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Anxiolytic effects of Plumeria rubra var. acutifolia (Poiret) L. flower extracts inthe elevated plus-maze model of anxiety in mice§

Manavi Chatterjee a, Rajkumar Verma a, Vijai Lakshmi b, Shibani Sengupta a, Anil Kumar Verma a,Abbas Ali Mahdi b, Gautam Palit a,*a Division of Pharmacology, Central Drug Research Institute, CSIR, Lucknow 226001, Uttar Pradesh, Indiab Department of Biochemistry, Chattrapati Shahuji Maharaj Medical University, Lucknow 226003, Uttar Pradesh, India

A R T I C L E I N F O

Article history:

Received 29 March 2012

Received in revised form 3 September 2012

Accepted 6 September 2012

Available online xxx

Keywords:

Anxiety

Elevated plus maze

Plumeria rubra

Gross behaviour

Rotarod

A B S T R A C T

Interest in alternative medicine and plant-derived medications that affect the ‘‘mind’’ is growing rapidly

since last two decades. The aim of the present study was to investigate the effects of ethanolic extract of

flower of Plumeria rubra (PR) along with its fractions in the elevated plus-maze (EPM) model of anxiety.

The P. rubra extract or its fractions was administered orally to male Swiss mice, at graded doses, 1 h prior

to behavioural assessment. The PR extract at the dose of 100 mg/kg p.o., significantly increased the time

spent in the open arms of the EPM. Further, the anxiolytic properties of hexane, chloroform and butanolic

soluble and insoluble fractions at one-fifth of the original dose were also observed in the EPM task. Out of

which butanol insoluble fraction showed significant anxiolytic activity comparable to standard

anxiolytic drug, diazepam. Further, pretreatment with crude ethanolic extract and butane insoluble

fraction showed no significant effects in the horizontal activity, total distance travelled and stereotypy

count in the animal activity monitor and had no motor in-coordination side effects in the rotarod test in

mice. These observations suggest that the flower extract of P. rubra and its insoluble butanolic fraction

might possess significant anxiolytic potential to be pursued further for drug development process.

� 2012 Elsevier B.V. All rights reserved.

Contents lists available at SciVerse ScienceDirect

Asian Journal of Psychiatry

jo u rn al h om epag e: ww w.els evier .c o m/lo cat e/a jp

1. Introduction

Anxiety is one of the most prevalent psychiatric disorders. Inany given year, approximately 40 million adults are affected byanxiety disorder and can also precipitate or aggravate cardiovas-cular and behavioural disorders (Weissman et al., 1990). Approxi-mately two-thirds of the anxious patients respond to the currentlyavailable treatments but the magnitude of improvement is stilldisappointing, besides, they also produce various systemic sideeffects and exhibit dependence and tolerance on chronic treatmentwhich now have become a major concern about the use ofcurrently used medicines. At present, the benzodiazepines (BZDs)are the most commonly employed medicinal treatments foranxiety. BZDs produce their pharmacological actions via specifichigh affinity binding sites on a supramolecular complex composedof GABA-A and a BZD receptor coupled with a chloride ion channel.Besides, the potential for drug dependence and side effects, BZDsalso cause drug interactions by potentiating the effect of other CNSdepressants such as alcohol, hypnotics, and neuroleptics if taken

§ CDRI communication no: 8334.

* Corresponding author. Tel.: +91 522 2612411–418x4303;

fax: +91 522 2623405/2623938.

E-mail addresses: gpalitcdri@gmail.com, meetmanavi@gmail.com (G. Palit).

Please cite this article in press as: Chatterjee, M., et al., Anxiolytic effecelevated plus-maze model of anxiety in mice. Asian J. Psychiatry (2

1876-2018/$ – see front matter � 2012 Elsevier B.V. All rights reserved.

http://dx.doi.org/10.1016/j.ajp.2012.09.005

together. Other anti-anxiety medications include antidepressants,buspirone and b-blockers which though effective in many cases,also possess side effects like nausea, light headedness, dizziness,headache, dry mouth, constipation, diarrhea, etc. (Smith et al.,2012). Therefore, there is an urgent need of drug which possessesgreater efficacy, lesser undesirable effects with minimum or notolerance and dependence. In order to overcome these adverseeffects, investigations has been extended for the search of noveland better biocompatible molecules from plant sources.

Herbs are widely accepted sources of medicine, which play animportant role in health care programme worldwide (Verma et al.,2010). The search for novel pharmacotherapy from medicinalplants for psychiatric illnesses has progressed significantly in thepast decade and their therapeutic potential has been assessed in avariety of animal models (Carlini, 2003; Zhang, 2004; Chatterjeeet al., 2012). Our previous studies also involved evaluation of someIndian medicinal plants like Ocimum sanctum (Chatterjee et al.,2011b) and Bacopa monera (Chatterjee et al., 2010) for theiranxiolytic effects. In this study, we extended our search for suchherbal medications to evaluate the neuropharmacological effectsof flower extract of another Indian medicinal plant, Plumeria rubra.P. rubra (PR) syn. Plumeria acutifolia is also known as Plumeria tree,temple tree, West Indian jasmine or frangipani. This shrub or smalltree is native to Mexico, and is well-known for its strongly

ts of Plumeria rubra var. acutifolia (Poiret) L. flower extracts in the012), http://dx.doi.org/10.1016/j.ajp.2012.09.005

Fig. 1. Diagrammatic representation of extraction procedure of P. rubra flowers.

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perfumed flowers that can be of several colors. Various species ofPlumeria has been previously shown to possess antioxidant (Ruiz-Teran et al., 2008), hypolipidemic (Merina et al., 2010), hypogly-cemic (Zaheer et al., 2010), antimicrobial (Rasool et al., 2008) andcytotoxic activities (Kardono et al., 1990). The medicinal propertiesof plant also include protection against ulcers, skin diseases,inflammation, arthritis and constipation (Hamburger et al., 1991;Gupta et al., 2006; Zaheer et al., 2010).

Despite the widely popular use of this plant, the availablescientific information about the potential effects of PR in animalmodels of psychiatric disorders especially in anxiety is limited.Therefore, the present study was conducted to evaluate theanxiolytic effect of PR in different models in mice.

2. Materials and methods

2.1. Animals

All experimental protocols were approved by our InstitutionalEthical Committee following the guidelines of Committee for thePurpose of Control and Supervision of Experiments on Animals(CPCSEA). Albino male Swiss mice weighing 20–25 g wereemployed in the study. Mice were housed in six per cage atconstant room temperature (22 � 2 8C) and 12-h light/12-h dark(8:00 a.m.–8:00 p.m.). Mice were fed standard laboratory food andwater was given ad libitum. Each animal was used once in thebehaviour tests.

2.2. Preparation of extract

2.2.1. Collection of the flowers

Fresh flowers of P. rubra were collected from Lucknow botanicalgardens during the month of October and identified by Dr. R.K.Sharma, Head, Botany Division of the Central Drug ResearchInstitute (CDRI), Lucknow. The specimen has been preserved in theBotany Division and has been allotted the CDRI extract no 117.

2.2.2. Extraction and fractionation procedure

Fresh flowers (500 g) were extracted with 90% ethanol(4 � 700 ml) at room temperature. The combined ethanolic extractwas filtered and concentrated in a rotavapour below 50 8C to aviscous mass which was dried under high vacuum to remove thelast traces of the solvent to get crude extract (12.6 g). The ethanolicextract (10.0 g) was successively fractionated with hexane (0.9 g),chloroform (1.2 g), n-butanol soluble (3.8 g) and n-butanolinsoluble (4.1 g) fractions (Fig. 1). All these fractions wereconcentrated under reduced pressure below 50 8C separately ina rotavapour and all were submitted for anxiolytic activity. The n-butanol soluble fraction and n-butanol insoluble fractions werefound showing some similar spots on TLC plates. Therefore thesefractions were mixed and chromatographed over a column of silicagel, rechromatography of some column fractions yielded 4 purecompounds, which were identified by co-TLC with the authenticsamples and also by comparison of physicochemical data providedin literature (Dubois et al., 2005; Kuigoua et al., 2010).

2.3. Drugs and treatment schedule

Diazepam (DZP) was obtained from M/s. Sigma (St. Louis, MO,USA). All compounds were suspended in 0.5% gum acacia. Micewere either treated with vehicle or the extract or fractions daily for3 days prior to the experiment. Drugs were prepared fresh dailybefore administration. DZP was administered once at a dose of1.5 mg/kg, 1 h prior to the experiment. Compounds wereadministered per orally at a volume of 0.1 ml/10 g body weight

Please cite this article in press as: Chatterjee, M., et al., Anxiolytic effeelevated plus-maze model of anxiety in mice. Asian J. Psychiatry (2

2.4. Behavioural observations

Mice were randomly divided into following groups of 8 miceeach.

(a) Control group: mice were treated with vehicle (0.5% gumacacia).

(b) Treatment group: mice treated with graded doses of extract orfractions.

(c) Standard drug treated group: mice were treated with diazepam(1.5 mg/kg p.o.).

2.5. Procedures

2.5.1. Elevated plus maze

This test has been widely used to measure anxiety in rodents(Lister, 1987). Mice were treated daily for 3 days with extract orfractions or DZP (single dose) 1 h prior to the experiment. Eachanimal was placed at the centre of the maze, facing one of the openarms. The time spent in enclosed and open arms was recorded for5 min test. The movement of animals across the arms is calculatedby interruption of beams which was analysed by maze trackingsoftware (M/s Columbus Instruments, USA). After each test, themaze was carefully cleaned up with a wet tissue paper (70%ethanol solution).

2.5.2. Spontaneous motor activity

Gross open field activity was studied using Digiscan InfraredPhotocell system [Omnitech Electronics, Columbus, Ohio] in42 � 42 � 30 cm Plexiglass arenas, fitted into infrared beamcontaining metallic grid. Activity of animals was observed bythe interruption of infrared beams (Chatterjee et al., 2011a).

(a) Horizontal activity: the total number of beam interruptionsthat occurred in the horizontal sensor in the duration of 2 min.

(b) Total distance travelled: it is the distance travelled by theanimal in a given sample period, indicated in centimetres. Total

cts of Plumeria rubra var. acutifolia (Poiret) L. flower extracts in the012), http://dx.doi.org/10.1016/j.ajp.2012.09.005

Table 1Effect of PR extract and fractions in the elevated plus maze test.

Groups Doses (mg/kg, p.o.) Mean � SEM

Control – 18.35 � 0.78

PR ethanolic extract 50 66.88 � 15.30**

100 81.84 � 10.33***

200 26.17 � 8.969

n-Butane insoluble 10 39.42 � 10.39

20 59.8 � 10.21***

40 25.02 � 1.994

Hexane 20 42.38 � 7.10*

Chloroform 20 37.34 � 10.77

n-Butane soluble 20 29.18 � 8.86

DZPM 1.5 68.35 � 4.240***

* P < 0.05 vs control.** P < 0.01 vs control.*** P < 0.001 vs control.

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distance travelled is a more accurate indicator of ambulatoryactivity.

(c) Stereotypy counts: if the animal breaks the same beam (or setof beams) repeatedly then the monitor considers that theanimal is exhibiting stereotypy, which typically involvesgrooming, licking, head bobbing etc.

Prior to the experiment, animals were habituated in the TestBox for 15 min. After the initial habituation process, the activity ofthe animal were treated and then monitored for 2 min at 15 minintervals for 2 h.

2.5.3. Rotarod test in mice

Rotarod test was used for evaluation of neuromuscularcoordination in mice treated with various extracts of the plantaccording to the protocol as described by previously (Dunham andMiya, 1957) and studied in the Rotamex 4/8 apparatus (M/sColumbus Instruments, USA). Basically the rotarod consists of a rodwhich is coated with polypropylene foam to provide friction and toprevent animals from slipping off the rod. The distance betweenthe rod and floor is kept 15 cm to avoid intentional jumping ofmice. The rod is driven by a motor and the rotational speed can beregulated which is maintained at 8 rpm in our study. Animals weretrained on the rotarod for duration of 2 min per trial, with 3 trialsper day for two days. On the third day, mice were given trialsbefore and after treatment of extract. The extracts were used at thehighest dose of 200 mg/kg p.o. in our studies, to evaluate anydefects in motor coordination.

3. Statistical analysis

The results were expressed as mean � SEM. The statisticalsignificance was determined by One-Way Analysis of Variance(ANOVA) followed by Dunnett’s test, using Prism software version5.0. P < 0.05 was considered to be statistically significant.

4. Results

4.1. Effect of ethanolic flower extract of P. rubra in elevated plus maze

test in mice

Graded doses (50–200 mg/kg, p.o.) of crude ethanolic flowerextract was studied in the elevated plus maze model. As shown in

Fig. 2. Bar diagram showing effects of crude PR flower extract in the elevated plus

maze model in mice. ***P < 0.001, **P < 0.01 vs control.

Please cite this article in press as: Chatterjee, M., et al., Anxiolytic effecelevated plus-maze model of anxiety in mice. Asian J. Psychiatry (2

the Fig. 2 (Table 1), a significant increase in the time spent in openarms was observed at both doses of 50 mg/kg (P < 0.01) and100 mg/kg, p.o. (P < 0.001). However, no such effects wereobserved with the higher dose of 200 mg/kg. The results werecompared with the standard drug diazepam (1.5 mg/kg, p.o.)

4.2. Effect of P. rubra fractions in elevated plus maze test in mice

We further evaluated the effects of hexane, chloroform,butanolic soluble and insoluble PR fraction in the EPM task, atone fifth of the initial dose of the crude extract, i.e. 20 mg/kg, p.o.Our findings indicate a significant anxiolytic activity of hexane(P < 0.05) and n-butanol insoluble fractions (P < 0.001), but notin the chloroform and butanolic soluble fractions (Fig. 3 andTable 1).

4.3. Effect of graded doses of n-butanol insoluble fraction of P. rubra in

elevated plus maze test in mice

To further confirm the activity of n-butanol insoluble fractions,a graded dose study (10–40 mg/kg, p.o.) was carried out in mice. Asshown in the Fig. 4, we observed an increase in the time spent inopen arms at 10 mg/kg, p.o. as compared to vehicle treated groups,however it showed significant difference only at 20 mg/kg, p.o.(P < 0.001). At higher doses of 40 mg/kg, p.o. the effect declined tonear control levels (Fig. 4 and Table 1).

Fig. 3. Bar diagram showing effects of fractions of PR flower extract in the elevated

plus maze model in mice. ***P < 0.001, **P < 0.01 vs control.

ts of Plumeria rubra var. acutifolia (Poiret) L. flower extracts in the012), http://dx.doi.org/10.1016/j.ajp.2012.09.005

Fig. 4. Bar diagram showing the effects of graded doses of active fractions of PR

flower extract in the elevated plus maze model in mice. ***P < 0.001 vs control.

Table 2Effect of PR extract and n-butane insoluble fraction on locomotor activity counts.

Control Ethanolic extract n-Butanol insoluble fraction

0 min 238.33 � 42.96 214.2 � 36.99 256.0 � 20.89

15 min 126 � 22.47 103.7 � 10.28 121.6 � 18.36

30 min 124.6 � 18.08 123.2 � 17.82 107.1 � 30.73

45 min 91.5 � 19.37 39.5 � 22.42 146.1 � 15.14

60 min 55.6 � 18.34 64.5 � 18.93 125.6 � 34.54

75 min 40.17 � 6.44 39.0 � 22.43 78.6 � 16.30

90 min 71.8 � 24.49 100.7 � 42.16 52.5 � 23.75

105 min 31.3 � 19.87 20.7 � 11.42 28.5 � 19.62

120 min 20.5 � 8.76 35.0 � 9.30 19.5 � 10.41

135 min 38.5 � 25.44 50.5 � 22.23 13.5 � 5.97

150 min 25.1 � 14.57 13.2 � 5.17 13.8 � 8.41

165 min 37.5 � 15.23 0.7 � 0.47 41.8 � 16.77

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4.4. Effect of ethanolic flower extract of P. rubra and its n-butanol

insoluble fraction in Digiscan animal activity monitor in mice

PR extract at a dose 100 mg/kg showed no significant differenceon the horizontal activity in comparison to control mice (Fig. 5a

Fig. 5. Line graph showing the effects of crude flower extract ant the active fraction

in (a) horizontal activity counts (b) distance travelled and (c) stereotypy counts.

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and Table 2). Treatment with PR butane insoluble fraction (F005) atthe dose of 20 mg/kg also did not show any significant alterationsin the horizontal activity counts in mice. Further, as shown in theFig. 5b (Table 3) and c (Table 4), we observed no significantalterations in the total distance travelled and stereotypy counts inmice treated with PR extract and fractions as compared to vehicletreated group (see Fig. 6).

4.5. Effect of ethanolic flower extract of P. rubra and its n-butanol

insoluble fraction in the rotarod test in mice

To further evaluate the effects of PR extract and fractions asmuscle relaxant, rotarod test was carried out in mice. Both PRcrude extract and the n-butanol insoluble fraction treated miceshowed 100% performance in the rotarod test as compared tocontrol. No significant change in motor coordination was observedwhen compared with control groups (data not shown).

Table 3Effect of PR extract and n-butane insoluble fraction on total distance travelled (cm).

Control Ethanolic extract n-Butanol insoluble fraction

0 min 134.5 � 25.92 168.5 � 29.25 194.3 � 27.68

15 min 26.7 � 10.32 24.3 � 11.09 44.3 � 11.41

30 min 42.2 � 9.52 67.3 � 11.50 44.7 � 12.98

45 min 6.5 � 2.70 23.0 � 12.51 16.0 � 7.72

60 min 14.3 � 4.24 31.0 � 9.23 30.8 � 8.74

75 min 10.5 � 2.67 14.5 � 2.50 20.8 � 1.08

90 min 12.8 � 5.32 54.3 � 21.38 17.3 � 6.28

105 min 12.8 � 7.55 9.0 � 1.22 16.5 � 9.83

120 min 9.5 � 3.15 17.8 � 8.55 9.3 � 3.41

135 min 21.7 � 12.61 18.8 � 14.20 5.3 � 1.67

150 min 13.2 � 8.45 11.8 � 4.97 6.5 � 2.31

165 min 7.7 � 2.64 3.8 � 1.25 19.5 � 12.60

Table 4Effect of PR extract and n-butane insoluble fraction on stereotypy counts.

Control Ethanolic extract n-Butanol insoluble fraction

0 min 80.8 � 24.51 54.5 � 27.15 82.2 � 8.07

15 min 51.5 � 12.68 44.3 � 9.81 51.5 � 9.88

30 min 36.8 � 8.27 32.3 � 13.36 49.7 � 14.85

45 min 43.8 � 9.30 25.8 � 9.00 65.7 � 15.04

60 min 18.7 � 7.14 15.0 � 7.49 58.5 � 16.00

75 min 21.5 � 8.33 28.5 � 19.55 35.7 � 14.77

90 min 41.8 � 18.45 22.0 � 10.86 24.7 � 10.75

105 min 10.5 � 6.89 9.5 � 7.35 11.5 � 7.10

120 min 9.7 � 5.23 3.8 � 2.59 6.8 � 3.85

135 min 5.5 � 3.59 7.8 � 4.52 8.5 � 4.68

150 min 12.2 � 8.34 3.0 � 1.78 9.8 � 6.36

165 min 16.3 � 7.28 0.3 � 0.20 16.8 � 5.88

cts of Plumeria rubra var. acutifolia (Poiret) L. flower extracts in the012), http://dx.doi.org/10.1016/j.ajp.2012.09.005

Fig. 6. Structure of active components of fraction F005.

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5. Discussion

The study analysed the therapeutic potential of P. rubra flowerextract in experimental models of anxiety in mice. The PR extractshowed promising anxiolytic effects without causing any neuro-muscular side effects.

To study the anxiolytic profile of the extract we used theelevated plus maze model, which is currently one of the mostfrequently used models of animal anxiety (Hogg, 1996). Theelevated plus maze is considered to be an etiologically valid animalmodel of anxiety because it uses natural stimuli (fear of a novelopen space and fear of balancing on a relatively narrow, raisedplatform) that can induce anxiety in humans (Dawson andTricklebank, 1995). An anxiolytic agent increases the frequencyof entries into the open arms and increases the time spent in openarms of the EPM. The indices of anxiety in this test, percent of openarm entries and time spent in the open arm are sensitive toanxiolytic agents. Diazepam increases the percentage of open armentries and the time spent in the open arms (Crawley andGoodwin, 1980), confirming its anxiolytic effects. The flowerextract of PR (100 mg/kg) had similar effects on these parameters.A graded dose study of ethanolic flower extract reveals maximumanxiolytic activity of PR extract at 100 mg/kg, p.o. (P < 0.001),however the lower dose of 50 mg/kg (P < 0.01) also showedsignificant anxiolytic effects at the elevated plus maze model inmice. The anxiolytic effects of the drug saturated at higher doses of200 mg/kg as they did not show significant activity.

The flowers of P. rubra are reported to contain resin, quercetin,traces of kamempferol and cyanidin diglycoside, seven volatileconstituents like 2-methylbutan-1-ol, b-phenylethyl alcohol,nanodecane, heneicosane, benzyl salicylate, tetradecanoic acid,phenylacetaldehyde, and phytoconstituents like tannins, flava-noid, terpenoid, reducing sugar, phlobatannins, saponins, steroidsetc. which might show a diverse array of biological activities(Hamburger et al., 1991; Ye et al., 2008; Zaheer et al., 2010).

In this study the crude ethanolic flower extract of P. rubra wasfurther fractionated using hexane, chloroform and n-butanol andfurther studied, at much lower doses (one-fifth of its crude dose forits anxiolytic effects). It was observed that both hexane (P < 0.05)

Please cite this article in press as: Chatterjee, M., et al., Anxiolytic effecelevated plus-maze model of anxiety in mice. Asian J. Psychiatry (2

and n-butanol insoluble (P < 0.001) fractions possessed significantanxiolytic effects. However hexane fraction showed mild (�2-fold)activity compared to the n-butanol insoluble fraction (�3-fold),crude ethanolic extract (�4.5-fold) and diazepam (�3.7-fold)activity.

In order to study their adverse effect profile the effects of crudeethanolic extract and n-butanol insoluble extracts were observedfor gross locomotor activity changes. Crude ethanolic extract aswell as the butanolic insoluble fraction were observed for 2 h posttreatment. Both compounds did not show any significant altera-tions in the horizontal activity counts, total distance travelled andstereotypic counts at their effective doses. Both compounds did notcause alteration in motor coordination on the rotarod test in theprotocol studied, the extract and its fractions shows any defects inneuromuscular coordination.

Various molecular targets have been identified for thetreatment of anxiety disorders, however GABAergic and seroto-nergic systems are reported to be the major players in this disorder(Handley, 1995; Kent et al., 2002). The chloride current flowingthrough the integral ion channel of the GABAA receptor is gated bythe endogenous agonist g-aminobutyric acid (GABA), and can bemodulated by a number of different classes of compoundsincluding the barbiturates, the benzodiazepines, the cyclopyrro-lones, ethanol, zinc, and the steroids (Sieghart, 1995). Further, aninvolvement of histamine neurotransmission has also emerged inthe pathophysiology of anxiety disorders (Kuchibhotla et al., 2007).In our study, the n-butanol insoluble fraction contains b-sitosterol,b-sitosterol-b-D-glucoside plumeride like constituent (Fig. 2)which has been reported previously to possess anxiolytic activity(Aguirre-Hernandez et al., 2007).

Therefore, in this study we identified PR flower extract alongwith its butanol insoluble fraction enriched in b-sitosterol, b-sitosterol-b-D-glucoside plumeride to be a potential anxiolyticagent. The phytoconstituent(s) responsible for the observedcentral effects has to be isolated and identified in future studies.

Role of funding source

The study was supported by the Council of Scientific andIndustrial Research, New Delhi, India.

Contributors

M.C. carried out experiments, undertook the statistical analysisand prepared the first draft. M.C. and R.V. managed the literaturesearches, reviewed and finalized the draft. V.L. and A.M preparedand purified the plant extract. S.S. and A.V. carried out experi-ments. G.P. designed the study. All authors contributed to and haveapproved the final manuscript. M.C., R.V. and G.P. revised themanuscript.

Conflict of interests

Authors declare that they have no conflict of interests.

Acknowledgement

Authors are grateful to CSIR, New Delhi, India for financialsupport.

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