**3. Results and Discussion**

#### *3.1. The Chemical Composition of the Essential Oil*

The chemical composition of the REO was identified by GC-MS analysis (Figure 2) and is presented in Table 1. Seventy-seven chemicals, corresponding to 73.48% of the total oil, were identified in the REO. The most abundant chemical classes of the oil components were monoterpene hydrocarbons (40.14%), followed by oxygenated monoterpenes (26.44%), sesquiterpene hydrocarbons (4.74%), and other compounds (2.16%). The major components of the REO were eucalyptol (26.02%), α-pinene (19.89%), camphor (16.71%), camphene (8.67%), β-myrcene (3.97%), β-caryophyllene (3.11%), borneol (2.50%), and limonene (2.16%). Our results are supported by Bouyahya et al. [31], who reported eucalyptol (23.673%), camphor (18.743%), borneol (15.46%), and α-pinene (14.076%) as the major chemical components of the REO. In addition, Elyemni et al. [32] reported the presence of eucalyptol

(32.18%), camphor (16.20%), α-pinene (15.82%), camphene (9.16%), and α-terpineol (7.36%) in the chemical composition of the REO. Based on these studies, our essential oil exhibits a chemical composition commensurate to those reported by other authors who presume its memory-enhancing and antioxidant activity.

**Figure 2.** Gas chromatography-mass spectrometry (GC–MS) profile of the *Rosmarinus o*ffi*cinalis* essential oil (numbers refer to Table 1).




**Table 1.** *Cont.*


**Table 1.** *Cont.*

a The numbering refers to the elution order, and values (relative peak area percent) represent averages of three determinations; b Retention Index (KI) relative to a standard mixture of *n*-alkanes on the SPBTM-5 column; c co-elution with an authentic sample.

#### *3.2. E*ff*ects on Anxiety-Like Behavior in NTT Test and on Y-Maze Response to Novelty and Spatial Memory*

Figure 3 reports the effects of Sco (100 μM) and REO (25, 150, and 300 μL/L) treatment on anxiety-like behavior in the NTT test. Representative locomotion tracking pattern (Figure 3A) illustrates the differences in swimming traces among the top and bottom zones and shows that the Sco-treated group traveled a greater distance in the bottom zone, suggesting an anxiogenic profile. Additionally, Sco treatment increased the time spent in the bottom zone of the tank (*p* < 0.0001) (Figure 3B) along with decreasing the time spent in the top zone of the tank (Figure 3B) (*p* < 0.0001) as compared to control. Reducing the time spent in the top zone of the tank suggests the anxiogenic-like effect of Sco. Sco treatment produced a hypolocomotor effect, by decreasing total distance traveled (*p* < 0.001) (Figure 3C) and average velocity (i.e., magnitude and direction of zebrafish speed, *p* < 0.01) (Figure 3D) compared to control. By contrast, increasing the time spent in the top zone of the tank (Figure 3B) suggests the anxiolytic-like effect of REO. Moreover, treatment with REO prevents the anxiogenic-like effect of Sco, in a dose-dependent manner, as evidenced through increasing of total distance traveled (*p* < 0.0001) (Figure 3C) and average velocity (*p* < 0.001) (Figure 3D) as compared to Sco-alone treated fish.

**Figure 3.** *Rosmarinus o*ffi*cinalis* essential oil (REO: 25, 150, and 300 μL/L) improved locomotion pattern and decreased anxiety in the NTT test. (**A**) Representative locomotion tracking pattern of the control, Sco (100 μM), and REO (25, 150, and 300 μL/L) treated groups. (**B**) The time spent by zebrafish in the top/bottom zone of the tank in different groups. (**C**) The total distance traveled by zebrafish in the tank in different groups. (**D**) The average velocity of zebrafish in the tank in different groups. Values are means ± S.E.M. (*n* = 10). For Tukey's post hoc analyses: (**B**) Control vs. Sco (100 μM): ### *p* < 0.0001, Sco vs. REO (25 μL/L): ## *p* < 0.001, Sco vs. REO (150 μL/L): ## *p* < 0.001, and Sco vs. REO (300 μL/L): ## *p* < 0.001; (**C**) Control vs. Sco (100 μM): ### *p* < 0.0001, Control vs. REO (300 μL/L): # *p* < 0.01, Sco vs. REO (25 μL/L): ### *p* < 0.0001, Sco vs. REO (150 μL/L): ### *p* < 0.0001, and Sco vs. REO (300 μL/L): ### *p* < 0.0001; and (**D**) Control vs. Sco (100 μM): # *p* < 0.01, Control vs. REO (300 μL/L): ## *p* < 0.001, Sco vs. REO (25 μL/L): ### *p* < 0.0001, Sco vs. REO (150 μL/L): ### *p* < 0.0001, and Sco vs. REO (300 μL/L): ### *p* < 0.0001.

Figure 4 shows the effects of Sco (100 μM) and REO (25, 150, and 300 μL/L) treatment on Y-maze response to novelty and spatial memory. Representative locomotion tracking pattern (Figure 4A) illustrates the differences in swimming traces among the Y-maze arms and shows that Sco treated group traveled a greater distance in the other arm, suggesting memory deficits. In addition, Sco administration significantly altered novel arm exploration (*p* < 0.0001) (Figure 4B) as compared to control zebrafish. The reduced percentage of time spent in novel arm suggests a memory impairment effect of Sco. The administration of Sco affects locomotion, as evidenced by the decreasing of total distance traveled (*p* < 0.0001) (Figure 4C) and turn angle (*p* > 0.05) (Figure 4D) compared to control. REO treatment significantly counters the Sco action induced hypolocomotion and memory deficits by improving the novel arm exploration (*p* < 0.001) (Figure 4B), while total distance traveled (*p* < 0.01) (Figure 4C) and turn angle (*p* < 0.01) (Figure 4D) was significantly increased at the high doses of REO (300 μL/L) as compared to Sco-alone treated zebrafish. Our results demonstrate that REO exhibited anxiolytic and memory-enhancing profile, which could be due to the presence of major active constituents shown in Table 1. The obtained results are in perfect agreemen<sup>t</sup> with those obtained by other groups that demonstrated anti-amnesic effects along with in vitro antioxidant and acetylcholinesterase and butyrylcholinesterase inhibition potential of *Rosmarinus o*ffi*cinalis* in scopolamine-induced memory impairment in mice [10]. Ozarowski et al. [33] demonstrated that *Rosmarinus o*ffi*cinalis* leaf extract improved long-term memory in rats, which can be partially explained by its inhibition of AChE activity in rat brain. Noori Ahmad Abadi et al. [34] reported that the hydroalcoholic extract of *Rosmarinus o*ffi*cinalis* L. leaf reduced anxiety in mice, probably due to the presence of flavonoids in this plant and their antioxidant property. Additionally, Abdelhalim et al. [35] attributed the anxiolytic effects of *Rosmarinus o*ffi*cinalis* with its effect on gamma-aminobutyric acid (GABA) receptors. Despite extensive knowledge about the effects of various *Rosmarinus o*ffi*cinalis* extracts on memory, anxiety, and AChE

activity in the rodent brain, we demonstrated for the first time the cognitive-enhancing, anxiolytic, and antioxidant profile of REO in the scopolamine zebrafish model. Furthermore, our study demonstrated that zebrafish is rapidly becoming one of the main organisms in translational neuroscience research, successfully completing rodent models for the study of dementia-related conditions.

**Figure 4.** *Rosmarinus o*ffi*cinalis* essential oil (REO: 25, 150, and 300 μL/L) improved locomotion pattern and memory in the Y-maze test. (**A**) Representative locomotion tracking of the control, Sco (100 μM), and REO (25, 150, and 300 μL/L) treated groups. (**B**) The time spent in each arm (start, novel, and novel arms) in different groups. (**C**) The total distance traveled by zebrafish in the tank in different groups. (**D**) The turn angle of zebrafish in the tank in different groups. Values are means ± S.E.M. (*n* = 10). For Tukey's post hoc analyses: (**B**) Control vs. Sco (100 μM): ### *p* < 0.0001, Sco vs. REO (25 μL/L): ## *p* < 0.001, Sco vs. REO (150 μL/L): ## *p* < 0.001, and Sco vs. REO (300 μL/L): ## *p* < 0.001; (**C**) Control vs. Sco (100 μM): ### *p* < 0.0001, Control vs. Sco (100 μM): ## *p* < 0.001, and Sco vs. REO (300 μL/L): # *p* < 0.01; and (**D**) Sco vs. REO (300 μL/L): # *p* < 0.01.
