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Article

Impact of Bird Cherry (Prunus padus) Extracts on the Oxidative Stability of a Model O/W Linoleic Acid Emulsion

by
Przemysław Siejak
1,*,
Grażyna Neunert
1,
Wojciech Smułek
2 and
Krzysztof Polewski
1
1
Department of Physics and Biophysics, Faculty of Food Science and Nutrition, Poznan University of Life Sciences, Wojska Polskiego 38/42, 60-637 Poznań, Poland
2
Institute of Chemical Technology Engineering, Poznan University of Technology, Berdychowo 4, 60-965 Poznań, Poland
*
Author to whom correspondence should be addressed.
Appl. Sci. 2023, 13(17), 9560; https://doi.org/10.3390/app13179560
Submission received: 30 June 2023 / Revised: 11 August 2023 / Accepted: 16 August 2023 / Published: 24 August 2023
(This article belongs to the Special Issue Bioactive Compounds: From Extraction to Application)
Browse Figures
Versions Notes

Abstract

:
The delivery and uptake of adequate doses of a number of active compounds, including selected saturated and unsaturated fatty acids (frequently in the form of emulsion systems), is crucial to maintaining a healthy diet. The susceptibility of acids to oxidation and the time stability of emulsions are factors limiting their shelf life and storage time. Those parameters could be improved using selected additives, including antioxidants. In this study, we examined the influence of different bird cherry extracts (varying in the content amounts of bioactive compounds) on the oxidative stability of a model O/W linoleic acid emulsion, using C11-BODIPY581/591 as a fluorescent indicator. We also examined the effect of these extracts on the physicochemical properties of the emulsions and the time stability of the produced emulsion using the dynamic laser scattering technique. The antioxidative efficacy of extracts differed significantly, depending on the extraction method and conditions. The observed differences in the results could be attributed to variations in the specific compositions of the extracts used, which were more or less rich in terms of antioxidants or their synergistic effects. Our results indicated that acetone extract was the most effective with regard to both the oxidation stability and time degradation tests of the emulsions produced. Moreover, the addition of gallic acid did not always have a positive effect on the abovementioned properties.

1. Introduction

Emulsions and emulsion-like systems are widespread in a number of aspects in everyday life, including medicine, cosmetics, food and beverages, and many more areas. The most commonly used emulsions are oil-in-water (O/W)-like or water-in-oil (W/O)-like systems (e.g., butter, beverages, or milk). It is, however, worth noting that specific requirements must be fulfilled for the safe usage of these kinds of product and that other additional conditions influence their quality. In terms of foods and beverages, the main recent interest has been their functional and bioactive properties, as well as the loss of those properties during storage (shelf life) [1,2,3,4,5]. Among those properties, oxidation stability is considered one of the most important. It determines consumer choices, especially when it comes to “healthy diet” or bioactive diet supplements, as well as many skin cosmetics, etc. Oxidative degradation, especially when it originates from lipid peroxidation in O/W- or W/O emulsion-like systems; susceptibility to this emulsion phenomenon is therefore the main obstacle to be overcome for the improvement of products based on or containing emulsions, without regarding the purpose of their usage. The other problem is degradation due to the aging of emulsions, which causes the worsening of droplet parameters [4,5,6,7]. Numerous attempts to improve both emulsion bioactive properties and time stability have been made, and many new formulations have been developed in order to avoid emulsion degradation, including the use of additives to prevent the association and coagulation of droplets of the dispersed phase and/or to prevent fatty acids from undergoing peroxidation [8,9].
To maintain a healthy diet, it is important to take in a number of active compounds, including selected saturated and unsaturated fatty acids, at an optimal ratio. It has been proven that certain groups of omega-3 and omega-6 fatty acids are the most important for human nutrition, and that the optimal ratio for these ranges from 1:2 to 1:6 [10]. Omega-6 fatty acids are mainly represented by linoleic acid (LA; C18:2n6), whereas omega-3 fatty acids are represented by alpha-linolenic acid (ALA; C18:3n3). LA is present in nature in the seeds of most plants except for coconut, cocoa, and palm. ALA, on the other hand, is found in high amounts in some plant oils, particularly flax, rape, chia, perilla, and in walnut oil [11,12].
It is commonly known that food products are complex systems, often including oils and fats, consisting of different fatty acids. The content of unsaturated fatty acids, such as LA or ALA, which are more sensitive to oxidation than saturated fatty acids, has the greatest impact on the oxidation process. Moreover, in a complex food product, numerous interactions between compounds may lead to different global effects, especially oxidation processes and products. Therefore, oxidation processes in emulsions are influenced not only by pH or homogenizer type, but also by many other factors, such the kind of emulsifiers or the presence of antioxidants [13,14,15,16]. The most popular antioxidant agents are water-soluble and lipophilic vitamins, such as tocopherol (Toc), or edible polyphenolic compounds. One of them is gallic acid (GA), for which the protective function against lipid peroxidation, oil oxidation, and the positive impact on dispersed droplets has been reported in selected emulsion systems [17,18,19].
In recent years, beyond the polar paradox theory [20], different hypotheses for the effect of antioxidants on emulsion oxidation process have been suggested, including the cut-off effect theory [21,22]. However, it seems that the former is still the main issue when it comes to lipid-based systems [23,24,25].
Based on our previous results on the antioxidant potential of bird cherry (Prunus padus) extract [26], we assume that aqueous solutions of those extracts are good candidates for the abovementioned natural additives improving the oxidative stability of lipids in micellar and emulsion systems. Those extracts are rich in phenolic compounds and flavonoids, known for their strong antioxidant properties. Moreover, we have previously identified that the antioxidant properties of extracts can differ significantly, depending on the extraction method and conditions. These differences in the antioxidative properties of extracts may arise from different specific compositions of extracts used, more or less rich in antioxidants or its synergistic effects.
The aim of our study was to evaluate whether bird cherry extracts either alone or in combination with GA are able to improve the oxidative stability of linoleic acid in a model emulsion system. One of the common forms of unsaturated fatty acid is LA; therefore, we decided to prepare a simple model of only this acid in water to examine the potential antioxidant activity of selected bird cherry extracts and the expected influence of GA presence in the emulsion system on the susceptibility to oxidation. We examined the influence of natural compounds from bird cherry as additives to the model O/W linoleic acid emulsion system on the resistance to thermal lipid peroxidation for freshly produced formulations and over 2 weeks of storage.
Considering the abovementioned potential influence of any additive on oxidation tests, we also decided to refrain from assessing any emulsifier which could affect the oxidation process [13,27]. This enabled us to assess the impact of fruit extracts on the emulsion oxidation stability.
We aimed to develop knowledge on the extracts’ influence on emulsion stability, in addition to the oxidation studies we carried out with the DLS measurements which yielded the information on droplet size distributions and droplet zeta potentials during estimated storage time.

2. Materials and Methods

2.1. Chemicals

Methanol, ethanol, chloroform, acetone, gallic acid (GA), and DL-α-tocopherol (Toc) were purchased from Merck (Darmstadt, Germany). Linoleic acid (LA) was obtained from Sigma-Aldrich (Steinheim, Germany). Fluorescent probe C11-BODIPY581/591 [4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid] was purchased from Invitrogen (Carlsbad, CA, USA). The used water was purified using a MicroPure Water System (TKA, Niederelbert, Germany).

2.2. Preparation of Extracts

The detailed procedure of the extract preparation was thoroughly described in our previous article [26]. In short, after grinding the lyophilized fruit, solvent (acetone, water, ethanol or methanol) was added to the fruit at a rate of 60 mL per 10 g of fruit and incubated for 1 h at 50 °C temperature in an ultrasound bath. Then, the liquid phase was filtered and vacuum-dried to obtain solid material. The eluents: methanol, ethanol, water, and acetone, were used separately. The molecule compositions in the obtained dry extracts were determined by HPLC and GG-TOFMS in our previous report [26]. It was also shown that the yield of extraction strongly depends on the eluent used; the yields were estimated as 38%, 30%, 26%, and 6.5% for methanol, ethanol, water, and acetone, respectively.

2.3. Preparation of Emulsions

The oil-in-water (O/W) emulsions were formulated at room temperature using a two-step homogenization process. In the first stage, LA (57 μL) and aqueous phase (95 mL) were mixed using a hand-held homogenizer CAT X120 fitted with a T10 shaft for 5 min (RPM speed approximately 22,000), followed by homogenization using a Sonoplus sonicator (Bandelin, Berlin, Germany) fitted with a TS109 probe in the following conditions: a duration of 4 min, with 10 s of action followed by 5 s of rest cycles, and an amplitude of 60%. The total energy of sonication was 6104 kJ. The final LA content was estimated as 0.60 μL/mL.
The relationship of oil to water was estimated experimentally to enable spectroscopic examination. Since one of the purposes of this study was to measure the fatty acid oxidation dynamics in water with the use of a spectrofluorometer (see Section 2.7 “Oxidation tests”), it was crucial to use an emulsion system that did not exhibit significant scattering of incident light, which could affect the results and their interpretation. Therefore, we prepared a set of emulsions with different oil-to-water ratios and checked them for scattering. The most suitable was an emulsion with an LA concentration in water of 0.60 μL/mL.
To examine the extracts’ impact on LA time and oxidative stability, we maintained all the experimental conditions, including concentrations, added volumes, etc., as identical to those in our earlier study [26] whenever possible. Therefore, the initial aqueous extract solutions were prepared in the same way as previously, namely, each crude (solid state) extract was dissolved in water to obtain the solution at a concentration of 10 mg/mL. Regarding the possible synergistic effect during autooxidation, gallic acid (GA) or tocopherol (Toc) were added during emulsion formulations. For this reason, the measurements in emulsions were performed with and without added GA. Then, 15 μL of each aqueous extract (or aliquot volume of GA water solution or Toc methanol solution, to obtain a final concentration of this compound equal to 12 μM) was added to 2 mL of emulsion and left for 20 min under stirring (magnetic stirrer at the rate of 150 RPM) without further agitation. The non-doped emulsion was considered as the reference sample.

2.4. Measurement of Mean Particle Size (Z-Ave), Zeta Potential (ZP), and Polydispersity Index (PDI)

The properties of the prepared emulsions with added extracts were characterized by dynamic light scattering (DLS) measurements, which determined the droplet size distribution, mean droplet diameter (Z-ave), polydispersity index (PDI), and zeta potential (ZP).
The samples were measured at a stabilized temperature of 20 °C using a Zetasizer Nano (Malvern Instruments, Worcestershire, UK) at 90°, following the methodology described by Rehman et al. [28]. Each analysis was carried out three times.

2.5. Microscopic Observation

Each of the prepared emulsions was observed under an optical microscope (Axio Vert.A1 Carl Zeiss, Shanghai, China) with an Axiocam 208 color camera (Carl Zeiss, Suzhou, China). The samples were introduced into a Cuvette 1 μ-Slide V1 0.1 (ibidi GmbH, Grafelfing, Germany) and analyzed at 40-fold magnification. The droplet diameter of the inner phase was marked for each emulsion; then, pictures of the microscopic image were taken. The images were recorded with AxioVision Rel. 4.8 software (Carl Zeiss Microscopy GmbH, Jena, Germany).

2.6. Storage Stability of Emulsions

All prepared emulsions were packed into glass vials immediately after formation and kept at 4 °C in the refrigerator in darkness for 14 days. The measurements of Z-ave, ZP, PDI, microscopy images, and oxidative stability of each sample were carried out directly after preparation, and then after 7 and 14 days.

2.7. Oxidation Tests

The level of thermal oxidation of LA in prepared emulsion systems was accomplished in the same way as previously described [26]. Briefly, after 10 min incubation of 2 mL of each sample, including the reference sample at 40 °C under constant stirring (magnetic stirrer at the rate of 150 RPM), 10 μL of methanol solution of C11-BODIPY581/591 (0.5 mg/mL) was added. The progression of oxidation in the emulsion was monitored by observing the rise in fluorescence intensity of C11-BODIPY581/591 at the wavelength λobs = 520 nm, with excitation at λex = 505 nm. The measurements were taken at 10 min intervals until the fluorescence intensity stabilized or exhibited a noticeable decline. The fluorescence spectra were collected in a quartz cuvette (10 × 10 mm) with the use of a Shimadzu RF 5001PC fluorimeter (Kyoto, Japan) equipped with a thermostated cell compartment.

2.8. Data Evaluation and Statistical Analysis

Each sample was measured at least three times and average values with standard deviations (when necessary) were calculated. The statistical calculations were performed using OriginPro Software for Windows, Version 2023b, OriginLab Corporation, Northampton, MA, USA.

3. Results and Discussion

3.1. Physiochemical Properties of Emulsions

The stability properties of the emulsions are usually characterized in terms of droplet size, polydispersity index (PDI), and zeta potential (ZP) [9]. Droplet size is one of the main factors influencing the physicochemical stability of emulsions [15]. Smaller droplets correspond to the better stability of systems because of the reduction in interfacial energy. PDI describes the broadness of size distribution of droplets, with smaller PDI values indicating a smaller difference between droplet sizes [28]. ZP is one of the other determinants of emulsion stability. A high positive or negative ZP value (>±30 mV) indicates a stable emulsion, while ZP values approaching zero may trigger degradation processes including coalescence, aggregation, or flocculation [29,30]. The obtained results of the DLS examination of extract-doped and non-doped linoleic acid (LA) emulsions are presented in Table 1, Figure 1, Figure S1 and Figure S2.

3.1.1. Freshly Prepared Emulsion Systems

The results obtained from the DLS measurements as mean droplet size by intensity (intensity-based overall average size, determined by cumulant method; Z-ave) and dominant size by number (the size of the most numerous droplets), PDI, and ZP for freshly prepared samples are presented in Table 1.
The results of droplet sizes determined by intensity or number analysis exhibited multimodal size distribution (Figures S1 and S2). The DLS results for freshly prepared samples show that each system, except for acetone extract, consists of at least two populations of droplets smaller than 1000 nm, with a mean size of 330–600 nm, depending on the extract added to the emulsion. Moreover, the size distribution by intensity revealed the presence of large droplets (2000–10000 nm in diameter); however, the amounts of these were too low to manifest in size distribution by number. However, the large droplets were clearly identified in microscopic investigations (Figure 2).
The PDI values of freshly prepared emulsions are relatively high, ranging between 0.250 and 0.414, depending on the specific composition of the sample (Table 1). The emulsions formulated are not homogenous, which can be seen from the obtained distributions and PDI values. This is quite understandable and expected, since no designed emulsifier was used during sample preparation.
Considering the influence of extract type on the parameters of freshly prepared emulsions, it is worth noticing that each bird cherry extract causes a decrease in the droplet diameter regardless of the analysis type. Such an effect is not seen for tocopherol (Toc) (Figure 1, day 0). For such cases, the decrease in droplet size should usually be accompanied by a decrease in PDI and an increase in ZP [17,31]. The investigated systems containing bird cherry extracts show that behavior. The effects are the most pronounced for acetone extract addition. There is a significant decrease in both Z-ave (form 511.0 nm for the undoped emulsion to 302.2 nm) and dominant droplet diameter (260.6→187.1 nm), accompanied by a decrease in PDI (0.414→0.308) and an increase in (negative) ZP value (−26.5→−41.1 mV). This is consistent with the results obtained for oxidation tests (Section 3.2.1). Moreover, our previous study revealed that the antioxidant properties of the acetone extract of bird cherry are the strongest among the investigated extracts and arise from different compositions of acetone extract [26]. This indicates that the use of acetone extract with its active compounds is the most effective way to increase emulsion stability. We may assume that the addition of any of the examined extracts to the emulsion is expected to improve the emulsion stability. However, compared with acetone extract, the changes observed for the other extracts are less noticeable. The detailed origin of the observed effect, including specific molecular interactions, remains unclear and requires further in-depth investigation. However, given the significant differences in results depending on the type of extract used, it is plausible to assume that the overall outcome involves synergistic action. Moreover, our previous study demonstrated that the acetone extract, which contains malic acid [26], known for its high bioactive and gelling activity [32,33], differs from all other extracts. Therefore, the role of this compound seems important in terms of synergistic interactions. Notably, emulsions with water and ethanolic extracts exhibit low stability. These extracts contain a very low amount of chlorogenic acid [26]. Hence, the contribution of this compound to emulsion stability should also be taken into consideration.
Interestingly, the presence of Toc in the emulsion system increases the droplet Z-ave and does not significantly influence the PDI and ZP, regardless of gallic acid (GA) presence in the emulsion. This can suggest strong interactions of fat-soluble Toc with fatty acid leading to the formation of bigger emulsion droplets. The effect of Toc on the physiochemical properties of emulsions that contain fatty fractions (such as oils, glycerides, etc.) is ambiguous and relies on both the ratio of Toc to fatty compounds and the specific compounds present. In certain formulations and specific ratios, a slight decrease in droplet size was observed [34], while in others, a significant increase occurred [35].
On the other hand, the Toc addition led to a decrease in PDI and an increase in the negative value of ZP, which is a good perspective application for the stability of these types of formulations. The positive effect of Toc on nanoemulsion stability has been also reported previously [34].
The GA presence in the formulations was expected to increase the quality of emulsion systems [17,18], including the improvement of each of the parameters discussed above (decrease in droplet diameter, PDI, and ZP). However, the effect of GA on the examined emulsion was ambiguous. A slight decrease in the droplet size for freshly prepared emulsions was observed for all samples, but at the same time, minor changes in PDI and ZP take place. The only noticeable decrease in droplet size was observed for water extract, but this effect was not unequivocal, because the ZP value for the sample with GA was worse (less negative) compared with that same sample without GA.
DLS results analyzed in the size distribution mode (Figure S2) show that, in the samples, there were also larger droplets in the range of 1000 nm and higher. Thus, it was possible to visualize each sample using optical microscopy. The 40-fold magnification objective was used, and the images were recorded with a CCD camera. The results are presented in Figure 2. The inhomogeneity of recorded droplets was highest for the emulsion system containing Toc. For this sample, the largest droplet sizes were observed. Additionally, large, but more homogenous droplets were observed for emulsion without any additive. The lowest droplet sizes were recorded for the emulsion with acetone extract. The presence of GA in the samples did not influence the droplet sizes. The visualization of emulsion samples presented in Figure 2 confirms the results obtained from the DLS measurements.

3.1.2. Storage Stability of Produced Formulations

The DLS parameters changes in emulsions during the storage are given in Figure 1, Figure S1 and Figure S2. Considering the stability of the investigated samples over 14 days, we found that the quality of each sample had decreased (Figure 1). This included an increase in sizes of droplets, PDI, and ZP values for each sample. However, the addition of extracts to the emulsion system prevented rapid degradation of the emulsion, as indicated by lower dynamics of Z-ave and increasing PDI values, especially for acetone-extract-doped samples. Such results agree with those reported earlier [26], as well as with the oxidative tests presented in this study. The presence of GA in emulsion does not always cause positive outcomes. In the presence of GA in most samples, the parameters describing the quality of the emulsion were less favorable compared with those without added GA. Only the reference sample was a clear exception, for which the GA presence did not cause a significant drop in quality factor for fresh samples. This effect was clearly visible for the samples after 7 days of storage, and partially after 14 days. For that time, only Z-ave increased. For the samples with one of the following added extracts, ethanolic, acetone, or water, the positive effect of GA addition on ZP values could be observed. The effect remained throughout the whole storage time (14 days); however, it was not accompanied by either a decrease in droplet size or PDI. Therefore, it can be concluded that even when GA positively influences the ZP value, it is not enough to prevent the degradation of the emulsion.
Analyzing the direct size distribution (Figures S1 and S2), as a function of time and the presence of GA, we can conclude that both of those factors affect both populations within the 100–1000 nm range. In general, both fractions tend to separate with the storage time, and the addition of GA does not prevent this separation, which confirms the changes in PDI presented in Figure 1. The effect of size separations is the most pronounced for the emulsion system without any additives, and the formulation containing acetone extract is almost insensitive to both factors: GA addition and storage time.
For each sample, on the 7th and 14th day of storage, the microscopic images are presented in Figure S3 and Figure S4, respectively. The conclusions support the results of the DLS analysis that the presence of GA does not affect the large-sized droplets.
Concluding this part of the study, we claim that for produced formulations, GA does not act synergistically with any of the used extracts. The globally observed interaction produces no effect or, for selected formulations, is rather anti-synergistic. It also shows that the size of observed droplets is mainly determined by the type of extract.

3.2. Oxidation Tests

Lipid oxidation is very sensitive to temperature [36,37]; in different oxidation experiments in emulsions, especially those rich in unsaturated lipids, this process could be accelerated by increasing temperatures. It is known that C11-BODIPY 581/591 is oxidized by lipid radicals as part of the propagation reaction. Free-radical-induced oxidation results in the modification of fluorescent properties, manifesting an increase in fluorescence emission observed at approximately 520 nm [38]. Thereby, this fluorescence probe has commonly been used to determine lipid peroxidation in liposomes and mammalian cells [26,37,39,40,41]. It is also widely recognized that “chain-breaking” antioxidants (such as Toc or phenolic compounds) decelerate the process of oxidation by effectively scavenging free radicals [42]. In our study, the oxidation in LA emulsion and the influence of bird cherry extracts on this process was measured with C11-BODIPY581/591 probe used as a fluorescence indicator.

3.2.1. Oxidative Stability of Extract-Loaded Emulsions

The antioxidant properties of the studied extracts were determined using a C11-BODIPY581/591 fluorescent probe. The thermal auto-oxidation process of fatty acids in the emulsion was observed with the increasing C11-BODIPY581/591 fluorescence intensity at 520 nm (Figure 3). The presence of Toc in solution very efficiently prevented fatty acid oxidation and almost completely suppressed oxidation in the LA emulsion. Among the extracts, the fastest oxidation occurred in the presence of water extract with kinetics comparable with a reference sample. In the presence of the other extracts the rate of oxidation decreased. The observed antioxidant activity ranked the extracts in the following order: acetone > ethanolic > methanolic > water. Our earlier study [26] confirmed the presence of phenolics and flavonoids in the investigated extracts. However, the kind and amount of compounds with antioxidant potential varied based on the eluent used. For this reason, water extract, mainly containing sugars and phenolic acids, demonstrates the least effective antioxidant performance in LA emulsion. The best among the extracts was acetone extract, which exhibited a reduced oxidation rate after 320 min at approximately 35%. This extract contains tocopherols and flavonoids, which are well-documented inhibitors of free radical oxidation.
The presence of GA in the emulsion increased oxidation by approximately 40%. Similar pro-antioxidant properties of GA were also observed in camellia oil emulsion [43]. This fact was explained as a result of the accumulation of oxidation products at the interface where, together with phenolic compounds and emulsifier, they can catalyze further lipid oxidation. It is also known that a high surface/volume ratio of emulsion (for smaller droplets) makes them more susceptible to oxidation [15,43,44]. Our DLS analysis showed that the presence of GA in pure emulsion only resulted in a slight decrease in droplet size. Moreover, in the presence of the extracts, the size of the droplets was in contrast to their antioxidant properties in LA emulsion (Figure 3 and Table 1). Thus, in our extract-doped emulsion systems, the surface/volume ratio was not the dominant factor; instead, the “polarity effect” may be responsible for the antioxidant activity of the extracts, as they are known to contain different amount of lipophilic antioxidants, such as tocopherols or flavonoids [26,45,46]. Only in the case of Toc, which demonstrated the best antioxidant activity in emulsion with the largest particle size, could it be the predominant phenomenon. For GA in linoleic emulsion, its pro-antioxidant properties might share a similar origin as those described above for camellia oil emulsion.
In the emulsions containing extracts and GA, the oxidation process was faster, except for the acetone extract. The presence of GA did not evidently improve the emulsion stabilization parameters (see Section 3.1). At the same time, it caused a deterioration in the antioxidant properties of the tested systems. It seems that these pro-antioxidant properties were dominated in some extract emulsions, as may be observed for water extract, which contains very few lipophilic antioxidants, and reached almost the same oxidation value as GA alone [26]. The exception was acetone extract, for which the oxidation rate decreased by almost 50% in the presence of GA, indicating the synergistic effect between GA and some components of the extract. In the case of Toc, the presence of GA had practically no influence on its antioxidation power (Figure 3); this indicates that this synergistic effect in the freshly prepared sample had occurred between GA and other acetone extract components.

3.2.2. Storage Stability of Extract-Loaded Emulsions

C11-BODIPY581/591 fluorescence changes after 320 min of oxidation as a function of storage time are presented in Figure 4. During storage, only in the case of acetone and methanolic extracts was a significant improvement observed in the oxidative stability of the emulsion. In turn, for water and ethanolic extracts, no significant changes in the oxidation behavior of LA emulsion were observed. On the other hand, clear acceleration of the oxidation process with storage time was observed in the presence of Toc. The presence of GA in the emulsion does not change this trend, with the exception of water and acetone extracts, for which, after 14 days of storage, clearly increased the emulsion oxidation rate by about 10% (similar results were obtained for GA alone). Notably, in an emulsion containing both GA and Toc, the oxidation process was slower compared with the rate of oxidation in the presence of Toc alone. We can therefore conclude that, during storage, a synergistic effect between Toc and GA was observed.
The abovementioned dominant pro-antioxidant effect of GA seems to be correct in the case of water and ethanolic extracts, for which the oxidation rate of the emulsion in the presence of GA exceeded the values for the reference sample after 14 days of storage.
On the other hand, methanolic and acetone extracts showed an improvement in antioxidant properties with time of storage, although the course of oxidation for both extracts in the presence of GA during storage demonstrated opposite behavior. For the methanolic extract, the protection against oxidation increased both for the extract itself and in the presence of GA, while for the acetone extract in the presence of GA, the protection slightly decreased.
Interestingly, in the case of acetone extract after 7 days of storage, the degree of protection of LA against oxidation was comparable to that of Toc and a sample containing GA. This significant improvement in oxidation stability was not reflected in the DLS measurements. These results have shown that other mechanisms may also take place during the oxidation processes of freshly prepared LA emulsion and during storage.
In order to explain this observation, we performed an experiment where acetone extract was added to the prepared emulsion immediately after its preparation and to the emulsion after 7 days of storage (Figure 5). For a freshly prepared emulsion, acetone extract demonstrated approximately 40% protection against oxidation. After 7 days of storage, its protection increased to approximately 67%, a similar value as obtained for Toc (Figure 4). When acetone extract was added to the emulsion after 7 days, its protective properties only showed approximately 30% effectiveness. Moreover, the addition of GA did not improve these properties, as was observed in the case of the “fresh” emulsion.
The same experiments were also performed for Toc. In this case, the emulsion oxidation process was even faster than for the acetone extract, and in the presence of GA, it was accelerated even more. This effect is in contrast to the synergistic effect observed with Toc and GA present in the emulsion from the beginning.
The above results show that in the case of acetone extract, the aging of the emulsion may affect how components of the extract interact with LA. A possible explanation for this phenomenon is the activation of a different protective mechanism or the migration of some acetone extract components closer to the oil–water interphase, where the oxidation takes place. Suppressing the oxidation of emulsions as a result of modifying the physical location of antioxidants was observed in the case of Toc in O/W emulsion prepared from canola oil [27]. However, in this case, the factor causing this phenomenon was the emulsifier. Previous reports also demonstrated that some additives could improve the antioxidant properties of lipophilic antioxidants in O/W emulsion [13,47]. Our findings suggest that the emulsion aging process might enhance the protective properties of certain antioxidant compounds.

4. Conclusions

The presented results show that acetone extract has the best inhibited thermal autooxidation in the linoleic acid emulsion. As the impact of droplet size is rather inconclusive, we can attribute the observed effect to the composition of the acetone extract. This confirms our previous observations, suggesting the high antioxidant potential of acetone extract in homogenous (bulk solutions) and heterogenous systems (liposomes). Our findings also suggest that the emulsion aging process might enhance the protective properties of certain antioxidant compounds that come from the extract. At the same time, the DLS results clearly indicate that this extract has also improved the stabilizing properties of both the freshly prepared emulsion and during storage.
Our studies have revealed that the presence of GA, whether as an emulsifier or antioxidant additive in LA emulsion systems containing bird cherry extracts as the primary antioxidant agent, does not consistently yield positive effects. The end quality of the systems, encompassing time stability and the resistance of LA to oxidation in emulsion systems, mainly depends on the components originating from the extracts used. It is particularly important for the enhancement of antioxidant efficiency, because the yield of the extraction process for the most promising extract, acetone, is relatively low (6.5%) compared with other eluents (38% for methanol, 30% for ethanol, and 26% for water).
Based on the obtained results, it can clearly be seen that acetone extract is a good prospective candidate for the further development of complex systems containing bioactive compounds. Notably, good results were also observed for methanolic extract, which exhibited the highest extraction efficiency. However, intensive study is needed to fully clarify the mechanisms leading to the observed effects.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/app13179560/s1, Figure S1: Size distributions of examined formulations by number. Figure S2: Size distributions of examined formulations by intensity. Figure S3: Microscopic images of linoleic acid emulsions in water after 7 days of storage, and the impact of water solutions of bird cherry extracts. The influence of gallic acid presence on the size of droplets with a diameter greater than 1000 nm. Figure S4: Microscopic images of linoleic acid emulsions in water after 14 days of storage, and the impact of water solutions of bird cherry extracts. The influence of gallic acid presence on the size of droplets with a diameter greater than 1000 nm.

Author Contributions

Conceptualization, P.S., K.P. and G.N.; methodology, P.S., K.P. and G.N.; software, K.P., G.N. and P.S.; validation, K.P., G.N. and P.S.; formal analysis, P.S., K.P. and G.N.; investigation, P.S., W.S. and G.N.; resources, K.P.; data curation, P.S., K.P. and G.N.; writing—original draft preparation, P.S., K.P. and G.N.; writing—review and editing, P.S., W.S., K.P. and G.N.; visualization, P.S., K.P. and G.N.; supervision, K.P. All authors have read and agreed to the published version of the manuscript.

Funding

This publication was co-financed within the framework of the Polish Ministry of Science and Higher Education’s program: “Regional Excellence Initiative” in 2019–2023 (No. 005/RID/2018/19), financing amount: PLN 12,000,000,00.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are available from the corresponding author upon reasonable request.

Conflicts of Interest

The authors declare no conflict of interest.

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Applsci 13 09560 g001
Figure 1. Mean size (z-ave), polydispersity index (PDI), and zeta potential (ZP) changes as a function of storage time and gallic acid addition.
Figure 1. Mean size (z-ave), polydispersity index (PDI), and zeta potential (ZP) changes as a function of storage time and gallic acid addition.
Applsci 13 09560 g001
Applsci 13 09560 g002aApplsci 13 09560 g002b
Figure 2. Microscopic images of freshly prepared linoleic acid emulsions in water and the impact of water solutions of bird cherry extracts. The influence of gallic acid presence on the size of droplets with diameters greater than 1000 nm. Bars in all images represent 20 μm.
Figure 2. Microscopic images of freshly prepared linoleic acid emulsions in water and the impact of water solutions of bird cherry extracts. The influence of gallic acid presence on the size of droplets with diameters greater than 1000 nm. Bars in all images represent 20 μm.
Applsci 13 09560 g002aApplsci 13 09560 g002b
Applsci 13 09560 g003
Figure 3. C11-BODIPY581/591 fluorescence intensity changes as a function of time (excitation 505 nm; emission 520 nm). Legend: reference, non-doped emulsion; GA, gallic acid; Toc, tocopherol; E, ethanolic extract; A, acetone extract; W, water extract; M, methanolic extract.
Figure 3. C11-BODIPY581/591 fluorescence intensity changes as a function of time (excitation 505 nm; emission 520 nm). Legend: reference, non-doped emulsion; GA, gallic acid; Toc, tocopherol; E, ethanolic extract; A, acetone extract; W, water extract; M, methanolic extract.
Applsci 13 09560 g003
Applsci 13 09560 g004
Figure 4. C11-BODIPY581/591 fluorescence intensity at 520 nm after 320 min of oxidation as a function of storage time (excitation 505 nm; emission 520 nm). Legend: reference, non-doped emulsion; GA, gallic acid; Toc, tocopherol; E, ethanolic extract; A, acetone extract; W, water extract; M, methanolic extract.
Figure 4. C11-BODIPY581/591 fluorescence intensity at 520 nm after 320 min of oxidation as a function of storage time (excitation 505 nm; emission 520 nm). Legend: reference, non-doped emulsion; GA, gallic acid; Toc, tocopherol; E, ethanolic extract; A, acetone extract; W, water extract; M, methanolic extract.
Applsci 13 09560 g004
Applsci 13 09560 g005
Figure 5. C11-BODIPY581/591 fluorescence intensity changes as a function of time for non-doped emulsion (reference) and the emulsion with or without gallic acid (GA) and: acetone extract (A) or tocopherol (Toc), added to fresh emulsion and added after 7 days after preparation of emulsion (excitation 505 nm; emission 520 nm).
Figure 5. C11-BODIPY581/591 fluorescence intensity changes as a function of time for non-doped emulsion (reference) and the emulsion with or without gallic acid (GA) and: acetone extract (A) or tocopherol (Toc), added to fresh emulsion and added after 7 days after preparation of emulsion (excitation 505 nm; emission 520 nm).
Applsci 13 09560 g005
Table 1. DLS results of freshly prepared linoleic emulsions with the addition of different bird cherry extracts (PDI, polydispersity index; ZP, zeta potential). The superscript letters indicate significant differences at the 0.05 level. Values in columns that do not share the same letter are significantly different.
Table 1. DLS results of freshly prepared linoleic emulsions with the addition of different bird cherry extracts (PDI, polydispersity index; ZP, zeta potential). The superscript letters indicate significant differences at the 0.05 level. Values in columns that do not share the same letter are significantly different.
Agent/Extract AddedMean Droplet Size by Intensity Z-Ave
(nm)		Dominant Droplet Diameter by Number
(nm)		PDIZP
(mV)
Without Gallic AcidWith Gallic AcidWithout Gallic AcidWith Gallic AcidWithout Gallic AcidWith Gallic AcidWithout Gallic AcidWith Gallic Acid
No addition (reference sample)511.0 ± 13.1 b496.5 ± 21.4 b,c260.6 ± 74.7 a268.4 ± 27.4 a0.414 ± 0.072 a0.406 ± 0.028 a−26.5 ± 3.1 e,f−22.3 ± 1.8 f
tocopherol587.5 ± 38.2 a587.8 ± 41.5 a280.9 ± 44.1 a294.8 ± 120.1 a0.379 ± 0.048 a,b0.385 ± 0.055 a,b−28.8 ± 1.9 c,d,e−28.4 ± 3.6 d,e
Bird Cherry ExtractsEthanolic433.2 ± 28.2 e441.5 ± 40.2 d,e208.4 ± 83.9 a225.1 ± 27.9 a0.343 ± 0.035 a,b,c0.397 ± 0.055 a,b−32.1 ± 4.0 b,c,d−33.5 ± 3.9 b,c
Acetone302.2 ± 5.2 f,g269.0 ± 6.4 g187.1 ± 27.4 a203.2 ± 43.2 a0.308 ± 0.012 b,c0.255 ± 0.027 c−41.1 ± 5.8 a−40.5 ± 5.0 a
Water494.8 ± 17.6 b,c,d442.3 ± 26.4 c,d,e230.8 ± 70.8 a181.9 ± 55.9 a0.376 ± 0.035 a,b0.364 ± 0.052 a,b−32.8 ± 4.0 b,c,d−28.7 ± 5.4 c,d,e
Methanolic357.0 ± 14.3 f330.3 ± 12.5 f214.9 ± 44.4 a216.1 ± 30.3 a0.342 ± 0.020 a,b,c0.339 ± 0.013 a,b,c−36.3 ± 4.0 a,b−34.2 ± 3.5 b
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MDPI and ACS Style

Siejak, P.; Neunert, G.; Smułek, W.; Polewski, K. Impact of Bird Cherry (Prunus padus) Extracts on the Oxidative Stability of a Model O/W Linoleic Acid Emulsion. Appl. Sci. 2023, 13, 9560. https://doi.org/10.3390/app13179560

AMA Style

Siejak P, Neunert G, Smułek W, Polewski K. Impact of Bird Cherry (Prunus padus) Extracts on the Oxidative Stability of a Model O/W Linoleic Acid Emulsion. Applied Sciences. 2023; 13(17):9560. https://doi.org/10.3390/app13179560

Chicago/Turabian Style

Siejak, Przemysław, Grażyna Neunert, Wojciech Smułek, and Krzysztof Polewski. 2023. "Impact of Bird Cherry (Prunus padus) Extracts on the Oxidative Stability of a Model O/W Linoleic Acid Emulsion" Applied Sciences 13, no. 17: 9560. https://doi.org/10.3390/app13179560

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