<abstract xmlns="http://www.w3.org/1999/xhtml">

<p>The subject of the research was the oleogel structure of the OraMAF oral suspension, containing a mixture of active substances in which the oleogel environment is formed by purified olive oil in combination with soy lecithin (SL) and sorbitan tristearate (STS). The objective of the research was the development of an optimal methodology for the work process, enabling microscopic observation of the structure of the oleogel suspension. Purified olive oil structured with a combination of gelators SL and STS with the addition of a solid phase of fucoidan (F) and chondroitin sulfate (CS) powders was used for the study as an alternative to OraMAF suspension. The sedimentation method of separation of the medium and staining of solid phases of the suspension brought the expected results, allowing the observation of the network structure of the gel, which consisted of interlaced gelator fibers assembling into star formations. The most interesting structures were clearly the star-like structures created from the star-shaped microtubules trapping the CS and F solid particles, colored blue.</p>
</abstract>

The subject of the research was the oleogel structure of the OraMAF oral suspension, containing a mixture of active substances in which the oleogel environment is formed by purified olive oil in combination with soy lecithin (SL) and sorbitan tristearate (STS). The objective of the research was the development of an optimal methodology for the work process, enabling microscopic observation of the structure of the oleogel suspension. Purified olive oil structured with a combination of gelators SL and STS with the addition of a solid phase of fucoidan (F) and chondroitin sulfate (CS) powders was used for the study as an alternative to OraMAF suspension. The sedimentation method of separation of the medium and staining of solid phases of the suspension brought the expected results, allowing the observation of the network structure of the gel, which consisted of interlaced gelator fibers assembling into star formations. The most interesting structures were clearly the star-like structures created from the star-shaped microtubules trapping the CS and F solid particles, colored blue.


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<sec id="j_afpuc-2023-0001_s_001"><div>INTRODUCTION</div>
<p>Oleogels are a specific part of the category of gels characterized by the presence of an internal three-dimensional network, capable of capturing either liquid or solid particles in a lipophilic environment. Research into the microstructure of oleogels aims to bring new knowledge about the physical properties of systems that find wide application in the food industry as well as in the formulation of innovative drug forms (<italic><a ref-type="bibr" href="#j_afpuc-2023-0001_ref_001">Co et Marangoni, 2012</a></italic>). Based on the analysis of the internal oleogel structure and thanks to the knowledge of the mechanisms of its formation, we can better evaluate and use suitable gelators in practice. In terms of the minimum concentration inducing gel formation, oleogelators meeting all the requirements that could be functional at the minimum concentration were looked for. The preparation of such gels requires correct procedures, and through research, we tried to achieve the efficiency of the available methods. We try to investigate the mechanisms by which solid particles of potential drugs can be retained in a specific structure and distinguish in which formulations the best fixation is ensured. Depending on the type of applied oleogelator, several colloidal structures can be assembled in the oleogel, each of which has its own specific formation mechanism (<a ref-type="fig" href="#j_afpuc-2023-0001_fig_001">Fig. 1</a>; <italic><a ref-type="bibr" href="#j_afpuc-2023-0001_ref_002">Li et al., 2022</a></italic>).</p>
<figure id="j_afpuc-2023-0001_fig_001" position="float" fig-type="figure"><h2>Figure 1.</h2>
<figCaption><p>Mechanisms of oleogelation (<a ref-type="bibr" href="#j_afpuc-2023-0001_ref_002">Li et al., 2022</a>).</p></figCaption>
<img xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="graphic/j_afpuc-2023-0001_fig_001.jpg" src="https://sciendo-parsed.s3.eu-central-1.amazonaws.com/6470851571e4585e08a9f136/j_afpuc-2023-0001_fig_001.jpg?X-Amz-Algorithm=AWS4-HMAC-SHA256&amp;X-Amz-Date=20240629T032712Z&amp;X-Amz-SignedHeaders=host&amp;X-Amz-Expires=18000&amp;X-Amz-Credential=AKIA6AP2G7AKP25APDM2%2F20240629%2Feu-central-1%2Fs3%2Faws4_request&amp;X-Amz-Signature=2d9cf83550a5d9234bf1e04e97247c3a388e3e5b5140cd961f12089c89006b91" class="mw-100"/>
</figure>
<p>The physical properties of oleogels prepared using low-molecular-weight gelators can be optimized by combining several low-molecular-weight gelators. Such mixtures used for the gelation of edible oils form crystals with mixed self-assembled structures. Typical representatives include combinations of lecithin and sorbitan tristearate (STS). It is a way of influencing the thermal behavior of the oleogel, providing an improvement of the rheological and textural properties by changing the ratio of the components involved in the formation of the gel (<italic><a ref-type="bibr" href="#j_afpuc-2023-0001_ref_004">Toro-Vazquez et al., 2007</a>; <a ref-type="bibr" href="#j_afpuc-2023-0001_ref_003">Patel, 2015</a></italic>).</p>
<sec id="j_afpuc-2023-0001_s_001_s_001"><div>Sorbitan and lecithin esters</div>
<p>An example of a two-component mixture of gelators is lecithin–STS oleogels. None of these substances can independently formulate a gel from oil. However, when their mixture is used in the right proportions, a synergistic connection occurs, leading to successful oleogelation. If this is the minimum concentration of the mixture inducing the formation of a gel, a mass concentration of 4% is sufficient.</p>
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<sec id="j_afpuc-2023-0001_s_002"><div>MATERIALS AND METHODS</div>
<p>In the present work, an oral suspension OraMAF was used for microscopic observation of the gel structure. As an alternative to OraMAF suspension, for observation, we used the suspension of purified olive oil structured with a combination of gelators soy lecithin (SL) and STS with a solid phase of fucoidan (F) and chondroitin sulfate (CS) powders.</p>
<sec id="j_afpuc-2023-0001_s_002_s_001"><div>Oleogel suspension</div>
<p>The subject of the research was the oleogel structure of the oral suspension OraMAF, containing a mixture of active substances, in which the oleogel environment is created by purified olive oil in combination with SL and STS, performing the role of gelling agents. Other components of the investigated mixture were glycosaminoglycans, fructooligosaccharides, and heteropolysaccharides, low-molecular-weight CS, F, inulin, dextrose, cholecalciferol, and live bacterial strains of <italic>Lactobacillus delbrueckii</italic>, subsp. <italic>bulgaricus</italic> and <italic>Streptococcus thermophilus</italic>. The oleogel environment protects suspended substances from oxygen and moisture, thus providing increased microbiological stability (<italic><a ref-type="bibr" href="#j_afpuc-2023-0001_ref_005">Zima, 2021</a></italic>).</p>
<p>The goal of the research was the development of an optimal methodology for the work process, enabling microscopic observation of the structure of the oleogel suspension.</p>
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<sec id="j_afpuc-2023-0001_s_003"><div>RESULTS AND DISCUSSION</div>
<p>Microscopic observation of the structure of the untreated suspension sample did not yield the expected results. The sample contained a large number of air bubbles and residues of insoluble lecithin components interfering with the observation (<a ref-type="fig" href="#j_afpuc-2023-0001_fig_002">Fig. 2</a>). Due to the matching color, there was a lack of contrast resolution, making it impossible to observe the created network structures in the oil environment and the method of trapping particles.</p>
<figure id="j_afpuc-2023-0001_fig_002" position="float" fig-type="figure"><h2>Figure 2.</h2>
<figCaption><p>A sample of OraMAF suspension in olive oil, observed with a light microscope, magnification 400×.</p></figCaption>
<img xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="graphic/j_afpuc-2023-0001_fig_002.jpg" src="https://sciendo-parsed.s3.eu-central-1.amazonaws.com/6470851571e4585e08a9f136/j_afpuc-2023-0001_fig_002.jpg?X-Amz-Algorithm=AWS4-HMAC-SHA256&amp;X-Amz-Date=20240629T032712Z&amp;X-Amz-SignedHeaders=host&amp;X-Amz-Expires=18000&amp;X-Amz-Credential=AKIA6AP2G7AKP25APDM2%2F20240629%2Feu-central-1%2Fs3%2Faws4_request&amp;X-Amz-Signature=5c83d99efad1b62f9a0e7a35e275a976ff538e3eadf09b4582d91fbd4967d66e" class="mw-100"/>
</figure>
<p>It was not possible to distinguish the gel structure. The observation was invalidated by the presence of a number of air bubbles created during preparation by high-speed mixing. Irregularly shaped brown areas represent insoluble lecithin residues (<a ref-type="fig" href="#j_afpuc-2023-0001_fig_002">Fig. 2</a>).</p>
<sec id="j_afpuc-2023-0001_s_003_s_001"><div>Dispersion of STS and SL in olive oil using filtration</div>
<p>We tried to separate the insoluble lecithin residues by the filtration method. Gelators STS and SL were dissolved in olive oil by heating to a temperature of 80°C. The mixture was stirred during heating until the target temperature was reached with a common laboratory stirrer to prevent the formation of air bubbles. Subsequently, the mixture was allowed to cool to room temperature. To separate the insoluble residues of SL, filtration under ordinary pressure using a cellulose filter partition was subsequently used. The mentioned preparation method did not prove successful as the dispersion showed considerable viscosity and insoluble residues of SL and the gel structure clogged the filter partition, which made normal filtration impossible.</p>
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<sec id="j_afpuc-2023-0001_s_003_s_002"><div>Dispersion of STS and SL in olive oil using sedimentation separation</div>
<p>Batches of STS and SL were dissolved in olive oil by heating to a temperature of 80°C. The mixture was stirred with a high-speed stirrer during heating until the target temperature was reached. Subsequently, the dispersion was allowed to slowly cool to room temperature. The dispersion prepared in this way, containing air bubbles and crushed insoluble residues of SL, was allowed to settle for 14 days at room temperature. The clear supernatant of the dispersion was separated from the sediment containing the remains of insoluble SL by the mentioned procedure. This method proved to be technically undemanding, practical, and repeatable and was suitable for the preparation of clear oleogel dispersion as an optimal environment for microscopic observation of the gel structure and the way solid particles are trapped in the gel structure.</p>
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<sec id="j_afpuc-2023-0001_s_003_s_003"><div>Staining of solid samples</div>
<p>To increase the contrast of the observed dispersion and capture of solid particles, colorless powder samples of low-molecular-weight CS and F were colored using organic dyes. Both investigated substances are well soluble in water. It follows that it was not possible to use an organic dye soluble in water, since after mixing an aqueous solution of the dye with a solid substance, the solid substance itself would dissolve. Therefore, dyes soluble in an ethanol/propanol mixture in a volume ratio of 1:2 were used, while solid substances did not dissolve in the said mixture. Solutions of methylene blue and organic red were used to stain solids. The dye solution was mixed with the solid and then the solvent was evaporated. Using this procedure, colored samples of solids suitable for microscopic observation were obtained.</p>
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<sec id="j_afpuc-2023-0001_s_003_s_004"><div>Microscopic observation of the gel structure</div>
<p>The sedimentation method of separation of the oleogel medium and the coloring of the solid phase of the suspension with organic dyes brought the expected results, allowing the observation of the network structure of the gel capturing solid particles (<a ref-type="fig" href="#j_afpuc-2023-0001_fig_003">Fig. 3</a>).</p>
<figure id="j_afpuc-2023-0001_fig_003" position="float" fig-type="figure"><h2>Figure 3.</h2>
<figCaption><p>Microscopic observation of 3% soy lecithin + 3% STS + fucoidan + LMWCS in olive oil, magnification 400×.</p></figCaption>
<img xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="graphic/j_afpuc-2023-0001_fig_003.jpg" src="https://sciendo-parsed.s3.eu-central-1.amazonaws.com/6470851571e4585e08a9f136/j_afpuc-2023-0001_fig_003.jpg?X-Amz-Algorithm=AWS4-HMAC-SHA256&amp;X-Amz-Date=20240629T032712Z&amp;X-Amz-SignedHeaders=host&amp;X-Amz-Expires=17999&amp;X-Amz-Credential=AKIA6AP2G7AKP25APDM2%2F20240629%2Feu-central-1%2Fs3%2Faws4_request&amp;X-Amz-Signature=564a69a3a0723c14a7735b69701cfab0326632172edbd22e9c2975a9b41ebd08" class="mw-100"/>
</figure>
<p>Investigation of the dispersion formed by SL and STS brought insight into the direct mechanism of gel network formation. The most interesting structures were clearly the star-like shapes created from the microtubules of the STS and lecithin complex, colored blue. The coloring was provided by the added dye methylene blue, which was partially adsorbed on the gelling substances, thus enhancing the contrast and enabling the observation itself. The results of the observation confirm that a combination of STS and SL forms fibrous three-dimensional structures of various lengths, intertwined and connected in some places into typical star-shaped structures (<a ref-type="fig" href="#j_afpuc-2023-0001_fig_003">Fig. 3</a>). It can be assumed that the growth of fibers starts at central points and further branches out in several directions. The initial step is thus the nucleation of nuclei. From the point of view of the capture of solid particles, it was possible to identify individual particles of F and CS, distributed over the entire surface of the sample. Macroscopically, it was also possible to observe that these solid components do not sediment because they are stabilized in the formed solid gel network. The stability is disturbed when the temperature increases when the gel melts.</p>
<p>The main goal of the experiment was to prepare and optimize the procedure for preparing an oleogel suspension using combined gelators suitable for microscopic observation. Sedimentation separation of the medium and staining of the solid phase of the suspension enabled the microscopic observation of the three-dimensional structure of the oleogel, which consisted of interlaced gelator fibers assembling into star formations. Solid connections also provide an excellent basis for maintaining solid particles, which can be represented by similar bioactive molecules. In pharmaceutical technology, the full potential of oleogelation is still not sufficiently utilized and the transfer of knowledge from the theoretical level to common practice is lacking. However, in the researched area, new findings on the incorporation of drugs are increasing very rapidly and ways of influencing the liquid oil environment continue to be an attractive topic for many scientific researchers.</p>
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Optimization of the Microscopic Method of Observing of the Oleogel Structure

Faculty of Pharmacy, Department of Galenic Pharmacy

Faculty of Natural Sciences, Department of Microbiology and Virology

European Pharmaceutical Journal

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

{"article-title":"Optimization of the Microscopic Method of Observing of the Oleogel Structure"}

The subject of the research was the oleogel structure of the OraMAF oral suspension, containing a mixture of active substances in which the oleogel environment...

Optimization of the Microscopic Method of Observing of the Oleogel Structure

Article Category: Original Paper

Published Online: Aug 24, 2023

Page range: 58 - 61

Received: Jun 22, 2023

Accepted: Jun 23, 2023

DOI: https://doi.org/10.2478/afpuc-2023-0001

Keywords
oleogel, oleogelators, lecithin, chondroitin sulfate, fucoidan, oleosuspension structure, microscopic method

© 2023 J. Zima et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Optimization of the Microscopic Method of Observing of the Oleogel Structure

Article Category: Original Paper

Published Online: Aug 24, 2023

Page range: 58 - 61

Received: Jun 22, 2023

Accepted: Jun 23, 2023

DOI: https://doi.org/10.2478/afpuc-2023-0001

Keywordsoleogel, oleogelators, lecithin, chondroitin sulfate, fucoidan, oleosuspension structure, microscopic method

© 2023 J. Zima et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Keywords
oleogel, oleogelators, lecithin, chondroitin sulfate, fucoidan, oleosuspension structure, microscopic method