Metabolomics provides novel understanding of Melissa officinalis mechanism of action ensuring its calming effect on dogs

Abstract

Background: Animal welfare encompasses both its physical and mental states. This latter could be altered by several psychological-related disorders including stress and anxiety. To address these issues, Melissa officinalis, a Lamiaceae plant, ensuring the anxiolytic-type effects, is widely used. In this study, the main aim was to explore the effect of a commercial hydro-alcoholic Melissa officinalis extract (MOE) and its major compound rosmarinic acid (RA) on dogs' behavior and metabolome. To do so, twenty healthy beagle dogs were randomly assigned to 4 dietary supplements (5 dogs/group): the first group received a placebo supplemented with maltodextrose (200 mg/kg), the second group was supplemented with MOE (200 mg/kg), the third group received RA at a dose of 10.6 mg/kg, and the fourth group was administered α-casozepine (AC) at a dose of 225 mg in capsule form. Dogs' behavior was monitored after 4 weeks of treatment using a standardized evaluation grid developed by Oniris. In addition, 4-hydroxybyturic acid (GHB) was quantified to study the effect of the supplementations on the metabolites of γ-aminobutyric acid (GABA) biosynthetic pathway. Moreover, the impact of all supplementations on dogs' metabolome was assessed using untargeted metabolomics at the end of the supplementation period.

Results: Results demonstrated significant differences between the mean behavioral score of placebo group (-3.4) compared to MOE (2.0), RA (1.4), and AC (0.8) groups. In addition, GHB measurement revealed a decrease in its quantity in all supplemented groups compared to the control. Moreover, untargeted metabolomics uncovered several metabolic pathways, that were impacted by MOE supplementation linked to lipids and bile acids metabolism. Furthermore, RA supplementation impacted fatty acids and lipids metabolism pathways while supplementation with AC affected pathways linked to lysine and sphingolipids metabolism.

Conclusions: Our study demonstrated a calming effect of MOE on beagles and proposes a novel hypothesis that sheds new light on its potential mechanism of action. This study underlines metabolomics as an effective tool for gaining deep insights into the metabolic changes associated with supplementation.

Keywords: Melissa officinalis; Behavior; Dog’s welfare; Metabolomics.

Fig. 1

Changes in (A) overall behavior of healthy beagle dogs, in (B) the kennel and (C) during walking before and after 4 weeks of supplementation with MOE, RA or AC. The scores of each dog in each group were added and then averaged, the means of the scores of week 4 were subtracted from those of week 0. The graph represents the difference in total behavior score between week 0 and week 4 (N = 20, n = 5; *: p < 0.05; ns: not significant)

Fig. 2

Chromatogram of mixed analytical standards of hydroxycinnamic acids (A) and chromatogram of MOE extract recorded in same conditions (B)

Fig. 3

Plasma metabolites levels (Creatinine, Urea, ALT, ALP, and AST) on dogs before and after 4 weeks of supplementation with placebo, Melissa officinalis extract (MOE), rosmarinic acid (RA) and α-casozepine (AC). The dosage of the different markers was measured in the plasma of the dogs in each group at 0 (before supplementation) and 4 weeks after supplementation (N = 20, n = 5 per group, ns: not significant)

Fig. 4

Evolution of the GHB level in plasma of dogs supplemented with placebo, MOE, RA and AC after 4 weeks of supplementation. The plasma GHB level for each group was performed before and after 4 weeks of supplementation and a significant decrease of plasma GBH level was observed for MOE and AC groups (N = 20, n = 5; **: p < 0.01; *: p < 0.05; ns: not significant)

Fig. 5

Metabolomic profiles of beagle dogs’ plasma generated by OPLS-DA of HPO extracts in ESI⁻ mode. Placebo and MOE comparison; placebo and RA comparison