research article phytochemical analysis, antioxidant...

Research ArticlePhytochemical Analysis Antioxidant Antistress andNootropic Activities of Aqueous and Methanolic Seed Extracts ofLadies Finger (Abelmoschus esculentus L) in Mice

Sathish Kumar Doreddula1 Srinivasa Reddy Bonam12 Durga Prasad Gaddam34

Brahma Srinivasa Rao Desu1 Nadendla Ramarao1 and Vijayapandi Pandy5

1 Department of Pharmacology Chalapathi Institute of Pharmaceutical Sciences Guntur Andhra Pradesh 522 034 India2 Vaccine Immunology Laboratory Natural Product Chemistry Division CSIR-Indian Institute of Chemical TechnologyTarnaka Hyderabad 500007 India

3 Department of Pharmaceutical Sciences Birla Institute of Technology Mesra Ranchi Jharkhand 835215 India4Center for Chemical Biology CSIR-Indian Institute of Chemical Technology Tarnaka Hyderabad 500007 India5 Department of Pharmacology Faculty of Medicine University of Malaya 50603 Kuala Lumpur Malaysia

Correspondence should be addressed to Vijayapandi Pandy pandiphdgmailcom

Received 29 April 2014 Revised 13 August 2014 Accepted 11 September 2014 Published 21 October 2014

Academic Editor Joao B T Da Rocha

Copyright copy 2014 Sathish Kumar Doreddula et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Abelmoschus esculentus L (ladies finger okra) is a well-known tropical vegetable widely planted fromAfrica to Asia and from SouthEurope to America In the present study we investigated the in vitro antioxidant capacity and in vivo protective effect of the aqueousandmethanolic seed extracts ofAbelmoschus esculentus against scopolamine-induced cognitive impairment using passive avoidancetask and acute restraining stress-induced behavioural and biochemical changes using elevated plus maze (EPM) and forcedswimming test (FST) in mice Our results demonstrated that the pretreatment of mice with aqueous and methanolic seed extractsof Abelmoschus esculentus (200mgkg po) for seven days significantly (119875 lt 001) attenuated scopolamine-induced cognitiveimpairment in the passive avoidance test In addition these extracts significantly reduced the blood glucose corticosteronecholesterol and triglyceride levels elevated by acute restraint stress and also significantly increased the time spent in open armin EPM and decreased the immobility time in FST It has also been revealed that these extracts showed a significant antioxidantactivity and no signs of toxicity or death up to a dose of 2000mgkg poThese results suggest that the seed extracts ofAbelmoschusesculentus L possess antioxidant antistress and nootropic activities which promisingly support themedicinal values of ladies fingeras a vegetable

1 Introduction

Stress is ldquoa condition or feeling experienced when a personperceives that demands exceed the personal and socialresources that the individual is able to mobilizerdquo In our day-to-day life every human faces a stressful situation whichis characterized by a combination of physiologic neuroen-docrine behavioural and emotional responses to novel orthreatening stimuli Stress causes disturbance in the bodyrsquosnormal physiological equilibrium and results in threatened

homeostasis and there is increasing evidence that overstressaffects cognitive functions and contributes to the develop-ment of many neuropsychiatric and neurodegenerative dis-orders including depression and anxiety Alzheimerrsquos diseaseand Parkinsonrsquos disease [1] Depression and anxiety disordersare extremely common in general population [2] Stressresponse activates bodyrsquos hypothalamic-pituitary-adrenal(HPA) axis resulting in elevated corticosterone hormone lev-els Previous studies have shown that acute restraint stress inrodents increases anxiety and depression-like behaviours [3]

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 519848 14 pageshttpdxdoiorg1011552014519848

2 The Scientific World Journal

An animal model that generates depression and anxiety-likebehaviours in response to stressful stimuli is very useful indetermining antidepressant and anxiolytic efficacy The ele-vated plus maze (EPM) is a well-established model for study-ing anxiety-like behaviour and the immobility of the animalin forced swimming test (FST) reflects a state of ldquobehaviouraldespairrdquo used to evaluate depression-like behaviour [2 4]Restraint stress or immobilization has been used extensivelyas a stressor for the study of stress-related biological bio-chemical and physiological responses in animals and iscommonly used because it is less severe than other physicalstressors such as a foot shock but is still capable of activatingthe stress response In this type of stressor movement is lim-ited by placement in a Plexiglas chamber or immobilizationbag

Dementia is a mental chronic disorder characterizedby the loss of intellectual ability sufficiently severe as toinvolve impairment of memory The most common causeof dementia is Alzheimerrsquos disease which is a progressiveneurodegenerative disorder associated with loss of neuronsin brain specific areas especially in the hippocampus [5] Thecentral cholinergic system plays an important role in learn-ing and memory processes Centrally acting antimuscarinicdrugs such as scopolamine impairs learning and memoryboth in animals and in human beings Dementia is largely ahidden problem as per the epidemiological studies in IndiaPrevalence rates for dementia increase exponentially withadvancing age Since allopathic system of medicine is yetto provide a radical cure it is worthwhile to look into newdirections which would minimize the incidents of memoryloss seen in elderly patients [6]

Medicinal plants are important sources of biologicallyactive antioxidants Natural antioxidants which are ubiqui-tous in fruits and vegetables have also received great attentionand have been studied extensively since they are effectivefree radical scavengers and are assumed to be less toxic thansynthetic antioxidants [7] Green leafy vegetables provide ahigh amount of carotene ascorbic acid and microelementswhich play important roles in nutrient metabolism andslowing down of degenerative diseases [8] Abelmoschusesculentus L (Family Malvaceae) also known as Hibiscusesculentus is an important vegetable widely distributed fromAfrica to Asia Southern Europe and America that is morecommonly known as ladies finger okra or gumbo [9]The fibres in ladies finger help to stabilize blood sugar byregulating the rate at which sugar is absorbed from theintestinal tract Previous studies reported that ladies fingerpolysaccharide possesses hepatoprotective [10] antidiabetic[11] antiulcer [12] anticancer [13 14] anti-inflammatorylaxative antihyperlipidemic antifungal and analgesic activ-ities [15] Recently some quercetin derivatives well-knownantioxidants were identified and isolated from ladies finger[16] Nutritionally the richest part of the ladies finger plantis the dried seeds The oil of ladies finger seeds is edible andthe residual meal after oil extraction is rich in protein Withthis background the present investigation aims at exploringthe natural antioxidant antistress and nootropic activitiesof the aqueous and methanolic seed extracts of Abelmoschusesculentus (AE ME)

2 Materials and Methods

21 Chemicals and Reagents Diazepam and piracetam wereobtained from Cipla Ltd Mumbai India 120573-Carotenelinoleic acid and butylated hydroxytoluene (BHT) werepurchased from Hi-Media (Mumbai India) DPPH (22-Diphenyl-1-(246-trinitrophenyl)hydrazyl) and (-)-scopola-mine hydrobromide trihydrate were purchased from Sigma-Aldrich (St Louis MO USA) TLC-solvents were of HPLCgrade obtained from Merck Mumbai India All remainingreagents were used of analytical grade

22 Plant Material The seeds of Abelmoschus esculentuswere collected from Nallaguntla Prakasam Andhra Pradesh(AP) India The seeds were identified and authenticated byDr Khasim Head of Department of botany Acharya Nagar-juna University Guntur AP India and a copy of voucherspecimen was deposited in the herbarium of the Departmentof Pharmacognosy Chalapathi Institute of PharmaceuticalSciences Guntur AP India for the future reference Afterdue authentication seeds were cleaned and shade dried at30 plusmn 5∘C and pulverized using a mechanical grinder Thecoarsely ground powder was passed through sieve number40 and stored in an airtight container at minus20∘C until extrac-tion

23 Preparation of Plant Extracts

231 Methanolic Extract of Abelmoschus esculentus Seeds(ME) The powdered material (500 g) in 80 vv methanolwas sonicated for 1 h and then extracted with 80 vvmethanol in a Soxhlet extractor (1000mL) for 72 h at roomtemperature with constant stirring The extract was filteredand the filtrate was concentrated at 30∘C under reducedpressure in a rotary evaporator until a crude solid extract wasobtained which was then freeze-dried for complete solventremoval The percentage yield obtained was 13 ww

232 Aqueous Extract of Abelmoschus esculentus Seeds (AE)The 500 g seed powder was macerated and extracted withdistilled water for 72 h at room temperature Chloroform wasadded to the mixture to prevent microbial contaminationAfter completion of extraction it was filtered and the solventwas removed by evaporation in a rotary evaporator and thesolid mass was freeze-dried The percentage yield obtainedwas 17 ww

24 Phytochemical Screening Preliminary phytochemicaltests for the detection of carbohydrates phenols flavonoidsalkaloids terpenoids steroids tannins saponins and cardiacglycosides were conducted using standard phytochemicalmethods as described elsewhere [17]

241 Thin Layer Chromatography (TLC) Analysis AE andME were loaded on silica plate (Merck Aluminium sheetmdashsilica gel 60 F 254) A mixture of hexane chloroform methanol (8 2 1) was used as the solvent system The TLCplate was kept in iodine chamber for one minute and under

The Scientific World Journal 3

UV light (254 nm) to visualize bands on chromatogram [1819]

242 Fourier Transform Infrared (FTIR) Fingerprint AnalysisFourier transform infrared (FTIR) spectrophotometer wasused to identify the characteristic functional groups in theseed extracts The AE and ME (5mg) respectively werethoroughlymixed with potassium bromide (KBr) in amortarand pressed at pressure of 6 bars within 2min in order toprepare a thin translucent sample discs The FT-IR spectrumwas obtained using Perkin Elmer 2000 spectrophotometersystemwith a scan range from400 to 4000 cmminus1 and analysedusing Bruker OPUS software [19ndash21]

25 In Vitro Antioxidant Activity

251 Ferric Reducing Antioxidant Power (FRAP) AssayFerric-reducing antioxidant power of AE and ME was mea-sured by the direct reduction of Fe3+(CN)

6to Fe2+(CN)

6and

was determined by measuring absorbance resulting from theformation of the Perl Prussian blue complex following theaddition of excess ferric ions (Fe3+) [22 23] Briefly variousconcentrations of AE and ME (25mL) were mixed with25mL of 200mM sodium phosphate buffer (pH 66) and25mL of 1 wv potassium ferricyanide [K

3Fe(CN)

6] The

mixture was incubated at 50∘C for 20min and then 25mLof 10 vv trichloroacetic acid was added This mixture wascentrifuged at 1000 rpm for 10min The supernatant (5mL)was mixed with an equal volume of deionized water and05mL of 01 wv ferric chloride and the absorbance wasmeasured spectrophotometrically at 700 nmThe assays werecarried out in triplicate and the results are expressed asmean plusmn standard error Increased absorbance of the reactionmixture indicates greater reduction capability [24]

252 DPPH Radical Scavenging Assay The stable 11-diphenyl-2-picryl hydrazyl (DPPH) free radical scavengingactivity of the AE and ME was determined by the methoddescribed by Blois with slight modification [25] One mLof 02mM DPPH solution in methanol was mixed withthe 1mL extracts of 125 250 500 1000 and 2000120583gmLconcentrations respectively The mixture was incubated indark for 20min at room temperature and the absorbance wasmeasured at 517 nm The free radical scavenging activity ofAE and ME was determined by comparing its absorbancewith that of a blank solution (no sample) Butylated hydroxy-toluene (BHT)was used as a reference standardThe ability toscavenge theDPPH radical was calculated using the followingequation

of DPPH radical scavenging activity

=(1198600minus 1198601)

1198600

times 100(1)

where 1198600is the absorbance of the blank and 119860

1is the

absorbance of the sample or standard

253 120573-Carotene-Linoleic Acid Assay Antioxidant activitywas determined by measuring the inhibition of volatileorganic compounds and the conjugated diene hydroper-oxides arising from linoleic acid oxidation described byWettasinghe and Shahidi [26]OnemLof120573-carotene solution(02mgmL in chloroform)was pipetted into a round-bottomflask containing 002mL of linoleic acid and 02mL of Tween20Themixture was then evaporated at 40∘C for 10min usinga rotary evaporator to remove chloroform Then 100mL ofoxygenated distilled water was added slowly with vigorousshaking to form an emulsion 5mL aliquots of the emulsionwere transferred into different test tubes containing 02mLof various concentrations (05ndash200mgmL) of AE and MEin methanol and the mixture was then gently mixed andplaced in a water bath at 50∘C for 2 h The same procedurewas repeated with the positive control BHT and a blank Theabsorbance of the mixtures was measured at 470 nm using aspectrophotometer until the 120573-carotene colour disappearedAll determinations were performed in triplicate

The 120573-carotene bleaching rate (119877) was calculated accord-ing to

119877 = ln (119886119887)119905 (2)

where ln = natural log 119886 = absorbance at time 119905(0) and 119887 =absorbance at time (120min)

The total antioxidant activity (AA) was calculated as thepercent inhibition relative to the control using

AA = [(119877control minus 119877sample)

119877control] times 100 (3)

254 Chelating Effects on Ferrous Ions The chelating effectwas determined according to the method of Dinis et al [27]Briefly 01mL of various concentrations (0063ndash10mgmL)of AE and ME in methanol was added to 01mL of 2mMFeCl2 The reaction was initiated by adding 02mL of 5mM

ferrozine (3-[2-Pyridyl]-5 6-diphenyl-1 2 4-triazine-4 41015840-disulfonic acid sodium-salt) and the mixture was incubatedat 37∘C for 10min After adding 15mL of double distilledH2O to themixture the absorbance wasmeasured at 562 nm

Ultrapure water instead of Ferrozine solution was used as ablank andBHTwas used as reference standardThe inhibitionpercentage of the ferrozine-Fe2+ complex formation wascalculated using the following formula

Ferrous ion-chelating ability ()

= [(119860control minus 119860 sample)

119860control] times 100

(4)

where 119860control is the absorbance of the control (control con-tained FeCl

2and ferrozine complex formation molecules)

and 119860 sample is the absorbance of the test compound

26 Experimental Animals Adult male Swiss albinomice (22plusmn 2 g) were housed under standard laboratory conditions of


temperature (22plusmn 3∘C) humidity (60plusmn 5) and illumination(12-h lightdark cycle) in polypropylene cages and were fedwater and standard laboratory food ad libitumThemice wereinitially acclimatized to the experimental housing conditionsand animal handlers for 3 days prior to all experiments inorder to minimize handling stress during the test Healthymice were selected based on the swimming ability rotarodtest and normal social behaviour All experiments were con-ducted in accordancewithNational Guidelines (CPCSEA) onthe Proper Care and Use of Animals in Laboratory Research(Indian National Science Academy New Delhi 2000) andwere approved by the Institutional Animal Ethics Committee(IAEC) (Approval number 6IAECCPSMPHARM2013-14)

27 Acute Toxicity Study Acute oral toxicity of Abelmoschusesculentus L seed extracts was determined as per Organiza-tion for Economic Cooperation and Development (OECD)guidelines 423 [28] A single oral dose (5 50 300 and2000mgkg) of extracts was administered to four micegroups (119899 = 6) and was observed individually at least onceduring the first 30min periodically during the first 24 hwith special attention given during the first 4 hours anddaily thereafter for a total of 14 days for abnormal signsdiarrhea and food and water intakeThe animal body weightand locomotor activity score using actophotometer weremeasured at 0th (initial) and 14th (final) daysOn the 14th daythe mice were anesthetized through intraperitoneal injectionof a cocktail containing ketamine (80mgkg) and xylazine(10mgkg) Blood samples were collected by cardiac punctureand hematological parameters were analysed using ABXMicros-60 hematology analyser All the vital organs werecollected andweighedTheheart liver and kidney of themicetreatedwithAE andME (2000mgkg po) were processed forhaematoxylin and eosin (HampE) histopathological stainingMice did not showany signs of toxicity or death up to a dose of2000mgkg hence 110th of the dose 200mgkg was taken asan effective dose for in vivo pharmacological studies [29 30]

28 Experimental Design The mice were randomly dividedinto two sets eachwith five groups (119899 = 6) First set of animalswere divided as Group I control (saline) Group II negativecontrol (scopolamine) Group III positive control (scopo-lamine + piracetam) Group IV scopolamine + aqueousextract (AE) andGroupV scopolamine +methanolic extract(ME) used for evaluation of nootropic activity Another setwas divided into Group I saline control (not exposed tostress) Group II negative control (untreated stress-induced)Group III positive control (diazepamimipramine treatedstress-induced) Group IV aqueous extract (AE) + stress-induced Group V methanolic extract (ME) + stress-inducedused for antistress screening The aqueous and methanolicextracts (200mgkg po) or piracetam (200mgkg ip) wereadministered once daily for seven days Memory impair-ment was induced by scopolamine treatment (04mgkgip) 30min before the passive avoidance task Diazepam(2mgkg ip) or imipramine (12mgkg ip) was given30min before the stress induction

29 Evaluation of Nootropic Activity

291 Passive Avoidance Task Passive avoidance task wascarried out as described elsewhere [31 32] The apparatus(Gemini Avoidance System San Diego CA USA) consistedof equally sized light and dark compartments (20 cm times 20 cmtimes 20 cm) separated by a guillotine door (5 cm times 5 cm)The illuminated compartment contained a 50W bulb andthe floor of nonilluminated compartment was composedof 2mm stainless steel rods spaced 1 cm apart Mouse wasgently placed in the illuminated compartment for the acqui-sition trial and the door between the two compartmentswas opened 10 s later When the mouse entered the darkcompartment the door automatically closed and an electricalfoot shock (05mA) of 2 s duration was delivered through thestainless steel rods Twenty-four hours after this acquisitiontrial the mouse was again placed in the illuminated com-partment for a retention trial The time taken for a mouse toenter the dark compartment after its door was opened wasdefined as step-down latency (SDL) for both acquisition andretention trials Latency for entering the dark compartmentwas recorded up to 300 s If a mouse did not enter thedark compartment within 300 s the mouse was removed andassigned a latency score of 300 s

210 Evaluation of Anti-Stress Activity

2101 Acute Restraint Stress Acute restraint stressmodel wasused according to Masood et al with minor modifications[33] All the animals were pretreated with the respectivevehicle or extract or standard drug for 7 days before theinduction of stress After 30min of pretreatment on day 7mice were exposed to stressful stimuli induced by restrain-ing the animals in polyvinyl chloride (PVC) restrainers of105mm length and 32mm diameter for a period of 4 hoursFollowing the induction of stress the mice were evaluated forbehavioural changes using the EPM and FST A different setof mice treated as above and the blood was withdrawn fromthe retro orbital plexus and plasma corticosterone glucosetotal protein cholesterol and triglycerides were measured[34]

2102 Elevated Plus Maze Task (EPM) EPM is a validatedbehavioural assay for measuring anxiolytic and anxiogenic-like activities of pharmacological agents in rodents [35] Inbrief the apparatus consisted of two opposite open arms (30times5 cm) and two enclosed arms (30 times 5 times 15 cm) extending froma common central platform (5 times 5 cm) The maze was 40 cmabove the floor and was constructed from plexiglass Micewere placed individually onto the center of the apparatusfacing an open arm The time spent in each arm and thenumber of entries into each arm were manually recorded byblind observer for 5min After each trial themaze was wipedclean with 10 vv ethanol solution and dried An entry wasdefined as all four paws having crossed the line between thearm and the central area Anxiolytic action was defined byincreased time in andor number of entries to open armsTheentries in closed arms and total entries were used to reflect themotor component of exploratory activity


2103 Forced Swimming Test (FST) FST was carried outaccording to the method described by Deepika et al withslight modifications [36 37] The mice were individuallyplaced into transparent plexiglass cylinders (25 cm height times18 cm in diameter) filled with water to a 15-cm depth at 25∘CEach mouse was placed into the water and forced to swimfor 6min The total duration of immobility (119904) was recordedduring the last 4min of a single 6-minute test session Micewere considered immobile when they made no attempts toescape with the exception of the movements necessary tokeep their heads above the water

211 Statistical Analysis Theresults are expressed as themeanplusmn SEM The data were analysed for statistically significanceusing two-way analysis of variance (ANOVA) followed byPost hoc multiple comparisons using Bonferroni test forantioxidant assays and passive avoidance test One-way anal-ysis of variance (ANOVA) followed by Post hoc multiplecomparisons using Tukey test was performed for plasmaparameters elevated plus maze test and forced swimmingtest 119875 lt 005 was considered to be statistically significant

3 Results

31 Phytochemical Screening Preliminary phytochemicalstudies of AE and ME revealed the presence of alkaloidscarbohydrates flavonoids phenols proteins terpenoids tan-nins and sterols Saponins and cardiac glycosides were foundto be absent in AE and ME

311 Thin Layer Chromatography Profiling Several bandswere observed during partitioning of extract componentswith solvent system indicating separation of phytocon-stituents depending on polarity Carbohydrates phenols andterpenoids were identified on chromatogram after sprayingspecific detecting agents as shown in Figure 1 The 119877

119865values

of carbohydrates phenols and terpenoids were found to be068 070 and 085 for aqueous extract and 065 073 and084 for methanolic extract respectively

312 Fourier Transform Infrared Fingerprint Analysis FTIRspectroscopic studies revealed the presence of various func-tional groups such as alkyl ketone aldehyde carboxylicacids esters and amide in aqueous and methanolic extractsof Abelmoschus esculentus respectively (Table 1 and Figures2(a) and 2(b))

32 Antioxidant Activity

321 Ferric Reducing Antioxidant Power (FRAP) Assay Thereducing power of aqueous (AE) and methanolic (ME) seedextracts of Abelmoschus esculentus compared with referenceBHT in FRAP assay has been shown in Figure 3(a) Thereducing power of these extracts was observed in a dose-dependent manner The maximum reducing power of theseextracts was determined at 1mgmL

Table 1 FTIR spectral peak values and functional groups obtained

Extract Peak values (cmminus1) Functional groups

Aqueous extract ofA esculentus

1375 1454 CndashH bending1638 C=C group1743 C=O carbonyl group

2854 2923 CndashH stretching3285 ndashOH group

Methanolic extractof A esculentus


2360 2691 CndashH stretching2853 2923 CndashH stretching3284 3379 ndashOH group

Figure 1 Thin layer chromatogram of Abelmoschus esculentus seedextracts A aqueous extract M methanolic extract The extractswere chromatographed in solvent system containing hexane chloroform methanol (8 2 1) followed by spraying of visualizationreagents for carbohydrates (ACampMC) phenols (APampMP) andterpenoids (ATampMT)

322 DPPH Radical Scavenging Assay Free radical scaveng-ing effects of theAE andMEonDPPHradicals increasedwithincrease in concentration At 0125 to 20mgmL the scav-enging activities of AE andME onDPPH radical ranged from905 to 7535 and 911 to 8242 respectively (Figure 3(b))Similarly the reference BHT (012 to 20mgmL) showed sig-nificant free radicals scavenging activities dose-dependentlyranging from 2199 to 8766

323 Antioxidant Activity against 120573-Carotene-Linoleic AcidFigure 4(a) shows the antioxidant activities of the AE andME compared to BHT At 025 to 100mgmL the antioxidantactivities of AE ME and BHT ranged from 3584 to 92764068 to 9776 and 55 to 9921 respectively

324 Chelating Effects on Ferrous Ions The ability of ferrousion chelating by the AE and ME was increased in a dose-dependentmanner as illustrated in Figure 4(b)The strongestchelating effect (7760)was obtained fromMEat 10mgmLAt this concentration the lowest chelating effect was exhib-ited byAE (7011) and the highest onewas exhibited by BHT(8420)


100015002000250030003500

394761

390364

385649 374039

328522

301025

292322

285469

174357

163892

153859

145479

137555

123023

115046

105083

84034

70895

100

95

90

85

80

75Tran

smitt

ance

()

Wavenumber (cmminus1)

(a)

390193

385257

381873

374425

367385362041

343631

337934

328454

300558

292336

285380

269113

236009

225198

174250

161840 152058

145416

136826

122793

114944

109601

102999

69505

64954

62145

100

98

96

94

92

90

88

86

84

100015002000250030003500


Tran

smitt

ance

()

(b)

Figure 2 FTIR spectrum of (a) aqueous (b) methanolic seed extract of Abelmoschus esculentus

100 200 400 800 100000

05

10

15

20

$

AEME

BHT

Concentration (120583gmL)

Abso

rban

ce at

700

nm

lowast

lowastlowast

(a)

125 250 500 1000 20000

20

40

60

80

100

D

PPH

radi

cal i

nhib

ition

()

$$$

$$

$$

AEME

BHT


(b)

Figure 3 (a) Ferric reducing power (b) DPPH radical scavenging activity of BHT AE and ME seed extracts of Abelmoschus esculentusData are representative of three independent experiments Values are mean plusmn SEMThe significance of differences was analysed by two-wayANOVA followed by Bonferroni multiple comparisons test (AE versus ME lowast119875 lt 005 lowastlowast119875 lt 001 ME versus BHT 119875 lt 005 119875 lt 0001AE versus BHT $119875 lt 005 $$119875 lt 001 $$$119875 lt 0001) (a) Treatment F (2 30) = 2062119875 lt 00001 concentration F (4 30) = 12239119875 lt 00001interaction (treatment times concentration) F (8 30 = 479 119875 lt 0001) (b) Treatment F (2 30) = 3582 119875 lt 00001 concentration F (4 30) =15848 119875 lt 00001 interaction (treatment times concentration) F (8 30 = 112 ns)

33 Acute Oral Toxicity Study In the acute oral toxicity studymice did not show any signs of toxicity up to 2000mgkg andhence the LD

50cut-off value might exceed 2000mgkg The

spontaneous locomotor activity score obtained for 10minuteswhen mice were administered with 2000mgkg of AE andME seed extracts respectively at 0th day and 14th day wasfound to be 31851 plusmn 265 32751 plusmn 250 and 325 plusmn 413 33236plusmn 27 respectively The initial and final body weight of micetreated with 2000mgkg of AE and ME was 2310 plusmn 1822584 plusmn 164 and 2394 plusmn 176 2424 plusmn 193 g respectively Themean relative organ weights and hematological parametersof mice are shown in Tables 2 and 3 respectively The HampEhistopathological staining of heart liver and kidney of micetreated with 2000mgkg AE and ME is shown in Figure 5

These studies revealed no significant abnormal changes in AEand ME treated mice when compared to control

34 Nootropic Activity

341 Passive Avoidance Task The step-down latency ofscopolamine (794 plusmn 15 s) treated mice in the passive avoid-ance task was significantly (119875 lt 0001) shorter thanthat of saline control mice (26902 plusmn 23 s) as shown inFigure 6 The shorter step-down latency of scopolamine wassignificantly reversed by pretreatment with reference drugpiracetam (2232 plusmn 161 s) Similarly AE (17101 plusmn 186 s) andME (18212 plusmn 175 s) significantly reversed the scopolamine-induced memory impairment (Figure 6)


250 500 2000 8000 100000

20

40

60

80

100

120

AEME

BHT

Ant

ioxi

dant

activ

ity (

)

$$$

$$$

$


(a)

625 125 250 500 10000

20

40

60

80

100

Ferr

ous i

on ch

elat

ing

abili

ty (

)

$$$

$$$

$$$$$

$$

AEME

BHT


(b)

Figure 4 (a) Antioxidant activity using 120573-carotene bleaching rate (b) Chelating effect on ferrous ion of BHT AE andME seed extracts ofAbelmoschus esculentus Data are representative of three independent experiments Values are mean plusmn SEM The significance of differenceswas analysed by two-way ANOVA followed by Bonferroni multiple comparisons test (AE versus ME lowast119875 lt 005 lowastlowast119875 lt 001 ME versus BHT119875 lt 005 119875 lt 0001 AE versus BHT $119875 lt 005 $$119875 lt 001 $$$119875 lt 0001) (a) Treatment F (2 30) = 2902 119875 lt 00001 concentrationF (4 30) = 19543 119875 lt 00001 interaction (treatment times concentration) F (8 30 = 151 ns) (b) Treatment F (2 30) = 5846 119875 lt 00001concentration F (4 30) = 20734 119875 lt 00001 interaction (treatment times concentration) F (8 30 = 118 ns)

Table 2 Mean relative organ weights of mice ingested with2000mgkg aqueous and methanolic seed extracts of A esculentusEach value is expressed as organ-to-body weight ratio

Organ Control AE MELiver 693 plusmn 034 622 plusmn 029 634 plusmn 025Heart 056 plusmn 005 046 plusmn 004 049 plusmn 005Kidney 166 plusmn 018 149 plusmn 013 150 plusmn 014Lungs 016 plusmn 009 015 plusmn 006 016 plusmn 003Spleen 038 plusmn 004 035 plusmn 003 036 plusmn 004Brain 198 plusmn 014 183 plusmn 011 189 plusmn 016

35 Antistress Activity

351 Effect of Seed Extracts of Abelmoschus esculentus onAcute Restraint Stress-Induced Changes in Biochemical Param-eters The AE and ME showed significant reduction inimmobilization stress when compared to the stress per segroup (Figure 7) The acute restraint stress-induced groupshowed a marked increase in serum glucose [F (4 25) = 856119875 lt 0001] corticosterone [F (4 25) = 1434 119875 lt 00001]cholesterol [F (4 35) = 9107 119875 lt 00001] and triglycerides[F (4 35) = 1379 119875 lt 00001] in mice These stress-inducedelevated levels of biochemical parameters were significantlyreversed by AE and ME for 7 days once daily and withreference drug diazepam (2mgkg ip) pretreatment asshown in Figure 7

352 Effect of Seed Extracts of Abelmoschus esculentus onEPM The one-way ANOVA results revealed that there wasa significant difference between the treatment groups on the

Table 3 Hematological parameters of mice ingested with2000mgkg aqueous (AE) and methanolic (ME) seed extractsof A esculentus

Haematologicalparameter Control AE ME

WBC (103mm3) 1025 plusmn 072 181 plusmn 427 179 plusmn 228Lymphocyte () 84 plusmn 052 786 plusmn 199 792 plusmn 086Monocyte () 2208 plusmn 027 169 plusmn 162 1766 plusmn 123Granulocytes () 1328 plusmn 073 1094 plusmn 177 1004 plusmn 235RBC (106mm3) 790 plusmn 077 745 plusmn 148 771 plusmn 098Platelet (103mm3) 7026 plusmn 212 6845 plusmn 52 655 plusmn 1384MPV (120583m3) 88 plusmn 069 87 plusmn 193 91 plusmn 204Hb (gdL) 1199 plusmn 047 123 plusmn 125 1228 plusmn 154Hematocrit () 319 plusmn 094 3805 plusmn 447 425 plusmn 696MCV (120583m3) 48 plusmn 161 525 plusmn 262 5055 plusmn 662Values are mean plusmn SD 119899 = 6 MCV mean corpuscular volume MPV meanplatelet volume Hb Hemoglobin

time spent [F (4 25) = 1180 119875 lt 00001] and number ofentries [F (4 25) = 1060 119875 lt 00001] in the open arms ofthe elevated plus maze (Figures 8(a) and 8(b)) The stressedmice group spent significantly (119875 lt 001) less time in theopen arms (9788 plusmn 117 s) when compared to the unstressedcontrol group Pretreatment with AE and ME significantly(119875 lt 005 and 119875 lt 001 resp) increased the time spentin the open arms (15731 plusmn 1128 s and 16943 plusmn 1207 s resp)when compared to the stress per se group (9788 plusmn 1170 s)The reference drug diazepam (2mgkg ip) also significantly(119875 lt 0001) increased the number of entries and time spent inopen arms of the elevated plus maze (Figures 8(a) and 8(b))


Heart

(a)

Liver

(b)

Kidney

(c)

(d) (e) (f)

(g) (h) (i)

Figure 5 Hematoxylin and eosin (HampE) stained photo micrographs of mice heart liver and kidney tissues (20times magnification) (a) (b)and (c) indicate control (d) (e) and (f) (g) (h) and (i) show no abnormality in mouse group treated with AE and ME (2000 mgkg po)respectively

353 Effect of Seed Extracts of Abelmoschus esculentus on theDuration of Immobility in the FST The one-way ANOVAresults revealed that there was a significant differencebetween the treatment groups on the immobility time [F (425) = 7272 119875 lt 0001] in FST The duration of immobilityin stressed mice was significantly (119875 lt 0001) increasedwhen compared to the control group as shown in Figure 9Pretreatment with AE and ME significantly (119875 lt 005)decreased the immobility time (9780 plusmn 960 s and 9280 plusmn890 s resp) when compared to the stress per se group (1427plusmn 122 s) The reference drug imipramine (12mgkg ip) alsosignificantly (119875 lt 001) decreased the immobility time in theforced swimming test (Figure 9)

4 Discussion

In worldwide search for novel therapeutic products for thetreatment of neurological disorders ethnopharmacological

approach has been progressed constantly demonstrating thetherapeutic effectiveness of different plant species in varietyof animal models Increasing evidences indicate the possibleassociation of oxidative stress with several diseases especiallycentral nervous system disorders including neurodegenera-tive diseases such as Alzheimerrsquos disease Parkinsonrsquos diseaseDownrsquos syndrome and neuropsychiatric diseases such asanxiety schizophrenia and major depressive disorder [38]The brain which utilizes 20 of oxygen consumed by thebody is highly vulnerable to oxidative stress due to its highO

2

consumption modest antioxidant defences and a high lipid-rich constitution [38] The lipid-rich constitution of brainfavours lipid peroxidation by the reactive oxygen species(ROS) formed primarily by reduction of molecular oxygenfrom superoxide anions [39 40]

Natural antioxidants which are ubiquitous in fruits veg-etables and medicinal plants have received great attentionand have been studied extensively since they are effective


0

50

100

150

200

250

300

Acquisition trialRetention trial

Step

-dow

n lat

ency

(s)

Con

trol

Scop

Scop

+Pi

r

Scop

+A

E

Scop

+M

E

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

Figure 6 The effect of AE and ME seed extracts of Abelmoschusesculentus on scopolamine-induced learning and memory impair-ment in the passive avoidance test Control indicates saline-treatedgroup Scop indicates scopolamine-treated group (04mgkg ip)Scop + Pir Scop + AE and Scop + ME indicate piracetam (Pir)aqueous (AE) and methanolic (ME) seed extracts of A esculentus(200mgkg po) respectively treated groups for 7 days once dailyValues are mean latency plusmn SEM (119899 = 6)

119875 lt 0001 whencompared to control group and lowastlowastlowast119875 lt 0001 when compared toScop-treated group Acquisition-retention trials F (1 50) = 35192119875 lt 00001 treatments F (4 50) = 1404 119875 lt 00001 interaction(acquisition-retention trials times treatments) F (5 50 = 1568 119875 lt00001)

free radical scavengers and are assumed to be less toxicthan synthetic antioxidants [7] The present study is a steptowards the exploration of natural antioxidant potentialantistress and nootropic activities of the seed extracts ofAbelmoschus esculentus as it is an important vegetable andwidely consumed across the world

The antioxidant activity of methanolic seed extracts ofAbelmoschus esculentus has been studied and reported in theliterature [10 41] In the present study the antioxidant activityof aqueous and methanolic seed extracts of Abelmoschusesculentus has been compared by using ferric reducingantioxidant power DPPH radical scavenging 120573-carotene-linoleic acid assay and chelating effect on ferrous ions TheIC50

values of methanolic seed extract were found to be542 416 and 374 120583gmL and for aqueous extract 693 695and 437 120583gmL respectively in DPPH radical scavenging 120573-carotene-linoleic acid assay and chelating effect on ferrousionsThese results revealed that bothmethanolic and aqueousseed extracts showed very weak antioxidant activity obtainedat 1 to 10mgmL

In acute toxicity study the oral administration of Abel-moschus esculentus seed extracts (up to 2000mgkg) did notshowany signs of toxicity andmortality up to 14 daysAppear-ance and behaviour of the extracts-treated animals weresimilar to the vehicle control group during the observationperiod Moreover the spontaneous locomotor activity of theanimals treated with extracts AE andME (up to 2000mgkg)

was not significantly altered Spontaneous locomotor activ-ity is an apical test of neuromotor function representingthe peak of neural integration which has been used fordecades to evaluate effects of chemical treatment [42] Inaddition normal body weight gains were observed in theextracts treated groups and therewas no significant differencebetween relative organ weights haematological parametersandHampE histopathological staining among treatment groups(Tables 2 and 3 Figure 5) It has been previously reportedthat potentially toxic substances can drastically reduce thebody weight gain and internal organ weights [43] It hasalso been demonstrated that the alteration of haematologicalparameters is a risk evaluation of higher predictive value forhuman toxicity when the data are translated from animalstudies [44] Therefore the present oral acute toxicity studysuggest that the aqueous and methanolic seed extracts ofAbelmoschus esculentus are safe at a dose level of 2000mgkgbody weight However the present study did not evaluatethe different vital organs toxicity of the extracts AE andME using various biochemical parameters Further acute andsubchronic (28 days) oral dose toxicity studies are warrantedto investigate the potential toxicity after single and 28-dayrepeated oral dosing of Abelmoschus esculentus extracts inexperimental animals

Stress-induced psychiatric disorders such as anxiety anddepression are commonest psychiatric diagnosis in patientsattending psychiatric clinics [45] Approximately two-thirdsof the anxious or depressed patients respond to the currentlyavailable treatments but the magnitude of improvementis still disappointing Although there are many effectiveantidepressants available today current armamentarium oftherapy is often inadequate with unsatisfactory results inabout one-third of all subjects treated [46] Prolonged severestress creates ineffective adaptation which results in reducedstamina andmoodThe concept of ldquoadaptogensrdquo as a separategroup of medicinal substances was first developed by Lazarevin the year 1958 [47] Since the introduction of adaptogensseveral plants that had once been used as tonics have beeninvestigated in Ayurvedic medicine due to their adaptogenicand rejuvenating properties The acute restraint stress is awidely used behavioural model to study the molecular basisof stress-related issues [4 48] Typically a stress response ischaracterized by the activation of hypothalamus-pituitary-adrenal axis resulting in an increase in blood corticos-terone levels which in turn lead to an increase in serumtriglycerides levels and hyperglycemia [49] The presentstudy results revealed that administration of Abelmoschusesculentus seed extracts (AE andME) significantly counteredthe acute restraint stress-induced elevated blood glucosecorticosterone cholesterol and triglycerides levels in miceThis demonstrated stress-relieving potential of ladies fingerseeds

EPM and FST are widely used animal models of anxietyand depression respectively for the screening of antistressactivity of drugs The FST model is based on despair orhelplessness behaviour in response to some inescapableand confined space and is sensitive to various antidepres-sant drugs Recently Ebrahimzadeh et al demonstrated theantidepressant activity of single dose of Hibiscus esculentus


0

25

50

75

100

125

150

175Pl

asm

a glu

cose

(mg

dL)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowastlowastlowast

lowastlowast

(a)

0

50

100

150

200

250

Plas

ma c

ortic

oste

rone

(ng

mL)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowastlowastlowast

lowastlowastlowast

(b)

0

25

50

75

100

125

150

Plas

ma c

hole

stero

l (m

gdL

)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowast

lowastlowastlowast

(c)

0

25

50

75

100

125

Plas

ma t

rigly

cerid

es (m

gdL

)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowast

lowastlowastlowast

lowastlowast

(d)

Figure 7 Effect of AE and ME seed extracts of Abelmoschus esculentus on acute restraint stress-induced changes in biochemical parameters(a) Plasma glucose (b) plasma corticosterone (c) plasma cholesterol (d) plasma triglycerides Mice were treated 7 days once daily withdiazepam (2mgkg ip) AE and ME (200mgkg po) The values shown are the mean plusmn SEM (119899 = 6) 119875 lt 0001 when compared tocontrol group lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 when compared to stress per se group

methanolic seed and leaf extracts using FST and tail suspen-sion tests (TST) [50] To further support these results we haveevaluated the antistress potential of seven days pretreatmentof aqueous and methanolic seed extracts of Abelmoschusesculentus by FST after acute restraint stress induction Theseed extracts (AE andME) treatedmice significantly reversedthe acute restraint stress-induced immobility in FST as shownin Figure 9

The EPM paradigm has been described as a robustmethod for assessing anxiety responses of rodents Thepresent study revealed that the acute restraint stressed micespent significantly less time and less number of entries intothe open arms when compared to the control Whereas micepretreated with AE and ME significantly increased the timespent and number of entries into open arms these results

further demonstrated the stress-relieving potential of ladiesfinger seeds

The loss of cholinergic neurons in the basal forebrain andhippocampus and pharmacological blockade of cholinergicneurons in these areas causes impairment of learning andmemory in experimental animals [51 52] Several stud-ies have employed scopolamine a nonselective muscarinicreceptor antagonist used to impair memory in laboratoryanimals served as a test model of cognitive function andthis model has been widely used to investigate the memoryenhancing properties of new test substances [46 53ndash57]In present study Abelmoschus esculentus seed extracts (AEand ME) were found to be enhancing cognitive functionin mouse model of scopolamine-induced amnesia usingpassive avoidance test Recently it has been reported that


0

50

100

150

200

250Ti

me s

pent

in o

pen

arm

(s)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowast

lowastlowastlowast

lowastlowast

(a)

0

5

10

15

Num

ber o

f ent

ries i

n op

en ar

m

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowast

lowastlowastlowast

lowastlowast

(b)

Figure 8 (a) Time spent and (b) number of entries in the open arms in elevated plus maze test The Diaz AE and ME groups were dailytreated with the diazepam (2mgkg ip) aqueous and methanolic seed extracts of Abelmoschus esculentus (200mgkg po) for 7 daysrespectively once daily and then stress was induced The values shown are the mean plusmn SEM (119899 = 6) 119875 lt 005 119875 lt 001 when comparedto control group lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 when compared to stress per se group

0

25

50

75

100

125

150

175

Imm

obili

ty ti

me (

s)

lowastlowast

lowastlowast

Con

trol

Stre

ss

Stre

ss+

Imp

Stre

ss+

AE

Stre

ss+

ME

Figure 9 Effect of aqueous and methanolic seed extracts ofAbelmoschus esculentus on the duration of immobility in the forcedswimming test in mice The Imp AE and ME groups were dailytreated with imipramine (12mgkg ip) aqueous and methanolicseed extracts (200mgkg po) respectively for 7 days once dailyand then stress was induced after 30min of last dose The valuesshown are the mean plusmn SEM (119899 = 6) 119875 lt 0001 when comparedto control group lowast119875 lt 005 and lowastlowast119875 lt 001 when compared tostressed group

Abelmoschus esculentus L fruit extract and its bioactive prin-ciples (quercetin and rutin) protected neuronal function andimproved learning and memory deficits caused by prolongedtreatment (21 days) of 60mgkg dexamethasone using theMorris water maze task [58] Thus the present results areconsistent with the earlier reports on the cognitive function

and demonstrated the nootropic activity of ladies fingerseeds

Inhibition of acetylcholinesterase (AChE) is still consid-ered as the main therapeutic strategy against Alzheimerrsquosdisease (AD) and cognitive deficits Many plant derivedphytochemicals claimed for nootropic activity have shownAChE inhibitory effect andor antioxidant activity [59] Toour knowledge based on thorough literature search therewas no report on AChE inhibitory effect of Abelmoschusesculentus However its major bioactive principles quercetinand rutin have been reported for AChE inhibitory effect[60] Nasal administration of quercetin (in the form ofliposomes 05mg of quercetin once daily 3 weeks) in aAF64A (ethylcholine mustard aziridinium) rat model ofAlzheimerrsquos disease improvedmemory deficits (spatial learn-ing and memory) studied in Morris water maze test [61]Further studies in this direction are currently underway inour laboratory to further explore the precise mechanisminvolved in the nootropic activity of Abelmoschus esculentusseed extracts

In conclusion the present study results provide evidencethat the aqueous andmethanolic seed extracts ofAbelmoschusesculentus possess antioxidant antistress (adaptogenic) andnootropic activities This study also provides scientific evi-dence of the traditional claim ofAbelmoschus esculentus fruitsin stress-related disorders and dementia Further investiga-tions are required to characterize the active constituent(s)responsible for the observed activities and to elucidate thedetailed mechanism of action at the cellular and molecularlevels

Conflict of Interests

Theauthors have declared that there is no conflict of interests


Authorsrsquo Contribution

Sathish Kumar Doreddula Srinivasa Reddy Bonam andDurga Prasad Gaddam contributed equally to this work

Acknowledgments

The Authors are indebted to the All India Council for Tech-nical Education (AICTE) Government of India New Delhifor providing amajor financial supportThey are also gratefulto the University of Malaya for providing financial assistance(HIR MOHE Project no UMC6251HIRMOHEMED05(H-20001-E000088) and UMRG Grant no RG495-13HTM)to one of the authors (Vijayapandi Pandy) The authors arealso grateful to the Chalapathi Institute of PharmaceuticalSciencesGuntur Andhra Pradesh India for providing all thenecessary lab facilities to carry out these studies

References

[1] T Joshi S P Sah and A Singh ldquoAntistress activity of ethanolicextract of Asparagus racemosus willd roots in micerdquo IndianJournal of Experimental Biology vol 50 no 6 pp 419ndash424 2012

[2] L Jin F Wu X Li et al ldquoAnti-depressant effects of aqueousextract from Acanthopanax senticosus in micerdquo PhytotherapyResearch vol 27 no 12 pp 1829ndash1833 2013

[3] G Patki N Solanki F Atrooz F Allam and S SalimldquoDepression anxiety-like behavior and memory impairmentare associated with increased oxidative stress and inflammationin a rat model of social stressrdquo Brain Research vol 1539 pp 73ndash86 2013

[4] H J Park Y K Hyun K H Yoon S K Kyung and I ShimldquoThe effects of Astragalus membranaceus on repeated restraintstress-induced biochemical and behavioral responsesrdquo KoreanJournal of Physiology and Pharmacology vol 13 no 4 pp 315ndash319 2009

[5] M Peizhong ldquoOxidative stress and its clinical applicationsin dementiardquo Journal of Neurodegenerative Diseases vol 2013Article ID 319898 15 pages 2013

[6] Y C Yadav A Jain and L Deb ldquoA review neuropharmaco-logical screening techniques for pharmaceuticalsrdquo InternationalJournal of Pharmacy and Pharmaceutical Sciences vol 2 no 2pp 10ndash14 2010

[7] M Z Gul L M Bhakshu F Ahmad A K Kondapi I AQureshi and I A Ghazi ldquoEvaluation ofAbelmoschusmoschatusextracts for antioxidant free radical scavenging antimicrobialand antiproliferative activities using in vitro assaysrdquo BMCComplementary and Alternative Medicine vol 11 article 642011

[8] C Olaiya and J Adebisi ldquoPhytoevaluation of the nutritionalvalues of ten green leafy vegetables in South-Western NigeriardquoThe Internet Journal of Nutrition andWellness vol 9 no 2 2010

[9] K Panadda T Walairorn P Noppakun S Maitree and CPiyanete ldquoAntioxidative activities and phenolic content ofextracts from Okra (Abelmoschus esculentus L)rdquo ResearchJournal of Biological Sciences vol 5 no 4 pp 310ndash313 2010

[10] L HuW Yu Y Li N Prasad and Z Tang ldquoAntioxidant activityof extract and its major constituents from okra seed on rathepatocytes injured by carbon tetrachloriderdquo BioMed ResearchInternational vol 2014 Article ID 341291 9 pages 2014

[11] V Sabitha S Ramachandran K R Naveen and K Pan-neerselvam ldquoAntidiabetic and antihyperlipidemic potential ofAbelmoschus esculentus (L)Moench in streptozotocin-induceddiabetic ratsrdquo Journal of Pharmacy and Bioallied Sciences vol 3no 3 pp 397ndash402 2011

[12] T A Olorunnipa C C Igbokwe T O Lawal B A Adeniyi andG B Mahady ldquoAnti-helicobacter pylori activity of Abelmoschusesculentus L moench (okra) an in vitro studyrdquo Clinical Micro-biology 2013

[13] Y-F Chu J Sun X Wu and R H Liu ldquoAntioxidant andantiproliferative activities of common vegetablesrdquo Journal ofAgricultural and Food Chemistry vol 50 no 23 pp 6910ndash69162002


[15] B N Shah A K Seth K M Maheshwari and R V DesaildquoScreening of abelmoschus esculentus fruits for its analgesicactivityrdquo Pharmacology Online vol 2 pp 17ndash21 2010

[16] G Shui and L L Peng ldquoAn improved method for the analysisof major antioxidants of Hibiscus esculentus Linnrdquo Journal ofChromatography A vol 1048 no 1 pp 17ndash24 2004

[17] S Dibyajyoti J Bindu and K V Jain ldquoPhytochemical evalu-ation and characterization of hypoglycemic activity of variousextracts of Abelmoschus esculentus linn fruitrdquo InternationalJournal of Pharmacy and Pharmaceutical Sciences vol 3 no 2pp 183ndash185 2011

[18] V Asha D Sudhanshudhar and S Namrata ldquoSpectral analysisof steroidal saponin isolated and purified from leaves extractof Asparagus racemosus (FamilymdashAsparagaceae)rdquo AmericanJournal of Advanced Drug Delivery vol 1 no 5 pp 770ndash7762013

[19] A Das Talukdar M C Dutta M Chakraborty and B K DuttaldquoPhytochemical screening and TLC profiling of plant extractsof Cyathea gigantea (Wall Ex Hook) Haltt and Cyatheabrunoniana Wall ex Hook (Cl amp Bak)rdquo Assam UniversityJournal of Science amp Technology vol 5 no 1 pp 70ndash74 2010

[20] RAshokkumar andMRamaswamy ldquoPhytochemical screeningby FTIR spectroscopic analysis of leaf extracts of selected Indianmedicinal plantsrdquo International Journal of Current Microbiologyand Applied Sciences vol 3 no 1 pp 395ndash406 2014

[21] T S Antony M P John Peter and J Y Raj ldquoPhytochemicalAnalysis of Stylosanthes fruticosa using UV-VIS FTIR andGC-MSrdquo Research Journal of Chemical Sciences vol 3 no 11 pp 14ndash23 2013

[22] M Oyaizu ldquoStudies on product of browning reaction preparedfrom glucose aminerdquo Japanese Journal of Nutrition vol 44 pp307ndash315 1986

[23] I F Benzie and J J Strain ldquoThe ferric reducing ability of plasma(FRAP) as a measure of ldquoantioxidant powerrdquo The FRAP assayrdquoAnalytical Biochemistry vol 239 no 1 pp 70ndash76 1996

[24] M E Buyukokuroglu I Gulcin M Oktay and O IKufrevioglu ldquoIn vitro antioxidant properties of dantrolenesodiumrdquo Pharmacological Research vol 44 pp 491ndash495 2011

[25] M S Blois ldquoAntioxidant determinations by the use of a stablefree radicalrdquo Nature vol 181 no 4617 pp 1199ndash1200 1958

[26] M Wettasinghe and F Shahidi ldquoAntioxidant and free radical-scavenging properties of ethanolic extracts of defatted borage


(Borago officinalis L) seedsrdquo Food Chemistry vol 67 no 4 pp399ndash414 1999

[27] T C Dinis V M C Madeira and L M Almeida ldquoActionof phenolic derivatives (acetaminophen salicylate and 5-aminosalicylate) as inhibitors of membrane lipid peroxidationand as peroxyl radical scavengersrdquo Archives of Biochemistry andBiophysics vol 315 no 1 pp 161ndash169 1994

[28] OECD (Organization for Economic Cooperation and Develop-ment)Acute oral Toxicity-Acute Toxic Class Method ChemicalsTesting Guidelines No 423 Organization for Economic Coop-eration and Development Paris France 2011

[29] U Idorenyin O Samson B Eno-obong and U Noah ldquoHis-tomorphological study of the effect of chronic consumptionof Abelmoschus esculentus and Piper guineense on the gastricmucosa of albino wistar ratsrdquo International Journal of Pharma-ceutical Research amp Allied Sciences vol 2 no 3 pp 31ndash37 2013

[30] K B Ilango R L A Pradeep D Vetrivel P Brinda andM Mishra ldquoSafety evaluation of abelmoschus esculentuspolysacchariderdquo International Journal of Pharmaceutical Sci-ences Review and Research vol 10 no 2 pp 106ndash110 2011

[31] K Abhinav M Jogender K Madhusudana V G M Naidu YK Gupta and S Ramakrishna ldquoAnti-amnesic activity of Vitexnegundo in scopolamine induced amnesia in ratsrdquo Pharmacol-ogy amp Pharmacy vol 1 no 1 pp 1ndash8 2010

[32] A Anand M K Saraf S Prabhakar and K L KhandujaldquoBacopa monniera attenuates scopolamine-induced impair-ment of spatial memory in micerdquo Evidence-Based Complemen-tary and Alternative Medicine vol 2011 Article ID 236186 10pages 2011

[33] A Masood B Banerjee V K Vijayan and A Ray ldquoModulationof stress-induced neurobehavioral changes by nitric oxide inratsrdquo European Journal of Pharmacology vol 458 no 1-2 pp135ndash139 2003

[34] A Raghu Ram S N Sri Harsha D Yashwanth Kumar and KS S N Neelima ldquoEffect of Trichopus zeylanicus leaf extract onacute stress induced anxiety in micerdquo Global Journal of Medicalresearch vol 13 no 2 2013

[35] A A Walf and C A Frye ldquoThe use of the elevated plusmaze as an assay of anxiety-related behavior in rodentsrdquoNatureProtocols vol 2 no 2 pp 322ndash328 2007

[36] R Deepika K Hemamalini and U Vasireddy ldquoAdaptogenicactivity of methanolic extract of anogeissus latifolia wall andtabebuia rosea (Bertol) DC on different experimental modelsrdquoInternational Journal of Pharmacy and Pharmaceutical Sciencesvol 5 no 4 pp 457ndash463 2013

[37] R D Porsolt M Le Pichon and M Jalfre ldquoDepression a newanimal model sensitive to antidepressant treatmentsrdquo Naturevol 266 no 5604 pp 730ndash732 1977

[38] J Bouayed H Rammal and R Soulimani ldquoOxidative stress andanxiety relationship and cellular pathwaysrdquoOxidativeMedicineand Cellular Longevity vol 2 no 2 pp 63ndash67 2009

[39] K Fukui N-O Omoi T Hayasaka et al ldquoCognitive impair-ment of rats caused by oxidative stress and aging and itsprevention by vitamin Erdquo Annals of the New York Academy ofSciences vol 959 pp 275ndash284 2002

[40] S Kumar A Mishra and A K Pandey ldquoAntioxidant mediatedprotective effect of Parthenium hysterophorus against oxidativedamage using in vitro modelsrdquo BMC Complementary andAlternative Medicine vol 13 article 120 9 pages 2013

[41] P Khomsug W Thongjaroenbuangam N Pakdeenarong MSuttajit and P Chantiratikul ldquoAntioxidative activities and

phenolic content of extracts fromOkra (Abelmoschus esculentusL)rdquo Research Journal of Biological Sciences vol 5 no 4 pp 310ndash313 2010

[42] V C Moser ldquoFunctional assays for neurotoxicity testingrdquoToxicologic pathology vol 39 no 1 pp 36ndash45 2011

[43] S Teo D Stirling S Thomas A Hoberman A Kiorpesand V Khetani ldquoA 90-day oral gavage toxicity study of D-methylphenidate and DL-methylphenidate in Sprague-Dawleyratsrdquo Toxicology vol 79 no 3 pp 183ndash196 2002

[44] H Olson G Betton D Robinson et al ldquoConcordance ofthe toxicity of pharmaceuticals in humans and in animalsrdquoRegulatory Toxicology and Pharmacology vol 32 no 1 pp 56ndash67 2000

[45] M Gautam M Agrawal M Gautam P Sharma A Gautamand S Gautam ldquoRole of antioxidants in generalised anxietydisorder and depressionrdquo Indian Journal of Psychiatry vol 54no 3 pp 244ndash247 2012

[46] M H Yang K D Yoon Y-W Chin et al ldquoNeuroprotectiveeffects of Dioscorea opposita on scopolamine-inducedmemoryimpairment in in vivo behavioral tests and in vitro assaysrdquoJournal of Ethnopharmacology vol 121 no 1 pp 130ndash134 2009

[47] N V Lazarev ldquoGeneral and specific effects of drugsrdquo Far-makologiia i Toksikologiia vol 21 no 3 pp 81ndash86 1958

[48] R Duraisami V A Mohite and A J Kasbe ldquoAnti stressadaptogenic activity of standardized dried fruit extract ofaegle marmelos against diverse stressorsrdquo Asian Journal ofPharmaceutical and Clinical Research vol 3 no 4 pp 1ndash3 2010

[49] B Wang Y Shaham D Zitzman S Azari R A Wise andZ-B You ldquoCocaine experience establishes control of midbrainglutamate and dopamine by corticotropin-releasing factor arole in stress-induced relapse to drug seekingrdquo Journal ofNeuroscience vol 25 no 22 pp 5389ndash5396 2005

[50] M A Ebrahimzadeh S M Nabavi and S F Nabavi ldquoAntide-pressant activity of Hibiscus esculentus Lrdquo European Review forMedical and Pharmacological Sciences vol 17 no 19 pp 2609ndash2612 2013

[51] P W Tak J G Howland J M Robillard et al ldquoHippocampallong-term depression mediates acute stress-induced spatialmemory retrieval impairmentrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 104 no27 pp 11471ndash11476 2007

[52] M E Hasselmo ldquoThe role of acetylcholine in learning andmemoryrdquo Current Opinion in Neurobiology vol 16 no 6 pp710ndash715 2006

[53] D B Hodges Jr M D Lindner J B Hogan K M Jonesand E J Markus ldquoScopolamine induced deficits in a batteryof rat cognitive tests comparisons of sensitivity and specificityrdquoBehavioural Pharmacology vol 20 no 3 pp 237ndash251 2009

[54] H K Dong H Y Byung Y-W Kim et al ldquoThe seedextract of Cassia obtusifolia ameliorates learning and memoryimpairments induced by scopolamine or transient cerebralhypoperfusion in micerdquo Journal of Pharmacological Sciencesvol 105 no 1 pp 82ndash93 2007

[55] L Zhu L Zhang L Zhan et al ldquoThe effects of Zibu Piyin Recipecomponents on scopolamine-induced learning and memoryimpairment in the mouserdquo Journal of Ethnopharmacology vol151 no 1 pp 576ndash582 2014

[56] I D Pena S Y Yoon H J Kim et al ldquoEffects of ginseol k-g3 an rg3-enriched fraction on scopolamine-induced memoryimpairment and learning deficit in micerdquo Journal of GinsengResearch vol 38 no 1 pp 1ndash7 2014


[57] W Tongjaroenbuangam N Ruksee P Chantiratikul NPakdeenarong W Kongbuntad and P Govitrapong ldquoNeuro-protective effects of quercetin rutin and okra (Abelmoschusesculentus Linn) in dexamethasone-treated micerdquo Neurochem-istry International vol 59 no 5 pp 677ndash685 2011

[58] M Mathew and S Subramanian ldquoIn vitro screening for anti-cholinesterase and antioxidant activity ofmethanolic extracts ofayurvedic medicinal plants used for cognitive disordersrdquo PLoSONE vol 9 no 1 Article ID e86804 2014

[59] D Szwajgier ldquoAnticholinesterase activities of selectedpolyphenolsmdasha short reportrdquo Polish Journal of Food andNutrition Sciences vol 64 no 1 pp 59ndash64 2014

[60] T Tong-Un S Muchimapura W Phachonpai and J Wat-tanathorn ldquoNasal administration of quercetin liposomes mod-ulate cognitive impairment and inhibit acetylcholinesteraseactivity in hippocampusrdquoAmerican Journal of Neuroscience vol1 no 1 pp 21ndash27 2010

[61] S Sreemantula S Nammi R Kolanukonda S Koppula andK M Boini ldquoAdaptogenic and nootropic activities of aqueousextract of Vitis vinifera (grape seed) an experimental study inrat modelrdquo BMC Complementary and Alternative Medicine vol5 article 1 2005

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


An animal model that generates depression and anxiety-likebehaviours in response to stressful stimuli is very useful indetermining antidepressant and anxiolytic efficacy The ele-vated plus maze (EPM) is a well-established model for study-ing anxiety-like behaviour and the immobility of the animalin forced swimming test (FST) reflects a state of ldquobehaviouraldespairrdquo used to evaluate depression-like behaviour [2 4]Restraint stress or immobilization has been used extensivelyas a stressor for the study of stress-related biological bio-chemical and physiological responses in animals and iscommonly used because it is less severe than other physicalstressors such as a foot shock but is still capable of activatingthe stress response In this type of stressor movement is lim-ited by placement in a Plexiglas chamber or immobilizationbag

Dementia is a mental chronic disorder characterizedby the loss of intellectual ability sufficiently severe as toinvolve impairment of memory The most common causeof dementia is Alzheimerrsquos disease which is a progressiveneurodegenerative disorder associated with loss of neuronsin brain specific areas especially in the hippocampus [5] Thecentral cholinergic system plays an important role in learn-ing and memory processes Centrally acting antimuscarinicdrugs such as scopolamine impairs learning and memoryboth in animals and in human beings Dementia is largely ahidden problem as per the epidemiological studies in IndiaPrevalence rates for dementia increase exponentially withadvancing age Since allopathic system of medicine is yetto provide a radical cure it is worthwhile to look into newdirections which would minimize the incidents of memoryloss seen in elderly patients [6]

Medicinal plants are important sources of biologicallyactive antioxidants Natural antioxidants which are ubiqui-tous in fruits and vegetables have also received great attentionand have been studied extensively since they are effectivefree radical scavengers and are assumed to be less toxic thansynthetic antioxidants [7] Green leafy vegetables provide ahigh amount of carotene ascorbic acid and microelementswhich play important roles in nutrient metabolism andslowing down of degenerative diseases [8] Abelmoschusesculentus L (Family Malvaceae) also known as Hibiscusesculentus is an important vegetable widely distributed fromAfrica to Asia Southern Europe and America that is morecommonly known as ladies finger okra or gumbo [9]The fibres in ladies finger help to stabilize blood sugar byregulating the rate at which sugar is absorbed from theintestinal tract Previous studies reported that ladies fingerpolysaccharide possesses hepatoprotective [10] antidiabetic[11] antiulcer [12] anticancer [13 14] anti-inflammatorylaxative antihyperlipidemic antifungal and analgesic activ-ities [15] Recently some quercetin derivatives well-knownantioxidants were identified and isolated from ladies finger[16] Nutritionally the richest part of the ladies finger plantis the dried seeds The oil of ladies finger seeds is edible andthe residual meal after oil extraction is rich in protein Withthis background the present investigation aims at exploringthe natural antioxidant antistress and nootropic activitiesof the aqueous and methanolic seed extracts of Abelmoschusesculentus (AE ME)

2 Materials and Methods

21 Chemicals and Reagents Diazepam and piracetam wereobtained from Cipla Ltd Mumbai India 120573-Carotenelinoleic acid and butylated hydroxytoluene (BHT) werepurchased from Hi-Media (Mumbai India) DPPH (22-Diphenyl-1-(246-trinitrophenyl)hydrazyl) and (-)-scopola-mine hydrobromide trihydrate were purchased from Sigma-Aldrich (St Louis MO USA) TLC-solvents were of HPLCgrade obtained from Merck Mumbai India All remainingreagents were used of analytical grade

22 Plant Material The seeds of Abelmoschus esculentuswere collected from Nallaguntla Prakasam Andhra Pradesh(AP) India The seeds were identified and authenticated byDr Khasim Head of Department of botany Acharya Nagar-juna University Guntur AP India and a copy of voucherspecimen was deposited in the herbarium of the Departmentof Pharmacognosy Chalapathi Institute of PharmaceuticalSciences Guntur AP India for the future reference Afterdue authentication seeds were cleaned and shade dried at30 plusmn 5∘C and pulverized using a mechanical grinder Thecoarsely ground powder was passed through sieve number40 and stored in an airtight container at minus20∘C until extrac-tion

23 Preparation of Plant Extracts

231 Methanolic Extract of Abelmoschus esculentus Seeds(ME) The powdered material (500 g) in 80 vv methanolwas sonicated for 1 h and then extracted with 80 vvmethanol in a Soxhlet extractor (1000mL) for 72 h at roomtemperature with constant stirring The extract was filteredand the filtrate was concentrated at 30∘C under reducedpressure in a rotary evaporator until a crude solid extract wasobtained which was then freeze-dried for complete solventremoval The percentage yield obtained was 13 ww

232 Aqueous Extract of Abelmoschus esculentus Seeds (AE)The 500 g seed powder was macerated and extracted withdistilled water for 72 h at room temperature Chloroform wasadded to the mixture to prevent microbial contaminationAfter completion of extraction it was filtered and the solventwas removed by evaporation in a rotary evaporator and thesolid mass was freeze-dried The percentage yield obtainedwas 17 ww

24 Phytochemical Screening Preliminary phytochemicaltests for the detection of carbohydrates phenols flavonoidsalkaloids terpenoids steroids tannins saponins and cardiacglycosides were conducted using standard phytochemicalmethods as described elsewhere [17]

241 Thin Layer Chromatography (TLC) Analysis AE andME were loaded on silica plate (Merck Aluminium sheetmdashsilica gel 60 F 254) A mixture of hexane chloroform methanol (8 2 1) was used as the solvent system The TLCplate was kept in iodine chamber for one minute and under






6to Fe2+(CN)

6and


3Fe(CN)

6] The




=(1198600minus 1198601)

1198600

times 100(1)


1is the




119877 = ln (119886119887)119905 (2)











(4)

















3 Results



119865values


















100015002000250030003500

394761

390364

385649 374039

328522

301025

292322

285469

174357

163892

153859

145479

137555

123023

115046

105083

84034

70895

100

95

90

85

80

75Tran

smitt

ance

()


(a)

390193

385257

381873

374425

367385362041

343631

337934

328454

300558

292336

285380

269113

236009

225198

174250

161840 152058

145416

136826

122793

114944

109601

102999

69505

64954

62145

100

98

96

94

92

90

88

86

84

100015002000250030003500


Tran

smitt

ance

()

(b)


100 200 400 800 100000

05

10

15

20

$

AEME

BHT


Abso

rban

ce at

700

nm

lowast

lowastlowast

(a)

125 250 500 1000 20000

20

40

60

80

100

D

PPH

radi

cal i

nhib

ition

()

$$$

$$

$$

AEME

BHT


(b)









250 500 2000 8000 100000

20

40

60

80

100

120

AEME

BHT

Ant

ioxi

dant

activ

ity (

)

$$$

$$$

$


(a)

625 125 250 500 10000

20

40

60

80

100

Ferr

ous i

on ch

elat

ing

abili

ty (

)

$$$

$$$

$$$$$

$$

AEME

BHT


(b)












Heart

(a)

Liver

(b)

Kidney

(c)

(d) (e) (f)

(g) (h) (i)



4 Discussion



2




0

50

100

150

200

250

300


Step

-dow

n lat

ency

(s)

Con

trol

Scop

Scop

+Pi

r

Scop

+A

E

Scop

+M

E

lowastlowastlowast












0

25

50

75

100

125

150

175Pl

asm

a glu

cose

(mg

dL)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowastlowastlowast

lowastlowast

(a)

0

50

100

150

200

250

Plas

ma c

ortic

oste

rone

(ng

mL)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowastlowastlowast

lowastlowastlowast

(b)

0

25

50

75

100

125

150

Plas

ma c

hole

stero

l (m

gdL

)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowast

lowastlowastlowast

(c)

0

25

50

75

100

125

Plas

ma t

rigly

cerid

es (m

gdL

)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowast

lowastlowastlowast

lowastlowast

(d)







0

50

100

150

200

250Ti

me s

pent

in o

pen

arm

(s)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowast

lowastlowastlowast

lowastlowast

(a)

0

5

10

15

Num

ber o

f ent

ries i

n op

en ar

m

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowast

lowastlowastlowast

lowastlowast

(b)


0

25

50

75

100

125

150

175

Imm

obili

ty ti

me (

s)

lowastlowast

lowastlowast

Con

trol

Stre

ss

Stre

ss+

Imp

Stre

ss+

AE

Stre

ss+

ME











Acknowledgments


References






































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of






6to Fe2+(CN)

6and


3Fe(CN)

6] The




=(1198600minus 1198601)

1198600

times 100(1)


1is the




119877 = ln (119886119887)119905 (2)











(4)

















3 Results



119865values


















100015002000250030003500

394761

390364

385649 374039

328522

301025

292322

285469

174357

163892

153859

145479

137555

123023

115046

105083

84034

70895

100

95

90

85

80

75Tran

smitt

ance

()


(a)

390193

385257

381873

374425

367385362041

343631

337934

328454

300558

292336

285380

269113

236009

225198

174250

161840 152058

145416

136826

122793

114944

109601

102999

69505

64954

62145

100

98

96

94

92

90

88

86

84

100015002000250030003500


Tran

smitt

ance

()

(b)


100 200 400 800 100000

05

10

15

20

$

AEME

BHT


Abso

rban

ce at

700

nm

lowast

lowastlowast

(a)

125 250 500 1000 20000

20

40

60

80

100

D

PPH

radi

cal i

nhib

ition

()

$$$

$$

$$

AEME

BHT


(b)









250 500 2000 8000 100000

20

40

60

80

100

120

AEME

BHT

Ant

ioxi

dant

activ

ity (

)

$$$

$$$

$


(a)

625 125 250 500 10000

20

40

60

80

100

Ferr

ous i

on ch

elat

ing

abili

ty (

)

$$$

$$$

$$$$$

$$

AEME

BHT


(b)












Heart

(a)

Liver

(b)

Kidney

(c)

(d) (e) (f)

(g) (h) (i)



4 Discussion



2




0

50

100

150

200

250

300


Step

-dow

n lat

ency

(s)

Con

trol

Scop

Scop

+Pi

r

Scop

+A

E

Scop

+M

E

lowastlowastlowast












0

25

50

75

100

125

150

175Pl

asm

a glu

cose

(mg

dL)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowastlowastlowast

lowastlowast

(a)

0

50

100

150

200

250

Plas

ma c

ortic

oste

rone

(ng

mL)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowastlowastlowast

lowastlowastlowast

(b)

0

25

50

75

100

125

150

Plas

ma c

hole

stero

l (m

gdL

)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowast

lowastlowastlowast

(c)

0

25

50

75

100

125

Plas

ma t

rigly

cerid

es (m

gdL

)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowast

lowastlowastlowast

lowastlowast

(d)







0

50

100

150

200

250Ti

me s

pent

in o

pen

arm

(s)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowast

lowastlowastlowast

lowastlowast

(a)

0

5

10

15

Num

ber o

f ent

ries i

n op

en ar

m

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowast

lowastlowastlowast

lowastlowast

(b)


0

25

50

75

100

125

150

175

Imm

obili

ty ti

me (

s)

lowastlowast

lowastlowast

Con

trol

Stre

ss

Stre

ss+

Imp

Stre

ss+

AE

Stre

ss+

ME











Acknowledgments


References






































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of













3 Results



119865values


















100015002000250030003500

394761

390364

385649 374039

328522

301025

292322

285469

174357

163892

153859

145479

137555

123023

115046

105083

84034

70895

100

95

90

85

80

75Tran

smitt

ance

()


(a)

390193

385257

381873

374425

367385362041

343631

337934

328454

300558

292336

285380

269113

236009

225198

174250

161840 152058

145416

136826

122793

114944

109601

102999

69505

64954

62145

100

98

96

94

92

90

88

86

84

100015002000250030003500


Tran

smitt

ance

()

(b)


100 200 400 800 100000

05

10

15

20

$

AEME

BHT


Abso

rban

ce at

700

nm

lowast

lowastlowast

(a)

125 250 500 1000 20000

20

40

60

80

100

D

PPH

radi

cal i

nhib

ition

()

$$$

$$

$$

AEME

BHT


(b)









250 500 2000 8000 100000

20

40

60

80

100

120

AEME

BHT

Ant

ioxi

dant

activ

ity (

)

$$$

$$$

$


(a)

625 125 250 500 10000

20

40

60

80

100

Ferr

ous i

on ch

elat

ing

abili

ty (

)

$$$

$$$

$$$$$

$$

AEME

BHT


(b)












Heart

(a)

Liver

(b)

Kidney

(c)

(d) (e) (f)

(g) (h) (i)



4 Discussion



2




0

50

100

150

200

250

300


Step

-dow

n lat

ency

(s)

Con

trol

Scop

Scop

+Pi

r

Scop

+A

E

Scop

+M

E

lowastlowastlowast












0

25

50

75

100

125

150

175Pl

asm

a glu

cose

(mg

dL)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowastlowastlowast

lowastlowast

(a)

0

50

100

150

200

250

Plas

ma c

ortic

oste

rone

(ng

mL)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowastlowastlowast

lowastlowastlowast

(b)

0

25

50

75

100

125

150

Plas

ma c

hole

stero

l (m

gdL

)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowast

lowastlowastlowast

(c)

0

25

50

75

100

125

Plas

ma t

rigly

cerid

es (m

gdL

)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowast

lowastlowastlowast

lowastlowast

(d)







0

50

100

150

200

250Ti

me s

pent

in o

pen

arm

(s)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowast

lowastlowastlowast

lowastlowast

(a)

0

5

10

15

Num

ber o

f ent

ries i

n op

en ar

m

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowast

lowastlowastlowast

lowastlowast

(b)


0

25

50

75

100

125

150

175

Imm

obili

ty ti

me (

s)

lowastlowast

lowastlowast

Con

trol

Stre

ss

Stre

ss+

Imp

Stre

ss+

AE

Stre

ss+

ME











Acknowledgments


References






































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


100015002000250030003500

394761

390364

385649 374039

328522

301025

292322

285469

174357

163892

153859

145479

137555

123023

115046

105083

84034

70895

100

95

90

85

80

75Tran

smitt

ance

()


(a)

390193

385257

381873

374425

367385362041

343631

337934

328454

300558

292336

285380

269113

236009

225198

174250

161840 152058

145416

136826

122793

114944

109601

102999

69505

64954

62145

100

98

96

94

92

90

88

86

84

100015002000250030003500


Tran

smitt

ance

()

(b)


100 200 400 800 100000

05

10

15

20

$

AEME

BHT


Abso

rban

ce at

700

nm

lowast

lowastlowast

(a)

125 250 500 1000 20000

20

40

60

80

100

D

PPH

radi

cal i

nhib

ition

()

$$$

$$

$$

AEME

BHT


(b)









250 500 2000 8000 100000

20

40

60

80

100

120

AEME

BHT

Ant

ioxi

dant

activ

ity (

)

$$$

$$$

$


(a)

625 125 250 500 10000

20

40

60

80

100

Ferr

ous i

on ch

elat

ing

abili

ty (

)

$$$

$$$

$$$$$

$$

AEME

BHT


(b)












Heart

(a)

Liver

(b)

Kidney

(c)

(d) (e) (f)

(g) (h) (i)



4 Discussion



2




0

50

100

150

200

250

300


Step

-dow

n lat

ency

(s)

Con

trol

Scop

Scop

+Pi

r

Scop

+A

E

Scop

+M

E

lowastlowastlowast












0

25

50

75

100

125

150

175Pl

asm

a glu

cose

(mg

dL)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowastlowastlowast

lowastlowast

(a)

0

50

100

150

200

250

Plas

ma c

ortic

oste

rone

(ng

mL)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowastlowastlowast

lowastlowastlowast

(b)

0

25

50

75

100

125

150

Plas

ma c

hole

stero

l (m

gdL

)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowast

lowastlowastlowast

(c)

0

25

50

75

100

125

Plas

ma t

rigly

cerid

es (m

gdL

)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowast

lowastlowastlowast

lowastlowast

(d)







0

50

100

150

200

250Ti

me s

pent

in o

pen

arm

(s)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowast

lowastlowastlowast

lowastlowast

(a)

0

5

10

15

Num

ber o

f ent

ries i

n op

en ar

m

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowast

lowastlowastlowast

lowastlowast

(b)


0

25

50

75

100

125

150

175

Imm

obili

ty ti

me (

s)

lowastlowast

lowastlowast

Con

trol

Stre

ss

Stre

ss+

Imp

Stre

ss+

AE

Stre

ss+

ME











Acknowledgments


References






































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


250 500 2000 8000 100000

20

40

60

80

100

120

AEME

BHT

Ant

ioxi

dant

activ

ity (

)

$$$

$$$

$


(a)

625 125 250 500 10000

20

40

60

80

100

Ferr

ous i

on ch

elat

ing

abili

ty (

)

$$$

$$$

$$$$$

$$

AEME

BHT


(b)












Heart

(a)

Liver

(b)

Kidney

(c)

(d) (e) (f)

(g) (h) (i)



4 Discussion



2




0

50

100

150

200

250

300


Step

-dow

n lat

ency

(s)

Con

trol

Scop

Scop

+Pi

r

Scop

+A

E

Scop

+M

E

lowastlowastlowast












0

25

50

75

100

125

150

175Pl

asm

a glu

cose

(mg

dL)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowastlowastlowast

lowastlowast

(a)

0

50

100

150

200

250

Plas

ma c

ortic

oste

rone

(ng

mL)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowastlowastlowast

lowastlowastlowast

(b)

0

25

50

75

100

125

150

Plas

ma c

hole

stero

l (m

gdL

)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowast

lowastlowastlowast

(c)

0

25

50

75

100

125

Plas

ma t

rigly

cerid

es (m

gdL

)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowast

lowastlowastlowast

lowastlowast

(d)







0

50

100

150

200

250Ti

me s

pent

in o

pen

arm

(s)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowast

lowastlowastlowast

lowastlowast

(a)

0

5

10

15

Num

ber o

f ent

ries i

n op

en ar

m

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowast

lowastlowastlowast

lowastlowast

(b)


0

25

50

75

100

125

150

175

Imm

obili

ty ti

me (

s)

lowastlowast

lowastlowast

Con

trol

Stre

ss

Stre

ss+

Imp

Stre

ss+

AE

Stre

ss+

ME











Acknowledgments


References






































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


Heart

(a)

Liver

(b)

Kidney

(c)

(d) (e) (f)

(g) (h) (i)



4 Discussion



2




0

50

100

150

200

250

300


Step

-dow

n lat

ency

(s)

Con

trol

Scop

Scop

+Pi

r

Scop

+A

E

Scop

+M

E

lowastlowastlowast












0

25

50

75

100

125

150

175Pl

asm

a glu

cose

(mg

dL)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowastlowastlowast

lowastlowast

(a)

0

50

100

150

200

250

Plas

ma c

ortic

oste

rone

(ng

mL)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowastlowastlowast

lowastlowastlowast

(b)

0

25

50

75

100

125

150

Plas

ma c

hole

stero

l (m

gdL

)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowast

lowastlowastlowast

(c)

0

25

50

75

100

125

Plas

ma t

rigly

cerid

es (m

gdL

)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowast

lowastlowastlowast

lowastlowast

(d)







0

50

100

150

200

250Ti

me s

pent

in o

pen

arm

(s)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowast

lowastlowastlowast

lowastlowast

(a)

0

5

10

15

Num

ber o

f ent

ries i

n op

en ar

m

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowast

lowastlowastlowast

lowastlowast

(b)


0

25

50

75

100

125

150

175

Imm

obili

ty ti

me (

s)

lowastlowast

lowastlowast

Con

trol

Stre

ss

Stre

ss+

Imp

Stre

ss+

AE

Stre

ss+

ME











Acknowledgments


References






































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0

50

100

150

200

250

300


Step

-dow

n lat

ency

(s)

Con

trol

Scop

Scop

+Pi

r

Scop

+A

E

Scop

+M

E

lowastlowastlowast












0

25

50

75

100

125

150

175Pl

asm

a glu

cose

(mg

dL)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowastlowastlowast

lowastlowast

(a)

0

50

100

150

200

250

Plas

ma c

ortic

oste

rone

(ng

mL)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowastlowastlowast

lowastlowastlowast

(b)

0

25

50

75

100

125

150

Plas

ma c

hole

stero

l (m

gdL

)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowast

lowastlowastlowast

(c)

0

25

50

75

100

125

Plas

ma t

rigly

cerid

es (m

gdL

)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowast

lowastlowastlowast

lowastlowast

(d)







0

50

100

150

200

250Ti

me s

pent

in o

pen

arm

(s)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowast

lowastlowastlowast

lowastlowast

(a)

0

5

10

15

Num

ber o

f ent

ries i

n op

en ar

m

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowast

lowastlowastlowast

lowastlowast

(b)


0

25

50

75

100

125

150

175

Imm

obili

ty ti

me (

s)

lowastlowast

lowastlowast

Con

trol

Stre

ss

Stre

ss+

Imp

Stre

ss+

AE

Stre

ss+

ME











Acknowledgments


References






































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0

25

50

75

100

125

150

175Pl

asm

a glu

cose

(mg

dL)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowastlowastlowast

lowastlowast

(a)

0

50

100

150

200

250

Plas

ma c

ortic

oste

rone

(ng

mL)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowastlowastlowast

lowastlowastlowast

(b)

0

25

50

75

100

125

150

Plas

ma c

hole

stero

l (m

gdL

)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowast

lowastlowastlowast

(c)

0

25

50

75

100

125

Plas

ma t

rigly

cerid

es (m

gdL

)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowast

lowastlowastlowast

lowastlowast

(d)







0

50

100

150

200

250Ti

me s

pent

in o

pen

arm

(s)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowast

lowastlowastlowast

lowastlowast

(a)

0

5

10

15

Num

ber o

f ent

ries i

n op

en ar

m

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowast

lowastlowastlowast

lowastlowast

(b)


0

25

50

75

100

125

150

175

Imm

obili

ty ti

me (

s)

lowastlowast

lowastlowast

Con

trol

Stre

ss

Stre

ss+

Imp

Stre

ss+

AE

Stre

ss+

ME











Acknowledgments


References






































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0

50

100

150

200

250Ti

me s

pent

in o

pen

arm

(s)

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowast

lowastlowastlowast

lowastlowast

(a)

0

5

10

15

Num

ber o

f ent

ries i

n op

en ar

m

Con

trol

Stre

ss

Stre

ss+

Dia

z

Stre

ss+

AE

Stre

ss+

ME

lowast

lowastlowastlowast

lowastlowast

(b)


0

25

50

75

100

125

150

175

Imm

obili

ty ti

me (

s)

lowastlowast

lowastlowast

Con

trol

Stre

ss

Stre

ss+

Imp

Stre

ss+

AE

Stre

ss+

ME











Acknowledgments


References






































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




Acknowledgments


References






































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of












































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

research article phytochemical analysis, antioxidant...

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