Use of an Animal Model to Evaluate Anxiolytic Effects of Dietary Supplementation with Tilia tomentosa Moench Bud Extracts

Abstract

Anxiety disorders are common and complex psychiatric syndromes affecting a broad spectrum of patients. On top of that, we know that aging produces an increase in anxiety vulnerability and sedative consumption. Moreover, stress disorders frequently show a clear gender susceptibility. Currently, the approved pharmacological strategies have severe side effects such as hallucinations, addiction, suicide, insomnia, and loss of motor coordination. Dietary integration with supplements represents an intriguing strategy for improving the efficacy and the safety of synthetic anxiolytics. Accordingly, a recent article demonstrated that glyceric bud extracts from Tilia tomentosa Moench (TTBEs) exert effects that are consistent with anxiolytic activity. However, the effects of these compounds in vivo are unknown. To examine this question, we conducted behavioral analysis in mice. A total of 21 days of oral supplements (vehicle and TTBEs) were assessed by Light Dark and Hole Board tests in male and female mice (young, 3 months; old, 24 months). Interestingly, the principal component analysis revealed gender and age-specific behavioral modulations. Moreover, the diet integration with the botanicals did not modify the body weight gain and the daily intake of water. Our results support the use of TTBEs as dietary supplements for anxiolytic purposes and unveil age and gender-dependent responses.

Keywords: anxiety; bud-derivatives; mice behaviour; mood disorders; nutraceuticals; polyphenols; principal component analysis; supplements.

Figure 1

UV-Vis spectra (a), bioactive compound quantification (b), and HPLC fingerprint (c,d) of the T. tomentosa bud-extracts.

Figure 2

In vitro noradrenaline release from cortical synaptosomes of young male mice: functional activity of native GABAA receptors in presence of TTBEs dilutions. (a) Counteracting effect of 1 µM bicucullin on muscimol evoked [3H]noradrenaline release (b). Effects of TTBEs on 10 µM muscimol evoked [3H]noradrenaline release. Data represent the mean ± S.E.M. from six young male mice. Statistical analysis was performed by applying ANOVA followed by the Tukey’s Multiple Comparison test. * p < 0.05, ** p < 0.01 vs. 10 µM muscimol.

Figure 3

Scree plot: % explained variance of each PCs.

Figure 4

(a) PC1-PC2 score plot and biplot of the D_16,8 data matrix. (b) PC1-PC2 biplot (scores plus loadings plot). Animals are categorized by age: old mice are reported in black and young ones in red, respectively. (c) Loading plot on PC1. (d) Loading plot on PC2. YMW: young male water, YFW: young female water, YMVehi: young male vehicle, YFVehi: young female vehicle, YMTTBEs: young male Tilia tomentosa Moench Buds Extracts, YFTTBEs: young female Tilia tomentosa Moench Buds Extracts, ZeroYMW: day 0 young male water, ZeroYFW: day 0 young female water, OMW: old male water, OFW: old female water, OMVehi: old male vehicle, OFVehi: old female vehicle, OMTTBEs: old male Tilia tomentosa Moench Buds Extracts, OFTTBEs: old female Tilia tomentosa Moench Buds Extracts, ZeroOMW: day 0 old male water, ZeroOFW: day 0 old female water, HD: n° head dippings, %AE: % of area explored, %C: % of entries into center, tL: time in light, T: n° of transitions, Av Sp: average speed, RAE: exploration rate %, D: total distance.

Figure 5

PC1-PC2 score plot (a) and biplot (b) of the M_16,8 data matrix. PC1-PC2 score plot (c) and biplot (d) of the F_16,8 data matrix. tL_1: time in light day 21-day 0, HD_1: head dippings day 21-day 0, AE_1: % of area explored day 21-day 0, RAE_1: exploration rate % day 21-day 0, %C_1: % of entries into center day 21-day 0, Av Sp_1: average speed day21-day0, T_1: n° of transitions day 21-day 0, D_1: distance day 21-day 0, W: water, Vehi: vehicle, TTBEs: Tilia tomentosa Moench Buds Extracts.

Figure 6

Hole Board test in young and old male mice: effects 21 days of dietary integration with vehicle, TTBEs respect to water. Behavioral skills were quantified as (a) n° head dippings (%HD), (b) % of area explored (%AE), (c) % of entries into center (%C). Data represent the mean ± S.E.M. (n = 6 (W), 14 (Vehi) 12 (TTTBEs) young and (n = 6 (W), 6 (Vehi) 6 (TTTBEs) old mice analyzed in three different trials. Statistical analysis was performed by applying ANOVA followed by the Tukey’s multiple comparison test. * p < 0.05; p < 0.01 vs. water; ^§ p < 0.05 vs. vehicle.

Figure 7

Hole Board test in young and old female mice: effects 21 days of dietary integration with vehicle, TTBEs respect to water. Behavioral skills were quantified as (a) n° head dippings (%HD), (b) % of area explored (%AE), (c) % of entries into center (%C). Data represent the mean ± S.E.M. (n = 6 (W), 11 (Vehi) 14 (TTTBEs) young and (n = 8 (W), 9 (Vehi) 9 (TTTBEs) old mice analyzed in three different trials. Statistical analysis was performed by applying ANOVA followed by the Tukey’s multiple comparison test. * p < 0.05 water.

Figure 8

Light Dark test in young and old male mice: effects 21 days of dietary integration with vehicle, TTBEs respect to water. Behavioral skills were quantified as (a) time in light (tL), (b) n° of transitions (T), (c) average speed (Av Sp), (d) exploration rate % (RAE) and (e) total distance (D). Data represent the mean ± S.E.M. (n = 9 (W), 12(Vehi) 12 (TTTBEs) young and (n = 6 (W), 6 (Vehi) 6 (TTTBEs) old mice analyzed in three different trials. Statistical analysis was performed by applying ANOVA followed by the Tukey’s multiple comparison test. * p < 0.05 water; ^§ p < 0.05 vs. vehicle.

Figure 9

Light Dark test in young and old female mice: effects 21 days of dietary integration with vehicle, TTBEs respect to water. Behavioral skills were quantified as (a) time in light (tL), (b) n° of transitions (T), (c) average speed (Av Sp), (d) exploration rate % (RAE) and (e) total distance (D). Data represent the mean ± S.E.M. (n = 8 (W), 9 (Vehi) 12 (TTTBEs) young and (n = 6 (W), 9 (Vehi) 9 (TTTBEs) old mice analyzed in three different trials. Statistical analysis was performed by applying ANOVA followed by the Tukey’s multiple comparison test. * p < 0.05 water.