Antioxidant Activity and Physicochemical Stability of Nanospheres: Evaluation in Vitro and Applied to Biodiesel

doi:10.21203/rs.3.rs-3888971/v1

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Antioxidant Activity and Physicochemical Stability of Nanospheres: Evaluation in Vitro and Applied to Biodiesel

https://doi.org/10.21203/rs.3.rs-3888971/v1

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Biodiesel is a fuel from renewable sources which has several environmental advantages. However, due to structural characteristics, it becomes susceptible to oxidation, requiring the use of antioxidants. In this way, antioxidants added to biodiesel could be degraded when exposed to environmental conditions, reducing their capacity. Therefore, carrier systems, such as nanospheres, are an alternative to providing protection and controlled release of antioxidants. In this work, poly-ε-caprolactone nanospheres containing tert-butyl-hydroquinone and ascorbic acid antioxidants were developed, and their physical-chemical stability and in vitro antioxidant activity were evaluated for 150 days. The nanospheres to biodiesel were also carried out to evaluate the antioxidant activity. Nanospheres with negative zeta potential, polydispersion index less than 0.3, and nanometric scale were obtained. Regarding the in vitro antioxidant activity, the ascorbic acid nanospheres showed results greater than 50%, while for the tert-butyl-hydroquinone nanospheres, the results were greater than 80% during 150 days. The addition of nanospheres containing antioxidants to biodiesel did not present satisfactory results, since it reduced oxidation stability of biodiesel.

Oxidation

nanoparticles

ascorbic acid

TBHQ

Biodiesel has advantages, mainly in the environmental context, which justifies the government's effort to encourage its production and use. However, due to the chemical properties of the biofuel, undesirable processes occur, which contribute to the depreciation of the biodiesel quality, bringing losses to resellers and consumers of this product [1, 2].

One of the undesirable processes is oxidation, which is mainly related to the unsaturation of fatty acids and triglycerides, which are the raw material for biodiesel production. Those substances have sites sensitive to oxidation by heat, light, metals, moisture, and oxygen exposure. These factors trigger a radical reaction, promoting oxidation and peroxides formation acting as propagators of the reaction. Consequently, there is polymer generation, causing clogging, for example [2, 3, 4].

Antioxidants are added to biodiesel to minimize or inhibit oxidation, in most cases neutralizing free radicals. The first can also reduce oxygen concentration and remove ions present [5]. Classified as synthetic or natural, according to their origin, with the former being more used due to their high efficiency, especially butylhydroxyanisole (BHA), butylhydroxytoluene ( BHT), and tert -butyl-hydroquinone ( TBHQ) [2].

Among natural antioxidants, studies showed the potential of tocopherols, phenols and flavonoids, substances that can be found in some teas and fruits, such as saffron and cashew fruit [6, 7]. In addition, vitamin C has stood out due to its ability to inhibit free radicals by donating electrons, demonstrating high antioxidant capacity [8].

However, when added to biodiesel, these substances are exposed to temperature, which can contribute to the degradation and consequent antioxidant activity reduction. An alternative to this challenge is encapsulating antioxidant substances [9].

Nanoparticles are used in a range of knowledge fields, mainly in the pharmaceutical area, and in the release of the optimal drugs. Polymeric nanoparticles are divided into nanocapsules and nanospheres, differing from each other by the structure of the formed sphere. While the first has a polymeric membrane at the outer limits of the sphere and an oily core, where the active principle is confined. On the other hand, nanospheres differ from the first ones in that they have only the polymeric structure, which comprises the entire body of the nanosphere, where the active principle is retained, without the presence of the oily core [10].

Nanoparticles have some advantages that can optimize their use in biodiesel, such as protecting the active against environmental stress and controlling the release of the active. The protection of the antioxidant by the polymer used in the production of the nanosphere and the controlled release of the activities tend to maximize the efficiency of the antioxidant.

In this way, the development of nanoparticles containing antioxidants aimed at application in biodiesel is still little explored, and there are no works on this subject in the literature. Therefore, the objectives of this work were to develop, physicochemically characterize, and evaluate the antioxidant capacity of polymeric nanoparticles containing the antioxidants ascorbic acid and TBHQ in biodiesel.

Preparation of TBHQ polymeric nanoparticles

Nanospheres ( NE) were prepared using the emulsification protocol followed by solvent evaporation, described by Mainardes and Evangelista [11], with modification. According to Hagemann and collaborators, the preparation of the emulsion has two phases: organic and the other aqueous [12]. Five formulations of TBHQ nanospheres were performed, and in two of these, the amount of all reagents was multiplied by 3, and added to a 100 mL volumetric flask made up of volume with distilled water.

Preparation of Active-free Polymeric Nanoparticles

To NE without active (white NE), the emulsification protocol with the evaporation of the solvent was used, as well as the NE of TBHQ. The only alteration was that the white NEs do not contain an active principle so the organic phase only contains poly-ε-caprolactone (PCL) dissolved in 2 mL of dichloromethane.

Preparation of polymeric nanoparticles Ascorbic acid

Nanospheres were produced using the double emulsion technique with solvent evaporation [11]. For this, two aqueous and one organic phase. The organic phase consisted of 50 mg of PCL dissolved in 1.8 ml of dichloromethane, which was poured into 10 mg of ascorbic acid (AA) dissolved in 0.2 ml of dimethylsulfoxide (DMSO). The first emulsion was formed by spraying the organic phase into the aqueous phase 1, which consisted of 4 mL of a 0.2% solution of polyvinyl alcohol (PVA), by dripping and under sonication, as was done for NE from TBHQ. To the second emulsion, the first emulsion was poured into aqueous phase 2, consisting of 4 mL of a 1% PVA solution under the same conditions as above. The dichloromethane was removed by rotary evaporation at 37°C for 20 minutes.

Ten formulations of AA nanospheres were prepared, added to 100 mL volumetric flask, and subsequently completed with distilled water. This sample was divided into three 30 mL aliquots used in the oven to stability study.

Determination of the encapsulation efficiency of the TBHQ nanosphere :

The encapsulation efficiency (EE) was indirectly determined by quantifying non-encapsulated active present in the supernatant after centrifuging a 2 mL aliquot of each NE suspension at 15000 rpm for 50 minutes [12]

The actives were quantified via Uv -Vis ( Thermo Scientific Evolution 201) at wavelengths of 287 nm, using a previously prepared calibration curve. To the curve, aqueous solutions of TBHQ were used at concentrations of 2, 4, 6, 10 and 20 mg L ^− 1, obtaining R ² = 0.9991 and y = 0.0199 x + 0.0053. The encapsulation efficiency was obtained according to Eq. 1.

$$EE\left(\%\right)=\left[ \frac{ (Ativo total- ativo sobrenadante)}{ativo total} \right]*100$$

Equation 1

Determination of the encapsulation efficiency of the AA nanosphere :

The EE of the AA NE solution was determined using an indirect method, in the same way as the TBHQ and under the same conditions [12]. The non-incorporated active was quantified via UV -vis at a wavelength of 264 nm. A calibration curve was constructed using aqueous solutions of AA at concentrations of 1, 2, 5, 7, and 10 mg.L. ¹. The curve obtained showed R ² = 0.9997 and y = 0.0766 X + 0.0255. Eq. 1 shown above was used to determine the percentage of AA incorporated into NE.

Production of aqueous solutions for the oven stability test:

For the oven stability test, aqueous solutions of TBHQ, ascorbic acid NE, control solutions of TBHQ and powdered ascorbic acid were prepared. For preparating aqueous solutions of TBHQ NE, after determining the EE of TBHQ NE, the initial suspension obtained (100mL) was adjusted to a concentration of 500 ppm of TBHQ NE, with the addition of 17.74 mL of distilled water. The resulting solution was divided into three 35 mL samples that were placed in the oven for the stability study. An AA NE solution at a concentration of 180 ppm was prepared and then divided into 3 samples of 30 mL.

Three formulations of NE without active were prepared, which were later added to a 100 mL volumetric flask whose volume was completed with distilled water. This solution was divided into 3 samples of 30 mL.

Aqueous solutions of the active principles AA and TBHQ were prepared at the same concentration as the nanosphere solutions of the respective antioxidants. For the TBHQ solution, 100 mL of a solution of 500 ppm was prepared and later divided into 3 samples of 30 mL and for AA, 100 mL of an aqueous solution of AA at a concentration of 180 ppm was produced, which was divided into 3 samples of 30 mL.

Stability study in an oven at 30°C

The NE solutions were divided into triplicates and stored in a SOLAB SL-100 oven for 150 days at 30°C. They were analyzed for size, polydispersion index, zeta potential, pH, and antioxidant activity from 0 to 150 days during this period. For TBHQ NE, samples were collected on days 0, 3, 10, 30, 45, 60, 75, 102 and 150. For AA and White NE samples, the times were 0, 3, 10, 30, 45, 60, 75, 90, 105, 150.

For comparison, the same study was carried out with aqueous solutions of antioxidant substances. However, these were analyzed only for antioxidant activity and pH.

Physicochemical characterization of NE

Polydispersion index and zeta potential

The analyzes for polydispersion index and zeta potential were performed in triplicate using the Zetasizer equipment. Malvern ^®. For samples preparation, it was diluted in distilled water at a ratio of 1:100 (V/V).

pH

The Tecnopon pH meter mpa 210 was used to measure the pH of aqueous solutions immersing the electrode into this.

Antioxidant activity

To evaluate the antioxidant activity of aqueous and NE solutions, the method used was the reduction of the DPPH radical (2,2-diphenyl-1-picryl-hydrazyl), adapted from Sousa et al. [13]. To carry out this test, the NE was previously centrifuged at 15,000 rpm for 50 minutes, and the supernatant was collected.

Sample absorbances were measured at 515 nm, and Eq. 2 was applied to convert absorbances into the antioxidant activity percentage.

$$AA\left(\%\right)=\left[ \frac{ (Abs controle - (Abs amostra-Abs branco)}{Abs controle} \right]*100$$

Equation 2.

Biodiesel production

The biodiesel production technique was the transesterification of commercially obtained soybean oil using methanol and potassium hydroxide catalyst, as described by Gallina et al. [3].

Preparation of NE DE TBHQ and AA in addition to biodiesel

Two formulations of each NE studied were prepared, and their addition to biodiesel occurred by two different means. Firstly, biodiesel was washed with the NE suspension, replacing the aqueous washing. The second formulation was the addition of powdered NE, obtained via lyophilization of the NE suspension, using a lyophilizer equipment Terroni Enterprise model II. These procedures were carried out to evaluate their effects on the oxidation stability of biodiesel.

Evaluation of biodiesel induction time

To assess the oxidation stability of biodiesel, its induction time at 110°C was measured, with and without the addition of NE antioxidants, using the Rancimat 873 equipment from Metrohm ^® .

Statistical analysis of data

For data analysis of size, zeta potential and polidispersion index, t-test statistical analyses were performed using Microsoft Excel software.

The homogeneity in the distribution of NE particle sizes is measured by the polydispersion index (IPD), which, for reference, considers values between 0 and 1. Results close to zero indicate greater homogeneity of the system, and results near to 1 reflect a heterogeneous system [12, 14]. According to the results of Table 1, the greatest homogeneity was obtained for the TBHQ NE, followed by the NE without active. On the other hand, the NE with the least homogeneity was that of AA.

Regarding the size of NE, in this work, we consider the result obtained to be adequate, as they are close to the range of values usually obtained for polymeric nanoparticle systems. Furthermore, applying nanoparticles obtained in biodiesel does not require a specific size range for EN.

Even without the size requirement, the results obtained for the AA nanosphere, with the largest diameter, are very close to those of Liu and collaborators [15] when producing PLGA nanospheres (poly(lactic acid- co - glycolic acid) containing daunorubicin through the double emulsification method using dichloromethane as a solvent, where the nanoparticles obtained by them showed a size of 416 nm. Mahmoud and McConville (2021), obtained nanospheres with a size of approximately 200 nm, using the same method and polymer in this assay[16].

Concerning EE, the results are acceptable for the method used since the greater the affinity of the substance for the aqueous phase, the lower the tendency to encapsulate [17]. Several factors can affect EE, such as the polymer amount, organic solvent, stabilizer used, and the chemical characteristics of the active ingredient.

Table 1 presents the physical-chemical results of the White NE, TBHQ, AA and the encapsulation efficiency (EE).

Table 1

Results of zeta potential, polydispersion, size and EE for White, TBHQ and AA nanospheres .
Sample	Zeta Potential	IPD	Size	pH	EE
White	-10.25 ± 0.65	0.189 ± 0.032	277.07 ± 2.90	4.76	-
TBHQ	-12.50 ± 0.42	0.158 ± 0.028	267.25 ± 4.03	6.20 / 7.7	64.33
AA	-11.63 ± 0.21	0.245 ± 0.035	401.20 ± 9.37	3.26/3.57	18.80

In Fig. 1, are showed the SEM images for the NE. From the SEM images, it was impossible to accurately identify the NE, probably due to loading in the sample by the microscope's electron beam, making it difficult to get closer to the sample and, consequently, not reaching the nanometric scale. However, it is possible to verify the presence of relatively agglomerated polymers, which is a negative factor for the system's stability. Still, this phenomenon was also expected since for the insertion of the sample in the SEM it must be without the presence of solvent that would contribute to greater particles dispersion.

Figure 1 Scanning electron microscopy images of EN containing antioxidants: (A) White and (B) TBHQ, the latter with the approximation of three thousand times, and (C) AA, with the approximation of two thousand times.

Oven stability test

Figure 2 shows the results of the physical-chemical tests, the antioxidant activity of TBHQ and white NE, and the TBHQ aqueous solution.

Fig. 2 Results referring to the tests

(A) IDP, (B) Size, (C) zeta potential, (D) antioxidant activity (bar) and pH (line) for TBHQ nanospheres, blank and TBHQ aqueous solution.

The IDP tests shown in Fig. 2A indicate no changes over the 150 days for the white NE and the TBHQ. The nanospheres maintained homogeneity, even with thermal stress. Also, the particles size (Fig. 2B) for both NE did not show significant changes over time.

Regarding the zeta potential (Fig. 2C), there are divergences between the behavior of white NE and TBHQ over time, suggesting the difference is related to TBHQ's release, changing the nanoparticle's surface charge. When only the NE of TBHQ is observed, there is a significant change in the zeta potential on day 60 (-9.69 ± 0.48 mV) and on day 75 (-25.43 ± 1.13 mV). Zeta potential may be related to the adsorption of free TBHQ on the NE surface, which has already been reported by Varan and Bilensoy [18], who found the presence of hydroxypropyl - 𝛽 - cyclodextrin on the surface of the nanoparticle caused alterations at the zeta potential value.

In addition to the above, according to the pH results (Fig. 2D), the NE remained stable throughout the study since the decrease in pH was not significant, except on day 150 when there was a drop in pH, due the acid character of the polymer degradation product.

As for the antioxidant activity (AAO) shown in Fig. 2D, the stability of the TBHQ aqueous solution over the time studied is highlighted since even under thermal stress, it presented values greater than 80% of antioxidant activity.

Regarding the TBHQ EN, the AAO from day 0 to day 10 showed values at the same level as the TBHQ solution due to the presence of free TBHQ in the nanosphere formulation. From day 31, there was a significant decrease in the AAO of TBHQ NE, indicating was no release of encapsulated antioxidants, but consumption of free TBHQ in the formulation. The increase in the antioxidant activity of the TBHQ NE occurred gradually from day 45 until day 105, suggesting in this time was a release of TBHQ. From day 105, the decrease in AAO is related to the degradation of TBHQ. The observed behavior is according with the literature, where NE from PCL tends not to initiate release instantly, in addition to presenting a slow liberation over time, completing this until 1 year [19]. Thus, the increase in the antioxidant activity of the TBHQ nanosphere after day 45 is coupled to the slow release of the antioxidant. However, the AAO of the NE was still lower afterwards free solution, probably due to the concentration in the solution being greater than the TBHQ concentration after the liberation. Another phenomenon which would justify the decrease in AAO over 150 days, is the product of PCL degradation is consuming the antioxidant.

Figure 3 UV-Vis absorption spectra for NE white (a), TBHQ solution (b), and TBHQ NE (c) samples.

The UV-Vis absorption spectroscopy images, Fig. 3a, illustrate that no band within the studied spectrum can be referred to as the NE polymer. The characteristic band of TBHQ is at the wavelength of 287 nm, this is well evidenced both in the aqueous solution and the NE spectra of TBHQ, Fig. 3b and 3c, mainly on day zero. From day 3, regardless of the solution, the insertion of a band at 250 nm is observed, with greater emphasis on solutions with TBHQ NE. The appearance of a new band in the UV-Vis spectrum indicates the presence of another compound, probably originating from the thermal degradation of TBHQ, which may present several structures with potential absorption in the UV-Vis region, as reported by Hamama and colaborators [20]. This degradation and the slow release of TBHQ may have contributed to the lower levels of AAO from the TBHQ NE throughout the assay compared to the TBHQ aqueous solution.

When analyzing Fig. 3C, it is observed that the characteristic bands of TBHQ at 287 nm decrease over time, being more evident on days 0 and 3, and after these times, the bands at 250 nm are more relevant. This result, together with the AAO assays in Fig. 3D, suggests that this possible compound from the degradation of TBHQ has high values of DPPH radical capture. Therefore the antioxidant capacity can be associated with this degradation product. These observations emphasize the constant and high AAO throughout the 150-day test under thermal stress of 30°C, justifying TBHQ's large-scale and efficient use as an antioxidant.

In Fig. 3b, there are bands with high absorption values at 250 and 287 nm, regardless of the days studied. In addition, the free TBHQ solutions showed the highest values and stability related to AAO, indicating that the association of the two compounds (TBHQ and the degradation product) has a superior effect on antioxidant capacity.

Figure 4 shows the results of the physical-chemical tests and the antioxidant activity of the NE of AA and white, together with the aqueous solution of AA.

Figure 4 Results to the IDP tests (A), size (B), zeta potential (C), pH (line), and antioxidant activity (bar) (D) for AA nanospheres, blank, and AA aqueous solution.

First, the effectiveness of the protection and release of NE from AA should be highlighted when compared to the aqueous solution of AA, presenting AAO above 50% over 150 days. Furthermore, the AAO of the AA nanosphere reached values greater than 90%, indicating excellent potential for use in biodiesel, as shown in Fig. 4d.

The results of the IDP and particle size tests (Figs. 4A and 4B) indicate a homogeneity of the NE solution during the 150 days. Regarding the zeta potential (Fig. 4C), there are divergences between the behavior of white NE and AA over time, especially on days when there is an increase in antioxidant activity, indicating the release of AA. It is observed that AA nanospheres tend to become more stable (zeta potential greater in modulus) in the same periods that there is stability or an increase in antioxidant activity. The results of IDP, particle size, and pH values did not show significant changes indicating a sharp degradation of the polymer used to produce nanospheres. It should be a considerable increase in the pH of the free solution due to AA degradation, which was not observed for the AA nanosphere, indicating the protection of the active by the NE. Regarding the size and zeta potential of the AA NE, similar results were obtained by Amin and collaborators [21] when they produced poly-ε-caprolactone nanoparticles containing AA, reaching a value of -11 mV for the zeta potential and a particle size of 321.8 nm.

Figure 5 Scan of samples of a) NE white, b) Free AA solution, and c) NE AA

The characteristic peak of AA is located at 264 nm, could be observed in the solution spectra of free AA and AA NE. In the case of the free AA solution, the peak disappears after day 0, evidencing the degradation of the compound, and confirming by the sudden drop in antioxidant activity (Fig. 5A, 5B, 5C). The AA NE solution, maintains the peak until day 3, and after that, it is no longer observed in the spectrum. However, the antioxidant activity remains throughout the period of 150 days, even if in smaller percentages compared to the initial days. This fact indicating that the degradation of the AA is no complete and the appearance of a peak at approximately 205 nm, near to presented by the white NE (< 212 nm ), may indicate the formation of an AA-PCL structure, since the products of AA degradation would be present in the absorption region between 230 and 300 nm according to Yuan and Chen [22].

The intensity of this peak is practical of the same magnitude for all the days which the samples were collected, except for day 150 presenting the lowest intensity, with a smaller amount of AA in the AA-PCL structure, corroborating the AAO tests (Fig. 5A, 5B, 5C).

Oxidation stability studies

After the in vitro analysis of AAO of the NE produced, they were added to the biodiesel intending verify the antioxidant effect. First the addition was made in a solution in the last biodiesel washing step. Second, after biodiesel production and washing, in powdered form (lyophilized), under mechanical agitation. The results referring to the rancimat tests are shown in Table 2. According to Table 2, the presence of NE accelerated the biodiesel oxidation since the induction time (IT) was below compared of biodiesel without the antioxidants addition, oposite to the studies of antioxidant activity in the greenhouse, which showed satisfactory AAO by the radical DPPH.

This behavior suggests was no release of the antioxidant as expected,that could be explained by the polarity of the medium. In the test carried out in a greenhouse, the solution was aqueous and organicin the case of biodiesel. The NE dissolution medium contributes to a greater or lesser release of the active. Another reason for not observing the increase in induction time may also be related to the method of adding NE to biodiesel, as the insertion was performed without the aid of methods that would enhance the complete homogenization of NE, such as heating and ultrasound.

Table 2

Induction time for biodiesel samples and with the addition of nanospheres containing antioxidants.
Sample	Lyophilized	Not lyophilized
AA NE	3.56 ± 0.01	4.71 ± 0.13
TBHQ NE	3.48 ± 0.1	4.77 ± 0.04
Pure Biodiesel	5.87 ± 0.14

It was possible to produce NE from PCL containing TBHQ and AA with an encapsulation efficiency of 64.33% and 18.80%, respectively. The physical-chemical characterization of NE showed values consistent with similar studies in the literature, such as negative zeta potential and relative stability for all NE. The PDI and particle size confirmed the homogeneity of NE and nanometer size.

About the AAO and protective capacity of the NE over time in the oven test at 30°C, the NE from TBHQ and AA showed AAO values of 84.35% for the NE from TBHQ and 89.14% for the NE of AA on day 0, with some stability over time, obtaining a value of 45.90% and 74.73% for the NE of TBHQ and AA, respectively in the time of 150 days.

Antioxidant activity was not significantly observed in the rancimat test, indicating the need for a more efficient method for inserting the antioxidant into biodiesel. Concerning the biodiesel oxidation kinetics in the presence of NE, all samples showed second-order kinetics, differing from pure biodiesel, which is first-order.

Statements and Declarations

The authors declare that there are no conflicts of interest.

Ethical approval

Not applicable

Competing Interests and Funding

This work was financially supported by the National Council for Scientific and Technological Development (CNPQ).

Availability of data and materials

Not applicable

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No competing interests reported.

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Antioxidant Activity and Physicochemical Stability of Nanospheres: Evaluation in Vitro and Applied to Biodiesel

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Abstract

Figures

Introduction

Materials and methods

Preparation of TBHQ polymeric nanoparticles

Preparation of Active-free Polymeric Nanoparticles

Preparation of polymeric nanoparticles Ascorbic acid

Determination of the encapsulation efficiency of the TBHQ nanosphere :

Determination of the encapsulation efficiency of the AA nanosphere :

Production of aqueous solutions for the oven stability test:

Stability study in an oven at 30°C

Physicochemical characterization of NE

Polydispersion index and zeta potential

pH

Antioxidant activity

Biodiesel production

Preparation of NE DE TBHQ and AA in addition to biodiesel

Evaluation of biodiesel induction time

Statistical analysis of data

Results and discussion

Oven stability test

Oxidation stability studies

Conclusions

Declarations

References

Additional Declarations

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