Anxiolytic- and anxiogenic-like effects of Montanoa tomentosa (Asteraceae): Dependence on the endocrine condition

Journal of Ethnopharmacology 241 (2019) 112006

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Anxiolytic- and anxiogenic-like effects of Montanoa tomentosa (Asteraceae): Dependence on the endocrine condition

T

Erika Estrada-Camarenaa, Isabel Sollozo-Dupontb, Dannia Islas-Preciadoa, María Eva González-Trujanoc, Miguel Carro-Juárezd,1, Carolina López-Rubalcavab,∗ Laboratorio de Neuropsicofarmacología, Dirección de Neurociencias, Instituto Nacional de Psiquiatría “Ramón de la Fuente Muñíz”, Calzada México Xochimilco 101, Col San Lorenzo Huipulco, Delegación Tlalpan, Ciudad de México, Mexico b Departamento de Farmacobiología, CINVESTAV-Sede Sur. Calzada de los Tenorios 235, Col Granjas Coapa, Delegación Tlalpan, Ciudad de México, Mexico c Laboratorio de Neurofarmacología de Productos Naturales, Dirección de Neurociencias, Instituto Nacional de Psiquiatría “Ramón de la Fuente Muñíz”, Calzada México, Xochimilco 101, Col San Lorenzo Huipulco, Delegación Tlalpan, Ciudad de México, Mexico d Facultad de Veterinaria, Universidad autónoma de Tlaxcala, Tlaxcala, Mexico a

A R T I C LE I N FO

A B S T R A C T

Keywords: Anxiolytic-like effect Estrous cycle Progesterone withdrawal Elevated plus-maze Montanoa tomentosa GABAA receptor

Ethnopharmacological relevance: Montanoa tomentosa Cerv. (MT) is a native plant from Mexico used in traditional medicine as a remedy for reproductive impairments and relaxing effects. In previous studies, it has been shown that the endocrine state could modify the antianxiety-like actions of anxiolytic compounds. Although women are the primary user of MT, no studies have evaluated the potential impact of the endocrine milieu on its antianxiety actions. Aims of the study:Ascertain the antianxiety effects of M. tomentosa in rats with different hormonal conditions, and to analyze the participation of the GABAA receptor in ovariectomized rats treated with MT. Materials and methods: The animal model of anxiety used was the elevated plus-maze (EPM). Rats’ endocrine conditions were: a) Low hormone levels (rats in diestrus I and II phases); b) High hormone levels (proestrus/ estrus phases); c) No hormones (ovariectomized rats); and d) Rats under progesterone withdrawal (PW). To evaluate the participation of the GABAA receptor in the anxiolytic-like action of MT the antagonist picrotoxin was used. Results: Results showed that MT induced dose-dependent anxiolytic-like actions in rats with low hormone level conditions. Also, MT reduced anxiety-like behavior in female rats under PW, in contrast to diazepam which was ineffective. MT's anxiolytic-like effect was blocked by picrotoxin, suggesting the participation of the GABAA receptor complex. However, increased anxiety-like behavior was observed in rats with a high hormone level condition and low doses of MT. Conclusions: Beneficial anxiolytic-like actions of MT are observed under low hormone conditions, particularly in the PW challenge (a condition that can be related to a premenstrual period). Furthermore, the participation of the GABAA receptor is evidenced. However, hormonal variations could induce the opposite effects, hence women should be cautious.

1. Introduction Montanoa frutescens, Montanoa grandiflora, and Montanoa tomentosa are small trees native from Mexico that are also known as “zoapatle” or “cihuapatli.” These plants are cherished for their medicinal properties (Gallegos, 1985). “Cihuapatli” means “women's medicine” in the Nahuatl language (Rodriguez-Landa et al., 2014b). The tea prepared from

the leaves of these three species (alone or combined) has been used in Mexican folk medicine as a remedy for menstrual cycle-related disorders and to treat some nervous disorders (Gallegos, 1985; Levine et al., 1981; Rodriguez-Landa et al., 2014b; Villa-Ruano et al., 2009). Recently, we reported that the aqueous extract of Montanoa tomentosa (MT) induces anxiolytic-like effects in male rats tested in different animal models of anxiety such as the “elevated plus-maze” (EPM), the

∗

Corresponding author. E-mail addresses: [email protected] (E. Estrada-Camarena), [email protected] (I. Sollozo-Dupont), [email protected] (D. Islas-Preciado), [email protected] (M.E. González-Trujano), [email protected] (C. López-Rubalcava). 1 In memoriam. https://doi.org/10.1016/j.jep.2019.112006 Received 11 February 2019; Received in revised form 28 May 2019; Accepted 29 May 2019 Available online 30 May 2019 0378-8741/ © 2019 Elsevier B.V. All rights reserved.

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“hole-board,” and the “burying behavior” tests (Sollozo-Dupont et al., 2015). In that study, we analyzed the participation of the GABAA receptor in MT's anxiolytic-like action in male rats (Sollozo-Dupont et al., 2015). The results supported the idea that MT interacts with the GABAA receptor complex in several binding sites to induce its anxiolytic-like actions. Similarly, finasteride (a compound that blocks the rate-limiting enzyme in the production of neurosteroids) prevented the antianxiety effects of MT, suggesting that neurosteroids also contributed to its antianxiety-like effects (Sollozo-Dupont et al., 2015). Even though the prevalence of anxiety is more significant in women than in men (Bekker and van Mens-Verhulst, 2007), there are no studies that analyze the influence of different hormonal conditions (premenstrual, postpartum, or menopause) on MT's anxiolytic-like actions. Moreover, some preclinical reports indicated that the endocrine state could modify the antianxiety-like actions of anxiolytic compounds like diazepam or buspirone (Fernandez-Guasti et al., 2001; FernandezGuasti and Picazo, 1990, 1997; Olvera-Hernandez and FernandezGuasti, 2011). Furthermore, it was reported that the anxiolytic effects of M. frutescens and M. grandiflora depended on the estrous cycle phase. Both plants reduced the anxiety-like behavior in female rats tested in diestrus I/II but not in proestrus-estrus phases (Rodriguez-Landa et al., 2014b). As to MT, Rodríguez-Landa et al. proved antianxiety actions on long-term ovariectomized rats (Rodriguez-Landa et al., 2014a). However, no studies have analyzed the effect of this plant in female rats under different endocrine conditions. Because GABAA receptors mediate MT's anxiolytic-like effect in male rats (Sollozo-Dupont et al., 2015), and ovarian hormonal fluctuations along the estrous cycle can modulate the affinity (Wilson, 1992) and expression of the GABAA receptors subunits (Lovick, 2006). It is feasible that ovarian hormone fluctuations can also influence MT's actions. Therefore, the primary objective of the present study was to ascertain the antianxiety effects of M. tomentosa in rats with different hormonal conditions. Furthermore, the participation of the GABAA receptor was evaluated in ovariectomized rats treated with MT. It should be mentioned that the EPM test is one of the few anxiety models that are sensitive enough to detect the effects of different endocrine conditions on the anxiety-like behavior of rats (Frye et al., 2000; Gulinello et al., 2003; Gulinello and Smith, 2003; Marcondes et al., 2001; Molina-Hernandez et al., 2013; Mora et al., 1996; Sayin et al., 2014). For example, a low anxiety-like behavior is observed when rats are tested in the proestrus and estrus phases, characterized by high levels of estradiol and progesterone (Frye et al., 2000; Marcondes et al., 2001). In contrast, during diestrus I and diestrus II phases (when low estradiol and progesterone levels are present) high anxiety levels are observed (Marcondes et al., 2001). Moreover, the EPM can detect increases in anxiety-like behavior induced by the PW (Gulinello et al., 2003; Gulinello and Smith, 2003). Therefore, in the present study, we selected the EPM to evaluate the effects of MT in female rats tested under different endocrine conditions. On the other hand, phytochemical studies have reported that MT contains several classes of chemical constituents including terpenoids such as grandiflorenic acid, zoapatanol, and montanol (Enriquez et al., 1996; Guzman-Duran et al., 1988; Marcelle et al., 1985). In addition, flavonoids such as nicotiflorin and isoquercitrin were isolated from a polar fraction of the MeOH extract; while acid hydrolysis showed quercetin, rutinose, and kaempferol (Oshima et al., 1986). In the present study, we analyzed the chromatographic profile of the aqueous extract of MT focusing mainly in the later compounds, since their presence is poorly described for this species.

specialist from the Herbarium of the Universidad Autónoma de Tlaxcala (voucher specimens-serial Number: MT UATX10). First, the dried leaves of M. tomentosa were ground into a fine powder. Twenty grams of the powder were mixed with 200 mL of distilled water and warmed up (just before boiling) approximately for 10 min. Then, the infusion was filtered into glass flasks and frozen using dry ice (−78 °C). After that, it was placed in a vacuum chamber (−50 °C and 0.9 mbars of pressure) for 4–5 h. A total of 2 g (10%) of lyophilized material was obtained (Sollozo-Dupont et al., 2015). 2.2. Animals Adult (3-months old) female Wistar rats from our breeding facilities were used. Animals were group-housed (5–6 per cage) in polycarbonate cages in a room with constant temperature (23 ± 2 °C) and inverted light: dark cycle conditions (lights on at 22:00–10:00 h). Rats had free access to water and rat chow (Harlan®, Mexico, S.A. de C.V.). This study was carried out in accordance with the recommendations of the National Institutes of Health Guide for the care and use of Laboratory Animals (NIH Publications No. 85–23, revised 1985) and the Mexican Official Norm for animal handling and care (NOM-062ZOO-1999). The protocol was approved by the Ethics Committee of the CINVESTAV-IPN No. 379–07 (23/04/2007) and INPRFM No. CEI-200 (10/12/2015). 2.3. Drugs Drugs and solvents used in the present study were: picrotoxin, diazepam, progesterone and propylene glycol. All compounds were purchased from Sigma-Aldrich (Toluca, México). Picrotoxin was dissolved in saline solution while Diazepam was dissolved in propylene glycol 40%. Both drugs were administered intraperitoneally (i.p.) in a volume of 2.0 ml/kg (Fernandez-Guasti and Picazo, 1990). MT was dissolved in physiologic saline solution (0.9%) and administered orally in a volume of 2.0 ml/kg (Sollozo-Dupont et al., 2015). Progesterone was dissolved in corn oil and injected subcutaneously in a final volume of 1 ml/kg. 2.4. Endocrine conditions/manipulations 2.4.1. Estrous cycle phases The estrous cycle phases were determined by vaginal cytology of at least three consecutive cycles. It has been demonstrated that serum levels of gonadal hormones (estradiol and progesterone) directly correlate with specific vaginal cytology along the estrous cycle in rodents (Butcher et al., 1974; Goldman et al., 2007; Walmer et al., 1992). Thus, the four estrous cycle phases observed were: a) The proestrous phase, characterized by the predominance of nucleated epithelial cells (distinctly round and visibly nucleated). b) The estrous phase, in which the cells that predominate are cornified squamous epithelial cells with an irregular shape and without an observable nucleus. c) The diestrus-I phase (also called metestrus), where the dominant cell type is the leukocyte (small cells with granular cytoplasm) and a significant number of nucleated epithelial cells is present. d) The diestrus-II phase, which presents mainly leukocytes cells (Fernandez-Guasti et al., 1999; Marcondes et al., 2002). Fig. 1 shows typical wet unstained vaginal smears obtained from rats under different estrous cycle phases.

2. Materials and Methods

2.4.2. Ovariectomy as a state resembling menopause Rats were ovariectomized under anesthesia with 2% tribromoethanol (0.1 mL/kg); later, a ventral incision was made to expose and remove the ovaries. After suturing the muscles and skin, the wound

2.1. Plant material The leaves of Montanoa tomentosa Cerv (Family: Asteraceae) were collected in Tlaxcala, Mexico. The plant was authenticated by a 2

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number of arm crosses. The parameters considered as indexes of anxiety were the % of time spent in open, the number of entries to open arms, and the ratio of the time spent in closed arms vs time spent in open arms (Brummelte et al., 2012; Gobinath et al., 2016; Pellow et al., 1985), while the total arm crosses was related to rat's general activity (Lister, 1990). 2.5.2. Locomotor activity All treatments studied in the EPM were also analyzed to detect any alteration in locomotor activity that could influence the response observed in the EPM. The apparatus consists of an opaque box (40 × 30 × 20 cm) with the floor divided into 12 equal squares. Each rat was set in one corner of the box, and the behavior was videotaped from above for 5 min. After each test, the box was thoroughly cleaned with a cleaning solution made from ethanol, ammonia, isopropanol, and extran®. The number of squares crossed by each rat was recorded by an observer unaware of the drug treatment. When there is a decrease in the number of counts, it shows a reduction in general motor activity (Estrada-Camarena et al., 2002). 2.6. Biochemical and chemical determinations

Fig. 1. Representative wet unstained vaginal smears obtained from rats under different estrous cycle phases. Proestrus, the predominance of nucleated epithelial cells which are distinctly round and visibly nucleated. Estrus, the predominance of cornified squamous epithelial cells with irregular shapes and without an observable nucleus. Diestrus-I, the dominant cell type is the leukocyte which appears along with a significant number of nucleated epithelial cells; leukocytes are small cells with granular cytoplasm. Diestrus-II, with mainly leukocytes cells. N = nucleated cell; C = cornified squamous cell; L = leukocytes.

2.6.1. Serum determination of progesterone levels Progesterone concentration in serum was determined from the following groups: ovariectomized, PW, proestrus, and diestrus (5–8 rats per group were used). Immediately after the behavioral test, rats were decapitated, and their trunk blood was collected. Specific commercial ELISA reagents were used following the manufacturer's instructions (Enzo Life Sciences, USA). All samples were assayed in duplicate. Intra and interassay variations were < 7.6% and < 6.8% respectively; the sensitivity was 8.57 pg/mL.

was treated with an antiseptic solution and analgesics. Rats were returned to their home cage and had a 3-week recovery period during which they were monitored daily to verify that no open wounds or infections were present. After recovery, rats were randomly assigned to the experimental groups (Estrada-Camarena et al., 2003, 2004). This period is considered enough to drop out the gonadal hormone levels (Liu et al., 2010).

2.6.2. Ultra-high performance liquid chromatography (UHPLC) analysis of flavonoids present in the aqueous extract of M. tomentosa Chromatographic analysis was performed using an Acquity UHPLCH class with PDA eλ detector (UHPLC, Acquity Waters, Singapore) equipped with a Symmetry C18 column (100 Å, 150 mm × 4.6 mm, 5 mm, Waters, Ireland) with the thermostat at 43 °C. The mobile phase consisted of acidified water at 0.1% phosphoric acid in Milli Q water (A) and methanol (B) (HPLC grade). The initial gradient elution was 80% A: 20% B to reach 100% B (17 min). The initial condition was regenerated for 3 min (80% A: 20% B). The constant flow rate was 1.0 mL/min with an elution curve of 6. The extract (3 mg/mL) was diluted in methanol/Milli Q water (50:50) and injected directly after filtration through a 0.2 mm filter (GHP, acrodisc-13, Waters) in a volume of 10 μL. Data acquisition, data handling, and instrument control were performed by the Empower® 3 software (Waters, Milford, MA, USA). The present analysis was focused on detecting mainly flavonoids, and the chromatographic analysis was done twice to corroborate the presence of these compounds. Because the main compound found was rutin, the molecular weight of rutin standard and its peak in the M. tomentosa extract was determined by MS (DART) [M+H]+. The peak profile was identified at UVλ max 254.9 and 354.6 nm.

2.4.3. Progesterone withdrawal (PW) challenge To study the antianxiety actions of MT in conditions in which a sudden decline of progesterone occurs (a condition related to the premenstrual syndrome), we induced the PW challenge. Thus, ovariectomized rats were injected subcutaneously with progesterone at 2.0 mg/kg or vehicle once a day for five consecutive days. Twenty-four hours after the last injection rats were subjected to the behavioral tests (Islas-Preciado et al., 2016; Saavedra et al., 2006). It is reported that 24 h latency is enough to induce anxiety-like behavior and alterations in the GABAA receptors’ function due to the changes in the expression of specific subunits (Gallo and Smith, 1993; Gulinello et al., 2002; Hsu and Smith, 2003). All treatments were administered between 9 and 10 a.m. 2.5. Behavioral tests 2.5.1. Elevated plus-maze (EPM) test The EPM test consists of a plus-maze shaped apparatus that is elevated 50 cm above the floor and placed in the center of a room illuminated by two red bulbs (40 W). Each arm of the maze is 50 cm (length) × 10 cm (width). Two opposing arms have plastic walls (40 cm high –closed arms) while the other two lack walls (open arms). The behavior of each animal was video-recorded from above the maze over a 10-min session. At the beginning of the test, the animal was placed in the center of the maze facing a closed arm; an arm entry was taken into account once the rat put all four paws on it. The parameters registered were: (a) the cumulative time spent in the open arms, expressed as % of time; (b) the cumulative time in the closed arms; (c) the number of open arm entries; and (d) the total

2.7. Experimental design Experiment 1. Effect of diazepam and MT on anxiety-like behavior in ovariectomized and intact cycling rats. On the day when the behavioral experiments were performed, after determining the rats’ hormonal condition by vaginal cytology, rats were assigned to one of the four phases of the estrous cycle and tested. After that, data were subsequently pooled into two categories: proestrus/estrus rats (characterized by high hormone concentrations) (n = 8–12) or diestrus-I/-II rats (characterized by low hormone concentration) 3

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Fig. 2. Schematic representation of the experimental design. * Intraperitoneal administration; ** oral administration, MT = M. tomentosa, Picro = picrotoxin, veh = vehicle, PW = progesterone withdrawal, OVX = ovariectomized.

The following experiment was designed to test if the GABAA receptor participates in MT's effects in female ovariectomized rats. Thus, rats were divided into four groups (n = 7–9 per group): control (vehicle treated), MT (1.5 mg/kg), picrotoxin (0.25 or 0.5 mg/kg), and MT plus picrotoxin (1.5 mg/kg (MT) plus 0.25 or 0.5 mg/kg (PTX)). After drug latencies, animals were tested in the locomotor activity test and the EPM (see Fig. 2, lower panel).

(n = 8–12) (Molina-Hernandez et al., 2013; Rodriguez-Landa et al., 2014b). An independent group consisted of ovariectomized rats (n = 10–14). All groups were administered with either MT (0.38, 0.75, 1.5 or 3.0 mg/kg, −1 h), diazepam (2.0 mg/kg, −30 min), or vehicle and tested in the EPM followed by the locomotor activity test. Diazepam was used as a positive control group (Bitran et al., 1991). See Fig. 2 (upper panel) for the schematic representation of the experimental design. Experiment 2. Effect of diazepam and MT on anxiety-like behavior in rats under progesterone withdrawal challenge. Ovariectomized rats were subjected to vehicle or PW challenge and treated with diazepam (0.125, 0.25, 0.5 or 1.0 mg/kg) or MT (0.38, 0.75, 1.5, or 3.0 mg/kg). Diazepam was used as an anxiolytic reference drug to compare the effect of MT under PW challenge. The doses and latencies of diazepam were taken from previous reports (FernandezGuasti and Picazo, 1990), whereas MT doses were taken from data obtained from experiment one (see Fig. 2, middle panel). Experiment 3. Participation of GABAA receptors in the anxiolyticlike action of MT.

2.8. Data analysis All data are expressed as the mean ± S.E.M. The experiments were analyzed using a One-Way Analysis of Variance (ANOVA) on ranks followed by Dunn's test as a post hoc analysis; the significance level was set at p < 0.05. Specific paired comparisons were done using the Mann-Whitney U test.

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3.2. Experiment 2. effect of diazepam and MT on anxiety-like behavior in rats under progesterone withdrawal challenge

Table 1 Effect of the different doses of M. tomentosa and diazepam on the total number of squares crossed in the open-field motor activity test in ovariectomized, DI/II, proestrus-estrus and progesterone withdrawal treated rats. M. tomentosa (mg/kg)

Ovariectomized

Vehicle 30.0 ± 3.0 (VEH) 0.38 43.5 ± 3.4** 0.75 42.8 ± 2.7** 1.50 48.3 ± 2.3**&,#,% 3.00 36.8 ± 7.0 Diazepam (mg/kg) 0.125 40.9 ± 8.2 0.25 38.7 ± 11.2 0.50 41.8 ± 9.6 1.00 38.5 ± 8.7 2.00 38.3 ± 6.9

DI/II

Proestrus-estrus

Progesterone Withdrawal

33.9 ± 1.0

35.6 ± 1.38

33.6 ± 1.0

33.1 34.0 34.1 32.4

36.0 35.0 35.7 36.0

33.8 33.7 37.6 34.8

± ± ± ±

1.5 3.9 1.5 1.5

31.4 30.5 31.3 32.8 ND

± ± ± ±

1.0 0.5 0.6 1.0

± ± ± ±

1.3 1.3 1.4 1.0

ND ND ND ND 30.8 ± 3.8

± ± ± ±

1.4 0.5 1.0 1.0

ND ND ND ND 27.4 ± 11.3

Fig. 4 shows the effect of diazepam (right side) and MT (left side) in rats under vehicle or PW challenge on the percentage of time spent in open arms (panel A), the total number of open arms entries (panel B) and the ratio of time in closed/open arms (panel C). Diazepam, at doses of 0.5 and 1.0 mg/kg, increased the % of time in open arms (p < 0.05) and reduced the anxiety index in control (vehicle treated) rats. There were no changes in the number of open arms entries at any dose. In contrast, diazepam in rats under PW challenge did not affect any of the parameters registered. In contrast to diazepam, MT at lower doses induced an anxiolyticlike effect in both OVX rats treated with vehicle and rats under the PW challenge condition. Thus, Fig. 3 (panel A, left side) shows that MT at a dose of 1.5 mg/kg in vehicle-treated rats, and 0.38 and 0.75 mg/kg in rats under PW challenge increased the % of time spent in open arms. In parallel, there was an increase on the number of entries to open arms (panel B), as well as a decrease in the ratio of time spent in closed/open arms (panel C) in both vehicle-treated rats (p < 0.05) and under PW challenge condition (p < 0.005). High doses of MT did not affect rats under the PW challenge in contrast to respective control-group in the same endocrine condition (Fig. 4).

Data are expressed as mean ± S.E.M of the number of squares crossed in a 5min test session. One-Way ANOVA on ranks followed by Dunn's test. *p < 0.05; **p < 0.005 vs. VEH; & p < 0.01 vs. DI/II; # = p < 0.01 vs. proestrus-estrus; % = p < 0.01 vs. progesterone withdrawal.

3. Results 3.3. Experiment 3. participation of GABAA receptors in the anxiolytic-like action of MT

3.1. Experiment 1. effect of diazepam and MT on anxiety-like behavior in rats with different endocrine conditions

Fig. 5 shows the effect of picrotoxin (an antagonist of the chloride ion channel of the GABAA receptor) in OVX rats previously treated with MT. The plant extract per se increased the % of time spent in open arms (panel A) (p < 0.005), and the number of entries to open arms (p < 0.05) (panel B) while reducing the ratio of time spent in closed/ open arms (panel C). In all variables, the effect was completely blocked by both doses of picrotoxin (p < 0.05). By itself, picrotoxin at dose of 0.25 and 0.5 mg/kg did not modify the % time spent in open arms (1.83 ± 0.8; 2.89 ± 1.31 vs. 3.7 ± 0.7), the number of entries to open arms (1.0 ± 0.43, 1.62 ± 0.26 vs. 1.44 ± 0.29) and the ratio (240.86 ± 97.12; 273.0 ± 101.34 vs. 92.66 ± 63.24) in comparison to control group. Table 2 shows the effect of the different doses of MT and diazepam in the total number of crosses to the arms of EPM in ovariectomized, diestrus I/II, proestrus/estrus, and PW treated rats. No significant differences were detected with MT treatments, and diazepam at the highest dose (2.0 mg/kg) decreased this parameter in diestrus I/II rats. Table 3 shows the effect of picrotoxin on total crosses and locomotor activity of OVX rats treated with MT. Picrotoxin by itself or combined with MT reduced the number of total crosses in the EPM compared to the control group (p < 0.001). Furthermore, the group given picrotoxin at a dose of 0.5 mg/kg plus MT showed the lowest total number of crosses (p < 0.001). Regarding the locomotor activity, all treatments reduced the number of squares crossed when compared to the control group (p < 0.001). Table 4 shows the serum levels of progesterone in animals subjected to ovariectomy alone and under PW, proestrus, and diestrus. As was described earlier by other authors, the PW challenge promoted a sudden fall of progesterone 24 h after its last administration (Butcher et al., 1974; Smith, 1975). Therefore, the values of progesterone after the PW challenge were similar to those detected in OVX rats treated with vehicle and rats in diestrus-II.

Table 1 shows the effect of several doses of MT and diazepam on general activity. There were no differences detected within groups in intact cycling rats treated with MT in relation to the vehicle treated groups in the same endocrine condition. However, the aqueous extract, at doses of 0.38, 0.75 and 1.5 mg/kg, increased the number of squares crossed in ovariectomized rats (p < 0.05). Also, this increase was significant when compared with other endocrine conditions (p < 0.05). There were no differences observed in the number of squares crossed after diazepam or diazepam under PW challenge treatment in ovariectomized rats (Table 1). Diazepam and MT data are presented in separate graphs considering endocrine conditions. Fig. 3 shows the effect of diazepam (right side) and MT (left side) in ovariectomized, and intact cycling rats in diestrus I/II and proestrus/estrus on the percentage of time in open arms (panel A), the total number of open arms entries (panel B), and the ratio of time spent in closed/open arms (panel C). Under basal conditions (vehicle-treated groups), proestrus/estrus rats showed the highest % of time and total number of entries to open arms in comparison to other endocrine conditions (p < 0.001). Furthermore, these rats exhibited the lowest index of anxiety (ratio closed/open arms time) when compared with ovariectomized and DiestrusI/II rats (p < 0.001). Diazepam in all endocrine conditions induced anxiolytic-like actions by increasing the percentage of time spent in open arms reaching statistical significance in OVX and proestrus/estrus rats (p < 0.05), while a non-significant decrease in the total number of entries was observed in all conditions. Diazepam also reduced the anxiety index (ratio time closed/open arms) in OVX rats and showed a trend to reduce this parameter in high hormone condition (proestrus/estrus) when compared with vehicle-treated rats in the same hormone condition (p = 0.07). Regarding MT's effects, it was observed that the plant extract increased the % of time spent in open arms as well as the number of entries in ovariectomized and diestrus I/II rats at a dose of 1.5 mg/kg (p < 0.005). In contrast, in proestrus/estrus rats at low doses, M. tomentosa (0.38 and 0.75 mg/kg) significantly reduced the % time and the number of entries to the open arms (p < 0.05) and increased the anxiety index in proestrus/estrus rats (p = 0.001) in comparison to the saline-treated group in the same endocrine condition.

3.4. Experiment 4. chromatographic analysis of the aqueous extract of MT Fig. 6 shows the chromatographic profile of MT aqueous extract obtained by UHPLC. The analysis indicates the presence of the flavonoid rutin in the retention time (Rt) of 9.9 min with a spectrum UV max at 254.9 and 354.6 nm as described in the inserted square. 5

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Fig. 3. Effect of different doses of M. tomentosa and diazepam (2.0 mg/kg) on anxiety-like behavior tested in the EPM. Data are presented as the mean ± S.E.M of the % of time spent in open arms (panel A), the total number of entries to open arms (panel B) and the ratio of time in closed arms/time in open arms (panel C) of OVX rats, low diestrus (Di I/II) and high proestrus/estrus (P/E) hormonal levels. White bars represent vehicle-treated groups, and gray bars represent diazepam or MT treated groups. One-way ANOVA on ranks followed by Dunn's multiple comparison's test *p < 0.05, **p < 0.005 vs. vehicle. Mann-Whitney's U test & p < 0.05 && p < 0.02 diazepam vs. vehicle. OVX = ovariectomized.

conditions, i.e., diestrus I/II and OVX. Similar findings were reported with other species of Montanoa (M. frutenses and M. grandiflora) in cycling rats (Carro-Juarez et al., 2012; Rodriguez-Landa et al., 2014b) and after 12 weeks post-OVX (Rodriguez-Landa et al., 2014a). Interestingly, in the present study, rats that were subjected to PW were more responsive to the anxiolytic-like action of MT than rats under other endocrine conditions. In all cases, the administration of picrotoxin (an antagonist to the chloride channel of the GABAA/BZD receptor complex) canceled the anxiolytic-like action of MT, suggesting the

4. Discussion Under basal conditions, rats tested in proestrus/estrus showed less anxiety-like behavior than females in diestrus I/II and OVX rats. Present data are in line with previous reports (Marcondes et al., 2001; Rodriguez-Landa et al., 2014a) reinforcing the notion that the increase in ovarian hormones exerts anxiolytic-like actions in the EPM and other anxiety paradigms (Fernandez-Guasti and Picazo, 1992). MT reduced the anxiety-like behavior in rats under low endocrine 6

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Fig. 4. Effect of different doses of diazepam and M. tomentosa on % of time spent in open arms (panel A), the total number of entries to open arms (panel B) and the ratio of time spent in closed/open arms (panel C) of ovariectomized rats subjected to progesterone withdrawal (PW). Data are presented as mean ± S.E.M. One-way ANOVA on ranks followed by Dunn's multiple comparison's test *p < 0.05, **p < 0.005 vs. vehicle. Control group vs. progesterone withdrawal; Mann-Whitney's U test & p < 0.05.

bicuculline, and only partially by flumazenil (Sollozo-Dupont et al., 2015). The present study reinforces the notion that GABAA receptors participate in MT's anxiolytic-like actions because also picrotoxin prevents its anxiolytic actions in female rats. Nevertheless, the participation of another mechanism of action cannot be discarded. Specific experiments are necessary to provide evidence for this hypothesis. MT induced a biphasic effect depending on the endocrine condition. Hence, when low levels of hormones are present, anxiolytic-like actions of MT were observed. In contrast, when high hormone levels are present an anxiogenic-like effect was evident. Similar findings are reported with progesterone metabolites, benzodiazepines and barbituric drugs, which can induce both anxiolytic or anxiogenic-like effects depending on the dose used (Andreen et al., 2009; Backstrom et al., 2011) — reinforcing the notion that the GABAA receptor is one of the mediators of

participation of the GABAergic system. Some reports show that the gonadal hormonal milieu influences the sensitivity of GABAA receptors by modulating the expressions of their subunits (Lovick, 2006). When low levels of progesterone metabolites are present (for example late diestrus and PW challenge), a low response of GABAA receptors is reported and is associated with an anxiogenic state (Gulinello et al., 2001; Lovick, 2006). It is possible that plastic adaptations in the composition of GABAA subunits in response to the reduction or absence of gonadal hormones will favor the anxiolyticlike actions of MT. In a previous study, we demonstrated that MT's anxiolytic-like actions require the activation of GABAA receptors in male rats (SollozoDupont et al., 2015). In that study, the anxiolytic-like effect of MT in the EPM and hole-board tests was blocked entirely by picrotoxin and 7

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Fig. 5. Effect of picrotoxin (0.25 and 0.50 mg/kg) on anxiety-like behavior tested in the EPM. Data are presented as the mean ± S.E.M of % the time spent in open arms (panel A), the total number of entries to open arms (panel B) and the ratio of time in closed arms/open arm (panel C) of ovariectomized (OVX) rats. One-way ANOVA on ranks followed by Dunn's multiple comparison's test **p < 0.005 vs. vehicle, && p < 0.005 vs. Mt 1.5 mg/kg. Table 2 Effect of the different doses of M. tomentosa and diazepam on the total number of arm crosses in the Elevated Plus-Maze in ovariectomized, DI/II, proestrusestrus and progesterone withdrawal treated rats. M. tomentosa (mg/kg)

Ovariectomized

Vehicle (VEH) 0.38 0.75 1.50 3.00 Diazepam 0.125 0.25 0.50 1.00 2.00

11.0 ± 0.5

13.2 ± 2.5

13.8 ± 2.1

12.3 ± 1.2

11.0 11.1 11.2 11.0

13.8 11.2 10.9 11.9

14.3 10.9 14.4 15.2

15.1 12.8 14.0 13.0

± ± ± ±

1.2 2.0 1.6 2.1

13.4 12.5 12.5 13.1 ND

± ± ± ±

3.1 2.0 2.0 2.4

± ± ± ±

1.5 1.6 2.0 1.6

14.9 ± 2.8 15.8 ± 4.2 17.1 ± 8.7 16.5 ± 3.2 9.6 ± 1.2

DI/II

Proestrus-estrus

± ± ± ±

1.6 1.0 1.6 2.1

ND ND ND ND 2.42 ± 0.7**

± ± ± ±

2.4 2.6 3.8 4.2

ND ND ND ND 7.8 ± 2.0

Table 3 Effect of picrotoxin on total crosses in the Elevated Plus-Maze and on the number of squares crossed in the open-field motor activity test in ovariectomized rats.

Progesterone withdrawal

Treatment (mg/kg)

# of crosses

# of squares

Vehicle M. tomentosa Picrotoxin 0.25 Picrotoxin 0.50 Picro 0.25 + M. tomentosa 1.5 Picro 0.5 + M. tomentosa 1.5

5.40 ± 1.6 6.5 ± 1.2 2.1 ± 1.12 2.8 ± 1.1** 1.1 ± 0.14 1.6 ± 0.18##,

24.75 23.37 21.71 20.50 20.14 17.50

&

± ± ± ± ± ±

2.63 2.32 5.15 1.76 3.68 1.66

Data are expressed as mean ± S.E.M of the number of squares crossed in a 5min test session and the number of arm crosses in the Elevated Plus-Maze. *p < 0.05; **p < 0.005 vs VEH; & p < 0.05 versus M. tomentosa; ##p < 0.005 vs picrotoxin.

elucidated (Backstrom et al., 2014). The fact that finasteride (that inhibits steroid synthesis) blocked the anxiolytic effect of MT in male rats suggests that this plant could induce the synthesis of neurosteroids to produce its antianxiety effects (Sollozo-Dupont et al., 2015). Therefore, it is possible that at low doses, MT promotes the synthesis of neurosteroids that activate the GABAA/BZD receptor inducing an anxiolyticlike effect. Under PW challenge, MT induced an inverted U-shape that is not observed in the other endocrine conditions. At low doses, MT produced anxiolytic actions that are absent at high doses. It could be proposed

Data are expressed as mean ± S.E.M of the number of arm crosses scored in the Elevated Plus-Maze. ND-not determined. Mann-Whitney U test **P < 0.002 vs. vehicle group.

MT's actions. High levels of progesterone and its metabolite allopregnanolone have been associated with high levels of depression and irritability on fertile women with Premenstrual Dysphoric Disorder (Andreen et al., 2009; Backstrom et al., 2003). The exact mechanism that could explain these paradoxical effects of steroids remains to be 8

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better response than quercetin or rutin in an individual administration (Aguirre-Hernandez et al., 2016; Hernandez-Leon et al., 2017). Therefore, it is possible that rutin and its derivative isoquercitrin participate in the mediation of the anxiolytic-like effects of MT. The present study also analyzed the presence of flavonoids in the aqueous extract of MT and detected rutin at a concentration of 0.02 mg/ mg of the extract. This compound has been reported to exert anti-anxiety effects via the activation of GABAA receptors in a different site from that of the BZD site (Hernandez-Leon et al., 2017). Therefore, it is possible that rutin could participate in the mediation of the anxiolyticlike effects of MT. In a previous study in male rats, flumazenil (a specific antagonist to the BZD site) partially blocked the anxiolytic-like action of the aqueous extract of MT (Sollozo-Dupont et al., 2015). Studies of structure-activity relationships showed that flavonoids glycosides have low affinity for the benzodiazepine site of the GABAA receptor (Marder and Paladini, 2002). As far as we know, there is no information about rutin and a specific binding site (s) on the GABAA receptor. Therefore, future experiments are needed to elucidate the exact mechanism of action of both MT and rutin on the GABAA receptor activation. The effect of MT on the anxiety test appears to be specific because no significant changes in the total number of crosses in the EPM were observed. Also, the locomotor activity test was run to discard unspecific effects of pharmacological treatments. Albeit an increase of locomotor activity was observed in ovariectomized rats at a dose of 1.50 mg/kg. This dose did not modify the total number of crosses suggesting that the possible stimulatory effect of MT does not influence the anxiety-like behavior expression in the EPM. As to diazepam, only the high dose (2.0 mg/kg) decreased the total number of arm entries in the EPM test but did not affect general motor activity; this could be related to an initial sedative effect of diazepam (Bitran et al., 1991). No other treatment modified the locomotor activity. In conclusion, the beneficial anxiolytic-like actions of MT were observed in low hormone conditions, particularly in the PW challenge, a condition that can be related to a premenstrual period. It is worth mentioning that MT had better antianxiety actions in comparison to diazepam in these circumstances. This finding opens the possibility to study this plant as an alternative treatment for the anxiety related to PW disorders. However, hormonal variations could induce the opposite effect.

Table 4 Serum levels of progesterone of rats in different endocrine conditions. Condition

Progesterone ng/ml

OVX + Vehicle (n = 5) OVX + PW (n = 5) Proestrus (n = 8) Diestrus (n = 5)

4.15 ± 0.77 5.99 ± 1.23 17.18 ± 2.41 7.57 ± 0.85

Data are expressed as mean ± S.E.M of E2 and progesterone in the serum of 5–8 animals per group. PW= Progesterone withdrawal group; OVX = ovariectomized.

that PW challenge may induce changes in the GABAA subunits composition that could modify the receptor's sensitivity to the actions of MT. In this study, we did not explore the composition of the GABAA receptor. However, other models using progesterone withdrawal showed increases in the expression of GABAA receptors with the α4β1δ subunits conformation (Gulinello et al., 2002; Lovick et al., 2005; Smith et al., 1998). It is worth mentioning that the expression of the α4 subunit shows low affinity for benzodiazepines (Lovick et al., 2005). Therefore, it is possible that in the present study, the PW challenge favors the expression of a receptor form (probably α4β1δ) that is irresponsive to diazepam but highly responsive to other GABAergic compounds, which may be the case for MT extract. Since 1970, the isolation and structural elucidation of bioactive compounds of MT has recognized constituents from terpene nature (Caballero and Walls, 1970; Quijano et al., 1984, 1985a, 1985b). Moreover, some flavonoids such as kaempferol or quercetin and its derivatives like isoquercitrin have also been reported (Béjar et al., 2000). Isoquercitrin is a derhamnosylation product from rutin, a flavonoid identified in the aqueous extract of MT in this study. The uses of MT have not been unequivocally associated to a single phytochemical; but generally, the terpenoid compounds have been reported to possess uterotonic and uterorelaxant activities (Béjar et al., 2000; Quijano et al., 1985a). Whereas, flavonoids like isoquercitrin and rutin are well known to be related with depressant central nervous activity, partly mediated by GABAA receptors (Aguirre-Hernandez et al., 2016), such as the anxiolytic-like effect reported in MT. Biological activity of flavonoids like quercetin and rutin has been extensively studied in comparison to isoquercitrin which has been studied in a much lesser extent. Nevertheless, the anxiolytic-like activity of isoquercitrin demonstrated

Fig. 6. Chromatographic profile of the aqueous extract of M. tomentosa by UHPLC chromatogram at l = 220. Presence of rutin in retention time (Rt) of 9.9 min with a spectrum UV max at 254.9 and 354.6 nm as described in the inserted square. 9

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Authors contributions

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IS-D executed the experiments and participated in the data analysis; DI-P executed the experiments, participated in the data analysis and discussion. MC-J was responsible for obtaining the plant material and participated during the conception, and discussion process; E-GT performed the chemical analysis and participated in the discussion; EE-C and CL-R designed the study, analyzed the results, discussed and wrote the manuscript. Funding This work was supported by “Consejo Nacional de Ciencia y Tecnología(CONACyT),” grant CB-2010/155255 (to C.L.-R.). Conflicts of interest “The authors declare no conflict of interest.” Acknowledgments: Authors wish to thank José Juan Cruz Martínez, María Isabel Beltrán Villalobos, Mariana Yetlanezy Hernández and Nancy Cervantes for animal care and technical assistance; to Raúl Cardoso and José Luis Cardoso for their support with the illustrations. Also, the authors are thankful to Dr. Rosa Estrada-Reyes for her support during the lyophilization process. References Aguirre-Hernandez, E., Gonzalez-Trujano, M.E., Terrazas, T., Santoyo, J.H., GuevaraFefer, P., 2016. Anxiolytic and sedative-like effects of flavonoids from Tilia Americana var. Mexicana: GABAergic and serotonergic participation. Salud Ment 39, 37–46. Andreen, L., Nyberg, S., Turkmen, S., van Wingen, G., Fernandez, G., Backstrom, T., 2009. Sex steroid induced negative mood may be explained by the paradoxical effect mediated by GABAA modulators. Psychoneuroendocrinology 34, 1121–1132. Backstrom, T., Andreen, L., Birzniece, V., Bjorn, I., Johansson, I.M., Nordenstam-Haghjo, M., Nyberg, S., Sundstrom-Poromaa, I., Wahlstrom, G., Wang, M., Zhu, D., 2003. The role of hormones and hormonal treatments in premenstrual syndrome. CNS Drugs 17, 325–342. Backstrom, T., Bixo, M., Johansson, M., Nyberg, S., Ossewaarde, L., Ragagnin, G., Savic, I., Stromberg, J., Timby, E., van Broekhoven, F., van Wingen, G., 2014. Allopregnanolone and mood disorders. Prog. Neurobiol. 113, 88–94. Backstrom, T., Haage, D., Lofgren, M., Johansson, I.M., Stromberg, J., Nyberg, S., Andreen, L., Ossewaarde, L., van Wingen, G.A., Turkmen, S., Bengtsson, S.K., 2011. Paradoxical effects of GABA-A modulators may explain sex steroid induced negative mood symptoms in some persons. Neuroscience 191, 46–54. Béjar, E., Reyes-Chilpa, R., Jiménez-Estrada, M., 2000. Bioactive compounds from selected plants used in the xvi century Mexican traditional medicine. In: Atta ur, R. (Ed.), Studies in Natural Products Chemistry. Elsevier, pp. 799–844. Bekker, M.H., van Mens-Verhulst, J., 2007. Anxiety disorders: sex differences in prevalence, degree, and background, but gender-neutral treatment. Gend. Med. 4 (Suppl. B), S178–S193. Bitran, D., Hilvers, R.J., Kellogg, C.K., 1991. Ovarian endocrine status modulates the anxiolytic potency of diazepam and the efficacy of gamma-aminobutyric acid-benzodiazepine receptor-mediated chloride ion transport. Behav. Neurosci. 105, 653–662. Brummelte, S., Lieblich, S.E., Galea, L.A., 2012. Gestational and postpartum corticosterone exposure to the dam affects behavioral and endocrine outcome of the offspring in a sexually-dimorphic manner. Neuropharmacology 62, 406–418. Butcher, R.L., Collins, W.E., Fugo, N.W., 1974. Plasma concentration of LH, FSH, prolactin, progesterone and estradiol-17beta throughout the 4-day estrous cycle of the rat. Endocrinology 94, 1704–1708. Caballero, Y., Walls, F., 1970. Productos naturales del zoapatle (Montanoa tomentosa Cerv.). Bol. Inst. Quim. Univ. Nac. Auton. Mex. 22, 79–102. Carro-Juarez, M., Rodriguez-Landa, J.F., Rodriguez-Pena Mde, L., Rovirosa-Hernandez Mde, J., Garcia-Orduna, F., 2012. The aqueous crude extract of Montanoa frutescens produces anxiolytic-like effects similarly to diazepam in Wistar rats: involvement of GABAA receptor. J. Ethnopharmacol. 143, 592–598. Enriquez, R.G., Miranda, E., Ortiz, B., Leon, I., Magos, G., Pena, A., Reynolds, W.F., Gnecco, D., 1996. The unambiguous detection of kaurenic derivatives in aqueous infusions of Montanoa tomentosa by GC-MS and 2D-NMR spectroscopy: an answer to contradictory reports. Planta Med. 62, 569–571. Estrada-Camarena, E., Contreras, C.M., Saavedra, M., Luna-Baltazar, I., Lopez-Rubalcava, C., 2002. Participation of the lateral septal nuclei (LSN) in the antidepressant-like

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