Uip1000hdt

The preparation of the nanocarriers was carried out as described in patent P202230668 [27 ]. The synthesis of methacrylated chitosan was conducted according to the procedure proposed by Gupta and Gupta [20 (link)], but with modifications. In brief, the methacrylation of COS was performed by the addition of 420 mg of oligomers which were dispersed in a solution of methacrylic anhydride in tetrahydrofuran (THF), which was obtained by dispersing 0.5 mL of MA (ρ = 1.035 g·cm⁻³) in 25 mL of THF. The mixture was sonicated for 5 min (distributed in 1 min periods) using a probe-type ultrasonicator (model UIP1000hdT; 1000 W, 20 kHz; Hielscher Ultrasonics, Teltow, Germany). The co-encapsulating chemical species was a porous form of g-C₃N₄ resulting from the attack of 210 mg of g-C₃N₄ with MA in THF (0.5 mL in 25 mL). The methacrylated g-C₃N₄ solution was then added dropwise to the methacrylated COS solution, which was followed by sonication for 5 min (distributed in 1 min periods) to obtain a g-C₃N₄:COS weight ratio of 0.5:1 (with an unknown MA proportion). It should be clarified that the other assayed weight ratios did not result in the formation of NCs or lead to non-monodisperse size distributions. The excess MA was removed by agitation and successive washings.

The crushed, dried plant material was extracted with 70% (v/v) ethanol. The solvent/plant material ratio (30 mL/g) has been established following the European Pharmacopeia 9th Edition. Sonochemistry concepts were used to establish a technology for extracting and concentrating bioactive compounds from the L. selago. The extraction was done with a Hielscher ultrasonic processor (Hielscher UIP1000hdT Berlin, Germany), with diameters of 40 mm, 1000-Watt, 20 kHz adjustable amplitude (amplitude ratio 1:0.7). Lastly, the samples were centrifuged (2.500 × g for 5 min at room temperature), and the supernatant obtained was subjected to the elimination of alcohol at 35 °C. The lyophilization process was used to remove the residual water and alcohol traces from the sampling process. The extract has been maintained at 4 °C for further study.

Sambucus nigra samples were collected in June 2022, in Alerre (Huesca, Spain; 42°09′27.4″ N 0°27′50.6″ W). A voucher specimen, identified and authenticated by Prof. J. Ascaso, has been deposited at the herbarium of the Escuela Politécnica Superior, Universidad de Zaragoza (Huesca, Spain). Aerial parts from different specimens (n = 20) were thoroughly mixed to obtain (separate) flowers and leaves composite samples. The composite samples were shade-dried, pulverized to a fine powder in a mechanical grinder, homogenized, and sieved (1 mm mesh).
An aqueous ammonia solution was chosen to dissolve the bioactive compounds of interest. The flower extract was prepared according to the procedure described in [26 (link)]: the flowers powder (30 g) was first digested in an aqueous ammonia solution (140 mL H₂O + 10 mL NH₃) for 2 h, then sonicated in pulsed mode (with a 2 min stop every 2.5 min) for 10 min using a probe-type ultrasonicator (model UIP1000hdT; 1000 W, 20 kHz; Hielscher Ultrasonics, Teltow, Germany), and then allowed to stand for 24 h. It was then adjusted to neutral pH using acetic acid. Finally, the solution was centrifuged at 9000 rpm for 15 min, and the supernatant was filtered through Whatman No. 1 paper. The extraction procedure for leaf samples was identical.
Aliquots of both extracts were freeze-dried for Fourier transform infrared (FTIR) analyses.

Gum nanoparticles containing OPE were produced by modified methods [14 (link)]. The prepared gum solutions (solvent) were mixed at 800 rpm for 5 min. OPE (0.1%) and Tween 20 (0.5%) was dissolved in optimized amounts of ethanol (Antisolvent). Tween 20 was used for better dissolution of OPE in ethanol. Ethanolic OPE (0.5 mL/min) was added dropwise to the gum solution (Solvent Phase) using a syringe pump system (New Era, NE, USA). After adding the organic phase, the solution was stirred at 800 rpm for 10 min. Then, by using ultrasonic processor (Hielscher UIP1000hdT, Germany), ultrasonication of 100 W was applied to the solutions for 1 min (every 30 s wait 10 s) in an ice bath. The remained nanoparticle suspensions were centrifuged at 9000 rpm for 30 min and the supernatant was discarded and Tween 20 was removed by centrifugation. Nanoparticles were redispersed with 5 mL of distilled water and then freeze-dried without using cryoprotectants. The same experimental procedure without OPE and Tween 20 was applied for blank gum nanoparticle production.

The
desolvation technique was used to fabricate gum nanoparticles. The
gum nanoparticles were prepared through using a desolvation method
by dropwise addition of the desolvating agent (ethanol) continuously.
The modified version of the method described by Taheri et al.^{23 (link)} was used in this study.^{22 (link)} The gum solutions (solvent) were blended at 800 rpm for 5 min. OPE
(0.1%) and Tween 20 (0.5%) were added in optimized amounts of ethanol
(antisolvent). Tween 20 was used for the better dissolving of OPE
in ethanol. Ethanolic OPE (0.5 mL/min) was put dropwise to the gum
solution (solvent phase) using a syringe pump system (New Era, NE,
USA). After adding the ethanol, the solution was stirred at 800 rpm
for 10 min. Then, ultrasonication (Hielscher UIP1000hdT, Germany)
of 100 W was performed to the solutions for 1 min (every 30 s wait
for 10 s) in an ice bath. The nanoparticle suspensions were centrifuged
at 9000 rpm for 30 min. Nanoparticles were redispersed with 5 mL of
distilled water and then freeze-dried without using cryoprotectants.
The same experimental analysis without OPE and Tween 20 was performed
for blank gum nanoparticle fabrication.

An aqueous ammonia solution was chosen to dissolve the bioactive compounds of interest contained in the bark of holm oak. The bark extract was prepared according to the procedure described in [60 (link)]. Briefly stated, a probe-type ultrasonicator (model UIP1000hdT; 1000 W, 20 kHz; Hielscher Ultrasonics, Teltow, Germany) was used to sonicate the bark sample for 10 min in pulse mode with a 2 min break after every 2.5 min of sonication, and the sample was then left to settle for 24 h. Acetic acid was then used to change the pH to neutral. After 15 min of centrifuging the solution at 9000 rpm, the supernatant was filtered using Whatman No. 1 paper.