Myrcene

The GC–MS analysis was carried out with an Agilent 6890N–5973N GC–MS system (Santa Clara, CA, USA) on a sample of M. aquatica EO prepared by dilution to 1:100 with n-hexane. The instrument was operating in the EI mode at 70 eV and using a HP-5MS (5% phenylmethylpolysiloxane, 30 m, 0.25 mm i.d., film thickness 0.1 µm) (J&W Scientific, Folsom, CA, USA) capillary column.
The chromatographic parameters and chromatogram analysis were the same as those reported by Nkuimi Wandjou et al. [27 (link)]. Briefly, the analytical standards of α-pinene, sabinene, β-pinene, myrcene, α-terpinene, p-cymene, limonene, 1,8-cineole, (Z)-β-ocimene, (E)-β-ocimene, γ-terpinene, terpinolene, terpinene-4-ol, α-terpineol, (E)-caryophyllene, and α-humulene were purchased from Merck (Milan, Italy) and used for peak assignments based on retention time and mass spectrum (MS). Moreover, the combination of the calculated linear retention index (RI) and MS was used to confirm the identity of the other compounds. Semi-quantitative values (peak area percentages) were obtained by peak normalization without using correction factors.

High-purity ROT and myrcene were obtained from Sigma-Aldrich (Missouri, MO, USA). Radioimmunoprecipitation assay (RIPA) buffer, antibodies for inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and glial fibrillary acidic protein (GFAP) were obtained from Sigma-Aldrich (Missouri, MO, USA). Protease and phosphatase inhibitor cocktails were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The anti-tyrosine hydrolase (polyclonal rabbit) antibody was purchased from Merck (Darmstadt, Germany). LC3, p62, mTOR, phospho-mTOR, and p70S6 antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). The apoptotic polyclonal markers, Bax and Bcl-2, were purchased from Abcam (Cambridge, MA, USA). The monoclonal mouse anti-α-synuclein antibody was purchased from BD Biosciences (San Jose, CA, USA). Anti-ionized calcium-binding adapter molecule 1 (Iba-1) antibody was procured from Wako Chemicals (Richmond, VA, USA). Alexa Fluor 488, a fluorescent secondary antibody, was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Biotinylated goat anti-rabbit secondary antibody was procured from Jackson Immune Research Laboratories (Baltimore Pike, West Grove, PA, USA). Biochemical assays were performed using kits available commercially. All other analytical grade chemicals used in these experiments were procured from local distributors.

Reference standards (+) -β-Pinene (≥98.5%), 1-Octen-3-ol, borneol (≥95%), butyric acid (≥99.5%), citral (≥95%), cis-3-hexen-1-ol (≥98%), diacetyl, dimethyl sulphide, dimethyl trisulphide (≥98.5%), 3-methylbutanal (≥97%), 2-methylbutanal (≥95%), ethyl 2-methylbutanoate (99%), ethyl 2-methylpentanoate (internal standard, ≥99%), ethyl-3-methylbutanoate (≥98%), 3-methylbutyl-2-methylpropanoate (≥98%), 2-methylbutyl-2-methylpropanoate (≥95%), and ethyl cinnamate (99%), ethyl 2-methylpropanoate (≥99%), ethyl 3-methylbutanoate (≥98%), ethyl 4-methylpentanoate (≥97%), ethyl butanoate (≥99,5%), ethyl hexanoate (≥99.5%), eugenol (99.6%), furaneol (≥99%), geraniol (≥99%), hexanol (≥99%), 3-methylbutyl acetate (≥97%), linalool (97%), menthol (99%), methyl nonanoate (≥99.8%), myrcene (≥90%), geranyl acetate (≥97%), dimethyl disulphide (≥90%), limonene (97%), hexanal (≥98%), 3-hexenol (≥95%), 2-phenyl ethanol (≥99%), β-caryophyllene (≥80%), α-humulene (≥96%), humulene oxide, S-methyl 5-methylpentanthioate S-methyl hexanthioate, S-methyl 4-methylpentanoate and theaspirane (≥90%) were purchased from Sigma Aldrich (St. Louis, MO, USA).

Type 90 hop pellets were used with regard to a higher homogeneity of the hop samples. One gram of type 90 pellets of 65 different commercially available brewing hops (listed in Electronic Supplementary Material (ESM) in Table S1) was dried with liquid nitrogen and ground by mortar and pestle. A total of 100 ± 10 mg of the resulting hop powder was transferred to a 20-mL headspace vial, 2 mL of a sodium chloride solution (300 g/L deionized water) was added, and the vial was sealed with a screw cap. All samples were measured in duplicate. Prior to sample preparation, the hops were stored in vacuum seal bags at 4 °C protected from light.
Stock solutions of reference compounds were prepared in sunflower oil (GLOBUS-Holding GmbH & Co. KG, St. Wendel, Germany) as solvent and in the concentration range of 10 mg/g for myrcene (Sigma Aldrich GmbH, Steinheim, Germany), β-caryophyllene (Carl Roth GmbH & Co. KG, Karlsruhe, Germany), and α-humulene (Sigma Aldrich); 1 mg/g for linalool (Sigma Aldrich) and limonene (Sigma Aldrich), as well as 0.1 mg/g for α-pinene (Acros Organics™ by Thermo Fisher GmbH, Kandel, Germany), and β-pinene (Alfa Aeser by Thermo Fisher GmbH, Kandel, Germany).

Analytical standard solutions of limonene, myrcene, β-pinene, and GC grade 1-hexanol were purchased from Sigma–Aldrich Corp (St. Louis, MO, USA). limonene and myrcene standards were used to check the quality of the analytical method (data are not shown). These compounds were chosen since they have been reported as major compounds of bitter orange EO [8 (link),18 (link)].
The fruits of the local bitter orange cultivar were taken at four ripening stages (September, October, November, and December) in 2016 from a garden located in Haji Abad, Bandar Abbas (South of Iran). Following literature recommendations, the fruits were sampled depending on the time [5 (link)] and color of the bitter oranges [8 (link)]. The fruits were collected based on their colors (green, yellow, light orange, and finally orange) with a one-month interval. The pigmented layer (flavedo) of the peel was separated from the soft white layer (albedo) with a sharp knife.