Evaluation and Theoretical Study on the Anti-inflammatory Mechanism of 1-Nitro-2-phenylethane

• Introduction
• Materials and Methods
• Results and Discussion
• Acknowledgements
• References

Abstract

In this study, 1-nitro-2-phenylethane was evaluated with respect to its effects in edema models of acute inflammation induced with carrageenan, dextran, and croton oil. 1-Nitro-2-phenylethane produced inhibition of rat paw edema induced by carrageenan and dextran at the doses of 25 and 50 mg/kg. The same doses caused an inhibition of croton oil-induced ear edema in mice. Our results suggest that 1-nitro-2-phenylethane has anti-inflammatory activity, probably of peripheral origin, acting in the synthesis and/or release of inflammatory mediators. A conformational study of 1-nitro-2-phenylethane was carried out using density functional theory calculations, showing three different groups of conformers corresponding to energy minimum geometries. The stereoelectronic repulsions are responsible for conformational preferences and the one most stable conformer. The prostaglandin endoperoxide synthase mechanism is related more to electrophilic than nucleophilic properties.

Introduction

Aniba canelilla (H. B. K.) is an aromatic plant in the Amazon region and belongs to the Lauraceae family. In folk medicine, the decoction of bark wood is used as an antispasmodic, digestive stimulant, and a carminative [1]. This plant was confused with cinnamon trees during the voyage of Pizarro and Orellana from the Andes to the Amazon estuary in 1540. The same occurred during the Humbolt and Bonpland expedition (1800) who found the “famous cinnamon” of the Orinoco River [2].

The odoriferous principle of leaf, bark, and trunk wood of A. canelilla, responsible for the cinnamon odor, is 1-nitro-2-phenylethane (NPE). The percentage of content of this compound depends on the season. In the rainy period, this molecule reaches values near 95 %, in the dry period, NPE decreases to 39 % [3].

NPE has been reported in pharmacological papers. It was observed that NPE exhibited an antinociceptive effect in mice [4], and caused hypotension and bradycardia in normotensive rats [5], [6]. Nevertheless, its action mechanism for these activities is unknown.

In the present investigation, we evaluated the anti-inflammatory effect of NPE. In fact, this study is one of the first attempts to address such ethnopharmacological properties of NPE in a comprehensive manner. This work is complemented by the conformational analysis of NPE and its anti-inflammatory mechanism is proposed using a theoretical approach.

Materials and Methods

Male Swiss mice (25–30 g) and Wistar rats (160–220 g) were housed at 25 ± 2 °C under a 12/12-h light/dark cycle, and food and water were supplied ad libitum. The animals were obtained from colonies maintained at the Instituto Evandro Chagas (Belém, Brazil). The animals were housed in groups of 10 under environmentally controlled conditions and the handling and use of the animals were in accordance with the institutional guidelines (MED 010/2008 – February 1, 2008).

The following drugs and chemicals were used: cyproheptadine (Sigma), indomethacin (Sigma), ketamine hydrochloride, carrageenin (Fluka), dextran (T-70; MW 70 000; Pharmacia), and dexamethasone (Aché). All reagents used in this work were of analytical grade (≥ 98–99 % TLC/HPLC).

The bark wood of A. canelilla was collected in the area of the Cauaxi River, municipality of Ulionópolis, Pará State, Brazil, May 2005, during the rainy season. The specimen was identified by comparison with an authentic voucher (MG 174 904) of A. canelilla that is deposited in the herbarium of the Emílio Goeldi Museum, city of Belém, Pará State, Brazil.

The bark wood was air-dried, ground, and submitted to hydrodistillation (100 g, 4 h) using a Clevenger-type apparatus. The oil was dried over anhydrous sodium sulfate and the percentage of content was calculated on the basis of the plant dry weight. The moisture content of samples was calculated after the phase separation in a Dean-Stark trap (5 g, 30 min) using toluene.

To purify the main constituent (NPE), bark essential oil (15 g) was submitted to fractionation in silica gel chromatographic column using petroleum ether (isocratic elution) and thin-layer chromatography (hexane-ethyl acetate, 9 : 1; approximately 2.5 L). The percentage of content of NPE in the oil and in the purified fractions was obtained in a Thermo Focus GC/FID operated in the following conditions: WCOT DB-5ms (30 m × 0.25 mm; 0.25 µm film thickness) fused silica capillary column; temperature programmed, 60–240 °C (3 °C/min); injector and detector temperatures, 220 and 250 °C, respectively, (3 °C/min); carrier gas, nitrogen; injection type, split-less (2 µL, of a 1 : 1000 hexane sol.).

Animals were divided into 4 groups of 10 mice each and treated with vehicle (control, 0.9 % saline plus 1 % Tween 80, p. o.) or dexamethasone (10 mg/kg; p. o.), used as a positive control, or NPE (25 and 50 mg/kg, p. o.). Sixty minutes later, the animals were anesthetized with ketamine hydrochloride (145 mg/kg, intraperitoneally). Coetaneous inflammation was induced by applying 20 µL of croton oil in acetone (2.5 % v/v) to the inner surface of the right ear. The same volume of acetone was applied to the left ear using the method of Tubaro et al. [7]. At the maximum of the edematous response, i.e., 6 h later, mice were sacrificed and a plug was removed from both the treated (right) and the untreated (left) ears. The inflammatory response (edema) was monitored by measuring the differences in weight (mg) between the two plugs.

The NPE was suspended in 0.9 % NaCl (1 g/mL) for administration to the animals 30 min before the experiments. The right rear plantar region of the rats was injected with 1 mg per paw (0.1 mL) of carrageenin (Iota-Fluka Biochemika) or dextran (T-70; MW 70 000; Pharmacia). The left rear paw of each animal receiving any of the three drugs listed above was also injected with an equal volume of 0.9 % saline solution. The edema produced in each paw was determined by measuring the paw diameter using an analogic pakimeter (Vernier) after stimulations [8].

Results are expressed as mean ± s. e. m., according to the test. Statistical evaluations were made using ANOVA followed Student-Newman-Keuls, and values were considered significantly different when p < 0.05.

The molecular orbital calculations were carried out with the GAUSSIAN 03 W program [9], within the density functional theory (DFT) approach, using the B3LYP functional, which includes a mixture of Hartree–Fock (HF) and DFT exchange terms. The gradient-corrected correlation functional [10], [11] was used parameterized after Becke [12], [13], along with the double-zeta split valence basis sets 6–31 G* [14]. Molecular geometries were fully optimized by the Berny algorithm, using redundant internal coordinates [15]. In order to study the barriers of internal rotation, the geometries were optimized with different dihedral angles for O₂N-C₁-C₂-C₆H₅.

Since our interest is to understand the role played by the possible action mechanism of NPE, we adopted a systematic study comparing electronic properties of NPE with aspirin (ASP) and paracetamol (PAR). The better correlation was obtained using the lowest standard deviation (SD) values for NPE and ASP or NPE and PAR. In order to achieve this aim, we calculated the following properties: (i) Highest Occupied Molecular Orbital (HOMO), (ii) Lowest Unoccupied Molecular Orbital (LUMO), (iii) Ionization Potential (IP), and (iv) Electron Affinity (EA).

The IP was calculated as the energy difference between a neutral molecule and the respective cation free radical (Equation 1).

IP = EM^•+ – EM (Eq. 1)

The EA was determined by the calculation of one-electron reduction energy, as shown in Equation 2 as the energy difference between an anion free radical and the respective neutral molecule.

EA = EM – EM^•– (Eq. 2)

Other reactivity descriptors were obtained as described by Equations 3–6, i.e., electronegativity (χ), hardness (η), softness (S), and electrophilic index (ω) [16], [17], [18], [19].

χ ≈ (IP + EA)/2 (Eq. 3)

η ≈ (IP – EA)/2 (Eq. 4)

S = 1/(2η) (Eq. 5)

ω = µ ²/2η (Eq. 6)

Results and Discussion

NPE was isolated from the trunk wood oil by silica column chromatography, reaching 99 % purity. The hydrogen NMR spectrum furnished the signals δ 7.27 (m, 5H, monosubstituted aromatic ring), δ 4.61 (t, 2H, J = 7.5 Hz, α-position to the nitro group), and δ 3.33 (t, 2H, J = 7.5 Hz, α-position to the aromatic ring) as can be seen in [Fig. 1]. Analytical conditions, composition of the NPE used in this study, and retention indices of its constituents have been previously reported [20], [21], [22].

In the present study, the anti-inflammatory properties of NPE isolated from A. canelilla were evaluated by different experimental inflammatory models and the results show that NPE can play a significant role in the inhibition of inflammatory processes.

[Fig. 2] shows that NPE produced a dose-related inhibition of edema with topical application of croton oil, an inflammatory irritant. Topical applications of croton oil induced cutaneous inflammation characterized by edema, neutrophil infiltration, prostaglandin production, and the increasing of the vascular permeability [23], which caused a significant increase in ear plug weight of the right ear when compared to the untreated left ear. The advantage of the model of ear inflammation induced by croton oil is a good prognosis for screening topical anti-inflammatory activity and sensitivity to steroidal drugs [7], [24]. In this study, dexamethasone (10 mg/kg body wt.), used as a positive control, gave rise to a significant inhibition of 87.01 % in ear plug weight. The NPE shows strong inhibitory activity on the croton oil-induced ear edema in mice. The groups treated with 25 and 50 mg/kg of NPE were inhibited by 73.8 and 79.4 %, respectively. So, this compound may interfere with the process of generation of prostaglandins, generated by the arachidonic acid metabolism when croton oil is applied [25], [26].

The NPE showed a dose-dependence effect on rat paw edema induced by dextran. At a dose of 25 mg/kg, NPE was capable of preventing the development of edema in 15.58 %, 26.78 %, 44.92 %, and 30.07 % for 30, 60, 90, and 120 minutes, respectively. At a dose of 50 mg/kg, there was an inhibition of development of edema in 38.1 %, 61 %, 69.09 %, and 73.65 % at 30, 60, 90, and 120 minutes, respectively. Cyproheptadine was able to prevent the development of edema in 71.94 %, 67.29 %, 71.18 %, and 52.25 % at 30, 60, 90, and 120 minutes, respectively (see [Fig. 3]).

The rat paw edema induced by carrageenan, in the presence of NPE, also showed a dose-dependent inhibition. The NPE, at a dose of 25 mg/kg, was capable of preventing the development of edema in 26.83 %, 43.91 %, 41.6 %, and 39.85 % in 2, 3, 4, and 5 h, respectively. At a dose of 50 mg/kg, the NPE caused an inhibition of the development of edema in 51.76 %, 54.46 %, 47.2 %, and 49 % during 2, 3, 4, and 5 h, respectively. Indomethacin was able to prevent the development of edema in 41.07 %, 66.95 %, 61.73 %, 50.07 % and 64.99 % in 1, 2, 3, 4 and 5 h, respectively ([Fig. 4]).

The edema induced by carrageenan is a temporal and multimediated phenomenon, involving the participation of a diversity of mediators such as histamine, serotonin, bradykinin, nitric oxide, and prostaglandins, being characterized by an intense neutrophil infiltrate [27]. On the other hand, dextran is a proinflammatory agent that promotes the release of vasoactive amines, such as histamine and serotonin, causing an osmotic edema, characterized by the increase of the vascular permeability with low levels of protein and neutrophils [28]. Since NPE showed anti-inflammatory effects in the paw and ear edemas induced by Cg, dextran, and croton oil, we can suggest that it acts in different aspects and chemical mediators of inflammation, such as histamine, serotonin, bradykinin, and prostaglandins.

In addition, since ASP and PAR mechanisms are known, our interest is to understand the role played by NPE related to two classical nonsteroidal anti-inflammatory drugs (NSAIDs). For this purpose, we adopted a systematic study comparing conformational and electronic properties of NPE with ASP and PAR.

The conformational analysis of NPE was performed using the DFT/B3LYP method with basis sets 6–31 G(d) [29]. Our results show that this molecule can adopt different conformations, varying dihedral angles between phenyl and nitro groups linked at the C₁-C₂ carbon atoms. The flexibility degrees were analyzed with intervals of 5 to 5° and variations from 0° to 180° in order to search for the lowest energy conformers.

These conformations can be observed in [Fig. 5], where the most stable conformations were obtained with values lower than 20 kcal/mol. The arrangement related to the s-trans conformer with the dihedral angle of 180° for O₂N-C₁-C₂-C₆H₅ was significantly more stable, with a value of 19.38 kcal/mol compared to the s-cis conformer (angle 0°). In addition, other conformations such as 90° and 120° were observed by the rotation of C₁-C₂. The energy differences for these conformations were 2.6 and 1.4 kcal/mol, respectively.

Our theoretical results are in good agreement with other conformational studies of ethylene molecules [30], [31], [32]. In our study, the aromatic ring is coplanar to the nitro moiety. In this case, the rotational isomerism between the aromatic ring, ethylene, and nitro groups are influenced by different factors, mainly from steric hindrance, dipolar, mesomeric, and hyperconjugative effects. Nevertheless, no hydrogen bonding interactions were observed among these functional groups. H-type bonds are, in this particular molecule, energetically disfavored. Moreover, this compound has many stereoelectronic hindrances, mainly between oxygen and all hydrogen atoms of the methylene groups.

In fact, the greater stabilization of the 180° conformer may be explained due to the following factor: the stabilizing interaction due to the presence of seven geometries of lower energies in the dihedral angles around the O-N-C₁-C₂ bonds (see [Fig. 6]). Therefore, this molecule has several conformers of lower energies. This aspect is important in the action mechanism due to many interaction ways in the possible active site.

From the electronic and structural analysis, it is possible to determine the electrostatic interaction pathway and the action of the charge transfer mechanism between NPE and a possible biological receptor. Once the mechanisms of action of PAR and ASP on prostaglandin endoperoxide synthase (PGES) are known for inhibiting the tyrosyl 385 [33] and acetylation of serine 530 [34], respectively, then, the PGES inhibition possibly depends on the redox property of paracetamol [35], [36] and the electron-accepting character of aspirin [37], [38]. Therefore the PGES inhibition mechanism of NPE can be compared with these drugs.

The NPE showed a low HOMO value of − 9.87 eV, while the LUMO value was − 0.07 eV. These values have shown a possible relevance of the nitro group as an electron-withdrawing group, showing its better electrophilic character. The good electron-accepting character of NPE is observed from the electron reduction potential (electron affinity) and this is possibly related to the biological activity of the nitro moiety [39].

Other evaluated physicochemical properties for NPE, PAR, and ACS were the molecular hardness (χ), molecular softness (η), softness (S), electrophilic index (ω), ionization potential (IP), and electron affinity (EA) as described in [Table 1].

The results of physicochemical calculations showed a good correlation between NPE and ASP, since these compounds have the lowest SD difference for all theoretical properties. On the contrary, PAR showed the highest SD difference for these theoretical properties. Therefore, the correlation with ASP is better than PAR, indicating that NPE may have its mechanism of action related to an electron acceptance.

In summary, NPE showed an anti-inflammatory activity in the antiedematogenic paw edema tests induced by dextran and carrageenan in rats and croton oil in mice. This anti-inflammatory effect may occur through interference in the synthesis and/or release of kinins, prostaglandins, histamines, and serotonins. Theoretical studies were realized to elucidate these hypotheses and our results suggest that NPE has an anti-inflammatory activity of probably peripheral origin. The conformational study of NPE was carried out using density functional theory calculations, showing three different groups of conformers corresponding to the energy minimum geometries. The rotations that interconvert the most stable conformer and the stereoelectronic repulsions are responsible for conformational preferences. The possible PGES interaction is more related to electrophilic than nucleophilic properties of NPE.

Acknowledgements

The authors are grateful to CNPq and CAPES, Brazilian Government, for financial support.

Conflict of Interest

This research does not have any conflicts of interest.

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Correspondence

• References

• 1 Maia JGS, Zoghbi MGB, Andrade EHA. Plantas aromáticas na Amazônia e seus óleos essenciais. 1st. edition, Volume 1. Belém: Museu Paraense Emílio Goeldi; 2001
  Search in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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