research article evaluation of anxiolytic-like effect of

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 587260 10 pageshttpdxdoiorg1011552013587260

Research ArticleEvaluation of Anxiolytic-Like Effect of Aqueous Extract ofAsparagus Stem in Mice

Long Cheng1 Guo-feng Pan2 Xiao-bo Sun1 Yun-xiang Huang3

You-shun Peng4 and Lin-yan Zhou5

1 Institute of Medicinal Plant Development Chinese Academy of Medical Sciences Beijing 100094 China2Department of TCM Beijing Shijitan Hospital Affiliated to Capital Medical University Beijing 100038 China3 Asparagus Engineering Research Centers of Hebei Province Qinhuangdao 066008 China4Hebei Normal University of Science amp Technology Qinhuangdao 066004 China5 Institute of Agro-Products Processing Science amp Technology Chinese Academy of Agricultural Sciences Beijing 100193 China

Correspondence should be addressed to Xiao-bo Sun sun-xiaobo163com

Received 10 July 2013 Revised 19 September 2013 Accepted 3 October 2013

Academic Editor Sarang Bani

Copyright copy 2013 Long Cheng et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

There are few studies on the neuropharmacological properties of asparagus which was applied in Chinese traditional medicineas a tonic and heat-clearing agent The present study was designed to investigate the anxiolytic-like activity of the aqueous extractof asparagus stem (AEAS) using elevated plus maze (EPM) and Vogel conflict tests (VCT) in mice AEAS significantly increasedthe percentage of time spent in open arms in EPM when compared with control group In the Vogel conflict drinking test thenumbers of punished licks increased to 177 and 174 by the treatment of AEAS at the doses of 15 and 30 gkg (250 and 500mgsarsasapogenin per kilogram of body weight) compared with control group The serum cortisol level decreased significantly atthe same time In conclusion these findings indicated that the aqueous extract of asparagus stem exhibited a strong anxiolytic-likeeffect at dose of 15 and 30 gkg (250 and 500mg sarsasapogenin per kilogram of body weight) in experimental models of anxietyand may be considered an alternative approach for the management of anxiety disorder

1 Introduction

Anxiety and related disordersmost commonmental illnessesworldwide represent a prominent healthcare problem [1ndash4]Reports from theWorldHealthOrganization (WHO) suggestthat anxiety and related disorders will become the secondleading cause of disability in both developed and developingcountries by the year 2020 [5] Benzodiazepines are amongthe first line of drugs that have been extensively used totreat several forms of anxiety [6] Although benzodiazepineshavewell-known benefits their side effects are prominent [7]Therefore the search for new therapeutic agents continuesInterest in alternative medicine and plant-derived functionalfood to conquer stress and promote relaxation has increasedrecently For example the concept of a relaxation drink firstemerged in Japan in 2005 [8]

Asparagus officinalis L a well-known nutritious andhealthy vegetable was applied widely in the traditional

Chinese medicine clinic practice as a tonic heat-clearingantitussive and diuretic agent In recent years various health-ful effects of the asparagus have been scientifically verified[9ndash13] Asparagus industrial product development is in theascendant in China In early 2013 the Chinese governmentauthorized the aqueous extract of asparagus stem (AEAS) as anatural functional food and beverage ingredient The presentstudy investigated the potential anxiolytic effect of AEAS inmice

2 Materials and Methods

21 Plant Material Material was collected from cultivationin Hangu administration zone of Tangshan a vegetationzone that belongs to Qinhuangdao Changsheng AgriculturalTechnology Development Co Ltd Botanical samples wereidentified by Professor Peng of Hebei Normal University ofScience and Technology Qinhuangdao China

2 Evidence-Based Complementary and Alternative Medicine

40383634323028262422201816141210

864

2826242220181614121086420

(mV

)

(min)

26107

(a) The refer substance

(min)

(mV

)

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28

26150

185175165155145135125115105

958575655545352515

5

(b) The sample of AEAS

Figure 1 The chromatography of sarsasapogenin from AEAS

22 Extract Preparation Aqueous extract from the stemof Asparagus officinalis L was provided by QinhuangdaoChangshengAgricultural TechnologyDevelopmentCo LtdHebei Province China To obtain the aqueous extract fromthe woody stem of asparagus fresh plant material (600 kg)was cleaned crushed and then extracted with drinking water(plantwater ratio 1 8 wv water temperature 85 plusmn 5∘C) bydynamic maceration for 5 h After filtering the solution ofasparagus stem was treated with spray drying to drynessyielding extract powder (10 kg AEAS)

23 Extract Character The nutrient and chemical compo-nents of this studied extract were identified and quanti-fied (shown in Table 1) Amino acid composition analysiswas performed by a fully automatic amino acid analyzer(Hitachi L-8900 Japan) The data were shown in Table 2The main biologically active constituents of asparagus arethe steroidal saponins and flavonoids For quantitative deter-mination of the steroidal saponins the AEAS was refluxedwith hydrochloric acid solution The sarsasapogenin fromasparagus was determined by reversed phase high perfor-mance liquid chromatographicmethodwith evaporative lightscattering detection (RP-HPLC-ELSD) (shown in Figure 1)The content of sarsasapogenin was 1780 The fingerprintof the flavonoids from AEAS was established by HPLC-UV(shown in Figure 2 and Table 3) Asparagus extract solutionwas mixed with diethyl ether then stirred The diethyl etherlayer was separated and evaporated to dryness The resultingresidue was dissolved in methanol as sample solution forfingerprint analyses

The analyses were performed using a Zorbax C18column

and a diode array detector in an Agilent1100 HPLC systemThe mobile phase was 05 formic acid solution (solvent A)and methanol (solvent B) at a flow rate of 07mLmin Thegradient elutionwas 0ndash20min 80ndash70A 20ndash30B 20ndash45min 70ndash60 A 30ndash40 B 45ndash60min 60ndash50 A40ndash50 B 60ndash70min 50ndash30 A 50ndash70 B and 70ndash75min 30ndash0 A 70ndash100 B The UV detector wave-length was set at 320 nm Five compounds were identified by

Table 1 Nutrient and chemical compositions of the AEAS

No Compositions Content Unit1 Carbohydrates 444 g100 g2 Proteins 245 g100 g3 Lipids 30 g100 g4 Calcium 384 g100 g5 Potassium 112 g100 g6 Magnesium 033 g100 g7 Sodium 028 g100 g8 Ferrum 006 g100 g9 Copper 019 g100 g10 Zinc 009 g100 g11 Vitamin E 017 mg100 g12 Vitamin B1 039 mg100 g13 Vitamin B2 089 mg100 g14 Selenium 014 mg100 g15 Saponins 1780 g100 g16 Flavone 291 g100 g17 Polyphenol 593 g100 g18 Polysaccharide 1121 g100 g19 GABA 082 g100 gAEAS aqueous extract of asparagus stem GABA gamma-aminobutyricacid

the spectral feature of UV MS and MSMS (Table 3) Themain 8 peaks were quantified by comparing their peak areaswith the refer substance (Peak 1)

24 Drug Distilled drinking water was used as vehicleDiazepam (DZP)was used as the reference drug for anxiolyticactivities

25 Animals Male Imprinting Control Region mice (CD-1 ICR 18sim22 g) were purchased from Beijing Vital RiverLaboratories Co Ltd (Beijing China) Animals were housedin a controlled environment (temperature 23∘ plusmn 2∘C relative

Evidence-Based Complementary and Alternative Medicine 3

Table 2 Amino acid analyses of the AEAS

No Amino acid Content (g100 g)1 ASP 5952 THR 0293 SER 0514 GLU 4775 GLY 0516 ALA 0487 VAL 0538 MET 0079 ILE 01610 LEU 04111 TYR 02312 PHE 01513 LYS 02914 HIS 01115 ARG 0 4116 PRO 26017 TRP 01118 CYS 02819 Total 1786AEAS aqueous extract of asparagus stem

(min)

0102030405060

70

(mAU

)

0 10 20 30 40 50 60

DAD1 CSig = 320 4 Ref = off (FR-LUYIMI3000006D)

1

23 45

678

Figure 2 The HPLC-UV fingerprint of the flavonoids from AEAS

humidity 50 plusmn 10 12 h lightdark cycle) and allowed freeaccess to standard diet They were acclimatized for 5 daysprior to the initiation of the test

For the repeated dose toxicity study SpragueDawley (SD)rats were used The rats were housed in stainless steel cageskept under controlled conditions of temperature (23 plusmn 2∘C)relative humidity (55 plusmn 10) and ventilation (more than 10timeshour) with a 12-hour lightdark cycle and allowed freeaccess to food and water throughout both the acclimationand experimentation periods All animals weremaintained inaccordance with the Beijing Laboratory AnimalManagementRegulations

26 Elevated Plus Maze Evaluation of the possible anxiolyticeffect was performed according to the previous literature [14ndash16] Shanghai Manpu Biotechnology Co Ltd furnished theEPM apparatus (mode RD1108-EPM-M) which comprisedtwo open (30 cm times 5 cm) and two closed arms (30 cm times5 cm times 15 cm) that extended from a common central platform

(5 cm times 5 cm) To prevent mice from falling out of themaze aledge (2mmH) surrounded the open armsThe floor and thewalls of each arm were painted black The entire maze waselevated 60 cm above floor

Experiments were conducted in a dimly lit quiet roomWe pretreatedmalemice orally with vehicle or different doses(06 15 or 30 gkg) of AEAS (equivalent to 100 250 and500mg sarsasapogenin per kilogram of body weight) 30minbefore placing them on the EPM One group of animalsreceived diazepam (3mgkg po) as reference drug To begina test session mice were placed in the center of the mazefacing one of the open arms and allowed to walk freely for5min After each trial themaze was wiped clean with a dampsponge and dried with paper towels All the tests wererecorded by video camera

Entry into an arm was defined as the animal placing allfour paws over the line marking that area Standard spa-tiotemporal measures were scored the number of entries inthe open and the closed arms the total number of arm entriesand the time spent in the different parts of themaze (open andclosed arms) The ratio of open arm entries and closed armentries to the total number of arm entries was also calculatedduring a 5min test period for each animal

27 Vogel Conflict Test Mice were habituated for 5 daysand then divided randomly into five groups Control groupanimals received drinking water only DZP group received30mgkg of diazepam (po) AEAS groups received 06 15or 30 gkg AEAS (equivalent to 100 250 and 500mg sarsas-apogenin per kilogram of body weight) per day respectivelyThis procedure continued for 7 days At 830 am of day 8 ratswere treated with AEAS DZP or drinking water The Vogelconflict test was performed about 30min later

The Vogel conflict test apparatus (mode AES-340 Ani-Lab Software and Instruments Co Ltd) was applied essen-tially using the procedure originally described by Vogel et al[17] The VCT was performed in a Plexiglas box with a stain-less steel grid floor The metallic spout of a drinking bottlethat containedwater projected into the boxThe simultaneouscontact of the animal with the spout and the grid floor closedan electrical circuit controlled by a sensor producing sevenpulses of water per second whenever the animal was in con-tact with both components Each pulse was considered a lickAfter every 20 licks the animal received a 05mA footshockfor 2 s The sensor recorded the total number of licks andshocks delivered during the test period The entire apparatuswas located inside a sound-attenuated cage After completingthe test session all ratswere anesthetizedThe femoral arterialblood was collected and centrifuged to get serum sampleTheir brains were rapidly dissected (not more than 3min)on ice then hippocampus were dissected weighed andhomogenized in 01M phosphate buffer solution by manualhomogeniser centrifuged at 10000 rpm for 20min to removesupernatant and stored at minus20∘C until analysis

28 Neurochemical Assay The cortical levels in serumwere determined by commercially available radioimmunoas-say kits (Beijing North Institute of Biological Technol-ogy Co Ltd Beijing China) Catecholamine (epinephrine


Table 3 The spectrum data of the AEAS

No UV (nm) MS MSMS Chemical name Relative peak area1 270 nm 595 [595] 331 (100) 535 (1965) 287 (819) Kaempferol-3-O-120573-rutinoside 1002 270 nm 268 [268] 136 (100) 7-hydroxy-41015840-methoxyisoflavone 0083 330 260 611 [611] 591 (100) 467 (3897) Rutin 0654 340 260 317 [317] 298 (84) 198 (100) Isorhamnetin 0725 320 280 301 [301] 269 (100) 241 (99) Quercetin 0676 0637 0468 033

norepinephrine and dopamine) and 5-hydroxytryptamine(5-HT) levels in serum and tissue homogenate were deter-mined by enzyme immunoassay kits (Cusabio BiologicalTechnology Co Ltd Wuhan China)

29 Repeated Doses Toxicity For evaluation of the ediblesafety 13 weeks repeated dose oral toxicity study was per-formed in rats SD rats were assigned into four groups ran-domly Group I control group Group II treated with AEAS125 gkg (equivalent to 208mg sarsasapogenin per kilogramof body weight) Group III treated with AEAS 250 gkg(equivalent to 417mg sarsasapogenin per kilogram of bodyweight) Group IV treated with AEAS 500 gkg (equivalentto 835mg sarsasapogenin per kilogram of body weight) Thedoses were designed according to the previous acute studyresult and the recommended daily dose (equivalent to 25-50- and 100-fold of recommended dose 30 gd for adult)

Rat gross behavior mortality body weight gain and foodintake were recorded throughout the repeated treatmentAfter 13 weeks feeding all rats were deprived of food (but notwater) overnight and then were anesthetized The urine andblood sample were collected then detected respectively Theorgans (liver kidneys adrenal glands spleen lungs heartbrain and so forth) were harvested weighed and then pro-cessed for histopathological examination Hematologicalexamination was carried out by an automatic analyzer(Hitachi 7600-010 Hitachi Co Hyogo Japan) The urineanalyses were performed with urinalysis strips (Suzhu phar-maceutical Co Ltd Jiangsu China)

210 Statistical Analysis Data were processed to give groupmean values and standard deviations The quantitative datawere assessed by one-way analysis of variance (ANOVA)If the F distribution was significant a t-test was used tospecify differences between groups 119875 lt 005 was consideredstatistically significant The SPSS 110 software package (SPSSInc Chicago IL USA) was used for the statistical test

3 Results

31 Effect of AEAS in EPM Test The one-way ANOVArevealed a significant increase in the percent of time spent inopen arms with AEAS treatment (15 and 30 gkg) comparedwith the control (Figure 3) AEAS treatment increased theratio between the number of entries in open arm and totalentries but these changes were not statistically significant No

differences were observed in total arm entries As expectedDZP treatment yielded a significantly increased percent ofopen arm entries and time spent in open arms

32 Effect of AEAS in Vogel Test To confirm the anxiolytic-like effect of AEAS the Vogel conflict test was applied In thisexperiment one-way ANOVA revealed significant varianceamong the five groups Doses of 15 and 30 gkg significantlyincreased the number of punished licks compared withcontrols (Figure 4) As expected DZP (3mgkg) significantlyincreased the number of punished licks

33 Neurochemical Assay In the Vogel conflict test theserum cortisol and epinephrine level increased signifi-cantly compared with the control (Figure 5) AEAS treat-ment resulted in decreasing levels of serum cortisol How-ever AEAS treatment produced an opposite effect on 5-HT increasing the serum 5-HT level Epinephrine levelsdecreased in AEAS- and DZP-treated groups with nostatistical significance compared with the model groupWe observed no significant changes in norepinephrine anddopamine levels between these groups

The levels of epinephrine 5-HT norepinephrine anddopamine in hippocampus homogenate showed no signifi-cant changes between groups (data not shown)

34 Repeated Doses Toxicity All rats survived for the dura-tion of the feeding trial There were no notable changes inbehavior activity posture gait or external appearance in anyof the groups There was no significant difference in averagebody weights or average daily food intakes between the ratsin the control group and those in any of the treated groupsduring the study (for both sexes) All animals were subjectedto complete necropsy For the relative organweight statisticalanalysis result showed that there was no difference betweengroups (Table 4) The qualitative analyses of urobilinogenurine glucose urine acetone bodies urobilinogen urineprotein and urine nitrite were negative The urine pH wasin normal range in both of the control group and treatedgroups As shown in Table 5 there was no significant adverseeffect in any of the treated groups compared with controlgroup Serum biochemistry results (Table 6) demonstratedthat the parameters of biochemistry were within the normalrange and statistical analysis results showed that there wasno difference between groups In the histopathological study


Control DZP AEAS-L AEAS-M AEAS-H0

10

20

30

40

50 lowast

lowast

lowast

OT

()

Group

(a)


10

20

30

40

50 lowast

OE

()

Group

(b)

Figure 3The effect of AEAS on the EPM test inmice ((b) for OE and (a) for OT)The aqueous extract of asparagus stem (AEAS) elevatedplus maze (EPM) and diazepam (DZP) The percent of open arm entries to the total number of arm entries (OE) The percent of time spentin the open arm to the total time in the open and close arms (OT) The dose of aqueous extract of asparagus stem 06 gkg (AEAS-L) Thedose of aqueous extract of asparagus stem 15 gkg (AEAS-M) The dose of aqueous extract of asparagus stem 30 gkg (AEAS-H) lowast119875 lt 005statistically significant difference from control


200

400

600

800lowast

lowast

lowast

Num

ber o

f pun

ished

lick

s

Group

Figure 4The effect of AEAS on the Vogel conflict testThe aqueousextract of asparagus stem (AEAS) and diazepam (DZP)The percentof open arm entries to the total number of arm entries (OE) Thepercent of time spent in the open arm to the total time in the openand close arms (OT)The dose of aqueous extract of asparagus stem06 gkg (AEAS-L) The dose of aqueous extract of asparagus stem15 gkg (AEAS-M) The dose of aqueous extract of asparagus stem30 gkg (AEAS-H) lowast119875 lt 005 statistically significant differencefrom control

there were no treatment-related changes in the main organs(for both sexes) whereas only sporadic andor spontaneouslesions were observed similarly in the control and treatedanimals The current study indicates no observable adverseeffects on the rat reproductive system

4 Discussion

The literature survey revealed that the steroidal saponins arethe main biologically active constituents of Asparagus In theprevious pentobarbital-induced sleep test linear regression

analysis showed a correlation between asparagus saponinintake and increased sleeping time Reports suggest thatsarsasapogenin fromAnemarrhena asphodeloides Bunge (Lil-iaceae) exile antidepressant activity by mediation of the cen-tral monoaminergic neurotransmitter systems [15 16] Thecontent of steroidal saponinswas quantified by determinationof sarsasapogenin as an index of active constituents ofasparagus

The present study sought to analyze the behavioral effectsof AEAS in two animalmodelsThe results showed thatAEASexhibited anxiolytic-like activity and mediated the secretionof cortisol and 5-HT The neurochemical assay suggestedthat the anxiolytic effects of AEAS may be partly attributableto the modulation of neuronal and endocrine The presentfindings may also provide important scientific evidence forthe application and development of relaxation functionalfood to conquer the anxiety

The elevated plus maze is one of the most widely usedmodels and has been validated [18 19] which uses the naturalfear of rodents to avoid open and elevated places When theanimals were treated with the higher doses of AEAS (15 and30 gkg) anxiety-like behavior in the elevated plus maze wassignificantly attenuated increasing the percent of time spentin open arms

The Vogel test has become a standard for fast screeningthe potential anxiolytic properties of drugs In this pro-cedure drinking behavior is punished by mild electricalshocks leading to significantly reducedwater consumption indeprived animals To further validate these data we tested theanxiolytic-like effects of these treatments in the VCT whichinvolves the suppression of punished responses The behav-ioral suppression induced by shocks in the VCT is atten-uated by anxiolytic drugs The doses of 15 and 3 gkgAEAS increased the number of punished licks (ie inducedanxiolytic-like effects) to 177 and 174 In the present studydiazepam was used as a positive control As expected itincreased activity in the open arms of the elevated plus maze


Table4Eff

ecto

fAEA

Srepeated

doseso

nrelativeo

rgan

weights(

)inrats

Treatm

ent

Dose(gkg)

Gender

Heart

Liver

Spleen

Lung

Kidn

eyBrain

Adrenal

Thym

usTestis

Ovary

Con

trol

mdashD

081plusmn006429plusmn056035plusmn006075plusmn005092plusmn011105plusmn0060019plusmn0002004plusmn001120plusmn011

mdashC

088plusmn006374plusmn062034plusmn004071plusmn005088plusmn011095plusmn0060018plusmn0003003plusmn001

008plusmn001

AEA

S

125

D082plusmn004419plusmn049035plusmn004073plusmn004092plusmn012101plusmn0040021plusmn0002004plusmn001119plusmn013

125

C092plusmn006378plusmn058034plusmn006070plusmn004086plusmn010091plusmn0050019plusmn0002004plusmn001

008plusmn001

25


25


008plusmn001

50


50


008plusmn001

lowast

significantly

different

from

thec

ontro

lgroup

inthes

ameg

endera

t119875lt005

Results

aree

xpressed

asmeanplusmnSD

119899=10anim

alsgrou

p


Table5Eff

ecto

fAEA

Srepeated

doseso

nhaem

ogram

inrats

Treatm

ent

Dose(gkg)

Gender

WBC

(times10

9 L)

RBC(times10

12L)

HGB(gL)

PLT(times10

9 L)

Lymph

()

Neutro

phil(

)Mon

ocyte()

Acidop

hil()

Basoph

ils(

)

Con

trol

mdashD

896plusmn216711plusmn054132plusmn10326plusmn36801plusmn33

143plusmn30

41plusmn06

09plusmn04

06plusmn03

mdashC


151plusmn45

41plusmn06

07plusmn03

05plusmn03

AEA

S

125

D936plusmn313703plusmn046131plusmn10336plusmn53787plusmn36

159plusmn39

42plusmn06

06plusmn05

04plusmn03

125

C698plusmn255716plusmn052130plusmn11336plusmn53795plusmn30

155plusmn32

36plusmn05

08plusmn04

06plusmn03

25


149plusmn33

38plusmn06

06plusmn04

05plusmn04

25


140plusmn37

40plusmn06

07plusmn04

05plusmn02

50


154plusmn37

42plusmn06

08plusmn04

05plusmn03

50

C765plusmn285732plusmn057131plusmn7

342plusmn27786plusmn31

162plusmn34

41plusmn05

06plusmn03

05plusmn03

lowast

significantly

different

from

thec

ontro

lgroup

inthes

ameg

endera

t119875lt005

Results

aree

xpressed

asmeanplusmnSD

119899=10anim

alsgrou

pTh

eredbloo

dcellcoun

t(RB

C)h

emoglobinconcentration(H

GB)lym

phocyteconcentration

neutroph

ilcells

concentration

mon

ocyteconcentration

acidop

hiland

basoph

ilsconcentration

white

bloo

dcell

coun

t(WBC

)andplateletcoun

t(PL

T)


Table6Eff

ecto

fAEA

Srepeated

doseso

nbiochemicalparametersinrats

Treatm

ent

Dose

(gkg)

Gender

TP(gL)

ALB

(gL)

T-CH

O(m

molL)

TG(m

molL)

T-BIL(120583molL)

BUN(m

molL)

CRE(120583molL)

ALT

(IUL)

AST

(IUL)

GLU

(mmolL)

Con

trol

mdashD705plusmn45400plusmn34176plusmn021

103plusmn015156plusmn011658plusmn036385plusmn48363plusmn37128plusmn11643plusmn034

mdashC706plusmn38405plusmn31165plusmn022


AEA

S

125

D706plusmn44408plusmn34170plusmn039


125



25



25



50



50



lowast

significantly

different

from

thec

ontro

lgroup

inthes

ameg

endera

t119875lt005

Results

aree

xpressed

asmeanplusmnSD

119899=10anim

alsgrou

pTo

talprotein

(TP)album

in(A

LB)totalcho

leste

rol(T-CH

O)triglycerid

es(TG)totalbilirubin(T-BIL)bloo

durea

nitro

gen(BUN)creatin

ine(CR

E)aspartateam

inotransferase

(AST

)alaninea

minotransferase

(ALT

)andbloo

dsugarlevel(G

LU)


Con

trol

Mod

e

DZP

AEA

S-L

AEA

S-M

AEA

S-H

0

20

40

60

lowast

Group

CRO

T (n

gm

L)

(a)

Con

trol

Mod

e

DZP

AEA

S-L

AEA

S-M

AEA

S-H

lowast

Group

16

14

12

10

08

06

04

02

00

5-H

T (n

gm

L)

(b)

Figure 5 The serum biochemical levels in the Vogel conflict test ((a) for CROT and (b) for 5-HT) The aqueous extract of asparagus stem(AEAS) and diazepam (DZP) The percent of open arm entries to the total number of arm entries (OE) The percent of time spent in theopen arm to the total time in the open and close arms (OT) The dose of aqueous extract of asparagus stem 06 gkg (AEAS-l) The dose ofaqueous extract of asparagus stem 15 gkg (AEAS-m) The dose of aqueous extract of asparagus stem 30 gkg (AEAS-h) Cortisol (CORT)and 5-Hydroxytryptophan (5-HT) lowast119875 lt 005 statistically significant difference from control

119875 lt 005 statistically significant differencefrom model

and number of punished licks in the VCT confirming itsanxiolytic actions

As traditional medicine spreads extensively worldwideedible herbs have become an increasingly important ingredi-ent in functional foods and therapeutic drugs In India tra-ditional folk medicine uses the roots of Asparagus officinalisL to promote improvement in physical and mental health[11ndash13] Component analysis showed that asparagus are richin saponins from Asparagus officinalis (178) aspartic acid(61 accounting to one third of total amino acid) andgamma-amino butyric acid (10) Aspartic acid is an aminoacid that participates in hormone production and nervoussystem function Specifically aspartic acid contributes to fightfatigue and other symptoms related to stamina deficiencyThebrain converts glutamine into glutamic acid and increasesthe amount of gamma-aminobutyric acid (GABA) which isneeded to sustain proper brain function and mental activity[20 21] Naturally existing GABA in AEAS also participatesimportantly in relieving stress and exhibiting anxiolyticeffect

For the edible safety investigation the acute toxicologystudy suggesting the maxim tolerant dose was 200 gkg inthis experiment condition In the repeated dose oral toxicol-ogy study the safety dose was 50 gkg for 13 weeks

In summary the present study showed that AEAS exhib-ited strong anxiolytic-like effect and mediated the secretionof crotisol and 5-HT These data demonstrated that acutestress (open elevated plus maze and punished licks) [22]elicits anxious behaviors and increases serum cortisol levelTheAEAS treatment which decreases the secretion of crotisolsuggested that the anxiolytic effect of AEAS may be partlyattributable to the modulation of neuronal and endocrineTherefore AEAS is a promising candidate for the treatmentof anxiety-like disorders in the alternative medicine

Conflict of Interests

The authors declare that they have no competing financialinterests

Authorsrsquo Contribution

Long Cheng and Guo-feng Pan equally contributed to thispaper

Acknowledgments

This work was financially supported by the Special Fundfor Agro-scientific Research in the Public Interest (no201303079) The authors thank scientific editor KarenWilliams (Kwills Editing Services Weymouth MA USA) forproviding professional English language editing of this paper

References

[1] D H Barlow ldquoUnraveling the mysteries of anxiety and its dis-orders from the perspective of emotion theoryrdquo AmericanPsychologist vol 55 no 11 pp 1247ndash1263 2000

[2] R Lieb E Becker and C Altamura ldquoThe epidemiology ofgeneralized anxiety disorder in Europerdquo European Neuropsy-chopharmacology vol 15 no 4 pp 445ndash452 2005

[3] S K Bhattamisra V K Khanna A K Agrawal P N Singh andS K Singh ldquoAntidepressant activity of standardised extract ofMarsilea minuta Linnrdquo Journal of Ethnopharmacology vol 117no 1 pp 51ndash57 2008

[4] V Dutt V J Dhar and A Sharma ldquoAntianxiety activity ofGelsemium sempervirensrdquo Pharmaceutical Biology vol 48 no10 pp 1091ndash1096 2010

[5] httpwwwwhointmental healthevidenceenprevention ofmental disorders srpdf


[6] AD Jordan C P Kordik A B Reitz and P J Sanfilippo ldquoNovelanxiolytic agents-1994 to presentrdquo Expert Opinion onTherapeu-tic Patents vol 6 no 10 pp 1047ndash1060 1996

[7] H I Kaplan and B J Sadock Comprehensive Textbook ofPsychiatry Lippincot Williams and Wilkins New York NYUSA 8th edition 2005

[8] httparabiamsncomlifestylehealth-fitness1928481relaxa-tion-drinks

[9] H Li X Zhao S Liu and Y Peng ldquoEffects of instant asparaguspowder on immune function ofmicerdquo Journal of Anhui Agricul-tural Sciences vol 40 pp 10819ndash10820 2012 (Chinese)

[10] S Ma F Wang H Li X Chang Z Wang and Y HangldquoStudy on the sleep improvement effect of the instant asparaguspowderrdquo Science and Technology of Food Industry vol 33 pp359ndash362 2012 (Chinese)

[11] N N Rege U M Thatte and S A Dahanukar ldquoAdaptogenicproperties of six rasayana herbs used in Ayurvedic medicinerdquoPhytotherapy Research vol 13 pp 275ndash291 1989

[12] S A Dahanukar R A Kulkarni and N N Rege ldquoPharmacol-ogy of medicinal plants and natural productsrdquo Indian Journal ofPharmacology vol 32 no 4 pp S81ndashS118 2000

[13] R Ojha A N Sahu A V Muruganandam G K Singh and SKrishnamurthy ldquoAsparagus recemosus enhances memory andprotects against amnesia in rodent modelsrdquo Brain and Cogni-tion vol 74 no 1 pp 1ndash9 2010

[14] S M Korte and S F De Boer ldquoA robust animal model of stateanxiety fear-potentiated behaviour in the elevated plus-mazerdquoEuropean Journal of Pharmacology vol 463 no 1ndash3 pp 163ndash1752003

[15] L-X Ren Y-F Luo X Li D-Y Zuo and Y-L Wu ldquoAntide-pressant-like effects of sarsasapogenin from Anemarrhenaasphodeloides Bunge (Liliaceae)rdquo Biological and PharmaceuticalBulletin vol 29 no 11 pp 2304ndash2306 2006

[16] L X Ren Y F Luo X Li and Y L Wu ldquoAntidepressant activityof sarsasapogenin from Anemarrhena asphodeloides Bunge(Liliaceae)rdquo Pharmazie vol 62 no 1 pp 78ndash79 2007

[17] J R Vogel B Beer and D E Clody ldquoA simple and reliableconflict procedure for testing anti-anxiety agentsrdquo Psychophar-macologia vol 21 no 1 pp 1ndash7 1971

[18] S Hogg ldquoA review of the validity and variability of the elevatedplus-maze as an animal model of anxietyrdquo Pharmacology Bio-chemistry and Behavior vol 54 no 1 pp 21ndash30 1996

[19] S Pellow P Chopin S E File and M Briley ldquoValidation ofopen-closed arm entries in an elevated plus-maze as a measureof anxiety in the ratrdquo Journal of Neuroscience Methods vol 14no 3 pp 149ndash167 1985

[20] A M Abdou S Higashiguchi K Horie M Kim H Hatta andH Yokogoshi ldquoRelaxation and immunity enhancement effectsof 120574-Aminobutyric acid (GABA) administration in humansrdquoBioFactors vol 26 no 3 pp 201ndash208 2006

[21] A Yoto S Murao M Motoki et al ldquoOral intake of 120574-aminobu-tyric acid affects mood and activities of central nervous systemduring stressed condition induced by mental tasksrdquo AminoAcids vol 43 no 3 pp 1331ndash1337 2012

[22] M Komiya T Takeuchi and E Harada ldquoLemon oil vaporcauses an anti-stress effect via modulating the 5-HT and DAactivities in micerdquo Behavioural Brain Research vol 172 no 2pp 240ndash249 2006

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


40383634323028262422201816141210

864

2826242220181614121086420

(mV

)

(min)

26107

(a) The refer substance

(min)

(mV

)

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28

26150

185175165155145135125115105

958575655545352515

5

(b) The sample of AEAS

Figure 1 The chromatography of sarsasapogenin from AEAS

22 Extract Preparation Aqueous extract from the stemof Asparagus officinalis L was provided by QinhuangdaoChangshengAgricultural TechnologyDevelopmentCo LtdHebei Province China To obtain the aqueous extract fromthe woody stem of asparagus fresh plant material (600 kg)was cleaned crushed and then extracted with drinking water(plantwater ratio 1 8 wv water temperature 85 plusmn 5∘C) bydynamic maceration for 5 h After filtering the solution ofasparagus stem was treated with spray drying to drynessyielding extract powder (10 kg AEAS)

23 Extract Character The nutrient and chemical compo-nents of this studied extract were identified and quanti-fied (shown in Table 1) Amino acid composition analysiswas performed by a fully automatic amino acid analyzer(Hitachi L-8900 Japan) The data were shown in Table 2The main biologically active constituents of asparagus arethe steroidal saponins and flavonoids For quantitative deter-mination of the steroidal saponins the AEAS was refluxedwith hydrochloric acid solution The sarsasapogenin fromasparagus was determined by reversed phase high perfor-mance liquid chromatographicmethodwith evaporative lightscattering detection (RP-HPLC-ELSD) (shown in Figure 1)The content of sarsasapogenin was 1780 The fingerprintof the flavonoids from AEAS was established by HPLC-UV(shown in Figure 2 and Table 3) Asparagus extract solutionwas mixed with diethyl ether then stirred The diethyl etherlayer was separated and evaporated to dryness The resultingresidue was dissolved in methanol as sample solution forfingerprint analyses

The analyses were performed using a Zorbax C18column

and a diode array detector in an Agilent1100 HPLC systemThe mobile phase was 05 formic acid solution (solvent A)and methanol (solvent B) at a flow rate of 07mLmin Thegradient elutionwas 0ndash20min 80ndash70A 20ndash30B 20ndash45min 70ndash60 A 30ndash40 B 45ndash60min 60ndash50 A40ndash50 B 60ndash70min 50ndash30 A 50ndash70 B and 70ndash75min 30ndash0 A 70ndash100 B The UV detector wave-length was set at 320 nm Five compounds were identified by

Table 1 Nutrient and chemical compositions of the AEAS

No Compositions Content Unit1 Carbohydrates 444 g100 g2 Proteins 245 g100 g3 Lipids 30 g100 g4 Calcium 384 g100 g5 Potassium 112 g100 g6 Magnesium 033 g100 g7 Sodium 028 g100 g8 Ferrum 006 g100 g9 Copper 019 g100 g10 Zinc 009 g100 g11 Vitamin E 017 mg100 g12 Vitamin B1 039 mg100 g13 Vitamin B2 089 mg100 g14 Selenium 014 mg100 g15 Saponins 1780 g100 g16 Flavone 291 g100 g17 Polyphenol 593 g100 g18 Polysaccharide 1121 g100 g19 GABA 082 g100 gAEAS aqueous extract of asparagus stem GABA gamma-aminobutyricacid

the spectral feature of UV MS and MSMS (Table 3) Themain 8 peaks were quantified by comparing their peak areaswith the refer substance (Peak 1)

24 Drug Distilled drinking water was used as vehicleDiazepam (DZP)was used as the reference drug for anxiolyticactivities

25 Animals Male Imprinting Control Region mice (CD-1 ICR 18sim22 g) were purchased from Beijing Vital RiverLaboratories Co Ltd (Beijing China) Animals were housedin a controlled environment (temperature 23∘ plusmn 2∘C relative




(min)

0102030405060

70

(mAU

)

0 10 20 30 40 50 60


1

23 45

678


















3 Results









10

20

30

40

50 lowast

lowast

lowast

OT

()

Group

(a)


10

20

30

40

50 lowast

OE

()

Group

(b)



200

400

600

800lowast

lowast

lowast

Num

ber o

f pun

ished

lick

s

Group



4 Discussion







Table4Eff

ecto

fAEA

Srepeated

doseso

nrelativeo

rgan

weights(

)inrats

Treatm

ent

Dose(gkg)

Gender

Heart

Liver

Spleen

Lung

Kidn

eyBrain

Adrenal

Thym

usTestis

Ovary

Con

trol

mdashD


mdashC


008plusmn001

AEA

S

125


125


008plusmn001

25


25


008plusmn001

50


50


008plusmn001

lowast

significantly

different

from

thec

ontro

lgroup

inthes

ameg

endera

t119875lt005

Results

aree

xpressed

asmeanplusmnSD

119899=10anim

alsgrou

p


Table5Eff

ecto

fAEA

Srepeated

doseso

nhaem

ogram

inrats

Treatm

ent

Dose(gkg)

Gender

WBC

(times10

9 L)

RBC(times10

12L)

HGB(gL)

PLT(times10

9 L)

Lymph

()

Neutro

phil(

)Mon

ocyte()

Acidop

hil()

Basoph

ils(

)

Con

trol

mdashD


143plusmn30

41plusmn06

09plusmn04

06plusmn03

mdashC


151plusmn45

41plusmn06

07plusmn03

05plusmn03

AEA

S

125


159plusmn39

42plusmn06

06plusmn05

04plusmn03

125


155plusmn32

36plusmn05

08plusmn04

06plusmn03

25


149plusmn33

38plusmn06

06plusmn04

05plusmn04

25


140plusmn37

40plusmn06

07plusmn04

05plusmn02

50


154plusmn37

42plusmn06

08plusmn04

05plusmn03

50



162plusmn34

41plusmn05

06plusmn03

05plusmn03

lowast

significantly

different

from

thec

ontro

lgroup

inthes

ameg

endera

t119875lt005

Results

aree

xpressed

asmeanplusmnSD

119899=10anim

alsgrou

pTh

eredbloo

dcellcoun

t(RB

C)h


GB)lym


neutroph

ilcells

concentration

mon

ocyteconcentration

acidop

hiland

basoph

ilsconcentration

white

bloo

dcell

coun

t(WBC

)andplateletcoun

t(PL

T)


Table6Eff

ecto

fAEA

Srepeated

doseso


Treatm

ent

Dose

(gkg)

Gender

TP(gL)

ALB

(gL)

T-CH

O(m

molL)

TG(m

molL)

T-BIL(120583molL)

BUN(m

molL)

CRE(120583molL)

ALT

(IUL)

AST

(IUL)

GLU

(mmolL)

Con

trol





AEA

S

125



125



25



25



50



50



lowast

significantly

different

from

thec

ontro

lgroup

inthes

ameg

endera

t119875lt005

Results

aree

xpressed

asmeanplusmnSD

119899=10anim

alsgrou

pTo

talprotein

(TP)album

in(A

LB)totalcho

leste

rol(T-CH

O)triglycerid


durea

nitro

gen(BUN)creatin

ine(CR

E)aspartateam

inotransferase

(AST

)alaninea

minotransferase

(ALT

)andbloo

dsugarlevel(G

LU)


Con

trol

Mod

e

DZP

AEA

S-L

AEA

S-M

AEA

S-H

0

20

40

60

lowast

Group

CRO

T (n

gm

L)

(a)

Con

trol

Mod

e

DZP

AEA

S-L

AEA

S-M

AEA

S-H

lowast

Group

16

14

12

10

08

06

04

02

00

5-H

T (n

gm

L)

(b)











Acknowledgments


References





























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















(min)

0102030405060

70

(mAU

)

0 10 20 30 40 50 60


1

23 45

678


















3 Results









10

20

30

40

50 lowast

lowast

lowast

OT

()

Group

(a)


10

20

30

40

50 lowast

OE

()

Group

(b)



200

400

600

800lowast

lowast

lowast

Num

ber o

f pun

ished

lick

s

Group



4 Discussion







Table4Eff

ecto

fAEA

Srepeated

doseso

nrelativeo

rgan

weights(

)inrats

Treatm

ent

Dose(gkg)

Gender

Heart

Liver

Spleen

Lung

Kidn

eyBrain

Adrenal

Thym

usTestis

Ovary

Con

trol

mdashD


mdashC


008plusmn001

AEA

S

125


125


008plusmn001

25


25


008plusmn001

50


50


008plusmn001

lowast

significantly

different

from

thec

ontro

lgroup

inthes

ameg

endera

t119875lt005

Results

aree

xpressed

asmeanplusmnSD

119899=10anim

alsgrou

p


Table5Eff

ecto

fAEA

Srepeated

doseso

nhaem

ogram

inrats

Treatm

ent

Dose(gkg)

Gender

WBC

(times10

9 L)

RBC(times10

12L)

HGB(gL)

PLT(times10

9 L)

Lymph

()

Neutro

phil(

)Mon

ocyte()

Acidop

hil()

Basoph

ils(

)

Con

trol

mdashD


143plusmn30

41plusmn06

09plusmn04

06plusmn03

mdashC


151plusmn45

41plusmn06

07plusmn03

05plusmn03

AEA

S

125


159plusmn39

42plusmn06

06plusmn05

04plusmn03

125


155plusmn32

36plusmn05

08plusmn04

06plusmn03

25


149plusmn33

38plusmn06

06plusmn04

05plusmn04

25


140plusmn37

40plusmn06

07plusmn04

05plusmn02

50


154plusmn37

42plusmn06

08plusmn04

05plusmn03

50



162plusmn34

41plusmn05

06plusmn03

05plusmn03

lowast

significantly

different

from

thec

ontro

lgroup

inthes

ameg

endera

t119875lt005

Results

aree

xpressed

asmeanplusmnSD

119899=10anim

alsgrou

pTh

eredbloo

dcellcoun

t(RB

C)h


GB)lym


neutroph

ilcells

concentration

mon

ocyteconcentration

acidop

hiland

basoph

ilsconcentration

white

bloo

dcell

coun

t(WBC

)andplateletcoun

t(PL

T)


Table6Eff

ecto

fAEA

Srepeated

doseso


Treatm

ent

Dose

(gkg)

Gender

TP(gL)

ALB

(gL)

T-CH

O(m

molL)

TG(m

molL)

T-BIL(120583molL)

BUN(m

molL)

CRE(120583molL)

ALT

(IUL)

AST

(IUL)

GLU

(mmolL)

Con

trol





AEA

S

125



125



25



25



50



50



lowast

significantly

different

from

thec

ontro

lgroup

inthes

ameg

endera

t119875lt005

Results

aree

xpressed

asmeanplusmnSD

119899=10anim

alsgrou

pTo

talprotein

(TP)album

in(A

LB)totalcho

leste

rol(T-CH

O)triglycerid


durea

nitro

gen(BUN)creatin

ine(CR

E)aspartateam

inotransferase

(AST

)alaninea

minotransferase

(ALT

)andbloo

dsugarlevel(G

LU)


Con

trol

Mod

e

DZP

AEA

S-L

AEA

S-M

AEA

S-H

0

20

40

60

lowast

Group

CRO

T (n

gm

L)

(a)

Con

trol

Mod

e

DZP

AEA

S-L

AEA

S-M

AEA

S-H

lowast

Group

16

14

12

10

08

06

04

02

00

5-H

T (n

gm

L)

(b)











Acknowledgments


References





























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























3 Results









10

20

30

40

50 lowast

lowast

lowast

OT

()

Group

(a)


10

20

30

40

50 lowast

OE

()

Group

(b)



200

400

600

800lowast

lowast

lowast

Num

ber o

f pun

ished

lick

s

Group



4 Discussion







Table4Eff

ecto

fAEA

Srepeated

doseso

nrelativeo

rgan

weights(

)inrats

Treatm

ent

Dose(gkg)

Gender

Heart

Liver

Spleen

Lung

Kidn

eyBrain

Adrenal

Thym

usTestis

Ovary

Con

trol

mdashD


mdashC


008plusmn001

AEA

S

125


125


008plusmn001

25


25


008plusmn001

50


50


008plusmn001

lowast

significantly

different

from

thec

ontro

lgroup

inthes

ameg

endera

t119875lt005

Results

aree

xpressed

asmeanplusmnSD

119899=10anim

alsgrou

p


Table5Eff

ecto

fAEA

Srepeated

doseso

nhaem

ogram

inrats

Treatm

ent

Dose(gkg)

Gender

WBC

(times10

9 L)

RBC(times10

12L)

HGB(gL)

PLT(times10

9 L)

Lymph

()

Neutro

phil(

)Mon

ocyte()

Acidop

hil()

Basoph

ils(

)

Con

trol

mdashD


143plusmn30

41plusmn06

09plusmn04

06plusmn03

mdashC


151plusmn45

41plusmn06

07plusmn03

05plusmn03

AEA

S

125


159plusmn39

42plusmn06

06plusmn05

04plusmn03

125


155plusmn32

36plusmn05

08plusmn04

06plusmn03

25


149plusmn33

38plusmn06

06plusmn04

05plusmn04

25


140plusmn37

40plusmn06

07plusmn04

05plusmn02

50


154plusmn37

42plusmn06

08plusmn04

05plusmn03

50



162plusmn34

41plusmn05

06plusmn03

05plusmn03

lowast

significantly

different

from

thec

ontro

lgroup

inthes

ameg

endera

t119875lt005

Results

aree

xpressed

asmeanplusmnSD

119899=10anim

alsgrou

pTh

eredbloo

dcellcoun

t(RB

C)h


GB)lym


neutroph

ilcells

concentration

mon

ocyteconcentration

acidop

hiland

basoph

ilsconcentration

white

bloo

dcell

coun

t(WBC

)andplateletcoun

t(PL

T)


Table6Eff

ecto

fAEA

Srepeated

doseso


Treatm

ent

Dose

(gkg)

Gender

TP(gL)

ALB

(gL)

T-CH

O(m

molL)

TG(m

molL)

T-BIL(120583molL)

BUN(m

molL)

CRE(120583molL)

ALT

(IUL)

AST

(IUL)

GLU

(mmolL)

Con

trol





AEA

S

125



125



25



25



50



50



lowast

significantly

different

from

thec

ontro

lgroup

inthes

ameg

endera

t119875lt005

Results

aree

xpressed

asmeanplusmnSD

119899=10anim

alsgrou

pTo

talprotein

(TP)album

in(A

LB)totalcho

leste

rol(T-CH

O)triglycerid


durea

nitro

gen(BUN)creatin

ine(CR

E)aspartateam

inotransferase

(AST

)alaninea

minotransferase

(ALT

)andbloo

dsugarlevel(G

LU)


Con

trol

Mod

e

DZP

AEA

S-L

AEA

S-M

AEA

S-H

0

20

40

60

lowast

Group

CRO

T (n

gm

L)

(a)

Con

trol

Mod

e

DZP

AEA

S-L

AEA

S-M

AEA

S-H

lowast

Group

16

14

12

10

08

06

04

02

00

5-H

T (n

gm

L)

(b)











Acknowledgments


References





























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















10

20

30

40

50 lowast

lowast

lowast

OT

()

Group

(a)


10

20

30

40

50 lowast

OE

()

Group

(b)



200

400

600

800lowast

lowast

lowast

Num

ber o

f pun

ished

lick

s

Group



4 Discussion







Table4Eff

ecto

fAEA

Srepeated

doseso

nrelativeo

rgan

weights(

)inrats

Treatm

ent

Dose(gkg)

Gender

Heart

Liver

Spleen

Lung

Kidn

eyBrain

Adrenal

Thym

usTestis

Ovary

Con

trol

mdashD


mdashC


008plusmn001

AEA

S

125


125


008plusmn001

25


25


008plusmn001

50


50


008plusmn001

lowast

significantly

different

from

thec

ontro

lgroup

inthes

ameg

endera

t119875lt005

Results

aree

xpressed

asmeanplusmnSD

119899=10anim

alsgrou

p


Table5Eff

ecto

fAEA

Srepeated

doseso

nhaem

ogram

inrats

Treatm

ent

Dose(gkg)

Gender

WBC

(times10

9 L)

RBC(times10

12L)

HGB(gL)

PLT(times10

9 L)

Lymph

()

Neutro

phil(

)Mon

ocyte()

Acidop

hil()

Basoph

ils(

)

Con

trol

mdashD


143plusmn30

41plusmn06

09plusmn04

06plusmn03

mdashC


151plusmn45

41plusmn06

07plusmn03

05plusmn03

AEA

S

125


159plusmn39

42plusmn06

06plusmn05

04plusmn03

125


155plusmn32

36plusmn05

08plusmn04

06plusmn03

25


149plusmn33

38plusmn06

06plusmn04

05plusmn04

25


140plusmn37

40plusmn06

07plusmn04

05plusmn02

50


154plusmn37

42plusmn06

08plusmn04

05plusmn03

50



162plusmn34

41plusmn05

06plusmn03

05plusmn03

lowast

significantly

different

from

thec

ontro

lgroup

inthes

ameg

endera

t119875lt005

Results

aree

xpressed

asmeanplusmnSD

119899=10anim

alsgrou

pTh

eredbloo

dcellcoun

t(RB

C)h


GB)lym


neutroph

ilcells

concentration

mon

ocyteconcentration

acidop

hiland

basoph

ilsconcentration

white

bloo

dcell

coun

t(WBC

)andplateletcoun

t(PL

T)


Table6Eff

ecto

fAEA

Srepeated

doseso


Treatm

ent

Dose

(gkg)

Gender

TP(gL)

ALB

(gL)

T-CH

O(m

molL)

TG(m

molL)

T-BIL(120583molL)

BUN(m

molL)

CRE(120583molL)

ALT

(IUL)

AST

(IUL)

GLU

(mmolL)

Con

trol





AEA

S

125



125



25



25



50



50



lowast

significantly

different

from

thec

ontro

lgroup

inthes

ameg

endera

t119875lt005

Results

aree

xpressed

asmeanplusmnSD

119899=10anim

alsgrou

pTo

talprotein

(TP)album

in(A

LB)totalcho

leste

rol(T-CH

O)triglycerid


durea

nitro

gen(BUN)creatin

ine(CR

E)aspartateam

inotransferase

(AST

)alaninea

minotransferase

(ALT

)andbloo

dsugarlevel(G

LU)


Con

trol

Mod

e

DZP

AEA

S-L

AEA

S-M

AEA

S-H

0

20

40

60

lowast

Group

CRO

T (n

gm

L)

(a)

Con

trol

Mod

e

DZP

AEA

S-L

AEA

S-M

AEA

S-H

lowast

Group

16

14

12

10

08

06

04

02

00

5-H

T (n

gm

L)

(b)











Acknowledgments


References





























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table4Eff

ecto

fAEA

Srepeated

doseso

nrelativeo

rgan

weights(

)inrats

Treatm

ent

Dose(gkg)

Gender

Heart

Liver

Spleen

Lung

Kidn

eyBrain

Adrenal

Thym

usTestis

Ovary

Con

trol

mdashD


mdashC


008plusmn001

AEA

S

125


125


008plusmn001

25


25


008plusmn001

50


50


008plusmn001

lowast

significantly

different

from

thec

ontro

lgroup

inthes

ameg

endera

t119875lt005

Results

aree

xpressed

asmeanplusmnSD

119899=10anim

alsgrou

p


Table5Eff

ecto

fAEA

Srepeated

doseso

nhaem

ogram

inrats

Treatm

ent

Dose(gkg)

Gender

WBC

(times10

9 L)

RBC(times10

12L)

HGB(gL)

PLT(times10

9 L)

Lymph

()

Neutro

phil(

)Mon

ocyte()

Acidop

hil()

Basoph

ils(

)

Con

trol

mdashD


143plusmn30

41plusmn06

09plusmn04

06plusmn03

mdashC


151plusmn45

41plusmn06

07plusmn03

05plusmn03

AEA

S

125


159plusmn39

42plusmn06

06plusmn05

04plusmn03

125


155plusmn32

36plusmn05

08plusmn04

06plusmn03

25


149plusmn33

38plusmn06

06plusmn04

05plusmn04

25


140plusmn37

40plusmn06

07plusmn04

05plusmn02

50


154plusmn37

42plusmn06

08plusmn04

05plusmn03

50



162plusmn34

41plusmn05

06plusmn03

05plusmn03

lowast

significantly

different

from

thec

ontro

lgroup

inthes

ameg

endera

t119875lt005

Results

aree

xpressed

asmeanplusmnSD

119899=10anim

alsgrou

pTh

eredbloo

dcellcoun

t(RB

C)h


GB)lym


neutroph

ilcells

concentration

mon

ocyteconcentration

acidop

hiland

basoph

ilsconcentration

white

bloo

dcell

coun

t(WBC

)andplateletcoun

t(PL

T)


Table6Eff

ecto

fAEA

Srepeated

doseso


Treatm

ent

Dose

(gkg)

Gender

TP(gL)

ALB

(gL)

T-CH

O(m

molL)

TG(m

molL)

T-BIL(120583molL)

BUN(m

molL)

CRE(120583molL)

ALT

(IUL)

AST

(IUL)

GLU

(mmolL)

Con

trol





AEA

S

125



125



25



25



50



50



lowast

significantly

different

from

thec

ontro

lgroup

inthes

ameg

endera

t119875lt005

Results

aree

xpressed

asmeanplusmnSD

119899=10anim

alsgrou

pTo

talprotein

(TP)album

in(A

LB)totalcho

leste

rol(T-CH

O)triglycerid


durea

nitro

gen(BUN)creatin

ine(CR

E)aspartateam

inotransferase

(AST

)alaninea

minotransferase

(ALT

)andbloo

dsugarlevel(G

LU)


Con

trol

Mod

e

DZP

AEA

S-L

AEA

S-M

AEA

S-H

0

20

40

60

lowast

Group

CRO

T (n

gm

L)

(a)

Con

trol

Mod

e

DZP

AEA

S-L

AEA

S-M

AEA

S-H

lowast

Group

16

14

12

10

08

06

04

02

00

5-H

T (n

gm

L)

(b)











Acknowledgments


References





























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table5Eff

ecto

fAEA

Srepeated

doseso

nhaem

ogram

inrats

Treatm

ent

Dose(gkg)

Gender

WBC

(times10

9 L)

RBC(times10

12L)

HGB(gL)

PLT(times10

9 L)

Lymph

()

Neutro

phil(

)Mon

ocyte()

Acidop

hil()

Basoph

ils(

)

Con

trol

mdashD


143plusmn30

41plusmn06

09plusmn04

06plusmn03

mdashC


151plusmn45

41plusmn06

07plusmn03

05plusmn03

AEA

S

125


159plusmn39

42plusmn06

06plusmn05

04plusmn03

125


155plusmn32

36plusmn05

08plusmn04

06plusmn03

25


149plusmn33

38plusmn06

06plusmn04

05plusmn04

25


140plusmn37

40plusmn06

07plusmn04

05plusmn02

50


154plusmn37

42plusmn06

08plusmn04

05plusmn03

50



162plusmn34

41plusmn05

06plusmn03

05plusmn03

lowast

significantly

different

from

thec

ontro

lgroup

inthes

ameg

endera

t119875lt005

Results

aree

xpressed

asmeanplusmnSD

119899=10anim

alsgrou

pTh

eredbloo

dcellcoun

t(RB

C)h


GB)lym


neutroph

ilcells

concentration

mon

ocyteconcentration

acidop

hiland

basoph

ilsconcentration

white

bloo

dcell

coun

t(WBC

)andplateletcoun

t(PL

T)


Table6Eff

ecto

fAEA

Srepeated

doseso


Treatm

ent

Dose

(gkg)

Gender

TP(gL)

ALB

(gL)

T-CH

O(m

molL)

TG(m

molL)

T-BIL(120583molL)

BUN(m

molL)

CRE(120583molL)

ALT

(IUL)

AST

(IUL)

GLU

(mmolL)

Con

trol





AEA

S

125



125



25



25



50



50



lowast

significantly

different

from

thec

ontro

lgroup

inthes

ameg

endera

t119875lt005

Results

aree

xpressed

asmeanplusmnSD

119899=10anim

alsgrou

pTo

talprotein

(TP)album

in(A

LB)totalcho

leste

rol(T-CH

O)triglycerid


durea

nitro

gen(BUN)creatin

ine(CR

E)aspartateam

inotransferase

(AST

)alaninea

minotransferase

(ALT

)andbloo

dsugarlevel(G

LU)


Con

trol

Mod

e

DZP

AEA

S-L

AEA

S-M

AEA

S-H

0

20

40

60

lowast

Group

CRO

T (n

gm

L)

(a)

Con

trol

Mod

e

DZP

AEA

S-L

AEA

S-M

AEA

S-H

lowast

Group

16

14

12

10

08

06

04

02

00

5-H

T (n

gm

L)

(b)











Acknowledgments


References





























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table6Eff

ecto

fAEA

Srepeated

doseso


Treatm

ent

Dose

(gkg)

Gender

TP(gL)

ALB

(gL)

T-CH

O(m

molL)

TG(m

molL)

T-BIL(120583molL)

BUN(m

molL)

CRE(120583molL)

ALT

(IUL)

AST

(IUL)

GLU

(mmolL)

Con

trol





AEA

S

125



125



25



25



50



50



lowast

significantly

different

from

thec

ontro

lgroup

inthes

ameg

endera

t119875lt005

Results

aree

xpressed

asmeanplusmnSD

119899=10anim

alsgrou

pTo

talprotein

(TP)album

in(A

LB)totalcho

leste

rol(T-CH

O)triglycerid


durea

nitro

gen(BUN)creatin

ine(CR

E)aspartateam

inotransferase

(AST

)alaninea

minotransferase

(ALT

)andbloo

dsugarlevel(G

LU)


Con

trol

Mod

e

DZP

AEA

S-L

AEA

S-M

AEA

S-H

0

20

40

60

lowast

Group

CRO

T (n

gm

L)

(a)

Con

trol

Mod

e

DZP

AEA

S-L

AEA

S-M

AEA

S-H

lowast

Group

16

14

12

10

08

06

04

02

00

5-H

T (n

gm

L)

(b)











Acknowledgments


References





























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Con

trol

Mod

e

DZP

AEA

S-L

AEA

S-M

AEA

S-H

0

20

40

60

lowast

Group

CRO

T (n

gm

L)

(a)

Con

trol

Mod

e

DZP

AEA

S-L

AEA

S-M

AEA

S-H

lowast

Group

16

14

12

10

08

06

04

02

00

5-H

T (n

gm

L)

(b)











Acknowledgments


References





























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article evaluation of anxiolytic-like effect of

Documents