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Phytomedicine 18 (2011) 1255– 1261

Contents lists available at ScienceDirect

Phytomedicine

j ourna l ho mepage: www.elsev ier .de /phymed

ntidepressant effect and pharmacological evaluation of standardized extract ofavonoids from Byrsonima crassifolia

. Herrera-Ruiza, A. Zamilpaa, M. González-Cortazara, R. Reyes-Chilpab, E. Leónc,

.P. Garcíaa, J. Tortorielloa, M. Huerta-Reyesa,∗

Centro de Investigación Biomédica del Sur, Instituto Mexicano del Seguro Social (IMSS), Argentina No. 1, 62790, Xochitepec, Morelos, MexicoInstituto de Química, Departamento de Productos Naturales, Universidad Nacional Autónoma de México, Ciudad Universitaria, Coyoacán, 04510, México, D.F., MexicoHerbario Nacional de México (MEXU), Instituto de Biología, Departamento de Botánica, Universidad Nacional Autónoma de México, Apartado Postal 70-367,el. Coyoacán, 04510, México, D.F., Mexico

r t i c l e i n f o

eywords:yrsonima crassifoliaentral Nervous Systemcute toxicityntidepressantutinesperidin

a b s t r a c t

Byrsonima crassifolia (Malpighiaceae) has been used in traditional medicine for the treatment of somemental-related diseases; however, its specific neuropharmacological activities remain to be defined. Thepresent study evaluates the anxiolytic, anticonvulsant, antidepressant, sedative effects produced by theextracts of Byrsonima crassifolia, and their influence on motor activity in ICR mice. Additionally, we deter-mine the acute toxicity profiles of the Byrsonima crassifolia extracts and the presence of neuroactiveconstituents. Our results show that the methanolic extract of Byrsonima crassifolia produces a signifi-cant (P < 0.05) antidepressant effect in the forced swimming test in mice at 500 mg/kg dose. However,it does not possess anxiolytic, sedative, or anticonvulsant properties, and does not cause a reductionof mice locomotion (P > 0.05). Although the main compound of the methanolic extract was identifiedas quercetin 3-O-xyloside (12 mg/kg), our findings suggest that flavonoids, such as rutin (4.4 mg/kg),

quercetin (1.4 mg/kg) and hesperidin (0.7 mg/kg), may be involved in the antidepressant effects. To thebest of our knowledge, the present study constitutes the first report on the presence of the flavonoids withneuropharmacological activity rutin and hesperidin in Byrsonima crassifolia. In conclusion, the presentresults showed that the methanolic extract standardized on flavonoids content of Byrsonima crassifoliapossesses potential antidepressant-like effects in the FST in mice, and could be considered as relativelysafe toxicologically with no deaths of mice when orally administered at 2000 mg/kg.

ntroduction

Byrsonima crassifolia (Malpighiaceae) is a tropical tree widelyistributed in Mexico, Central and South America. The pharmaco-

ogical activities of Byrsonima crassifolia extracts as a bactericide,ungicide, leishmanicide, and as a topical anti-inflammatoryMaldini et al. 2009) have been described. Byrsonima crassifolias popularly known as “nanche” and it has been used medicinallyince prehispanic times, mainly to treat gastrointestinal afflictionsnd gynecological inflammation (Heinrich et al. 1998). Some othereports indicate that Byrsonima crassifolia has been employed in the

reatment of nervous excitement and to induce a pleasant dizzinessMaldonado 2008). A preliminary screening on the central nervousystem (CNS) showed effects on gross behavior produced by aque-

∗ Corresponding author at: Centro de Investigación Biomédica del Sur, Institutoexicano del Seguro Social, Argentina No. 1, Col. Centro, C.P. 62790, Xochitepec,orelos, Mexico. Tel.: +52 777 3 61 21 55; fax: +52 777 3 61 21 94.

E-mail address: chilanguisima@yahoo.com (M. Huerta-Reyes).

944-7113/$ – see front matter © 2011 Elsevier GmbH. All rights reserved.oi:10.1016/j.phymed.2011.06.018

© 2011 Elsevier GmbH. All rights reserved.

ous extracts of the leaves and bark of Byrsonima crassifolia (Moraleset al. 2001). In addition, the triterpens betulin, betulinic acid andlupeol, the flavonoids catechin, epicatechin, guiaverin, quercetinand its 3-O-�-d-glucopyranoside have been isolated in the leavesand bark of Byrsonima crassifolia (Béjar et al. 1995, 2000). How-ever, although previous studies, as well as its traditional uses thatsuggest the possible ability to modulate the CNS physiology of thistree, the specific neuropharmacological activity and toxicity of Byr-sonima crassifolia as same as the identification of the neuroactiveconstituents remain uninvestigated.

Our study focuses on the neuropharmacological activities ofByrsonima crassifolia, with respect to understanding its traditionalmedicinal applications, its medicinal uses in the modern society,and potential uses in drug development. The present study wasdesigned to evaluate the anxiolytic, antidepressant, sedative,and anticonvulsant effects produced by extracts of Byrsonima

crassifolia in ICR mice by using different models, such as theelevated plus maze, the forced swimming test, the pentobarbitalpotentiation test, and the seizures-induced pentylenetetrazol. Fur-thermore, their influence on motor activity test was also studied.

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dditionally, by using the isolated guinea pig ileum model, theffects of Byrsonima crassifolia extracts on the enteric nervousystem were evaluated. The safety of “nanche” was evaluatedy determination of the acute toxicity profile of the Byrsonimarassifolia extracts while the extract were characterized by meansf the presence and quantification of main constituents.

aterials and methods

lant material and preparation of the extracts

Byrsonima crassifolia (L.) Kunth sens. lat. (Malpighiaceae) aerialarts were collected in the state of Morelos, Mexico. The identifi-ation of the plant was authenticated by one expert in the field oflant taxonomy, who is also one of the authors (E. León). A voucheras deposited at the Mexican Institute of Social Security Medicinalerbarium (IMSSM) under the number 15441. The plant materialas dried in the dark at room temperature. Then, it was pow-

red (2100 g) and successively extracted with n-hexane (4×) andethanol (4×). The extraction volumes were 7.5 l of solvent per

ach kg of plant material. The samples were dried by removal ofolvent under vacuum. The hexane (BcHx) (22.4 g) and methano-ic (BcMeOH) (302.3 g) extracts of Byrsonima crassifolia were thensed in the pharmacological experiments.

nimals

The animal experiments were performed observing the officialequirements of the Mexican Regulations of Experimental Ani-al Care (NOM-062-ZOO-1999). The experimental protocol was

pproved by the institutional research and ethics committee (Reg-stry number 2007-1701-8). For each neuropharmacological assay,ndependent and unique groups of eight ICR albino mice weigh-ng 30–36 g each was utilized (Harlan México S.A. de C. V., Mexicoity, Mexico). The animals were housed in community cages andaintained under regular laboratory conditions (25 ± 2 ◦C, 12-h

ight-dark cycle, free access to water and standard rodent chow:018S, Harlan Tekland). All animals were acclimatized for 3 weeksrior to initiation of the test. The experiments were carried out in

special adjacent noise-free room with controlled illumination.

hemicals

Imipramine hydrochloride (IMI), pentylenetetrazole (PTZ) andhe standard compounds for HPLC analysis rutin, hesperidin,uercetin, chrysin, hesperetin, kaempferol and naringin were pur-hased from Sigma–Aldrich Chemical Co. (St. Louis, MO, USA).iazepam (DZP) and carboxymethyl cellulose (CMC) were obtained

rom Cryopharma S.A. de C. V. (Guadalajara, Jal, Mexico). Sodiumentobarbital (PEN) was purchased from Pfizer Inc. (New York, NY,SA).

orced swimming test (FST)

The FST is the most widely used pharmacological in vivo modelor assessing antidepressant activity. The development of immo-ility when mice are placed in an inescapable cylinder filled withater reflects the cessation of persistent escape-directed behav-

or (Porsolt et al. 1977). The apparatus utilized to perform the FSTonsisted of a clear glass cylinder (20 cm high × 12 cm diameter)ith water filled to a depth of 15 cm (24 ± 1 ◦C). The mice were

reated with BcHx, BcMeOH (500 mg/kg, experimental treatments, = 8) and CMC 1% (500 mg/kg, vehicle; control group, n = 8) at 48,6, 24, 18, and 1 h prior to the test. IMI (15.0 mg/kg, positive control,

= 8) was administered 24 h, 18 h, and 30 min before the test. Prior

icine 18 (2011) 1255– 1261

to the administration schedule, the mice were subject to a pre-test session, in which every animal was individually placed intothe cylinder for 15 min. During the test session, a trained observerrecorded the immobility time.

Open field test

The open field area was comprised of transparent acrylic wallsand a black floor (30 cm × 30 cm × 15 cm) divided into nine squaresof equal size. One hour before the test, the mice were treatedwith BcMeOH, BcHx (500 mg/kg, experimental treatments, n = 8)and CMC (500 mg/kg, vehicle, control group, n = 8). The open fieldtest was used to evaluate the locomotor activity of mice that hadpreviously been subjected to the FST and EPM tests. The observedparameters included the number of squares crossed (with fourpaws) and the number of rearings (Archer 1973).

Elevated plus-maze test (EPM)

The EPM test is the most frequently employed model for theassessment of the anxiolytic activity of novel substances (Lister1987). The maze was constructed of Plexiglas and consisted of acentral platform (5 cm × 5 cm) with two open (30 cm × 5 cm) andtwo closed arms (30 cm × 5 cm) and 25 cm high walls. The mazewas elevated 38.5 cm from the room’s floor. The mice were treated30 min prior to the test with DZP (1.0 mg/kg, positive control, n = 8),while CMC (500 mg/kg, vehicle, control group, n = 8) BcHx, BcMeOH(500 mg/kg, experimental treatments, n = 8) were administrated 1 hprior to the test. Each animal was placed at the center of the mazefacing one of the open arms. The number of entries and the timespent in the enclosed and open arms were recorded during the5 min test. Other ethologically derived measures (grooming, rear-ing, stretched attend postures, head dipping) were also registered.All of the test sessions were recorded by video camera. After eachtest, the maze was carefully cleaned with wet tissue paper (10%ethanol solution).

Pentylenetetrazole (PTZ)-induced seizures

BcHx, BcMeOH (500 mg/kg, experimental treatments, n = 8) andCMC (500 mg/kg, vehicle, control group, n = 8) were administered1 h before the PTZ (75.0 mg/kg), while DZP (1.0 mg/kg, positive con-trol, n = 8) was administered only 30 min prior to the PTZ. Followingthe administration of PTZ, mice were placed in separate transpar-ent Plexiglas cages (25 cm × 15 cm × 10 cm) and were observed forthe occurrence of seizures over a 30 min time span. The time priorto the onset of clonic convulsions and the percentage of mortalityprotection was recorded (Williamson et al. 1996).

Pentobarbital-induced hypnosis

BcHx, BcMeOH (500 mg/kg, experimental treatments, n = 8) andCMC (500 mg/kg, vehicle, control group, n = 8) were administrated1 h prior to the test, and DZP (1.0 mg/kg, positive control, n = 8)was administered 30 min before the test. In this experiment, afteradministration of PEN (30.0 mg/kg), the mice were placed sep-arately in transparent Plexiglas cages (25 cm × 15 cm × 10 cm) toobserve the hypnotic effect, which was considered as the timeinterval between disappearance (latency) and reappearance (dura-tion) of the righting reflex (Williamson et al. 1996).

Enteric nervous system in isolated guinea pig ileum

The effects of Byrsonima crassifolia extracts on the enteric ner-vous system were evaluated according to the method describedelsewhere (Lozoya et al. 1990). EC50 values were determined for

tomedicine 18 (2011) 1255– 1261 1257

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Fig. 1. Effect of oral administration of Byrsonima crassifolia extracts on immobil-ity time of ICR mice exposed to FST. *P < 0.05 with ANOVA followed by post hoc

Pentylenetetrazole (PTZ)-induced seizures

Even though treatment with BcHx induced a protection of33.3%, treatment with BcMeOH offered no anticonvulsant protec-

M. Herrera-Ruiz et al. / Phy

apaverine (Pap, positive control). BcHx, BcMeOH were tested atoncentrations ranging from 10 to 100 �g/ml. All treatments wereissolved in Tyrode’s solution but BcHx was previously dissolved inolyvinylpyrrolidone (PVP) and evaporated to dryness. The valuesbtained correspond to the mean of three independent experi-ents.

oxicity assay

Acute toxicity test were only conducted on extracts hav-ng been deemed active by the previous neurobehavioral assaysnd performed according to the OECD guidelines for the test-ng of chemicals (OECD 2009). Nine ICR female mice (26–28 g,

weeks old) were acclimatized under regular laboratory condi-ions (same as those describe above). Food was withheld for 1 hefore the administration. The animals were caged in groups ofhree and doses of 500 and 2000 mg/kg of BcMeOH and 100 �l/10 gf CMC 1% (vehicle, control group) were administered orally byavage. Thirty minutes after administration, the animals wereubjected to an initial period of observation of several CNS activity-ssociated behavioral parameters: locomotor activity, tremors, griptrength, bizarre behavior, convulsions, abdominal contortions,ait incapacity, piloerection, palpebral closure, and constipation.his procedure was carried out three times weekly for 2 weeks, dur-ng which animal deaths, animal weights, and food consumption

ere also recorded.

PLC analysis

One gram of the BcMeOH was dissolved in 100 ml of 100%ethanol for the pattern analysis using HPLC (Waters 2695; Waters

o., Milford, MA, USA), with a photodiode array detector (Waters996). Separation was carried out using a RP C-18 SuperspherMerck) column (120 mm × 4 mm; 5 �m) with the following sol-ent ratios for the mobile phase, where solvent A is water andolvent B is acetonitrile: A:B = 100:0 (0–1 min); 90:10 (2–4 min);0:20 (5–9 min); 70:30 (10–15 min); 60:40 (16–18 min); 40:6019–20 min); 0:100 (21–23 min); 100:0 (24–25 min). The detec-ion wavelength was scanned at 190–400 nm with 1.0 ml/min ofow. The peak analysis and assignment were performed usinghe standard compounds, which were identified in accordanceith their UV spectra and retention time (tR) in the HPLC chro-atogram. Quantification of main flavonoids was calculated byeans of calibration curves which were separately constructed for

he commercial standards (Herrera-Ruiz et al. 2006).Purification of the main compound detected in extract

as carried on by successive column chromatography from3.5 g of BcMeOH under the following conditions: silica gel 6040–63 �m), Hex:EtOAc = 7:3. Fraction 9 (300 mg) was subjecto chromatography: silica gel RP-18 (40–63 �m), H2O/TFA 0.5%pH = 3.01):CHCN3 = 8:2. Pure quercetin 3-O-xyloside was detectedn Fraction 9 (38.5 mg).

tatistical analysis

Data were analyzed by one-way ANOVA followed by a post hocunnett test using the SPSS 11.0 program. Differences betweenxperimental groups were considered statistically significant when

< 0.05.

esults

ST

BcMeOH induced a significant antidepressant effect in the FSTecause it significantly diminished the immobility time compared

Dunnett test (mean ± S.D.). BcHx, Hexane extract of Byrsonima crassifolia; BcMeOH,Methanolic extract of Byrsonima crassifolia; IMI, Imipramine hydrochloride; Veh,Vehicle.

with the control (P < 0.05) (Fig. 1). On the contrary, BcHx did notinduce this behavior. In addition to the effects observed for the500 mg/kg dose, the 1000 mg/kg dose caused a significant increasein immobility time (P < 0.0) in the FST with respect to control group(Veh). However, the 2000 mg/kg doses did not induce a similarbehavior (Fig. 2).

Open field

BcHx and BcMeOH did not show a significant decreased in totaltime spent by mice on the periphery and on the number of crossingsin the open field test (P < 0.05) (Fig. 3).

EPM

Mice treated with BcHx and BcMeOH did not induce changes inthe percentage of Time that mice spent in Open Arms (TOA) and thepercentage of Entries into the Open Arms (EOA) with respect to thecontrol group (Veh) (P > 0.05) (Fig. 4). Consequently, no anxiolyticproperties were observed.

Fig. 2. Effect of oral administration of different doses of BcMeOH on immobility timeof ICR mice exposed to FST. *P < 0.05 with ANOVA followed by post hoc Dunnett test(mean ± S.D.). BcMeOH, Methanolic extract of Byrsonima crassifolia; IMI, Imipraminehydrochloride; Veh, Vehicle.

1258 M. Herrera-Ruiz et al. / Phytomedicine 18 (2011) 1255– 1261

Fig. 3. Effect of oral administration of Byrsonima crassifolia extracts on the number of total

ANOVA followed by post hoc Dunnett test (mean ± S.D.). BcHx, Hexane extract of ByrsonimVeh, Vehicle.

Fig. 4. Effect of oral administration of Byrsonima crassifolia extracts on the percent-age of Time spent in Open Arms (TOA) and Entries into Open Arms (EOA) by miceeBs

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xposed to EPM. *P < 0.05 in ANOVA followed by post hoc Dunnett test (mean ± S.D.).cHx, Hexane extract of Byrsonima crassifolia; BcMeOH, Methanolic extract of Byr-onima crassifolia; DZP, Diazepam; Veh, Vehicle.

ion. These treatments did not change the onset of seizures, andhese effects are not different as compared with those observed forhe control group (Table 1).

entobarbital-induced hypnosis

Because the pentobarbital dose administered (30 mg/kg) wasub-hypnotic, mice that received vehicle (Veh) exhibited nohanges in their behavior, while, mice treated with DZP (a sedative

able 1nticonvulsant effect of B. crassifolia extracts on PTZ-induced seizures in ICR mice.

Treatment (mg/kg) Onset of seizures (s) Mortality protection (%)

BcHx (500) 65.3 ± 4.7 33.3BcMeOH (500) 90.8 ± 26.0 0.0DZP (1.0) 0.0 ± 0.0* 100.0Veh (100 �l/10 g) 156.5 ± 100.0 0.0

ata presented as the mean ± S.D. with n = 8.* P < 0.05 compared to vehicle using ANOVA and post hoc Dunnett test.

crossings and rearings of ICR mice exposed to the open field paradigm. *P < 0.05 witha crassifolia; BcMeOH, Methanolic extract of Byrsonima crassifolia; DZP, Diazepam;

drug) evidenced pentobarbital potentiation when hypnotic effectand time to fall asleep were significantly different as compared withthe control group (P < 0.05). In opposite fashion, animals treatedwith 500 mg/kg of BcHx and BcMeOH did not evidence this effect(P > 0.05).

Enteric nervous system in isolated guinea pig ileum

As shown in Table 2, BcHx and BcMeOH did not produce relax-ation of ileon tissue at maximum concentration, in contrast withPap, which presented an EC50 of 18.5 �g/ml.

Toxicity assay

Normal behavior was observed in CMC 1%-treated mice becauseanimals did not exhibit alterations in the parameters analyzed.No changes in the weight of the animals and in the food con-sumed were detected. There were no deaths of mice treated with2000 mg/kg of BcMeOH after 24 h; however, these mice presentedconstipation and decrease in locomotor activity and grip strength,while the 500 mg/kg dose also induced changes, but lower in inten-sity (Table 3).

HPLC analysis

We analyzed BcMeOH by HPLC for detection and quantifica-tion of major constituents of active extract. Fig. 5 shows the HPLCprofile of BcMeOH recorded at 360 nm where presences of peak 1-peak 7 were detected. Peak 1, peak 4 and peak 6 were overlapped

Table 2Effects of B. crassifolia extracts on the enteric nervous system in isolated guinea pigileum.

Extract Concentration (�g/ml)

BcHx >100BcMeOH >100Pap (control) 18.5*

EC50 value (*). Mean values corresponding to three independent experiments. n = 9.

M. Herrera-Ruiz et al. / Phytomedicine 18 (2011) 1255– 1261 1259

Table 3Toxicity of 500 and 2000 mg/kg doses of BcMeOH administered by oral route in ICR mice.

Time 10 min 30 min 2 h 3 h 24 h

Tx1 Tx2 Tx3 Tx1 Tx2 Tx3 Tx1 Tx2 Tx3 Tx1 Tx2 Tx3 Tx1 Tx2 Tx3

Abdominal contortions − − − − − − − − − − − − − − −Piloerection − − − − − − − − − − − − − − −Palpebral closure − − − − − − − − − − − − − − −Locomotor activity + − − − − − − − − − − − − − −Grip strength + − − + − − − − − ++ + − ++ ++ −Tremors − − − − − − − − − − − − − − −Gait incapacity − − − − − − − − − − − − − − −Convulsions − − − − − − − − − − − − − − −Bizarre behavior − − − − − − − − − − − − − − −Constipation − − − − − − − − − − − − ++ + −Death − − − − − − − − − − − − − − −

+ Low; ++ Moderate; +++ Severe; − Absence; Tx1 = 2000 mg/kg of BcMeOH; Tx2 = 500 mg/kg of BcMeOH; Tx3 = Veh.

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ig. 5. HPLC chromatogram of the active antidepressant BcMeOH extract. Peaks wernd 7. unknown.

ith the commercial standards of rutin (tR = 8.4 min), quercetintR = 14.6 min) and hesperidin (tR = 11.5 min) respectively, althoughhe detection wavelength was scanned at 190–400 nm. Mainompound of BcMeOH, peak 3 (tR = 10.4 min), was isolated anddentified as quercetin 3-O-xyloside since spectroscopic data wereound to be in good agreement with the literature values (Yant al. 2002). This glycoside was present in 12 mg/kg in the BcMeOHested in neuropharmacological assays, while rutin, quercetinnd hesperidin were present in 4.4 mg/kg, 1.4 mg/g and 0.7 mg/gespectively, as determined from the calibration curve (linearegression where r2 > 0.9800). On the other hand, the peak 2tR = 8.8 min; UV absorption = 204, 256, 348 nm; 238, 320 nm), theeak 5 (tR = 12.5 min; UV absorption: 210, 260, 365 nm) and theeak 7 (tR = 19.9 min; UV absorption: 205, 268, 336 nm) remainsnknown (Fig. 5), since its tR and its UV absorption values were not

n agreement with any of the commercial standards employed addi-ionally such as hesperetin (tR = 7.6 min; UV absorption = 207, 227,80 nm); chrysin (tR = 9.3 min; UV absorption = 202, 256, 278 nm),aringin (tR = 10.8 min; UV absorption = 193, 282, 329 nm) andaempferol (tR = 18.3 min; UV absorption = 206, 266, 364 nm).

iscussion

Although Byrsonima crassifolia has been used to treat someental-related diseases in traditional medicine, its specific neu-

opharmacological activities have not been demonstrated yet.The findings of the current investigation show for the first

ime that BcMeOH standardized in its content of flavonoids withoses of quercetin 3-O-xyloside (12 mg/kg), rutin (4.4 mg/kg),uercetin (1.4 mg/kg) and hesperidin (0.7 mg/kg) induced a signif-

cant antidepressant effect in the FST. In this assay, mice are forced

tin, 2. unknown, 3. quercetin 3-O-xyloside, 4. hesperidin, 5. unknown, 6. quercetin,

to swim in a restricted space from which there is no escape, andthey develop a state of despair characterized by a lowered motiva-tion for escaping, as evidenced by increased periods of immobility.It is well known that clinically effective antidepressants (such asIMI) typically increase the swimming efforts of the animal seek-ing a solution to the problem and, therefore, they decrease theduration of immobility in the forced swimming test (Porsolt et al.1977; Sánchez-Mateo et al. 2005). Because the effective dose ofother plant species widely recognized as antidepressants, suchas Valeriana officinalis, Paeonia lactiflora, and several Hypericumspecies (Sánchez-Mateo et al. 2002, 2005, 2009), is between 500and 1000 mg/kg of plant extracts, the dose-dependent assay showthat Byrsonima crassifolia appears to be within a similar rangeof potency. It is noteworthy that in the FST test, false positiveresults can be obtained for agents that stimulate locomotor activ-ity (Bourin et al. 2001). Therefore, the observation that BcMeOH didnot increase the number of crossings and rearings in the open fieldtest confirms the assumption that the antidepressant-like effect ofthe extract in the FST is specific (Sánchez-Mateo et al. 2005).

While behavioral models used in this study cannot clarify themechanisms of action involved in the effect by which the extract isexerting its antidepressant effect, it is suggested that activity is duein part to its rich content of flavonoids, since these compounds haveexhibited important effects on central nervous system, includingantidepressant effects (Butterweck et al. 2000). Rutin, for example,has been shown to play an essential role in the antidepressant activ-ity of plant extracts widely recognized as antidepressant, such asHypericum perforatum, with an average concentration of rutin about

3%. It has also been reported that rutin may directly or indirectlyenhance the bioavailability of other constituents present in theextracts that may be necessary to confer the full biological activityin the FST such as phenylpropanes, naphtodianthrones and possibly

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260 M. Herrera-Ruiz et al. / Phy

henylpropanoid-compounds (Nölder and Schötz 2002) that werelso detected in BcMeOH. In addition, in order to ascertain the pos-ible mechanism of action, it has shown that rutin in a dose rangef 5–80 mg/kg orally produces changes in the frequency of electri-al activity of rat brain, a pattern behavior similar to that recordedor antidepressants such as moclobemide (monoamine oxidase A-

AO) (Dimpfel 2009). The lowest dose of rutine used in that work,ay be correlated with the 4.4 mg/kg, contained in the BcMeOHith antidepressant activity. Also, when this flavonoid, isolated

rom the ethanolic extract of Schinus molle, was administered to.3–3.0 mg/kg, induced an antidepressant effect in mice in the tailuspension test but not in FST, with a possible mechanism of actionhrough of serotonergic and noradrenergic systems (Machado et al.008). These doses were lower than those employed in this study ofyrsonima crassifolia. It is likely then, that the antidepressant effecthown by BcMeOH is due largely to its content of rutine, where theresence of this flavonoid is essential to exercise the antidepressantffect in the FST (Wurlics and Schubert-Zsilavecz 2006).

On the other hand, hesperidin was administered in the BcMeOHt a dose of 0.7 mg/kg, a substance considered as neuroactiveavonoid, for its sedative and sleep-enhancing properties. Hes-eridin is frequently detected as a racemic mixture in plantxtracts, and the CNS active isomer is the 2S (−) form. Consequently,he low concentration of hesperidin present in BcMeOH may haveimited the role of hesperidin in the antidepressant effect observedn the FST. The sedative effect of 2S-hesperidin was observed atoses of 5 mg/kg in mice tested in the hole board and the sodiumhiopental-induced sleep assays (Martínez et al. 2009). Since thisose is higher than those calculated for hesperidin present in thecMeOH, it is probably that the sedative effect of Byrsonima cras-ifolia was not observed. This assumption is strengthened whennalyzed in those study that rutin at doses of 10 and 20 mg/kg has

sedative effect on the potentiation of barbiturates test. Therefore,igher doses of the extract and consequently, higher doses of rutinnd hesperidin, may induce a sedative effect from BcMeOH.

Although quercetin and some of its glycosides had been foundn Byrsonima crassifolia, the presence of quercetin 3-O-xylosides the main compound in BcMeOH had not been reported pre-iously. Quercetin 3-O-xyloside is frequently found in bark andature fruits of cultivars with commercial importance where

ntioxidant properties are perceived as health benefits includingnticancer, anti-inflammatory, and vasoprotective effects (Ozga etl. 2007). However, no evidence of neuroactivity has been reported.n the case of quercetin, it was found inactive when tested in FST,evertheless, quercetin dose-dependently reduced the immobilityeriod in diabetic mice (Anjaneyulu et al. 2003). Recent studieshowed that daily oral administration of quercetin for a period of

days induced in mice an increase in the time of immobility in theST indicating an antidepressant effect of this substance (Chimentit al. 2006). This fact does not preclude the active involvementf quercetin on the antidepressant effect of BcMeOH, due to thedministration schedule used in this study (five administrations)nhances the accumulation of quercetin is necessary to exert anntidepressant effect, which is consistent with reports where thentidepressant drugs require cumulative doses for take effect. Fur-hermore, quercetin has an effect of inhibiting the activity of thenzyme monoamine oxidase-A, which is one of the main mecha-isms of action of antidepressant substances (Racagni and Popoli010).

As many reports indicate, the role of the quercetin and its gly-osides in the antidepressant effects is still unclear. However, it is

well-known fact in phytochemistry that crude plant extracts are

sually more powerful medicines than pure isolated compoundsay be due on the multiple actions of complex mixtures but could

lso arise from synergistic interactions among their componentsFernández et al. 2005).

icine 18 (2011) 1255– 1261

Although peak 2 remains unknown, its tR and UV absorption val-ues suggest the presence of a complex mixture of at least a coupleof glycosylated flavone-type compounds (204, 256, 348 nm) anda phenylpropanoid (238, 320 nm), possibly a ferulate-type com-pounds, while in case of peak 5 and peak 7, may belong to theflavonols and flavone-type compounds (Lin and Harnly 2007). Pre-viously, glycolipids, proanthocyanidins and triterpens (Béjar et al.2000) have been reported to be the main compounds in Byrson-ima crassifolia, and thus, the best of our knowledge, this is the firstreport of the presence of rutin and hesperidin flavonoids in the Byr-sonima crassifolia species. Our findings suggest that flavonoid-typecompounds may be involved in the antidepressant effects pro-duced by BcMeOH. Nonetheless, because concentrations of otherflavonoid-type compounds that also exhibited neuropharmacolog-ical properties as kaempferol (0.02–1.0 mg/kg o.p.) isolated fromApocynum venetum (up to 125 mg/kg doses) (Grundmann et al.2009) are similar to those found in the present study for hes-peridin (0.7 mg/kg o.p.) from Byrsonima crassifolia (up to 500 mg/kgdoses), it cannot be ruled out that plant extracts which con-tain these flavonoid-type compounds exert also antidepressantactivities since these studies are limited for animal models. Thus,the establishment of the antidepressant effects of BcMeOH inmore complex in vivo models as well as the precise role andcombination of the principal active components requires furtherinvestigation.

In the present acute toxicity study our results contrast withthose reported for aqueous extracts, where piloerection and palpe-bral ptosis is observed (Morales et al. 2001), as well as those forethanolic and acetic acid extracts where ear blanching, catalepsy,and strong hypothermia have been reported (Béjar and Malone1993). It is important to note that in the present work, these signs oftoxicity were observed primarily in the highest dose of 2000 mg/kgin which also, no mortality in mice occurred. Some reports indicatethat substances with an LD50 of 1000 mg/kg body weight/oral routeare regarded as safe or as having low toxicity (Adeneye et al. 2006)then, BcMeOH could be considered as relatively safe toxicologi-cally when orally administered. The apparent lack of clinical signsof acute toxicities in human when the extract was orally adminis-tered may be a reflection of the oral route of administration, lowdose administration as well as short duration of exposure (Gazdaet al. 2006). The constipation that was observed in mice after 24 hof administration is in agreement with biphasic effects reported inrat jejunum and ileum of ethanolic and acetic acid extracts (Béjarand Malone 1993).

In the remains neuropharmacological assays, our resultsshowed that Byrsonima crassifolia extracts do not posses anxi-olytic nor sedative nor anticonvulsant properties, nor effects onthe enteric nervous system at the dose studied.

In conclusion, the present results showed that the methanolicextract standardized on flavonoids content of Byrsonima crassifoliapossesses potential antidepressant-like effects in the FST in mice,and could be considered as relatively safe toxicologically whenorally administered. Our findings suggest that the flavonoids rutin,hesperidin and quercetin could be involved in the antidepressanteffects. To the best of our knowledge, the present study constitutesthe first report of the presence of the neuroactive flavonoids rutinand hesperidin in Byrsonima crassifolia.

Acknowledgments

Ciencia Básica 2007, Mexico (to M. Huerta-Reyes) and grantFIS/IMSS/PROT/C2007/040 from the FIS, Instituto Mexicano delSeguro Social, Mexico (to M. Huerta-Reyes). The authors wish tothank Arturo Pérez M. for technical assistance.

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