Myrtenol

For identification of volatiles the following analytical standards were purchased from Sigma-Aldrich (Milan, Italy): α-pinene, β-pinene, p-cymene, limonene, γ-terpinene, linalool, trans-pinocarveol, terpinen-4-ol, α-terpineol, myrtenol, nerol, carvone, geraniol, geranial, eugenol, (E)-caryophyllene, α-humulene, (E)-β-farnesene, (E)-β-ionone, (E)-nerolidol, caryophyllene oxide, n-pentacosane, n-heptacosane; (E)-Phytol was previously isolated from Onosma echioides [43 (link)]. For retention-index (RI) determination, a mixture of hydrocarbons, ranging from octane (C₈) to triacontane (C₃₀) (Supelco, Bellefonte, PA, USA) was used and run under the experimental conditions reported below. All compounds were of analytical standard grade. Analytical-grade hexane solvent was purchased from Carlo Erba (Milan, Italy); it was successively distilled through a Vigreux column before use.

The standards, including β-Myrcene (90.0%), Limonene (99.0%), terpinolene (94.0%), cis-rose oxide (96.0%), cis-furan linalool oxide (98.0%), linalool (96.0%), 4-terpinenol (95.0%), menthol (98.0%), β-citronellal (99.0%), myrtenol (95.0%), α-terpineol (99.0%), nerol (98.0%), neral, geraniol (97.0%), and geranial (98.0%), were obtained from Sigma-Aldrich (St. Louis, MO, USA). In total, 98% of 4-methyl-2-pentanol (internal standard) was also obtained from Sigma-Aldrich. The purified water used in this research was obtained from a Master Touch purification system (Shanghai, China). Polyvinylpolypyrrolidone (PVPP) was a product of huayueyang (Beijing, China). SYBR^® Premix Ex TaqTM and Plant Total RNA Kit were respectively purchased from TaKaRa Bio (Otsu, Shiga, Japan) and Gene-Better Life Science (Beijing, China). The reverse transcription system kit was purchased from Promega (Madison, WI, USA). All other reagents used in this study were obtained from the Beijing Chemical Works (Beijing, China), unless otherwise specified.

In the tested process, the following reagents were used for oxidation carried out with the use of titanium-silicate TS-1 catalysts: α-pinene (98%, Sigma Aldrich, Poznań, Poland), and oxygen (99.99%, Messer, Szczecin, Poland). Moreover, the following compounds were used as standards for chromatographic analyzes: α-pinene oxide (97%, Sigma Aldrich, Poznań, Poland), verbenol (95%, Sigma Aldrich, Poznań, Poland), verbenone (≥93%, Sigma Aldrich, Poznań, Poland), trans-pinocarveol (≥96%, Sigma Aldrich, Poznań, Poland), myrtenal (98%, Sigma Aldrich, Poznań, Poland), myrtenol (95%, Sigma Aldrich, Poznań, Poland), carveol (≥95%, Sigma Aldrich, Poznań, Poland), carvone (98%, Sigma Aldrich, Poznań, Poland), pinanediol (99%, Sigma Aldrich, Poznań, Poland). Campholenic aldehyde was identified by GC-MS method.

Myrtenol was purchased from Sigma-Aldrich Corporation (W343900, CAS-No.19894-97-4, purity ≥95%; Figure 1). Seventy-five SD rats were randomly divided into five groups (n = 15): sham group: rats were treated with saline but without middle cerebral artery occlusion (MCAO). MCAO group: rats were given with saline alone with cerebral ischaemia/reperfusion (I/R) surgery. MCAO + Myr groups: rats were intraperitoneally injected with 10, 30, or 50 mg/kg Myrtenol after cerebral I/R surgery, respectively (Britto et al. 2018 (link)), once daily for seven consecutive days. The remaining 60 SD rats were randomly divided into four groups (n = 15): sham group, MCAO group, MCAO + Myr (50 mg/kg) group: rats were treated as above, MCAO + Myr + U0126 group: rats were intracerebroventricularly injected with 10 μL U0126 (Cell Signalling Technology, USA), a highly selective inhibitor of MEK (Ahnstedt et al. 2015 (link)), into the cerebral ischaemia side at 30 min prior to Myrtenol treatment, once daily for 7 consecutive days. The intracerebral ventricular injection and the dose of U0126 were based on the work of Ye et al. (2018 (link)).

Hydrochloric acid, acetic acid, methanol, sodium carbonate, iron chloride, ammonium acetate and Folin–Ciocalteu reagent were purchased from the manufacturer Kemika (Zagreb, Croatia). Trolox was purchased from Sigma (Darmstadt, Germany). 2,2’-azinobis (3-ethylbenzothiozolinesulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Fluka (Darmstadt, Germany). 2,4,6,-tri (2-pyridyl)-s-triazine (TPTZ), gallic acid, procyanidin B2 and myrtenol were purchased from Sigma, Germany. Neocuproine and copper chloride are products of Gram-mol Zagreb, Croatia). Sucrose was purchased from Gram-mol (Zagreb, Croatia), and maltose and trehalose were obtained from Hayashibara doo (Nagase group, Tokyo, Japan). Citrus fibers were obtained from Biesterfeld AG (Zagreb, Croatia).

Apple fibers were obtained from Biesterfeld AG (Zagreb, Croatia). 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 4-(dimethylamino)-cinnamaldehyde (DMAC), trolox, 2,2-diphenyl-1-picrylhydrazyl (DPPH), rutin, quercetin, phloretin, phlorizin, chlorogenic, caffeic, p-coumaric, gallic, ellagic acids, myrtenol and α-amylase (from the porcine pancreas) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Potassium persulfate, Folin–Ciocalteu reagent and sodium carbonate were purchased from Kemika (Zagreb, Croatia). Neocuproine, 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ) and copper (II) chloride were bought from Acros Organics (Geel, Belgium). Methanol (HPLC grade) was from J.T. Baker (Deventer, Netherlands) and orthophosphoric acid (HPLC grade > 85%) was from Fisher Scientific (Loughborough, UK). Iron (III) chloride hexahydrate, sodium acetate, ethanol, ammonium acetate and starch were bought from Gram-mol (Zagreb, Croatia). Cyanidin 3-glucoside was from Extrasynthese (Genay, France) and 3,5-dinitrosalicylic acid from Alfa Aesar (Kandel, Germany).

Trolox, 2,4,6,-tri (2-pyridyl)-s-triazine (TPTZ), gallic acid, procyanidin B2 and myrtenol were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hydrochloric acid, acetic acid, methanol, sodium carbonate, iron chloride, ammonium acetate and Folin-Ciocalteu reagent were purchased from Kemika (Zagreb, Croatia). 2,2′-azinobis (3-ethylbenzothiozolinesulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Fluka (Buchs, Switzerland). Neocuproine and copper chloride were obtained from Gram-mol (Zagreb, Croatia). Sucrose was purchased from Gram-mol (Zagreb, Croatia), while trehalose was obtained from Hayashibara doo (Okayama, Japan). Citrus fibers were obtained from Biesterfeld AG (Zagreb, Croatia).