 7.65as singlet for H-4, d 7.38 as doublet for H-8, d 6.67 as doublet forH-9 and d 5.67 as doublet of doublet for H-10 (Abu Bakar et al.,2007; Rahman and Gray, 2005).

3.2. Acute toxicity studies

MKA extract up to 300 mg/kg (through oral route) did not pro-duce any mortality. Therefore, three doses 10, 20 and 30 mg/kgwere fixed for further pharmacological evaluation.

3.3. Effect on transfer latency (TL)

A significant reduction in TL value of retention (on 16th day)indicated improvement in memory. The young and aged micetreated orally with 20 and 30 mg/kg showed remarkable reduction(p < 0.05) in TL value of retention as compared to the control group(Fig.3). Oral administration with MKA at the lowest dose (10 mg/kg) however, did not show any significant changes in TL on 16thday. Piracetam (used as a positive control) at the dose 400 mg/kg, i.p. improved memory (p < 0.001) of both young and aged miceand reversed the amnesia induced by scopolamine and diazepam.Scopolamine (0.4 mg/kg, i.p.) and diazepam (1 mg/kg, i.p.) injectedbefore training significantly increased (p < 0.001) TL on 16th day

Hz, CDCl3) of MKA extract.

N OR3

R2

H

R1 1

2

3

45

6

7

8

9

10

11

Fig. 2. General structure of carbazol alkaloids.

V. Mani et al. / Food and Chemical Toxicology 50 (2012) 1036–1044 1039

indicating impairment in memory but MKA (20 and 30 mg/kg, p.o)successfully reversed the memory deficits induced (Fig.4). Thehigher dose (30 mg/kg, p.o.) of MKA reversed the memory deficitabsolutely in aged, scopolamine and diazepam-induced experi-mental models.

3.4. Effect on step-down latency (SDL)

Significant increase in SDL value indicated improvement inmemory. Ageing process remarkably (p < 0.001) reduced SDL of agedmice. MKA (20 and 30 mg/kg) treated orally in young (p < 0.05) andaged (p < 0.01) mice for 15 days markedly increased the SDL as com-pared to the respective control groups (Fig.5). Significant (p < 0.001)decrease in SDL of scopolamine (0.4 mg/kg, i.p.) and diazepam(1 mg/kg, i.p.) groups indicated impairment of memory (amnesia).The high doses (20 and 30 mg/kg, p.o.) of MKA however notablyreversed the amnesia induced by both scopolamine and diazepam(Fig.6). The groups of mice treated with piracetam (400 mg/kg, i.p.)showed improvement (p < 0.001) in memory of young as well asaged mice. The same dose of piracetam also reversed amnesia in-duced by scopolamine and diazepam.

Fig. 3. Effect of MKA (10, 20 and 30 mg/kg) or piracetam (mg/kg) administered orally formaze. Values are in mean ± SEM (n = 6). ⁄p < 0.05 and ⁄⁄⁄p < 0.001 as compared to controlmice.

3.5. Effect on brain cholinesterase activity

The lowest dose of MKA (10 mg/kg, p.o.) did not produce any ef-fect on cholinesterase activity in young mice. However, at higherdoses (20 and 30 mg/kg, p.o.) MKA showed a remarkable reduction(p < 0.01) in brain cholinesterase activity in young mice, as com-pared to the respective control groups using Ellman’s kinetic color-imetric method (Fig.7). All the doses (10, 20 and 30 mg/kg, p.o.) ofMKA showed a dose dependent reduction (p < 0.05) in brain cho-linesterase activity in aged mice. In young mice, the percentageof decline in AChE activity was 21.18% at the dose of 20 mg/kg(p < 0.001), and 30.62% at the dose of 30 mg/kg (p < 0.001) throughoral route. In the aged mice, the AChE activity declined from19.42%, 30.83% and 43.99% at 10, 20 and 30 mg/kg, respectively.Metrifonate (50 mg/kg, i.p.) used as a standard drug showed a sig-nificant (p < 0.001) 36.93% and 45.50% reduction of brain cholines-terase activity in young and aged mice, respectively.

3.6. Effect on BACE1 inhibitory activity

The MKA exhibited BACE1 inhibitory activity with an IC50 valueof 1.7 lg/mL (Fig.8). Plots of the initial velocity versus MKA in thepresence of different substrate concentrations gave a family ofstraight lines. The inhibition pattern of MKA (inhibition constant,Ki = 1 mg/mL) against BACE1 from the Dixon plot (Fig.9), was foundto be non-competitive with the substrate at the active site ofBACE1. It is a non-competitive inhibitor because increasing thesubstrate concentration resulted in lines, which declined with acommon intercept on the x-axis.

4. Discussion

Cognition includes all aspects of perceiving, learning, thinkingand remembering. The cognitive dysfunctions include delirium,behavioral disorders, and dementia. Dementia is a common disorder

15 successive days on transfer latency of young and aged mice using elevated plusgroup of young mice. ⁄p < 0.05 and ⁄⁄⁄p < 0.001 as compared to control group of aged

Fig. 4. Reversal of scopolamine (0.4 mg/kg, i.p.) or diazepam (1 mg/kg, i.p.) induced amnesia by MKA (10, 20 and 30 mg/kg, p.o.) in young mice using elevated plus maze.Values are in mean ± SEM (n = 6). ⁄⁄⁄p < 0.001 as compared to control group of young mice. ⁄p < 0.05 and ⁄⁄⁄p < 0.001 as compared to scopolamine (Sco) alone. jp < 0.05 andjjjp < 0.001 as compared to diazepam (Dia) alone.

1040 V. Mani et al. / Food and Chemical Toxicology 50 (2012) 1036–1044

of elderly individuals. Currently, there are no satisfactory therapeu-tic regimens available for the management of cognitive dysfunc-

Fig. 5. Effect of MKA (10, 20 and 30 mg/kg) administered orally for 15 successive daysValues are in mean ± SEM (n = 6). ⁄p < 0.05 and ⁄⁄⁄p < 0.001 as compared to control group

tions. Therefore, in the present study, the effect of total alkaloidextract of an edible medicinal plant, M. koenigii leaves on memory

on step-down latency of young and aged mice using passive avoidance paradigm.of young mice. ⁄⁄p < 0.01 and ⁄⁄⁄p < 0.001 as compared to control group of aged mice.

Fig. 6. Reversal of scopolamine (0.4 mg/kg, i.p.) or diazepam (1 mg/kg, i.p.) induced amnesia by MKA (10, 20 and 30 mg/kg, p.o.) in young mice using passive avoidanceparadigm. Values are in mean ± SEM (n = 6). ⁄⁄⁄p < 0.001 as compared to control group of young mice. ⁄p < 0.05 and ⁄⁄⁄p < 0.001 as compared to scopolamine (Sco) alone.jjp < 0.01 and jjjp < 0.001 as compared to diazepam (Dia) alone.

V. Mani et al. / Food and Chemical Toxicology 50 (2012) 1036–1044 1041

and reversal of memory deficit using experimental animals wereexplored.

Alkaloids are a group of naturally occurring chemical com-pounds, which mostly contain basic nitrogen atoms. Several reportshave supported the role of alkaloids in treatment of Alzheimer’sdisease. Galanthamine, nicotine, caffeine and huperzine A have beenestablished to improve Alzheimer’s disease (Desilets et al., 2009;Prvulovic et al., 2010; Levin and Rezvani, 2002; Botton et al.,2010). Jung et al. (2009) reported six protoberberine alkaloids(berberine, palmatine, jateorrhizine, epiberberine, coptisine, andgroenlandicine) and one aporphine alkaloid (magnoflorine) fromCoptidis rhizoma have been established as useful compounds forthe prevention of Alzheimer’s disease through inhibition of bothacetylcholinesterase and beta-amyloid formation. Recently, piper-ine from black pepper and berberine from Hydrastis canadensis alsofurther strengthened the role of alkaloids in the management ofAlzheimer’s disease (Chonpathompikunlert et al., 2010; Kulkarniand Dhir, 2010). M. koenigii leaves are a major source of carbazolealkaloids that have been reported for various biological activities(Iyer and Devi, 2008). In the present study, we documented theutility of total alkaloid extract in the management of Alzheimer’sdisease. Amnesia was induced in mice by intraperitoneal injectionof scopolamine or diazepam, in addition to the natural process ofageing-induced amnesia. The higher doses of MKA (20 and 30 mg/kg, p.o) significantly improved the memory in young and aged miceand successfully reversed the amnesia induced by scopolamine,diazepam and ageing, when administered 15 days.

Cholinergic neurons in the central cholinergic system play animportant role in the cognitive deficits associated with aging andneurodegenerative diseases. The deficiency in acetylcholine levelin cerebral cortex is one of the major features seen in Alzheimer’sdisease (Gauthier, 2010). Selective loss of cholinergic neurons anddecrease in cholinacetyltransferase activity was reported to be acharacteristic feature of senile dementia of the Alzheimer’s type.

According to the cholinergic hypothesis, memory impairments inpatients with the senile dementia are due to a selective and irre-versible deficiency in the cholinergic functions in the brain (Overket al., 2010). Acetylcholinesterase (AChE) modulates ACh to properlevels by degradation. However, excessive AChE activity leads toconstant ACh deficiency, memory, and cognitive impairments(Pepeu and Giovannini, 2010). In the present study, MKA reducedthe brain cholinesterase activity in a dose dependent manner intreated young and aged mice which may facilitate the improve-ment of memory. Previous research findings using Glycyrrhizaglabra, Myristica fragrance, ascorbic acid, Lepidium meyenii, Zingiberofficianale, Thespesia populnea, and Daucus carota also havedisplayed a link between memory improving effect and cholines-terase activity (Vasudevan and Parle, 2006a,b; Joshi and Parle,2006; Rubio et al., 2007).

Impairment of central cholinergic system negatively affectslearning and memory in a variety of paradigms. Scopolamine, a non-selective muscarinic cholinergic antagonist, is a well known cen-trally acting cholinergic probe, which interferes with memory andcognitive function in human and experimental animals (Kanwallet al., 2010). This experimental animal model of scopolamine-in-duced amnesia has been widely used in research to screen for drugswith potential therapeutic value in dementia. Moreover, it disruptsacquisition of new tasks and performance on previously learnedtasks (Aigner and Mishkin, 2001). Huperzine A and galantamineare well known useful plant-based alkaloids for Alzheimer’s diseasethat are able to reverse the scopolamine-induced memory deficit invarious animal models (Gao et al., 2000; de Bruin and Pouzet, 2006).Total alkaloids from Uncaria tomentosa also showed a beneficialeffect on memory impairment induced with scopalamine by thedysfunction of cholinergic systems in the brain using passive avoid-ance test. In the present study, mice treated with scopolamineshowed larger TL and shorter SDL in control groups. The animalstreated with MKA extract (20 and 30 mg/kg, p.o.) showed inhibitory

Fig. 7. Effect of MKA (10, 20 and 30 mg/kg) administered orally for 15 successive days on brain cholinesterase (AChE) activity of young and aged mice. Values are inmean ± SEM (n = 6). ⁄⁄⁄p < 0.001 as compared to control group of young mice. ⁄p < 0.05 and ⁄⁄⁄p < 0.001 as compared to control group of aged mice (One-way ANOVA followedby Tukey–Kramer multiple comparisons test).

Fig. 8. Effect of MKA on the inhibitory activity of BACE1.

1042 V. Mani et al. / Food and Chemical Toxicology 50 (2012) 1036–1044

effects against scopolamine-induced memory impairment in theelevated plus-maze and passive avoidance paradigm maze modelsby shortening the TL and improving the SDL values.

Benzodiazepines are well established as inhibitory modulators ofmemory processing. The amnestic effects of benzodiazepines inclu-ding diazepam are mediated by the inhibitory neurotransmitter ‘‘c’’-

amino butyric acid (GABA) acting at the GABAA receptor complex(Rudolph et al., 1999). Clinical and experimental studies have shownthat diazepam causes temporary memory impairment (Costa et al.,2010; Saraf et al., 2008). It is also known to block long-term poten-tiation (LTP) in slices of the hippocampus and piriform cortexand believed to potentiate the amplitude and prolong the decay of

Fig. 9. Dixon plots from inhibition of MKA on BACE1 at different concentrations.

V. Mani et al. / Food and Chemical Toxicology 50 (2012) 1036–1044 1043

GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs)in neurons of rat hippocampus (Xu and Sastry, 2005). Diazepam,which is also frequently prescribed for disorders such as seizure,insomnia, and anesthesia, may result in cognitive dysfunction. Thereis a pharmacological evidence that intraseptal injection of the GABAreceptor agonist muscimol reduces ACh release, acetylcholine turn-over rate and high-affinity choline uptake (HAChU) in the hippo-campus (Walsh et al., 1993). The MKA (20 and 30 mg/kg, p.o.)treated groups showed decrease in TL and elevated SDL values high-lighting the reversal of memory impairment induced by diazepam.The protective effect offered against diazepam-induced amnesiaby MKA may due to GABAergic inhibitory response and indirectfacilitation of ACh activity in brain.

Normal ageing is associated with a decline in functions such asmemory formation, retention and retrieval. The present studyshowed that there is marked impairment in the cognitive perfor-mance of aged mice (12–15 months) by increased TL value andshortened SDL values. Oxidative damage is considered a likely causeof age-associated brain dysfunction because the brain is believed tobe particularly vulnerable to oxidative stress due to a relatively highrate of oxygen free radical generation without commensurate levelsof antioxidative defenses (Coyle and Puttfarken, 1993). Socci et al.(1995) focused that the chronic treatment of antioxidants were alle-viated in age-associated cognitive deficits in animals. Moreover, theprevious findings exposed that levels of thiobarbituric acid reactivesubstances (TBARS) and conjugated dienes are markedly increasedby oxidative stress through aging in the hippocampus of rats, whichmodulates cognitive performance like learning ability and memoryretention in animal models (Fukui et al., 2002). Interestingly, MKAreversed ageing-induced amnesia, when administered for 15 days.Moreover, antioxidant activity of carbazole alkaloids like euchres-tine B, bismurrayafoline E, mahanine, mahanimbicine, mahanim-bine, koenimbine, O-methylmurrayamine A, O-methylmahanine,isomahanine, bismahanine, and bispyrayafoline from M. koenigiileaves strengthen the reversal of memory deficit by natural ageingprocess (Tachibana et al., 2001).

b-Secretase or beta-site APP-cleaving enzyme 1 (BACE1) is a keyenzyme that selectively metabolize the amyloid precursor protein(APP) and generate the toxic b-amyloid (Ab) in brain (Choi et al.,2011). It is well known that Ab plays a critical role in AD pathogen-

esis; moreover, aggregations of Ab trigger a complex pathologicalcascade which leads to neurodegeneration (Golde et al., 2006).Knockout of the BACE1 gene in mice abolishes the cerebral Ab for-mation and amyloid deposition, and the mice were free from Alz-heimer’s associated pathological changes including neuronal lossand certain memory deficits (Luo et al., 2001, 2003). A furtherstudy demonstrated that the reduction of Ab levels with BACE1gene deletion prevent memory impairment and hippocampal cho-linergic dysfunction in BACE1(�/�). Tg2576(+) bigenic mice model(Ohno et al., 2004). These results suggest that inhibition of BACE1is a valid therapeutic strategy for AD and may not encounter severemechanism-based toxicity. In our study, MKA exhibited BACE1non-competitive inhibitory activity with an IC50 value of 1.7 lg/mL. Moreover, Ghribi et al. (2006) showed that the co-localizationcholesterol–BACE1 in the hippocampus of cholesterol-treated ani-mals was accompanied by an increase in BACE1 protein level, anincrease in b-APP cleavage, and an overproduction of Ab peptide.The b-APP and BACE1 are upregulated by the cholesterol diet,resulting in an increase in the cleavage of b-APP to Ab peptide. Ex-tracts of M. koenigii leaves and it is marker carbazole alkaloidmahanimbine significantly reduced the body weight gain, plasmatotal cholesterol and triglyceride levels in high fat diet induced ob-ese rats (Birari et al., 2010). Not only hypercholesterolemia, otherneuronal toxic like hypoxia, mitochondrial dysfunction, oxidativestress, and injury are also play a significant role in elevation ofBACE1 activity (Cole and Vassar, 2007). Antioxidant activity of car-bazole alkaloids also helped the BACE1 inhibitory activity and im-proved memory in the present study. Therefore, MKA which showspotential BACE1 inhibitory activity in vitro may require furtherinvestigation in vivo to confirm the role as a BACE 1 inhibitor foranti-Alzheimer therapy.

5. Conclusion

In the present study, we observed that total alkaloids extract ofM. koenigii leaves elevated acetylcholine level in brain and ulti-mately improved memory of both young and aged mice. In vitroit showed BACE1 inhibition and was found to be a non-competitiveinhibitor. In light of the above, it may be worthwhile to explore thepotential of this plant in the management of Alzheimer patients.

1044 V. Mani et al. / Food and Chemical Toxicology 50 (2012) 1036–1044

Conflict of Interest

The authors declare that there are no conflicts of interest.

References

Abu Bakar, N.H., Sukari, M.A., Rahmani, M., Sharif, A.M., Khalid, K., Yusuf, U.K., 2007.Chemical constituents from stem barks and roots of Murraya koenigii(Rutaceae). Malays. J. Anal. Sci. 11, 173–176.

Aigner, T.G., Mishkin, M., 2001. The effects of physostigmine and scopolamine onrecognition memory in monkeys. Behav. Neural. Biol. 45, 81–87.

Birari, R., Javia, V., Bhutani, K.K., 2010. Antiobesity and lipid lowering effects ofMurraya koenigii (L.) Spreng leaves extracts and mahanimbine on high fat dietinduced obese rats. Fitoterapia 81, 129–133.

Botton, P.H., Costa, M.S., Ardais, A.P., Mioranzza, S., Souza, D.O., da Rocha, J.B.T.,Porciuncula, L.O., 2010. Caffeine prevents disruption of memory consolidationin the inhibitory avoidance and novel object recognition tasks by scopolaminein adult mice. Behav. Brain Res. 214, 254–259.

Choi, C.W., Choi, Y.H., Cha, M.R., Kim, Y.S., Yon, G.H., Hong, K.S., Park, W.K., Kim, Y.H.,Ryu, S.Y., 2011. In vitro BACE-1 inhibitory activity of resveratrol oligomers fromthe seed extract of Paeonia lactiflora. Planta Med. 77, 374–376.

Chonpathompikunlert, P., Wattanathorn, J., Muchimapura, S., 2010. Piperine, themain alkaloid of Thai black pepper, protects against neurodegeneration andcognitive impairment in animal model of cognitive deficit like condition ofAlzheimer’s disease. Food Chem. Toxicol. 48, 798–802.

Cole, S.L., Vassar, R., 2007. The Alzheimer’s disease b-secretase enzyme, BACE1. Mol.Neurodegener. 22, 1–25.

Costa, J.C., Costa, K.M., do Nascimento, J.L., 2010. Scopolamine- and diazepam-induced amnesia are blocked by systemic and intraseptal administration ofsubstance P and choline chloride. Peptides 31, 1756–1760.

Coyle, J.T., Puttfarken, P., 1993. Oxidative stress, glutamate, and neurodegenerativedisorders. Science 262, 689–695.

Dasgupta, T., Rao, A.R., Yadava, P.K., 2003. Chemomodulatory action of curry leaf(Murraya koenigii) extract on hepatic and extrahepatic xenobiotic metabolizingenzymes, antioxidant levels, lipid peroxidation, skin and forestomachpapillomagenesis. Nutr. Res. 23, 1427–1446.

de Bruin, N., Pouzet, B., 2006. Beneficial effects of galantamine on performance inthe object recognition task in Swiss mice: deficits induced by scopolamine andby prolonging the retention interval. Pharmacol. Biochem. Behav. 85, 253–260.

Desilets, A.R., Gickas, J.J., Dunican, K.C., 2009. Role of huperzine A in the treatment ofAlzheimer’s disease. Ann. Pharmacother. 43, 514–518.

Ecobichon, D.J., 1997. The Basis of Toxicology Testing. CRC Press, New York.Ferreira, A., Proenc, C., Serralheiro, M.L.M., Araújo, M.E.M., 2006. The in vitro

screening for acetylcholinesterase inhibition and antioxidant activity ofmedicinal plants from Portugal. J. Ethnopharmacol. 108, 31–37.

Fukui, K., Omoi, N., Hayasaka, T., Shinkai, T., Suzuki, S., Abe, K., 2002. Cognitiveimpairment of rats caused by oxidative stress and aging, and its prevention byvitamin E. Ann. N Y Acad. Sci. 959, 275–284.

Gao, Y., Tang, X.C., Guan, L.C., Kuang, P.Z., 2000. Huperzine A reverses scopolamine-and muscimol-induced memory deficits in chick. Acta Pharmacol. Sin. 21,1169–1173.

Gauthier, M., 2010. State of the art on insect nicotinic acetylcholine receptorfunctions in learning and memory. Adv. Exp. Med. Biol. 683, 97–115.

Ghribi, O., Larsen, B., Schrag, M., Herman, M.M., 2006. High cholesterol content inneurons increases BACE, b-amyloid, and phosphorylated tau levels in rabbithippocampus. Exp. Neurol. 200, 460–467.

Golde, T.E., Dickson, D., Hutton, M., 2006. Filling the gaps in the abeta cascadehypothesis of Alzheimer’s disease. Curr. Alzheimer Res. 3, 421–430.

Gupta, S., George, M., Singhal, M., Garg, V., 2009. Wound healing activity ofmethanol extract of Murraya koenigii leaves. Pharmacologyonline 3, 915–923.

Hardy, J., Selkoe, D.J., 2002. The amyloid hypothesis of Alzheimer’s disease: progressand problems on the road to therapeutics. Science 297, 353–356.

Itoh, J., Nabeshima, T., Kameyama, T., 1990. Utility of an elevated plus-maze for theevaluation of memory in mice. effect of nootropics, scopolamine andelectroconvulsive shock. Psychopharmacology 101, 27–33.

Iyer, D., Devi, P.U., 2008. Phyto-pharmacology of Murraya koenigii (L.). Pharmacog.Rev. 2, 180–184.

Joseph, S., Peter, K.V., 1985. Curry leaf: perennial nutritious, leafy vegetable. Econ.Bot. 39, 68–73.

Joshi, H., Parle, H., 2006. Zingiber officinale: evaluation of its nootropic effect in mice.Afr. J. Trad. CAM. 3, 64–74.

Jung, H.A., Min, B.S., Yokozawa, T., Lee, J.H., Kim, Y.S., Choi, J.S., 2009. Anti-Alzheimerand antioxidant activities of Coptidis rhizoma alkaloids. Biol. Pharm. Bull. 32,1433–1438.

Kanwall, A., Mehla, J., Kunchal, M., Naidul, V.G.M., Gupta, Y.K., Sistla, R., 2010. Anti-amnesic activity of Vitex negundo in scopolamine induced amnesia in rats.Pharmacol. Pharm. 1, 1–8.

Kashani, A., Lepicard, E., Poirel, O., Videau, C., David, J.P., Fallet-Bianco, A., Simon, A.,Delacourte, A., Giros, B., Epelbaum, J., Betancur, C., El Mestikawy, S., 2008. Lossof VGLUT1 and VGLUT2 in the prefrontal cortex is correlated with cognitivedecline in Alzheimer disease. Neurobiol. Aging 29, 1619–1630.

Kovarik, Z., Radic, Z., Berman, H.A., Simeon-Rudolf, V., Reiner, E., Taylor, P., 2003.Acetylcholinesterase active centre and gorge conformations analysed by

combinatorial mutations and enantiomeric phosphonates. Biochem. J. 373,33–40.

Krishna, S., Usha, P.T.A., 2009. Hypoglycaemic effect of a combination of Pleurotusostreatus, Murraya koenigii and Aegle marmelos in diabetic rats. Indian J. Anim.Sci. 79, 986–987.

Kulkarni, S.K., Dhir, A., 2010. Berberine: a plant alkaloid with therapeutic potentialfor central nervous system disorders. Phytother. Res. 24, 317–324.

Kumar, N.S., Mukherjee, P.K., Bhadra, S., Saha, B.P., Pal, B.C., 2010.Acetylcholinesterase inhibitory potential of a carbazole alkaloid,mahanimbine, from Murraya koenigii. Phytother. Res. 24, 629–631.

Levin, E.D., Rezvani, A.H., 2002. Nicotinic treatment for cognitive dysfunction. Curr.Drug Targets CNS Neurol. Disord. 1, 423–431.

Luo, Y., Bolon, B., Damore, M.A., Fitzpatrick, D., Liu, H., Zhang, J., Yan, Q., Vassar, R.,Citron, M., 2003. BACE1 (beta-secretase) knockout mice do not acquirecompensatory gene expression changes or develop neural lesions over time.Neurobiol. Dis. 14, 81–88.

Luo, Y., Bolon, B., Kahn, S., Bennett, B.D., Babu-Khan, S., Denis, P., Fan, W., Kha, H.,Zhang, J., Gong, Y., Martin, L., Louis, J.C., Yan, Q., Richards, W.G., Citron, M.,Vassar, R., 2001. Mice deficient in BACE1, the Alzheimer’s beta-secretase, havenormal phenotype and abolished beta-amyloid generation. Nat. Neurosci. 4,231–232.

Mandal, S., Nayak, A., Kar, M., Banerjee, S.K., Das, A., Upadhyay, S.N., Singh, R.K.,Banerji, A., Banerji, J., 2010. Antidiarrhoeal activity of carbazole alkaloids fromMurraya koenigii Spreng (Rutaceae) seeds. Fitoterapia 81, 72–74.

Manoj, N.G., Archana, R.J., Sachin, S.S., 2009. Evaluation of anti-inflammatoryactivity of methanol extract of Murraya koenigi leaves by in vivo and in vitromethods. Pharmacol. Online 1, 1072–1094.

Maurice, T., Junien, J.L., Privat, A., 1997. Dehydroepiandrosterone sulfate attenuatesdizocilpine-induced learning impairment in mice via a1-receptors. Behav. BrainRes. 83, 159–164.

Ningappa, M.B., Dhananjaya, B.L., Dinesha, R., Harsha, R., Srinivas, L., 2010. Potentantibacterial property of APC protein from curry leaves (Murraya koenigii L.).Food Chem. 118, 747–750.

Ohno, M., Sametsky, E.A., Younkin, L.H., Oakley, H., Younkin, S.G., Citron, M., Vassar,R., Disterhoft, J.F., 2004. BACE1 deficiency rescues memory deficits andcholinergic dysfunction in a mouse model of Alzheimer’s disease. Neuron 41,27–33.

Overk, C.R., Felder, C.C., Tu, Y., Schober, D.A., Bales, K.R., Wuu, J., Mufson, E.J., 2010.Cortical M1 receptor concentration increases without a concomitant change infunction in Alzheimer’s disease. J. Chem. Neuroanat. 40, 63–70.

Pepeu, G., Giovannini, M.G., 2010. Cholinesterase inhibitors and memory. Chem.Biol. Interact. 187, 403–408.

Prvulovic, D., Hampel, H., Pantel, J., 2010. Galantamine for Alzheimer’s disease.Expert Opin. Drug Metab. Toxicol. 6, 345–354.

Rahman, M.M., Gray, A.I., 2005. A benzoisofuranone derivative and carbazolealkaloids from Murraya koenigii and their antitumor activity. Pytochemistry 66,1601–1606.

Rubio, J., Dang, H., Gong, M., Liu, X., Chen, S., Gonzales, G.F., 2007. Aqueous andhydroalcoholic extracts of Black Maca (Lepidium meyenii) improve scopolamine-induced memory impairment in mice. Food Chem. Toxicol. 45, 1882–1890.

Rudolph, U., Crestani, F., Benke, D., Brünig, I., Benson, J.A., Fritschy, J.M., Martin, J.R.,Bluethmann, H., Mohler, H., 1999. Benzodiazepine actions mediated by specificgamma-aminobutyric acid (GABA) receptor subtypes. Nature 401, 796–800.

Rujjanawate, C., Kanjanapothi, D., Panthong, A., 2003. Pharmacological effect andtoxicity of alkaloids from Gelsemium elegans Benth. J. Ethnopharmacol. 89, 91–95.

Saraf, M.K., Prabhakar, S., Pandhi, P., Anand, A., 2008. Bacopa monniera amelioratesamnesic effects of diazepam qualifying behavioral–molecular partitioning.Neuroscience 155, 476–484.

Sathaye, S., Bagul, Y., Gupta, S., Kaur, H., Redkar, R., 2010. Hepatoprotective effects ofaqueous leaf extract and crude isolates of Murraya koenigii against in vitroethanol-induced hepatotoxicity model. Exp. Toxicol. Pathol. 63, 587–591.

Shah, A.S., Wakade, A.S., Juvekar, A.R., 2008. Immunomodulatory activity ofmethanolic extract of Murraya koenigii (L) Spreng. leaves. Indian J. Exp. Biol.46, 505–509.

Socci, D.J., Crandall, B.M., Arendash, G.W., 1995. Chronic antioxidant treatmentimproves the cognitive performance of aged rats. Brain Res. 693, 88–94.

Tachibana, Y., Kikuzaki, H., Lajis, N.H., Nakatani, N., 2001. Antioxidative activity ofcarbazoles from Murraya koenigii leaves. J. Agric. Food Chem. 49, 5589–5594.

Tachibana, Y., Kikuzaki, H., Lajis, N.H., Nakatani, N., 2003. Comparison ofantioxidative properties of carbazole alkaloids from Murraya koenigii leaves. J.Agric. Food Chem. 51, 6461–6467.

Vasudevan, M., Parle, M., 2006a. Pharmacological actions of Thespesia populnearelevant to Alzheimer’s disease. Phytomedicine 13, 677–687.

Vasudevan, M., Parle, M., 2006b. Pharmacological evidence for the potential ofDaucus carota in the management of cognitive dysfunctions. Biol. Pharm. Bull.29, 1154–1161.

Vasudevan, M., Parle, M., 2009. Antiamnesic potential of Murraya koenigii leaves.Phytother. Res. 23, 308–316.

Walsh, T.J., Stackman, R.W., Emerich, D.F., Taylor, L.A., 1993. Intraseptal injection ofGABA and benzodiazepine receptor ligands alters high-affinity cholinetransport in the hippocampus. Brain Res. Bull. 31, 267–271.

Willem, M., Lammich, S., Haass, C., 2009. Function, regulation and therapeuticproperties of b-secretase (BACE1). Semin. Cell Dev. Biol. 20, 175–182.

Xu, J.Y., Sastry, B.R., 2005. Benzodiazepine involvement in LTP of the GABA-ergicIPSC in rat hippocampal CA1 neurons. Brain Res. 1062, 134–143.