Effect of high-pressure homogenization on formulation of TPGS loaded nanoemulsion of rutin – pharmacodynamic and antioxidant studies

Abstract

Polyphenolic bioflavonoid, Rutin possesses wide range of pharmacological activities. However, it shows poor bioavailability when administered orally. The aim of this study was to formulate and compare the potential of nanoemulsions for the solubility enhancement of rutin (RU) by using different techniques. RU-loaded nanoemulsions were prepared by spontaneous emulsification method and high-pressure homogenization (HPH) technique using sefsol 218 and tocopheryl polyethylene glycol 1000 succinate (TPGS) (1:1), solutol HS15 andtranscutol P as oil phase, surfactant and co-surfactant, respectively. The prepared formulations were compared for various parameters like droplet size, percentage transmittance, zeta potential, viscosity, refractive index and in vitro release. The HPH nanoemulsions showed smaller droplet size and increased in vitro release when compared to nanoemulsions prepared by spontaneous emulsification method. The optimized formulation showed spherical globules with average globule diameter of 18 nm and zeta potential of −41 mV. Cumulative percentage drug released obtained for RU, PF6 (spontaneous emulsification formulation F6) and HF6 (HPH formulation F6) were 41.5 ± 0.04%, 49.5 ± 0.06% and 94.8 ± 0.03%, respectively, after 6 h. The permeability of RU from HF6 was found to be ≈4.6 times higher than RU suspension during ex vivo everted gut sac studies. Antioxidant activity was determined by using DPPH assay and reducing power assay method. Results showed a high scavenging efficiency toward DPPH radicals by HF6. Anti-inflammatory effect of RU as determined by carrageenan-induced rat paw edema method was found to be higher (75.2 ± 4.8%) when compared to RU suspension (46.56 ± 3.5%). It can be inferred that TPGS-loaded nanoemulsion of RU serve as an effective tool in increasing solubility and permeability of RU.

• DPPH assay
• everted gut sac method
• high-pressure homogenization
• nanoemulsion
• rutin

Introduction

Bioflavonoids belong to group of natural substances widely distributed in plant kingdom in fruits, vegetables, grains, bark, roots, stems, flowers, tea and wine (Nijveldt et al., 2001). More than 4000 varieties of flavonoids have been identified so far. Chemically, they are benzo-γ-pyrone derivatives and classified into various groups depending on the chemical structure and properties. Their name has been derived from the Latin word flavus meaning yellow, and many of these compounds are responsible for the coloration of flowers, yolks or leaves in autumn (Williams et al., 2004). They have been known for their beneficial effects on health due to their antioxidant and free radical scavenging properties (Williams et al., 2004).

Rutin (2-(3,4-dihydroxyphenyl)-4,5-dihydroxy-3-[3,4,5-trihydroxy-6-[(3,4,5-trihydroxy-6 methyl-oxan-2-yl)oxy-methyl]oxan-2-yl]oxy-chromen-7-one) also known as quercetin-3-rutinoside or sophorin is a flavonol glycoside comprising of the flavonol quercetin and the disaccharide rutinose (Calabro et al., 2005; Sharma et al., 2013). Rutin (RU) is a strong antioxidant molecule and has significant scavenging properties on oxidizing species such as OH radical, superoxide radical and peroxyl radical. Therefore, it shows several pharmacological properties including antiallergic, anti-inflammatory and vasoactive, antitumor, antibacterial, antiviral and antiprotozoal properties. Moreover, it has also been reported that RU has other therapeutic effects such as hypolipidemic, anticarcinogenic and antidiabetic effects (Sharma et al., 2013). RU offers an advantage over myricetin, quercetagenin and other flavonoids, which on some occasions behave as prooxidant agents and catalyze oxygen radical production. RU is also advantageous over aglycones whose use as pharmaceutical agents is restricted due to their mutagenic and cytotoxic activity. The major disadvantage associated with this molecule is poor solubility in aqueous media, being the reason for its poor bioavailability (Sharma et al., 2013).

In order to overcome the poor solubility problem of RU, various approaches such as complexation using cyclodextrin and phospholipids has been researched and documented (Sri et al., 2007; Jain et al. 2012). Over the past few decades, use of lipid-based approaches have found significant acceptance in the market for improving the aqueous solubility thereby increasing the oral bioavailability (Khan et al., 2012). These formulations disperse or solubilize the drug in the dosage form and facilitate emulsification in gastrointestinal tract thereby increasing absorption. In the past, nanoemulsions have been used for improving the solubility of poorly soluble drugs. Nanoemulsions have several advantages over conventional emulsions due their droplet size below 200 nm, large surface area to volume ratio and thermodynamic behavior which make them suitable for several applications in personal care products, cosmetic and health care (Kotta et al., 2012; Sharma et al., 2012. They are produced by spontaneous emulsification, phase inversion and high-pressure homogenization (HPH) techniques.

The aim of this work was to prepare a tocopheryl polyethylene glycol 1000 succinate (TPGS) loaded RU nanoemulsion of small droplet size and low polydispersity index displaying increased absorption and greater bioavailability.

Materials and methods

Materials

RU hydrate and TPGS were purchased from Sigma Aldrich Pvt Ltd (Bangalore, India). Sefsol was purchased from Nikko Chemicals (Tokyo, Japan). Solutol HS15 was obtained as a gift sample from Signet Chemicals (Mumbai, India). Transcutol® HP (diethylene glycol monoethyl ether) was gifted by Gattefosse (Saint Priest, Cedex, France). Water was obtained from Milli-Q-water purification system (Millipore, MA). All other chemicals and reagents were of analytical grade and procured from Merck (Mumbai, India) and S.D. Fine Chem. (Mumbai, India).

Analytical methodology

RU was assayed by reversed-phase high-performance liquid chromatography (RP-HPLC) method using a HPLC system (Shimadzu , Kyoto, Japan). The analysis was carried out on a LiChrospher® C18 (150 mm × 4.6 mm i.d., 5 µm particle size; Merck) column. The mobile phase used was methanol–water (60:40). The flow rate was 1 ml/min and the retention time was 3.12 min. The detection was performed at 360 nm. The eluents were filtered (pore size, 0.45 µm) before use and then degassed by sonication in an ultrasonic bath. The assays were performed at ambient temperature (25 ± 1 °C). Class VP 5.03 version software (Shimadzu, Kyoto, Japan) was used to process the chromatograms (Vesna et al., 2007).

Formulation development and optimization

Screening of components

High drug loading and stability are important criteria for screening of components for formulation of nanoemulsions. Phase solubility/miscibility studies were done to determine the most suitable oil, surfactant and cosurfactant for the preparation of nanoemulsions of RU. Three milliliters of selected oils [olive oil, TPGS, capmul 90, propylene glycol monocaprylic ester (sefsol 218), sefsol + TPGS (1:1)] and surfactants [tween 80, labrasol, solutol HS15 and cremophor RH 40] were taken in small vials (5.0 ml capacity) and excess amount of drug was added in the oils and surfactants and kept in biological shaker (Nirmal International, Delhi, India) for 72 h at a constant temperature (25 ± 1.0 °C) to reach to an equilibrium (Sharma et al., 2012). The equilibrated samples were removed from the shaker and centrifuged at 3000 rpm for 15 min. The supernatant was taken and filtered through a 0.45-μm membrane filter and concentration of drug was determined by taking absorbance using UV double beam spectrophotometer at 258 nm after dilution with methanol. Further miscibility of surfactant with the oil showing maximum solubility was also done as one of selection parameter. Co-surfactant was also selected on the basis of miscibility studies. Observations were done visually for miscibility. The mixtures which were clear/transparent in the 1:1 ratio were considered for further studies.

Spontaneous emulsification method

The pseudoternary phase diagrams were constructed using the selected oil, S_mix (surfactant and co-surfactant) and double-distilled water using aqueous phase titration method. S_mix were used in different volume ratios (1:0, 1:1, 2:1, 3:1, 4:1, 5:1) with increasing amount of surfactant with respect to co-surfactant. Sixteen different combinations of oil and S_mix (1:9, 1:8, 1:7, 1:6, 1:5 1:4, 1:3.5, 1:3, 3:7, 1:2, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1) were made so that maximum ratio could be covered for the study to delineate the boundaries of the phases formed precisely in the phase diagrams. Slow titration with the aqueous phase was done for each weight ratio of oil and S_mix, and visual observations were made for transparent and easily flowable oil-in-water nanoemulsions. Samples were vigorously shaken by vortexing after each addition of titrating water and left for 24 h to attain equilibrium and to screen the metastable compositions. After constructing the phase diagram, various formulations compositions were selected from each phase diagram plotted for different S_mix ratios so that the oil concentration was such that it dissolved the drug easily and surfactant concentration was minimum. These formulations were then prepared by dissolving 10 mg/ml of RU and subjected to physical stability tests [centrifugation stress (5000 rpm, 30 min), heating cooling stress (0 and 45 °C, eight cycles) and freeze-thawing stress (−21 and 25 °C, ≥48 h)] (Baboota et al., 2007).

Homogenized nanoemulsion

Selected formulations produced by spontaneous emulsification (SE) method which passed the stability studies as discussed in above section were also subjected to homogenization where pressure and number of cycles were varied and their effect on droplet size, percentage transmittance (%TR), zeta potential, viscosity, refractive index (RI) and in vitro release was determined. The homogenization pressure and number of cycles were optimized by giving single treatments cycle using different pressure which ranged between 1000 and 2500 bar (100–250 MPa). Optimized formulations was further stressed with zero to five cyclic treatment at optimum homogenization pressure (STANSTED® pressure Cell Homogeniser, Harlow, Essex, UK) to see the effects on nanosizing (Mustafa et al., 2012; Ledet et al., 2013).

The possible differences between the nanoemulsion produced by spontaneous emulsification and homogenization technique were assessed by using various quantitative parameters like droplet size, percentage transmittance (%TR), zeta potential, viscosity, RI and in vitro release.

Characterization of nanoemulsions

Droplet size and zeta potential

Droplet size of the nanoemulsions was determined by photon correlation spectroscopy using Zetasizer 1000 HS (Malvern Instruments, Worcestershire, UK) which is based on the principle of dynamic light scattering. The formulations were diluted with distilled water and filtered through 0.22-µm membrane filter in order to eliminate multiscattering phenomena and experimental errors. The measurements were performed using a He–Ne laser at 633 nm by using Avalanche photo diode detector (Hawthorne, CA). Light scattering was monitored at 25 °C at a 90° angle. Droplet size distribution studies were performed at RI of 1.5 because the RI for all formulation was in this range. Zeta potential was measured by applying an electric field across the dispersion. Particles within the dispersion with a zeta potential migrated toward the electrode of opposite charge with a velocity proportional to the magnitude of the zeta potential. The velocity was measured by using M3PALS (phase analysis light scattering).

Viscosity and RI determination

Viscosity of nanoemulsion was determined by using Brookfield DV III ultra V6.0 RV cone and plate rheometer (Brookfield Engineering Laboratories, Middleboro, MA). The optimized parameters used were: sample size/wt: 0.5 g; speed: 30 rpm; data interval: 1.0; loop start: 1; wait time: 30 min; temperature: 25 ± 0.3 °C; shear rate: 60 s⁻¹. RI was determined for different nanoemulsion formulations by using Abbe’s refractometer (Nirmal International, Delhi, India) at 25 °C in triplicate.

Percentage Transmittance

The percentage transmittance of nanoemulsions was determined spectrophotometrically by using UV–Vis spectrophotometer (Shimadzu, Kyoto, Japan). One milliliter of the formulation was diluted 500 times using methanol and analyzed at 630 nm using methanol as blank (Table 3).

Surface morphology by TEM

Morphology and structure of the nanoemulsion were studied using Morgagni 268D transmission electron microscopy (TEM) (FEI, Hillsbro, OR) operating at 70 kV and capable of point to point resolution. Combination of bright field imaging at increasing magnification and diffraction modes was used to reveal the form and size of nanoemulsion droplets. In order to perform the TEM observations, a drop of nanoemulsion was applied on carbon-coated grid with 2% phosphotungstic acid and was left for 30 s. The dried coated grid was taken on a slide and covered with a cover slip. The slide was observed under the electron microscope (Baboota et al., 2007).

In vitro release studies

The in vitro release studies were done to compare the release of RU from nanoemulsions prepared by SE and HPH and RU suspension prepared with 1% w/v sodium carboxy methyl cellulose. The dialysis bag (MWCO 1200 g/mol, Sigma Aldrich, St. Louis, MO) was treated as per instruction of Sigma Aldrich. One milliliter of NE formulations (10 mg/ml) and suspension (10 mg/ml) was filled in treated dialysis bag which was tied using nylon thread. Integrity of bag was assessed visually. The release studies were performed in 900 ml of phosphate buffer (pH 6.8) using USP apparatus 1 (Basket), at 100 rpm, 37 ± 0.5 °C (Hanson Research SR8 plus, Chatsworth, CA). The dialysis bag was kept inside the basket. Five millliter samples were withdrawn at regular time intervals (10, 20, 30, 60, 120, 180 and 360 min) and aliquot amount of phosphate buffer was replaced in order to maintain sink condition. The samples were analyzed for the drug content using HPLC method.

Ex vivo release studies

Ex vivo release studies were done for the optimized nanoemulsion formulation in comparison with the RU suspension. All animal experiments were carried out after approval of the protocol by Jamia Hamdard, Institutional Animal Ethics Committee, New Delhi and their guidelines were adhered for the whole study. For carrying out the experiment, rats were sacrificed and duodenum was taken out, cut into parts and washed with saline to remove any excretory product present in the duodenum by flushing. One milliliter of NE (nanoemulsion) formulation (10 mg/ml) and suspension (10 mg/ml) were filled in duodenum separately which were then tied properly using nylon thread and kept inside the basket. The release study was performed in 100 ml of Tyrode’s solution. The gut sac bath was surrounded by an outer water jacket to control the temperature of the bath at 37 ± 5 °C. Two milliliter samples were withdrawn at regular time intervals (10, 20, 30, 60, 120, 180, 240, 300 and 360 min) and aliquot amount of buffer was replaced in order to maintain sink condition. The samples were analyzed for the drug content using HPLC method.

Ex vivo everted gut sac method

An improved everted gut sac model was used as an in vitro tool to study the permeation of rutin. For this study, male Wistar rats (200–250 g), after being fasted for 10–12 h with free access to tap water, were anesthetized by excessive ether inhalation. Following a midline incision in the abdomen, the small intestine was excised at two positions, at 4 cm distal to the stomach and at the ileocecal junction. The entire length of the small intestine was carefully removed and, before tissue preparation, placed in Tyrode’s solution continuously aerated with the aid of an electrical aerator. Medial jejuna/duodenum segments (≈4 cm) were used for the permeation studies. These segments were cut and washed six to eight times with Tyrode’s solution, ligated with nylon thread at one end, and carefully everted on the glass rod. The everted gut sac was filled with 1 ml of Tyrode’s solution, ligated and placed inside the conical flask containing 20 ml of the test solution (10 mg/ml of NE and RU suspension) continuously bubbled with atmospheric air at 13–19 bubbles/min separately. The gut sac bath was surrounded by an outer water jacket to control the temperature of the bath at 37 ± 5 °C. The amount of RU permeated across the intestine was determined using HPLC method after predetermined time period (0.5, 1, 1.5, 2 h) (Kumar et al., 2012). Permeability coefficient (P_app) of RU was calculated from mucosal to serosal direction according to the equation: where the dQ/dt is the rate of drug permeation from the tissue, A is the cross-sectional area (≈4.52 cm²) of the tissue and C₀ is the initial RU concentration in the donor compartment at t = 0.

Antioxidant activity

DPPH method

The antioxidant activity of the optimized formulation was compared with ascorbic acid (standard antioxidant) and pure RU suspension using DPPH assay method. This test is based on the free radical scavenging activity of the stable DPPH free radical as described by Braca et al. (2001). Stock solutions (1.0 mg ml⁻¹) in methanol were made for formulation, RU and ascorbic acid. Serial dilutions (1–20 µg/ml) were made for all the samples. One milliliter of the samples was added to 1 ml of MeOH solution of DPPH (0.004% w/v). The absorbance was measured after 30 min at 515 nm. Methanol (95%) was used as blank. Percent inhibition was calculated by using following formula: where A₀ was the absorbance of the control (blank, without extract) and A₁ was the absorbance of the extract or standard. The mean percentage inhibition by each concentration was plotted against the log concentration. The 50% inhibitory dose (IC₅₀ value) was found by interpolation by using graph pad (Prism 6 software, San Diego, CA) and compared with standard.

Reducing power assay

The reducing power of all samples was estimated as per the previously established methods (Athukorala et al., 2006). One milliliter of different concentrations (5.0–100 μg ml⁻¹) of ascorbic acid (as standard antioxidant) in distilled water and sample solutions were mixed with 2.5 ml of 0.2 M phosphate buffer (pH 6.6) and 2.5 ml of 10% potassium ferricyanide [K₃Fe(CN)₆]. The mixture was incubated at 50 °C for 20 min and 2.5 ml of 10% trichloroacetic acid was added, which was then centrifuged at 3000 rpm for 10 min. The upper layer of the solution (2.5 ml) was separated; mixed with distilled water (2.5 ml) and 0.1% ferric chloride (0.5 ml). The absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicated more reducing power. Ascorbic acid was used as a reference standard and phosphate buffer (pH 6.6) was used as blank solution.

In vivo anti-inflammatory study

The protocol to carry out in vivo anti-inflammatory efficacy studies was approved by the Institutional Animal Ethics Committee Jamia Hamdard (New Delhi, India). The committee’s guidelines were followed for the studies. The anti-inflammatory activity of the optimized formulation and RU suspension was evaluated by the carrageenan-induced hind paw edema method by using digital plethysmometer (UGO Basile, Comerio VA, Italy) in Wistar rats of either sex weighing 180–200 g. RU suspension 1% (w/v CMC) and formulation were administered as a single dose of 100 mg/kg body weight, 1 h before the injection of 0.1 ml of carrageenan 1% (w/v) in the subplantar region, i.e. hind paw (Winter et al., 1962). Rats of control group were injected with only the vehicle (CMC 1%). The edema was assessed by measuring paw volume at 0, 1, 2, 3, 4, 5 and 6 h after carrageenan injection. The amount of paw swelling was determined for 6 h and expressed as percent edema relative to the initial hind paw volume. Percent inhibition of edema was calculated for placebo and drug-loaded group with respect to control group using the following formula:

Statistical analysis

Each experiment was conducted in triplicate and data were analyzed using Excel 2007 (Microsoft Office, Microsoft Inc., Redmont, Washington) and expressed as a mean ± standard deviation (SD). Comparison between the differences of means was performed by using paired t test for paired comparisons where p values of 0.05 or less were considered significant.

Results and discussion

Screening of components

Drug loading per formulation is a very critical design factor in the development of nanoemulsion systems for poorly water soluble drugs, which is dependent on the drug solubility in oil phase. Oil represents one of the most important excipient in the nanoemulsions formulation, which can solubilize sufficient amount of the drug and help the nanoemulsion to maintain the drug in solubilized form (Holm et al., 2002). After screening the oils for RU solubility, it was observed that RU exhibited maximum solubility in sefsol 218 and TPGS 1:1 combination (95.76 ± 0.12 mg/ml). Therefore, it was selected as the oil phase for the development of nanoemulsion. Moreover, TPGS is having inherent antioxidant activity which will potentiate the antioxidant activity of RU (Yan et al., 2007). In the nanoemulsion formulations, surfactants are required to lower the interfacial tension to a very small value to aid dispersion process and to provide a flexible film that can readily deform around the droplets. The presence of co-surfactants allows the interfacial film sufficient flexibility to take up different curvatures required to form nanoemulsion over a wide range of composition (Wennerstrom & Olsson, 2009). While selecting surfactants, safety is an important criterion followed by selection of the right blend of low and high HLB (hydrophile lipophile balance) surfactant which leads to the formation of a stable nanoemulsion formulation. Non-ionic surfactants are considered to be less toxic than ionic surfactants. Therefore, solubility and miscibility of sefsol 218 + TPGS was performed with different surfactants and co-surfactants (Table 1). On the basis of solubility/miscibility study solutol HS15 and transcutol P were selected as surfactant and co-surfactant respectively.

Table 1. Solubility of RU in oil and surfactants and miscibility with surfactants and co-surfactants.

Solubility of RU in oils		Solubility of RU in surfactants		Miscibility of sefsol + TPGS in surfactant/cosurfactant
Oil	Solubility (mg/ml) ± SD (n = 3)	Surfactant	Solubility (mg/ml) ± SD (n = 3)	Surfactant/cosurfactant	Inference
Sefsol 218	46.46 ± 0.12	Solutol HS15	115.45 ± 0.40	Solutol HS15	Clear
Olive oil	14.41 ± 0.05	Tween 80	75.64 ± 0.31	Transcutol P	Clear
TPGS	48.18 ± 0.06	Labrasol	81.68 ± 0.06	Plurol Oleique	Turbid
Sefsol + TPGS	95.76 ± 0.12	Cremophor RH 40	52.18 ± 3.21	PEG 400	Slightly turbid
Capmul 90	10.78 ± 0.01

Preparation of nanoemulsions

Spontaneous emulsification method

Nanoemulsions were prepared with sefsol + TPGS (1:1), Solutol HS15 and transcutol P as oil, surfactant and cosurfactant respectively using aqueous phase titration method. The phase behavior of nanoemulsion system was studied with the aid of pseudoternary phase diagrams in which each corner of the diagram represents 100% of that particular component. The amount of aqueous phase added was varied to produce a water concentration in the range of 5–95% of total volume at around 5% intervals. Slow titration with the aqueous phase was performed for each combination of oil and S_mix, separately. Care was taken to ensure that observations were not made on metastable system. The existence of nanoemulsion region whether large or small depends on the capability of that particular surfactant or surfactant cosurfactant mixture to solubilize the oil phase (Baboota et al., 2007). The area of nanoemulsion isotropic region changed slightly as the ratio of surfactant in S_mix was increased. In the phase diagrams, the existence of large or small nanoemulsion region depends on the capability of the particular S_mix to solubilize the oil phase. The extent of solubilization results in a greater area with the formation of more clear and homogenous solution. In Figure 1, the S_mix ratio 1:1 (Figure 1A) had a low nanoemulsion area. Thus, oil phase was solubilized to a lesser extent implying that the S_mix was not able to reduce the interfacial tension of the oil droplets to sufficiently low level and thus was not able to reduce the free energy of the system to ultra low level desired to produce nanoemulsions. As the surfactant concentration was increased in S_mix ratio 2:1 (Figure 1B) a higher nanoemulsion region was observed, perhaps because of further reduction of interfacial tension, increased the fluidity of the interface, which resulted in increased entropy of system. As the surfactant concentration was further increased to 3:1 and 4:1 (Figure 1C and D), nanoemulsion region further increased as compared to S_mix ratio 1:1 and 2:1 and was found to be maximum in case of S_mix ratio 4:1. When S_mix ratio 5:1 was studied, nanoemulsion region decreased slightly as compared to S_mix 4:1 which might be due to insufficient amount of co-surfactant for emulsification and required HLB value was not obtained. No nanoemulsion regions were found in S_mix ratios 1:2 and 1:3. From phase diagram having maximum nanoemulsions area, fixed concentration of oil and different concentration of surfactant and co-surfactant, which solubilized the drug, were selected.

Figure 1. Pseudoternary phase diagrams showing nanoemulsion area (a–f). (a) Smix ratio (1:1); (b) Smix ratio (2:1); (c) Smix ratio (3:1); (d) S_mix ratio (4:1); (e) S_mix ratio (5:1); (f) S_mix ratio (1:0).

Physical stability studies

Nanoemulsions are considered to be kinetically stable systems which are formed at a particular concentration of oil, surfactant/co-surfactant and water, with no phase separation, creaming or cracking. Different formulations were selected from phase diagrams and subjected to different stress stability testing like heating cooling cycle, centrifugation and freeze thaw cycles (Table 2). During physical stability testing some formulations became turbid and in some phase separation occurred. One reason of this instability in nanoemulsions may be due to the Ostwald ripening in which molecules move as a monomer and coalescence of small droplets takes place, resulting in the formation of large droplets by diffusion processes driven by the gain in surface free energy. The other reason may be that when temperature quench occurs during stress stability study, instability of nanoemulsion occurs due to separation of oil phase and droplet distribution of smaller size is favored by the change in curvature free energy (Wennerstrom & Olsson, 2009). Only those formulations, which showed no phase separation, creaming, cracking, coalescence and phase inversion during stress stability tests, were taken for further studies.

Table 2. NE formulations, composition and physical stability studies.

F. code	Smix ratio	% Oil	% Smix	% Water	H/C	F/T	Cent	Inference
F1		10	37.5	52.5	✓	✓	✓	Passed
F2		10	40	50	✓	✓	✓	Passed
F3		12	45	43	✓	×	−	Failed
F4	4:1	8	35	57	✓	✓	×	Failed
F5		12	42	46	×	−	−	Failed
F6		10	45	45	✓	✓	✓	Passed
F7	3:1	8	35	57	✓	✓	×	Failed
F8		9	40	51	×	−	−	Failed
F9	2:1	8	40	52	✓	×	−	Failed
F10		10	45	45	✓	✓	×	Failed
F11	1:1	8	40	57	✓	✓	×	Failed
F12		8	45	47	×	−	−	Failed

H/C, heating cooling cycle; F/T, freeze thaw, Cent, centrifugation.

High-pressure homogenization

HPH technique was used to attain the desired particle size in nanometric size range. Homogenization at high pressures with multiple cycles has always been considered problematic, especially for the thermo labile molecules due to rise in temperature on increasing pressure. In order to optimize the pressure and number of cycles, minimum treatment cycles with homogenization pressures (100250 MPa) were applied for optimization to produce nanoemulsions with least globule size. It was observed that droplet size was reduced significantly as the pressure was increased from 100 to 200 MPa (Figure 2a). But further increase in pressure from 200 to 250 MPa did not lead to further drop in droplet size. Keeping the pressure constant at 200 MPa, effect of number of cycles on droplet size was observed. Significant decrease in droplet size was seen as homogenization cycles were increased from 1 to 4 (Figure 2b). Further increase in number of cycles did not lead to decrease in droplet size. During HPH, increase in temperature was observed as pressure was increased which might be due to increase in kinetic energy of the system (Schultz et al., 2004). Immediate cooling of the products after passing through HPH was done to prevent drug degradation. This evidence has been supported by a research work recently carried out by Baspinar et al. (2010) where they have assessed the effect of homogenization on prednicarbate stability. The findings concluded that HPH pressure and no of cycles played important role in decreasing the globule size to nanometric size. Although, HPH pressure and number of cycles played an important role in size reduction but the overall composition of formulations is also important which is mainly dependent on Smix and oil ratio. The homogenized formulations were further characterized for various parameters.

Figure 2. Effect of homogenization pressure (a) and homogenization cycles (b) on globule size.

Characterization of formulations

Size and surface

The average droplet size of nanoemulsions was found in the range between 15 and 280 nm (Table 3). The globule size of nanoemulsions prepared by SE and HPH method was measured immediately. The significant changes (p > 0.05) in droplet size from 275 → 45 (F1), 176 → 26 (F2) and 146 → 18 (F6) showed the significance of high energy impact in getting kinetically stabilized droplets. There are chances of agglomeration of droplets after homogenization, which might be due to the insufficient level of stabilizer for the newly created surface. But, we had selected only the stable formulations which contained an adequate amount of stabilizer and passed the physical stability tests to overcome the problem of destabilization. The increase in surface charges 25.27 → 38.23 (F1), 30.38 → 33.61 (F2), 29.57 → 32.07 (F6) is an assertion of increased stability profile of newly created surface. The nanoemulsions prepared by spontaneous emulsification showed non-homogenous distribution with larger size range (146 ± 2.32 to 275 ± 5.18 nm) but the same composition after passing through homogenizer showed significantly smaller size range (18 to 46 ± 2.87 nm) with low polydispersity index. As discussed earlier, both homogenization pressure and number of cycles played a role in decreasing the droplet size in case of homogenized formulations. In case of formulations prepared by SE S_mix ratio played a significant role in reducing the globule size. As concentration of S_mix ratio increased, the droplet size decreased (Sharma et al., 2012). The surface morphology, the droplet size distribution and zeta potential of formulation F6 are shown in Figure 3.

Figure 3. Surface morphology of formulation F6 by TEM. (a) Prepared by SE; (b) Prepared by HPH; (c) Globule size distribution (HF6); and (d) zeta potential determination (HF6).

Table 3. Comparison of parameters between nanoemulsions prepared by SE and HPH method.

	DS (nm) ± SD (n = 3)		ZP ± SD (n = 3)		PDI ± SD (n = 3)		Viscosity ± SD (n = 3)		%TR ± SD (n = 3)		RI ± SD (n = 3)		%DR ± SD (n = 3)
F. code	SEF	HPHF	SEF	HPHF	SEF	HPHF	SEF	HPHF	SEF	HPHF	SEF	HPHF	SEF	HPHF
F1	275 ± 0.01	45 ± 0.01	−22 ± 0.02	−36 ± 0.04	0.431 ± 0.02	0.167 ± 0.06	18.9 ± 0.01	14.6 ± 006	93.78 ± 0.01	98.21 ± 0.02	1.46 ± 0.02	1.45 ± 0.03	41.6 ± 0.04	69.2 ± 0.02
F2	176 ± 0.06	26 ± 0.02	−25 ± 0.01	−28 ± 0.04	0.567 ± 0.01	0.114 ± 0.03	15.6 ± 0.02	12.6 ± 0.04	94.45 ± 0.02	98.76 ± 0.03	1.45 ± 0.05	1.43 ± 0.02	42.5 ± 0.05	79.36 ± 0.01
F6	148 ± 0.03	18 ± 0.01	−16 ± 0.02	−41 ± 0.02	0.682 ± 0.02	0.125 ± 0.04	11.3 ± 0.06	9.34 ± 0.06	95.31 ± 0.03	99.89 ± 0.01	1.43 ± 0.01	1.41 ± 0.08	49.5 ± 0.06	94.8 ± 0.03

SEF, spontaneous emulsification formulation; HPHF, high-pressure homogenization; DS, droplet size; ZP, zeta potential; PDI, polydispersity index; %TR, % transmittance; RI, refractive index; %DR, % drug released in 6h.

Percentage transmittance

The percentage transmittance was determined to have an idea about the size of the droplets as droplet size is directly proportional to the percentage transmittance of the formulation. It also indicates about the isotropicity of formulations. The percentage transmittance approaching 100% indicates isotropy of the formulations. It was observed that transmittance ranged from 93.78 to 95.31 for spontaneous nanoemulsions, whereas 98.2–99.89 for homogenized nanoemulsions (Table 3). The maximum TR was observed for F6 (99.89); however the minimum was for F1 (98.2).

RI and viscosity

RI being an optical property is used to characterize the isotropic nature of the nanoemulsions and basically signifies the chemical interaction among drug and excipients. No significant differences in the RI were observed, so it was concluded that the NE formulations were chemically stable and remained isotropic in nature, thus showing no interaction between excipient and drug. The RI of all the formulations was in the range of 1.41 ± 0.08 to 1.46 ± 0.02 (Table 3). RI is also affected by the size of the oils globules, as the globules size increased the RI increased as observed for the formulation (Table 3). The viscosity of the nanoemulsions is given in Table 3. The viscosity of all nanoemulsion formulations was very low as expected as one of the characteristics. It was observed from Table 3 that viscosity of all the formulations was less than 19 cps. HPH formulations had minimum viscosity, i.e. 9.34 ± 0.06 cps. Results also revealed that the viscosity was directly proportional to the concentration of oil and surfactants used in the formulation.

In vitro release studies

In vitro release studies were performed to compare the release of RU from nanoemulsions prepared from SE (PF1, PF2, PF6) and HPH (HF1, HF2 and HF6). A significant (p < 0.05) increase in percentage drug release was achieved in the case of homogenized nanoemulsions formulations as compared to formulations prepared by SE and RU suspension. The concentration was determined by the extrapolation of calibration curve and graph was plotted between time and percent cumulative release (Figure 4). Maximum release was obtained in case of HF6 (94.8%) which was significantly greater than SE formulation (PF6) which showed only 49.5% release. The reason might be due to decrease in droplet size by HPH. The minimum release was observed in case of F1 formulation, may have been due to bigger globule size resulting in slow release of the drug from nanoemulsion formulation. All the nanoemulsion formulations showed better results as compared to RU suspension (41.5%) because of small droplet size and low polydispersity values. As observed from the graph 85% of the drug was released within 1 h which may have been because of small droplet size leading to high high surface area, thus permitting faster rate of drug release.

Figure 4. Comparative in vitro release profile of nanoemulsions prepared by SE (PF1, PF2 and PF6) and HPH (HF1, HF2 and HF6) and RU suspension in phosphate buffer (pH 6.8).

Ex vivo release studies

Ex vivo studies were done to compare the permeation of RU from optimized formulation (HF6) and RU suspension using a small fragment (≈4 cm) of small intestine of rat as a diffusional barrier. Maximum release of RU (96.75%) was detected for formulation HF6 which was significantly higher (p < 0.05) than the RU suspension (46.6%) after 4 h of study (Figure 5). This was due to size reduction of formulation to nanometric range which has a remarkable enhancing potential on drug release from nanoemulsion. These studies were found in correlation with the in vitro release studies where same pattern was observed. It might be due to availability of larger surface area for partitioning of the drug across the intestinal membrane.

Figure 5. Ex vivo comparative permeation studies of RU and HF6 formulation in rat intestinal membrane in Tyrode's solution.

Ex vivo everted gut sac method

Ex vivo everted gut sac system is an inexpensive and relatively simple technique with considerable potential as an in vitro tool to study the enhancement of drug transport across the small intestine (Barthe et al., 1998) RU loaded NE produced P_app up to 5.68 × 10⁻⁵ cm/s with flux of 0.568 µg/cm²/h at 120 min, whereas drug suspension had P_app 1.24 × 10⁻⁵ cm/s with flux of 0.124 µg/cm²/h (Table 4). The results indicated that RU in suspension form poorly permeated across the intestinal membrane since RU was not available in the soluble form in the donor compartment. But when RU was loaded in the nanoemulsions formulation, the droplet size significantly decreased and RU was easily solubilized in the donor compartment and simply permeated from the intestinal membrane. This indicated the better absorption of RU. The permeability of RU from NE was found to be significantly higher (≈4.6 times) when compared to drug suspension.

Table 4. Comparative permeation profile of RU suspension and HF6 Formulation using everted gut sac model.

	Drug permeated/Area (µg/cm^2) ± SD (n = 3)
	Time (min)
	30	60	90	120	Flux (µg/cm2/h) ± SD (n = 3)	Papp (cm/s)
Drug suspension	2.31 ± 0.01	4.56 ± 0.09	9.7 ± 0.12	12.37 ± 0.15	0.543 ± 0.01	5.43 × 10^−5
Formulation HF6	28.06 ± 0.21	36.07 ± 0.25	49.59 ± 0.15	82.41 ± 0.23	0.124 ± 0.03	1.24 × 10^−5

Antioxidant activity

DPPH activity

The effect of antioxidants on DPPH radical scavenging is believed to be due to their hydrogen donating ability. DPPH is a stable free radical and accepts an electron or hydrogen radical to become a stable diamagnetic molecule. The reduction capability of DPPH radicals was determined by the decrease in its absorbance at 517 nm induced by antioxidants. It was visually noticeable as a discoloration from purple to yellow. Figure 6 illustrates a significant decrease in the concentration of DPPH radical due to the free radical scavenging ability of the RU, nanoemulsion formulation and ascorbic acid (standard antioxidant). The percentage inhibition obtained in case of RU, HF6 and ascorbic acid were 87.21%, 96.48% and 95.3%, respectively. Significant (p < 0.05) antioxidant activity was observed in the formulation HF6 which may be due to combined effect of RU and TPGS and size reduction of the formulation. These results were found in accordance with Baydar et al. (2007) who reported that RU exhibited a high scavenging efficiency toward DPPH radicals. These results clearly indicated that nanoemulsion formulation HF6 had a noticeable effect on scavenging of free radicals.

Figure 6. Comparison of antioxidant activity of Ascorbic acid, RU and HF6 formulation by DPPH assay.

Reducing power assay

The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity. The reducing ability of a compound generally depends on the presence of reductants, which have exhibited antioxidative potential by breaking the free radical chain, donating a hydrogen atom. Many reports have shown that there is a direct correlation between antioxidant activities and reducing power of components of some plants (Yildirim et al., 2001). In this assay, the presence of reductants (i.e. antioxidants) in the methanol extracts of all drugs caused the reduction of the Fe³⁺ ferricyanide complex to the ferrous form. Therefore, the Fe²⁺ was monitored by measuring the formation of Perl’s Prussian blue at 700 nm (Athukorala et al., 2006). Figure 7 shows the reductive capability of RU in comparison with the NE formulation and ascorbic acid (standard antioxidant). Higher absorbance of the reaction mixture indicated greater reducing power. The results showed that the reducing power of RU was in a concentration-dependent manner. At all the concentrations, reductive capability of RU was similar to ascorbic acid. The nanoemulsion formulations showed more reducing power ability than the pure drug. It might be due to the combined effect of RU and TPGS (having inherent antioxidant activity). These results suggested that nanoemulsion formulation HF6 had a remarkable potency to donate electron to reactive free radicals, converting them into more stable non-reactive species and terminating the free radical chain reaction.

Figure 7. Reductive capabilities of ascorbic acid, RU and HF6 formulation.

In vivo anti-inflammatory activity

Rat paw edema, as a standard model of acute inflammation, was used for testing the anti-inflammatory activity of RU. The anti-inflammatory effect of RU suspension was compared with the nanoemulsions formulation HF6. The rat’s left footpad became edematous soon after the subplanter injection of carrageenan 1% and reached maximal volume in the control group in 5 h. In the control group, rat paws showed a slight swelling, and recovered their initial volume after 1 h. The results showed that HF6 formulation has significant (p < 0.05) effect on rat paw edema, compared with the RU and control group. Formulation HF6 exhibited significant (p < 0.05) inhibition activity (75.2 ± 4.8%) determined at 6 h after edema induction. This inhibition was higher than that obtained with RU suspension, (46.56 ± 3.5%) inhibition (Figure 8). These results have been found in accordance with the work done by Selloum et al. where they had compared the anti-inflammatory activity of RU in comparison with aspirin after oral administration. This indicates that formulation HF6 is an effective anti-inflammatory agent and found to be more effective than the pure drug. The enhanced anti-inflammatory effects of nanoemulsion formulation of RU were achieved due to size reduction and increased absorption of RU into the systemic circulation.

Figure 8. Anti-inflammatory study of RU suspension and HF6 formulation.

Conclusion

TPGS-loaded nanoemulsion of RU was successfully formulated by HPH method. The optimized formulation showed significant improvement in solubility, in vitro release and ex vivo permeation as compared to RU suspension. Surface morphology studies demonstrated the spherical shape of the globules. The permeability of RU from NE was found to be significantly higher (≈4.6 times) than the RU suspension during ex vivo everted gut sac studies. Antioxidant activity of the optimized nanoemulsion formulation was found to be higher than pure RU, when compared with the ascorbic acid as a standard antioxidant. During pharmacodynamic studies, anti-inflammatory effect of RU suspension was compared with the nanoemulsion and was found to be significantly higher (p < 0.05). Thus, it can be inferred that TPGS-loaded NE of RU have the potential to improve the permeability of RU, and consequently increase its oral bioavailability.

Declaration of interest

The authors are thankful to Council of Science and Industrial Research (CSIR) India, for providing financial assistance to this work.

The authors state no conflict of interest and have received no payment in preparation of this manuscript.