Cellular and Molecular Biology Phytol anti-inflammatory activity: Pre-clinical assessment and possible mechanism of action elucidation

: Phytol (PHY) is an acyclic natural diterpene alcohol and a chlorophyll constituent that exhibits several pharmacological effects, such as anticancer, antioxidant, and antimicrobial. Here, we aimed to assess the PHY anti-inflammatory effect in vitro and in vivo , and to deepen knowledge on the possible mechanism of action. For this purpose, egg albumin ( in vitro ) test was performed by using acetyl salicylic acid (ASA) as a standard nonsteroidal anti-inflammatory drugs (NSAID). For in vivo test, male Wistar albino rats were treated (intraperitoneally) with 100 mg/kg of PHY and/or standard NSAIDs ASA (100 mg/kg) and diclofenac sodium (Diclo-Na, 10 mg/kg) to evaluate the combined effect of PHY in formalin-induced paw edema model. Furthermore, an in silico (CADD) study was accomplished to assess the effect of PHY against cyclooxygenase (COX)-1 and 2 enzymes, nuclear factor kappa B (NF-κB), and interleukin-1β (IL-1β). Results revealed that PHY exhibits dose-dependent anti-inflammatory effect using the egg albumin method. PHY (100 mg/kg) co-treated with ASA and/or Diclo-Na reduced paw edema better than PHY alone or NSAIDs individual groups. Computational study showed that PHY efficiently interacts with COX-1 and 2, NF-κB, and IL-1β. In conclusion, PHY exhibits anti-inflammatory activity, possibly via COX-1 and 2, NF-κB, and IL-1β dependent pathways.


Introduction
Phytol (PHY) is an acyclic natural diterpene alcohol and a constituent of chlorophyll abundantly found in nature (1). Although it is used mainly as a fragrance component, PHY possesses numerous important biological effects, drowning the attention of scientists for possible biomedical applications (2).
Research findings have shown that several inflammatory mediators, among them interleukin 1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α), are involved in the nociceptive response to chemical stimulus in experimental animals (3,4). In this context, prostaglandins synthesis displays a key role in both inflammatory and febrile response to endogenous (e.g., cytokines) or exogenous (e.g., lipopolysaccharide) pyrogens (5). On the other hand, cyclooxygenase (COX) enzymes are involved in inflammation-and pain-related symptoms (6). Along this line, nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin (acetyl salicylic acid, ASA) and diclofenac (e.g., Diclofenac sodium, Diclo-Na) exert their effect by inhibiting the COX activity. Brain endothelium COX-2 induction exerts a crucial role in inflammation (7). COX-2 pathways transcription and induction are activated by cytokines and TNF, triggering the central mechanisms that act via the transcription factors such nuclear factor kappa B (NF-κB) and signal transducer and activator of transcription (STAT-3) (8,9). In male ICR and BALB/c mice, PHY inhibit allergic cytokine expression through NF-κB and activator protein (AP)-1 regulation (10). Moreover, Silva et al. (2014) suggested that PHY exhibits antiinflammatory effect in carrageenan, compound 48/80-, histamine-, serotonin-, bradykinin-and PGE2-induced paw edema models (11). Also, PHY inhibit neutrophil migration through reducing IL-1β and TNF-α levels and oxidative stress (11). Thus, in light of the wide range of PHY pharmacological profile, this study aimed to assess its anti-inflammatory potential both in vitro and in vivo. Additionally, the anti-inflammatory mechanism of action was investigated through combination of PHY with ASA or Diclo-Na and by an in-silico study. Results of this study may have some pharmacological implications as to the use of PHY as anti-inflammatory.

Drugs, chemicals and reagents
ASA and Diclo-Na were from Zenith Pharmaceuticals Ltd., Bangladesh. PHY and other analytical grade chemicals or reagents were acquired in Sigma Aldrich, USA.

Animals and diets
Adult male Wistar albino rats (120-150 g) were from the animal resource branch of Bangladesh Council of Scientific and Industrial Research (BCSIR), Chittagong. Animals were housed at standard environmental conditions (temperature: 24±1 ○ C, humidity: 50±5%, and 12 h light/dark cycles), had free access to standard pellets (basal diet) and water ad libitum. Acclimatization phase was of 7 days before the beginning of the study. Then, animals were randomly divided into experimental (EG) and control (CG) groups, and food was withdrawn 12 h before the experiment's onset. Committee on Animal Research at the Federal University of Piauí (UFPI, #109/2014) approved all the procedures, which were done in accordance with the Brazilian (COBEA-Colégio Brasileiro de Experimentação Animal) and International Standards on experimental animals care and use (Directive 2010/63/EU of the European Parliament and of the Council).

Egg albumin test (in vitro)
This study was accomplished following the procedure of Ullah et al. (2014), with some modifications (12). Briefly, PHY (mixed with the vehicle 0.05% Tween 80 dissolved in 0.9% NaCl solution) was used at 12.5, 25, 50, 75 and 100 µg/mL. ASA (a standard) was dissolved in distilled water and employed at 100 µg/ mL, while 0.05% Tween 80 (a vehicle) was dissolved in 0.9% NaCl solution. Reaction mixture (5 mL) was composed by egg albumin (0.2 mL), PBS (2.8 mL; pH 6.4), and test sample/controls (2 mL). Then, mixtures were incubated at 37±2 °C in a BOD incubator (Lab line Technologies) for 15 min and afterwards heated at 70 °C for 5 min. After cooling, absorbance was measured at 660 nm (LABOCON MODEL: LUVS-201, monochromatic beam) using the vehicle as a blank. The percentage of protein denaturation inhibition was determined using the following formula: % Inhibition = [(A control -A test ) ÷ A control ] × 100

Formalin-induced paw edema test (in vivo)
The method of Chau (1989) was used to this experiment, with slight changes (13). Paw edema was induced through injection of 0.2 mL of 1% w/v formalin solution suspended in distilled water into the rat sub-plantar tis-sues of the left hind paw. For this purpose, 40 rats were divided into 8 groups with 5 animals each, as follows: G1 (formalin), G2 (formalin + Vehicle), G3 (ASA), G4 (Diclo-Na), G5 (PHY), G6 (PHY + ASA), G7 (PHY + Diclo-Na) and G8 (PHY + ASA + Diclo-Na). The last one was used for further confirmation of the co-treatment effects of PHY and standard NSAIDs. In all of these treatments (except formalin), animals were administered i.p. at 10 mL/kg (Table 2). Treatments were given 15 min before injecting the formalin solution. Thickness of the paw was measured in each rat by using a vernier caliper in mm.

Molecular docking study (in silico)
Four-dimensional crystal structures of COX-1 (PDB ID: 2OYE), COX -2 (PDB ID: 6COX), NF-κB (PDB ID: 5LDE), and IL-1β (PDB ID: 3O4O) were downloaded from the protein data bank; structures were prepared and refined by using Schrödinger-Maestro v10.1. Bond orders and charges were assigned, hydrogen moieties were added, selenomethionines were converted to methionines, and water molecules other than those present in the active site were removed. Optimization of amide groups on asparagines, imidazole ring and glutamines on histidines, protonation states on histidines, glutamic and aspartic acids was performed at neutral pH (7.0). Furthermore, prepared proteins were minimized to the RMSD value of 0.30 Å using the force field OPLS_2005. Docking grids of 17 Å × 17 Å × 17 Å were generated in glide around the active site residues (reference ligand active site) keeping the Van der Waals scaling factor 1.00 and charge cutoff 0.25 subjected to OPLS 2001 force field. The target compound, PHY, was retrieved from the Pubchem database (CID: 5280435) and the 3D structure was created by Schrödinger-Maestro v10.1 with OPLS 2005 force field, whereas PHY ionization states were generated at pH 7.0.
Flexible docking was achieved using the Glide of Schrödinger-Maestro v10.1 and applying penalties for non-cis/trans amide bonds and keeping all docking parameters as standard. No binding restrictions were given during the docking calculations. Ligand pose generated for the input molecule (PHY) and the PHY binding affinity to the target bio-macromolecules were calculated as docking glide score using Monte Carlo random search algorithm, performing post-docking minimization with the field OPLS 2005 force.

Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) analysis
To evaluate whether PHY is biologically suitable to be considered as a drug, its pharmacokinetic parameters were analyzed. For this purpose, Swiss ADMET analysis (http://www.swissadme.ch/) was employed for estimating PHY ADMET.

Statistical analysis
Data were expressed as mean±SEM and analyzed using one-way analysis of variance (ANOVA), followed by Tukey post hoc multiple comparison test. GraphPad Prism software (v. 6.0, San Diego, California, USA) was used for statistical analysis, with p<0.05 considered statistically significant. and conventional hydrogen bond.
Furthermore, we examined the drug-like property

Results
Results revealed that PHY, at all doses tested, markedly inhibits heat-induced protein denaturation in egg albumin method. ASA, at 100 µg/mL, exhibited the highest protein denaturation inhibition (78.08±1.08), whereas the same dose of PHY led to 62.12±0.78 inhibition. On the other hand, the vehicle showed negligible protein denaturation inhibition as shown in Table 1.
Results from Table 2 suggest that PHY and both NSAIDs significantly (p<0.05) reduce paw edema in a time-dependent manner comparing with control and vehicle groups. Diclo-Na was found to reduce more paw volume than ASA and PHY groups. Interestingly, PHY when co-administered with ASA or Diclo-Na was found to be more potent in reducing paw edema than PHY, ASA, and Diclo-Na groups. However, a significant and best reduction of paw edema was observed in PHY + ASA and in PHY + Diclo-Na groups. The highest reduction of paw volume was 0.9 ± 0.12 mm on the 3 rd day in PHY + ASA + Diclo-Na group.
Results from in silico study suggests that PHY strongly interacts with COX-2 (binding affinity: -3.778, followed by NF-κB, COX-1, and IL-1β with binding affinities of -1.979, -1.583, and -0.379, respectively. PHY was docked in the reference binding pockets of these proteins ( Figure 1) and the interacting bond types are shown in Table 3. In addition, results suggest that, PHY interacts with 10 amino acids within the reference binding pocket when docked with COX-2, whereas it interacts with 8, 5, and 5 amino acid residues, respectively, when docked with NF-κB, COX-1, and IL-1β. These interactions involved 4 types of attractive forces, including alkyl bond, pi-alkyl bond, carbon-carbon bond,    Table 4. It was found that PHY can be considered a drug-like molecule upon analyzing its molecular weight, capacity of hydrogen bond donating or accepting, lipophilicity, and molar refractivity according to Lipinski's rule of 5.

Discussion
Natural products, whether in the form of pure compounds or standardized extracts of various sources, have gained much attention in the field of drugs discovery and development (14). In this context, diterpenes have been coming into the spotlight given their multiple bioactivities (15). PHY is a diterpenoid known for its fragrance indication and diverse pharmacological effects (1).
On the other hand, oxidative stress leads to inflammation, a key triggering factor of some known diseases, such as atrial fibrillation in obesity and diabetes (16), and in neurological diseases and disorders (17,18). Currently available NSAIDs have many side effects, including combinational risks, cardiovascular diseases, erectile dysfunction, gastrointestinal complications, inflammatory bowel disease, renal diseases, photosensitivity, pregnancy disorders, allergic or allergic-like hypersensitivity reactions, anastomosis raised liver enzymes, headache, dizziness, hyperkalemia, confusion, complicated breathing, and aseptic meningitis, among others. In addition, diterpenes, such as carnosol (19), paclitaxel (20), and andographolide and its derivatives (21) have shown anti-inflammatory activity in various test systems. Generally, diterpenes are cytoprotective in nature and possess diverse pharmacological effects (15).
On the other side, oxidative stress leading to inflammation can damage cell macromolecules, such as carbohydrates, proteins, lipids, and genetic materials (e.g., DNA, RNA) (22). Results obtained from this study indicated that PHY, at all doses (in vitro), inhibits protein denaturation, which might be related to its antioxidant (22)(23)(24) and anti-inflammatory 11 activities. Furthermore, the in vivo data suggested that PHY alone or co-administered with NSAIDs exhibit anti-inflammatory effects in rats. In addition, research findings showed that PHY can led to a substantial reduction of IFN-γ, IL-4, and IL-10 production in mouse splenocytes stimulated by T-cell mitogens (25). PHY is also able to inhibit IL-17A production, a promoter of neuro-inflammation and cognitive dysfunction in animals through NF-κB signaling pathway activation (26). In this respect, a recent study by Goncalves and colleagues found that a betulin derivative inhibits IFN-γ and modulates COX-2 expression (27). In a similar fashion, the Ephedra gerardiana hidroethanolic extract and fractions down-regulated PGE2, COX-2, IL-1β, IL-6, TNF-α, and NF-κB, and upregulated IL-4 and IL-10 expression in Sprague Dawley rats (28). Thus, the immunomodulatory (10) and anti-inflammatory (11) effects of PHY in experimental animals may be interrelated. In a study,  showed that PHY exerts anxiolytic-like effects in mice possibly through GABAergic transmission which is similar to the effect of diazepam (29). Along this line, diazepam was found to enhance the neuroprotective activity of NS-398, a COX-2 inhibitor in male Sprague-Dawley rats (30). Therefore, the PHY-mediated neuroprotective effects (29,(31)(32)(33) may be linked with each other. In this study, PHY co-treated with ASA and Diclo-Na exhibited more potent anti-inflammatory effect than PHY and NSAIDs individual groups, where both of these NSAIDs act through COX enzymes.
The in silico study indicated that PHY strongly interacts with COX-2, the enzyme responsible for inflammatory prostaglandins generation (9). Additionally, PHY showed significant interaction with NF-κB, a regulatory protein that mediates COX-2, TNF-α, and IL-1β expression (34). Furthermore, our results reveal a noticeable interaction of PHY with COX-1 and IL-1β. These results agree with that of Silva et al. (2014) that proposed IL-1β and TNF-α levels reduction, triggered by PHY, as a potential mechanism of its anti-inflammatory action (11). Possible anti-inflammatory mechanism of PHY is depicted in Figure 2.
Evaluation of the pharmacokinetic parameters (AD-MET) is an important factor in drug discovery, since it reduces the likelihood of failures in clinical phases (35), and the computer-based approach is a reliable and convenient strategy to do so (36). In our study, PHY was found to be a potential drug-like molecule for oral administration due to its lower molecular weight and lower hydrogen bonding capacity. Low molecular weight and low hydrogen bonding capacity contribute to better per-