Anethole

A Waters ACQUITY ultra-performance LC system (USA) was utilized to conduct the ultra-performance liquid chromatography (UPLC). A Waters ACQUITY^TM photodiode array detector (PDA) and HPLC column (Waters ACQUITY^TM BEH C₁₈ columns, 1.7 µm, 2.1 × 100), along with the software Empower, were employed for the analysis. The experiment involved the use of methanol (HPLC grade, Junsei, Tokyo, Japan), acetonitrile (HPLC grade, JT-BAKER, Radnor, PA, USA), and tertiary distilled water as reagents. The standard preparations of this experiment were obtained from Anethole (Sigma-Aldrich, St. Louis, MO, USA), R-(a)-phellandrene (Sigma-Aldrich, St. Louis, MO, USA), and 4-Methoxybenzoic acid (ChemFaces, Wuhan, China).

For our experiments, eugenol and estragole were purchased from Alfa Aesar—Thermo Fisher Scientific (Karlsruhe, Germany) and anethole from Sigma-Aldrich (Milan, Italy), all in liquid form and with purity ≥ 98%, and they were dissolved in assay buffer (Tris 50 mM, pH 7).

Unless otherwise mentioned, the culture media, ascorbic acid, anethole, eugenol and other chemicals used in the present experiments were purchased from Sigma-Aldrich (USA).

Human bone marrow derived mesenchymal stem cells (hMSCs, Cat# PT-2501) were obtained from Lonza (Walkersville, MD, USA) and cultured according to the manufacturer’s instructions. For adipocyte differentiation, cells were cultured for 4 weeks in high-glucose (25 mM) Dulbecco’s Modified Eagles Medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, 200 μM indomethacin, 1 μM dexamethasone, 10 μg insulin, and 0.5 mM isobutylmethylxanthine. DMEM, FBS, and penicillin-streptomycin were purchased from Corning (Oneonta, NY, USA). Components of adipogenic induction media and all reagents including anethole were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Lippia graveolens was collected from Cuatro Ciénegas, Coahuila de Zaragoza (26°59′ N 102°03′59″ OE), México. The specimens were identified in the Herbarium of the School of Biological Sciences, Universidad Autónoma de Nuevo León, México. Methanol, acetone (Tedia, Fairfield, OH, USA), and isopropyl alcohol (Chromadex, Los Angeles, CA, USA) were of HPLC grade. Purified water was from a Milli-Q water-purification system (Veolia, Boston, MA, USA). The standard solution n-alkanes (C₈–C₂₀, C₂₂, and C₂₄), myrcene (≥99.5%), p-cymene (≥97%), carvacrol (≥98%), anethole (99%) GC grade, and sodium alginate were purchased from Sigma-Aldrich, St. Louis, MO, USA. Eudragit L100-55 polymer (1:1 methacrylic acid: ethyl acrylate) was purchased from Evonik Industries, Essen, Germany.

Leaves of V. globosa were collected in Caatinga vegetation in Alagoinha country in the state of Pernambuco (Brazil). The collection site is located at latitude 08°27′59″ south and at longitude 36°46′33″ west. Taxonomic identification was carried out by experts at the Instituto Agronômico de Pernambuco (IPA) and the voucher specimens were deposited at the herbarium under the reference number 93.137. Legal registration for accessing and studying Brazilian genetic heritage was done in the Sistema Nacional de Gestão do Patrimônio Genético e do Conhecimento Traditional Associado (SisGen-A80E074). The material was hydrodistilled using a Clevenger apparatus to obtain the essential oil. Twenty grams of dried (in an oven with air circulation at 60 °C for 48 h) and crushed leaves were placed in 250 mL of de-ionized water with some pumice stones and the distillation procedure took 3 h. We recovered the essential oil droplet in 1 mL of xylene.
Anethole (99% purity) and caryophyllene (>98.5% purity) were provided by Sigma-Aldrich (Saint-Quentin Fallavier, France).
All stimuli were diluted in mineral oil and 10 μL of each solution was applied on a filter paper inserted into a Pasteur pipette. Pure mineral oil was used as a control stimulus.

Anethole was purchased from Sigma-Aldrich (Oakville, ON, Canada). It was diluted in methanol to reach a stock solution of 3 mM and used at various concentrations (0, 0.3, 3 and 30 μM).