FORMULATION AND IN VITRO EVALUATION OF SELF NANO EMULSIFYING DRUG DELIVERY SYSTEM OF QUERCETIN FOR ENHANCEMENT OF ORAL BIOAVAILABILITY

Objective: The study aims to develop a Self-nanoemulsifying drug delivery system (SNEDDS) of Quercetin to enhance its oral bioavailability. 
Methods: In the present study, Quercetin was formulated into SNEDDS using various oils, surfactants, and co-surfactants. The developed formulations were subjected to various studies like drug content analysis, droplet size and thermodynamic stability, and in vitro drug release studies. 
Results: From the screening of oils, surfactant and cosurfactant, the combination of Triacetin as oil phase, Tween 20 as surfactant and Ethanol as co-surfactant was selected for the development of SNEDDS of Quercetin. The composition of the formulation was optimized using pseudo ternary phase diagram. The optimized formulation has been evaluated and found to have good physical stability and improved in vitro drug release. 
Conclusion: A stable SNEDDS of Quercetin was developed, and results indicated substantial enhancement in the dissolution of the drug when formulated as a self-nano emulsifying drug delivery system, indicating its potential to enhance oral solubility and bioavailability of the drug.


INTRODUCTION
Quercetin ( fig. 1) is a polyphenolic flavonoid, which is the safe, most abundant, and commonly ingested dietary phytochemical which possess a wide spectrum of pharmacological action, mainly antiviral, antidiabetic, anti-inflammatory, neuroprotection and antiproliferative [1]. However, clinical applications of quercetin are limited due to its hydrophobicity and poor gastrointestinal absorption [2]. Several attempts have been made to improve the poor bioavailability of Quercetin, including a Self-emulsifying drug delivery system (SEDDS) [3,4].

Fig. 1: Structure of quercetin
Self-emulsifying drug delivery system (SEDDS) formulation are isotropic mixture of an oil, surfactant, co-surfactant and drug, that has the ability to form emulsion with water under gentle agitation as in the gastrointestinal tract. This spontaneous emulsion formation in vivo presents the drug in the solubilized form and the small particle size of the droplets provide a large interfacial area, which promote higher rate and extent of absorption [5,6]. Self-Nano Emulsifying Drug Delivery System (SNEDDS) are much more stabler form of SEDDS that have received particular attention as a means of enhancing oral bioavailability of poorly absorbed drug [7].
In the present study, Quercetin was formulated into Self-Nano Emulsifying Drug Delivery System (SNEDDS) to overcome its poor bioavailability and evaluated in vitro.

Materials
Quercetin was purchased from TCI chemicals (India), Pvt. Ltd. All the other chemicals and solvents used in the study were of analytical grade.

Solubility study of quercetin in different oils, surfactant, and cosurfactant
For determination of the solubility of Quercetin in various oils, surfactant and co-surfactant, excess amount of drug was suspended separately in 1 ml of each solvent (oil/surfactant/co-surfactant) at room temperature in tightly closed centrifuge tubes and shaken in a bath shaker (NSW-128, Remi Equipments, Mumbai, India) for 24 h. The samples were then centrifuged, and the supernatant was estimated for drug content spectrophotometrically using validated method [8].

Preliminary screening of oils, surfactants, and co-surfactant
The mixtures containing selected oils and surfactants were gradually heated at 50 ℃ for homogenization of the components. Each mixture was then diluted with distilled water to 100 ml in a stoppered conical flask. Ease of emulsification was judged by the number of flask inversions required to yield homogenous emulsion. Emulsions were allowed to stand for 2 h and their % transmittance was evaluated at 638 nm by UV-Visible spectrophotometer (UV1800, Shimadzu, Japan) using distilled water as a blank. Emulsions were furthermore observed visually for any turbidity or phase separation [9].

Solubility of quercetin in the screened mixture
The selected oil phase and surfactant were used for further screening of the different co-surfactants for their drug solubility ability. An estimated amount of Quercetin was added in the solution mixture of optimized oil and surfactant along with the selected cosurfactant. Each solvent was kept at room temperature in tightly closed culture tubes and shaken in a bath shaker for 24 h. The samples were then centrifuged, and the supernatant was estimated for solubilized drug [10].

Pseudo ternary phase diagram
Phase diagrams involve plotting the three components oil (triacetin), surfactant: co-surfactant (Smix i.e., tween 20: ethanol) and water content, each of them representing an apex of triangle. The required amounts of the components (oil and Smix) were weighed accurately and then sonicated for 3 min. The mixture was then gently heated at 45-50 °C and vortex to form a homogenous mixture. To this mixture, distilled water was added drop by drop until a transparent solution was formed. The surfactant and co-surfactant (Smix) were varied in mass ratios 1:1, 1:2, 1:3, 2:1 and 3:1. Pseudo ternary mixtures were formed in these ratios and then the quantity of water forming transparent solution was plotted with other components in the pseudo-ternary phase diagram [11].

Preparation of liquid SNEDDS
Different formulations were carefully chosen from the zone of nanoemulsions of each constructed phase diagram, based on objective that the oil phase concentration should be such that it would be capable to dissolve 0.05% w/v of Quercetin. Quercetin was dissolved in two different concentrations one being 0.015% w/v and 0.05% w/v in oil, the mixture of surfactant and cosurfactant was mixed in oil phase in appropriate quantity while deionized water was further added in dropwise way with constant vortexing until the formation of a clear transparent monophasic liquid state.

Percentage drug content
The selected formulations were evaluated for drug content. Then the formulation was diluted with methanol and absorbance was measured at 374 nm by UV Spectrophotometer [12].

pH of SNEDDS
SNEDDS (100 µl) was dissolved in 5 ml of distilled water. pH of the nanoemulsion was measured by using pH meter (ST3100M, Ohaus, USA)at room temperature. pH of SNEDDS was measured before dilution and after dilution with aqueous phase [13].

Self-Emulsification time and robustness to dilution
To determine the emulsification time (time needed to reach the emulsified and homogeneous mixture, upon dilution), formulation containing Quercetin (0.015% w/v and 0.05% w/v) was added to 100 ml of 0.1N HCl, 100 ml of Phosphate Buffer pH 6.8 and 100 ml of distilled water at 37 °C with gentle agitation using a magnetic stirrer (.100MHPS0515000, Remi Equipments, Mumbai, India). d The formulation was assessed visually according to the rate of emulsification and the final appearance of the emulsion [14].

Thermodynamic stability studies
Optimized formulation was subjected to thermodynamic stability studies to access any phase separation and stability of the formed nanoemulsion [15].

Centrifugation study
The formulation was centrifuged in a centrifuge (CPR-30-PLUS, Remi Equipments, Mumbai, India) at 18000 rpm for 30 min. The resultant formulation was then checked for any instability problem, such as phase separation, creaming, or cracking.

Heating and cooling cycle
The liquid SNEDDS formulations were subjected to a heating-cooling test using six refrigerator cycles at 45 °C and 4 °C temperatures separately for 48 h in an incubator (Remi, Mumbai, India). Afterward, it was assessed for phase separation.

Cloud point measurement
The cloud point temperature of the diluted formulation was determined by gradual heating on a water bath and the temperature at which cloudiness appears was denoted using the thermometer. The formulation was diluted with distilled water in the ratio of 1:100. The diluted samples were placed in a water bath and its temperature was increased gradually. Cloud point was determined as the temperature at which there was a sudden appearance of cloudiness [16].

Viscosity studies
The viscosity of the optimized formulation was measured using small sample adapter of Brookfield viscometer (ViscoQC100, Anton Paar, Gurugram, India) at 12 rpm at room temperature (25±1 °C), repeated in triplicate [17].

Particle size
The particle size of the selected formulation was determined by using particle size analyzer (Litesizer 500, Anton Paar, Gurugram, India). The measurements were performed at 25 ℃ at a fixed angle of 90°. Aliquots of the formulation, serially diluted with purified water, were employed to assess the particle size using a particle size analyzer [18].

Transmission electron microscopy (TEM)
Transmission electron microscopy (H-600, Hitachi, Japan) was employed to study the morphology of the resulting nanoemulsion. Prior to the analysis, the SNEDDS samples were diluted 1000 times with water to form an emulsion, stained with 2% (w/v) phosphotungstic acid for 30 s and placed on 400-mesh copper grids with films for observation [19].

In vitro drug release
The relative in vitro dissolution behavior of quercetin from pure quercetin and SNEDDS filled capsule in 0.1 N hydrochloric acid (900 ml; pH 1.2; 37±0.5 ℃ ) was assessed using USP type I apparatusbasket type (DS8000, Labindia Ltd. Mumbai, India)at a rotation speed of 50 rpm. At predetermined time points (0, 5, 10, 15, 20, 25 and 30 min), an aliquot of 5 ml was withdrawn with equal volumes of fresh dissolution medium replacements to maintain the medium volume constant. All the samples were filtered, diluted and the concentration of quercetin dissolved was estimated spectrophotometrically at 374 nm. In case of oils, the solubility of Quercetin was found in Capmul MCM>Triacetin>Labrafil 2125 CS>Acconon MCB-2>Maisine CC>Labrafac MCM 1944 CS and was found to be more than 2 mg/ml.

Solubility study of quercetin in different oils, surfactant, and cosurfactant
In case of Surfactants, the solubility of Quercetin was found in maximum in the case of Tween 20, followed by Labrasol ranging from 4.232±0.005 to 27.078±0.133 mg/ml.
In case of Co-surfactants, the solubility of Quercetin was found in maximum in case of Ethanol, followed by PEG 400 and PEG 200 ranging from 6.229±0.002 to 20.000±0.030 mg/ml.

Preliminary screening of oils, surfactants and co-surfactant
Different combinations (F1 to F15) of selected oils and surfactants (ratio 3:2) were subjected to an emulsification efficiency study to select which one tends to have the maximum soluble content of quercetin. It was found ( fig. 5) that the F4 with oil phase as Triacetin and surfactant phase as Tween 20 and F8 with oil phase as Acconon MCB-2 and surfactant phase as Tween 80 showed maximum transmittance without any evident turbidity and phase separation. The provided two combinations were further subjected to screening of co-surfactant study. The mixture were further subjected to emulsification study with the addition of different co-surfactant (PEG 200, Koliphor HS15, PEG 400, Glycerol and Ethanol) with each of the selected mixture in the ratio oil: surfactant: co-surfactant ie 3:2:2 to produce formulations F16 to F25. It was found that the transmittance ( fig. 6) was to be maximum in case of F20, where Ethanol was used as a co-surfactant with Triacetin (oil) and tween 20 (surfactant). Furthermore, it didn't show any evident turbidity and phase separation.

Solubility of quercetin in screened mixture
When around 0.05% w/v of the drug was added to the mixture solution, the solubility was found to be was found to be 33.31±0.457 mg/ml. From the screening of surfactant and cosurfactant the combination Triacetin as oil phase, Tween 20 as surfactant and Ethanol as co-surfactant was selected for the preparation of pseudo ternary phase diagram.

Pseudo ternary phase diagram
In this provided combination of oil, surfactant and co-surfactant, formulations as shown in the shaded portion of the pseudo ternary phase diagrams ( fig. 7, fig. 8, fig. 9, fig. 10, fig. 11) was transparent on addition of water. While the remaining formulation were found to be turbid upon the addition of water up to the amount of 5 ml. The above-mentioned formulations remain transparent even after 24 h of water addition. However, the ones having turbid appearance doesn't exhibit any change even after 24 h of water addition.

Percentage drug content of SNEDDS
The prepared formulation was subjected to percentage drug content analysis. From the fig. 12 it can be inferred that most of the formulations were having high drug content, comprising about 97.98±0.105 to 100.88±0.457 percent of Quercetin loaded into the formulation.

pH of liquid SNEDDS containing quercetin
On pH study of selected Liquid SNEDDS containing Quercetin, pH of all the formulations was found to be in a range of 6.05±0.040 to 6.86±0.015.

Self-emulsification time and robustness to dilution
Initially, the drug incorporated for the preparation of nanoemulsion was 0.05% w/v of the emulsion and precipitation was witnessed in all these formulation with 0.1N HCl and Phosphate Buffer pH 6.8 solution.
However, upon reduction of the fed Quercetin to 0.015% w/v, all the formulation form nanoemulsion within 8 seconds upon diluting it into 0.1N HCl and Phosphate Buffer pH 6.8 solution (table 2). However, as far as the stable homogeneity and transparency is concerned, the formulation NE4, NE10 and NE22 was found to have clear transparent appearance even after 24 h. In case of distilled water, all the formulations were found to have a clear, yellowish transparent appearance.

Centrifugation study
Upon centrifugation study, all three selected SNEDDS formulations (NE4, NE10 and NE22) were found to be stable after centrifugation. No sign of phase separation was seen in the formulations.  The nanoemulsions formed from the optimized SNEDDS formulations are found to be thermodynamically stable systems without phase separation, creaming and cracking.

Heating and cooling cycle
The optimized SNEDDS examined for centrifugation and heatingcooling cycle, passed these tests and no phase separation, creaming or cracking were observed.

Cloud point measurement
Estimation of cloud points is an important factor for the stability of the self-emulsifying formulation. The cloud point is the temperature above which dehydration of self-emulsifying ingredients occurs and turns a clear dispersion to a cloudy one which in turn may affect drug absorption. Hence, cloud point of the self-emulsifying formulation should be above body temperature (37 °C). The cloud point of NE4, NE10 and NE22 indicated the formed SNEDDS at the physiological temperature will be a stable one.

Viscosity studies
Estimation of viscosity was carried out of the enlisted formulations and was found to be in the range of 187.67±2.309 to 275.67±1.528 cP. Also, the lower viscosity of SNEDDS is mainly due to the smaller droplet size. NE22 203.87±1.629 Determination of particle size Fig. 13-15, shows the particle size and PDI of optimized SNEDDS formulation

Transmission electron microscopy of SNEDDS
As shown in the TEM photograph ( fig. 16), the diluted preparation (nanoemulsion) appears spherical and homogeneous with a large population of the smaller droplet in the size range of less than 125 nm, which is consistent with the distribution data obtained from particle size measurement.