Biological investigation of a supersaturated self-nanoemulsifying drug delivery system of Piper cubeba essential oil

Abstract

Piper cubeba essential oil (PCEO) is used in many ailments but its mechanism of action is not very well reported especially in the case of pain and inflammation. Therefore, in this work, the mechanism of its anti-inflammatory activity alone and as a supersaturated self-nanoemulsifying drug delivery system (S-SNEDDS) was evaluated. S-SNEDDS formulations of PCEO were developed by an aqueous phase titration method. Thermodynamically stable S-SNEDDSs were characterized based on their droplet size, polydispersity index, zeta potential, viscosity, refractive index, % transmittance and surface morphology. Based on the best physicochemical parameters, the S-SNEDDS F1 was selected for biological investigations in rats. The dose of pure PCEO was 400 mg kg⁻¹ body weight while the S-SNEDDS F1 was administered in two different doses i.e. 40 mg kg⁻¹ and 80 mg kg⁻¹ body weight. The results of this work indicated that pretreatment of PCEO and the S-SNEDDS F1 reduced the exudate volume and polymorphonuclear cell number significantly. Moreover, the levels of MPO, NO and proinflammatory cytokines (TNF-α and IL-β) were also reduced by PCEO and the S-SNEDDS F1 and this observation was also supported by histological observation. The results of the S-SNEDDS F1 were superior compared to PCEO alone even at significantly lower doses. These results indicate the potential of a developed S-SNEDDS in enhancing the therapeutic efficacy of PCEO.

---

1. Introduction

Essential oils are commonly used in cosmetics and perfumes, as flavoring agents in food and beverages and in pharmaceutical industries.¹ They have been investigated for a variety of medicinal properties such as antimicrobial, antibacterial, antifungal, antibiotic, anti-leukemic, analgesic, anti-inflammatory, antiviral, anticancer and antioxidant.^2–4 The Piper cubeba plant belongs to the Piperaceae family which is commonly known as ‘Kababa’ in Arab countries. A literature survey revealed the gastroprotective activity of this plant.^5,6 However, no scientific data are available to validate the traditional claim of the anti-inflammatory effects of P. cubeba. Neutrophils play an important role in the inflammatory response.⁷ However, polymorphonuclear (PMN) cells have an important role in lung damage.⁸ Analgesic and anti-inflammatory effects of methanolic and hydroalcoholic extracts of P. cubeba have been reported in the literature.^9,10

In the last decade, various lipid based nanosized formulations such as nanoemulsions, microemulsions, self-microemulsifying drug delivery systems (SMEDDSs) and self-nanoemulsifying drug delivery systems (SNEDDSs) have been investigated successfully to enhance the therapeutic efficacy of various anti-inflammatory drugs.^11–17 SNEDDSs are transparent mixtures of a drug, an oil/lipid, a surfactant and a cosurfactant which upon agitation with water or gastrointestinal (GI) fluids produce very fine nanoemulsions (droplet size < 100 nm).^18,19 If these SNEDDSs produce nanoemulsions with a size that is too fine or ultrafine (<20 nm), these systems would be considered supersaturated-SNEDDSs (S-SNEDDSs).²⁰ As a drug delivery carrier, the S-SNEDDS offers several advantages such as thermodynamic stability, self-nanoemulsification efficiency, ease of preparation (spontaneous emulsification), minimal adverse effects and nanosized droplets (less than 50 nm), which could result in the rapid absorption of PCEO and finally enhanced anti-inflammatory effects compared to unstable dispersions.^18–20 Because of the ultra low size of these systems, they are known to enhance the therapeutic efficacy and bioavailability of several poorly water-soluble drugs.^15–20 Since no scientific data are available on the anti-inflammatory mechanism of Piper cubeba essential oil (PCEO), attempts were made to develop S-SNEDDS formulations of PCEO and to evaluate its anti-inflammatory effects compared to pure PCEO and standard indomethacin treatment against carrageenan-induced pleurisy in a rat model. Various S-SNEDDS formulations of PCEO were developed by a spontaneous emulsification method using Sefsol-218 (oil phase), Triton-X100 (surfactant), Transcutol-HP (cosurfactant) and water (aqueous phase). SNEDDSs are systems which are able to self-emulsify with GI fluids when administered orally. Due to their potential for self-emulsification, these systems can only be administered by an oral route. In order to provide similar conditions for statistical comparisons, PCEO was also administered orally. Moreover, mice were not used as an animal model in this work because mice are small animals and they do not have a sufficient amount of GI fluids for the self-nanoemulsification of a SNEDDS. All of the components of the S-SNEDDS are nontoxic and generally regarded as falling under the safe category of excipients.

2. Materials and methods

2.1. Materials

The Piper cubeba (P. cubeba) plant was purchased from a local market in Riyadh, Saudi Arabia. The plant was authenticated by the taxonomist Dr Mohammed Y. Yaqoob at the College of Pharmacy, King Saud University, Riyadh, Saudi Arabia.

PCEO was extracted and characterized using a gas chromatography-mass spectrometry technique in the laboratory. Propylene glycol monocaprylic ester (Sefsol-218) was procured from Nikko Chemicals (Tokyo, Japan). Highly purified diethylene glycol monoethyl ether (Transcutol-HP) was obtained as a kind gift sample from Gattefossé (Lyon, France). Iso-octylphenoxypolyethoxyethanol (Triton-X100), dimethyl sulfoxide (DMSO) and ethanol were procured from Sigma Aldrich (St. Louis, MO). Ultra-pure chromatography grade water (deionized water) was collected from a Milli-Q water purification system (Berlin, Germany). All other materials and reagents used were of analytical grade and used without any further purification.

2.2. Extraction of essential oil from Piper cubeba

The dried fruits of P. cubeba were coarsely pulverized and hydro-distilled for 3 h using a clevenger-type apparatus according to the European Pharmacopoeia. The obtained oil was dried over anhydrous sodium sulfate, filtered and stored at 4 °C until further use. The detailed procedure of its extraction and characterization is given in our previous article.²¹

2.3. Selection of components for S-SNEDDS preparation of PCEO

For the selection of components when preparing the SNEDDS, the solubility of a biological molecule in different components is one of the most important criteria.²⁰ Nevertheless, solubility studies were not performed in this work because the biological molecule in this work was PCEO, which is easily miscible in oils and surfactants. Therefore, safety and nontoxicity were the main criteria for selection of the oil phase, surfactant and cosurfactant in this work. Based on the above criteria, Sefsol-218, Triton-X100 and Tarnscutol-HP and water were selected as principal components in order to develop a suitable S-SNEDDS of PCEO.¹⁶

2.4. Construction of pseudo-ternary phase diagrams

For preparation of the S-SNEDDS of PCEO, pseudo-ternary phase diagrams were constructed by an aqueous phase titration method as reported in the literature.^11,12 Briefly, Triton-X100 and Transcutol-HP were mixed in the mass ratios of 1 : 0, 1 : 2, 1 : 1, 2 : 1, 3 : 1 and 4 : 1. The total stock of the mixture of surfactant and cosurfactant (S_mix) was 20 g. Sefsol-218 and a particular S_mix were then mixed at different mass ratios (i.e. 1 : 9 to 9 : 1). Pseudo-ternary phase diagrams were constructed by a spontaneous emulsification method. In this method, the mixture of Sefsol-218 and a specific S_mix was titrated by the slow addition of water and visual observations were recorded on the basis of its clarity.^12,22 The clear, transparent and easily flowable SNEDDS zones were plotted on each phase diagram with one axis representing water, the second Sefsol-218 and the third representing the S_mix.

2.5. Formulation development of PCEO

From pseudo-ternary phase diagrams, it was observed that the maximum SNEDDS zones were exposed by a 1 : 1 S_mix ratio, hence a 1 : 1 mass ratio was chosen for preparation of the SNEDDS of PCEO. From the phase diagram, different SNEDDSs with formulation codes of F1–F9 were precisely selected. Almost the entire region of SNEDDS zones were taken into account. In formulations F1–F5, the concentration of the oil phase (Sefsol-218) was kept constant at 5% w/w and the concentration of the S_mix varied from 10–50% w/w. However, in formulations F6–F9, the concentration of the S_mix was kept constant at 40% w/w and the concentration of the oil phase varied from 10–25% w/w in order to cover the entire SNEDDS zones in the phase diagram. After selection of a blank SNEDDS from the phase diagram, 5% w/w of PCEO was incorporated in each SNEDDS by vortexing at 1000 rpm and 25 °C for about 5 min. The composition of the PCEO loaded SNEDDS is shown in Table 1.

Table 1 Formulation components of S-SNEDDS formulations of PCEO (F1–F9) prepared using Sefsol-218, Triton-X100, Transcutol-HP and water

Code	Formulation composition (% w/w)					Smix ratio
	PCEO	Sefsol-218	Triton-X100	Transcutol-HP	Water
F1	5	5	25	25	40	1 : 1
F2	5	5	20	20	50	1 : 1
F3	5	5	15	15	60	1 : 1
F4	5	5	10	10	70	1 : 1
F5	5	5	5	5	80	1 : 1
F6	5	10	20	20	45	1 : 1
F7	5	15	20	20	40	1 : 1
F8	5	20	20	20	35	1 : 1
F9	5	25	20	20	30	1 : 1

---

2.6. Thermodynamic stability and self-nanoemulsification tests

Thermodynamic stability tests on the developed PCEO loaded SNEDDS (F1–F9) were performed to remove any unstable or metastable formulation. These tests were performed viz. centrifugation, heating and cooling cycles and freeze–pump–thaw cycles. The detailed procedures of these tests are given in our previously published articles.^20,22 The purpose of the self-nanoemulsification test was to investigate any phase separation or precipitation upon dilution with water, acid buffer (0.1 N HCl) and phosphate buffer (pH 7.4). This test was carried out by diluting 1 ml of each PCEO loaded SNEDDS (F1–F9) with water, 0.1 N HCl and phosphate buffer (pH 7.4) in the dilution ratio of 1 : 500. The self-nanoemulsification efficiency of each formulation was assessed visually with the help of the following grading systems:^20,22

Grade A: rapid/spontaneously forming clear nanoemulsion.

Grade B: rapid/spontaneously forming bluish slightly less clear nanoemulsion.

Grade C: slowly forming turbid emulsion.

Grade D: dull, grayish slowly forming turbid emulsion.

Grade E: turbid emulsion with the presence of oil globules at the surface.

2.7. Physicochemical characterization of the PCEO–S-SNEDDS

Developed S-SNEDDSs of PCEO were physicochemically characterized to assess their droplet size distribution, polydispersity index (PI), zeta potential (ZP), viscosity, refractive index (RI), percentage of transmittance (% T) and surface morphology using transmission electron microscopy (TEM). The mean droplet size, PI and ZP of the prepared formulations (F1–F9) were determined with the help of a Malvern Particle Size Analyzer (Holtsville, NY) at 25 ± 1. The detailed procedure for the measurement of droplet size, PI and ZP is presented in our previous article.²⁰ The viscosity of the PCEO–S-SNEDDSs (F1–F9) was measured using a Brookfield Viscometer (Brookfield Engineering Laboratories, Middleboro, MA) at 25 ± 1 °C as reported previously.²² However, RIs of the PCEO–S-SNEDDSs (F1–F9) were measured using an Abbes type Refractometer (Precision Testing Instruments Laboratory, Germany) at 25 ± 1 °C as reported previously.²⁰ The % T of the PCEO–S-SNEDDSs (F1–F9) was determined spectrophotometrically at 550 nm as reported in the literature.²⁰

The surface morphology and structure of droplets of the optimized PCEO–S-SNEDDS formulation F1 was evaluated using TEM (JEOL JEM-2100 F, USA). TEM analysis was carried out using electron microscopy operating at 100 KV. The detailed procedure for TEM analysis is presented in our previous article.¹²

2.8. Animals and study protocol

Male Albino Wistar rats (weighing in the range of 180–220 g) were collected from the Experimental Animal Care Center of the Institute. The animals were provided with controlled environmental conditions. All the rats were receiving free access to a standard pellet diet and tap water. The study protocol was approved by Research Ethics Committee of the Institute (Clearance no. 021656-0621). The institutional guidelines were strictly followed for these studies. Based on the best physicochemical properties of the PCEO S-SNEDDS, the F1 formulation was selected as the optimized formulation for in vivo investigations in rats. The rats were divided into six groups with six rats per group. Group-I: control, Group-II: carrageenan only, Group-III: PCEO, Group-IV: F1 (80 mg kg⁻¹), Group-V: F1 (40 mg kg⁻¹) and Group-VI: indomethacin (standard).

The F1 formulation at two different doses (i.e. 40 and 80 mg kg⁻¹) was administered orally once a day for the period of 4 days. However, control and carrageenan groups were given an equivalent volume of a 0.5% suspension of sodium carboxymethyl cellulose. Approximately 0.3 ml of saline or saline containing 1% carrageenan was injected directly into the pleural cavity of animals under anesthesia on the last day i.e. the fourth day. 4 h after the carrageenan injection, the animals were sacrificed, the chest was opened and the pleural cavity was rinsed with approximately 2 ml of a sterile saline solution with heparin (5 U ml⁻¹). The exudate was taken out by aspiration and the total volume was determined. The samples were centrifuged and the cell pellet was resuspended in a solution of phosphate buffer saline. The number of neutrophils in the exudates was counted with the help of an optical microscope after Giemsa staining.

2.9. Determination of nitric oxide (NO)

Production of NO was carried out by the quantification of its related end products such as nitrite/nitrate using the Griess reaction as reported previously.^23,24 The measurement was carried out using a 540 nm filter in a titrated Biotek ELISA reader.

2.10. Myeloperoxidase (MPO) level of pleural exudates

A MPO assay was performed for the measurement of neutrophil recruitment which was measured indirectly by means of the MPO activity. MPO activity was determined by adopting the procedure reported by Campos et al. (2002).²⁵ The measurements were performed at 690 nm as reported in literature.²⁵

2.11. Cytokine determination

The levels of cytokines such as TNF-α, IL-6 and IL-1β in lung tissues were determined colorimetrically using a commercial ELISA kit (R&D systems, USA).

2.12. Total RNA isolation and cDNA synthesis

4 h after carrageenan administration, lung samples were obtained and used for total RNA isolation. Total RNA isolation was performed using the manufacturer’s instructions for the Trizol reagent (Life Technologies, Inc., NY, USA). Following total RNA isolation, reverse transcription (from total RNA to cDNA) was processed by the cDNA reverse transcription kit of Applied Biosystems as per the manufacturer’s instructions.²⁶

2.13. Expression of iNOS-2 and COX-2 mRNA in lung tissue

The analysis of specific mRNA expression was carried out by RT-PCR by subjecting the resulting cDNA to PCR amplification using 96-well optical reaction plates in an ABI Prism 7500 System (Applied-Biosystems). The 25 μl reaction mixture contained 0.1 μl of a 10 μM forward primer and 0.1 μl of a 10 μM reverse primer (40 nM final concentration for each primer), 12.5 μl of a SYBR green universal master mix, 11.05 μl of nuclease-free water and 1.25 μl of the cDNA sample. Rat primers for the iNOS-2, COX-2 and β-actin genes were procured from Integrated DNA Technologies (IDT, Coralville, IA). The fold change in the level of mRNA between treated and untreated groups was corrected by the levels of β-actin. The RT-PCR data were analyzed using the relative gene expression, that is, the DDCT method, as described and explained previously.^27,28

2.14. Western blot analysis

This analysis was carried out according to the method proposed by Towbin et al.^29,30 The proteins were electrophoretically transferred to PVDF membranes, blocked in 5% skim milk in Tris buffer saline (TBS) containing 1% Tween-20 for a period of 2 h at room temperature and probed with polyclonal iNos (sc-651), COX-2 (sc-sc-1746) and β-actin (sc-47778) polyclonal rabbit anti-rat antibodies in a solution of PBS containing 1% Tween-20 on a shaker for 2 h at room temperature followed by horseradish peroxidase-(HRP-)conjugated secondary antibody incubation (1 : 3000) for 1 h and visualization using an enhanced chemiluminescence system (Santa Cruz Biotechnology, USA). β-Actin was used as the loading control for total protein and densitometric analysis of the immunoblots was performed with Image J software (NIH).

2.15. Histological examination of the lungs

The lung tissue was taken out from rats, fixed in 10% formalin for a period of 7 days at room temperature, dehydrated and finally embedded in paraffin blocks. Sections were deparaffinized using xylene and stained using hematoxylin–eosin. The sections of lung tissue were examined using light microscopy.

2.16. Data analysis

All values for biological investigations are expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed by ANOVA in which a value of P < 0.05 was considered statistically significant.

3. Results and discussion

3.1. Selection of components for the S-SNEDDS preparation of PCEO

As stated previously, the solubility of therapeutic drug molecules in different components is the main criteria for their selection. In this work, as we have not used any solid crystalline drug molecule, solubility studies were not performed. Moreover, the PCEO used in this study is easily miscible in oils and surfactants. Nevertheless, different components were selected on the basis of safety and nontoxicity of the oil phase, surfactant and cosurfactant in this work. Based on these criteria, Sefsol-218, Triton-X100, Tarnscutol-HP and water were selected as principal components in order to develop a suitable S-SNEDDS for PCEO.

3.2. Construction of pseudo-ternary phase diagrams and formulation development

For development of the PCEO–S-SNEDDS, pseudo-ternary phase diagrams were constructed using Sefsol-218, Triton-X100, Transcutol-HP and water. The results of this study are presented in Fig. 1. However, a brief summary of the results is listed in Table 2. From Fig. 1, it was clear that the S_mix ratio of 1 : 0 (surfactant alone) showed poor zones of the SNEDDS (Fig. 1A). The maximum amount of Sefsol-218 (oil phase) that was solubilized by a 1 : 0 ratio was observed as 9% w/w with respect to 82% w/w of the S_mix. But when the amount of Transcutol-HP (cosurfactant) was increased in comparison with Triton-X100 (surfactant) [S_mix ratio of 1 : 2], the SNEDDS zones were increased as compared to that of the 1 : 0 ratio (Fig. 1B). The maximum amount of Sefsol-218 that was solubilized by the 1 : 2 ratio was higher than that shown in Fig. 1A i.e. 14% w/w with respect to 55% w/w of the S_mix (Table 2). When the concentrations of the surfactant and cosurfactant were kept equal (S_mix ratio of 1 : 1), the SNEDDS zones increased significantly in comparison with that of the previous S_mix ratios (Fig. 1C). The maximum amount of Sefsol-218 that was solubilized by the 1 : 1 mass ratio was observed as 28% w/w with respect to 41% w/w of the S_mix (Table 2). However, when the S_mix ratio of 2 : 1 was studied, SNEDDS regions were found to be reduced in comparison with that of the 1 : 1 S_mix ratio (Fig. 1D). The maximum amount of Sefsol-218 that was solubilized by the 2 : 1 mass ratio was observed as 22% w/w with respect to 52% w/w of the S_mix (Table 2). When the concentration of Triton-X100 was further increased with respect to Transcutol-HP (S_mix ratio of 3 : 1), SNEDDS zones were found to decrease further as compared to those of the 1 : 1 and 2 : 1 ratios (Fig. 1E). The maximum amount of Sefsol-218 that was solubilized by the 3 : 1 ratio was recorded as 16% w/w by utilizing 67% w/w of the S_mix (Table 2). When the concentration of Triton-X100 was further increased with respect to Transcutol-HP (S_mix ratio of 4 : 1), SNEDDS zones were decreased further as compared to those of the 1 : 1, 2 : 1 and 3 : 1 ratios (Fig. 1F). The maximum amount of Sefsol-218 that was solubilized by the 4 : 1 ratio was recorded as 15% w/w by incorporating around 59% w/w of the S_mix (Table 2).

Fig. 1 Phase diagrams developed by the aqueous phase titration method for SNEDDS zones of PCEO (dotted area) for Sefsol-218, water, Triton-X100 and Transcutol-HP at S_mix ratios of (A) 1 : 0, (B) 1 : 2, (C) 1 : 1, (D) 2 : 1, (E) 3 : 1 and (F) 4 : 1.

Table 2 Summary of observations made during the aqueous phase titration of SNEDDSs

Figure	Smix ratio	Surfactant	Cosurfactant	Nanoemulsion zones	Oil phase solubilized^a (% w/w)	Smix solubilized^b (% w/w)
a The maximum amount of Sefsol-218 (oil phase) that was solubilized.b The maximum amount of the Smix (Triton-X100:Transcutol-HP) phase that was solubilized with respect to the maximum amount of the oil phase.
1A	1 : 0	Triton-X100	Transcutol-HP	Low	9	82
1B	1 : 2	Triton-X100	Transcutol-HP	Higher than 1A	14	55
1C	1 : 1	Triton-X100	Transcutol-HP	Highest	28	41
1D	2 : 1	Triton-X100	Transcutol-HP	Lower than 1C	22	52
1E	3 : 1	Triton-X100	Transcutol-HP	Lower than 1C & 1D	16	67
1F	4 : 1	Triton-X100	Transcutol-HP	Lower than 1C, 1D and 1E	15	59

---

Aqueous phase titration studies indicated that the highest SNEDDS zones were exposed by the S_mix ratio of 1 : 1 (Fig. 1C). Therefore, different SNEDDS formulations for PCEO were precisely selected from Fig. 1C. Almost the entire region of SNEDDS zones were taken into account in Fig. 1C. In the first five formulations (F1–F5), the concentration of Sefsol-218 was kept constant at 5% w/w and the concentration of the S_mix was varied from 10–50% w/w. However, in the next four formulations (F6–F9), the S_mix concentration was kept fixed (i.e. 40% w/w) and the Sefsol-218 concentration was changed from 10–25% w/w in order to cover the entire SNEDDS zone in Fig. 1C. After selection of the blank SNEDDS, 5% w/w of PCEO was incorporated in each SNEDDS by vortexing at 1000 rpm and 25 °C for about 5 min (Table 1).

3.3. Thermodynamic stability and self-nanoemulsification tests

The primary objective of thermodynamic stability tests was to remove any metastable/unstable SNEDDS because observations during phase titration studies were made visually. Hence, selected formulations of PCEO were subjected to various thermodynamic stability tests. The qualitative results of these studies are presented in Table 3. From Table 3, it can be seen that all developed SNEDDSs survived at all steps of the thermodynamic stability tests. Developed formulations of PCEO (F1–F9) were further subjected to a self-nanoemulsification efficiency test because it is mandatory for oral emulsifying formulations.²² The primary aim of this test was to investigate any drug precipitation or phase separation upon mild agitation with water, acid buffer (0.1 N HCl) and phosphate buffer (pH 6.8) and qualitative results are presented in Table 3.^20,22 It was observed that all the SNEDDSs of PCEO survived this test with a grade of A in the presence of all three diluents. Since all the formulations remained transparent (passed the test with a grade of A), they were considered supersaturated SNEDDSs (S-SNEDDSs). Overall, these results indicated that PCEO was maintained in a solubilized form at the molecular state in developed SNEDDSs and the self-nanoemulsification behavior of all the formulations was independent of pH.³¹

Table 3 Results of thermodynamic stability and the self-nanoemulsification test in the presence of deionized water, 0.1 N HCl and phosphate buffer (pH 6.8)^a

Code	Self-nanoemulsification test grade*	Thermodynamic stability tests
		Cent.	H & T	FPT
a ✓ (passed the respective test), cent. (centrifugation), H & T (heating and cooling cycles), FPT (freeze–pump–thaw cycles), *(all the formulations passed this test with a grade of A in the presence of deionized water, 0.1 N HCl and phosphate buffer).
F1	A	✓	✓	✓
F2	A	✓	✓	✓
F3	A	✓	✓	✓
F4	A	✓	✓	✓
F5	A	✓	✓	✓
F6	A	✓	✓	✓
F7	A	✓	✓	✓
F8	A	✓	✓	✓
F9	A	✓	✓	✓

---

3.4. Physicochemical characterization of the PCEO–S-SNEDDS

The results obtained from physicochemical investigation of the developed PCEO–S-SNEDDS (F1–F9) are presented in Table 4. The droplet size of the SNEDDS is one of the most important characterization parameters. From Table 4, it can be seen that the droplet size of the PCEO–S-SNEDDS (F1–F9) was observed in the range of 7.53–52.45 nm. When the concentration of the oil phase (Sefsol-218) was kept constant at 5% w/w and the concentration of the S_mix was varied from 10–50% w/w (F1–F5), the droplet size of the S-SNEDDS was found to change slightly. The droplet size of the formulations decreased slightly with increasing concentration of the S_mix (Table 4). However, when the concentration of Sefsol-218 was varied from 10–25% w/w (F6–F9) and the concentration of the S_mix was fixed, the droplet size of the S-SNEDDS was found to change significantly. The droplet size of these formulations was found to rapidly enhance with an increase in Sefsol-218 concentration in the S-SNEDDS (Table 4). These results indicated that the S_mix had little impact on the droplet size of the PCEO–S-SNEDDS while Sefsol-218 had a greater impact on the droplet size of the PCEO–S-SNEDDS. Overall, the largest droplet size was observed in formulation F9 (52.45 ± 4.17 nm) which was probably due to the presence of the highest amount of Sefsol-218 (25% w/w) in F9. However, the droplet size of the PCEO–S-SNEDDS F1 was observed as the lowest (7.53 ± 0.56 nm), which was probably due to the presence of the lowest amount of Sefsol-218 (5% w/w) in F1.

Table 4 Various physicochemical parameters of the PCEO–S-SNEDDS (F1–F9)^a

Code	Characterization parameters
	Δdm ± SD (nm)	PI	ZP (mV)	η ± SD (cp)	RI ± SD	% T ± SD
a Mean droplet diameter (Δdm), polydispersity index (PI), viscosity (η), % transmittance (% T), zeta potential (ZP), refractive index (RI), standard deviation (SD).
F1	7.53 ± 0.56	0.119	−28.98	12.80 ± 1.09	1.337 ± 0.06	98.7 ± 0.3
F2	8.81 ± 0.84	0.187	−27.56	13.42 ± 1.21	1.338 ± 0.05	98.4 ± 0.2
F3	9.35 ± 0.90	0.198	−27.12	14.34 ± 1.41	1.339 ± 0.08	97.5 ± 0.1
F4	10.21 ± 0.95	0.202	−26.38	15.41 ± 1.53	1.341 ± 0.10	97.3 ± 0.4
F5	11.13 ± 0.98	0.210	−26.16	16.32 ± 1.74	1.342 ± 0.12	97.1 ± 0.3
F6	21.62 ± 1.21	0.209	−25.80	26.4 ± 2.12	1.343 ± 0.09	96.7 ± 0.5
F7	33.45 ± 2.42	0.234	−25.64	38.62 ± 3.13	1.344 ± 0.07	96.3 ± 0.2
F8	41.32 ± 3.45	0.242	−25.32	48.76 ± 4.61	1.345 ± 0.08	96.2 ± 0.3
F9	52.45 ± 4.17	0.247	−25.14	61.24 ± 5.21	1.347 ± 0.11	96.0 ± 0.5

---

Measurement of the PI is useful to evaluate the uniformity of the droplets in the developed SNEDDS. PIs of the PCEO–S-SNEDDSs (F1–F9) were recorded as 0.119–0.247 as shown in Table 4. The least PI was observed in the PCEO–S-SNEDDS F1 (0.119), indicating higher uniformity of the droplet size distribution as compared to other PCEO–S-SNEDDSs. However, the highest PI value was observed in the PCEO–S-SNEDDS F9 (0.247). Overall, the PIs were less than 0.3 in all formulations, indicating good uniformity of the droplet size distribution in all formulations.

Measurement of the ZP is important to evaluate the net surface charge and stability of the developed SNEDDS. The values of the ZP for formulations (F1–F9) were recorded as −28.98 to −25.14 mV (Table 4). The lowest ZP value was recorded in the PCEO–S-SNEDDS F1 (−28.98 mV). However, the highest ZP value was recorded in the PCEO–S-SNEDDS F9 (−25.14 mV). The negative ZP values in all PCEO–S-SNEDDSs were possibly due to the presence of negatively charged fatty acid esters in Sefsol-218.³² The experimental ZP values in the magnitude of ±30 mV indicated the stable formation of the PCEO–S-SNEDDS.²⁰

Measurement of viscosity is important to evaluate the flow behavior of the developed SNEDDS. The viscosity for all formulations (F1–F9) was recorded as 12.80–61.24 cp (Table 4). The viscosity results were correlated with formulation compositions and droplet sizes. It was observed that when the Sefsol-218 concentration was kept fixed (5% w/w) and the S_mix concentration was varied from 10–50% w/w (F1–F5), viscosity of the PCEO–S-SNEDDS was found to be changed slightly. The viscosity of these formulations (F1–F5) was found to decrease slightly with respect to the S_mix concentration (Table 4). However, when the amount of the S_mix was fixed and the amount of Sefsol-218 was varied from 10–25% w/w (F6–F9), the viscosity of the PCEO–S-SNEDDS was found to change significantly. The viscosity of these formulations (F6–F9) was found to increase significantly with increasing concentration of Sefsol-218 (Table 4). Moreover, the viscosity of all PCEO–S-SNEDDSs was also found to reduce with a decrease in the droplet size of the formulations. These results indicated that the S_mix had little impact on viscosity of the PCEO–S-SNEDDS. However, Sefsol-218 and the droplet size had a greater impact on viscosity of the PCEO–S-SNEDDS. Overall, the highest and lowest viscosities were also recorded in the PCEO–S-SNEDDS F9 (61.24 ± 5.21 nm) and F1 (12.80 ± 1.09 cp), respectively. Overall, the lower values for viscosity in all PCEO formulations were responsible for the free flowing behavior of these formulations.

Measurement of the RI is useful to evaluate the transparent behavior and type of nanoemulsion. The RIs of the PCEO–S-SNEDDSs (F1–F9) were observed in the range of 1.337–1.347 (Table 4). The highest value for the RI was observed in the PCEO–S-SNEDDS F9 (1.347 ± 0.11). However, the lowest value of the RI was recorded in the PCEO–S-SNEDDS F1 (1.337 ± 0.06). The RIs of all formulations were very close to the RI of water (1.333), indicating the transparent nature and oil-in-water type behavior of all PCEO–S-SNEDDSs.

Measurement of % T is also useful to evaluate the transparent behavior of the developed SNEDDS. The % T of developed formulations (F1–F9) was recorded as 96.0–98.7% (Table 4). The formulation PCEO–S-SNEDDS F1 showed the highest value of % T (98.7 ± 0.3%). While, the formulation PCEO–S-SNEDDS F9 showed the lowest value of % T (96.0 ± 0.5%). These results indicate the transparent behavior of all PCEO–S-SNEDDSs.

TEM evaluation was conducted to investigate the morphological characteristics of the optimized PCEO–S-SNEDDS F1. TEM images of the F1 formulation were taken and evaluated for its surface morphology and droplet size (Fig. 2). The size of all the droplets of F1 was observed within the nanometer range (Fig. 2). The shape of the droplets of F1 was observed as spherical. The spherical shape of the F1 formulation could be due to the presence of Sefsol-218 and Triton-X100.²²

Fig. 2 TEM image of the optimized PCEO–S-SNEDDS (F1) having spherical shaped droplets in the nanometer range.

3.5. Effect of PCEO and its F1 S-SNEDDS on carrageenan-induced pleurisy

As shown in Fig. 3, the exudate volume was found to increase from 0.373 ± 0.012 to 1.27 ± 0.383 ml per rat after 4 h in the pleural cavity of rats induced by carrageenan (P < 0.01). In addition, the number of neutrophils were found to increase from 2.71 ± 0.098 to 62.41 ± 3.35 × 10⁶ cells per rat (P < 0.01). Pretreatment with PCEO and the F1 formulation showed a significant reduction in the exudate volume in a dose dependent manner which indicated that PCEO has an anti-inflammatory effect while the F1 formulation was highly comparable with standard indomethacin treatment (Fig. 3). Moreover, the number of polymorphonuclear (PMN) cells were also reduced significantly by the F1 formulation in a dose dependent manner (P < 0.01).

Fig. 3 Effect of PCEO and the optimized PCEO–S-SNEDDS (F1) on carrageenan-induced pleurisy in rats; values are the mean ± SEM of 6 rats in each group; P values: * <0.05, ** <0.01, and *** <0.001 compared with the toxic control (carrageenan).

In this study, the results showed that PCEO and its S-SNEDDS formulation could mitigate the carrageenan-induced lung inflammation response. Further studies showed that PCEO and its S-SNEDDS formulation could decrease the degree of lung inflammation in a dose dependent manner in rats. This observation was probably due to a reduction of PMN cell infiltration and release of inflammatory factors. Carrageenan in the plural cavity elicited an acute inflammatory reaction characterized by accumulation of the pleural exudate with a large amount of PMN cell infiltration. Neutrophils were recruited to the site of aggregation and can be highly activated by a wide array of ligands. The results showed that PCEO and the S-SNEDDS formulation could attenuate the number of polymorphonuclear cells and the exudate volume. These results suggest that PCEO and its S-SNEDDS formulation could inhibit carrageenan-induced pleurisy in rats.^33,34 Neutrophils are known to adhere to the endothelial layer which could be activated by mediators released locally.³³ The pathological changes in the rat lung were also examined. In comparison to rats treated with vehicle, the rats treated with carrageenan showed pathological changes in the lungs including inflammatory cell infiltration and local edema.

3.6. MPO level of pleural exudates

Furthermore, we evaluated the MPO activity in this work. MPO is an indicator of polymorphonuclear leukocyte accumulation in pleural exudates. The MPO level was measured with the help of a colorimetric, commercial kit. As shown in Fig. 4, the level of MPO was found to increase from 36.71 ± 1.0 to 260.08 ± 9.13 U l⁻¹ in pleural exudates of carrageenan-induced rats (P < 0.01). The result showed that PCEO and its S-SNEDDS formulation could decrease the MPO level to 210.45 ± 5.55, 196.88 ± 3.96 and 176.80 ± 3.39 in a dose dependent manner. The indomethacin group showed potent inhibition of the MPO level. These investigations further confirm the anti-inflammatory effect of PCEO and its S-SNEDDS formulation. MPO, the principal enzyme released following PMN cell stimulation is used as a biomarker for measuring acute inflammation.³⁵ MPO may lead to tissue damage during inflammation.³⁶ The results showed that PCEO and its S-SNEDDS formulation inhibit MPO levels in pleural exudates of carrageenan treated rats in a dose dependent manner. Acute inflammation in rats results in a significant infiltration of inflammatory cells in the lung tissue. Our results demonstrated that treatment with PCEO and its S-SNEDDS formulation, along with the standard drug indomethacin, reduced infiltration of these inflammatory cells.

Fig. 4 Effect of PCEO and the optimized PCEO–S-SNEDDS (F1) on nitric oxide and MPO levels in rats; values are the mean ± SEM of 6 rats in each group. P values: * <0.05, ** <0.01, and *** <0.001 compared with the toxic control (carrageenan).

3.7. Cytokine levels in the lungs

As shown in Fig. 5, the levels of TNF-α and IL-1β were found to increase in the lungs of carrageenan-induced rats (P < 0.01). PCEO and its S-SNEDDS formulation could significantly decrease the release of TNF-α and IL-1β in a dose-dependent manner (P < 0.05, P < 0.01). Inflammation is mediated by a variety of soluble factors, including a group of secreted polypeptides known as cytokines.³⁷ Cytokine activation has been reported as the key process in an inflammatory reaction.³⁸ The release of cytokines can induce chemotaxis to attract migrating granulocytes or leukocytes which further release cytokines and inflammatory mediators.³⁷ TNF-α, IL-β and IL-6 are associated with lung inflammation in animal models.^38–41 The activated neutrophils in the lung are known to release pro-inflammatory cytokines upon endotoxin administration.³⁴ This study demonstrated that PCEO and its S-SNEDDS formulation significantly attenuated the levels of TNF-α IL 6 and IL-1β in the lungs of carrageenan-injected rats leading to an anti-inflammatory effect. Therefore, the anti-inflammatory action of PCEO may be due to attenuation of chemotaxis and inhibition of TNF-α and IL-1β in a dose dependent manner.

Fig. 5 Effect of PCEO and the optimized PCEO–S-SNEDDS (F1) on cytokine levels in carrageenan-induced pleurisy in rats; values are the mean ± SEM of 6 rats in each group. P values: * <0.05, ** <0.01, and *** <0.001 compared with the toxic control (carrageenan).

3.8. mRNA expression and protein expression of iNOS and COX-2 in the lungs

A significant increase in iNOS and COX-2 mRNA and protein expression 4 h after carrageenan injection was detected in the lungs obtained from rats subjected to carrageenan-induced pleurisy as shown in (Fig. 6). The pretreatment with PCEO and its S-SNEDDS formulation showed dose dependent inhibition of iNOS and COX-2 expression following carrageenan induced pleurisy. This further confirms the anti-inflammatory potential of PCEO and its S-SNEDDS formulation. NO is very important in the regulation of vascular permeability and cell migration induced by pro-inflammatory agents such as carrageenan. To further elucidate the mechanism underlying the preventive action of PCEO and its S-SNEDDS formulation on lung tissue, we examined the mRNA and protein expression levels of inflammatory mediators in lung tissue using RT-PCR and western blot analysis. The mRNA levels of iNOS and COX-2 were significantly increased in the carrageenan injected rats, whereas treatment with PCEO and its S-SNEDDS formulation attenuated the mRNA and protein expression of these pro-inflammatory mediators. The activation of iNOS catalyzes the formation of a large amount of NO, which plays a key role in the pathogenesis of a variety of inflammatory diseases.⁴²

Fig. 6 mRNA expression and protein expression of iNOS and COX-2 by PCEO and the optimized PCEO–S-SNEDDS (F1) in lungs; values are the mean ± SEM of 6 rats in each group. P values: * <0.05, ** <0.01, and *** <0.001 compared with the toxic control (carrageenan).

Inhibitors of iNOS activity reduce the increase of carrageenan induced inflammation.⁴³ Induction of the COX-2 gene is responsible for the increase of PGE2, a key mediator of inflammation, and inhibitors of COX-2 may attenuate inflammation in the lungs. We further demonstrate here that the increase in the expression levels of iNOS and COX-2 caused by carrageenan in the lung tissue is attenuated by PCEO and its S-SNEDDS formulation, deregulating PGE2 and inhibiting its synthesis, leading to the attenuation of the classical signs of inflammation including redness, swelling and pain. The results of the NO level analysis clearly showed that there is up-regulation of NO in carrageenan induced rats while PCEO and the S-SNEDDS formulation attenuate NO synthesis. These results clearly indicate that PCEO and its S-SNEDDS formulation have anti-inflammatory activity.

3.9. Histological analysis of lung tissues

As compared to the vehicle group (Fig. 7A), the lung tissues treated with carrageenan showed pathological changes including inflammatory cell infiltration and local edema (Fig. 7B). The results showed that PCEO and its S-SNEDDS formulation could decrease inflammation in the lungs of rats treated with carrageenan (Fig. 7C–F). The histological observation further substantiates our findings. Therefore, PCEO and the S-SNEDDS formulation were able to mitigate acute inflammation in a dose dependent manner in carrageenan injected rats. There are reports indicating that some monoterpenes from essential oils are strong inhibitors of certain inflammatory mediators such as prostaglandins and other arachidonic acid metabolites. The presented GC-MS investigation revealed that monoterpenes, e.g. sabinene, 4-terpineol, γ-terpinene and α-thujene, were the major components in PCEO which could be responsible for the observed activities.^44,45

Fig. 7 Effect of PCEO and the PCEO–S-SNEDDS (F1) on lung inflammation (HE ×100); a lung section of a rat treated with normal saline (A); a lung section of a rat treated with carrageenan (B); a lung section of a rat treated with carrageenan and PCEO (C and D) and a lung section treated with carrageenan and the S-SNEDDS (F1) (E and F).

4. Conclusions

In this work, various S-SNEDDSs of PCEO were prepared, characterized and investigated for their anti-inflammatory effects as compared to pure PCEO and standard indomethacin treatment. Based on the lowest droplet size (7.53 nm), lowest PI (0.119), lowest viscosity (12.80 cp), optimal values of ZP (−28.98 mV) and RI (1.337) and highest % T (98.7%), the S-SNEDDS F1 containing 5% w/w of PCEO, 5% w/w of Sefsol-218, 25% w/w of Triton-X100, 25% w/w of Transcutol-HP and 40% w/w of water was selected for biological investigation in a rat model. The results indicated that pretreatment with PCEO and the S-SNEDDS F1 reduced the exudate volume and PMN cell number significantly. Moreover, the levels of MPO, nitric oxide and proinflammatory cytokines (TNF-α and IL-β) were also reduced by PCEO and the S-SNEDDS F1 and this observation was also supported by histological observation. The results of the S-SNEDDS F1 were superior compared to PCEO alone even at significantly lower doses. The results obtained in this work indicated that the prepared S-SNEDDS could be successfully administered orally to enhance the therapeutic efficacy of PCEO.

5. Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgements

The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for funding the work through the research group no. RGP-139.

References

1. M. G. Miguel, Molecules, 2000, 15, 9252–9287.
2. F. Bakkali, S. Averbeck, D. Averbeck and M. Idaomar, Food Chem. Toxicol., 2008, 46, 446–475.
3. R. Bos, O. Kayser and W. J. Quax, J. Essent. Oil Res., 2007, 19, 14–17.
4. B. Adorjan, Flavour Fragrance J., 2010, 1, 407–426.
5. T. Morikawa, H. Matsuda, I. Yamaguchi, Y. Pongpiriyadacha and M. Yoshikawa, Planta Med., 2004, 70, 152–159.
6. M. AlSaid, M. Raish, M. Al-Sobaihani, M. Al-Yahya and S. Rafatullah, Int. J. Biotechnol. Wellness Ind., 2014, 2, 173–181.
7. R. J. Folz, A. M. Abushamaa and H. B. Suliman, J. Clin. Invest., 1999, 103, 1055–1066.
8. W. L. Lee and G. P. Downey, Curr. Opin. Crit. Care, 2001, 7, 1–7.
9. E. M. Choi and J. K. Hwang, J. Ethnopharmacol., 2003, 89, 171–175.
10. F. F. Perazzo, I. V. Rodrigues, E. L. Maistro, S. M. Souza, N. P. D. Nanaykkara, J. K. Bastos, J. C. T. Carvalho and G. H. B. de Souza, Pharmacogn. J., 2013, 5, 13–16.
11. S. Baboota, F. Shakeel, A. Ahuja, J. Ali and S. Shafiq, Acta Pharm., 2007, 57, 315–332.
12. F. Shakeel, S. Baboota, A. Ahuja, J. Ali, M. Aqil and S. Shafiq, AAPS PharmSciTech, 2007, 8, E104.
13. F. Shakeel, S. Baboota, A. Ahuja, J. Ali and S. Shafiq, Pharmazie, 2009, 64, 258–259.
14. F. Shakeel, W. Ramadan, H. M. Gargum and R. Singh, Sci. Pharm., 2010, 78, 47–56.
15. F. Shakeel, W. Ramadan, M. S. Faisal, M. Rizwan, M. Faiyazuddin, G. Mustafa and S. Shafiq, Curr. Nanosci., 2010, 6, 184–198.
16. F. Shakeel, S. Shafiq, N. Haq, F. K. Alanazi and I. A. Alsarra, Expert Opin. Drug Delivery, 2012, 9, 953–974.
17. N. C. Obitte, K. C. Ofokansi and F. C. Kenechukwu, J. Pharm., 2014, 2014, E340486.
18. N. Thomas, R. Holm, A. Mullertz and T. Rades, J. Controlled Release, 2012, 160, 25–32.
19. A. M. Villar, B. C. Naveros, A. C. Campmany, M. A. Trenchs, C. B. Rocabert and L. H. Bellowa, Int. J. Pharm., 2012, 431, 161–175.
20. F. Shakeel, N. Haq, M. El-Badry, F. K. Alanazi and I. A. Alsarra, J. Mol. Liq., 2013, 180, 89–94.
21. M. AlSaid, R. Mothana, M. Raish, M. Al-Sobaihani, M. Al-Yahya, A. Ahmad, M. Al-Dosari and S. Rafatullah, BioMed Res. Int., 2015, 2015, E359358.
22. S. Shafiq, F. Shakeel, S. Talegaonkar, F. J. Ahmad, R. K. Khar and M. Ali, Eur. J. Pharm. Biopharm., 2007, 66, 227–243.
23. L. C. Green, D. A. Wagner, J. Glogowski, P. L. Skipper, J. S. Wishnok and S. R. Tannebaum, Anal. Biochem., 1982, 126, 131–138.
24. A. H. Ding, C. F. Nathan and D. J. Stuehr, J. Immunol., 1988, 141, 2407–2412.
25. M. M. Campos, G. E. de Souza, N. D. Ricci, J. L. Pesquero, M. M. Teixeira and J. B. Calixto, Br. J. Pharmacol., 2002, 135, 1107–1114.
26. P. Chomczynski and N. Sacchi, Nat. Protoc., 2006, 1, 581–585.
27. W. W. Pfaffl, Nucleic Acids Res., 2001, 29, E45.
28. M. W. Pfaffl, G. W. Horgan and L. Dempfle, Nucleic Acids Res., 2002, 30, E36.
29. H. Towbin, T. Staehelin and J. Gordon, J. Clin. Chem. Clin. Biochem., 1989, 27, 495–501.
30. H. Towbin, T. Staehelin and T. Gordon, Biotechnology, 1992, 24, 145–149.
31. F. Shakeel, N. Haq, F. K. Alanazi and I. A. Alsarra, Pharm. Dev. Technol., 2016, 21, 131–139.
32. M. K. Anwer, S. Jamil, E. O. Ibnouf and F. Shakeel, J. Oleo Sci., 2014, 63, 347–354.
33. A. B. Lentsch and P. A. Ward, Respir. Physiol., 2001, 128, 17–22.
34. D. N. Severino, R. L. Pereira, I. Knysak, D. M. Candido and F. H. Kwasniewski, Inflammation, 2009, 32, 57–64.
35. R. J. Goiffon, S. C. Martinez and D. A. Piwnica-Worms, Nat. Commun., 2015, 6, 6271.
36. A. J. McCabe, M. Dowhy, B. A. Holm and P. L. Glick, J. Pediatr. Surg., 2001, 36, 334–347.
37. C. A. Feghali and T. M. Wright, Front. Biosci., 1997, 2, 12–26.
38. I. Utsunomiya, M. Ito and S. Ohishi, Cytokines, 1998, 10, 956–963.
39. J. C. Leemans, M. J. Vervoordeldonk, S. Florquin, K. P. van Kessel and T. van der Poll, Am. J. Respir. Crit. Care Med., 2002, 165, 1445–1450.
40. J. A. Frank, J. F. Pittet, C. Wray and M. A. Matthay, Thorax, 2008, 63, 147–153.
41. M. T. Ganter and J. Roux, et al., Circ. Res., 2008, 102, 804–812.
42. R. Korhonen, A. Lahti, H. Kankaanranta and E. Moilanen, Curr. Drug Targets: Inflammation Allergy, 2005, 4, 471–479.
43. S. Cuzzocrea, B. Zingarelli, P. Hake, A. L. Salzman and C. Szabo, Free Radical Biol. Med., 1998, 24, 450–459.
44. T. Marchi, G. Magarotto, G. Della Valle and P. G. Nardi, Med. Lav., 1992, 83, 318–325.
45. U. R. Juergens, M. Stober, L. Schmidt-Schilling, T. Kleuver and H. Vetter, Eur. J. Med. Res., 1998, 3, 407–412.

---