Percutaneous Absorption Enhancer Properties of Lavandula Angustifolia Essential Oil on Percutaneous Absorption of Naproxen Sodium from Topical Gel

The naproxen bioavailability via percutaneous route is low, and several technologies have been used to overcome the problem. Although the permeation enhancer properties of natural essential oils have been reported, there is no study to show the effectiveness of Lavandula angustifolia essential oil on the percutaneous absorption of naproxen sodium from a topical gel to overcome poor percutaneous absorption of naproxen. To this end, the naproxen topical gel was formulated using Carbopol 940 (as a gelling agent), and several vehicles. The results showed a greater level of penetration into (222.19 ± 24.87 vs. 107.65 ± 6.38 µg/cm 2 ) and across (22.07 ± 4.42 vs. 13.14 ± 2.87µg/cm 2 ) the skin layers for the formulation containing essential oil in comparison with the naproxen gel ( P < 0.05) . A signicant antinociceptive property was observed in naproxen topical gel containing 0.5% essential oil in both the rst and late phase of the formalin test. The effect was observed in the late phase of the tail-ick test too. In conclusion, the study demonstrated that Lavandula angustifolia essential oil signicantly enhanced the percutaneous absorption of naproxen and the analgesic effects.


Introduction
The human skin is an acceptable and suitable route for topical, local, or systemic drug delivery. Nevertheless, diffusion via the skin is controlled by the stratum corneum, which causing a barrier for transdermal delivery of chemicals [1]. In order to achieve a better transdermal delivery, it is important to overcome skin barriers, particularly the stratum corneum [2]. Different strategies have been used and developed for enhancing percutaneous drug absorption. One of the acceptable and suitable strategies to increase chemicals delivered via the skin is the use of permeation enhancers [1,3]. Permeation enhancer properties of natural essential oils have been reported in many studies [4][5][6][7][8]. The owers and essential oils of Lavandula angustifolia (LA) known as "Ostokhoddous", are used in traditional medicines to treat several disorders and conditions. Many studies have reported antinociceptive and anti-in ammatory properties of different parts of LA [9]. The plant essential oil's major components are camphor, 1,8cineole, and endo-borneol [10], which showed percutaneous enhancer effects in studies [11][12][13].
Naproxen sodium is an anti-in ammatory and antinociceptive drug that is used at an increasing rate in painful positions. The drug has high protein binding and may cause gastritis and peptic ulcer disease.
The use of a percutaneous route could reduce the naproxen side effects. Nonetheless, the naproxen bioavailability via percutaneous route is low, and several technologies have been used to overcome the problem [14].
As there is no study on the percutaneous enhancing effects of Lavandula angustifolia essential oil (LAEO) on percutaneous absorption, the present study was aimed to investigate the percutaneous enhancing effects of LAEO on percutaneous absorption of naproxen sodium from a topical gel. In addition, anti-nociceptive and anti-in ammatory effects of the gel were measured in mice using tail-ick and formalin tests.

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.

Gas chromatography-mass spectrometry (GC-MS) analysis
Perkin-Elmer 8500 equipped with a DB-5 capillary column (30 m ´ 0.25 mm; lm 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 ow 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].

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].

Viscosity measurement and rheological behavior study
The viscosity was measured at different speeds (5, 10, 20, 50,  The formulations were assessed for physical appearance, color, and phase separation by visual observation. Homogeneity and texture were evaluated by sensations between the ngers, and on the skin. The consistency (determines its "feel" and "body" and judge proper consistency) and presence of particles was also evaluated [18].

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].

Animals
To do tail-ick 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 rst 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).

Tail ick test
The tail-ick studies were done with a tail-ick 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].

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].

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 cm 2 ), and the dermis faced the receiver medium. Ethanol 50% was used as receiver uid at 32±0.5 º C (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 uid 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 uid was ltered through a lter paper (Whatman lter paper grade 591) and selected for analysis. All samples were subject to ltration by a syringe lter (0.22 µm) and were analyzed for naproxen content [16].

Statistical analysis
To measure differences between groups, ANOVA followed by the Newman-Keuls test was used and pvalue equal to 0.05 was considered signi cant.

Results
In the preliminary stage, several formulations were prepared with different solvents and gelling agents, and the best formulation (F4) was chosen based on the physical stability as main naproxen sodium gel 1% w/w containing essential oil 0.5% w/w Table 1.
The formulation has a smooth texture with a clear and transparent appearance, homogeneous with the odor of LA. The characteristics remained constant for 6 months, and there was no difference in the aspect of the formulation before and after a freeze-thaw cycle. The pH values of the formulation were found to be 5.7 ± 0.02. The spreadability percent of the formulation was 210%. Table 3 shows the rheological behavior of the formulation at different speeds. Figure 1 showed the rheological behavior of the formulation. The viscosity decreases as the shear rate increases (shear thinning). Based on the GC-MS analysis, 1,8-cineol (22.3%), Linalool (11.2%), camphor (7.9%), β-pinene (5.8%), α-terpineol (4.9%), αpinene (4.6%), Terpinen-4-ol (4.2%), borneol(4.0%) were the major constituents of the essential oil; these compounds were also identi ed in Table 2. Figure 2 shows the permeated (transdermal delivery) amount of naproxen through the skin from both gels. Figure 3 shows the cumulative percentage of plots of naproxen penetrated (dermal delivery) and permeated after 24 h (transdermal delivery) for both gels. The amount of naproxen permeated through the skin and penetrated to skin layers was higher for F4. The concentration of naproxen quanti ed in the receptor chamber was 22.07 ± 4.42 µg/cm 2 for the formulation containing essential oil and was 13.14 ± 2.87 µg/cm 2 for naproxen gel. The amount of naproxen deposited in the skin for the formulation containing essential oil was 222.19 ± 24.87 µg/cm 2 , and 107.65 ± 6.38 µg/cm 2 for naproxen gel. The tail-ick study showed more analgesic effect after 45 min for the naproxen sodium formulation, which containing LAEO (p<0.05) than other formulations ( Figure 4). The formalin test results showed the analgesic effect of formulations containing naproxen (f3 and f4) compared with the vehicle Figures 5 and 6. However, the analgesic effect of formulation containing LAEO was more. In vitro percutaneous absorption was performed, to compare the e cacy of naproxen-essential oil gel (as a sample) and standard naproxen gel (as control).
Many studies have reported the anti-in ammatory and antinociceptive effects of different parts of LA [9,[21][22][23]. Silva et al. studied the anti-in ammatory and antinociceptive activity of LAEO and reported LAEO has an anti-edematogenic effect similar to dexamethasone. They also reported that LAEO has an antinociceptive activity similar to tramadol, and dose-dependent antioxidant activity [22]. Husseini et al. have assessed Lavandula o cinalis extract analgesic and anti-in ammatory effects by formalin test where their effects were similar to the effects of morphine, dexamethasone, and indomethacin [23].
Cardia et al. have reported that topical application of LAEO had signi cant effects on acute in ammatory responses in different models. They also reported LAEO at below concentration of 10 µg/ml did not show in vitro cytotoxicity [10]. Hajhashemi et al. evaluated the anti-in ammatory and antinociceptive effects of essential oil, polyphenolic fraction, and hydroalcoholic extract of the leaves of LA [9]. Terpenes are one of the promising clinically acceptable enhancers (at a concentration between 1-5%) Due to their minimal systemic toxicity and dermal irritation with high enhancement activity [7]. 1,8-Cineole is a terpene that is characterized as a main constituent of several aromatic plants' essential oil [24] is a major part of LAEO, which could be the suitable potential of LAEO as an enhancer in naproxen sodium gel. These results have been reported by other researchers previously [10,[25][26][27][28]. The formulations were prepared with Carbopol 934 as the gelling agent at the concentration of 1% Table 1. pH, physical stability, spreadability, and organoleptic characteristics of formulations were examined. The results approved suitable properties of carbopol 940 as the gelling agent. Topical dermal or transdermal formulations were evaluated for skin penetration into and across the skin, respectively. Even though the human's skin can serve as a more valid model, most studies were performed on rat's skin [19]. Similarly, we used rat's skin as a model to evaluate the e cacy of LAEO. Figure 2 shows the permeated (transdermal delivery) amount of naproxen through the skin from both gels. Results show that after 8 h, naproxen permeated more from F4. This indicates that LAEO is suitable for enhancing transdermal delivery too. Figure 3 shows the cumulative percentage of plots of naproxen penetrated (dermal delivery) and permeated after 24 h (transdermal delivery) for both gels. The amount of naproxen permeated through the skin and penetrated to skin layers was higher for F4 (p < 0.05). Naproxen concentration measured in the receptor chamber was 22.07 ± 4.42 µg/cm 2 for the formulation containing essential oil and was 13.14 ± 2.87 µg/cm 2 for naproxen gel(p < 0.05). The deposited concentration of naproxen in the skin for the formulation containing essential oil was 222.19 ± 24.87 µg/cm 2 , and 107.65 ± 6.38 µg/cm2 for naproxen gel it was (p < 0.05). The results showed that the LAEO increases the drug permeation and also increases the accumulation of naproxen in the horny layer. Naproxen topical gel containing essential oil 0.5% induced a signi cant analgesic effect in the early phase of the formalin test. Moreover, this effect was increased in the late phase. The result of tail-ick test was consistent with the formalin test. Latency time in tail-ick test was increased in the group that recieved naproxen topical preparations with essential oil. As any signi cant analgesic and antiin ammatory effect was not observed in both formalin and tail-ick tests for the gel, which have only the essential oil. The authors concluded that the increasing anti-in ammatory effects of naproxen topical gel containing 0.5% essential oil is due to the enhancement percutaneous absorption of the drug in the presence of essential oil, and the essential oil by itself has no anti-in ammatory effect. It is suggested the use of a higher concentration of LAEO could show more analgesic and anti-in ammatory activity because of both enhancement and antinociceptive effects of the essential oil. Low concentrations of LAEO was selected, as the study aim was to evaluate the enhancement effects of LAEO. Okabe et al. studied the enhancement activity of cyclic monoterpenes on the transdermal absorption of indomethacin gel [29]. They reported that d-limonene could increase the percutaneous absorption of indomethacin. They also evaluated the d-limonene skin pretreatment, and observed no enhancement of d-limonene on skin barrier function, which suggest that d-limonene might have reversible alteration on the skin [29]. 1,8-Cineol, Linalool, and camphor, the major constituents of Lavandula O cinalis essential oil accelerate transepidermal absorption of naproxen sodium. Xie et al. evaluated the penetration-enhancing pro le of (+)-camphor via in vitro percutaneous experiments. They reported that camphor could signi cantly increase the transdermal absorption of drugs with different hydrophobicity, but maximum penetrationenhancing e ciency was for hydrophilic drugs. They also reported relatively low skin irritation of camphor [13]. Monoterpenes are safe compounds as they have reversible effects on skin lipids and relatively low cutaneous irritation at concentrations (1-5%w/v). For formulations containing limonene, linalool, anethole, thymol, carvacrol, and menthol, no skin irritation in humans was reported [30]. Availability of data and materials:

Conclusion
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Tables   Table 1 Composition (w/w %) of various gels.

Figure 2
The accumulated amount of naproxen permeated through the rat skin within 24 hours (data are shown as mean ± SD, n = 3; the ANOVA test followed by the Tukey test showed a signi cant impact of time and formulation on the permeation of naproxen p < 0.05). The sample represents 1 percent naproxen with 0.5 percent essential oil, and the standard formulation is 1 percent naproxen gel.

Figure 4
Tail ick latency in different topical preparation. Values are presented as Mean ± SD (n=5 animals per group).

Figure 5
First phase (0-5 min) of formalin test in topical preparations. Data are presented as Mean ± SD (n=5 animals per group) mean±SD (n=5 animals per group). P<0.05 for the rst phase (Tukey test).