R 210

Ficus carica (L.), (white variety: Lembdar labiad) was identified and collected from Sefrou city in Morocco (33°50′57″ N, 4°51′40″ W) in 2020. The samples are available at the herbarium of the Faculty of Sciences Dhar El Mehraz, Fez, Morocco, under the following voucher names: (WL20) for Ficus carica (L.) leaves and (WB21) for Ficus carica (L.) buds.
The leaves were cleaned and air-dried for 15 days, then turned into fine powder. Fresh buds were put in the fridge at 4 °C until it was used to prepare extracts used in the experiments.
The extracts of the leaves and buds of Ficus carica (L.) were prepared as follows: 500 g of the plant (dried leaves or fresh buds) were macerated in hydro-methanolic solution (70% v/v) for one week under agitation at 150 rpm at room temperature. The obtained solution was filtered using a Whatman filter paper no. 1 and then concentrated in a rotary evaporator (Büchi R-210, Flawil, Switzerland) at 40 °C under reduced pressure to obtain a solid residue [29 (link)]. This later was dissolved in distilled water to obtain the desired concentrations selected for the in vitro and in vivo experiments.

The determination of the drug content of the NPs was carried out as previously described^{12 (link)} . Around 5 mg of lyophilized NPs were accurately weighed using a high-precision analytical balance (d = 0.01 mg; Model CP 225D; Sartorius AG, Göttingen, Germany). Following, 5 mL of acetone were added and the mixture was accurately vortexed to dissolve the particles in the organic phase. Acetone was then evaporated using a rotavapor (Büchi R/210, BüchiLabortechnik AG, Flawil, Switzerland). Next, 1 mL of ethanol was added and the Δ⁹-THC content solved under sonication (Branson 50 W, Branson Ultrasonics Corporation, Danbury, CT, USA) and vortexing for 5 min. The obtained drug solution, was then filtered (0.22 µm syringe filter, Millex® GV, Millipore, Barcelona, Spain) and injected into the HPLC system for Δ⁹-THC detection following a validated method¹⁰ .
The drug content was expressed as entrapment efficiency (EE %) and drug loading (DL %) following the Eqs. (1 and 2): $$EE\%=\left(\frac{actualamountof\mathrm{\Delta }9-THCloadedinNPs}{theoreticalamountof\mathrm{\Delta }9-THCinNPs}\right)\times 100$$ $$DL\%=\left(\frac{massof\mathrm{\Delta }9-THCinNPs}{massofNPsrecovered}\right)\times 100$$

The chitosan-coated nanocapsules were prepared as previously described with modifications [17 ,19 ]. Briefly, for the in vitro experiments, 400 μl of a 100 mg/ml ethanolic lecithin solution (Epikuron 145 V, Cargill texturing solutions Deutschland GmbH &Co. KG, Hamburg, Germany) was mixed with 530 μl of the capsaicin stock solution (24 mg/ml). This was supplemented with 125 μl Miglyol 812 N (Sasol GmbH, Witten, Germany) and 9.5 ml ethanol. The organic solution was immediately poured into 20 ml chitosan in the aqueous phase (0.5 mg/ml in 5% stoichiometric excess of HCl). The milky mixture was concentrated in a rotavapor (Büchi R-210, Büchi Labortechnik GmbH, Essen, Germany) at 50°C until 3.5–4.0 ml remained and the volume was topped up to 4.0 ml with milliQ water if necessary to yield a final capsaicin concentration of ~10 mM. The nanoemulsions were prepared using the same procedure but without including chitosan. Unloaded nanocapsules and nanoemulsions were prepared by replacing the capsaicin solution with ethanol. The physical characteristics of the nanocapsules have been documented in a previous study [17 ]. Briefly, the size of the formulations was around 200 nm, the zeta potential was highly positive (~ +60 mV) and the polydispersity index had a value of ~ 0.2. These values were obtained by dynamic light scattering.