Potential application of munguba butter in the formulation of nanoemulsified systems

Potencial aplicação da manteiga de munguba no desenvolvimento de sistemas nanoemulsionados

Abstract

Munguba butter has bioactive compounds such as vitamin E and phytosterols, which has valued its application in the development of new products, with advantages in its use in emulsified formulations. Therefore, the objective was to develop and evaluate the stability of a nanoemulsion containing munguba butter as the oily phase. Munguba butter was extracted by the ultrasound assisted method and its HLB (hydrophilic-lipophilic balance) was determined. Next, formulations varying the concentration of butter from 1-40% were developed and classified into liquid or solid emulsion and phase separation. Liquid emulsions were evaluated for hydrodynamic particle diameter, polydispersity index (PDI), Zeta potential (ζ), rheological characterization, and stability assays. The butter had an HLB of 6.98. The NE 1.0% formulation was selected and demonstrated to be unstable at high temperatures (45 ± 2 °C) and remained stable at room temperature, refrigeration and light radiation for 90 days. Munguba butter, because it has high amounts of saturated fatty acids, hinders its application in the development of new products. However, the success in the development of the NE 1.0% formulation is noteworthy, remaining stable when exposed to refrigeration, room temperature and light radiation.

Keywords:  particle diameter; infrared; butter; nanoemulsion; Pachira aquatica Aublet

Resumo:

A manteiga de munguba apresenta compostos bioativos como vitamina E e fitoesteróis o que tem valorizado sua aplicação no desenvolvimento de novos produtos, havendo vantagens em sua aplicação em formulações emulsionadas. Logo, o objetivo foi desenvolver e avaliar a estabilidade de uma nanoemulsão contendo como fase oleosa a manteiga de munguba. A manteiga de munguba foi extraída pelo método de ultrassom e seu EHL (equilíbrio hidrófilo-lipófilo) foi determinado. Em seguida, foram desenvolvidas formulações variando as concentrações de manteiga de 1-40% sendo classificadas em emulsão líquida ou sólida e separação de fases. As emulsões líquidas foram avaliadas quanto ao diâmetro das partículas hidrodinâmicas (DLS), o índice de polidispersividade (PDI), potencial Zeta (ζ), caracterização reológica e ensaios de estabilidade. A manteiga apresentou EHL de 6,98. A formulação NE 1.0% foi selecionada e mostrou-se instável em alta temperatura (45 ± 2 °C) e manteve-se estável em temperatura ambiente, refrigeração e radiação luminosa durante 90 dias. A manteiga de munguba, por possuir altas quantidades de ácidos graxos saturados, dificulta sua aplicação no desenvolvimento de novos produtos. Entretanto, ressalta-se o êxito no desenvolvimento da formulação NE 1.0% sendo estável quando exposta a refrigeração, a temperatura ambiente e a radiação luminosa.

Palavras-chave:  diâmetro de partícula; infravermelho; manteiga; nanoemulsão; Pachira aquatica Aublet

1. Introduction

Munguba (Pachira aquatica Aublet), also known as fake cocoa and wild cocoa (Dourado et al., 2015), is a fruit with numerous seeds that can be used for application in different fields such as food, biofuel, pharmaceutical and cosmetics (Rezende et al., 2021). Its seeds are rich in proteins, carbohydrates, fibers, and lipids, the latter being composed mainly of fatty acids that are liquid at elevated temperatures and solid at room temperature (Rodrigues and Pastore, 2021). However, although several authors define it as an oil, it is noteworthy that, because it is in solid or semisolid form at room temperature, 25°C, it is classified as a vegetable fat (FAO, 2023; Brasil, 2021). Thus, we call it munguba butter.

Munguba butter is predominantly composed of saturated fatty acids, mostly palmitic, and has important properties derived from bioactive compounds such as vitamin E and phytosterols (Rodrigues and Pastore, 2021). Therefore, some studies have evaluated the presence of phenolic compounds and the antioxidant activity of munguba butter (Rezende et al., 2021; Rodrigues et al., 2019; Raiser et al., 2020; Silva et al., 2020), valorizing its application in developing new products.

Raiser et al. (2018), when developing a cosmetic cream containing munguba butter observed stability at room temperature, maintenance of pH close to the skin's pH, and good spreadability of the product, in addition to antioxidant activity suggesting the use of munguba butter as an alternative to synthetic antioxidants. Still, according to Rodrigues and Pastore (2021), although munguba butter cannot be used in emulgel formulations due to the greater presence of saturated fatty acids, which gives it a sticky feeling, the results obtained in their studies showed advantages in its application in emulsified formulations.

Within this context, nanotechnology has been used in several industrial fileds in order to optimize and/or obtain safer, more effective and quality products. Therefore, the nanoemulsion is characterized by presenting nanometric particles, on the order of 1-500 nm, stabilized through the action of surfactant molecules (Kumar et al., 2021; Moghassemi et al., 2022).

Nanoemulsified systems have advantages such as modified release profile, lower toxicity, kinetic stability, and good skin permeation (Kong et al., 2011; Mahboobian et al., 2019). They are composed of a polar phase (usually water) and an apolar phase (usually oil), the latter can be composed of lipid soluble vitamins, essential oils and vegetable oils (Ozogul et al., 2022).

Combining the advantages of using munguba butter due to its fatty acids and bioactive compounds with the advantages of developing nanoemulsified systems, we sought to develop a new product that will serve as a basis for incorporating pharmaceutical and/or cosmetic substances. Therefore, the aim of the present study was to develop and evaluate the stability of a nanoemulsion containing munguba butter as the oily phase.

2. Materials and Methods

2.1. Plant material

Munguba fruits were collected in the municipality of Sinop/MT, Brazil (S11 50’06,20”; 30’02,30”) and taken to the Quality Control Laboratory (LaCQ). The exsiccata was registered at the Centro-Norte-Mato-Grossense Herbarium (CNMT) located at the Federal University of Mato Grosso, Sinop Campus, under registration number 4506.

2.2. Munguba butter obtention

The extraction process was performed according to Raiser et al. (2020) using munguba flour and hexane as solvent in the ratio of 1:5 (weight/volume [w/v]). The extraction was performed using the ultrasound assisted method, with an ultrasonic bath (Cristófolo®), for 2 hours at a frequency of 45 kHz and a temperature of 35 ± 2 °C. Then, the mixture was vacuum filtered and the butter was concentrated on a rotary evaporator (450-5, Fisatom®) at 50 ± 2 °C.

2.3. Determination of hydrophilic-lipophilic balance (HLB)

The HLB of munguba butter was determined according to Fiori et al. (2017), with adaptations. The serial emulsion samples were prepared, in triplicate, by heating in a water bath at 75 ± 2 °C, in which the aqueous phase (polyoxyethylene sorbitan monooleate (Tween 80® - TW 80) and water) and oily phase (sorbitan monooleate (Span® 80 – SP 80) and munguba butter) were prepared separately. Then, they were mixed and stirred under heating for 5 minutes in an ultrasonic processor (VCX-130, Sonics & Materials®) at 40% amplitude and a frequency of 20 kHz. After that, the stirring was kept for another 5 minutes at room temperature. After 24 hours of preparation, the samples were evaluated using a BEL® Photonics microscope at 40x magnification and the globules were photographed to determine the area using Image J Software.

2.4. Development of the nanoemulsified systems

The emulsified formulations consisted of distilled water, methylparaben and TW 80 as aqueous phase and munguba butter and SP 80 as oily phase. Forty-one formulations were developed, varying the concentration of munguba butter between 1 and 40%. The aqueous and oily phases were placed separately in the ultrasonic bath at 35 °C to aid the solubilization of the constituents and provide energy in the form of heat. Then, the aqueous phase was poured over the oily phase and the formulation was homogenized in an ultrasonic processor (VCX-130, Sonics & Materials®) at a frequency of 20 kHz, with an amplitude of 40%, for 1 hour.

2.5. Dynamic light scattering, polydispersity index and zeta potential

The hydrodynamic particle diameter and the polydispersity index (PDI) of the formulations were determined using dynamic light scattering (DLS), while the Zeta potential (ζ) was determined by electrophoretic measurements (Zetasizer® Nano ZS Instrument (Malvern Instruments)). The formulations were evaluated in triplicate, at 25 °C, in a 1:400 (v/v) dilution.

2.6. Rheological characterization

The rheological characterization was performed in a rheometer (MCR 102, Anton Paar® GmbH, Ostfildern, BH, Germany) coupled to the Rheoplus V3.61 Software and, according to Ribeiro et al. (2020), using 600 µL aliquots of formulation on the surface of the reading plate. For the flow and viscosity curves, the shear stress was determined with a variation from 0 to 5 Pa for the upward curve and from 5 to 0 for the downward curve, at 25 ± 2 °C, with 75 readings.

2.7. Quality control: stability tests

2.7.1. Preliminary stability

Alternating temperature cycles were performed at 5 and 45 °C, every 24 hours for a period of 14 days. Physicochemical parameters were evaluated at the beginning and end of the 14 days cycle.

2.7.2. Accelerated stability

New formulations were prepared and divided into four groups according to storage place: group G (5 °C), group TA (25 °C), group E (45 °C) and group RL (light radiation). Physicochemical parameters were evaluated at 24 hours after preparation, 30, 60 and 90 days.

2.7.3. Physicochemical parameters evaluated

• Centrifugation: 10g aliquots of each formulation were tested at 25 °C in a centrifuge (Quimis®) at 3000 rpm (rotation per minute) for 30 minutes;

• Organoleptic characteristics: the formulations were evaluated for their visual appearance and odor and categorized as N (normal) or M (changed), at each analysis period;

• pH determination: pH was performed by directly inserting the electrode into the formulation in order to determine the pH of the formulations and monitor possible changes that may indicate degradation. The pH meter (Tecnopon®, Brazil) was calibrated with standard buffer solutions of pH equal to 4 and 7;

• Determination of refractive index: the refractive index was determined on the Abbe refractometer (Polax WYA-2S) according to the Brazilian Pharmacopoeia (Brasil, 2019) in order to detect deviations in the incident light scaterring on the formulations and thus, observe signs of instability.

2.8. Fourier-transform infrared spectroscopy (FTIR)

The infrared spectrum was performed according to Geetanjali et al (2021), with adaptations. The test took place in a Fourier-transform spectrophotometer (Shimadzu Iraffinity-1 (Shimadzu)) applying the sample directly to KBr tablets, at a wavelength ranging from 4000 to 500 cm^-1, resolution of 4 cm^-1 and 32 scans.

3. Results

The obtained munguba butter was analyzed by the LaCQ at the Federal University of Mato Grosso. The results corroborated those obtained by Raiser et al. (2020), in conformity with the legislation for oils and fats, enabling its use in the development of formulations.

The HLB of the munguba butter was determined by mixing the surfactants TW 80 and SP 80 with distilled water for emulsification. Then, the coefficient of variation of the globules area was calculated, resulting in the HLB of 6.98.

From the HLB, 32 emulsified formulations were prepared by varying the concentrations of the aqueous phase, oily phase, and surfactant mixture in order to verify the best concentration regions of each phase in the formation of liquid emulsified system. After 24 hours of preparation, the formulations were categorized into EML (liquid emulsion), EMS (solid emulsion) and SF (phase separation). The samples designated as EMS and SF were discarded from the study and only one formulation was classified as EML, which had 10% of munguba butter, 5% of surfactants (TW and SP) and 85% of water in its composition. The formulation went on to preliminary stability assay, however, lump formation was observed during the assay. Thus, new formulations were prepared, this time varying the concentration of munguba butter from 1.0 to 5.0%, which when analyzed in the centrifugation test showed no instability, and were then submitted to the preliminary stability test. However, only the formulations containing 1.0, 1.5 and 2.0% of munguba butter maintained their stability during the 15-day period (Table 1).

Table 1
Preliminary stability of emulsified formulations containing 1.0, 1.5 and 2.0% of munguba butter.

Formulation	Time (days)	Appearance, Color and Odor	pH (value)	Refractive index
NE 1.0%	1	N	5.42 ± 0.02	1.3500 ± 0.00
	15	N	5.29 ± 0.06	1.3500 ± 0.00
NE 1.5%	1	N	5.52 ± 0.05	1.3500 ± 0.00
	15	N	5.04 ± 0.09	1.3500 ± 0.00
NE 2.0%	1	N	5.51 ± 0.06	1.3500 ± 0.00
	15	N	5.13 ± 0.13	1.3500 ± 0.00

• Key: Appearance: N = Normal; NE = Nanoemulsion.

In order to classify the formulations and detect the best one to proceed with the quality control tests, the NE 1.0%, 1.5% and 2.0% formulations were subjected to determination of particle size, polydispersity index and zeta potential. The results were 155.1, 164.7, and 176.2 nm for the DLS analysis, PDI was equal to 0.216, 0.197, and 0.214 and ζ of -38.8, -39.2, and -39.1 mV, respectively.

Figure 1 shows the results obtained in the rheological characterization showing that the formulations containing different concentrations of munguba butter have a non-Newtonian profile, all of which are classified as pseudoplastic. Moreover, in Figure 2, a reduction in viscosity was observed with the increasing of the shear rate. Therefore, the results showed no differences between the nanoemulsions with different concentrations of butter, as shown by the occurrence of an overlap between them

Figure 1
Rheological characterization of nanoemulsions containing munguba butter. Key: NE = Nanoemulsion.

Figure 2
Viscosity variation of nanoemulsions containing munguba butter. Key: NE = Nanoemulsion.

The NE 1.0% formulation was chosen to continue in the experiment and was prepared again and subjected to the accelerated stability test, for 90 days. The physicochemical parameters were evaluated at 1, 30, 60 and 90 days. The results are shown in Table 2.

Table 2
Accelerated stability evaluation of the NE 1.0% sample in the groups exposed to 25, 5 and 45 °C and light radiation.

Formulation	Time (days)	Appearance, Color and Odor	pH (value)	Refractive index	RL
NE 1.0% 25 °C	1	N	5.15 ± 0.13	1.3500 ± 0.00	ASF
	30	N	5.10 ± 0.42	1.3500 ± 0.00	ASF
	60	N	4.99 ± 0.29	1.3500 ± 0.00	ASF
	90	N	4.98 ± 0.27	1.3500 ± 0.00	ASF
NE 1.0% 5 °C	30	N	5.28 ± 0.04	1.3500 ± 0.00	-
	60	N	5.53 ± 0.20	1.3500 ± 0.00	-
	90	N	5.56 ± 0.28	1.3500 ± 0.00	-
NE 1.0% 45 °C	30	N	4.00 ± 0.45	1.3500 ± 0.00	-
	60	N	3.40 ± 0.00	1.3500 ± 0.00	-
	90	M	-	-	-

• Key: Appearance: NE = Nanoemulsion; N = Normal; M = Modified; RL = Exposure to Light Radiation; ASF = absence of phase separation.

Formulations NE 1.0% from the groups G and TA showed normal organoleptic characteristics and stable refractive index. Concerning the pH values, the formulations from group G showed pH variation of 5.28 ± 0.04 and 5.56 ± 0.28 and those from the TA group had pH variations of 4.98 ± 0.27 and 5.15 ± 0.13. Regarding the RL group, the formulation demonstrated maintenance of the organoleptic characteristics during the 90 days. However, the formulations from group E showed a decrease in pH values and phase separation after 90 days.

IR characterization (Figure 3) was performed in order to evaluate the quality and maintenance of the characteristics of NE 1.0% LR, exposed to light radiation for 90 days, compared to NE 1.0%, without light incidence, after 24 hours of preparation. The formulations showed the same profile with similar bands, close to 1627 nm and 3462 nm.

Figure 3
Infrared absorption spectrum of NE 1.0% and NE 1.0% RL nanoemulsions. Key: (a) NE 1.0%; (b) NE 1.0% RL (light radiation). NE = Nanoemulsion.

4. Discussion

Munguba butter has a solid appearance at room temperature, yellowish color and characteristic odor. Furthermore, studies have reported the presence of γ-tocopherol, phytosterols and flavonoids, bioactive compounds that perform important functions such as antioxidant, anti-inflammatory and anti-hyperglycemic actions, in addition to preventing cardiovascular diseases (Rodrigues and Pastore, 2021; Silva et al., 2020; Lopes et al., 2020), demonstrating potential application in the development of new food, pharmaceutical and cosmetic products.

The determination of the HLB made it possible to establish the appropriate proportions of surfactants to be used to prepare the formulations. From the resulting measurements of the formed globules, it was observed that the lowest coefficient of variation was 0.00013% and an area of 0.042 mm². The emulsified system was homogeneous and had no aggregation, resulting in the HLB of 6.98 for the munguba butter. Also, according to Fiori et al (2017), microscopic evaluations for the determination of HLB of vegetable oils have been used in the literature, however, there are few studies regarding the determination of the area of the globules.

After the HLB value determination, 32 emulsified formulations were developed and made possible to identify the most appropriate concentrations to estimate the stability of the product. However, after the development and preliminary tests, the presence of clusters was identified in all formulations, suggesting solidification of munguba butter due to its fatty acid composition, in which there is a predominance of saturated chains. Therefore, new formulations were prepared to prevent solidification and instability of the formulation.

The preliminary stability tests of the formulations NE 1.0%; NE 1.5% and NE 2.0% showed that the decrease in the concentration of munguba butter favored the development, since the organoleptic characteristics remained unchanged, and no signs of clusters, precipitates, and coalescence were found. Regarding the pH, its determination makes it possible to identify possible hydrolytic degradation, which was not observed since there was no significant change in its values. The refractive index aims to identify changes in the samples as the scattering of the incident light changes, which was not identified in the test showing that the formulations maintained their initial physicochemical characteristics.

The particle size determination and the polysdispersity index assays allow to classify the formulations and identify homogeneity or heterogeneity of the system. The analyzed formulations presented an average particle size below 200 nm and low PDI, those being characteristics of monodisperse systems and indicative of homogeneity. Therefore, the formulations were referred to as nanoemulsified systems (Moghassemi et al., 2022). The use of butter in nanoemulsion systems can lead to larger globule sizes and instability due to the saturation of their chemical chains. In this way, the efficiency in the development of the formulations containing munguba butter is emphasized, since it was classified as a nanoemulsion. The zeta potential is used to predict the stability of a formulation and its value depends on the physicochemical properties of the formulation constituents, as well as the presence of electrolytes (Gurpreet and Singh, 2018). The nanoemulsions presented values greater than - 30 mV, which generated electrostatic and steric stability, ensuring non-coalescence.

The pseudoplastic characteristics and viscosity reduction with increased shear rate in the formulations (Figure 1 and Figure 2) are advantages collaborating to spreadability and fluidity of formulations, facilitating the flow of the product in industrial pipelines, for example.

Also, Raiser et al (2018) when preparing and evaluating the stability of emulsions containing 3 and 5% of munguba butter observed that the emulsion with a higher concentration of butter had its spreadability and stability affected, since a greater amount of butter increases the consistency and solidity of the product.

Therefore, the NE 1.0% formulation was chosen for the accelerated stability test, due to its smaller globule diameter (155.1 nm) and lower content of munguba butter (1.0%). In this assay, the results (Table 2) showed that the formulation cannot be stored at high temperatures as it leads to coalescence and phase separation. However, exposure to low temperatures, room temperature and light radiation did not compromise the stability of the formulation over the 90 days. Thus, it is an advantageous formulation for industrial application, as it maintained its stability, in addition to using low concentrations of surfactants and munguba butter, improving yield and production costs.

From the FTIR, two main bands were observed in the formulations exposed to radiation, the band at 3465 nm indicating the presence of water molecules, which intensity due to the high amount of water in the formulations (≅ 94%), and a band close to 1664 nm corresponding to the presence of ketones and aldehydes, suggestive of fatty acids (C=O) (Raiser et al., 2020; Ranjbar et al., 2023). Additionally, the FTIR allowed to verify the preservation of the characteristics of NE 1.0%, even after light exposure for 90 days. The stability under light radiation becomes a great advantage since it allows the use of diverse packaging and reduces storage difficulties.

The developed nanoemulsion showed to be promising as it presented excellent stability, both thermal and against light radiation, valuing its potential for its application in different industrial fields. It also consisted of a high concentration of water and a low content of surfactants, reducing the toxicity of the formulation, in addition to using munguba butter, which is rich in antioxidant molecules, which can provide benefits for the formulations and for the consumer.

5. Conclusion

Nanoemulsions have advantages such as greater permeation and absorption of active ingredients and lower toxicity due to the low surfactant content, in addition to enabling their application in the development of new products such as food, pharmaceuticals and cosmetics. It is noteworthy the success in the development of the NE 1.0% formulation, which consisted of a high concentration of water and low content of surfactants, variables that present a greater risk of triggering instabilities and phase separation. The formulation maintained physicochemical stability for 90 days exposed to 5 and 25°C and maintained its organoleptic characteristics even in the face of light radiation.

Acknowledgements

This study was supported by The Federal University of Mato Grosso, Sinop Campus.

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• RODRIGUES, A.P., PEREIRA, G.A., TOMÉ, P.H.F., ARRUDA, H.S., EBERLIN, M.N. and PASTORE, G.M., 2019. Chemical composition and antioxidant activity of Monguba (Pachira aquatica) seeds. Food Research International, vol. 121, pp. 880-887. http://doi.org/10.1016/j.foodres.2019.01.014 PMid:31108821.
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  » http://doi.org/10.33448/rsd-v9i7.4391

Publication Dates

• Publication in this collection
  20 May 2024
• Date of issue
  2024

History

• Received
  08 Dec 2023
• Accepted
  03 Apr 2024