2.1 Materials
Naproxen sodium was purchased from Tehran Daroo pharmaceutical Co. (Tehran, Iran). PEG 400 and ethanol were purchased from Merck (Merck Co., Germany). Methylparaben was acquired from Sigma-Aldrich, and propylparaben was obtained from Acros Organics. Carbopol 934P was from BF Goodrich, (Cleveland, OH), and LAEO was obtained from Barij Essence, Tehran, Iran.
2.2 Gas chromatography-mass spectrometry (GC-MS) analysis
Perkin-Elmer 8500 equipped with a DB-5 capillary column (30 m ´ 0.25 mm; film thickness 0.25 mm) equivalent to USP phase G27 was used for GC analysis. The FID detector programmed at 60 °C for 5 min and then up to 220 °C at 4 °C/min. Helium in a constant flow rate (2 mL/min) was used as carrier gas, and the split ratio was 1:30. GC-MS analysis was done on Hewlett Packard 6890 series (Hewlett-Packard Enterprise, Palo Alto, CA) using electron energy of 70 eV with a scans time of 1 sec in split mode with 1:40 ratio. The acquisition mass range was 40-400 m/z. Column and carrier gas was set similar to the gas chromatographic analysis [15].
2.3 Preparation of gels
The details of formulation compositions showed in Table 1. Carbopol (5% w/w) was dispersed in water and kept 24h to prepare the plain gel. Naproxen sodium solved in PEG400 at 40 ◦C by a heater stirrer (phase 1) and other components solved in ethanol at room temperature (phase 2). Two phases were mixed at room temperature and then was mixed with carbopol 5% gel under propeller homogenizer at 400 rpm (unneutralized Carbopol gel) [16].
2.4 Viscosity measurement and rheological behavior study
The viscosity was measured at different speeds (5, 10, 20, 50, 100 rpm) at 25°C using spindle S5 by Brookfield viscometer (Brookfield, DV-II +, USA) [17].
2.5 Physical Evaluation
2.5.1 Organoleptic Characteristics.
The formulations were assessed for physical appearance, color, and phase separation by visual observation. Homogeneity and texture were evaluated by sensations between the fingers, and on the skin. The consistency (determines its "feel" and "body" and judge proper consistency) and presence of particles was also evaluated [18].
2.5.2. Spreadability.
Spreadability was measured by spreading the diameter of the sample (1g) on a premarked circle (2 cm diameter) between two glass plates (n=3). The weight 500 g applied to the upper plate for 5 minutes [19].
% spreadability= (𝐴2/𝐴1) × 100, (1)
𝐴1 = 2 cm and 𝐴2 = after spreading
2.5.2 pH Values.
The gel (1 gram) was dispersed in deionized water (25 mL), and the pH was measured (n=3) by a pH meter (Mettler-Toledo Ingold Inc., Billerica, MA) [20].
2.6 Physical Stability
The formulation in triplicate was submitted to stability tests with tubes of 30 grams. The formulation was kept under three different temperatures for 6 months (4, 25, and 40 ◦C). At the temperature of 40 ◦C the humidity was selected at 75% in climate chamber HPPeco (Memmert) [18].
2.6.1 Freeze-Thaw Cycle.
The formulations for 12 days remained in two different temperatures (4 ∘C and 40 ∘C) for a period of 24 hours in six cycles [18].
2.6.2 Centrifugation Test.
10 g of the sample was added in a test tube and was subjected to 3000 rpm for 30 minutes at 20 ◦C ( 3-30K, Sigma, Germany) [18].
2.7 Animals
To do tail-flick and formalin studies, four groups were selected: (1) naproxen-essential oil gel, (2) naproxen gel, (3) essential oil gel (placebo), and (4) base gel (control). For percutaneous studies, only two first groups were selected. The research method was acceptable with the Ethical Guidelines for Investigations in Laboratory Animals. In the studies, male Swiss–Webster mice weighing 25–30 g and male Wistar rats (weighing 120-150 g) were used [19]. This study was approved and supervised by the ethics committee of Baqiyatallah University of Medical Sciences (No. IR.BMSU.REC.1398.403).
2.8 Tail flick test
The tail-flick studies were done with a tail-flick instrument (Model TF-1435; Technic Azma; Tabriz, Iran). 0.5 gram of the sample was rubbed 50 times on the animals' tails (n=5 in each group). The heat was applied on proximal 2 cm of the tail base. The delay in pain responses were considered as an indication of nociception. Ten seconds was selected as maximal exposure time to avoid tissue damage. The study was done every 5 minutes until 1 hour [19].
2.9 Formalin test
To start the test, the formulations were applied topically to the plantar surfaces of the left hind paws (n=5 in each group) by rubbing 50 times. Animals were placed in an observation chamber, immediately after injection of formalin 2.5 % (50 μL) under the dorsal surface of the left hind paw, and the time spent on licking, shaking, and biting of the injected paw was measured and considered as an indication of pain. The early and the last phase was from 0 to 5 min and between 15 to 60 min after the injection, respectively [19].
2.10 In vitro skin permeation study
Male Wistar rats were anesthetized by injecting ketamine and xylazine 87 and 13 mg /kg, respectively. To remove abdominal skin, chloroform was used to kill rats after 48 h of the shaved time. The subcutaneous skin fat was cleaned, and then it was placed in contact with a normal saline solution (0.9%) for 24 h.
The skin was used between the Franz cells halves (with an area of 3.8 cm2), and the dermis faced the receiver medium. Ethanol 50% was used as receiver fluid at 32±0.5 ºC (approximate normal skin conditions) and stirred at 150 rpm throughout the study.1g (equal to 10000 µg naproxen) of formulations (f3 and f4) were spread out uniformly on the skin surface as donor compartment. 4 ml of the fluid were withdrawn at predetermined times (2, 6, 8, 10, and 24 h) and fresh medium was replaced immediately after withdrawing the sample. The skin was removed and washed at the end of the study and cut into small pieces, and put in a tube for 24 h in contact with 15 ml ethanol 50% and then sonicated for 1 h with bath sonicator. The fluid was filtered through a filter paper (Whatman filter paper grade 591) and selected for analysis. All samples were subject to filtration by a syringe filter (0.22 µm) and were analyzed for naproxen content [16].
2.11 HPLC
HPLC waters 2695 equipped with the Prodigy™ 5 µm ODS-3 100 Å, LC Column 125 x 4.6 mm, Ea was used in permeation study to detect naproxen at 230 nm. The mobile phase was 40:20:40 acetonitrile, methanol, and acetic acid (1% v/v). The flow rate was 0.7 ml/min, and the naproxen peak retention time was 6 minutes [14].
2.12 Statistical analysis
To measure differences between groups, ANOVA followed by the Newman– Keuls test was used and p-value equal to 0.05 was considered significant.