Introduction

Nanotechnology is characterised by a multidisciplinary approach and involves the creation and usage of different systems nanometric proportions (De Villiers et al. 2009). Nanoformulations have a wide variety of applications (e.g. the food industry, cosmetics, medicines and pesticides) (Assis et al. 2012; Patel and Velikov 2011; Duncan 2011; Brumfiel 2006; Irache et al. 2011; Wang et al. 2007). Among nanoformulations, nanoemulsions are systems formed by two immiscible liquids and one or more stabilising liquids, which enable the formation of small droplets (McClements 2012).

Nanoemulsions are characterised by their thermodynamic stability and possess drop sizes between 20 and 200 nm (Ostertag et al. 2012). Those formulations have a wide variety of industrial applications (Izquierdo et al. 2002; Tadros et al. 2004) (e.g. as an adjuvant in foods, medicines and agricultural products), and they exhibit a high economic potential.

Essentials oils are complex mixtures of volatile substances extracted from plants. They typically contain monoterpenes, sesquiterpenes and other low molecular weight substances in addition to phenylpropanoids in some cases (plants used in drugs). The essentials oils are used in the food and pharmaceutical industries as flavouring agents and can be larval, antibacterial, antifungal, anticancer, antimutagenic, antidiabetic, antiviral, anti-inflammatory and antiprotozoal (Raut and Karuppayil 2010).

There is an active field research involving the anti-inflammatory drugs in nanoformulations. This field principally focuses on active compounds from natural products, which is the case for Z-ligustilide. This substance is isolated and purified from the essential oil of Angelica sinensis, and it attenuates inflammatory pain behaviour in mice (Kuang et al. 2006; Yu et al. 2008; Du et al. 2007).

Rosmarinus officinalis L. is a medicinal plant of the Lamiaceae family (Lorenzi and Matos 2002) that is commonly used for medicinal purposes. It is ingested as a tea (Marchiori 2004). This species is a copious producer of essential oils, and it has been studied thoroughly. It possesses several biological activities, including spasmolytic (Ventura-Martinez et al. 2011), antioxidant (Raškovic et al. 2014; Ojeda-Sana et al. 2013), antibacterial (Ojeda-Sana et al. 2013), anti-inflammatory (Melo et al. 2011), antidepressive (Machado et al. 2013) and antifungal (Gauch et al. 2014; Cleff et al. 2012) properties. The chemical composition of the essential oil of Rosmarinus officinalis L. (EORO) can vary according to several factors, such as climate, soil, sun exposure and extraction procedure. However, the chemotypes most commonly reported are cineoliferum, composed primarily of 1,8-cineole, and camphoric acid, where camphor prevails (Napoli et al. 2015).

EORO is mainly composed of terpenoids, more specifically monoterpenes. This group of terpenoids are biologically active, and many of these terpenoids possess anti-inflammatory activity (Souza et al. 2014). One of the components of this essential oil, 1,8-cineol/1,8-cineole/eucalyptol, had already been tested in a double-blind, placebo-controlled trial in patients with acute asthma and allergic inflammation. The results suggested that EORO exhibited anti-inflammatory activity in asthma and that it could be used as a mucolytic agent in upper and lower airway diseases (Juergens et al. 2003).

Here, we sought to obtain a nanoemulsion using EORO and to evaluate its anti-inflammatory potential in in vivo assays. We also evaluated the effects of the nanoemulsion on inflammation models and outlying pain in animals.

Methods and materials

EORO acquisition

We acquired the EORO from the Florien Company (Sao Paulo, Brazil). It had been botanically authenticated as being Rosmarinus officinalis L., and it possessed organoleptic, physicochemical and microbiological characteristics. It also contained essential oil from the rosemary plant, which was obtained from leaves collected in Brazil, lot 056757-LC02062016DA.

Analysis of EORO by GC–MS

Coupled gas chromatography–mass spectrometry (GC–MS) analyses were performed on a Shimadzu system/GC 2010 coupled to a self-gun Shimadzu/AOC-5000 and mass detector (Shimadzu MS2010 Plus) with electron impact (70 eV) equipped with a fused silica column of DB-5MS (Agilent Advanced J & W; 30 m × 0.25 mm × 0.25 µm). The parameters of the X were as follows: split ratio, 1:30; helium as the carrier gas (65 kPa); injection volume, 1.0 µl; injector temperature, 250 °C; detector temperature, 250 °C; initial column temperature, 60 °C for 1 min; heating rate, 3 °C min−1 to 290 °C. The total analysis time was 76.67 min, and we calculated retention indices (RI) via interpolation to the retention times for a mixture of aliphatic hydrocarbons (C9–C30) analysed under the same conditions. The MS fragmentation pattern of the compounds was also compared with NIST mass spectrum libraries (National Institute of Standards and Technology).

Nanoemulsion preparation

The nanoemulsions were prepared using a low-energy load methodology that has been described by Fernandes et al. (2013). For a final mass of 50 g, we used 90% water, 5% EORO and 5% Tween 20. Initially, an organic phase was prepared by adding EORO and the tensioactive Tween into a beaker. The mixture was agitated using a magnetic agitator (750 rpm) for 30 min. Next, the aqueous phase was added at a flow rate of 0.5 mL/min with continuous agitation for 60 min. The stability of the emulsions was evaluated 1, 30 and 60 days after the preparation using macroscopic analysis (colour, visual aspect, phase separation, creaming and sedimentation) (Falcão et al. 2007). During this period, the emulsions were maintained at room temperature (25 ± 2 °C) in screw-capped glass test tubes.

Droplet size analysis

We determined droplet size and polydispersity with photon correlation spectroscopy using a Zetasizer 5000 (Malvern Instruments, Malvern, UK). Each emulsion was diluted using ultrapure Milli-Q water (1:25), and the measurements were performed in triplicate. The average droplet size was expressed as the mean diameter (Orafidiya and Oladimeji 2002).

Evaluation of EORO and NEORO on rat paw oedema, writhing test in mice and production of H2S in rat stomachs

Experimental animals

Male Wistar rats (body weight: 180–200 g) were used, along with Swiss mice (body weight: 20–25 g) from the Central Biotério of the Department of Pharmacy of Facultad de Química da Universidad Autonoma de Mexico. The animals were housed in polyethylene boxes in groups of five, and their access to food was removed 12 h prior to the experiments. They were given free access to water.

This research was approved by the Ethics Committee for the Use of Animals of Amapá Federal University (Authorisation No. 0021/2015). After the experiments, the animals were euthanised according to the guidelines for the euthanasia of animals (AVMA, American Veterinary Medical Association 2013).

Rat paw oedema induced by carrageenan

This test was carried out according to the method described by Winter et al. (1962). Groups of animals (n = 5) each received different doses of EORO (100 and 300 mg/kg) and NEORO (498 µg/kg) 30 min prior to the application of the inflammatory agent (carrageenan 1000 mg/paw, 01 mL, kappa, Sigma Company, Sao Paulo, Brazil). We administered 0.1 mL of saline solution in the plantar space of the left hind paw and the same volume containing the inflammatory agent in the right paw. We measured paw volume using a plethysmometer (Model 7540; Ugo Basile, Italy). The paws were measured every hour prior to the administration of the inflammatory substance and 4 h after the application of the carrageenan.

Acetic acid-induced writhing test

The acetic acid-induced writhing test was carried out in mice according to the method described by Koster et al. (1959). The different groups of animals were treated orally with EORO (100 mg/kg) and NEORO (166, 498 and 830 µg/kg), and the control animals were treated with 0.5 mL of Tween solution to 5%. Thirty minutes later, abdominal twitches (writhes) were induced intraperitoneally via (i.p.) administration of 1% acetic acid (0.25 mL). The writhing was observed, and we recorded the mean ± mean standard error of the number of writhes in an interval of 20 min.

Test of H2S production in rat stomachs

This assay was based on the methods described by Khan et al. (1980) and Eto and Kimura (2002). Wistar rats were used (n = 6 per group), which were anaesthetised with sodium pentobarbital (10 mg/kg, i.p, Anestesal™, MSD, Mexico) and treated orally with EORO (100 mg/kg) and NEORO (498 µg/kg). After 30 min, l-cysteine was administered (100 mg/kg, via oral (v.o.), Sigma Company). The rats then underwent a laparotomy to reveal the pyloric region, and the H2S microelectrodes were attached to an analyser system (Micro Hydrogen Sulfide Measurement System—microLazar Model ISM-146H2S-XS; Lazar, USA). We measured H2S levels every 5 min, and after 30 min we injected 200 μL of ethyl alcohol (PA) directly in the animal’s stomachs and recorded data for up to 60 min.

Molecular docking of the major chemical constituents of EORO

For the docking study, we downloaded files deposited in the Protein Data Bank (PDB) from the Research Collaboratory for Structural Bioinformatics (Li et al. 2007, 2008; Sandy and Butler 2009; Orlando and Malkowski 2016) with the coordinates of the crystallographic structures of the COX-1 therapeutic targets (PDB ID: 3N8X, resolution: 2.75 Å) complexed with the nimesulide inhibitor. COX-2 (PDB ID: 5IKQ, resolution: 2.41 Å) was complexed with meclofenamic acid and prostacyclin (PDB ID: 3B6H, resolution: 2.41 Å) was complexed with the minoxidil inhibitor.

To perform the molecular docking, we added hydrogen atoms and removed water molecules from the enzymes. The inhibitors that were complexed with each therapeutic target were extracted. Prior to performing the docking simulation, we validated our results by calculating the root-mean-square deviation (RMSD) between the experimental binder and the conformation of the binder that yielded the best posture after docking. To calculate the docking of the major phytochemical constituents of EORO, we used the following coordinates: cyclooxygenase-1 (COX-1): x: −21.43, Y: −50.79 and z: 1.42; cyclooxygenase-2 (COX-2): ν: 22.83, Y: 51.56 and z: 17.81; and prostacyclin (PGI-2): ν: 72.25, Y: 54.20 and z: 42.19.

To identify the interactions between the compounds and the therapeutic targets, it was necessary to identify the amino acids that make up the catalytic site of the enzymes: COX-1 (ARG120, TYR355, SER530 and ILE523), COX-2 (TYR385 and SER530) and PGI-2 (CYS441, TRY282, PHE483 and GLY482).

Statistical analysis

We applied analysis of variance (ANOVA) followed by the Tukey test. P values less than 0.05 were considered to be statistically significant. We plotted the data using GraphPad Prism 6.0.

Results and discussion

The chromatographic data indicated that the EORO used in this study to obtain the NEORO contained the following major compounds: 21.99% limonene, 33.70% 1,8-cineole and 27.68% camphor (Fig. 1; Table 1). These results are in accordance with those reported by Zaouali et al. (2010). These authors found that camphor and 1,8-cineol were the primary compounds of EORO from Tunisia. In studies conducted by Boix et al. (2010) and Fernandes et al. (2013), α-pinene, 1,8-cineol and camphor were the primary components of EORO samples from Brazil. Ribeiro et al. (2012) found α-pinene and 1,8 cineol to be among the major compounds of EORO from fresh rosemary leaves cultivated in northeastern Brazil.

Fig. 1
figure 1

Chromatogram obtained by analysis of EORO by coupled gas chromatography–mass spectrometry (GC–MS), where 10—limonene (21.99%), 11—1,8-cineole (33.70%) and 16—camphor (27.68%)

Full size image
Table 1 Chemical constituents of Rosmarinus officinalis L. essential oil (EORO) determined by GC–MS analysis
Full size table

The nanoemulsion, NEORO, prepared with EORO, presented a fluid appearance, a whitish coloration and a slightly bluish reflection, which are common macroscopic characteristics in this type of formulation. The mean droplet diameter remained below 200 nm in all emulsions, as described by Solans et al. (2005) and Solè et al. (2012). As shown in Fig. 2, the NEORO presented a distribution of monomodal droplet sizes, but none of the samples exhibited signs of instability, such as cremation and phase separation, as described by Duarte et al. (2015).

Fig. 2
figure 2

Particle size distribution of NEORO mean droplet—89.87 ± 0,083727 nm; polydispersity 0.193 ± 0.008 nm

Full size image

The carrageenan-induced rat paw oedema assay is widely used as a test for evaluating anti-inflammatory activity. This assay has become a standard model for experiments related to acute inflammation, this is described as a biphasic response because it has an initial phase in the first 2 h after the injection of carrageenan, which is related to the release of histamine and serotonin and another phase that comprises the production of prostaglandins, bradykinin and proteases (Patgiri et al. 2014). Carrageenan was used in this assay because it is devoid of apparent systemic effects (Ganguly et al. 2013).

In a study by De Faria et al. (2011) focusing on EORO, the authors noted ED50 values of 300 and 261 mg/kg in the writhing test in mice and for carrageenan-induced paw oedema in rats, respectively. Additionally, LD50 in the mice was greater than 2.0 g/kg.

Oral administration of NEORO at a dose of 498 μg/kg inhibited the maximum peak of oedema by 46%, and EORO administered at a dose of 300 mg/kg inhibited the maximum peak of oedema by 50%. This result demonstrates that EORO delivered as a nanoemulsion was much more effective on carrageenan oedema, inhibiting this oedema, with a dose six hundred times lower than that of EORO (Fig. 3). In the acetic acid-induced writhing test in mice, the oral administration of NEORO yielded a dose-dependent effect, and a dose of 830 μg/kg inhibited the algogenic process by 84%. EORO alone at a dose of 100 mg/kg inhibited the algogenic process by 55% (Fig. 4).

Fig. 3
figure 3

Effect of oral treatments with EORO (100 mg/kg) and NEORO (166, 498 and 830 μg/kg) on writhing in mouse induced by acetic acid. The points represent the mean ± SEM of n = 5/group. *p < 0.05, ANOVA followed by the Tukey test

Full size image
Fig. 4
figure 4

Effect of oral treatments with EORO (100 and 300 mg/kg) and NEORO (498 μg/kg) on rat paw oedema induced by carrageenan. The points represent the mean ± SEM of n = 5/group. *p < 0.05, ANOVA followed by the Tukey test

Full size image

The physiological functions of hydrogen sulphide (H2S) have been recognised and evidence is being sought that this endogenous, gaseous substance can modulate inflammatory processes. However, H2S donors have been shown to reduce oedema formation and the adhesion of leukocytes to vascular endothelium and inhibit the synthesis of proinflammatory cytokines (Wallace 2007). In addition, H2S donors can increase gastric mucosal resistance to injury and accelerate repair (Wallace 2007). These observations and others suggest that anti-inflammatory drugs that are modified with the ability to release H2S have improved anti-inflammatory efficacy and reduced toxicity (Wallace 2007).

In our analysis of how EORO and NEORO affected the l-cysteine-induced production of H2S in rat stomachs, we observed that NEORO at a dose of 498 μg/kg inhibited production at all measurement times. Additionally, EORO at a dose of 100 mg/kg inhibited production of H2S by 60% and potentiated the ethanol effect, thereby decreasing H2S production (Fig. 5).

Fig. 5
figure 5

Effect of oral treatments with EORO (100 mg/kg) and NEORO (498 μg/kg) on the production of H2S in the stomach of Wistar rats. The points represent the mean ± SEM of n = 5/group. *p < 0.05, ANOVA followed by the Tukey test

Full size image

The evidence suggests that H2S is a mediator of several aspects of the gastrointestinal function and liver. In addition, changes in H2S production may contribute to diseases of the gastrointestinal tract and liver, and non-steroidal anti-inflammatory drugs may reduce the production of H2S in the stomach, and this fact has been shown to contribute to the generation of mucosal injury (Fiorucci et al. 2005, 2006). However, Martínez et al. (2009) demonstrated the antinociceptive effect of EORO in arthritic pain in a rat model, suggesting the involvement of the serotonergic system via 5-HT1A receptors and endogenous opioids. Furthermore, De Faria et al. (2011) described the anti-inflammatory effect of EORO via the inhibition of cyclooxygenase; this effect persisted without causing gastric lesions or increasing mucus production. This fact may explain the observed results regarding H2S production: a consistent mucus layer could interfere with the release of this mediator and, therefore, not be detected by the electrode system, a hypothesis that should be explored further in future studies.

Molecular docking is a computational method currently widely used in the drug discovery process (Chandak et al. 2014). The benefit of docking is to identify the mode of interaction of the study molecules at the site of the enzyme or receptor through specific key interactions and to predict the binding affinity between the protein-binding complexes, in this case, the chemical constituents of EORO. The Genetic Optimisation for Ligand Docking (GOLD) program uses a genetic algorithm to conduct flexible docking experiments of ligands within protein-binding sites. The GOLD program has been used to investigate the modes of interaction between compounds and therapeutic targets (Chandak et al. 2014).

The RMSD value indicates the accuracy of the docking postures calculated by the GOLD docking algorithm compared with the experimentally determined poses of a compound bound to a biological target. An RMSD less than 2 Å is considered to be successful (i.e. to have justified validity) (Cole et al. 2005). In this study, the best RMSD values obtained with nimesulide, meclofenamic acid and minoxidil inhibitors were 0.87, 0.99 and 0.89 Å for the respective therapeutic targets COX-1, COX-2 and PGI-2.

We then performed docking between the therapeutic targets and the compounds limonene, 1,8-cineole and camphor. We selected the docking results that yielded the highest score for each therapeutic target limonene: 50.14 for COX-1, 45.85 for COX-2 and 38.21 for PGI-2; 1,8-cineole compound: 36.30 for COX-1, 37.76 for COX-2 and 33.71 for PGI-2; camphor compound: 33.49 for COX-1, 34.38 for COX-2 and 36.13 for PGI-2.

With the COX-1 therapeutic target, the docking of the limonene compound had two alkyl-type hydrophobic interactions with distances of 3.97 and 4.22 Å from the amino acid ILE523 (Fig. 6a). Alkyl groups are defined as a predominantly aliphatic amino acid side chains, and they include alanine, valine, leucine, isoleucine, methionine, selenomethionine, cysteine, proline, CB, CG and CD atoms of lysine and CB and CG arginine atoms. Hydrophobic groups in binders are contiguous sets of atoms that are not adjacent to charge concentrations (charged atoms or electronegative atoms). A group of atoms is considered to be hydrophobic if its surface area is equal to or greater than the area of a methyl group multiplied by the surface area scale factor, which corresponds to the surface area of a chlorine atom. The criteria for this type of interaction were met when the centre of the groups was within 5.5 Å of an alkyl centre (Wolber and Langer 2005).

Fig. 6
figure 6

Docking of the compound limonene performing interaction with a COX-1, b COX-2 and c PGI-2

Full size image

It is important to note that the limonene compound also exhibited numerous interactions with amino acids close to the amino acids of the active site of COX-1. Therefore, this compound can potentially modify the local structure, which can result in a biological effect. For the amino acid LEU352, we noted a hydrophobic interaction of the alkyl type with a distance of 5.13 Å. For the amino acid LEU384, we measured a hydrophobic interaction of the alkyl type with a distance of 4.91 Å. For the amino acid TYR385, we noted a hydrophobic interaction of the Pi–alkyl type with a distance of 4.21 Å. For the amino acid LEU387, we observed a hydrophobic P-alkyl-type interaction with a distance of 4.72 Å. For the amino acid PHE518, we observed two hydrophobic interactions, both of the Pi–alkyl type, with distances of 4.84 and 5.38 Å. For the amino acid ALA527, we noted two hydrophobic interactions of the alkyl type with distances of 3.75 and 4.78 Å.

For the COX-2 therapeutic target, the limonene compound presented a hydrophobic Pi–alkyl-like interaction with the amino acid residue TYR385 with a distance of 4.26 Å (Fig. 6b). Pi–sigma interactions are weak interactions between a hydrogen and a ring system Pi. Pi–alkyl interactions exist where the centres of an aromatic ring and an alkyl group are within the alkyl centroid limit with a maximum distance of 5.5 Å, and they have at least one pair of atoms within the same atom closest to Pi–Pi. For this interaction to occur, the following conditions are necessary: (a) hydrogens that act as donors may be implicit or explicit hydrogens; they must be connected to a non-aromatic carbon atom; (b) the distances between the hydrogen and the centre of the Pi ring must be within a maximum distance of approximately 4.0 Å; (c) the centre angle CH can deviate from linear by a maximum of 20°; (d) the angle between the C-centre and the normal plane of the ring must not exceed 45° (Fares et al. 2016).

Among the amino acids close to the site of action of COX-2, the limonene compound had the following interactions: a hydrophobic Pi–alkyl-type interaction with the amino acid PHE381 with a distance of 5.27 Å, two hydrophobic interactions of the alkyl type with the amino acid LEU352 with distances of 3.90 and 5.19 Å were presented, a hydrophobic interaction of the alkyl type with the amino acid LEU384 with a distance of 4.48 Å, a hydrophobic interaction of the Pi–alkyl type with the amino acid TRP387 with a distance of 4.85 Å, two hydrophobic Pi–alkyl-type interactions with the amino acid PHE518 with distances of 4.87 and 5.31 Å, three hydrophobic interactions of the alkyl type with the amino acid VAL523 with distances of 3.92, 3.93 and 5.25 Å.

With the PGI-2 therapeutic target, the limonene compound exhibited two hydrophobic interactions with the amino acid CYS441 (both alkyl type with distances of 4.6 and 5.2 Å) (Fig. 1c). Regarding the amino acids close to the active site, the docking presented an alkyl-type hydrophobic interaction with a distance of 4.46 Å with the amino acid LYS121.

Regarding the therapeutic target COX-1, the 1,8-cineole compound exhibited hydrophobic binding of the Pi–alkyl type with the amino acid TYR355 with a distance of 4.46 Å. The 1,8-cineole compound also showed two hydrophobic bonds of the alkyl type with a distance of 5.37 Å and 4.43 Å with the amino acid ILE523 (Fig. 7).

Fig. 7
figure 7

Docking of the compound 1,8-cineole performing interaction with a COX-1, b COX-2 and c PGI-2

Full size image

Compound-1,8-cineole docking resulted in amino acid linkages very close to the amino acids of the active site of COX-1: two hydrophobic interactions with the amino acid LEU352 of the alkyl type at distances of 4.81 and 5.01 Å and a hydrogen bridge-type interaction with a distance of 2.28 Å. There were also two hydrophobic interactions with the amino acid VAL349 of the alkyl type with distances of 4.35 and 4.94 Å and four hydrophobic interactions with the amino acid ALA527 of the alkyl type with distances of 3.71, 3.94, 4.14 and 4.68 Å.

The docking of compound 1,8-cineole did not present interaction results with the amino acids present in the active site of the COX-2 and PGI-2 therapeutic targets. However, it presented interactions with seven amino acids close to the active site of COX-2 and interactions with three amino acids close to the active site of PGE2.

In terms of the amino acids close to the active site of COX-2, the docking exhibited two hydrophobic alkyl-type interactions with the amino acid VAL349 with distances of 4.51 and 4.01 Å; three hydrophobic interactions of the alkyl type with the amino acid LEU352 with distances of 5.40, 5.21 and 3.61 Å; and a conventional hydrogen-bonding interaction with a distance of 2.93 Å.

The amino acids close to the active site of PGI-2 that showed an interaction included LYS121 (with a hydrophobic interaction of alkyl type with a distance of 5.17 Å), the amino acid HIS440, which exhibited a hydrogen-bonding interaction of the conventional type with a distance of 2.14 Å, and the amino acid LEU442 interaction of a conventional-type hydrogen bridge with a distance of 1.90 Å.

The compound that exhibited the largest number of interactions with the therapeutic targets was camphor. In the COX-1 therapeutic target, this compound exhibited an interaction of conventional hydrogen bonds with the ARG120 amino acid with a distance of 2.10 Å. Conventional hydrogen-bonding interactions may exist between a hydrogen-bonding donor atom and an acceptor atom such as N, O, P or S. The maximum accepted distance for this bond is 3.8 Å (Bissantz et al. 2010; Ziólkowski et al. 2006). For the same target, a hydrophobic interaction with the amino acid ILE523 of the alkyl type with a distance of 4.52 Å was presented. We also noted a hydrophobic interaction of the Pi–alkyl type with a distance of 4.36 Å with the amino acid TRY355 (Fig. 8a).

Fig. 8
figure 8

Docking of the camphor compound performing interaction with nearby amino acids and of the active site of COX-1, COX-2 and PGI-2

Full size image

With the amino acids present near the active site of COX-1, the docking presented three hydrophobic alkyl-type interactions with distances of 3.76, 4.18 and 4.80 Å with the amino acid VAL349; two hydrophobic interactions of the alkyl type with distances of 3.47 and 4.65 Å with the amino acid LEU352; and two hydrophobic alkyl-type interactions with distances of 3.33 Å and 4.94 Å with the amino acid ALA527 (Table 2).

Table 2 Interactions between amino acids nearby and of the active site of the biological target and distances where interactions occur
Full size table

In terms of the docking of the compound camphor with COX-2, this did not present interaction results with the amino acids present in the site. However, camphor presented interactions with amino acids close to the active site: an alkyl-type hydrophobic interaction with a distance of 4.16 Å with the amino acid VAL349; three hydrophobic alkyl-type interactions with distances of 3.66, 4.08 and 4.64 Å with the amino acid LEU352; a hydrophobic interaction of the Pi–alkyl type with a distance of 5.41 Å with the amino acid PHE518; three hydrophobic interactions of the alkyl type with distances of 3.84, 4.30 and 4.41 Å with the amino acid VAL523; two alkyl-type hydrophobic interactions with distances of 4.81 and 3.65 Å; and a hydrogen-bonding interaction with the amino acid ALA527.

Camphor docking with the therapeutic target PGI-2 yielded two hydrogen-bonding interactions: a conventional interaction with a distance of 2.09 Å and a carbon–hydrogen-type interaction with a distance of 2.09 Å. Both interactions occurred at amino acid CYS441 (Fig. 8b).

The docking results yielded the following interactions with the amino acids close to the active site: two hydrophobic alkyl-type interactions with the amino acid MET124 with distances of 4.93 and 4.95 Å; a hydrophobic alkyl-type interaction with a distance of 4.15 Å with the amino acid ALA283; an alkyl-type hydrophobic interaction with a distance of 5.07 Å; a conventional hydrogen-bonding interaction with a distance of 4.72 Å with the amino acid LEU 442; a conventional hydrogen-bonding interaction with the amino acid GLY 443 with a distance of 2.08 Å; and a metal–receptor type interaction with a distance of 2.61 Å (Fig. 8c).

Conclusion

Our results demonstrate the importance of nanotechnology as an alternative for the delivery of drugs. Nanotechnology is capable of increasing the bioavailability of active principles from vegetable drugs and improving its action on certain target systems. The nanoemulsion (NEORO) obtained from EORO was able to reduce rat paw oedema induced by carrageenan with a dose 600 times lower than the effective dose of EORO, and it produced a dose–response effect in the algesic test. NEORO accordingly demonstrated a potent antialgic effect. With the results obtained in the molecular docking study, we observed that among the primary compounds of EORO the camphor molecule presented the largest number of interactions with therapeutic targets related to the inflammatory process. This finding suggests that the camphor molecule is responsible for the anti-inflammatory and antialgic effects observed in the experimental results with NEORO and EORO. However, additional studies are necessary to elucidate the mechanism of H2S production.