Journal of Ethnopharmacology 143 (2012) 604–610
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Potential behavioral and pro-oxidant effects of Petiveria alliacea L. extract in adult rats Thaı´s Montenegro de Andrade a, Ademar Soares de Melo a, Rui Guilherme Cardoso Dias b, Everton Luı´s Pompeu Varela b, Fa´bio Rodrigues de Oliveira a, Jose´ Luı´s Fernandes Vieira a, Marcieni Ataı´de de Andrade a, Ana Cristina Baetas a, Marta Chagas Monteiro a, Cristiane do Socorro Ferraz Maia a,n a b
´, Instituto de Ciˆencias da Sau ´, Brazil ´s-Graduac- a~ o em Ciˆencias Farmacˆeuticas, Universidade Federal do Para ´ de, Rua Augusto Corrˆea, N101, 66075–900 Bele´m, Para Programa de Po ´cia, Instituto de Ciˆencias da Saude, Universidade Federal do Para ´, 66075-900 Bele´m, Para ´, Brazil Faculdade de Farma
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 May 2012 Received in revised form 19 July 2012 Accepted 20 July 2012 Available online 7 August 2012
Ethnopharmacological relevance: Petiveria alliacea (Phytolaccaceae) is a perennial shrub indigenous to the Amazon Rainforest and tropical areas of Central and South America, the Caribbean, and sub-Saharan Africa. In folk medicine, Petiveria alliacea has a broad range of therapeutic properties; however, it is also associated with toxic effects. Aim of the study: The present study evaluated the putative effects of Petiveria alliacea on the central nervous system, including locomotor activity, anxiety, depression-like behavior, and memory, and oxidative stress. Materials and methods: Two-month-old male and female Wistar rats (n ¼ 7–10 rats/group) were administered with 900 mg/kg of hydroalcoholic extracts of Petiveria alliacea L. The behavioral assays included open-field, forced swimming, and elevated T-maze tests. The oxidative stress levels were measured in rat blood samples after behavioral assays and methemoglobin levels were measured in vitro. Results: Consistent with previous reports, Petiveria alliacea increased locomotor activity. It also exerted previously unreported anxiolytic and antidepressant effects in behavioral tests. In the oxidative stress assays, the Petiveria alliacea extract decreased Trolox equivalent antioxidant capacity levels and increased methemoglobin levels, which was related to the toxic effects. Conclusions: The Petiveria alliacea extract exerted motor stimulatory and anxiolytic effects in the OF test, antidepressant effects in the FS test, and elicited memory improvement in ETM. Furthermore, the Petiveria alliacea extract also exerted pro-oxidant effects in vitro and in vivo, inhibiting the antioxidant status and increasing MetHb levels in human plasma, respectively. & 2012 Elsevier Ireland Ltd. All rights reserved.
Keywords: Petiveria alliacea Anxiety Memory Depression Methemoglobin
1. Introduction Following the meeting in 1978, the World Health Organization (WHO) recognized the importance of medicinal plants and galenical preparations in curing diseases and the knowledge for the use of medicinal plants and recommended their use worldwide. Natural products from folk remedies have significantly contributed to the discovery of modern drugs that act on the central nervous system (CNS). Moreover, herbal medicines are often considered gentle and safe alternatives to synthetic drugs (Carlini, 2003). Today, a large number of these drugs are derived from plants, such as morphine from Papaver somniferum, ephedrine from Ephedra vulgaris, and atropine from Atropa belladonna (Prakash and Gupta, 2005).
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Corresponding author. Tel./fax: þ 55 91 3201 7201. E-mail address:
[email protected] (C.d.S.F. Maia).
0378-8741/$ - see front matter & 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2012.07.020
Petiveria alliacea L. (Phytolaccaceae) is a perennial shrub indigenous to the Amazon Rainforest and tropical areas of Central and South America, the Caribbean, and sub-Saharan Africa. This plant is popularly known as pˆenis-de-coelho, tipi, true-tipi, tamemister, mucuracaa´, eye’s herb, embayayendo, and ouoembo. In folk medicine, Petiveria alliacea has a broad range of therapeutic properties. Root decoction, powder, and leaf infusions are used as antileukemic, antispasmodic, antirheumatic (topical use), antitumor, anticancer, immunostimulant, anti-inflammatory, antinociceptive, and antimicrobial agents (Lima et al., 1991). Furthermore, the plant has been reported to have sudorific, antivenereal, diuretic, sedative, antihelminthic, emmenagogue, anesthetic, and depurative properties (Lima et al., 1991). Other studies have reported its use in religious ceremonies by the slaves, who called it ‘‘Remedy to tame the Master,’’ referring to its toxicity and sedative property (Gomes et al., 2008). The Para´ communities mixed the leaves of mucuracaa´ with alcohol and
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used the mixture for a body massage in order to treat seizures (Branch and Silva, 1983). Moreover, an infusion of the bark of Petiveria alliacea L. is used for headaches and stimulation, depending on the dose; however, this plant can also produce toxic effects on the CNS (Lima et al., 1991). According to Peckolt and Peckolt (1900), some popular accounts indicate that it caused madness when used continuously. Its poisoning is slow and causes hyperarousal, insomnia, and hallucination during the acute period. However, chronic use causes profound changes in the CNS and opposite symptoms such as imbecility, weakness, seizures, and death after 1 year, depending on the dose. Indeed, Gomes et al. (2008) and Blainski et al. (2010) reported a possible CNS depressant action after administration of a Petiveria alliacea L. solution and alteration on motor functions and anxiety. Besides behavioral effects, this plant has been reported to have other effects, such as oxidative stress modulation via inhibition of superoxide dismutase and malondialdehyde (MDA) (Chen et al., 2005). Many authors have reported a role for oxidative stress in anxiety-like behavior in rats, and studies have shown that induction of oxidative stress in the hypothalamus and amygdala occurs in parallel with anxiety in mice. Increased anxiety has been found to be positively correlated with increases in reactive oxygen species (ROS) in granulocytes (Salim, 2011). Curiously, induction of oxidative stress through non-pharmacological methods also leads to anxiety-like behavior in rats (Vollert et al., 2011). In addition, some studies suggest that various plants extracts or compounds exhibit antioxidant or pro-oxidant activity (Carlini, 2003); however, few studies have associated the behavioral effects with changes in oxidative stress. Therefore, drugs that interfere with oxidative stress could improve behavior or inhibit cell death. The purpose of the present study was to investigate the effects of Petiveria alliacea L. on the CNS in rats using different predictive behavioral tests that evaluate antidepressant and anxiolytic activities and effects on memory. To elucidate the ethnopharmacological indications of this extract, we also evaluated its antioxidant and pro-oxidant activities and its possible alteration through oxidative action on hemoglobin (Hb).
2. Material and methods 2.1. Collection, identification and preparation of crude extract of Petiveria alliacea Petiveria alliacea was collected from Sa~ o Raimundo, a village in Acara´ city (Para´ state, Amazon region) in March, 2010. The region is situated at latitude 01132.6840 and longitude 048123.9840 (geographic coordinates obtained using global positioning system [GPS] equipment). The botanical identification was performed by Dr. Ma´rio Jardim, a specialist from the Emilio Goeldi Museum (Para´-Brazil), and the sample was deposited as a voucher specimen under number MG94354. According to its popular use, approximately 9 g of the dried mucuracaa´ were added to 600 mL of water and boiled, filtered and should be taken orally 3 times a day (Ferraz et al., 1991). The plant material, including leaves, stems, and roots, was washed with tap water followed by 10% ethanol solution. The plant parts were dried at room temperature for 2 day, dried in an oven with forced air circulation at an average temperature of 40 1C for 6 day, and crushed in a knife mill to obtain the drug spray. After grinding, the dust was macerated for 5 day in 70% ethanol. The mash was dried in a rotator evaporator (Laborata 4000 efficient; Heidolph Instruments GmbH & Co. KG) at 45 1C, 1 atm pressure, and 120 rpm. To ensure the total removal of the solvent, the mash was taken to the oven and the water bath at
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40 1C. 96.65 g of the whole plant extract, corresponding to 1430 g of the dried plant, were obtained and submitted to animal treatment. 2.2. Animals Two-month-old, male and female Wistar rats (n¼ 7–10 rats/ group), weighing between 150 and 300 g, obtained from the Animal Facility, Biological Sciences Institute, Federal University of Para´ (UFPA), and Swiss female mice of the species Mus musculus (25–35 g; n ¼10 per group), obtained from the Animal Facility, Evandro Chagas Institute (IEC), were used as experimental models. The animals were kept under standard conditions of temperature, humidity, and a light/dark cycle of 12 h with water and food ad libitum. Fluorescent lights (12 lx) were used in the rooms where the behavioral experiments were performed. The research project was approved by the Research Ethics Committee of the Evandro Chagas Institute (CEPAN-IEC) under number 56/2009, and the study was conducted in accordance with the standards set by the Guide for the Care and Use of Laboratory Animals. The extract was prepared at the Extraction Central of Chemical and Pharmacognosy Laboratory and the experiments for oxidative stress evaluation were designed in the In vitro Activities Laboratory and performed in the Laboratory of Pharmacological Assays at the Federal University of Para´. As described by Blainski et al. (2010), each animal was orally administered (gavage) with 900 mg/kg of Petiveria alliacea L. hydroalcoholic extract dissolved in saline solution (0.9% NaCl). This oral dose was chosen because the strongest effects of the whole plant extract were observed with this concentration. The positive control treatments included diazepam (DZP: 7-chloro-1-methyl-5-phenyl1,3-dihydro-2H-1,4-benzodiazepin-2-one, Diazepamils; Hipolabor Laboratory, Brazil) 1 mg/kg, fluoxetine (FXT: N-methyl-3-phenyl3-[4-(trifluoromethyl)phenoxy]propan-1-amine hydrochloride, Fluxenes; Eurofarma Laboratory, Brazil), and caffeine (CAF: 1,3,7-trimethylxanthine; Sigma-Aldrich) at a concentration of 10 mg/kg each. Trolox equivalent antioxidant capacity (TEAC), nitric oxide (NO), and MDA levels were measured from blood samples of rats treated with saline (behavioral training stress [BTS] group) or Petiveria alliacea extract (BTSþ Petiveria alliacea) and subjected to a forced-swimming test for 5 min. The control group animals were not subjected to behavioral activities (Basal group). 2.3. Acute oral toxicity Acute oral toxicity was evaluated in Swiss albino female mice weighing 20–35 g (n ¼10 mice per group) using the guidelines for testing chemicals number 423 Organization for Economic Co-operation and Development-OECD. Each group was fasted for 12 h and 2000 mg/kg or 5000 mg/kg plant extract (doses more likely to cause death) diluted in 0.9% saline solution was administered by oral gavage through an orogastric tube. At 0 min, 10 min, 15 min, 30 min, 60 min, 2 h, 3 h, 24 h (1st day), 48 h (2nd days), and 72 h (3rd days), the animals were observed for 1 min after extract administration for evaluation of possible behavioral changes. According to the test described by Malone (1977), the behavioral parameters related to stimulant activity were: snout scratching, tremors, exophthalmia, attention, increased respiratory rate, paw licking, tail biting, arousal, spontaneous motor activity, lack of appetite, nasal discharge, piloerection, stereotyped movements, escape reaction, and convulsions. The parameters related to depressant activity were: alienation of the environment, analgesia and anesthesia, ataxia, catatonia, decreased respiratory rate, decreased motility, decreased corneal reflex, apathy, dyspnea, response to touch, ptosis, sedation, and dorsal tone. Other parameters observed were aggressiveness, writhing, pupil diameter, diarrhea, cyanotic, hyperemic, or pale
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ears, sweating, increased or decreased urination, grunting, tail tremors, tearing, sweating, coma, and death. After this period, the animals were treated with diet and water ad libitum and observed for an additional 11 day to verify the possible occurrence of death (OECD Organization of Economic Co-operation and Development, 2001). 2.4. Behavioral assays 2.4.1. Open-field (OF) test Animals were placed individually in the center of a wooden arena (100 100 40 cm) divided into 25 quadrants to evaluate the number of sections visited by the animal over a period of 5 min. During this period, the number of total and central squares crossed, and the frequencies of rearing and grooming were measured (adapted from Zeef et al. (2012). The placement of only 1, 2, or 3 paws in a square followed by a return to the previous square was not considered as a crossing. 2.4.2. Forced swimming (FS) test Rodents were placed individually into a cylindrical tank (50 cm in diameter and 70 cm high) containing water at 23 ( 71) 1C and left there for 5 min in a condition from which they could not escape. The water depth allowed the rats to swim or float without their hindlimbs touching the bottom of the tank. Immobility time was recorded during the last 3 min. The first 2 min were for habituation. The rats were judged as immobile whenever they stopped swimming and floated in an upright position for 2 s, making only small movements to keep their head above the water level. The animals were dried using a small towel as soon as the test was completed. Immobility time was used as an index for depression. Antidepressant activity was considered when greater immobility was observed (Porsolt et al., 1978).
In this study, TAS was evaluated by measuring the TEAC levels in blood samples of rats subjected to FS test for 5 min using the method developed by Rufino et al. (2007). This method is based on persulfate oxidation of 2,2-azinobis (3-ethylbenzothiazoline, 6-sulfonate) (ABTS2) by antioxidants present in the sample to a degree that is proportional to their concentration. The antioxidant capacities of the samples are expressed as TEAC using a calibration curve plotted with different amounts of Trolox and their absorbance measured at 740 nm (Re et al., 1999). 2.6. Determination of lipid peroxidation (TBARS) Lipid peroxidation was measured by quantifying MDA in blood samples of rats subjected to the FS test for 5 min using the thiobarbituric acid reactive substances (TBARS) assay. This method is considered a very useful, cheap, and easy assay for evaluating oxidative stress (Esterbauer, 1996). Briefly, lipoproteins were precipitated by adding 0.05 M trichloroacetic acid and 0.67% thiobarbituric acid (TBA) in 2 M sodium sulfate. The lipid peroxide-TBA reaction was facilitated by heating in a water bath for 90 min. The chromogen formed was extracted in n-butanol and measured at 535 nm. Lipid peroxidation is expressed as nanomoles of MDA/L. 2.7. Oxidative stress levels Oxidative stress is determined by the ratio between TBARS and TAS (Cohen et al., 2009). This ratio is affected by the antioxidant response of the organism against pro-oxidant production with higher values indicating higher oxidative stress. Researchers use this ratio because these variables are significantly correlated (Esterbauer, 1996). 2.8. Determination of serum nitrate concentration
2.4.3. Elevated T-maze (ETM) test The equipment originated from elevated plus-maze (EPM), as described previously. It consisted of a T-shaped wooden maze with 2 opposite open arms (50 10 cm) and 1 enclosed arm (50 10 40 cm), spreading out from a central platform of 10 10 cm, elevated to a height of 50 cm from the floor and internally painted with an impermeable dark epoxy resin to avoid urine impregnation. The apparatus was placed in a lit room. After each trial, the ETM was cleaned with an ethanol solution (10%, v/v). In accordance with Takahashi et al. (2005) and Maia et al. (2010), each animal was placed in the OF for 300 s to enhance ETM exploration, after which they were placed at the end of the enclosed arm facing the open space. To measure inhibitory avoidance acquisition, rats were allowed to explore the enclosed arm of the maze as many times as necessary to comply with the avoidance criterion, which determined that animals should remain there for 300 s. When a rat placed all 4 paws onto one of the open arms, the trial ended and the animal was returned to the arena for 30 s. After 24 h, the animals were subjected to 2 subsequent enclosed arm trials (called test [long-term memory] and retest [priming memory]), with a 30 s interval between trials. The number of trials required for inhibitory avoidance acquisition (the first trial [called the baseline] and the 2 consecutive trials [called avoidance 1 and 2]; and avoidance latency test and retest) was measured.
The nitrate (NO3 ) present in the serum samples was reduced to nitrite with nitrate reductase, and the nitrite concentration was determined using the Griess method (Granger et al., 1999). Briefly, 100 mL of the supernatant samples was incubated with an equal volume of Griess reagent for 10 min at room temperature. The absorbance was measured on a plate scanner (Spectra Max 250; Molecular Devices, Menlo Park, CA, USA) at 550 nm. The nitrite (NO2 ) concentration was determined using a standard curve generated using sodium nitrate (NaNO2). Nitrite production is expressed per mM.
2.5. Measurement of total antioxidant status (TAS)
Data were analyzed using one-way analysis of variance (ANOVA) followed by Newman–Keuls or Tukey’s tests for multiple comparisons of behavior or oxidative stress test results, respectively. Student’s t-test was used to compare 2 groups. A value of po0.05 was considered statistically significant. All experiments were performed at least 2 times.
The total antioxidant status (TAS) is a sensitive and reliable marker for detecting changes in in vivo oxidative stress that may not be detectable through the measurement of a single, specific antioxidant (Cohen et al., 2009).
2.9. Determination of methemoglobin levels Methemoglobin (MetHb) was measured in blood samples obtained from normal volunteers, according to the method of Hegesh et al. (1970). The MetHb content was evaluated from the change in absorbance at 632 nm caused by the addition of potassium cyanide (KCN) to the buffered hemolysate. A dilution of the hemolysate in which potassium ferricyanide (K3Fe[CN]6) was used to convert all possible forms of Hb to MetHb served as the reference solution. MetHb levels were measured in duplicate and values less than 2% were considered normal. 2.10. Statistical analysis
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3. Results In the acute toxicity test, animals treated with Petiveria alliacea at 2000 mg/kg (oral route) exhibited lethargy and drowsiness 30 and 60 min after administration of the extract, with no other changes up to 14 day. With the 5000 mg/kg dose, drowsiness and lethargy were more prominent at 30, 60, and 90 min. At 24 h with the doses mentioned above, the animals exhibited nose scratching, paw licking, vertical movements, and normal food intake. There were no changes thereafter up to 14 day. None of the treated animals died. Thus, the orally administered Petiveria alliacea extract exhibited low toxicity, which was consistent with the general pharmacological effects observed in mice (OECD Organization of Economic Co-operation and Development, 2001). In the OF test, Petiveria alliacea-treated rats exhibited increased total locomotor activity compared to the control group (F(2,27) ¼3.022; p o0.05) as well as diazepam (DZP) group (F(2,27) ¼3.659; p o0.05). The locomotion of the Petiveria alliacea group was predominantly in the central area of the arena (F(2,29) ¼6.045; p o0.001), similar to that of the DZP group (F(2,29) ¼5.778; p o0.05). The grooming and rearing behaviors were not different from that of the control group (Fig. 1). In the FS test, the Petiveria alliacea L. (F(2,29) ¼9.883; p o0.001) and fluoxetine (FXT) groups (F(2,29) ¼15.18; p o0.001) exhibited reduced immobility time than the control group; however, the reduction was different from that of the FXT group (F(2,29) ¼5.292; p o0.001) (Fig. 2). In the ETM test, the Petiveria alliacea L. group did not exhibit a difference in the number of re-exposures necessary to achieve inhibitory avoidance. Only the caffeine (CAF) group showed a reduction in this parameter compared to the control (F(2,24) ¼3.273; po0.05) and Petiveria alliacea (F(2,29) ¼ 4.973; po0.01) groups. Following the first 3 exposures, all groups exhibited similar behavior with no differences from baseline. Furthermore, all groups exhibited
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an increase in the time spent in the enclosed arm during the second exposure (avoidance 1), and this increase was maintained in the third exposure (avoidance 2) without any difference between the groups (Fig. 3). Fig. 4 represents the test and retest conducted 24 h after the exposures. In the test (T1) related to long-term memory, the Petiveria alliacea (F(2,28) ¼10.12; p o0.001) and CAF groups (F(2,28) ¼13.60; p o0.001) exhibited an increase in enclosed-arm time compared to the control group. Furthermore, the Petiveria alliacea group exhibited a lower latency in the enclosed arms than the CAF group (F(2,28) ¼ 3.741; p o0.05). Thirty seconds after T1, the animals were subjected to retest (R1), which is related to short-term memory. No differences were observed between the analyzed groups (Fig. 4). Fig. 5 shows that the BTS group exhibited a significant increase in TEAC, NO, and MDA levels, and treatment of the BTS group with 900 mg/kg of Petiveria alliacea (BTSþ Petiveria alliacea group) elicited a decrease in TEAC levels relative to that of the BTS group without treatment; this effect was not detected on the NO and MDA levels. Treatment of normal rats with 900 mg/kg of Petiveria alliacea (Basalþ Petiveria alliacea group) did not induce any changes in TEAC, NO, and MDA levels relative to those of the basal group. In the MetHb in vitro assay (Fig. 6), incubation of erythrocytes with Petiveria alliacea elicited an increase in MetHb levels in a dosedependent manner compared to primaquine (positive control).
4. Discussion and conclusion In this study, the effects of the whole Petiveria alliacea plant on behavioral activities, such as anxiety, locomotor activity, memory, and depression, were examined using OF, FS, and ETM tests to evaluate possible CNS activity.
Fig. 1. Locomotor activity (panel A), central locomotion (panel B), number of grooming (panel C) and rearing (panel D) of rats tested in the wooden arena for 5 min. Each value represents the mean (S.E.M.) of 5–10 animals (males and females). Groups: Control, Petiveria alliacea and diazepam (DZP). npo 0.05 compared to the control group treated with saline. #po 0.05 compared to the DZP group (ANOVA, Newman–Keuls’ test).
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Fig. 2. Immobility time of rats in the forced swimming test for 5 min. Each value represents the mean (S.E.M.) of 10 animals (males and females). Groups: Control, Petiveria alliacea, and fluoxetine (FXT). np o 0.05 compared to the control group treated with saline. np o 0.05 compared to the FXT group (ANOVA, Newman– Keuls test).
Fig. 3. Number of re-exposures (panel A) and time of enclosed arms in the first 3 expositions (panel B) of rats tested in the Elevated T Maze (ETM). Each value represents the mean (S.E.M.) of 7–10 animals (males and females). Groups: Control, Petiveria alliacea and caffeine (CAF). np o 0.05 compared to the control group treated with saline. #p o0.05 compared to the CAF group. þ po 0.05 compared to avoidance 1. ypo 0.05 compared to avoidance 2 (ANOVA, Newman–Keuls’ test).
Fig. 4. Enclosed arm time after 24 h after training session of rats tested in the Elevated T Maze (ETM). Each value represents the mean (S.E.M.) of 10 animals (5 males and 5 females). Groups: Control, Petiveria alliacea and caffeine (CAF). n po 0.05 compared to the control group treated with saline. #p o 0.05 compared to the CAF group (ANOVA, Newman–Keuls’ test).
In the initial evaluation of toxicological effects, animals treated with 2000 and 5000 mg/kg of Petiveria alliacea extract exhibited only drowsiness and reduced locomotor activity. No death
Fig. 5. Determination of TEAC (panel A); NO (panel B) and levels of MDA (panel C) in blood samples of rats submitted to forced swim test for 5 min. Each value represents the mean (S.E.M.) of 5 animals (3 males and 2 females). Groups: Control (basal), Basal treated with Petiveria alliacea; BTS (Behavioral Training Stress) treated with saline and BTS treated with Petiveria alliacea. np o 0.05 compared to the control group, without stress; #p o0.05 compared to the BTS group (ANOVA, Tukey’s test).
occurred after 14 day. These results are similar to those observed by Lima et al. (1991) in acute toxicity tests. Other study also suggested low acute toxicity and the lethal dose 50 may be higher than 3 or 4 g/kg (Gomes, 2006).
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Fig. 6. Percentage of methemoglobin in the blood sample obtained from normal volunteers. The dosage of MetHb was measured in duplicate. Groups: positive control (primaquine) and Petiveria alliacea. npo 0.05 compared to positive control (Student’s t-test).
The OF test is used as a measure of emotionality in rodents. Crossings and rearing, and grooming behavior are the most common parameters measured (Montgomery, 1955). The OF test results showed an increase in the total number of crossings and in the number of central quadrants crossed, suggesting anxiolytic activity. Similar result was reported by Blainski et al. (2010) who observed the same activity in OF and EPM tests conducted with extracts from different parts of the plant. The presence of metabolites such as polyphenols may have contributed to these results (Ariza et al., 2007). In this regard, Gomes et al. (2008) demonstrated that acute treatment with fractions from the root induced significant decrease in locomotor activity, and rearing and grooming frequency, suggesting a possible central depressant action. Our data are inconsistent with these results. In the FS test, the crude extract of Petiveria alliacea exhibited antidepressant activity, indicated by the decrease in immobility of rats. Our results are different from those of Gomes et al. (2008), who reported that the isolated fractions from roots increased the immobility time. However, the results of the present study are consistent with the pharmacological effects of the extract constituents, including coumarins, which modulate serotonergic/ noradrenergic transmissions that are related to mood and depressant effects (Ariza et al., 2007). In the ETM test, analysis of the re-exposures, first 3 exposures and retest stage, revealed no significant differences, suggesting that the extract did not interfere with learning, short-time, and priming memory, respectively. The analysis of enclosed-arm time after 24 h revealed improvement in long-term memory, demonstrated by higher enclosed-arm latency values (Takahashi et al., 2005). Previous work on Petiveria alliacea revealed the presence of triterpenoids, saponins, polyphenols, coumarins, benzaldehyde, benzoic acid, essential oils (Petiverina), flavonoids, fredelinol, pinitol and allantoin in the root, stems and leaves, respectively (Rocha and Da Silva, 1969). Recent studies have evaluated the activity of terpenoids on the CNS, and the activities evaluated in vivo are related to the popular use as sedative and anxiolytic. The molecular targets studied are mainly gamma-aminobutyric acid (GABA) neurotransmitter systems (Passos et al., 2009).
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Coumarins and flavonoids are a sub-group of phenolic substances in the benzopyran group. Ariza et al. (2007) examined the effects of coumarin on the CNS, and demonstrated possible sedative; anxiolytic (GABAergic and serotonergic/noradrenergic mechanisms); anticonvulsant (voltage-dependent sodium channels mechanism); and antidepressant effects (inhibition of monoamine oxidase) (Chen et al., 2005).With regard to flavonoids, Youdim et al. (2004) demonstrated the ability of these compounds to cross the blood–brain barrier and reach the CNS unchanged, which play an important role in the regulation of anxiety and epilepsy (mediated by GABA). Studies involving dibenzyl trisulfide (DTS) isolated from Petiveria alliacea have demonstrated hyper-phosphorylation of growth factor induced MAPKinases (erk 1 and erk 2) phosphorylation, which is critical for the improvement of long-term memory, and neuronal growth (Williams et al., 2007). Thus, the mnemonic effect induced by the Petiveria alliacea extract may be related to DTS acting via the MAPkinase pathway. In this study, in addition to behavioral evaluations, we also showed that the Petiveria alliacea extract exerted pro-oxidant effects in vitro and in vivo, as inhibited the antioxidant status and maintained NO levels in an animal model of behavioral stress, but would increase the concentration of other oxidant radicals, such as superoxide (O2 ) and hydrogen peroxide (H2O2), that in red blood cells would lead to Hb oxidation, and increased MetHb levels in the human plasma. MetHb is a potential biomarker for nitrite exposure and its formation directly correlates with plasma nitrite levels. A major cause of nitrite toxicity is the oxidation of blood Hb iron to its ferric state to form MetHb, a derivative incapable of binding oxygen, resulting in hypoxia and death (Williams and Eddy, 1987). In this regard, the level of lipid peroxidation, indicated by MDA levels, increased in our study; suggests lipid peroxidation and cell damage caused by the action of reactive species in the body, as well as oxidative processes in the membrane (Del-Rio et al., 2005). Non-enzymatic plasma proteins and enzymatic factors constitute a few of endogenous antioxidants that prevent MetHb formation. In the behavioral model of the present study, the extract inhibited TEAC levels; this effect may be related to a decrease in thiol and non-thiol antioxidants in rat plasma (Rahman et al., 2000). Recently, Salim et al. (2010a) showed that treatment with oxidative stress inducers cause anxiety-like behavior. The amygdala, locus coeruleus, and hippocampus are equally responsive to changes in oxidative stress (Salim et al., 2010a) with reflex in cognitive processes. Indeed, Petiveria alliacea L. contain biologically active substance(s) that may act on the CNS and have significant depressant and anticonvulsant potential (Gomes et al., 2008). On the other hand, studies have also demonstrated that the actions of natural polyphenols on biomembranes are dual and obscure, as they exhibit not only antioxidative effects but also pro-oxidative toxicity toward cells, which contributes to hemolysis along with glutathione and Hb oxidation (Galati et al., 2002). These substances are also found as secondary metabolites in Petiveria alliacea and they may be involved in neuronal mechanisms that potentially regulate anxiety, as well as oxidative stress. These data show that Petiveria alliacea extract exerted motor stimulatory and anxiolytic effects in the OF test, antidepressant effects in the FS test, and elicited memory improvement in ETM. Furthermore, the Petiveria alliacea extract also exerted pro-oxidant effects in vitro and in vivo, inhibiting the antioxidant status and increasing MetHb levels in human plasma, respectively.
Acknowledgements Thaı´s Montenegro de Andrade was supported by a Brazilian Government/Coordenac- a~ o de Aperfeic- oamento de Pessoal de
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