and Montanoa grandiflora - Hindawi Publishing …downloads.hindawi.com/journals/bmri/2014/938060.pdf · Montanoa frutescens and Montanoa grandiflora Extracts Reduce Anxiety-Like Behavior

Research ArticleMontanoa frutescens and Montanoa grandiflora ExtractsReduce Anxiety-Like Behavior during the Metestrus-DiestrusPhase of the Ovarian Cycle in Wistar Rats

Juan Francisco Rodriacuteguez-Landa12 Julio Vicente-Serna23

Luis Alfredo Rodriacuteguez-Blanco23 Mariacutea de Jesuacutes Rovirosa-Hernaacutendez1

Francisco Garciacutea-Orduntildea1 and Miguel Carro-Juaacuterez4

1 Instituto de Neuroetologıa Universidad Veracruzana Avenida Dr Luis Castelazo snColonia Industrial Animas 91001 Xalapa VER Mexico

2 Facultad de Quımica Farmaceutica Biologica Universidad Veracruzana 91190 Xalapa VER Mexico3 Programa de Tutorıa para la Investigacion Universidad Veracruzana 91001 Xalapa VER Mexico4 Laboratorio de Comportamiento Reproductivo Escuela de Medicina Veterinaria y ZootecniaUniversidad Autonoma de Tlaxcala 90000 Tlaxcala TLAX Mexico

Correspondence should be addressed to Juan Francisco Rodrıguez-Landa juarodriguezuvmx

Received 18 December 2013 Revised 17 February 2014 Accepted 27 February 2014 Published 1 April 2014

Academic Editor Corina O Bondi

Copyright copy 2014 Juan Francisco Rodrıguez-Landa 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

In previous studies the anxiolytic-like effects ofMontanoa tomentosa andMontanoa frutescens were reported in male rats but thepotential anxiolytic-like effects ofMontanoa plants during the different phases of the ovarian cycle in rats remain to be exploredTheanxiolytic-like effects of the aqueous crude extracts ofM frutescens (25 and 50mgkg) andM grandiflora (25 and 50mgkg) in theelevated plus maze were investigated in Wistar rats during the estrous cycle and compared with 2mgkg diazepam as a referenceanxiolytic drug To investigate any motor effect (ie hyperactivity no changes or hypoactivity) associated with the treatmentsthe rats were evaluated in the open field test The M frutescens (25 and 50mgkg) and M grandiflora (50mgkg) extracts exertedanxiolytic-like effects during the metestrus-diestrus phase similar to diazepam without disrupting spontaneous motor activityNo significant effects of the extracts were detected in either behavioral test during the proestrus-estrus phase whereas diazepamproduced motor hypoactivity in the open field test These results indicate that theM frutescens andM grandiflora extracts possessanxiolytic-like effects that depend on the ovarian cycle phase supporting the Mexican ancient medicinal use of these plants toameliorate anxiety disorders

1 Introduction

Generalized anxiety disorder is one of the most commonpsychiatric disorders affecting a high percentage of thegeneral population around the world [1] Anxiety disorderspossess marked gender differences and occur more oftenin women than in men [2ndash5] The reproductive cycle ofwomen exhibits fluctuations in plasma and brain steroidhormone concentrations (eg estradiol and progesteroneamong others) that are related to anxiety and mood swingsLow concentrations of steroid hormones are associated with

irritability anxiety or depressive symptoms and occur duringthe premenstrual postpartum and climacteric periods [6ndash8] Interestingly rats exhibit a predisposition to displaymore anxiety- and depressive-like states when lower con-centrations of ovarian hormones are observed during themetestrus-diestrus phase of the ovarian cycle During theovarian cycle phases (ie proestrus-estrus phase) when highconcentrations of steroid hormones are observed anxiety-and depressive-like behaviors diminish [9 10]

Variations in ovarian hormone levels across the estrouscycle in female rats permit the evaluation of anxiety- and

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 938060 9 pageshttpdxdoiorg1011552014938060

2 BioMed Research International

depressive-like behaviors under the presence of standardanxiolytic drugs [11ndash13] and plant extracts [14] with anxiolyticor antidepressant activity This has permitted investigators toexplore potential therapeutic regimens to ameliorate anxiety-or depressive-like behaviors associated with naturally occur-ring low plasma concentrations of steroid hormones

In ancientMexican traditional medicine somemedicinalplants have been reported to possess anxiolytic effects andare currently recommended for the treatment of ldquoanxiousstatesrdquo The Badianus Codex or Libellus de MedicinalibusIndorum Herbis written in 1552 [15] describes the use ofCihuapatli (ldquowomenrsquos medicinerdquo in the Nahuatl language)for the treatment of mood and ldquonervous disordersrdquo ananxiety-like behavior that is mentioned in the context ofancient Mexican culture Early reports by Ximenez (1615)stated ldquoCihuapatli resolves the mood changes and nerves inan admirable formrdquo [16] Cihuapatli is the common nameassigned to some plants included in the genus Montanoa(family Asteraceae tribe Heliantheae) in which M tomen-tosa M frutescens and M grandiflora are included Theaqueous crude extract from these plants has been used forcenturies in traditional Mexican medicine as a remedy forreproductive impairments anxiety and mood disorders [16ndash19] Recently the aqueous extracts of M tomentosa [20] andM frutescens [21] were reported to produce anxiolytic-likeeffects that are similar to diazepam in Wistar rats and theparticipation of 120574-aminobutyric acid-A (GABAA) receptorsin their anxiolytic-like effect was identified Nonethelessalthough some anxiolytic-like effects ofM frutescens extracthave been corroborated in male rats the anxiolytic-likeeffects ofM frutescens andM grandiflora in the female genusremain unexplored We hypothesized that M frutescens andM grandiflora extracts reduce anxiety-like behavior duringthe metestrus-diestrus phase of the estrous cycle in ratsTo test this hypothesis we used the elevated plus maze awell-accepted model that tests clinically effective anxiolyticdrugs [22 23] to evaluate the effects of several doses ofM frutescens and M grandiflora aqueous crude extracts onanxiety-like behavior during different phases of the ovariancycle in Wistar rats We also made comparisons with ananxiolytic dose of diazepam

2 Material and Methods

21 Animals Female Wistar rats were obtained from a localstrain supplied by Harlan (Mexico City Mexico) Adultfemale rats weighing 200ndash250 g were included in the exper-iments The rats were housed in Plexiglas cages (six to sevenrats per cage 44 cm width 33 cm length and 20 cm height)under a 12 h12 h lightdark cycle (lights on at 700 am)at an average temperature of 25∘C (plusmn2∘C) with ad libitumaccess to purified water and food (Harlan Mexico) All of theexperimental procedures were performed according to theGuide for the Care and Use of Laboratory Animals publishedby the National Institutes of Health [24] and Norma OficialMexicana para el Cuidado y Uso de Animales de Laboratorio[25] The general protocol received authorization (MVZ-18912) from the Comite Interno de Etica de la Escuela

de Medicina Veterinaria y Zootecnia de la UniversidadAutonoma de Tlaxcala

22 Preparation of M frutescens and M grandiflora ExtractsM frutescens and M grandiflora were collected in theirnatural habitat in the state of Tlaxcala Mexico in Septem-ber 2011 and were authenticated by a specialist from theHerbarium of the Universidad Autonoma de Tlaxcala wherevoucher specimens are preserved and the plants are cultivated(serial number of M frutescens UATX11 and M grandifloraUATX12) The doses of the aqueous crude extracts of Mfrutescens and M grandiflora used in the present study areequivalent to those used for humans and were preparedaccording to previous studies [21] in which 25 and 50mgkgbut not higher doses ofM frutescens produce anxiolytic-likeeffects Briefly the leaves ofM frutescens andM grandiflorawere collected and prepared for drying for 20 days Oncedried the material was ground into a fine powder (average1 g) which was mixed with 20mL of purified water Thismixture was warmed for approximately 10min just beforeboiling The obtained infusion was filtered and oven-driedat a temperature of 55∘C and the brownish residue of theextract yield was calculated as 80mg The dried extract ofthe plant was maintained at 3∘C and then used to preparethe stock solutions In the present study a 50mgmL solutionwas initially prepared and then diluted to obtain equivalentsolutions of 25mgmL Considering that in traditional Mex-ican medicine similar effects are attributed to M frutescensand M grandiflora extracts [17 18] we also tested the effectsof 25 and 50mgkg of the M grandiflora extract on anxiety-like behavior in female rats in the present study The extractsthat corresponded to each dose were prepared 40min priorto administration to avoid modifications in the chemicalproperties of the extracts

23 Behavioral Tests To evaluate the effects of the treatmentsthe same rats were evaluated in the elevated plusmaze (5min)and subsequently in the open field (5min) as previouslyreported [20 21]

231 Elevated Plus Maze The apparatus was constructedof wood and situated in a brightly lit room (40 lux) Theapparatus consisted of two opposite open and closed armsset in a plus configuration The dimensions of the openarms were 50 cm length times 10 cm width and the closedarms were 50 cm length times 10 cm width times 40 cm height Theentire maze was elevated 50 cm above the floor A digitalvideo camera (Sony DCR-SR42 40x optical zoom CarlZeiss lens) was installed above the apparatus to record theratsrsquo activity Later two independent observers measured thebehavioral variables until reaching at least 95 agreement inthe measurements

To evaluate the effects of the treatments the rats wereplaced in the center of the maze facing an open arm Theevaluated variables were (i) the time spent in the open arms(ii) the number of entries into the open arms (iii) the totalnumber of entries (open arms + closed arms) and (iv) thepercentage of open arm entries ((open entries)(total entries)

BioMed Research International 3

times 100) These variables were selected based on previousstudies in which these measures were shown to be reliableindicators of experimental anxiety [26 27] Rats that fell fromthe apparatus were discarded from subsequent data analysis

232 Open Field Test To evaluate the effects of the Mfrutescens andM grandiflora extracts and diazepam on spon-taneous motor activity the rats were individually subjectedto a 5min open field test The open field apparatus was anopaque Plexiglas cage (44 times 33 cm) with 20 cm high wallsThe floor was delineated into 12 squares (11 times 11 cm) Adigital video camera (Sony DCR-SR42 40x optical zoomCarl Zeiss lens) was installed above the cage to record theratsrsquo activity Later two independent observers measured thebehavioral variables General motor activity was examinedto determine whether the treatments caused hypoactivityhyperactivity or no motor changes which could interferewith behavioral activity in the elevated plusmazeAt the onsetof the test the rats were gently placed in one of the cornersof the cage and the following variables were measured (i)the number of squares crossed by the rat (ie when ananimal passed from one square to another with its rear legs)(ii) the time in seconds spent in rearing (ie when the ratacquired a vertical posture with respect to the cage floor)(iii) the time in seconds spent in grooming (ie paw lickingnoseface grooming (strokes along the snout) head washing(semicircularmovements over the top of the head and behindthe ears) body groomingscratching (body fur licking andscratching the body with the hind paws) leg licking andtailgenital grooming (licking of the genital area and tail))[28 29] and (iv) total resting time in seconds (ie when therat acquired a quiet posture on the floorwithoutmovements)After each test session the elevated plus maze apparatus andopen field cage were carefully cleaned with a 10 ethanolsolution to remove the scents of previously evaluated animalsFiveminutes elapsed between each test to allow the odors andcleaning solutions to dissipate

24 Vaginal Smears Before initiating the behavioral tests weobtained daily vaginal smears from the rats Only femaleswith three continuous regular cycles (4-5 days) were includedin the study The vaginal samples were gently collected byinserting the tip of a medicine dropper into the vaginaflushing saline in and out and placing the fluid ontomicroscope slides The estrous cycle stage was determinedimmediately by optical microscopy (40x magnification) Thesmears were classified by estimating the relative propor-tions of leukocytes nucleated epithelial cells and cornifiedepithelial cells [30] as follows diestrus (predominance oflarge numbers of leukocytes some nucleated and almost nocornified cells) proestrus (primarily large round nucleatedcells) estrus (many cornified cells) and metestrus (largenumbers of leukocytes some cornified cells and almostno nucleated cells) We classified the animals into twosubgroups for the statistical analysis (ie proestrus-estrusand metestrus-diestrus) by considering previous studies inwhich proestrus-estrus was characterized by low anxiety-like indicators associated with high concentrations of ovarian

hormones and metestrus-diestrus was characterized by highanxiety-like indicators associated with low concentrations ofovarian hormones [10 11 31]

25 Experimental Groups and Treatments A total of 104 ratswere included in the study and assigned to six independentgroups The control group (vehicle 119899 = 17) receivedpurified water (1mLkg) which was used to prepare theplant extracts Two groups received 25mgkgmL (119899 = 16)and 50mgkgmL (119899 = 17) of the aqueous extract of Mfrutescens Two groups received 25mgkgmL (119899 = 18)and 50mgkgmL (119899 = 19) of the aqueous extract of Mgrandiflora All of the doses of the extracts were administeredorally by gavage Purified water was used as the vehicleconsidering that it is used in ancient traditional medicineto prepare Montanoa extracts [15 16] An additional groupreceived 2mgkg diazepam ip (119899 = 17) as a referenceanxiolytic drug to validate the experimental conditions ofthe elevated plus maze and make comparisons with theeffects of the Montanoa extracts The 2mgkg dose ofdiazepam was selected according to previous studies thatreported anxiolytic-like effects of this dose in the elevatedplus maze [27 32] Diazepam was acquired from Labora-torios Cryopharma DF Mexico All of the treatments wereadministered in a single dosage in an equivalent volume of1mLkg Thirty minutes after the corresponding treatmentthe rats were evaluated in the elevated plus maze (5min) andsubsequently in the open field (5min) After the open fieldtest vaginal smears were obtained to assign each rat fromeach treatment group to its corresponding subgroup (ieproestrus-estrus and metestrus-diestrus groups)

26 Statistical Analysis All of the data were analyzed usingtwo-way analysis of variance (ANOVA) with independentgroups with treatment as the first factor and phase of theovarian cycle as the second factor Values of 119875 le 005 in theANOVA were followed by the Student-Newman-Keuls posthoc test The data are expressed as mean plusmn standard error

3 Results

31 Vaginal Smears The microscopic analysis of vaginalsmears permitted us to identify the phase of the ovariancycle in each rat to be included in each subgroup (ieproestrus-estrus and metestrus-diestrus phases) for the sta-tistical analysis The number of animals in each subgroupwas the following proestrus-estrus (119899 = 8-9 per group) andmetestrus-diestrus (119899 = 8minus10 per group)

32 Elevated Plus Maze The two-way ANOVA showedsignificant differences in the time spent in the open armsaccording to the phase of the ovarian cycle (119865

192= 1685

119875 lt 0001) and treatment (119865592= 1095 119875 lt 0001)

The interaction between factors was also significant (119865592=

874 119875 lt 0001) The post hoc test showed that similarto diazepam rats treated with 25 and 50mgkg of the Mfrutescens extract and 50mgkg of theM grandiflora extractsduring the metestrus-diestrus phase spent more time in the


open arms compared with rats in the metestrus-diestrusphase in the control group No other effects of the treatmentson the time spent in the open arms during the proestrus-estrus phase were detected

The two-wayANOVAof the percentage of entries into theopen arms did not reveal statistically significant differencesconsidering the phase of the ovarian cycle (119865

192= 064 119875 =

0425) but statistically significant differences were detectedamong treatments (119865

592= 344 119875 lt 0007) The post

hoc test showed that 50mgkg of the M frutescens extractand 2mgkg diazepam increased this variable compared withthe control group The interaction between factors was alsosignificant (119865

592= 359 119875 lt 0005) The post hoc test

showed that similar to diazepam rats treated with 25 and50mgkg of the M frutescens and M grandiflora extractsduring the metestrus-diestrus phase exhibited an increase inthe percentage of entries into the open arms compared withthe control group in the metestrus-diestrus phase No othereffects of the treatments on this variable were detected duringthe proestrus-estrus phase (Figure 1(b))

The analysis of the number of entries into the open armsdid not reveal statistically significant differences betweenphases of the ovarian cycle (119865

192= 068 119875 = 0413) or

among treatments (119865592= 054 119875 = 0748) or an interaction

between factors (119865592= 164 119875 = 0159) The analysis of the

total number of entries into the arms (open arms + closedarms) did not reveal significant differences between phasesof the ovarian cycle (119865

192= 001 119875 = 095) or treatments

(119865592= 208 119875 = 0074) or an interaction among factors

(119865592= 060 119875 = 0703 Table 1)

33 Open Field Test The two-way ANOVA did not showstatistically significant differences (119865

192= 107 119875 =

0303) in the number of crossings between phases of theovarian cycle but significant differences (119865

592= 410 119875 lt

0002) were detected among treatments The post hoc testrevealed that diazepam reduced (119875 lt 005) the number ofcrossings compared with all of the other treatment groupsThe interaction between factors was also significant (119865

592=

666 119875 lt 0001) The post hoc test showed that only ratstreated with diazepam during the proestrus-estrus phaseexhibited a significant reduction (119875 lt 005) of the number ofcrossings compared with all of the other treatment groups inthe proestrus-estrus andmetestrus-diestrus phases No othereffects of treatment on the number of crossings were detectedduring the metestrus-diestrus phase (Figure 2(a))

The analysis of rearing time did not show statisticallysignificant differences (119865

192= 309 119875 = 0082) between

phases of the ovarian cycle but significant (119865592= 485

119875 lt 0001) differences were detected among treatments Thepost hoc test revealed that diazepam reduced rearing timecompared with all of the other treatment groups (119875 lt 005)The interaction between factors was not significant (119865

592=

112 119875 = 0360) Rats in the proestrus-estrus phase treatedwith diazepam exhibited a significant reduction (119875 lt 005)of rearing time compared with all of the other treatmentgroups in the proestrus-estrus and metestrus-diestrus phases(Figure 2(b))

The analysis of grooming behavior revealed statisticallysignificant differences (119865

192= 404 119875 lt 0047) between

phases of the ovarian cycle and among treatments (119865592=

776 119875 lt 0001) and a significant interaction between factors(119865592= 1655 119875 lt 0001) The post hoc test showed that only

rats treated with diazepam during the proestrus-estrus phaseexhibited a significant reduction (119875 lt 005) of groomingtime compared with all of the other treatment groups in theproestrus-estrus ormetestrus-diestrus phaseNoother effectsof treatment on grooming behavior were detected during themetestrus-diestrus phase (Figure 2(c))The analysis of restingtime did not reveal statistically significant differences (119865

192=

066 119875 = 0416) between phases of the ovarian cycle butthe analysis of this variable revealed statistically significantdifferences among treatments (119865

592= 295 119875 = 0016) The

post hoc test revealed that diazepam increased resting timecompared with all of the other treatment groups (119875 lt 005)The interaction between factors was also significant (119865

592=

265 119875 = 0028) The post hoc test showed that only rats inthe proestrus-estrus phase treated with diazepam exhibited asignificant increase in resting time compared with all of theother treatment groups in the proestrus-estrus or metestrus-diestrus phase (119875 lt 005) No other effects of treatmenton resting time were detected during the metestrus-diestrusphase (Figure 2(d))

4 Discussion

The present study investigated the influence of ovarian cyclephases on anxiety-like behavior in the elevated plus mazein female rats treated with M frutescens and M grandifloraaqueous crude extracts and the results were comparedwith the effects of diazepam The extracts of M frutescens(25 and 50mgkg) and M grandiflora (50mgkg) producedanxiolytic-like effects similarly to 2mgkg diazepam duringthe metestrus-diestrus phase TheM frutescens andM gran-diflora extracts did not exert any behavioral effects during theproestrus-estrus phase but diazepam produced hypoactivityin the open field test during this phase of the ovarian cyclereflected by reductions of the number of crossings rearingtime and grooming time and an increase in resting timeThese findings indicate that similar to diazepam the Mfrutescens and M grandiflora extracts produced differentialanxiolytic-like effects that depended on the phase of theovarian cycle but lacked additional anxiolytic-like effectsduring the proestrus-estrus phase These data also indicatethat a lower dose of the M frutescens extract (25mgkg)was required to produce an anxiolytic-like effect comparedwith the M grandiflora extract (50mgkg) suggesting thedifferential composition of bioactive compounds containedin each plant

The elevated plus maze is a well-accepted and validatedanimal model for testing the effectiveness of anxiolyticdrugs [23 26 33] In this animal model rats or mice thatdisplay anxiety-like behavior usually show a reduction ofopen-arm exploration reflected by a decrease in the timespent in the open arms Animals treated with clinicallyeffective anxiolytic drugs (eg diazepam) [34ndash36] someneurosteroids with anxiolytic-like potency (eg progesterone


Proestrus-estrus phaseMetestrus-diestrus phase

0

50

100

150

200

250Ti

me i

n op

en ar

ms (

s)

VehM frutescens Diazepam

(mgkg)

+ ++

25 50 25 250M grandiflora

lowast

lowast

+

(a)

0

20

40

60

80

100

Entr

ies i

nto

the o

pen

arm

s (

)

+

+ ++

+



(mgkg)


lowast

(b)

Figure 1 Elevated plus maze (a) Time spent in the open arms and (b) percentage of entries into the open arms during the 5min testlowast

119875 lt 005 compared with proestrus-estrus phase in the same group +119875 lt 005 compared with metestrus-diestrus phase in the controlgroup (Veh) (two-way ANOVA followed by Student-Newman-Keuls post hoc test)

Table 1 Variables evaluated in the elevated plus maze without significant changes associated with treatment and phase of the ovarian cycle

Group Number of entries into the open arms Total number of entries into the arms (open + close)P-E M-D P-E M-D

Vehicle 1011 plusmn 186 563 plusmn 207 1322 plusmn 208 1450 plusmn 206Mf (mgkg)

25 775 plusmn 171 650 plusmn 095 1387 plusmn 356 1025 plusmn 23950 711 plusmn 091 800 plusmn 111 956 plusmn 160 1087 plusmn 179

Mg (mgkg)25 566 plusmn 067 767 plusmn 085 844 plusmn 061 967 plusmn 05750 711 plusmn 121 710 plusmn 096 1011 plusmn 077 950 plusmn 083

Diazepam (mgkg)2 878 plusmn 082 813 plusmn 097 1122 plusmn 121 1125 plusmn 149

No significant differences among subgroups were found two-wayANOVAMf M frutescensMgM grandiflora P-E proestrus-estrus phaseM-Dmetestrus-diestrus phase

and allopregnanolone) [37ndash39] and some extracts frommedicinal plants with reputed anxiolytic properties exhibitan increase in the total time spent in the open arms [4041] which is considered an anxiolytic-like effect at theexperimental level

The present study found that 25 and 50mgkg of theM frutescens extract and 50mgkg of the M grandifloraextract decreased anxiety-like behavior in the elevated plusmaze in female rats during the metestrus-diestrus phasewhen the concentrations of steroid hormones are lower andanxiety-like behavior increases During the proestrus-estrusphase when steroid hormones reach higher levels treatmentwith the M frutescens or M grandiflora extract did notproduce anxiolytic-like effects The effects exerted by the Mfrutescens and M grandiflora extracts during the metestrus-diestrus phase were comparable to those observed afterdiazepam administration supporting the possible anxiolytic-like profile of the aqueous crude extracts of these Montanoaplants

Similar to diazepam theM frutescens andM grandifloraextracts did not exert additional anxiolytic-like effects in theelevated plus maze during the proestrus-estrus phase Onlythe naturally observed anxiolytic-like effects in this phaseof the ovarian cycle (associated with high concentrations ofsteroid hormones [42]) were observed These data suggesta ldquoceiling effectrdquo in the elevated plus maze that preventsthe detection of additional anxiolytic-like effects during thisphase of the estrous cycle Other experimental models thatevaluate the effects of anxiolytic drugs may detect significantdifferences in the proestrus-estrus phase The present resultsshowed that diazepam but not the M frutescens or Mgrandiflora extract produced hypoactivity in the open fieldtest during the proestrus-estrus phase reflected by reductionsof the number of crossings rearing time and grooming timeand an increase in resting time Interestingly hypoactivitywas only identified in the open field test and not in theelevated plus maze in which the total number of entries intothe arms is considered an indicator of motor activity These


0

20

40

60Cr

ossin

g (n

)


(mgkg)


lowast

(a)

0

10

20

30

Rear

ing

(s)


(mgkg)


lowast

(b)

0

10

20

30

Gro

omin

g (s

) +

+

+

++



(mgkg)


lowast

(c)

0

50

100

150

200

250

Resti

ng (s

)



(mgkg)


lowast

(d)

Figure 2 Open field test (a) The number of crossings (b) rearing time (c) grooming time and (d) resting time during the 5min testlowast

119875 lt 005 compared with metestrus-diestrus phase in the same group +119875 lt 005 compared with metestrus-diestrus phase in the controlgroup (Veh) 119875 lt 005 compared with proestrus-estrus phase in the same group (two-way ANOVA followed by Student-Newman-Keulspost hoc test)

results are relevant because they show that the total numberof entries into the arms was not an adequate parameterfor identifying an effect on spontaneous motor activityTherefore the open field test should be specifically used toevaluate motor activity in parallel with the elevated plusmaze

In the present study the effect on general motor activitymay be related to a potential combined effect of diazepamand endogenous steroid hormones (eg progesterone orallopregnanolone) which together could act on GABAAreceptors and disrupt locomotor activityThis possibility maybe supported by previous studies inwhich the involvement ofGABAA receptors in the anxiolytic-like effects of the aqueouscrude extracts of M frutescens [21] and M tomentosa [20]was reported In those studies GABAA receptor antagonismblocked the anxiolytic-like effect of the Montanoa extractssuggesting that the GABAergic system mediates the actionsof these plants on the central nervous system

Considering that the proestrus-estrus phase is associatedwith higher circulating concentrations of steroid hormonesrecognized by the GABAA receptor [43] and that diazepameffectively targets GABAA receptors overstimulation of thisreceptor may have occurred and female rats may haveconsequently exhibited hypoactivity in the open field testThis effect on motor activity may be comparable to theeffect in the open field test in rats after administration ofhigher doses of diazepam [21 44] This finding suggests thatthe chemical compounds contained in the aqueous crudeextracts ofM frutescens andM grandiflorapossess low syner-gistic activity with steroid hormones on theGABAA receptorscomparedwith diazepam to provoke overstimulation of thesereceptors in central structures that mediate the reduction ofanxiety-like behavior Further studies of synergistic activityare needed to evaluate this possibility

Grooming behavior increases in rats subjected to mildto moderate stress [45ndash47] whereas animals subjected to


high stress exhibit a significant decrease in this behavior[48 49] compared with nonstressed animals The decreasein grooming behavior after high levels of stress can beprevented by anxiolytic drugs such as diazepam and othersubstances with anxiolytic potency which is considered anadditional indicator of anxiolytic-like activity at the experi-mental level [50ndash52] In the present study the vehicle-treatedgroup showed lower levels of grooming behavior duringthe metestrus-diestrus phase compared with the proestrus-estrus phase an effect possibly associated with stress inducedby exposure to the elevated plus maze and the reducedconcentration of steroid hormones during this phase of theovarian cycle A similar pattern of activity in the lightdarktest has also been reported in rats with long-term suppressionof ovarian hormones [52] Interestingly similar to diazepam25 and 50mgkg of the M frutescens extract and 50mgkgof the M grandiflora extract maintained grooming behaviorduring the metestrus-diestrus phase at a similar level asduring the proestrus-estrus phase in vehicle-treated ratsThese data support the hypothesis that M frutescens andM grandiflora possess distinctive anxiolytic-like profiles inthe elevated plus maze Additionally during the proestrus-estrus phase no effects of theMontanoa extracts on groomingand rearing behavior were detected Nevertheless diazepamduring this same phase of the ovarian cycle reduced bothbehaviors in the open field test whichmay be associated witha relaxation effect of diazepam that is enhanced by steroidhormones during this phase of the ovarian cycle

One limitation of the present study is that we did notidentify the bioactive compounds involved in the anxiolytic-like effects of the extracts or differences in the bioavailabil-ity of these bioactive compounds between different plantextracts Nonetheless the present results support the tradi-tional use of M frutescens and M grandiflora as potentialanxiolytic agents and further studies should be conductedto identify the chemical compounds and parameters relatedto their bioavailability that are involved in their anxiolytic-like effects We can offer a partial explanation of theanxiolytic-like activity exhibited by the extracts analyzedherein by considering phytochemical data on Montanoaplants Pentacyclic triterpenes and tetracyclic diterpenes(eg ent-kaurenoic acid grandiflorenic acid kauradienoicacid zoapatanol and montanol) sesquiterpene lactonesand flavonoids are the main biologically active compoundscontained in extracts ofMontanoa plants [53 54]Montanoaplants and other Heliantheae plants contain terpenes withremarkable pleiotropic features For example ent-kaurenoicacid possesses both uterotonic and hypoglycemic activity[55] We propose that the terpenes or flavonoids containedinMontanoa plants could be the active compounds involvedin the anxiolytic-like effects reported herein by targetingthe GABAergic system Some flavonoids and terpenes withanxiolytic-like activity target GABAA receptors [56ndash58]Other compounds such as sesquiterpene lactones can alsocontribute to the beneficial effects of M frutescens and Mgrandiflora extracts after they enter the nervous systemThus sesquiterpene lactones act as alkylating agents and wehave observed antitumoral effects on uterine leiomyomas inwomen after chronic treatment under specific circumstances

(Carro-Juarez et al in preparation) Toxicological studiesare necessary to investigate the safety profile of Montanoaextracts to avoid side effects similar to previous reports forother medicinal plants that act on the central nervous system[59 60]

In conclusion the present findings justify further studiesthat seek to standardize theM frutescens andM grandifloraextracts and identify the bioactive compounds that producetheir anxiolytic-like effects Additionally the present resultsmay impact the future development of natural therapeuticalternatives to ameliorate symptoms of anxiety in women inwhom changes in concentrations of steroid hormones havebeen reportedThe data presented herein further support thetraditional use ofM frutescens andM grandiflora describedby Ximenez in 1615 ldquoresolves the mood changes and nervesin an admirable formrdquo [16] for the treatment of ldquonervousrdquodisorders associated with the menstrual cycle in women

Conflict of Interests

There is no conflict of interests

Acknowledgments

The authors thank Michael Arends for revising and editingthe English of this paper This study was partially supportedby Universidad Autonoma de Tlaxcala (Cuerpo AcademicoUATX-CA-215) and Universidad Veracruzana (CuerpoAcademico UVE-CA-25 and Programa de FortalecimientoAcademico del Posgrado de Alta Calidad I0104582013C-7032013) that did not participate in the study design datacollection data analysis interpretation of the data writingof the paper or decision to submit the paper for publication

References

[1] R C Kessler A M Ruscio K Shear and H U Wittchen ldquoEpi-demiology of anxiety disordersrdquo Current Topics in BehavioralNeurosciences vol 2 pp 21ndash35 2010

[2] F R Schneier J Johnson C D HornigM R Liebowitz andMMWeissman ldquoSocial phobia comorbidity and morbidity in anepidemiologic samplerdquo Archives of General Psychiatry vol 49no 4 pp 282ndash288 1992

[3] R C Kessler K A McGonagle S Zhao et al ldquoLifetime and12-month prevalence of DSM-III-R psychiatric disorders in theUnited States results from the national comorbidity surveyrdquoArchives of General Psychiatry vol 51 no 1 pp 8ndash19 1994

[4] N Breslau L Schultz and E Peterson ldquoSex differences indepression a role for preexisting anxietyrdquo Psychiatry Researchvol 58 no 1 pp 1ndash12 1995

[5] M V Seeman ldquoPsychopathology in women and men focus onfemale hormonesrdquoThe American Journal of Psychiatry vol 154no 12 pp 1641ndash1647 1997

[6] R Bowen A Bowen M Baetz J Wagner and R PiersonldquoMood instability in women with premenstrual syndromerdquoJournal of Obstetrics and Gynaecology Canada vol 33 no 9 pp927ndash934 2011

[7] J Studd and R E Nappi ldquoReproductive depressionrdquoGynecolog-ical Endocrinology vol 28 no 1 pp 42ndash45 2012


[8] C N Soares ldquoDepression in peri- and postmenopausal womenprevalence pathophysiology and pharmacological manage-mentrdquo Drugs Aging vol 30 no 9 pp 677ndash685 2013

[9] C M Contreras M Molina M Saavedra and L Martınez-Mota ldquoLateral septal neuronal firing rate increases duringproestrus-estrus in the ratrdquo Physiology and Behavior vol 68 no3 pp 279ndash284 2000

[10] M J Zuluaga D Agrati M Pereira N Uriarte A Fernandez-Guasti and A Ferreira ldquoExperimental anxiety in the black andwhite model in cycling pregnant and lactating ratsrdquo Physiologyand Behavior vol 84 no 2 pp 279ndash286 2005

[11] A Fernandez-Guasti and O Picazo ldquoThe actions of diazepamand serotonergic anxiolytics vary according to the genderand the estrous cycle phaserdquo Pharmacology Biochemistry andBehavior vol 37 no 1 pp 77ndash81 1990

[12] C M Contreras L Martınez-Mota and M Saavedra ldquoDe-sipramine restricts estral cycle oscillations in swimmingrdquoProgress in Neuro-Psychopharmacology and Biological Psychia-try vol 22 no 7 pp 1121ndash1128 1998

[13] A Fernandez-Guasti L Martınez-Mota E Estrada-CamarenaC M Contreras and C Lopez-Rubalcava ldquoChronic treat-ment with desipramine induces an estrous cycle-dependentanxiolytic-like action in the burying behavior but not inthe elevated plus-maze testrdquo Pharmacology Biochemistry andBehavior vol 63 no 1 pp 13ndash20 1999

[14] M Molina-Hernandez N P Tellez-Alcantara M A Dıaz JPerez-Garcıa J I Olivera-Lopez andM T Jaramillo ldquoAnticon-flict actions of aqueous extracts of flowers ofAchilleamillefoliumL vary according to the estrous cycle phases in Wistar ratsrdquoPhytotherapy Research vol 18 no 11 pp 915ndash920 2004

[15] M de la Cruz in Libellus de Medicinalibus Indorum HerbisManuscrito Azteca de 1552 version Espanola con estudiosy comentarios por diversos autores Ediciones del InstitutoMexicano del Seguro Social Mexico City Mexico 1964

[16] F Ximenez Quatro libros de la naturaleza y virtudes de lasplantas y animales que estan recevidos en el uso de medicina enla Nueva Espana y la metodo y correccion y preperacion quepara administrarllas se requiere con lo que el doctor FranciscoHernandez escrivio en lengua latina Viuda de Diego LopezDavalos Mexico City Mexico 1615

[17] S D Levine D W Hahn M L Cotter et al ldquoThe Mexicanplant zoapatle (Montanoa tomentosa) in reproductivemedicinePast present and futurerdquo Journal of Reproductive Medicine forthe Obstetrician and Gynecologist vol 26 no 10 pp 524ndash5281981

[18] A J Gallegos ldquoThe zoapatle VIrdquo Contraception vol 31 no 5pp 487ndash497 1985

[19] M Carro-Juarez E Cervantes M Cervantes-Mendez andG Rodrıguez-Manzo ldquoAphrodisiac properties of Montanoatomentosa aqueous crude extract in male ratsrdquo PharmacologyBiochemistry and Behavior vol 78 no 1 pp 129ndash134 2004

[20] M I Sollozo-Dupont E Estrada-Camarena M Carro-Juarezand C Lopez-Rubalcava ldquoThe lyophilisate of M tomentosainduces anxiolytic-like effects in male rats through modulationof GABA-A receptor complexrdquo in Proceedings of the 42ndAnnual Meeting of the Society for Neuroscience New OrleansLa USA 2012

[21] M Carro-Juarez J F Rodrıguez-Landa M L Rodrıguez-PenaM J Rovirosa-Hernandez and FGarcıa-Orduna ldquoThe aqueouscrude extract of Montanoa frutescens produces anxiolytic-likeeffects similarly to diazepam in Wistar rats involvement of

GABAA receptorrdquo Journal of Ethnopharmacology vol 143 no2 pp 592ndash598 2012

[22] G Griebel D J Sanger and G Perrault ldquoThe use of therat elevated plus-maze to discriminate between non-selectiveand BZ-1 (1205961) selective benzodiazepine receptor ligandsrdquoPsychopharmacology vol 124 no 3 pp 245ndash254 1996

[23] G B Varty C A Morgan M E Cohen-Williams V LCoffin and G J Carey ldquoThe gerbil elevated plus-maze Ibehavioral characterization and pharmacological validationrdquoNeuropsychopharmacology vol 27 no 3 pp 357ndash370 2002

[24] National Research Council Guide for the Care and Use ofLaboratory Animals Publication no 85-23 National AcademyPress Washington DC USA 1985

[25] NOM-062-ZOO-1999 Especificaciones Tecnicas para la Pro-duccion Cuidado y Uso de los Animales de Laboratorio Sec-retarıa de Agricultura Ganaderıa Desarrollo Rural Pesca yAlimentacion

[26] 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

[27] C M Contreras J F Rodrıguez-Landa A G Gutierrez-Garcıa M R Mendoza-Lopez R I Garcıa-Rıos and J Cueto-Escobedo ldquoAnxiolytic-like effects of human amniotic fluid andits fatty acids inWistar ratsrdquo Behavioural Pharmacology vol 22no 7 pp 655ndash662 2011

[28] A V Kalueff and P Tuohimaa ldquoGrooming analysis algorithmfor neurobehavioural stress researchrdquo Brain Research Protocolsvol 13 no 3 pp 151ndash158 2004

[29] A V Kalueff and P Tuohimaa ldquoContrasting grooming phe-notypes in three mouse strains markedly different in anxietyand activity (129S1 BALBc and NMRI)rdquo Behavioural BrainResearch vol 160 no 1 pp 1ndash10 2005

[30] M E Rhodes E M Balestreire R K Czambel and R TRubin ldquoEstrous cycle influences on sexual diergism of HPAaxis responses to cholinergic stimulation in ratsrdquo Brain ResearchBulletin vol 59 no 3 pp 217ndash225 2002

[31] A A Walf C Koonce K Manley and C A Frye ldquoProestrouscompared to diestrous wildtype but not estrogen receptor betaknockout mice have better performance in the spontaneousalternation and object recognition tasks and reduced anxiety-like behavior in the elevated plus andmirrormazerdquo BehaviouralBrain Research vol 196 no 2 pp 254ndash260 2009

[32] N Karim N Gavande P Wellendorph G A R JohnstonJ R Hanrahan and M Chebib ldquo3-Hydroxy-21015840-methoxy-6-methylflavone a potent anxiolytic with a unique selectivity pro-file at GABAA receptor subtypesrdquo Biochemical Pharmacologyvol 82 no 12 pp 1971ndash1983 2011

[33] G Griebel R J Rodgers G Perrault and D J Sanger ldquoRiskassessment behaviour evaluation of utility in the study of 5-HT-related drugs in the rat elevated plus-maze testrdquo PharmacologyBiochemistry and Behavior vol 57 no 4 pp 817ndash827 1997

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

[35] A Fernandez-Guasti A Ferreira and O Picazo ldquoDiazepambut not buspirone induces similar anxiolytic-like actions inlactating and ovariectomized Wistar ratsrdquo Pharmacology Bio-chemistry and Behavior vol 70 no 1 pp 85ndash93 2001

[36] M A Wilson P R Burghardt K A Ford M B Wilkinsonand S D Primeaux ldquoAnxiolytic effects of diazepam and ethanol


in two behavioral models comparison of males and femalesrdquoPharmacology Biochemistry andBehavior vol 78 no 3 pp 445ndash458 2004

[37] CGomez A Saldivar-Gonzalez GDelgado andR RodrıguezldquoRapid anxiolytic activity of progesterone and pregnenolone inmale ratsrdquo Pharmacology Biochemistry and Behavior vol 72 no3 pp 543ndash550 2002

[38] D S Reddy B W OrsquoMalley and M A Rogawski ldquoAnxiolyticactivity of progesterone in progesterone receptor knockoutmicerdquo Neuropharmacology vol 48 no 1 pp 14ndash24 2005

[39] E Engin and D Treit ldquoThe anxiolytic-like effects of allopreg-nanolone vary as a function of intracerebral microinfusionsite the amygdala medial prefrontal cortex or hippocampusrdquoBehavioural Pharmacology vol 18 no 5-6 pp 461ndash470 2007

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

[41] M Herrera-Ruiz A Gonzalez-Carranza A Zamilpa EJimenez-Ferrer M Huerta-Reyes and V M Navarro-GarcıaldquoThe standardized extract of Loeselia mexicana possesses anx-iolytic activity through the 120574-amino butyric acid mechanismrdquoJournal of Ethnopharmacology vol 138 no 2 pp 261ndash267 2011

[42] F K Marcondes K J Miguel L L Melo and R C Spadari-Bratfisch ldquoEstrous cycle influences the response of female ratsin the elevated plus-maze testrdquo Physiology and Behavior vol 74no 4-5 pp 435ndash440 2001

[43] M E Freeman ldquoThe ovarian cycle of the female of the ratrdquo inThe Physiology of Reproduction E Knobil and J Neill Eds pp1893ndash1923 Raven Press New York NY USA 1988

[44] X Y Wu J L Zhao M Zhang F Li T Zhao and L QYang ldquoSedative hypnotic and anticonvulsant activities of theethanol fraction from Rhizoma Pinelliae Praeparatumrdquo Journalof Ethnopharmacology vol 135 no 2 pp 325ndash329 2011

[45] Y Singh A K Jaiswal M Singh and S K BhattacharyaldquoBehavioural effects of prenatal diazepam administration onanxiety patterns in ratsrdquo Indian Journal of Experimental Biologyvol 34 no 11 pp 1095ndash1099 1996

[46] A K Jaiswal ldquoEffect of prenatal alprazolam exposure onanxiety patterns in rat offspringrdquo Indian Journal of ExperimentalBiology vol 40 no 1 pp 35ndash39 2002

[47] A Moyaho and J Valencia ldquoGrooming and yawning traceadjustment to unfamiliar environments in laboratory Sprague-Dawley rats (Rattus norvegicus)rdquo Journal of Comparative Psy-chology vol 116 no 3 pp 263ndash269 2002

[48] H H van Dijken J A M van der Heyden J Mos and TJ H Tilders ldquoInescapable footshocks induce progressive andlong-lasting behavioural changes in male ratsrdquo Physiology andBehavior vol 51 no 4 pp 787ndash794 1992

[49] T S Perrot-Sinal A Gregus D Boudreau and L E KalynchukldquoSex and repeated restraint stress interact to affect cat odor-induced defensive behavior in adult ratsrdquo Brain Research vol1027 no 1-2 pp 161ndash172 2004

[50] T Hata Y Nishimura T Kita E Itoh and A Kawabata ldquoTheabnormal open-field behavior of SART-stressed rats and effectsof some drugs on itrdquo Japanese Journal of Pharmacology vol 48no 4 pp 479ndash490 1988

[51] P S DrsquoAquila A T Peana V Carboni and G SerraldquoExploratory behaviour and grooming after repeated restraintand chronic mild stress effect of desipraminerdquo EuropeanJournal of Pharmacology vol 399 no 1 pp 43ndash47 2000

[52] J F Rodrıguez-Landa J D Hernandez-Figueroa B CHernandez-Calderon and M Saavedra ldquoAnxiolytic-like effectof phytoestrogen genistein in rats with long-term absence ofovarian hormones in the black and white modelrdquo Progress inNeuro-Psychopharmacology and Biological Psychiatry vol 33no 2 pp 367ndash372 2009

[53] A J Gallegos ldquoThe zoapatle I A traditional remedy fromMexico emerges to modern timesrdquo Contraception vol 27 no3 pp 211ndash225 1983

[54] A Guzman-Duran M A Wens H Ponce-Monter N Pedronand A J Gallegos ldquoZoapatle XIII Isolation of water solublefractions fromMontanoa frutescens and some biological activi-tiesrdquoArchivos de InvestigacionMedica vol 19 no 2 pp 157ndash1631988

[55] DWHahn EWEricsonMT Lai andA Probst ldquoAntifertilityactivity ofMontanoa tomentosa (zoapatle)rdquo Contraception vol23 no 2 pp 133ndash140 1981

[56] A C Paladini M Marder H Viola C Wolfman C Wasowskiand J H Medina ldquoFlavonoids and the central nervous systemfrom forgotten factors to potent anxiolytic compoundsrdquo Journalof Pharmacy and Pharmacology vol 51 no 5 pp 519ndash526 1999

[57] P Zanoli R Avallone and M Baraldi ldquoBehavioral characteri-sation of the flavonoids apigenin and chrysinrdquo Fitoterapia vol71 supplement 1 pp S117ndashS123 2000

[58] S Khom B Strommer J Ramharter et al ldquoValerenicacid derivatives as novel subunit-selective GABAA receptorligandsmdashin vitro and in vivo characterizationrdquo British Journalof Pharmacology vol 161 no 1 pp 65ndash78 2010

[59] J F Rodrıguez-Landa and CM Contreras ldquoA review of clinicaland experimental observations about antidepressant actionsand side effects produced by Hypericum perforatum extractsrdquoPhytomedicine vol 10 no 8 pp 688ndash699 2003

[60] P Posadzki L K Watson and E Ernst ldquoAdverse effects ofherbal medicines an overview of systematic reviewsrdquo ClinicalMedicine vol 13 no 1 pp 7ndash12 2013

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


depressive-like behaviors under the presence of standardanxiolytic drugs [11ndash13] and plant extracts [14] with anxiolyticor antidepressant activity This has permitted investigators toexplore potential therapeutic regimens to ameliorate anxiety-or depressive-like behaviors associated with naturally occur-ring low plasma concentrations of steroid hormones

In ancientMexican traditional medicine somemedicinalplants have been reported to possess anxiolytic effects andare currently recommended for the treatment of ldquoanxiousstatesrdquo The Badianus Codex or Libellus de MedicinalibusIndorum Herbis written in 1552 [15] describes the use ofCihuapatli (ldquowomenrsquos medicinerdquo in the Nahuatl language)for the treatment of mood and ldquonervous disordersrdquo ananxiety-like behavior that is mentioned in the context ofancient Mexican culture Early reports by Ximenez (1615)stated ldquoCihuapatli resolves the mood changes and nerves inan admirable formrdquo [16] Cihuapatli is the common nameassigned to some plants included in the genus Montanoa(family Asteraceae tribe Heliantheae) in which M tomen-tosa M frutescens and M grandiflora are included Theaqueous crude extract from these plants has been used forcenturies in traditional Mexican medicine as a remedy forreproductive impairments anxiety and mood disorders [16ndash19] Recently the aqueous extracts of M tomentosa [20] andM frutescens [21] were reported to produce anxiolytic-likeeffects that are similar to diazepam in Wistar rats and theparticipation of 120574-aminobutyric acid-A (GABAA) receptorsin their anxiolytic-like effect was identified Nonethelessalthough some anxiolytic-like effects ofM frutescens extracthave been corroborated in male rats the anxiolytic-likeeffects ofM frutescens andM grandiflora in the female genusremain unexplored We hypothesized that M frutescens andM grandiflora extracts reduce anxiety-like behavior duringthe metestrus-diestrus phase of the estrous cycle in ratsTo test this hypothesis we used the elevated plus maze awell-accepted model that tests clinically effective anxiolyticdrugs [22 23] to evaluate the effects of several doses ofM frutescens and M grandiflora aqueous crude extracts onanxiety-like behavior during different phases of the ovariancycle in Wistar rats We also made comparisons with ananxiolytic dose of diazepam

2 Material and Methods

21 Animals Female Wistar rats were obtained from a localstrain supplied by Harlan (Mexico City Mexico) Adultfemale rats weighing 200ndash250 g were included in the exper-iments The rats were housed in Plexiglas cages (six to sevenrats per cage 44 cm width 33 cm length and 20 cm height)under a 12 h12 h lightdark cycle (lights on at 700 am)at an average temperature of 25∘C (plusmn2∘C) with ad libitumaccess to purified water and food (Harlan Mexico) All of theexperimental procedures were performed according to theGuide for the Care and Use of Laboratory Animals publishedby the National Institutes of Health [24] and Norma OficialMexicana para el Cuidado y Uso de Animales de Laboratorio[25] The general protocol received authorization (MVZ-18912) from the Comite Interno de Etica de la Escuela

de Medicina Veterinaria y Zootecnia de la UniversidadAutonoma de Tlaxcala

22 Preparation of M frutescens and M grandiflora ExtractsM frutescens and M grandiflora were collected in theirnatural habitat in the state of Tlaxcala Mexico in Septem-ber 2011 and were authenticated by a specialist from theHerbarium of the Universidad Autonoma de Tlaxcala wherevoucher specimens are preserved and the plants are cultivated(serial number of M frutescens UATX11 and M grandifloraUATX12) The doses of the aqueous crude extracts of Mfrutescens and M grandiflora used in the present study areequivalent to those used for humans and were preparedaccording to previous studies [21] in which 25 and 50mgkgbut not higher doses ofM frutescens produce anxiolytic-likeeffects Briefly the leaves ofM frutescens andM grandiflorawere collected and prepared for drying for 20 days Oncedried the material was ground into a fine powder (average1 g) which was mixed with 20mL of purified water Thismixture was warmed for approximately 10min just beforeboiling The obtained infusion was filtered and oven-driedat a temperature of 55∘C and the brownish residue of theextract yield was calculated as 80mg The dried extract ofthe plant was maintained at 3∘C and then used to preparethe stock solutions In the present study a 50mgmL solutionwas initially prepared and then diluted to obtain equivalentsolutions of 25mgmL Considering that in traditional Mex-ican medicine similar effects are attributed to M frutescensand M grandiflora extracts [17 18] we also tested the effectsof 25 and 50mgkg of the M grandiflora extract on anxiety-like behavior in female rats in the present study The extractsthat corresponded to each dose were prepared 40min priorto administration to avoid modifications in the chemicalproperties of the extracts

23 Behavioral Tests To evaluate the effects of the treatmentsthe same rats were evaluated in the elevated plusmaze (5min)and subsequently in the open field (5min) as previouslyreported [20 21]

231 Elevated Plus Maze The apparatus was constructedof wood and situated in a brightly lit room (40 lux) Theapparatus consisted of two opposite open and closed armsset in a plus configuration The dimensions of the openarms were 50 cm length times 10 cm width and the closedarms were 50 cm length times 10 cm width times 40 cm height Theentire maze was elevated 50 cm above the floor A digitalvideo camera (Sony DCR-SR42 40x optical zoom CarlZeiss lens) was installed above the apparatus to record theratsrsquo activity Later two independent observers measured thebehavioral variables until reaching at least 95 agreement inthe measurements

To evaluate the effects of the treatments the rats wereplaced in the center of the maze facing an open arm Theevaluated variables were (i) the time spent in the open arms(ii) the number of entries into the open arms (iii) the totalnumber of entries (open arms + closed arms) and (iv) thepercentage of open arm entries ((open entries)(total entries)








3 Results



192= 1685







192= 064 119875 =


592= 344 119875 lt 0007) The post





192= 068 119875 = 0413) or




192= 001 119875 = 095) or treatments


(119865592= 060 119875 = 0703 Table 1)


192= 107 119875 =


592= 410 119875 lt


592=



192= 309 119875 = 0082) between



592=



192= 404 119875 lt 0047) between




192=


592= 295 119875 = 0016) The


592=


4 Discussion





0

50

100

150

200

250Ti

me i

n op

en ar

ms (

s)


(mgkg)

+ ++


lowast

lowast

+

(a)

0

20

40

60

80

100

Entr

ies i

nto

the o

pen

arm

s (

)

+

+ ++

+



(mgkg)


lowast

(b)














0

20

40

60Cr

ossin

g (n

)


(mgkg)


lowast

(a)

0

10

20

30

Rear

ing

(s)


(mgkg)


lowast

(b)

0

10

20

30

Gro

omin

g (s

) +

+

+

++



(mgkg)


lowast

(c)

0

50

100

150

200

250

Resti

ng (s

)



(mgkg)


lowast

(d)














Acknowledgments


References





































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of








3 Results



192= 1685







192= 064 119875 =


592= 344 119875 lt 0007) The post





192= 068 119875 = 0413) or




192= 001 119875 = 095) or treatments


(119865592= 060 119875 = 0703 Table 1)


192= 107 119875 =


592= 410 119875 lt


592=



192= 309 119875 = 0082) between



592=



192= 404 119875 lt 0047) between




192=


592= 295 119875 = 0016) The


592=


4 Discussion





0

50

100

150

200

250Ti

me i

n op

en ar

ms (

s)


(mgkg)

+ ++


lowast

lowast

+

(a)

0

20

40

60

80

100

Entr

ies i

nto

the o

pen

arm

s (

)

+

+ ++

+



(mgkg)


lowast

(b)














0

20

40

60Cr

ossin

g (n

)


(mgkg)


lowast

(a)

0

10

20

30

Rear

ing

(s)


(mgkg)


lowast

(b)

0

10

20

30

Gro

omin

g (s

) +

+

+

++



(mgkg)


lowast

(c)

0

50

100

150

200

250

Resti

ng (s

)



(mgkg)


lowast

(d)














Acknowledgments


References





































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




192= 064 119875 =


592= 344 119875 lt 0007) The post





192= 068 119875 = 0413) or




192= 001 119875 = 095) or treatments


(119865592= 060 119875 = 0703 Table 1)


192= 107 119875 =


592= 410 119875 lt


592=



192= 309 119875 = 0082) between



592=



192= 404 119875 lt 0047) between




192=


592= 295 119875 = 0016) The


592=


4 Discussion





0

50

100

150

200

250Ti

me i

n op

en ar

ms (

s)


(mgkg)

+ ++


lowast

lowast

+

(a)

0

20

40

60

80

100

Entr

ies i

nto

the o

pen

arm

s (

)

+

+ ++

+



(mgkg)


lowast

(b)














0

20

40

60Cr

ossin

g (n

)


(mgkg)


lowast

(a)

0

10

20

30

Rear

ing

(s)


(mgkg)


lowast

(b)

0

10

20

30

Gro

omin

g (s

) +

+

+

++



(mgkg)


lowast

(c)

0

50

100

150

200

250

Resti

ng (s

)



(mgkg)


lowast

(d)














Acknowledgments


References





































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



0

50

100

150

200

250Ti

me i

n op

en ar

ms (

s)


(mgkg)

+ ++


lowast

lowast

+

(a)

0

20

40

60

80

100

Entr

ies i

nto

the o

pen

arm

s (

)

+

+ ++

+



(mgkg)


lowast

(b)














0

20

40

60Cr

ossin

g (n

)


(mgkg)


lowast

(a)

0

10

20

30

Rear

ing

(s)


(mgkg)


lowast

(b)

0

10

20

30

Gro

omin

g (s

) +

+

+

++



(mgkg)


lowast

(c)

0

50

100

150

200

250

Resti

ng (s

)



(mgkg)


lowast

(d)














Acknowledgments


References





































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0

20

40

60Cr

ossin

g (n

)


(mgkg)


lowast

(a)

0

10

20

30

Rear

ing

(s)


(mgkg)


lowast

(b)

0

10

20

30

Gro

omin

g (s

) +

+

+

++



(mgkg)


lowast

(c)

0

50

100

150

200

250

Resti

ng (s

)



(mgkg)


lowast

(d)














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































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

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and Montanoa grandiflora - Hindawi Publishing …downloads.hindawi.com/journals/bmri/2014/938060.pdf · Montanoa frutescens and Montanoa grandiflora Extracts Reduce Anxiety-Like Behavior