(W1030-12-78) Preparation of Propolis Extract and Its In Vitro Bioactivity Enhancement by Encapsulation

Purpose: Propolis is a natural product collected by honeybees with many promising biological activities. However, its poor solubility and low bioavailability reduce its effectiveness in vivo. To overcome these limitations, encapsulation techniques are frequently used. Spray drying is considered one of the most advantageous encapsulation techniques due to its abundant equipment availability, cost-effectiveness, and ability to maintain the stability and quality of the final product. Propolis has been already encapsulated by spray-drying in various carriers. Despite this, there have been rarely tested any biocompatible carriers from natural sources that could benefit from their own biological activity and consequently increase it even more. Carriers, such as glucan particles, can interact with the immune cells and promote immune responses. They are cheap and easily obtained from the cell walls of baker´s yeast. In addition, the use of these particles as a carrier improves dissolution properties. In our study, we aimed to prepare propolis extract and encapsulate it into glucan particles by spray-drying to enhance its bioactivity and dissolution properties.

Methods: Preparation of propolis extract: Crude propolis (Prague, Czech Republic, 2021) was frozen with liquid nitrogen and crushed. Solution of propolis in 70% ethanol (ratio 1:10) was prepared and stirred (300 rpm), at 30 °C. After 24 hours in the dark, the solution was sonicated and cleared of impurities by centrifugation and filtration. Then, the solvent was evaporated in a vacuum evaporator (200 rpm) at 30 °C and pure propolis sample was lyophilized. Dried and weighted extract was stored in amber glass vials at 4 °C. Preparation of glucan particles: Glucan particles (GPs) were obtained from baker´s yeast by a multistep procedure, based on a work of Saloň et al. This procedure involves the removal of internal organelles by alkali extraction, acidification and washing with deionized water, isopropanol, and acetone. The final powder was lyophilized and stored in a cold and dark place. Encapsulation by spray-drying: Homogenized ethanolic suspension of glucan particles (20 mg/ml) and pre-dissolved propolis was used as feed for the encapsulation. It was performed on a Mini Spray Dryer B‑290 (Buchi AG, Switzerland) with an ultrasonic nozzle under a nitrogen atmosphere. The inlet gas temperature was 120 °C, the flow rate of the suspension was 5 ml/min and the ultrasonic nozzle was set to 2.4 W. The outlet qas temperature was 70 °C. Parameters were set according to the work of Ruphuy et al. Characterization of glucan particles/propolis composites: Surface morphology and shape were obtained by scanning electron microscopy (Joel JCM-5700 microscope). Samples were coated before analysis with a thin layer (5 nm) of gold (Emitech K550X). The crystallinity of the samples was checked by X-ray diffraction using Panalytical X’Pert PRO with High Score Plus diffractometer. Samples were also measured using FTIR spectrometer Nicolet iZ10. Dissolution tests: The release of propolis from glucan particles was observed in single-phase dissolution using a 200 mL USP Apparatus II filled with a phosphate buffer (pH 6.8), at a stirring speed 150 rpm and a temperature of 37 °C. Samples were filtered collected at pre-defined time points (0 to 60 min). Anti-inflammatory testing: Nitric oxide production and cytokine release (TNFα, IL-6) was quantified using RAW 264.7 cells. In this work, 0.04 g/mL of Griess reagent in deionized water was used for NO quantification. Cytokines release was quantified after 24 h in the supernatant of LPS-stimulated cells by ELISA. The concentration of samples, that were cytotoxic, were excluded.

Results: Characterization of glucan particles/propolis composites: By scanning electron microscopy were characterized empty and loaded glucan particles. Both show the same ellipsoidal shape, wrinkled surface, and size particle between 2-5 μm. No mass was detected outside the glucan particles. XRD spectra showed that propolis extract, so as encapsulated one, is in amorphous form. The FTIR analysis was performed to observe the structural changes after encapsulation. Indication of some interactions appeared with a peak shift around 1550 cm^-1. Increasement in the intensity can be attributed to the successful encapsulation. Dissolution tests: A significant improvement in the dissolution rate of propolis extract after encapsulation occurred. In the case of propolis extract, the maximum concentration was reached within 5 minutes and remained stable (2.20 ± 0.01 mg/L). With loaded particles, there was a rapid release in the first 5 minutes and after 25 minutes reached its solubility limit (14.50 ± 2.00 mg/L) and remained constant. Anti-inflammatory effect: Nitric oxide production was significantly reduced by propolis formulation. The encapsulation increased anti-inflammatory activity of propolis solubilized in cultivation medium. This effect was comparable to usage of organic solvents.

Conclusion: Propolis was successfully encapsulated in glucan particles by spray-drying. By using this carrier, we achieved a sixfold increase in the solubility of propolis, and much higher anti-inflammatory activity. This improvement was also due to the additional contribution of the glucan particles’ own bioactivity. This approach may lead to the direct application of glucan particles/propolis composites.

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Acknowledgements:The financial support from the Specific University Research (MSMT), of the Czech Sceince Foundation (project No. GX19-26127X).

Preparation of propolis extract.

Dissolution kinetics of propolis extract and spray-dried glucan particles/propolis composites in 200 mL media (pH 6.8) at 37 °C and mini-paddles set as 150 rpm, 8 mg of dose.

Concentration of propolis and glucan halving production of NO, TNFα and IL-6 by LPS-stimulated macrophages (RAW 264.7).