Alpha-pinene preserves human dopaminergic SH-SY5Y cells against 6-hydroxydopamine-induced toxicity through its antioxidant and antiapoptotic properties and gamma-aminobutyric acid type A signaling

Mandana Moshrefi; Ali Mohammad Pourrahimi; Mehdi Abbasnejad; Mohammad Hadi Farjoo; Saeed Esmaeili-Mahani

doi:10.4103/bbrj.bbrj_51_22

ORIGINAL ARTICLE

Year : 2022 | Volume : 6 | Issue : 2 | Page : 255-260

Alpha-pinene preserves human dopaminergic SH-SY5Y cells against 6-hydroxydopamine-induced toxicity through its antioxidant and antiapoptotic properties and gamma-aminobutyric acid type A signaling

Mandana Moshrefi¹, Ali Mohammad Pourrahimi², Mehdi Abbasnejad³, Mohammad Hadi Farjoo⁴, Saeed Esmaeili-Mahani³
¹ Kerman Neuroscience Research Center, Laboratory of Molecular Neuroscience, Institute of Neuropharmacology, Kerman University of Medical Sciences; Department of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman, Kerman, Iran
² Kerman Neuroscience Research Center, Laboratory of Molecular Neuroscience, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran
³ Department of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman, Kerman, Iran
⁴ Department of Pharmacology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

Date of Submission	28-Feb-2022
Date of Acceptance	11-May-2022
Date of Web Publication	17-Jun-2022

Correspondence Address:
Saeed Esmaeili-Mahani
Department of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman, and Kerman Neuroscience Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, P.O. Box 76135-133, Kerman
Iran

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/bbrj.bbrj_51_22

Abstract

Background: Parkinson's disease (PD) is one of the most common neurodegenerative disorders which is characterized by progressive loss of dopaminergic neurons in substantia nigra. Therefore, drugs or natural agents that have suppressive effects on dopaminergic cell death may reduce the progression of such disorder. Here, the effect of natural product alpha-pinene was evaluated on 6-hydroxydopamine (6-OHDA)-induced damage in SH-SY5Y human dopaminergic cell line as an in vitro model of PD. Methods: The cells were incubated by 150 μM 6-OHDA alone or accompanied with different concentration of alpha-pinene (10–180 μM). Cell viability was determined by MTT assay. The amount of intracellular reactive oxygen species (ROS) and mitochondrial membrane potential were measured by fluorescence spectrophotometry. In addition, the components of molecular apoptotic pathway such as cytochrome c release, Bax, Bcl-2, and caspase-3 levels were measured by immunoblotting. Gamma-aminobutyric acid (GABA) antagonist, bicuculline, was used to find the role of GABA Type A (GABAA) receptors in the signaling of alpha-pinene. Results: The data showed that 6-OHDA produced cell damage, decreased mitochondrial membrane potential, increased intracellular ROS and cytochrome c release, as well as increased Bax/Bcl-2 ratio and caspase-3 activity. Moreover, alpha-pinene (70 μM) significantly inhibited cellular and molecular abnormalities. Blockage of GABAA receptor significantly suppressed the protective effect of alpha-pinene. Conclusion: The results suggest that alpha-pinene has a protective effect against dopaminergic toxicity, and at least in part, its antioxidant and antiapoptotic properties are probably involved in such protection.

Keywords: 6-hydroxydopamine, alpha-pinene, apoptosis, gamma-aminobutyric acid Type A receptors, Parkinson's disease, SH-SY5Y cells

How to cite this article:
Moshrefi M, Pourrahimi AM, Abbasnejad M, Farjoo MH, Esmaeili-Mahani S. Alpha-pinene preserves human dopaminergic SH-SY5Y cells against 6-hydroxydopamine-induced toxicity through its antioxidant and antiapoptotic properties and gamma-aminobutyric acid type A signaling. Biomed Biotechnol Res J 2022;6:255-60

How to cite this URL:
Moshrefi M, Pourrahimi AM, Abbasnejad M, Farjoo MH, Esmaeili-Mahani S. Alpha-pinene preserves human dopaminergic SH-SY5Y cells against 6-hydroxydopamine-induced toxicity through its antioxidant and antiapoptotic properties and gamma-aminobutyric acid type A signaling. Biomed Biotechnol Res J [serial online] 2022 [cited 2023 Jun 10];6:255-60. Available from: https://www.bmbtrj.org/text.asp?2022/6/2/255/347719

Introduction

Parkinson's disease (PD) has a life span risk of 2%, making it the second most common neurodegenerative disorder after Alzheimer's disease.^[1] PD is determined by the profound death of dopaminergic neurons in the substantia nigra pars compacta. The deprivation of dopamine causes characteristic movement disorder that is revealed by motor symptoms.^[2] However, the etiology of this disease is not well known, and many pathological mechanisms such as mitochondrial dysfunctions, oxidative stress, neuroinflammatory processes, apoptotic cell death, and the formation of pathological liability are introduced as possible causes of the diseases.^[3] It should be noted that current drug treatments are maintenance therapy. Therefore, protective mechanisms and neural preservation can be useful to prevent PD induction.^[4] The purpose of neuroprotective therapies is to decrease or prevent disease progression and decrease the loss of dopaminergic neurons in the brain. Naturally occurring bioactive compounds with high antioxidant capacity may reverse the neural damage in PD.^[5] Nowadays, there is no proven neuroprotective or an alternative for the treatment of PD,^[6] and further efforts are needed to find the optimal neuroprotective agents.

Current medications for PD are generally successful in reducing symptoms but have side effects. Therefore, there is a need to find new therapeutic agents with fewer side effects. The side effects of using synthetic drugs, along with the cost of purchasing and shortages, have recently increased people's interest in using medicinal plants and essential oils.

However, the scientific basis for many medicinal plants and natural products is still unclear.^[7],[8] Therefore, the need to clarify the scientific mechanism of medicinal plants that have a physiological effect is increasing rapidly.^[9] Besides, these medicinal plants are also an important source of new chemicals with potential therapeutic effects.

Alpha-pinene is found in the essential oils of many plant species, especially pine trees, and found in rosemary (Rosmarinus officinalis) essential oil.^[10] This agent has several biological effects such as antifungal, anti-inflammatory, antitumor, anticholinesterase, anticonvulsant, antinociceptive, cytoprotective, and neuroprotective effects.^{[10],[11],[12]}

In fact, α-pinene has a physiological effect on humans and therefore is taken into consideration.^[9],[13] Essential oils containing α-pinene have been used to treat several diseases.^[14] It has been reported that alpha-pinene exerts its neuroprotective effect through the restoration of antioxidant capacity and reduction of inflammation in the ischemic rat brain.^[15] The antioxidant and protective potential of the monoterpenes α-pinene have been confirmed in H₂O₂-induced oxidative stress in rat pheochromocytoma cells.^[16]

Since numerous evidence suggests that the progressive cell death is the main mechanism of PD, and natural component alpha-pinene has potential antioxidative and cell protective properties, the present study was designed to investigate the effects of alpha-pinene in 6-hydroxydopamine (6-OHDA)-induced toxicity in SH-SY5Y cells as an in vitro model of Parkinson's disease.

Materials and Methods

Materials

Ethic Committee of Kerman Neuroscience Research Center approved the experiments (Ethics Code: EC/98). Cell culture reagents, penicillin–streptomycin solution, trypsin EDTA, and fetal bovine serum (FBS) were obtained from Biosera Co. (East Sussex, UK). Culture flasks and dishes were acquired from SPL Life Sciences Inc. (Gyeonggi-Do, South Korea). 3 [4,5-dimethyl-2-thiazolyl]-2,5-di–phenyl-2-tetrazoliom bromide (MTT), 6-OHDA, 2,7-dichlorofluorescein diacetate (DCFH-DA), and rhodamine 123 (RH-123) were purchased from Sigma-Aldrich (St. Louis, Ml, USA). Primary polyclonal antibodies and secondary goat anti-mouse antibodies were obtained from Santa Cruz Biotechnology, Inc. (Delaware Ave. Santa Cruz, USA).

Cell culture

Human neuroblastoma SH-SY5Y cells were obtained from Iran National Cell Bank Pasteur Institute of Iran (Tehran, Iran). The cells were grown with Dulbecco Modified Eagle Medium supplemented with 10% FBS with penicillin (100 U/ml) and streptomycin (100 μg/ml) and maintained at 37°C in a CO₂ incubator. After two passages, SH-SY5Y cells were plated at the density of 5000 cells per well in a 96-well microplate for the MTT, mitochondrial membrane potential, and reactive oxygen species (ROS) assays. For protein extraction, cells were grown in a 6-well plate. Then, the cells were incubated with 6-OHDA and different concentrations of alpha-pinene for 48 h. Alpha-pinene was added 30 min before 6-OHDA.

Cell viability analysis

Cellular viability was assessed by the reduction of 2-(4,5-dimethylthiazol-2-y)-2,5-diphenyltetrazolium bromide (MTT) to formazan.^[17] MTT (0.5 mg/ml) was added to the culture, and after 2 h incubation at 37°C, the media were completely removed and DMSO (100 μl) was added to each well, and the absorbance (OD) values were determined at 490 nm by an automatic microplate reader (BioTeck, USA). Each experiment was performed six independent times. The results were expressed as percentages of control.

Measurement of intracellular reactive oxygen species formation

The level of intracellular ROS was determined with DCFH-DA probe and fluorescence spectrophotometry. DCFH-DA is converted to the highly fluorescent dichlorofluorescein in the presence of oxidants. At first, the cells were incubated with DCFH-DA (1 mM for 10 min at 37°C) and carefully washed with PBS and analyzed on the fluorescence plate reader (FLX 800, BioTek, USA). The fluorescence intensity was quantified at an excitation of 485 nm and emission of 538 nm. Each experiment was performed six independent times. The results were expressed as fluorescence percentage of control cells.^[18]

Measurement of mitochondrial membrane potential

Membrane potential was determined with RH-123 probe and fluorescence spectrophotometry. Different groups of cells were incubated with 10 μM RH-123 in the dark for 10 min at 37°C. After the incubation, the cells were washed (three time) with PBS and analyzed immediately on the fluorescence plate reader (Bioteck, USA). The fluorescence intensity of cells was quantified at an excitation of 485 nm and emission of 538 nm. Each experiment was performed six independent times. The results were expressed as fluorescence percentage of control cells.^[19]

Immunoblot analysis

SH-SY5Y cells were homogenized in ice-cold buffer (10 mM Tris-HCL PH 7.4, 1 mM EDTA, 0.15 SDS, 0.1% Na-deoxycholate, 1% NP-40) with protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 2.5 μg/ml of leupeptin, 10 μg/ml of aprotinin), and 1 mM sodium orthovanadate. The homogenate was centrifuged at 14,000 g for 15 min at 4°C the resulting supernatant was retained as the whole cell fraction. For the detection of cytochrome, the cells were lysed for 30 min on ice in buffer containing 20 mM HEPES, pH 7.6, 20% glycerol, 500 mM NaCl, 1.5 mM MgCl 2.0.2 mM EDTA, 0.1% Triton X-100, 1 mM dithiothreitol, and 0.5% sodium dodecyl sulfate. Lysates were centrifuged (30 min at 14,000 g at 4°C) and the supernatants removed and then the pellets re-suspended in a 50 μl volume of buffer. Protein concentrations were measured and equal amounts (40 μg) of protein resolved electrophoretically on a 9% SDS-PAGE gel and transferred to polyvinylidene difluoride membrane. After overnight blocking, the membranes were probed with primary antibodies to caspase-3, cytochrome c, Bax, and Bcl-2 (1:1000 overnight at 4°C). After washing, the blots were incubated for 60 min at room temperature with a horseradish peroxidase-conjugated secondary antibody (1:10,000, GE Healthcare Bio-Science Corp. NJ, USA). The antibody–antigen complexes were detected using the ECL system and exposed to Lumi-Film Chemiluminescent Detection Film (Roche, Germany). Lab work analyzing software (UVP, UK) was used to analyze the intensity of the expression. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) immunoblotting (1:1000) was used to control for loading. The immunoblotting experiments were performed 3–4 independent times for each protein.

Statistical analysis

The results were expressed as mean ± standard error of the mean; the differences in mean of the MTT, ROS, and mitochondrial membrane potential were determined by one-way analysis of variance (ANOVA), followed by Newman-Keuls test. The values of immunoblot band densities (obtained from band densitometry) were expressed as tested proteins/GAPDH ratio for each sample. The averages for different groups were compared by ANOVA, followed by the Newman-Keuls test. P < 0.05 was considered significant.

Results

The effect of alpha-pinene on 6-hydroxydopamine-induced cell toxicity

As shown in [Figure 1]a, 150 μM of 6-OHDA caused a significant decreased (about 50%) in SH-SY5Y cell viability. Alpha-pinene in concentration of 70 μM significantly inhibited the 6-OHDA-induced toxicity, while could not prevent cell damage in lower and upper concentrations. The cells were incubated with alpha-pinene, 30 min before 6-OHDA treatment.

Figure 1: The effects of different concentrations of alpha-pinene against 150 μM 6-OHDA-induced SH-SY5Y cell damage (a) and effect of bicuculline as GABAA antagonist on the alpha-pinene effects (b). Alpha-pinene was added 30 min before 6-OHDA. The cells were treated with 6-OHDA and different concentrations (μM) of alpha-pinene for 24 h. Data were expressed as mean ± SEM; n = 5–6 wells for each group. *P<0.05 and ***P<0.001 vs. control group, ^###P < 0.001 and ^##P < 0.01 versus6-OHDA-treated group. 6-OHDA: 6-hydroxydopamine, SEM: Standard error of the mean, GABAA: Gamma-aminobutyric acid Type A

Click here to view

For investigating the possible interaction between alpha-pinene and gamma-aminobutyric acid Type A (GABAA) receptors, a GABAA selective antagonist (bicuculline) was used. The antagonists were added 10 min before alpha-pinene treatment. The data showed that bicuculline significantly inhibited the protective effect of alpha-pinene [Figure 1]b. The antagonist alone had no significant effect on SH-SY5Y cells viability (data not shown).

As shown in [Figure 2], alpha-pinene alone had no significant effect on the cell viability.

Figure 2: Effects of different concentrations of alpha-pinene on SH-SY5Y cells viability. The cells were incubated with alpha-pinene for 24 h. Different doses of alpha-pinene did not show any toxic effect on SH-SY5Y cells. Data were expressed as mean ± SEM; n = 5–6 wells for each group. SEM: Standard error of the mean, GABAA: Gamma-aminobutyric acid Type A

Click here to view

The effect of alpha-pinene on 6-hydroxydopamine-induced intracellular reactive oxygen species formation

As shown in [Figure 3], the exposure of SH-SY5Y cells to the 6-OHDA led to an increase in the intracellular ROS levels as compared to the control cells (P < 0.001). The toxin-induced increased in ROS level was reduced significantly (P < 0.001) in 70 μM alpha-pinene treated cells, while 30 μM had no reducing effect (P > 0.05). Therefore, 70 μM (the most effective dose in MTT and ROS assays) was selected for using in the next steps of the experiment.

Figure 3: Effect of alpha-pinene on 6-OHDA-induced intracellular ROS production. The cells were pretreated with alpha-pinene (30 and 70 μM) for 30 min, and then 150 μM 6-OHDA was added and incubated for an additional 24 h. Data were expressed as mean ± SEM; n = 5–6 wells for each group. ***P < 0.001 versus control group, ^###P < 0.001 versus 6-OHDA group and ^$$$P < 0.001 versus 6-OHDA + alpha-pinene (30 μM). 6-OHDA: 6-hydroxydopamine, SEM: Standard error of the mean, GABAA: Gamma-aminobutyric acid Type A

Click here to view

Measurement of mitochondrial membrane potential in SH-SY5Y cells

As shown in [Figure 4], the mitochondrial membrane potential was measured in control, 6-OHDA-treated, and 6-OHDA + alpha-pinene-incubated cells. 6-OHDA significantly (P < 0.001) decreased mitochondrial membrane potential which was reversed to the control level by 70 μM alpha-pinene (P < 0.01).

Figure 4: The effect of alpha-pinene (70 μM) on mitochondrial membrane potential in the presence of 6-OHDA. ***P < 0.001 versus control group, ^##P < 0.01 versus 6-OHDA-treated cells. 6-OHDA: 6-hydroxydopamine

Click here to view

Western blot analysis of cytochrome c, Bax, Bcl-2, and cleaved caspase-3

As shown in [Figure 5], the toxin 6-OHDA could significantly promote the release of cytochrome c, which was markedly suppressed by alpha-pinene. Furthermore, 6-OHDA incubation caused a significant increase in the level of Bax/Bcl-2 ratio. Such increased level was not observed in 6-OHDA + alpha-pinene-treated cells [Figure 6].

Figure 5: Effect of alpha-pinene on 6-OHDA-induced cytochrome c release in SH-SY5Y cells. Each value represents the mean ± SEM band density ratio for each group. GAPDH was used as an internal control for loading. **P < 0.01 significantly different versus control cells, ⁺P < 0.05 versus 6-OHDA-treated cells. 6-OHDA: 6-hydroxydopamine, SEM: Standard error of the mean, GAPDH: Glyceraldehyde-3-phosphate dehydrogenase

Click here to view

Figure 6: Effect of 6-OHDA or 6-OHDA plus alpha-pinene on Bax and Bcl-2 expression in SH-SY5Y cells. Each value represents the mean ± SEM band density ratio for each group. GAPDH was used as an internal control for loading. ***P < 0.001 significantly different versus control cells, ⁺⁺P < 0.01 versus 6-OHDA-treated cells. 6-OHDA: 6-hydroxydopamine, SEM: Standard error of the mean, GAPDH: Glyceraldehyde-3-phosphate dehydrogenase

Click here to view

In addition, the amount of activated caspase-3 was significantly increased in the 6-OHDA-incubated cells as compared to the control cells. Alpha-pinene in dose of 70 could significantly reduce the 6-OHDA-promoted caspase-3 activation [Figure 7].

Figure 7: Effect of alpha-pinene on the activation of caspase-3 protein in 6-OHDA-treated SH-SY5Y cells. The cells were incubated with 70 μM alpha-pinene for 30 min, and then, 6-OHDA (150 μM) was added for an additional 24 h. Each value represents the mean ± SEM band density ratio for each group. GAPDH was used as an internal control. Each experiment was performed four independent times. *P < 0.05 significantly versus control cells. ⁺P < 0.05 versus 6-OHDA-treated cells. 6-OHDA: 6-hydroxydopamine, SEM: Standard error of the mean

Click here to view

Discussion

In this study, the possible protective effect of alpha-pinene was assessed in a cell model of PD. The ability of alpha-pinene, which is widely used as a food flavoring, to prevent apoptosis was evaluated in the 6-OHDA-induced human neuroblastoma cell model (SH-SY5Y).

The results showed that alpha-pinene significantly protects 6-OHDA-induced dopaminergic SH-SY5Y cell damage, suppresses oxidative stress, and down-regulates apoptotic signals (cytochrome c release, Bax/Bcl2 ratio, and activation of caspase 3), suggesting that alpha-pinene may have neuroprotective properties.

Our results showed that gamma-aminobutyric acid (GABA) antagonist (bicuculline) inhibited the protective effect of alpha-pinene in 6-OHDA-treated cells. It seems that GABA signaling may play an important role in the protective effects of alpha-pinene in such dopaminergic insult. It has been reported that GABA significantly ameliorates neuronal damage and oxidative stress in the corpus striatum of 6-OHDA lesion Parkinsonism rats.^[20] Surprisingly, Andersson et al. confirmed that GABAA receptors are expressed in SH-SY5Y cells.^[21] Based on the literature, this is the first report that alpha-pinene can reduce damages caused by 6-OHDA in SH-SY5Y cells. Therefore, our findings indicate that alpha-pinene has antiapoptotic and antioxidant activity in human dopaminergic neuroblastoma cells by recruiting GABAA receptors. It has been reported that alpha-pinene interacts with GABAA signaling and induces nonrapid eye movement sleep through GABAA-benzodiazepine receptors in mice.^[22] In addition, alpha-pinene reduces MK-801-induced abnormal behaviors.^[23]

However, several studies have reported the biological effects of alpha-pinene-containing extract.^{[14],[24],[25],[26]} Alpha-pinene enhances GABAA receptor function and increases postsynaptic GABA-dependent chloride flow in GABAA receptors.^[27] In addition, it has been reported that oral use of alpha-pinene, through binding with the GABA receptor benzodiazepine, produces a beneficial hypnotic agent.^[28] As previously reported, alpha-pinene is thought to affect the brain by stimulating the GABA receptor.^[29] Here, the results of bicuculline test also confirmed such issues.

In contrast, monoterpenes such as alpha-pinene, citronellal, citronellol, and myrcene have been reported as N-methyl-D-aspartate (NMDA) receptor antagonists.^[30] It has been demonstrated that NMDA antagonists can provide anti-Parkinsonian benefits in various experimental models.^[31] However, the interaction between alpha-pinene and NMDA receptors needs to be clarified in further studies.

It has been documented that oxidative stress plays a critical role in the pathogenesis of PD, causes molecular damage, and eventually leads to the dopaminergic neurodegeneration.^[32] Furthermore, 6-OHDA induces ROS production and causes phosphorylation of p38 and caspases, and finally cell death associated with caspase-3 in mesencephalic dopaminergic cells. Due to the release of cytochrome c from the mitochondria, some proteolytic enzymes, called caspases, are activated and eventually lead to DNA damage.^[33]

Deficiency in mitochondrial function can conduct the cell death system by releasing proapoptotic factors such as pro-caspases, caspase activators (i.e., cytochrome c), and caspase-independent factors. Then, cytochrome c release can induce apoptosis by activation of caspases, such as caspase-3.^[34] Regarding the above, oxidative stress is one of the main causes of apoptosis in Parkinson's disease. Numerous studies have been performed to reveal the usefulness of antioxidant agents in the prevention or treatment of PD. The antioxidant activity of monoterpenes and alpha-pinene has been demonstrated.^[35] It has been reported that alpha-pinene biological effects are directly related to its concentration.^[12]

In addition, studies have shown that the mechanism of action of terpenes such as alpha-pinene is based on the modulation of the apoptotic and antiapoptotic cascades, in addition, mitochondrial stress.^[10]

Conclusions

Taken together, these findings propose that the alpha-pinene protection against 6-OHDA-induced damage to dopaminergic SH-SY5Y cells may be mediated by its antioxidant and antiapoptotic activities via the reduction of ROS. Our data suggest that alpha-pinene exerts its effects mainly via pharmacological interfacing with GABAA receptors. However, further studies are needed to explore the details of alpha-pinene protective pathway.

Acknowledgment

The authors would like to thank the financial support from Kerman Neuroscience Research Center (KNRC#95-29).

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1.	Chaudhuri KR, Schapira AH. Non-motor symptoms of Parkinson's disease: Dopaminergic pathophysiology and treatment. Lancet Neurol 2009;8:464-74.
2.	Smeyne RJ, Jackson-Lewis V. The MPTP model of Parkinson's disease. Brain Res Mol Brain Res 2005;134:57-66.
3.	Tolleson CM, Fang JY. Advances in the mechanisms of Parkinson's disease. Discov Med 2013;15:61-6.
4.	Salamon A, Zádori D, Szpisjak L, Klivényi P, Vécsei L. Neuroprotection in Parkinson's disease: facts and hopes. J Neural Transm (Vienna). 2020; 127:821-9. doi: 10.1007/s00702-019-02115-8.
5.	Mazo NA, Echeverria V, Cabezas R, Avila-Rodriguez M, Tarasov VV, Yarla NS, et al. Medicinal plants as protective strategies against parkinson's disease. Curr Pharm Des 2017;23:4180-8.
6.	Borrajo A, Rodriguez-Perez AI, Villar-Cheda B, Guerra MJ, Labandeira-Garcia JL. Inhibition of the microglial response is essential for the neuroprotective effects of Rho-kinase inhibitors on MPTP-induced dopaminergic cell death. Neuropharmacology 2014;85:1-8.
7.	Zhang Y, Wu Y, Chen T, Yao L, Liu J, Pan X, et al. Assessing the metabolic effects of aromatherapy in human volunteers. Evid Based Complement Alternat Med 2013;2013:356381.
8.	Lis-Balchin M. Essential oils and 'aromatherapy': Their modern role in healing. J R Soc Health 1997;117:324-9.
9.	Falk A, Gullstrand E, Löf A, Wigaeus-Hjelm E. Liquid/air partition coefficients of four terpenes. Br J Ind Med 1990;47:62-4.
10.	Mbarki S, Alimi H, Bouzenna H, Elfeki A, Hfaiedh N. Phytochemical study and protective effect of Trigonella foenum graecum (Fenugreek seeds) against carbon tetrachloride-induced toxicity in liver and kidney of male rat. Biomed Pharmacother 2017;88:19-26.
11.	Burgess S, Ference BA, Staley JR, Freitag DF, Mason AM, Nielsen SF, et al. Association of LPA variants with risk of coronary disease and the implications for lipoprotein (a)-lowering therapies: A Mendelian randomization analysis. JAMA Cardiol 2018;3:619-27.
12.	Salehi B, Upadhyay S, Erdogan Orhan I, Kumar Jugran A, LD Jayaweera S, A Dias D, et al. Therapeutic Potential of α- and β-Pinene: A Miracle Gift of Nature. Biomolecules 2019;9.pii:E738. doi: 10.3390/biom9110738.
13.	Falk AA, Hagberg MT, Löf AE, Wigaeus-Hjelm EM, Wang ZP. Uptake, distribution and elimination of alpha-pinene in man after exposure by inhalation. Scand J Work Environ Health 1990;16:372-8.
14.	Mercier B, Prost J, Prost M. The essential oil of turpentine and its major volatile fraction (alpha- and beta-pinenes): A review. Int J Occup Med Environ Health 2009;22:331-42.
15.	Khoshnazar M, Bigdeli MR, Parvardeh S, Pouriran R. Attenuating effect of α-pinene on neurobehavioural deficit, oxidative damage and inflammatory response following focal ischaemic stroke in rat. J Pharm Pharmacol 2019;71:1725-33.
16.	Porres-Martínez M, González-Burgos E, Carretero ME, Gómez-Serranillos MP. In vitro neuroprotective potential of the monoterpenes α-pinene and 1,8-cineole against H2O2-induced oxidative stress in PC12 cells. Z Naturforsch C J Biosci 2016;71:191-9.
17.	Denizot F, Lang R. Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods 1986;89:271-7.
18.	Elyasi L, Eftekhar-Vaghefi SH, Esmaeili-Mahani S. Morphine protects SH-SY5Y human neuroblastoma cells against 6-hydroxydopamine-induced cell damage: involvement of anti-oxidant, calcium blocking, and anti-apoptotic properties. Rejuvenation Res 2014;17:255-63. doi: 10.1089/rej.2013.1473.
19.	Pouresmaeili-Babaki E, Esmaeili-Mahani S, Abbasnejad M, Ravan H. Protective effect of neuropeptide apelin-13 on 6-hydroxydopamine-induced neurotoxicity in SH-SY5Y dopaminergic cells: Involvement of its antioxidant and antiapoptotic properties. Rejuvenation Res 2018;21:162-7.
20.	Lipovsky A, Popa A, Pimienta G, Wyler M, Bhan A, Kuruvilla L, et al. Genome-wide siRNA screen identifies the retromer as a cellular entry factor for human papillomavirus. Proc Natl Acad Sci U S A 2013;110:7452-7.
21.	Andersson H, Björnström K, Eintrei C, Sundqvist T. Orexin A phosphorylates the γ-aminobutyric acid type A receptor β2 subunit on a serine residue and changes the surface expression of the receptor in SH-SY5Y cells exposed to propofol. J. Neurosci. Res 2015;93: 1748-55. 10.1002/jnr.23631.
22.	Yang H, Woo J, Pae AN, Um MY, Cho NC, Park KD, et al. α-Pinene, a major constituent of pine tree oils, enhances non-rapid eye movement sleep in mice through GABAA-benzodiazepine receptors. Mol Pharmacol 2016;90:530-9.
23.	Ueno H, Shimada A, Suemitsu S, Murakami S, Kitamura N, Wani K, et al. Attenuation effects of alpha-pinene inhalation on mice with dizocilpine-induced psychiatric-like behaviour. Evid Based Complement Alternat Med 2019;2019:2745453.
24.	Lv XN, Liu ZJ, Zhang HJ, Tzeng CM. Aromatherapy and the central nerve system (CNS): Therapeutic mechanism and its associated genes. Curr Drug Targets 2013;14:872-9.
25.	Ahmad A, Husain A, Mujeeb M, Khan SA, Najmi AK, Siddique NA, et al. A review on therapeutic potential of Nigella sativa: A miracle herb. Asian Pac J Trop Biomed 2013;3:337-52.
26.	Satou T, Kasuya H, Maeda K, Koike K. Daily inhalation of α-pinene in mice: Effects on behavior and organ accumulation. Phytother Res 2014;28:1284-7.
27.	Aoshima H, Hamamoto K. Potentiation of GABAA receptors expressed in Xenopus oocytes by perfume and phytoncid. Biosci Biotechnol Biochem 1999;63:743-8.
28.	Yang H, Woo J, Pae AN, Um MY, Cho NC, Park KD, et al. α-pinene, a major constituent of pine tree oils, enhances non-rapid eye movement sleep in mice through GABAA-benzodiazepine receptors. Mol Pharmacol 2016;90:530-9.
29.	Kasture VS, Chopde CT, Deshmukh VK. Anticonvulsive activity of Albizzia lebbeck, Hibiscus rosa sinesis and Butea monosperma in experimental animals. J Ethnopharmacol 2000;71:65-75.
30.	Guimarães AG, Quintans JS, Quintans LJ Jr., Monoterpenes with analgesic activity – A systematic review. Phytother Res 2013;27:1-15.
31.	Zhang Z, Zhang S, Fu P, Zhang Z, Lin K, Ko JK, et al. Roles of glutamate receptors in Parkinson's disease. Int J Mol Sci 2019;20:E4391.
32.	Blesa J, Trigo-Damas I, Quiroga-Varela A, Jackson-Lewis VR. Oxidative stress and Parkinson's disease. Front Neuroanat 2015;9:91.
33.	Oh K, Sarzi M, Schawinski K, Yi SK. Improved and quality-assessed emission and absorption line measurements in Sloan Digital Sky Survey galaxies. Astrophys J Suppl Ser 2011;195:13.
34.	Cunningham F, Achuthan P, Akanni W, Allen J, Amode MR, Armean IM, et al. Ensembl 2019. Nucleic Acids Res 2019;47:D745-51.
35.	Zamyad M, Abbasnejad M, Esmaeili-Mahani S, Mostafavi A, Sheibani V. The anticonvulsant effects of Ducrosia anethifolia (Boiss) essential oil are produced by its main component alpha-pinene in rats. Arq Neuropsiquiatr 2019;77:106-14.