Anxiolytic activity and active principles of Piper amalago (Piperaceae), a medicinal plant used by the Q’eqchi’ Maya to treat susto, a culture-bound illness

doi:10.1016/j.jep.2016.03.013

Journal of Ethnopharmacology

Volume 185, 5 June 2016, Pages 147-154

https://doi.org/10.1016/j.jep.2016.03.013 Get rights and content

Abstract

Ethnopharmacological relevance

The medicinal plant, Piper amalago L. (Piperaceae), is used traditionally by Q’eqchi’ Maya healers for the treatment of “susto” a culture-bound syndrome. Previous research suggests that susto symptoms may be a manifestation of anxiety. The objectives were to characterize the effect of ethanolic extract of P. amalago in behavioral assays of anxiety at doses representative of traditional use and to isolate active principles.

Materials and methods

Rats treated orally with low dose ethanolic extracts of P. amalago leaves (8–75 mg/kg) were tested in several behavioral paradigms including the elevated plus maze (EPM), social interaction (SI), and conditioned emotional response (CER) tests, and compared to diazepam, a positive control. The active anxiolytic principle was isolated by bioassay guided isolation using an in vitro GABA_A competitive binding assay.

Results

Extracts had significant anxiolytic activity in all behavioral tests, with the strongest activity in the SI and the CER paradigms. In an in vitro GABA_A competitive binding assay, a 66.5 µg/mL concentration of P. amalago ethanol extract displaced 50% of the GABA_A–BZD receptor ligand [³H]-Flunitrazepam. Bioassay-guided fractionation identified a furofuran lignan, a molecule with structural similarity to yangambin, with high affinity for the GABA_A-BZD receptor as the principle bioactive.

Conclusion

The results suggest that the ethnobotanical use of this plant may have a pharmacological basis in its anxiolytic activity, as demonstrated in animal behaviour tests

Graphical abstract

Piper amalago, a plant traditionally used by Q’eqchi’ Maya healers for the culture bound syndrome, susto, has significant anxiolytic effects mediated by a furofuran active principle.

Introduction

In an ethnobotanical survey of the Q’eqchi’ Maya of Belize we found high use of plants for neurological disorders including headaches, susto (a culture-bound illness associated with fright), and epilepsy (Bourbonnais-Spear et al., 2005). Screening of plants used to treat susto in animal behavioral assays of anxiety demonstrated a possible link between susto and anxiety. Specifically, plants used to treat susto significantly reduced anxiety-like behavior in rodents (Bourbonnais-Spear et al., 2007).

The neurobiology of anxiety and epilepsy involve the neurotransmitter γ-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the mammalian central nervous system (CNS). Reduction in CNS inhibition is associated with excessive activity, that is experienced as anxiety or in severe cases, epileptic seizures (Treiman, 2001). A therapeutic approach to counter this exacerbated neuronal activity is to increase GABA levels in the brain. This can be achieved either by inhibiting the activity of the catabolic enzyme in the GABA system, GABA-transaminase (GABA-T), or by increasing the affinity of GABA for its receptor, GABA_A, facilitated by the binding of GABA_A receptor agonists, such as benzodiazepines (BZD), to the GABA_A-BZD receptor (Kinrys et al., 2003, Mula et al., 2007, Zwanzger and Rupprecht, 2005, Yogeeswari et al., 2005)

We previously reported on screening plants identified by the Q’eqchi’ and Yanesha healers (Awad et al., 2009, Picard et al., 2014) for their effect on conditions associated with the GABAergic system. The model developed showed a positive correlation between healer consensus for plants used to treat susto, and extract interaction with the GABA_A receptor. The results suggest that the selection of a plant to treat susto may be linked to the plant's pharmacological interaction with the BZD site of the GABA_A receptor (Awad et al., 2009). One of the most active plants in the GABA_A-BZD competitive binding assay was Piper amalago L. (Piperaceae), known as Tziritok in Q’eqchi’, which translates to “small and fragile”.

P. amalago is identified as a plant to treat susto and epilepsy by the Q’eqchi and other healers in Belize (Arvigo and Balick, 1998, Balick and Arvigo, 2015, Bourbonnais-Spear et al., 2005). Leaves are crushed in water which are drunk as a sedative. The liquid is also used to bathe the patient to treat the culture bound syndrome “mal viento” or fatigue and sleeplessness.

In addition to the Q’eqchi’ Maya uses described above, P. amalago is used in traditional medicine from Mexico to Brazil, over its entire range. The Yucatec Maya consume a decoction for treatment of snake bite (Arnason et al. 1980). In Jamaica, stems and leaves are boiled and taken as a “blood tonic” (Morten, 1981). In Brazil it is traditionally used as a vermifuge (Carrara et al., 2014) and as diuretic for the treatment of urinary conditions (da Silva Novaes et al. 2014). Nicararguan midwives use a decoction of leaves and roots to reduce nervousness (Coe, 2008).

Based on the findings of Bourbonnais-Spear et al. (2005), a recent report (Lopes et al., 2012) has investigated the activity of P. amalago in the elevated plus maze (EPM). Very high doses (250 and 420 mg/kg i.p) had an anxiogenic (anxiety-increasing) effect; extract-dosed animals spent significantly more time in the closed arms of the maze when compared to diazepam controls. However, the LD₅₀ of P. amalago was estimated to be 2.5 g/kg suggesting a very low toxicity and high safety margin for the use of this plant. Our ethnobotanical data indicated a maximum dose of 100 g leaf/patient (estimated equivalent <100 mg/kg of alcohol extract or less), and suggested that study of the anxiolytic activity at lower dose may be justified. Phytochemical investigation (Achenbach et al., 1986; Carrera et al., 2011) has identified piperamides in P. amalago, but no investigation has been made of their effect on the GABA system.

As a result, the work described here had two objectives. The first was to characterize the effect of ethanolic extract of P. amalago in EPM, Conditioned emotional response (CER) and Social interaction (SI) behavioral assays of anxiety at doses more representative of traditional use. Since the extract has activity in an in vitro GABA_A-BZD receptor binding assay, the second objective was to isolate and identify the secondary metabolite(s) responsible for the activity using bioassay-guided fractionation.

Section snippets

Plant material

Fresh samples of wild P. amalago L. (Piperaceae) were collected under permit from the Itzamma ethnobotanical garden in the Toledo district, Belize. Samples were stored in 90% ethanol for shipping. Voucher specimens were identified (by L.P. and P.S.) and deposited in the JVR Herbarium, Universidad Nacional de Costa Rica, and the University of Ottawa Herbarium (UTT no. 19915). The plants were collected under a permit issued by the Ministry of Natural Resources of the Government of Belize (Ref #

Elevated plus maze (EPM)

Different doses (8–75 mg/kg) of the P. amalago crude extract were compared in the EPM and there was a dose dependent effect on time spent in open arms but not on the number of unprotected head (UH) dips (Fig. 1A and B). The medium (25 mg/kg) and high (75 mg/kg) doses showed significant increases in the percentage of time spent in the open arms (F (4, 41) 3.996=p<0.05 and p<0.01). The number of UH (Fig. 1b) showed significance in both low( 8 mg/kg) and high doses (75 mg/kg) (F (4, 41) 2.238=p<0.05).

Discussion

This study examined the effect of a P. amalgo extract in behavioural models of anxiety. P. amalago lowered anxiety-like behaviour in rats significantly in all three behavioral tests, although not at all doses. For example, the fear extinction process seen in the CER test was effective only at the highest dose. This result corroborates previous findings (Bourbonnais-Spear et al., 2007), that plants used to treat susto significantly reduced anxiety-like and/or fear behavior in rodents. Also the

Acknowledgments

This work was supported by the Natural Sciences and Engineering Research Council of Canada (Discovery Grant, G30360) to JTA.

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Anxiolytic activity and active principles of Piper amalago (Piperaceae), a medicinal plant used by the Q’eqchi’ Maya to treat susto, a culture-bound illness

Abstract

Ethnopharmacological relevance

Materials and methods

Results

Conclusion

Graphical abstract

Introduction

Section snippets

Plant material

Elevated plus maze (EPM)

Discussion

Acknowledgments

J. Ethnopharmacol.

J. Ethnopharmacol.

J. Ethnopharmacol.

J. Ethnopharmacol.

Pharmacol. Biochem. Behav.

Exp. Toxicol. Pathol.

Pharmacol. Biochem. Behav

J. Ethnopharmacol.

Pharmacol. Biochem. Behav.

Constituents of Tropical Medicinal Plants, IXX1 GC/MS-Investigations of the constituents of Piper amalago−30 new amides of the piperine-type

Planta Med.

Rainforest Remedies: One Hundred Healing Herbs of Belize

Messages From the Gods

Characterization of benzodiazepine binding to GABAA Receptors

Plant use by the Q’eqchi’ Maya of Belize in ethnopsychiatry and neurological pathology

Econ. Bot.

Characterization of benzodiazepine binding to GABA_A Receptors