Anti-anxiety and hypnotic effects of ethanolic and aqueous extracts of Lippia citriodora leaves and verbascoside in mice

Abstract

Objectives: The extract of Lippia citriodora and its main component, verbascoside, are known for their hypnotic effects in traditional medicine. In this study, the anxiolytic and hypnotic effects of L. citriodora leave extracts and verbascoside were evaluated in mice.

Materials and methods: Animals were divided into 11 groups of six mice each. Group I received normal saline, Group II received Diazepam (2 mg/kg) as positive control, Groups III, IV and V received L. citriodora ethanolic extracts (50, 100 and 200 mg/kg, respectively), Groups VI, VII and VIII received L. citriodora aqueous extracts (50, 100 and 200 mg/kg, respectively) and Groups IX, X and XI received Verbascoside (25, 50 and 100 mg/kg, respectively). All agents were administrated intraperitoneally. To evaluate hypnotic activity, pentobarbital sleeping test, and for anxiolytic activity, elevated plus-maze (EPM), locomotor activity, open field and motor coordination (rotarod test) tests were used. To understand the role of GABA_A receptor, flumazenil was also administered.

Results: The extracts and verbascoside increased the time spent and number of entries in the open arms of EPM. Moreover, these agents significantly increased the sleeping time induced by pentobarbital. In addition, the highest dose of extracts and verbascoside reduced time spent on the rod and total locomotion in the open field tests, respectively. Flumazenil inhibited the effects of extracts and verbascoside in EPM and hypnotic tests.

Conclusion: These results suggested that ethanolic and aqueous extracts of L. citriodora and verbascoside exhibit anxiolytic, hypnotic and muscle relaxant effects especially at the highest doses and these effects are partially due to the interaction with GABA_A receptor.

Keywords: Anti-anxiety; Elevated plus maze; Hypnotic; Lippia citriodora; Verbascoside.

Figure 1

Effects of ethanolic and aqueous extracts of L. citriodora and verbascoside on the percentage of the open arm entries of the elevated plus-maze (EPM) (A) and on the time spent in open arm of the elevated plus-maze (EPM) (B). Effect of flumazenil on the percentage of entries into open arms (C) and on the percentage of time spent in open arms (D) by mice treated with ethanolic and aqueous extracts of L. citriodora and verbascoside in EPM test. Data are presented as mean±SEM of a group of six mice. *, ** and *** indicate p<0.05, p<0.01 and p<0.001, respectively, compared to normal saline. ^#, ^## and ^### indicate p<0.05, p<0.01 and p<0.001, respectively, compared to diazepam. ⁺, ⁺⁺ and ⁺⁺⁺ indicate p<0.05, p<0.01 and p<0.001 respectively compared to group in the absence of flumazenil. Statistical analysis were performed using Tukey-Kramer test. Ns: normal saline; dz: diazepam; Ex aq: aqueous extract; Ex et: ethanolic extract; verb: verbascoside; flo: flumazenil

Figure 2

Effects of ethanolic and aqueous extracts of L. citriodora and verbascoside on the peripheral (A), central (B) and total (C) locomotion in open field test. Data are presented as mean ± SEM of a group of six mice. *, ** and *** indicate p<0.05, p<0.01 and p<0.001, respectively, compared to normal saline. ^#, ^## and ### indicate p<0.05, p<0.01 and p<0.001, respectively, compared to diazepam. Statistical analysis were performed using Tukey-Kramer test. Ns: normal saline; dz: diazepam; Ex aq: aqueous extract; Ex et: ethanolic extract; verb: verbascoside

Figure 3

Effects of ethanolic and aqueous extracts of L. citriodora and verbascoside on motor coordination in Rotarod test, 30 (A) and 60 min (B) after the IP administration of the extracts or diazapam. Data are presented as mean ± SEM of a group of six mice.*, ** and *** indicate p<0.05, p<0.01 and p<0.001, respectively, compared to normal saline. ^#, ^## and ^### indicate p<0.05, p<0.01 and p<0.001, compared to diazepam. Tukey-Kramer test. Ns: normal saline; dz: diazepam; Ex aq: aqueous extract; Ex et: ethanolic extract; verb: verbascoside

Figure 4

Effects of ethanolic and aqueous extracts of L. citriodora and verbascoside on sleep latency (A) and sleeping time (B) on the potentiation of pentobarbital sleep test. Effect of flumazenil on sleep latency (C) and sleeping time (D) induced by ethanolic and aqueous extracts of L. citriodora and verbascoside in the potentiation of pentobarbital sleep test. Data are presented as mean ± SEM of a group of six mice.*, ** and *** indicate p<0.05, p<0.01 and p<0.001, respectively, compared to normal saline. ^#, ^## and ^### indicate p<0.05, p<0.01 and p<0.001, respectively, compared to diazepam. ⁺, ⁺⁺ and ⁺⁺⁺ indicate p<0.05, p<0.01 and p<0.001, respectively, compared to group in the absence of flumazenil. Statistical analysis were performed using Tukey-Kramer test. Ns: normal saline; dz: diazepam; Ex aq: aqueous extract; Ex et: ethanolic extract; verb: verbascoside; flo: flumazenil

Figure 5

The HPLC fingerprints of the ethanolic (A) and aqueous (B) extracts of L. citriodora