Antidepressant effect and pharmacological evaluation of standardized extract of flavonoids from byronima crassifolia.
Byrsonima crassifolia (Malpighiaceae) has been used in traditional medicine for the treatment of some mental-related diseases; however, its specific neuropharmacological activities remain to be defined. The present study evaluates the anxiolytic, anticonvulsant, antidepressant, sedative effects produced by the extracts of Byrsonima crassifolia, and their influence on motor activity in ICR mice. Additionally, we determine the acute toxicity profiles of the Byrsonima crassifolia extracts and the presence of neuroactive constituents. Our results show that the methanolic extract of Byrsonima crassifolia produces a significant (P<0.05) antidepressant effect in the forced swimming test in mice at 500mg/kg dose. However, it does not possess anxiolytic, sedative, or anticonvulsant properties, and does not cause a reduction of mice locomotion (P>0.05). Although the main compound of the methanolic extract was identified as quercetin 3-O-xyloside (12mg/kg), our findings suggest that flavonoids, such as rutin (4.4mg/kg), quercetin (1.4 mg/kg) and hesperidin (0.7 mg/kg), may be involved in the antidepressant effects. To the best of our knowledge, the present study constitutes the first report on the presence of the flavonoids with neuropharmacological activity rutin and hesperidin in Byrsonima crassifolia. In conclusion, the present results showed that the methanolic extract standardized on flavonoids content of Byrsonima crassifolia possesses potential antidepressant-like effects in the FST in mice, and could be considered as relatively safe toxicologically with no deaths of mice when orally administered at 2000 mg/kg.
[c] 2011 Elsevier GmbH. All rights reserved.
Central Nervous System
Byrsonima crassifolia (Malpighiaceae) is a tropical tree widely distributed in Mexico, Central and South America. The pharmacological activities of Byrsonima crassifolia extracts as a bactericide, fungicide, leishmanicide, and as a topical anti-inflammatory (Maldini et al. 2009) have been described. Byrsonima crassifolia is popularly known as "nanche" and it has been used medicinally since prehispanic times, mainly to treat gastrointestinal afflictions and gynecological inflammation (Heinrich et al. 1998). Some other reports indicate that Byrsonima crassifolia has been employed in the treatment of nervous excitement and to induce a pleasant dizziness (Maldonado 2008). A preliminary screening on the central nervous system (CNS) showed effects on gross behavior produced by Aqueous extracts of the leaves and bark of Byrsonima crassifolia (Morales et al. 2001). In addition, the triterpens betulin, betulinic acid and lupeol, the flavonoids catchin, epicatechin, guiaverin, quercetin and its 3-0- [beta]-D-glucopyranoside have been isolated in the leaves and bark of Byrsonima crassifolia (Bejar et al. 1995, 2000). However, although previous studies, as well as its traditional uses that suggest the possible ability to modulate the CNS physiology of this tree, the specific neuropharmacological activity and toxicity of Byr sonima crassifolia as same as the identification of the neuroactive constituents remain uninvestigated.
Our study focuses on the neuropharmacological activities of Byrsonima crassifolia, with respect to understanding its traditional medicinal applications, its medicinal uses in the modern society, and potential uses in drug development. The present study was designed to evaluate the anxiolytic, antidepressant, sedative, and anticonvulsant effects produced by extracts of Byrsonima crassifolia in ICR mice by using different models, such as the elevated plus maze, the forced swimming test, the pentobarbital potentiation test, and the seizures-induced pentylenetetrazol. Furthermore, their influence on motor activity test was also studied. Additionally, by using the isolated guinea pig ileum model, the effects of Byrsonima crassifolia extracts on the enteric nervous system were evaluated. The safety of "nonce" was evaluated by determination of the acute toxicity profile of the Byronima Crassifolia extracts while the extract were characterized by means of the presence and quantification of main constituents.
Materials and methods
Plant material and preparation of the extracts
Byrsonima crassifolia (L) Knuth sens. lat. (Malpighiaceae) aerial parts were collected in the state of Morelos, Mexico. The identification of the plant was authenticated by one expert in the field of plant taxonomy, who is also one of the authors (E. Leon). A voucher was deposited at the Mexican Institute of Social Security Medicinal Herbarium (IMSSM) under the number 15441. The plant material was dried in the dark at room temperature. Then, it was powered (2100g) and successively extracted with n-hexane (4x) and methanol (4x). The extraction volumes were 7.51 of solvent per each kg of plant material. The samples were dried by removal of solvent under vacuum. The hexane (BcHx) (22.4g) and methanolis (BcMeOH) (302.3 g) extracts of Byrsonima crassifolia were then used in the pharmacological experiments.
The animal experiments were performed observing the official requirements of the Mexican Regulations of Experimental Animal Care (NOM-062-ZOO-1999). The experimental protocol was approved by the institutional research and ethics committee (Reg-istry number 2007-1701 -8). For each neuropharmacological assay, independent and unique groups of eight 1CR albino mice weighing 30-36g each was utilized (Harlan Mexico S.A. de C. V., Mexico City, Mexico). The animals were housed in community cages and maintained under regular laboratory conditions (25[+ or -]2[degrees]C, 12-h light-dark cycle, free access to water and standard rodent chow: 2018S, Harlan Tekland). All animals were acclimatized for 3 weeks prior to initiation of the test. The experiments were carried out in a special adjacent noise-free room with controlled illumination.
Imipramine hydrochloride (IMI), pentylenetetrazol (PTZ) and the standard compounds for HPLC analysis rutin, hesperidin, quercetin, chrysin, hesperetin, kaempferol and naringin were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Diazepam (DZP) and carboxymethyl cellulose (CMC) were obtained from Cryopharma S.A. de C. V. (Guadalajara, Jal, Mexico). Sodium pentobarbital (PEN) was purchased from Pfizer Inc. (New York, NY, USA).
Forced swimming test (FST)
The FST is the most widely used pharmacological in vivo model for assessing antidepressant activity. The development of immobility when mice are placed in an inescapable cylinder filled with water reflects the cessation of persistent escape-directed behavior (Porsolt et al. 1977). The apparatus utilized to perform the FST consisted of a clear glass cylinder (20 cm high x 12 cm diameter) with water filled to a depth of 15 cm (24 [+ or -]1 !C). The mice were treated with BcHx, BcMeOH (500 mg/kg, experimental treatments, n = 8) and CMC 1% (500 mg/kg, vehicle; control group, n = 8) at 48, 36,24,18, and 1 h prior to the test. IMI (15.0 mg/kg, positive control, n = 8) was administered 24 h, 18 h, and 30 min before the test. Prior To the administration schedule, the mice were subject to a pretest session, in which every animal was individually placed into the cylinder for 15 min. During the test session, a trained observer recorded the immobility time.
Open field test
The open field area was comprised of transparent acrylic walls and a black floor (30 cm x 30 cm x 15 cm) divided into nine squares of equal size. One hour before the test, the mice were treated with Cameo, Bach (500mg/kg, experimental treatments, n = 8) and CMC (500mg/kg, vehicle, control group, n = 8). The open field test was used to evaluate the locomotors activity of mice that had previously been subjected to the FST and EPM tests. The observed parameters included the number of squares crossed (with four paws) and the number of rearing (Archer 1973).
Elevated plus-maze test (EPM)
The EPM test is the most frequently employed model for the assessment of the anxiolytic activity of novel substances (Lister 1987). The maze was constructed of Plexiglas and consisted of a central platform (5 cm x 5 cm) with two open (30 cm x 5 cm) and two closed arms (30cm x 5 cm) and 25 cm high walls. The maze was elevated 38.5 cm from the room's floor. The mice were treated 30 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 h prior to the test. Each animal was placed at the center of the maze facing one of the open arms. The number of entries and the time spent in the enclosed and open arms were recorded during the 5 min test. Other ethologically derived measures (grooming, rearing, stretched attend postures, head dipping) were also registered. All of the test sessions were recorded by video camera. After each test, 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) and CMC (500 mg/kg, vehicle, control group, n = 8) were administered 1 h before the PTZ (75.0 mg/kg), while DZP (1.0 mg/kg, positive control, n = 8) was administered only 30 min prior to the PTZ. Following the administration of PTZ, mice were placed in separate transparent Plexiglas cages (25 cm x 15 cm x 10 cm) and were observed for the occurrence of seizures over a 30 min time span. The time prior to the onset of clonic convulsions and the percentage of mortality protection was recorded (Williamson et al. 1996).
BcHx, BcMeOH (500 mg/kg, experimental treatments, n = 8) and CMC (500 mg/kg, vehicle, control group, n = 8) were administrated 1 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, after administration of PEN (30.0 mg/kg), the mice were placed sep-arately in transparent Plexiglas cages (25 cm x 15cm x 10cm) to observe the hypnotic effect, which was considered as the time interval between disappearance (latency) and reappearance (dura-tion) of the righting reflex (Williamson etal. 1996).
Enteric nervous system in isolated guinea pig ileum
The effects of Byrsonima crassifolia extracts on the enteric nervous system were evaluated according to the method described elsewhere (Lozoya et al. 1990). EC50 values were determined for Palaverine (Pap, positive control). BcHx, BcMeOH were tested at concentrations ranging from 10 to 100 g/ml. All treatments were dissolved in Tyrode's solution but BcHx was previously dissolved in polyvinylpyrrolidone (PVP) and evaporated to dryness. The values obtained correspond to the mean of three independent experiments.
Acute toxicity test were only conducted on extracts having been deemed active by the previous neurobehavioral assays and performed according to the OECD guidelines for the testing of chemicals (OECD 2009). Nine ICR female mice (26-28 g, 8 weeks old) were acclimatized under regular laboratory conditions (same as those describe above). Food was withheld for 1 h before the administration. The animals were caged in groups of three and doses of 500 and 2000 mg/kg of Cameo and 100 |xl/10 g of CMC 1% (vehicle, control group) were administered orally by gavages. Thirty minutes after administration, the animals were subjected to an initial period of observation of several CNS activityassociated behavioral parameters: locomotors activity, tremors, grip strength, bizarre behavior, convulsions, abdominal contortions, gait incapacity, piloerection, palpebral closure, and constipation. This procedure was carried out three times weekly for 2 weeks, during which animal deaths, animal weights, and food consumption were also recorded.
One gram of the BcMeOH was dissolved in 100 ml of 100% methanol for the pattern analysis using HPLC (Waters 2695; Waters Co., Milford, MA, USA), with a photodiode array detector (Waters 2996). Separation was carried out using a RP C-18 Superspher (Merck) column (120 mm x 4 mm; 5(xm) with the following solvent ratios for the mobile phase, where solvent A is water and solvent B is acetonitrile: A:B= 100:0 (0-1 min); 90:10 (2-4min); 80:20 (5-9min); 70:30 (10-15min); 60:40 (16-18min); 40:60 (19-20min); 0:100 (21-23min); 100:0 (24-25min). The detection wavelength was scanned at 190-400nm with l.Oml/min of flow. The peak analysis and assignment were performed using the standard compounds, which were identified in accordance with their UV spectra and retention time (t/?) in the HPLC chromatogram. Quantification of main flavonoids was calculated by means of calibration curves which were separately constructed for the commercial standards (Herrera-Ruiz et al. 2006).
Purification of the main compound detected in extract was carried on by successive column chromatography from 13.5g of BcMeOH under the following conditions: silica gel 60 (40-63 fxm), Hex:EtOAc = 7:3. Fraction 9 (300 mg) was subject to chromatography: silica gel RP-18 (40-63 fxm), H20/TFA 0.5% (pH = 3.01): CHCN3 = 8:2. Pure quercetin 3-O-xyloside was detected in Fraction 9 (38.5 mg).
Data were analyzed by one-way ANOVA followed by a post hoc Dunnett test using the SPSS 11.0 program. Differences between experimental groups were considered statistically significant when P<0.05.
BcMeOH induced a significant antidepressant effect in the FST because it significantly diminished the immobility time compared With the control (P<0.05) (Fig. 1). On the contrary, BcHx did not induce this behavior. In addition to the effects observed for the 500 mg/kg dose, the 1000 mg/kg dose caused a significant increase in immobility time (P< 0.0) in the FST with respect to control group (Veh). However, the 2000 mg/kg doses did not induce a similar behavior (Fig. 2).
[FIGURE 1 OMITTED]
Bach and BcMeOH did not show a significant decreased in total time spent by mice on the periphery and on the number of crossings in the open field test (P<0.05) (Fig. 3).
Mice treated with BcHx and BcMeOH did not induce changes in the percentage of Time that mice spent in Open Arms (TOA) and the percentage of Entries into the Open Arms (EOA) with respect to the control group (Vet) (P>0.05) (Fig. 4). Consequently, no anxiolytic properties were observed.
Pentylenetetrazol (PTZ)-induced seizures
Even though treatment with BcHx induced a protection of 33.3%, treatment with BcMeOH offered no anticonvulsant Protection. These treatments did not change the onset of seizures, and these effects are not different as compared with those observed for the control group (Table 1).
Table 1 Anticonvulsant effect of B. crassifolia extracts on PTZ-induced seizures in ICR mice. Treatment Onset of seizures (mg/kg) Mortality(s) protection (%) BcHx (500) 653 [+ or333 -] 4.7 33.3 BcMeOH (500) 90.8 [+0.0 or -] 26.0 0.0 DZP(l.O) 0.0 [+ or -]0.0' 100.0 Veh(100[micro] 156.5 [+ or -100.0] 0.0 l/10g) Data presented as the mean [+ or -] S.D. with n = 8. * P<0.05 compared to vehicle using ANOVA and post hoc Dunnett test.
[FIGURE 2 OMITTED]
[FIGURE 3 OMITTED]
[FIGURE 4 OMITTED]
Because the pentobarbital dose administered (30mg/kg) was sub-hypnotic, mice that received vehicle (Vet) exhibited no changes in their behavior, while, mice treated with DZP (a sedative Drug) evidenced pentobarbital ostentation when hypnotic effect and time to fall asleep were significantly different as compared with the control group (P<0.05). In opposite fashion, animals treated with 500mg/kg of Bach and Cameo did not evidence this effect (P>0.05).
[FIGURE 5 OMITTED]
Enteric nervous system in isolated guinea pig ileum
As shown in Table 2, BaHx and BcMeOH did not produce relaxation of ileom tissue at maximum concentration, in contrast with Pap, which presented an EC50 of 18.5 [mu]g/ml.
Table 2 Effects of B. crassifolia extracts on the enteric nervous system in isolated guinea pig ileum. Extract Concentration (fxg/ml) BcHx >100 BcMeOH >100 Pap (control) 18.5 * EC50 value *. Mean values corresponding to three independent experiments, n = 9.
Normal behavior was observed in CMC 1 %-treated mice because animals did not exhibit alterations in the parameters analyzed. No changes in the weight of the animals and in the food consumed were detected. There were no deaths of mice treated with 2000mg/kg of BcMeOH after 24 h; however, these mice presented constipation and decrease in locomotor activity and grip strength, while the 500 mg/kg dose also induced changes, but lower in intensity (Table 3).
Table 3 Toxicity of 500 and 2000 mg/kg doses of BcMeOH administered by oral route in ICRmice. Time lOmin 30min 2h Txl Tx2 Tx3 Txl Tx2 Tx3 Txl Tx2 Tx3 Abdominal -- -- -- -- -- -- -- -- -- contortions Piloerection -- -- -- -- -- -- -- -- -- Palpebral -- -- -- -- -- -- -- -- -- closure Locomotor + -- -- -- -- -- -- -- -- activity Grip + -- -- + -- -- -- -- -- strength Tremors -- -- -- -- -- -- -- -- -- Gait -- -- -- -- -- -- -- -- -- incapacity Convulsions -- -- -- -- -- -- -- -- -- Bizarre -- -- -- -- -- -- -- -- -- behavior Constipation -- -- -- -- -- -- -- -- -- Death -- -- -- -- -- -- -- -- -- Time 3h 24 h Txl Tx2 Tx3 Txl Tx2 Tx3 Abdominal -- -- -- -- -- -- contortions Piloerection -- -- -- -- -- -- Palpebral -- -- -- -- -- -- closure Locomotor -- -- -- -- -- -- activity Grip ++ + -- ++ ++ -- strength Tremors -- -- -- -- -- -- Gait -- -- -- -- -- -- incapacity Convulsions -- -- -- -- -- -- Bizarre -- -- -- -- -- -- behavior Constipation -- -- -- ++ + -- Death -- -- -- -- -- -- + Low; ++ Moderate; +++ Severe; - Absence; Txl = 2000 mg/kg of BcMeOH; Tx2 = 500 mg/kg of BcMeOH; Tx3 - Veh.
We analyzed BcMeOH by HPLC for detection and quantification of major constituents of active extract. Fig. 5 shows the HPLC profile of BcMeOH recorded at 360 nm where presences of peak 1-peak 7 were detected. Peak 1, peak 4 and peak 6 were overlapped With the commercial standards of ruin ([t.sub.R] = 8.4min, quartering ([t.sub.R]=14.6 min) and hesperidins ([T.sub.R] = 11.5 min) respectively, although the detection wavelength was scanned at 190-400 nm. Main compound of Cameo, peak 3 ([t.sub.R] = 10.4 min), was isolated and identified as quartering 3-O-xyloside since spectroscopic data were found to be in good agreement with the literature values (Yam et al. 2002). This glycoside was present in 12 mg/kg in the Cameo tested in neuropharmacological assays, while rutin, quercetin and hesperidin were present in 4.4 mg/kg, 1.4mg/g and 0.7mg/g respectively, as determined from the calibration curve (linear regression where r2>0.9800). On the other hand, the peak 2 [(t.sub.R = 8.8 min); UV absorption = 204, 256, 348 nm; 238, 320 nm), the peak 5 ([t.sub.R=12.5min]; UV absorption: 210, 260, 365nm) and the peak 7 (t.sub.R = 19.9min); UV absorption: 205, 268, 336 nm) remains unknown (Fig. 5), since its tR and its UV absorption values were not in agreement with any of the commercial standards employed additionally such as hesperetin [(t.sub.R = 7.6 min); UV absorption = 207, 227, 280 nm); chrysin ([t.sub.R = 9.3min]; UV absorption = 202, 256, 278 nm), naringin (t^ = 10.8min; UV absorption = 193, 282, 329nm) and kaempferol [t.sub.R = 18.3 min]; UV absorption = 206,266, 364 nm).
Although Byrsonima crassifolia has been used to treat some mental-related diseases in traditional medicine, its specific neuropharmacological activities have not been demonstrated yet.
The findings of the current investigation show for the first time that BcMeOH standardized in its content of flavonoids with doses of quercetin 3-O-xyloside (12mg/kg), rutin (4.4mg/kg), quercetin (1.4 mg/kg) and hesperidin (0.7 mg/kg) induced a signif-icant antidepressant effect in the FST. In this assay, mice are forced To swim in a restricted space from which there is no escape, and they develop a state of despair characterized by a lowered motivation for escaping, as evidenced by increased periods of immobility. It is well known that clinically effective antidepressants (such as IMI) typically increase the swimming efforts of the animal seeking a solution to the problem and, therefore, they decrease the duration of immobility in the forced swimming test (Porosity et al. 1977; Sanchez-Mateo et al. 2005). Because the effective dose of other plant species widely recognized as antidepressants, such as Valerian officinal is, Paeonia lactiflora, and several Hypericum species (Sanchez-Mateo et al. 2002, 2005, 2009), is between 500 and 1000 mg/kg of plant extracts, the dose-dependent assay show that Byrsonima crassifolia appears to be within a similar range of potency. It is noteworthy that in the FST test, false positive results can be obtained for agents that stimulate locomotor activity (Bourin et al. 2001). Therefore, the observation that BcMeOH did not increase the number of crossings and rearings in the open field test confirms the assumption that the antidepressant-like effect of the extract in the FST is specific (Sanchez-Mateo et al. 2005).
While behavioral models used in this study cannot clarify the mechanisms of action involved in the effect by which the extract is exerting its antidepressant effect, it is suggested that activity is due in part to its rich content of falconoid, since these compounds have exhibited important effects on central nervous system, including antidepressant effects (Butteries et al. 2000). Ruin, for example, has been shown to play an essential role in the antidepressant activity of plant extracts widely recognized as antidepressant, such as Hyperacid perforate, with an average concentration of ruin about 3%. It has also been reported that ruin may directly or indirectly enhance the bioavailability of other constituents present in the extracts that may be necessary to confer the full biological activity in the FST such as phenylpropanes, naphtodianthrones and possibly phenylpropanoid-compounds (Nolder and Schotz 2002) that were also detected in BcMeOH. In addition, in order to ascertain the possible mechanism of action, it has shown that rutin in a dose range of 5-80mg/kg orally produces changes in the frequency of electrical activity of rat brain, a pattern behavior similar to that recorded for antidepressants such as moclobemide (monoamine oxidase A-MAO) (Dimpfel 2009). The lowest dose of rutine used in that work, may be correlated with the 4.4mg/kg, contained in the BcMeOH with antidepressant activity. Also, when this flavonoid, isolated from the ethanolic extract of Schinus molle1 was administered to 0.3-3.0 mg/kg, induced an antidepressant effect in mice in the tail suspension test but not in FST, with a possible mechanism of action through of serotonergic and noradrenergic systems (Machado et al. 2008). These doses were lower than those employed in this study of Byrsonima crassifolia. It is likely then, that the antidepressant effect shown by BcMeOH is due largely to its content of rutine, where the presence of this flavonoid is essential to exercise the antidepressant effect in the FST (Wurlics and Schubert-Zsilavecz 2006).
On the other hand, hesperidin was administered in the Cameo at a dose of 0.7mg/kg, a substance considered as retroactive flavonoid, for its sedative and sleep-enhancing properties. Hesperidin are frequently detected as a racemes mixture in plant extracts, and the CNS active isomer is the 2S (-) form. Consequently, the low concentration of hesperidins present in Cameo may have limited the role of hesperidins in the antidepressant effect observed in the FST. The sedative effect of 2S-hesperidin was observed at doses of 5mg/kg in mice tested in the whole board and the sodium thiopental-induced sleep assays (Martinez et al. 2009). Since this dose is higher than those calculated for hesperidins present in the Cameo, it is probably that the sedative effect of Byronism cras-sifolio was not observed. This assumption is strengthened when analyzed in those study that rutin at doses of 10 and 20 mg/kg has a sedative effect on the potentiation of barbiturates test. Therefore, higher doses of the extract and consequently, higher doses of rutin and hesperidin, may induce a sedative effect from BcMeOH.
Although quercetin and some of its glycosides had been found in Byrsonima crassifolia, the presence of quercetin 3-O-xyloside as the main compound in BcMeOH had not been reported previously. Quercetin 3-O-xyloside is frequently found in bark and mature fruits of cultivars with commercial importance where antioxidant properties are perceived as health benefits including anticancer, anti-inflammatory, and vasoprotective effects (Ozga et al. 2007). However, no evidence of neuroactivity has been reported. In the case of quercetin, it was found inactive when tested in FST, nevertheless, quercetin dose-dependently reduced the immobility period in diabetic mice (Anjaneyulu et al. 2003). Recent studies showed that daily oral administration of quercetin for a period of 9 days induced in mice an increase in the time of immobility in the FST indicating an antidepressant effect of this substance (Chimenti et al. 2006). This fact does not preclude the active involvement of quercetin on the antidepressant effect of BcMeOH, due to the administration schedule used in this study (five administrations) enhances the accumulation of quercetin is necessary to exert an antidepressant effect, which is consistent with reports where the antidepressant drugs require cumulative doses for take effect. Furthermore, quercetin has an effect of inhibiting the activity of the enzyme monoamine oxidase-A, which is one of the main mechanisms of action of antidepressant substances (Racagni and Popoli 2010).
As many reports indicate, the role of the quercetin and its glycosides in the antidepressant effects is still unclear. However, it is a well-known fact in phytochemistry that crude plant extracts are usually more powerful medicines than pure isolated compounds may be due on the multiple actions of complex mixtures but could also arise from synergistic interactions among their components (Fernandez etal. 2005).
Although peak 2 remains unknown, its tr and UV absorption Valuse suggest the presence of a complex mixture of at least a couple of glycosylated flavone-type compounds (204, 256, 348 nm) and a phenylpropanoid (238, 320 nm), possibly a ferulate-type compounds, while in case of peak 5 and peak 7, may belong to the flavonols and flavone-type compounds (Lin and Harnly 2007). Previously, glycolipids, proanthocyanidins and triterpens (Bejar et al. 2000) have been reported to be the main compounds in Byrsonima crassifolia, and thus, the best of our knowledge, this is the first report of the presence of rutin and hesperidin flavonoids in the Byrsonima crassifolia species. Our findings suggest that flavonoid-type compounds may be involved in the antidepressant effects produced by BcMeOH. Nonetheless, because concentrations of other flavonoid-type compounds that also exhibited neuropharmacological properties as kaempferol (0.02-1.0 mg/kg o.p.) isolated from Apocynum venetum (up to 125 mg/kg doses) (Grundmann et al. 2009) are similar to those found in the present study for hesperidin (0.7 mg/kg o.p.) from Byrsonima crossifolia (up to 500 mg/kg doses), it cannot be ruled out that plant extracts which contain these flavonoid-type compounds exert also antidepressant activities since these studies are limited for animal models. Thus, the establishment of the antidepressant effects of BcMeOH in more complex in vivo models as well as the precise role and combination of the principal active components requires further investigation.
In the present acute toxicity study our results contrast with those reported for aqueous extracts, where piloerection and palpebral ptosis is observed (Morales et al. 2001), as well as those for ethanolic and acetic acid extracts where ear blanching, catalepsy, and strong hypothermia have been reported (Bejar and Malone 1993). It is important to note that in the present work, these signs of toxicity were observed primarily in the highest dose of 2000 mg/kg in which also, no mortality in mice occurred. Some reports indicate that substances with an LD50 of 1000 mg/kg body weight/oral route are 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 signs of acute toxicities in human when the extract was orally administered may be a reflection of the oral route of administration, low dose administration as well as short duration of exposure (Gazda et al. 2006). The constipation that was observed in mice after 24 h of administration is in agreement with biphasic effects reported in rat jejunum and ileum of ethanolic and acetic acid extracts (Bejar and Malone 1993).
In the remains neuropharmacological assays, our results showed that Byrsonima crassifolio extracts do not posses anxiolytic nor sedative nor anticonvulsant properties, nor effects on the enteric nervous system at the dose studied.
In conclusion, the present results showed that the methanolic extract standardized on flavonoids content of Byrsonima crassifolio possesses potential antidepressant-like effects in the FST in mice, and could be considered as relatively safe toxicologically when orally administered. Our findings suggest that the flavonoids rutin, hesperidin and quercetin could be involved in the antidepressant effects. To the best of our knowledge, the present study constitutes the first report of the presence of the neuroactive flavonoids rutin and hesperidin in Byrsonima crassifolia.
This work was supported by grant 82588 from SEP-CONACyT-Ciencia Basica 2007. Mexico (to M. Huerta-Reyes) and grant FIS/IMSS/PROT/C2007/040 from the FIS, Instituto Mexicano del Seguro Social, Mexico (to M. Huerta-Reyes). The authors wish to thank Arturo Perez M. for technical assistance.
0944-7113/$ - see front matter [c] 2011 Elsevier GmbH. All rights reserved, doi: 10.1016/j.phymed.2011.06.018
Aden eye, A.A., Jawbone, O.P., Adel eke, T.I., Belo, S.O., 2006. Preliminary toxicity and photochemical studies of the stem bark aqueous extract of Musing cecropioides in rats. J. Ethnopharmacol. 105, 374-379.
Anjaneyulu, M., Chopra, K., Kaur, I., 2003. Antidepressant activity of quercetin, a bioflavonoid, in streptozotocin-induced diabetic mice. J. Med. Food 6,391-395.
Archer, J., 1973. Test for emotionality in rats and mice: a review. Anim. Behav. 21, 205-235.
Bejar, E., Amarquaye, A., Che, C.T., Malone, M.H., Fong, H.H.S., 1995. Constituents of Byrsonima crassifolia, and their spasmogenic activity. Int. J. Pharmacognosy 33, 25-32.
Bejar, E., Malone, M.H., 1993. Pharmacological and chemical screening of Byrsonima crassifolia, a medicinal tree from Mexico, Part. I.J. Ethnopharmacol. 39,141 -158.
Bejar, E., Reyes-Chilpa, R., Jimenez-Estrada, M., 2000. In: Atta-ur-Rahman (Ed.), Studies in Natural Products Chemistry, vol. 24. Elsevier Science Publisher, Amsterdam, pp. 799-844.
Bourin, M., Fiocco, A.J., Clenet, F., 2001. How valuable are animal models in defining antidepressant activity? Hum. Psychopharmacol. 16, 9-21.
Butterweck, V., Jurgenliemk, G., Nahrstedt, A., Winterhoff, H., 2000. Fiavonoids from Hypericum perforatum show antidepressant activity in the forced swimming test. Planta Med. 66,3-6.
Chimenti, F., Cottiglia, F., Bonsignore, L, Casu, L, Casu, M., Floris, C, Secci, D., Bolasco, A., Chimenti, P., Granese, A., Befani, O., Turini, P., Alcaro, S., Ortuso, F., Trombetta, G., Loizzo, A., Guarino, I., 2006. Quercetin as the active principle of Hypericum hircinum exerts a selective inhibitory activity against MAO-A: extraction, biological analysis, and computational study. J. Nat. Prod. 69,945-949.
Dimpfel, W., 2009. Rat electropharmacograms of the flavonoids rutin and quercetin in comparison to those of moclobemide and clinically used reference drugs suggest antidepressive and/or neuroprotective action. Phytomedicine 16,287-294.
Fernandez, S.P., Wasowski, C, Paladini, A., Marder, M., 2005. Synergistic interaction between hesperidin, a natural flavonoid, and diazepam. Eur. J. Pharmacol. 512, 189-198.
Gazda, V.E., Gomes-Cameiro, M.R., Barbi, N.S., Paumgartten, F.J.R., 2006. Tox-icological evaluation of an ethanolic extract from Chiococca alba roots. J. Ethnopharmacol. 105,187-195.
Grundmann, O., Nakajima, J.I., Kamata, K., Seo, S., Butterweck, V., 2009. Kaempferol from the leaves of Apocynum venetum possesses anxiolytic activities in the elevated plus maze test in mice. Phytomedicine 16,295-302.
Heinrich, MM Ankli, A., Frei, B., Weimann, C, Sticher, O., 1998. Medicinal plants in Mexico: healers' consensus and cultural importance. Soc. Sci. Med. 47, 1859-1871.
Herrera-Ruiz, M., Gonzalez Cortazar, M., Jimenez-Ferrer, E., Zamilpa, A., Alvarez, L. Ramirez, G., Tortoriello, J., 2006. Anxiolytic effect of natural galphimines from Galphimia glauca and their chemical derivatives. J. Nat. Prod. 69, 59-61.
Lin, L.Z., Harnly, J.M., 2007. A screening method for the identification of glycosylated fiavonoids and other phenolic compounds using a standard analytical approach for all plant materials. J. Agric. Food Chem. 55,1084-1096.
Lister, R.G., 1987. The use of a plus-maze to measure anxiety in the mouse. Psychopharmacology 92,180-185.
Latoya, X., Becerra, G., Martinez, M., 1990. Model of intraluminal perfusion of the
guinea pig ileum in vitro in the study of the antidiarrheal properties of the guava
(Psidium guajava). Arch. Inv. Med. (Mex.) 21,155-162. Machado, D.G., Bettio, LE., Cunha, M.P., Santos. A.R., Pizzolatti, M.G., Brighente,
I.M., Rodrigues, A.L, 2008. Antidepressant-like effect of rutin isolated from the ethanolic extract from Schinus molle L. in mice: evidence for the involvement of the serotonergic and noradrenergic systems. Eur. J. Pharmacol. 587,163-168.
Maldini, M., Sosa, S., Montoro. P., Giangaspero, A., Balick, M.J., Pizza, C, Delia Loggia,
R., 2009. Screening of the topical anti-inflammatory activity of the bark of Acacia
cornigera Willdenow, Byrsonima crassifolia Kunth. Sweetia panamensis Yakovlev
and the leaves of Sphagneticola trilobata Hitchcock. J. Ethnopharmacol. 122,
430-433. Maldonado, A., 2008. Busqueda de la actividad antidepresiva de diferentes plantas
medicinales mexicanas sobre modelos animates de depresion. Undergraduate
thesis, Institute Tecnologico de Zacatepec, Mexico. Martinez, M.C., Fernandez, S.P., Loscalzo, L.M., Wasowski, C, Paiadini, A.C., Marder,
M., Medina, J.H., Viola, H., 2009. Hesperidin, a flavonoid glycoside with sedative
effect, decreases brain pERK1/2 levels in mice. Pharmacol. Biochem. Behav. 92,
291-296. Morales, C. Gomez-Serranillos, M.P., Iglesias, I., Viliar del Fresno, A.M., 2001.
Neuropharmacological profile of ethnomedicinal plants of Guatemala. J.
Ethnopharmacol. 76,223-228. Nolder, M., Schotz, K., 2002. Rutin is essential for the antidepressant activity of
Hypericum perforatum extracts in the forced swimming test. Planta Med. 68, 577-580.
OECD/OCDE 423. OECD web. Consulted 22 May 2009. http://iccvam.niehs.nih.gov/SuppDocs/FedDocs/OECD/OECD_GL423.pdf.
Ozga, J.A., Saeed, A., Wismer. W., Reinecke, D.M., 2007. Characterization of cyanidin-and quercetin-derived flavonoids and other phenolics in mature saskatoon fruits (Ame/anc/rier alnifolia Nutt.). J. Agric. Food Chem. 55,10414-10424.
Porsolt, R.D., Bertin, A., Jalfre, M, 1977. Behavioral despair in mice: a primary screening test for antidepressants. Arch. Int. Pharmacodyn. Ther. 229,327-336.
Racagni, G., Popoli, M., 2010. The pharmacological properties of antidepressants. Int.
Clin. Psychopharmacol. 25,117-131. Sanchez-Mateo, C.C., Prado, B., Rabanal, R.M., 2002. Antidepressant effects of the methanol extract of several Hypericum species from the Canary Islands. J. Ethnopharmacol. 79,119-127.
Sanchez-Mateo, C.C., Bonkanka, CX, Prado, B., Rabanal, R.M., 2005. Antidepressant properties of some Hypericum canariense L and Hypericum gtandulosum Ait. Extracts in the forced swimming test in mice. J. Ethnopharmacol. 97, 541-547.
Sanchez-Mateo. C.C., Bonkanka, C.X., Rabanal, R.M., 2009. Hypericum grandifolium Choisy: a species native to Macaronesian Region with antidepressant effect. J. Ethnopharmacol. 121, 297-303.
Williamson, E., Okpako, D.T., Evans, F.J., 1996. Selection, Preparation and Pharmacological Evaluation of Plant Material. Wiley. Sussex, pp. 169-189.
Wurlics, M., Schubert-Zsilavecz, M., 2006. Hypericum perforatum: a "modern" herbal antidepressant: pharmacokinetics of active ingredients. Clin. Pharmacokinet. 45. 449-468.
Yan, X.. Murphy, B.T., Hammond, G.B., Vinson, J.A., Neto, C.C., 2002. Antioxidant activities and antitumor screening of extracts from cranberry fruit (Vaccinium macrocarpon). J. Agric. Food Chem. 50,5844-5849.
M. Herrera-Ruiza (a), A. Zamilpaa (a), M. Gonzalez-Cortazara (a), R. Reyes-Chilpa (b), E. Leon (c), M.P. Garcia (a), J. Tortoriello (a), M. Huerta-Reyesa (a), *
(a) Centro de Investigation Biomedical del Sur, Instituto Mexicano del Seguro Social (IMSS), Argentina No. 1, 62790, Xochitepec, Morelos, Mexico
(b) Instituto de Quimica, Departamento de Productos Naturales, Universidad Nacional Autonoma de Mexico, Ciudad Universitaria, Coyoacdn, 04510, Mexico, D.F., Mexico
(c) Herbario Nacional de Mexico (MEXU), Instituto de Biologia, Departamento de Botdnica, Universidad Nacional Autonoma de Mexico, Apartado Postal 70-367, Del Coyoacan, 04510, Mexico, D.F., Mexico
* Corresponding author at: Centro de Investigation Biomedical del Sure, Instituto Mexicano del Seguro Social, Argentina No. 1, Col. Centro, C.P. 62790, Xochitepec, Morelos, Mexico. Tel.: +52 777 3 61 21 55; fax: +52 777 3 61 21 94.
E-mail address: firstname.lastname@example.org (M. Huerta-Reyes).