(-)-cis-Carveol, a Natural Compound, Improves β-Amyloid-Peptide 1-42-Induced Memory Impairment and Oxidative Stress in the Rat Hippocampus

Alzheimer's disease (AD) could be considered a multifactorial neurodegenerative disorder characterized by the accumulation of the β-amyloid-peptide (Aβ) within the brain leading to cognitive deficits, oxidative stress, and neuroinflammation. The present work was carried out to investigate the neuroprotective effect of (-)-cis-carveol (1% and 3%, for 21 days) against the β-amyloid-peptide 1-42- (Aβ1-42-) induced AD. Twenty-five rats were divided into five groups (n = 5/group): the first group—control (sham-operated); the second group—Aβ1-42 (1 mM) that received donepezil treatment (5 mg/kg, as the positive reference drug in the Y-maze and the radial arm maze tests); the third group—Aβ1-42 (1 mM); the fourth and fifth groups—Aβ1-42 (1 mM) that received (-)-cis-carveol treatment groups (1% and 3%). The results of this study demonstrated that (-)-cis-carveol improved Aβ1-42-induced memory deficits examined by using Y-maze and radial arm maze in vivo tests. Also, the biochemical analyses of the hippocampus homogenates showed that (-)-cis-carveol reduced hippocampal oxidative stress caused by Aβ1-42. Our results suggested that the use of (-)-cis-carveol may be suitable for decreasing AD-related symptoms.


Introduction
Alzheimer's disease (AD) is considered an irreversible, progressive neurological disorder characterized by the accumulation of the extracellular β-amyloid-peptide (Aβ), intracellular neurofibrillary tangles, disruption of synapses, brain inflammation, and memory loss [1]. Also, there is some evidence that neuropsychiatric symptoms, particularly anxiety and depression, could be risk factors for cognitive impairment and AD dementia and are also associated with cortical Aβ deposition [2,3]. Furthermore, there is evidence between oxidative stress and neuronal dysfunction in AD [4]. Also, excessive Aβ may trigger stress-related signaling pathways through increasing Ca 2+ influx, increasing oxidative stress, and impairing energy metabolism [4,5]. At present, clinical drug treatments are limited to acetylcholinesterase inhibitors (AChEIs), such as donepezil and the antagonist of N-methyl-D-aspartic acid (NMDA) receptor, represented by memantine [6]. Unfortunately, recent failures and limited progress of therapeutics suggest that alternative strategies for AD treatment could be considered [7].
Essential oils are natural products that demonstrate high therapeutic potential against various types of pathologies, including central nervous system (CNS) disorders. Their chemical constituents exhibit various pharmacological activities such as anxiolytic [8], antidepressant [9], analgesic [10], anticonvulsant [11], and inhibitory effects against brain tumor cells [12]. Monoterpenes are compounds commonly found in essential oils that have received much attention as a potential therapy for AD [13]. It has been documented that carvacrol, a monoterpenoid phenol, significantly reduced the development of cerebral edema [14]. Linalool, terpene alco-hol, improved learning and memory in a transgenic mouse model of AD by reduction of the level of inflammation markers [15]. Khazdair et al. [16] reported that monoterpenes from various medicinal plants are active constituents with therapeutic potential in disorders associated with neuroinflammation and neurotransmitter deficiency such as AD and depression.

Animals and Drug
Administration. Twenty-five adult male Wistar rats were used in this study (300 ± 50 g; purchased from Cantacuzino Institute, Bucharest, Romania). The animals were kept in a temperature and lightcontrolled room (22°C, a 12 h cycle starting at 08:00 h) with free access to food and water. Rats were divided into five groups (n = 5/group): the first group-control (sham-operated); the second group-Aβ1-42 (1 mM) that received donepezil treatment (5 mg/kg, as the positive reference drug in the Y-maze and the radial arm maze tests); the third group-Aβ1-42 (1 mM); the fourth and fifth groups-Aβ1-42 (1 mM) received (-)-cis-carveol treatment groups (1% and 3%). The control, Aβ1-42+donepezil, and Aβ1-42 groups received 1% Tween 80 solution through inhalation. (-)-cis-Carveol was diluted with 1% Tween 80 (v/v), and exposure (200 μL, either 1% or 3%) was via an electronic vaporizer (KBAYBO). Regarding concentrations to be used in the pharmacological tests, we chose the dose of 1% for the carveol in the same way as it is used for the essential oil in aromatherapy and a higher dose (3%) to emphasize the effects [24]. Rats were pretreated by inhalation with (-)-ciscarveol (1% and 3%) for seven days before starting behavioral tests and continuously administered during behavior tests (21 days). Donepezil hydrochloride (Sigma-Aldrich, Germany) was dissolved in 0.9% physiological saline (5 mg/kg) and injected i.p., once daily, 30 min before the Y-maze and radial arm maze tasks. Also, we confirm that n = 5 animals/group is appropriate using InVivoStat and R-based statistical package [25]. Based on a significance level of 0.05, the power to detect a 20% biologically relevant change from control is 99%. All experimental procedures were strictly conducted by the Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals. The experimental process is indicated in Figure 1. Also, for the behavioral and biochemical parameter assays, we followed the methods of Postu et al. [26].

Neurosurgery.
Under sodium pentobarbital (50 mg/kg b.w., i.p., Sigma-Aldrich, Germany) anesthesia, 1 mM aggregated Aβ1-42 in sterile saline solution (Sigma-Aldrich, Germany) was intracerebroventricularly (i.c.v.) delivered to rats on day 0 as discussed by Postu et al. [26]. The infusion volume (4 μL) was injected gradually (1 μL/min) utilizing the following coordinates: 1.5 mm lateral to the midline and 7.4 mm ventral to the surface of the cortex [27]. The control group (sham-operated animals) got an identical volume of the saline solution rather than the Aβ1-42 solution. The behavioral tests were done from the 22 nd day (Y-maze) and the 24 th day (radial arm maze) after neurosurgery ( Figure 1) and were performed blind to the treatments by the observer.

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BioMed Research International 2.6. Behavioral Assays 2.6.1. Y-Maze Test. The impact of (-)-cis-carveol (1% and 3%) on the spontaneous alternation behavior was confirmed in a single-session Y-maze on the 22 th day postsurgery [26,28]. The Y-maze used in the present study was constructed of Plexiglas having the following dimensions: 25 cm high, 35 cm long, and 10 cm wide of each arm and an equilateral triangular area. 15 min after receiving (-)-cis-carveol (1% and 3%) by inhalation, each animal was put at the end of one arm and permitted to move openly for an 8 min session. Spontaneous alternation behavior was characterized as consecutive entry into all three arms on covering triplet sets. The spontaneous alternation percentage (SAP) was calculated as follows: SAP ð%Þ = ðnumber of alternation/ number of total arm entries − 2Þ × 100. The animal behavior was recorded using a Logitech HD Webcam C922 Pro Stream camera, and the videos were analyzed with ANY-maze® software (Stoelting CO, USA). The Y-maze was washed with a 10% ethanol solution in trials.

Radial Arm Maze
Test. The impacts of (-)-cis-carveol (1% and 3%) on the spatial memory were tested by employing a radial arm maze for 7 days, starting with the 24 th day postsurgery [26,29]. The maze, which consisted of eight arms numbered from 1 to 8 (48 cm × 12 cm), with a radial extension of 32 cm in diameter from the central field, had 50 mg of food pellets at the end of arms 1, 2, 4, 5, and 7. Four days of sessions on habituation were conducted. Rats have been instructed to move to the end of the arms and eat the pellet of food during 5 min sessions. After habituation, all rats were given just one trial per day. For working and reference memory assignments, each rat was separately put within the center of the maze, taking 15 min administration by inhalation of (-)-cis-carveol (1% and 3%   BioMed Research International addition of 1 ml of dichromate: acetic acid reagent. The tubes were immediately kept in a boiling water bath for 15 min, and the green color formed during the reaction was read at 570 nm by using a spectrophotometer. Control tubes, devoid of the enzyme, were also processed in parallel. The activity of the enzyme is expressed as μmol of H 2 O 2 consumed/min/mg protein. 2.7.4. Determination of the Hippocampal GPX Activity. For the assessment of the glutathione peroxidase (GPX, E.C. 1.11.1.9) activity, an approach previously described by Sharma and Gupta [33] was used. A reaction mixture consisting of 1 mL 0.4 mM phosphate buffer (pH 7.0) containing 0.4 mM EDTA, 1 mL of 5 mM NaN 3 , 1 mL of 4 mM glutathione (GSH), and 200 μL of supernatant was preincubated at 37°C for 5 min. Then, 1 mL of 4 mM H 2 O 2 was inserted and incubated for another 5 min at 37°C. The GSH excess was quantified using the DTNB method. One unit of GPX is specified as the amount of enzyme needed to oxidize for 1 nmol GSH/min. The enzyme activity was expressed as units/mg protein.

The Total Hippocampal Content of Reduced GSH.
For the measurement of the reduced glutathione (GSH) content, the method of Fukuzawa and Tokumura [34] was used. A 200 μL supernatant was applied to 1.1 mL of 0.25 M sodium phosphate buffer (pH 7.4), followed by the introduction of 130 μL DTNB 0.04%. Finally, the mixture was taken to a final volume of 1.5 mL with distilled water, and the absorbance was read at 412 nm using a spectrophotometer. Results were shown as μg GSH/μg protein.

Determination of Hippocampal Protein Carbonyl Level.
The extent of protein oxidation in the hippocampus was assessed by measuring the content of protein carbonyl groups, using a method described by Oliver et al. [35] and modified through Luo and Wehr [36]. The supernatant fraction was split into two equal aliquots each that contained around 2 mg of protein. Both aliquots were precipitated using 10% trichloroacetic acid (TCA (w/v), final concentration). Another sample was treated with 2 N HCl, and another sample was treated with 0.2% (w/v) DNPH in 2 N HCl at equivalent volume. Both samples were incubated at 25°C and then stirred at intervals of 5 min. The results had been expressed as nmol/mg protein.
2.7.7. Determination of Hippocampal MDA Level. The content of malondialdehyde (MDA), an indicator of lipid peroxidation, was measured via the usage of the approach previously described [37]. 200 μL of supernatant was applied and briefly mixed in 0.1 M HCl with 1 mL of 50% TCA in 0.1 M HCl and 1 mL of 26 mM thiobarbituric acid. Samples were held at 95°C for 20 min after vortex mixing. Samples were then centrifuged for 10 min at 960 × g, and the supernatants were read at 532 nm. The findings were presented as nmol/mg protein, as stated.

Estimation of Protein
Concentration. The evaluation of protein was accomplished by a bicinchoninic acid (BCA) protein assay kit from Sigma-Aldrich, Germany, following an approach described by Smith et al. [38].

Statistical
Analysis. Data are expressed as the means ± SEM. The statistical evaluation was done through a oneway analysis of variance (ANOVA) accompanied by Tukey's post hoc test. Results were analyzed with GraphPad Prism 7 software, and the values of F for which p < 0:05 had been considered to indicate statistical significance.

Results and Discussion
(-)-cis-Carveol was obtained with a high yield (80%; 53.46 mmol). Analytical and spectroscopic data and comparison with literature data confirmed the chemical structure of this monoterpene [22,23].
Our results are in line with previous studies where chronic treatment with carvacrol, a phenolic monoterpene abundantly presented in the essential oil of the Lamiaceae family, improved memory deficits in the 6-hydroxydopamine Parkinson's disease (PD) rat model [39]. Supporting evidence suggested many pharmacological effects of the carvacrol, including antibacterial, antifungal, antioxidant, antinociceptive, anti-inflammatory, antiapoptosis, and anticancer activi-ties [40]. Also, carvacrol exhibited AChE inhibition [41] as well as having anxiolytic [42] and antidepressant properties [43]. Moreover, our group demonstrated that monoterpenerich essential oil from Pinus halepensis improved memory   [26]. The present study showed that (-)-cis-carveol improves spatial memory impairment in an amyloidosis rat model.

Effects on the AChE Inhibition.
The modulatory activity of (-)-cis-carveol on AChE activity was also determined. AChE exhibited a secondary noncholinergic function, including the processing and deposition of Aβ. Currently, the therapeutic option for AD patients is the use of AChE inhibitors, which gives only a symptomatic relief [44]. Compared with the control group, Aβ1-42 injection promoted increasing of the hippocampal AChE activity (p < 0:01) (Figure 3). On the contrary, (-)-cis-carveol (1% and 3%), but especially the dose of 3%, significantly decreased the AChE activity in the rat hippocampus (p < 0:01 for (-)-ciscarveol 3%) as compared to Aβ1-42 rats.

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BioMed Research International after Aβ1-42 injection. Therefore, this may be one of the mechanisms of (-)-cis-carveol improving memory performance in the Y-maze and radial arm maze tests.

Effects on the Oxidative Stress Markers in the
Hippocampal Homogenates. Aβ-associated oxidative stress is known to play an essential role in the etiology and the pathogenesis of AD [47]. The Aβ1-42 group had a significant decrease of SOD-(p < 0:01) and GPX-(p < 0:01) specific activities along with a significant decrease of the total content of reduced GSH (p < 0:001) and significantly higher levels of protein carbonyl (p < 0:001) and MDA (p < 0:001) as a valid biomarker of lipid peroxidation when compared to the control group. Additionally, treatment of the Aβ1-42 group with (-)-cis-carveol (1% and 3%) significantly attenuated these alterations as compared to Aβ1-42-treated rats. Supporting data suggested that the downregulation of the antioxidants determines neurodegeneration [48]. Thus, the downregulation of SOD contributed to Aβ oligomerization and initiated cognitive impairment [49]. Also, GSH is reported to have an essential role in the brain detoxification of reactive oxygen species [50]. MDA is a crucial indicator of lipid peroxidation. Moreover, it was reported that Aβ1-42 induced an overt enhancement of oxidative stress characterized by an increase in MDA level [51]. In the present work, (-)-cis-carveol demonstrated antioxidant activity, as evidenced by enhanced activity of SOD and GPX, increased GSH level and reduced protein carbonyl and MDA level in the brain of AD rats.
Several studies demonstrated the antioxidant activities of single or multiple monoterpenes [52,53]. Postu et al. [26] showed antioxidant activity of the Pinus halepensis essential oil mainly attributed to its phytoconstituents. Kaur et al. [54] demonstrated that among all the tested compounds from Anethum graveolens L. essential oil for their antioxidant activity, (-)-cis-carveol and perillyl alcohol were most productive (IC 50 values < 0:16 mg/mL). Also, in a recent study, Ibrahim et al. [55] demonstrated antioxidant activity of the volatile components of the peppermint essential oil, including (-)-cis-carveol. Therefore, these results support the idea that (-)-cis-carveol is an effective antioxidant for AD prevention or treatment.
Regarding the limitations of our study, the levels of the (-)-cis-carveol in the blood-mediated observed effects in the experimental groups were still unknown. Furthermore, Jäger et al. [56] demonstrated that R-(-)-and S-(-)-carvone rapidly penetrated the skin of healthy subjects leading to significantly different blood levels. The highest concentration was found in the first 4 hours following exposure. Therefore, we presumed that (-)-cis-carveol exhibited the positive effects in mediating the improvement of the memory processes via decreasing brain oxidative stress and inhibiting the hippocampal AChE activity in the Sco-treated rats.

Conclusions
In summary, the present study demonstrated that (-)-cis-carveol was able to reverse the cognitive deficits resulting from the Aβ1-42 treatment. Additionally, we also established that the positive effects of (-)-cis-carveol could be mediated by decreasing brain oxidative stress and regulation of AChE activity. Therefore, the present work suggested that (-)-ciscarveol provides neuroprotection against Aβ1-42 and can be regarded an alternative therapeutic agent for dementiarelated neurological conditions, including AD.

Data Availability
The data used to support the findings of this study have been deposited in the website of the Federal University of Sergipe at https://http://www.sigaa.ufs.br/sigaa/public/ programa/defesas.jsf?lc=pt_BR&id=719 and published in file:///C:/Users/damia/Downloads/molecules-20-19649.pdf.

Conflicts of Interest
The authors declare that they have no conflicts of interest.