Babassu oil extraction is the main income source in nut breakers communities in northeast of Brazil. Among these communities, babassu oil is used for cooking but also medically to treat skin wounds and inflammation, and vulvovaginitis. This study aimed to evaluate the anti-inflammatory activity of babassu oil and develop a microemulsion system with babassu oil for topical delivery. Topical anti-inflammatory activity was evaluated in mice ear edema using PMA, arachidonic acid, ethyl phenylpropiolate, phenol, and capsaicin as phlogistic agents. A microemulsion system was successfully developed using a Span® 80/Kolliphor® EL ratio of 6 : 4 as the surfactant system (S), propylene glycol and water (3 : 1) as the aqueous phase (A), and babassu oil as the oil phase (O), and analyzed through conductivity, SAXS, DSC, TEM, and rheological assays. Babassu oil and lauric acid showed anti-inflammatory activity in mice ear edema, through inhibition of eicosanoid pathway and bioactive amines. The developed formulation (39% A, 12.2% O, and 48.8% S) was classified as a bicontinuous to o/w transition microemulsion that showed a Newtonian profile. The topical anti-inflammatory activity of microemulsified babassu oil was markedly increased. A new delivery system of babassu microemulsion droplet clusters was designed to enhance the therapeutic efficacy of vegetable oil.
Babassu palm tree (
Among the parts used for medicinal purposes, leaves, roots, and fruits should be highlighted. Leaves and roots are used as tea for pain and wound healing, while fruits are used in a much bigger scale: mesocarp floor and milk are used for the treatment of gastritis, hepatitis, osteoporosis, skin wounds, and leukorrhea; liquid albumen is used as eyedrops to treat conjunctivitis; and the seed oil is used as laxative, vermifuge, and anti-inflammatory and for the treatment of myiasis, mycosis, skin wounds, hemorrhoids, leukorrhea and female genital inflammation, and spider bites [
Vegetable oils such as olive, palm, and coconut oils are well known for their anti-inflammatory properties. Olive oil is rich in anti-inflammatory phenolics like oleocanthal and oleuropein glycosides [
The availability of babassu oil and its easy handling have enabled development of several types of formulations to enhance the therapeutic efficacy of the biologically active components. Targeted delivery can be achieved by applying pharmaceutical nanotechnology, which is based on the synthesis, application, and characterization of nanoscale therapeutic systems to provide a more controlled drug release. In this context, nanostructured systems, for example, microemulsions containing babassu oil, may act as new and potentially efficient therapies for benign prostatic hyperplasia due to their antiproliferative and apoptotic effects [
Microemulsion is a system of two immiscible fluids that is stabilized by an interfacial film of surfactants. It offers the advantages of spontaneous formation, thermodynamic stability, manufacturing simplicity, solubilization capacity of lipophilic and/or hydrophilic solutes, a large area per volume ratio for mass transfer, and the potential for permeation enhancement [
Phorbol 12-myristate 13-acetate (PMA), arachidonic acid (AA), ethyl phenylpropiolate (EPP), phenol, and capsaicin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The surfactants, Span 80 (sorbitan mono-oleate) and Tween® 80 (polyoxyethylene sorbitan monooleate), ethanol, 99.9% deuterated CDCl3, and Supelco® 37 Component FAME Mix were also obtained from Sigma Chemical Co. Kolliphor EL (polyoxyl castor oil) was from BASF SE (Ludwigshafen, Germany) and propylene glycol PA was purchased from Neon Comercial Ltda (São Paulo, Brazil).
Babassu fruits were collected from one palm tree in Araripe, located in the State of Ceará (Brazil), in July 2013. Botanical authentication was conducted by Olívia O. Cano, and a voucher specimen was deposited at the Herbarium of the Agronomic Institute of Pernambuco under number 90,472.
The oil was extracted by a well-established method that is practiced by farmers in forested areas. After harvesting, the seeds were ground with a stone grinder, and the obtained paste was mixed well and allowed to stand overnight. On the next day, cold water was added to the paste, and the upper part was collected and placed on a fire to heat it until it started boiling. Subsequently, the liquid phase was separated from the oil meal using a tissue and heated until the water completely evaporated. Finally, the oil was filtered and used [
Traditionally extracted babassu oil was immediately analysed for some physicochemical properties as described by Adolfo Lutz Institute [
The transesterification procedure of the oil was realized in accord with Metcalfe et al. [
The samples were analyzed using an Agilent Technologies (Palo Alto, CA, USA) 5975C single quadrupole GC-MS equipped with a nonpolar HP-5MS (Agilent) fused silica capillary column (30 m × 0.25 mm i.d.; film thickness 0.25 mm). The oven was initially held at 150°C for 2 min, increased to 230°C by 5°C/min (held for 7 min), and finally increased to 260°C by 4°C/min. The final temperature was maintained for 7.5 min. The carrier gas was helium supplied at a constant flow of 1 mL/min and the split/splitless injector was maintained at 230°C. The applied ionization potential was 70 eV; the scan range was from 35 to 450
The extracted oil (approximately 50 mg) was dissolved in 0.6 mL of CDCl3 and placed in a 5 mm NMR tube. The NMR analyses were performed on a Varian 400-VNMRS (Agilent Technologies, CA, USA) at 26°C, operating at the frequencies of 399.74 and 100.51 MHz for 1H and 13C, respectively. 1H NMR spectra were recorded with 2.5 s acquisition times, sweep widths of 6.4 kHz, 45° pulse angles, and 1 s delay times.
Male Swiss and BALB/c mice (25–30 g,
For evaluation of ear weight, mice were euthanized and 6 mm diameter samples were taken from both ears using a biopsy punch (Richter®, Brazil). Each biopsy was weighed on a semi-micro analytical balance (AUW-D 220, Shimadzu, Japan). Ear edema (EE) was expressed as the increase in ear sample weight, using the following formula:
For all treatments, animals were anesthetized with 1% halothane. Right ears were then challenged with different phlogistic agents diluted in acetone (20
Ear edema was induced by topical application of 20
For elucidation of the mechanisms underlying the topical anti-inflammatory activity of babassu oil on PMA-induced mice ear edema, different phlogistic agents were used to induce ear edema: arachidonic acid (2 mg/ear), ethyl phenylpropiolate 5% (20
After 1 h, mice were euthanized for ear edema measurement. Mice challenged with capsaicin were euthanized after 30 min of exposure. AA-induced ear edema was performed in BALB/c mice [
A series of oil-in-water emulsions with HLB values ranging from 4.5 to 15 was prepared with the surfactants Span 80 and Tween 80 at a 2% total blend concentration w/v, 93% water, and 5% babassu oil using the Griffin equation [
The droplet size distribution of the dispersed phase of the emulsions was determined by DLS using the Nanotrac Wave equipment (Microtrac Inc., Montgomeryville, PA, USA) with measurement capability from 0.8 to 6500 nanometers. The data were calculated using the manufacturer’s software. Each emulsion was diluted with aqueous propylene glycol (1 : 100) and analyzed in triplicate.
The emulsions were diluted 1 : 25 with aqueous propylene glycol, and the percentage of transmission (%
Babassu oil, Span 80, and Kolliphor EL were selected as the oil phase and surfactants, respectively. Surfactant mixtures were tested at a ratio of 6 : 4 (w/w), as defined by the Griffin equation. The pseudo-ternary phase diagrams were constructed using the water titration method at room temperature, and the results were plotted using Software Origin® Pro 8.0. For each phase diagram, mixtures of surfactants and oil were prepared at weight ratios of 1 : 9, 2 : 8, 3 : 7, 4 : 6, 5 : 5, 6 : 4, 7 : 3, 8 : 2, and 9 : 1 (
After data analyses of the pseudo-ternary phase diagrams, the microemulsion system was prepared by mixing babassu oil (12.2%) with the surfactants (48.8%) before adding the aqueous phase (39%) under magnetic stirring. After two days, the system was evaluated by complementary techniques, as described below.
EC was evaluated using a digital conductivity meter (mCA 150P, Tecnopon, São Paulo, Brazil) previously calibrated with a calibration solution (146.9
The sample was dropped onto a 300 mesh carbon-coated cooper grid and negatively stained with 2% phosphotungstic acid. The grid was analyzed in an FEI Tecnai Spirit Biotwin G2 microscope (Hillsboro, Oregon, USA) operated with 80 KV of accelerating voltage.
SAXS experiments were performed on the SAXS1 beamline of the Brazilian Synchrotron Light Laboratory (LNLS, São Paulo, Brazil), monitored with a photomultiplier, and detected on a Pilatus detector (300k Dectris) positioned at 836 mm that generated scattering wave vectors (
DSC analysis was performed using a Shimadzu DSC 50 (Kyoto, Japan) with 9-10 mg of sample under a nitrogen atmosphere with a flow rate of 50 mL/min. The melting temperature and enthalpy were calibrated with indium and zinc standards. The samples were analyzed from −50 to 60°C with a heating rate of 10°C/min.
The rheological behavior of the formulation was investigated on an Anton Paar Physica MCR502 oscillatory rheometer (Ashland, VA, USA) with cone and plate geometry (50 mm diameter). The gap between the cone and plate was set at 0.05 mm. The measurements were taken at 25°C over a shear rate (
Results were expressed as mean ± SEM and normal distributions were checked by Shapiro-Wilk test. Then, data were analyzed by one-way ANOVA followed by Tukey’s test using GraphPad Prism® 5.0 with significance set at
Physicochemical parameters of babassu oil and reference values are placed in Table
Physicochemical parameters of babassu oil from Chapada do Araripe, Brazil.
Physicochemical parameters | Babassu oil (unrefined) | Reference value (refined oil) |
---|---|---|
Relative density (g/mL) | 0.9210 | 0.9140–0.9170 |
Refractive index at 40°C | 1.458 | 1.448–1.451 |
Acid value (mgKOH/g) | 0.13 | Max. 4 |
Peroxide value (meq/kg) | nd | Max. 15 |
Rancidity | Absent | Absent |
nd: not detected.
Fatty acid composition of babassu oil from Chapada do Araripe, Brazil.
Skeleton | Compound | Area (%) ± St Dev |
---|---|---|
C12:0 | Dodecanoic acid | 40.78 ± 1.56 |
C13:0 | Tridecanoic acid | 0.03 ± 0.01 |
C14:0 | Tetradecanoic acid | 20.05 ± 0.27 |
C16:0 | Hexadecanoic acid | 12.26 ± 0.59 |
C18:2n6c | (Z,Z)-9,12-Octadecadienoic acid | 2.39 ± 0.29 |
C18:1n9c | (Z)-9-Octadecenoic acid | 21.35 ± 0.36 |
C18:0 | Octadecanoic acid | 2.64 ± 0.09 |
1H NMR and 13C NMR analyses were performed to characterize the components of the sample and determine the distribution of fatty acids in the glycerol backbone of babassu oil (Table
1H and 13C NMR chemical shift (
Hydrogen |
|
Carbon |
|
---|---|---|---|
C |
5.33 |
|
173.2 and 172.8 |
C |
5.22 |
|
129.9 and 129.6 |
C |
4.29 |
|
62.1 |
C |
4.15 |
|
68.8 |
C=C-C |
2.74 | C=C- |
27.2 |
C |
2.30 |
|
34.1 |
C |
2.01 |
|
31.8 |
C |
1.61 |
|
24.8 |
|
1.28 |
|
22.6 |
CH3 | 0.87 |
|
29.7–28.8 |
|
14.0 |
The signals at 5.33, 2.01, and 2.74 ppm were assigned to the olefinic hydrogens, the protons attached to the allylic carbons, and the protons attached to the
13C NMR spectra provide information regarding the positional isomerism of fatty acids in the glycerol backbones of triacylglycerols (TAG), diacylglycerols (DAG), and monoacylglycerols (MAG) [
PMA-induced ear edema is a useful model for screening topical anti-inflammatory compounds and/or plant extracts that act at a variety of levels. Skin inflammation induced by topical PMA administration is mediated trough protein kinase C (PKC) activation of NF-
Babassu oil (3 and 10
(a) Topical and (b) systemic anti-inflammatory activity of babassu oil in PMA-induced ear edema. Results are expressed as the mean ± SEM and analyzed by ANOVA followed by Tukey’s test with
It is worth to comment that the high content of lauric acid in babassu oil defines the importance of this study, since its antibacterial and anti-inflammatory activities are described in the literature [
To further elucidate the topical anti-inflammatory activity of babassu oil, different phlogistic agents-induced ear edema was performed. Arachidonic acid-induced ear edema is a useful tool to identify compounds that interfere in eicosanoid pathway, such as COX and LOX inhibitors. However, this model is not sensitive to PLA2 inhibitors such as glucocorticoids [
Topical anti-inflammatory activity of babassu oil and lauric acid in (a) arachidonic acid; (b) ethyl phenylpropiolate; and (c) phenol-induced ear edema. Results are expressed as the mean ± SEM and analyzed by ANOVA followed by Tukey’s test with
Barbosa et al. [
Phenol-induced skin inflammation is very like human contact dermatitis processes [
Topical administration of capsaicin releases proinflammatory mediators such as substance P and histamine, due to TRPV-1 activation, which result in an immediate vasodilation and erythema followed by edema. Maximum edema is achieved within 30 minutes after capsaicin administration [
The HLB number is a semiempirical scale for selecting surfactants [
The maximum turbidity values are the same HLB value at which the mean droplet diameter is minimal [
Using the HLB value required for the babassu oil (HLB 8.0), Span 80 and Kolliphor EL were selected as the surfactants at a ratio of 6 : 4, respectively, because this blend had an HLB value equal to that determined for the oil. The aqueous phase was composed of water and propylene glycol (1 : 3) and the oil phase was babassu oil. In most cases, single-chain surfactants alone are unable to reduce the interfacial tension sufficiently to form a microemulsion. Propylene glycol was added as a cosurfactant to decrease the interfacial tension and increase the fluidity of the interface. In this case, part of the propylene glycol content was incorporated into the surfactant layer, and the other part decreased the polarity of the water by dissolving in the water. However, a higher amount of propylene glycol molecules favors formation of a bicontinuous microemulsion and avoids rigid structures, such as gels and liquid-crystals [
The transparent liquid systems formed by the pseudo-ternary phase diagram can be used to obtain concentration ranges of babassu oil, emulsifiers, and the aqueous phase for microemulsion formulations. A system composed of 39% aqueous phase, 12.2% oil phase, and 48.8% surfactants (Figure
Pseudo-ternary phase diagram (8 : 2) of the babassu oil microemulsion. The black region includes the microemulsion systems (ME) and the selected formulation (white dot). OLE, opaque liquid emulsions; MLE, milk liquid emulsions.
Electrical conductivity is commonly used to characterize the microstructure transitions that occur in microemulsions, that is, transformation from water-entrapped systems to intermediate structures and then to water continuous microstructures. Conductivity is low for reverse structures in nonconducting oil media that have little interactions with each other. When more water is added to the system, the conductive droplets begin to contact one another and form other structures, resulting in increased EC [
The ultrastructure of the babassu microemulsion was investigated using transmission electron microscopy (Figure
The ultrastructure of the babassu microemulsion that shows clusters of nanodroplets filled with oil, some of which have merged with one another and are surrounded by the surfactant interface and aqueous phase.
The microemulsion composition and individual characteristics of components define the phase morphology of the system. As the concentration of water increases, the droplets increase in size and eventually form a cluster that is considered infinite. At this stage, the microemulsion possesses a bicontinuous structure. Further addition of the water phase transforms the bicontinuous system into an o/w microemulsion, where the droplets of the organic phase are surrounded by the water bath, and the interface is composed of surfactant species. During each of these transition steps, morphological phases in intermediate regions may be formed, and the stability of the system depends on thermodynamic conditions [
SAXS is a well-established technique used to investigate the morphology, shape, and size of a multiphase sample, namely, aggregates dispersed in liquids, to obtain structural information regarding inhomogeneities based on the difference of electron density in the samples. This technique provides them with a characteristic length on the order of tens to hundreds of Angstroms (Å) [
Figure
(a) Scattering intensity
The scattering length density difference between the shell and matrix (
The thermal behaviors of water in the microemulsion system were investigated by DSC and compared with that of pure water (Figure
DSC thermographs for the babassu microemulsion, showing a broad endothermic melt transition in two temperature regions (inset).
The presence of two melting peaks is a characteristic behavior of a bicontinuous microemulsion in situations where the oil phase is composed of a single fatty acid [
The rheological behavior of the babassu microemulsion is demonstrated in SM (Figure
The topical anti-inflammatory activity of the babassu microemulsion compared with pure babassu oil is shown in Figure
Topical anti-inflammatory activity of babassu oil and microemulsion in PMA-induced ear edema. Results are expressed as the mean ± SEM and analyzed by ANOVA followed by Tukey’s test with
Skin permeation enhancement by microemulsions has been widely studied for several anti-inflammatory drugs, for example, indomethacin, aspirin, and rofecoxib [
In this work, we determined the chemical composition of the main fatty acids of babassu oil using GC-MS and the results were confirmed by 1H and 13C NMR spectra. Moreover, 13C NMR provided additional information, showing that triacylglycerol was the only positional fatty acid isomer in the glycerol backbone.
Babassu oil and lauric showed topical anti-inflammatory activity in different phlogistic agents-induced ear edema in mice, probably due to inhibition of AA metabolism and prostaglandin biosynthesis and/or action, release of histamine and serotonin, and inhibition of preformed cytokine release.
The developed babassu nanosystem was characterized as a phase transition microemulsion, which can be considered similarly bicontinuous as an o/w phase in contrast with classical microemulsion systems. Addition of more aqueous phase should form a well-defined o/w microemulsion.
The babassu oil microemulsion obtained here displayed advantages due to the combined features of bicontinuous and o/w microemulsions, including very low interfacial tension, high fluctuating interface, and high solubilizing properties. This system may have the ability to incorporate hydrophilic and/or lipophilic drugs that could be released faster than globular microemulsions with superior stability against the aqueous biological environment.
Topical anti-inflammatory activity of babassu oil was enhanced by microemulsification, reaching the same ear edema inhibition as pure babassu oil at a much lower concentration. This synthesized nanocarrier represents a new promising strategy for diseases treatment because babassu oil contains fatty acids with important biological properties, such as antioxidant, anti-inflammatory, antitumor, and antimicrobial.
Arachidonic acid
Babassu oil
Cyclooxygenase
Diacylglycerols
Dexamethasone
Dynamic light scattering
Differential scanning calorimetry
Electrical conductivity
Ethyl phenylpropiolate
Gas chromatography-mass spectrometry
Hydrophilic-lipophilic balance
Interleukin
Indomethacin
Lauric acid
Brazilian synchrotron light laboratory
Monoacylglycerols
Microemulsion
Nuclear magnetic resonance
Oil-in-water
Prostaglandin
Phospholipase A2
Phorbol 12-myristate 13-acetate
Reactive oxygen species
Small-angle X-ray scattering
Triacylglycerols
Transmission electron microscopy
Tetramethylsilane
Tumor necrosis factor
Water-in-oil.
The authors declare no conflicts of interest.
Mysrayn Y. F. A. Reis, Simone M. dos Santos, Rafael M. Ximenes, Giovanna Machado, and Karina L. A. Saraiva designed the research. Mysrayn Y. F. A. Reis, Simone M. dos Santos, Danielle R. Silva, Márcia V. Silva, Maria Tereza S. Correia, Daniela M. A. Ferraz Navarro, Geanne K. N. Santos, Fernando Hallwass, Otávio Bianchi, Alexandre G. Silva, Janaína V. Melo, and Alessandra B. Mattos performed the research. Mysrayn Y. F. A. Reis, Simone M. dos Santos, Daniela M. A. Ferraz Navarro, Fernando Hallwass, Otávio Bianchi, Rafael M. Ximenes, Giovanna Machado, and Karina L. A. Saraiva analyzed the data. Mysrayn Y. F. A. Reis, Simone M. dos Santos, Rafael M. Ximenes, Giovanna Machado, and Karina L. A. Saraiva wrote the manuscript. All authors reviewed and edited the manuscript. Mysrayn Y. F. A. Reis and Simone M. dos Santos contributed equally to this work. Rafael M. Ximenes, Giovanna Machado, and Karina L. A. Saraiva also contributed equally to this work.
This work was supported by FACEPE (Grant no. APQ-1067-4.03/15), CAPES, and CNPq. Maria Tereza S. Correia, Daniela M. A. Ferraz Navarro, Otávio Bianchi, and Giovanna Machado are grateful for CNPq Productivity Research Fellowships. The authors also thank the Brazilian Synchrotron Light Laboratory (LNLS) for the use of scientific installations (SAXS1 beamline).
As supplementary material one can find the GC-MS chromatogram and also H1 and C13 MNR spectra of babassu oil. Data regarding droplet size distribution, shear stress, and stability studies can also be found. SAXS equations used in the measurement of babassu microemulsion are described in detail in this file. Fig. S1: droplet size and turbidity as a function of HLB. The values from 8 to 10 showed the smallest droplet size and the highest turbidity, which is indicative of more stable emulsions. Fig. S2: GC-MS chromatogram of the babassu oil fatty acids. Fig. S3: 1H NMR spectrum of the babassu oil sample in CDCl3. Fig. S4: 13C NMR spectrum of the babassu oil sample in CDCl3. Fig. S5: microemulsion rheological curve showing Newtonian behavior. Table S1: centrifugation study to investigate the stability of Babassu microemulsion. Table S2: heating stress applied to babassu microemulsion to check stability in the temperature range 40–80°C. Table S3: heating-cooling cycles to check the effect of temperature variations on the stability of babassu microemulsion.