INTRODUCTION:

Dermatitis is a common inflammatory disorder of the skin. It is also known as eczema which is a word derived from the Greek origin meaning 'boil over' or 'break out' (ekzein, ek-out, zein-boil). The words 'dermatitis' and 'eczema' are synonymously used as dermatitis is also derived from Greek origins and refers to skin inflammation (derma-skin, itis-inflammation) ¹.

Dermatitis is best characterized by atypical fluid secretions in the epidermal layer due to spongiosis. These secretions cause intercellular swelling, formation of intra-epidermal vesicles and juicy papules. Another symptom of this dermal disorder which intensifies the skin's degradation is constant itching or pruritus. Pruritus makes the skin thick or leather-like which reflects that it leads to 'lichenification'. Dermatitis has become a major stress causing factor among different diversity of patients and their families. It results from multiple factors like the kind of genetic predisposition a person has, environmental aspects, improper functioning of the immune system and skin (as a barrier). As an immune disorder dermatitis can also make the patient hypersensitive and susceptible to allergic disorders like rhinitis, asthma, etc ².

Since it majorly affects paediatrics, there is a 50% chance that the young ones suffering may develop asthma and 75% suffer from the risk of getting high fever. Therapeutic treatment of eczema has always been a major challenge for the pharmaceutical field. Commonly used conventional therapies include topical or systemic corticosteroids, topical calcineurin inhibitors, phototherapy, antibiotics, antihistamines and immunosuppressive agents. Although these therapies dominate the anti-dermatitis medications sectors, but they can lead to multiple local / systemic adversities. This is due to the fact that dermatitis patients require long-term therapeutic assistance. Topical treatment strategies are very effective but difficult to develop due to the barrier function of stratum corneum which balances the skin’s integrity. It functions as an effective barrier to skin penetration of drugs and it is necessary to employ enhancement strategies. There are various innovative researches exploiting strategies for penetration enhancement. There are various penetration enhancement techniques like iontophoresis, sonophoresis, micro-needles, and electroporation. These holds promise for the use of these drugs in a better and successful manner that is patient-friendly. The novel drug delivery systems (NDDS) can also be significantly used to combat the drawbacks and side effects of conventional drug delivery systems. Conventional medication requires multiple-dose therapy that holds innumerable issues. It becomes complicated for the above-mentioned conventional systems to deliver the perfect number of active agents to the right target because each medication needs to be delivered in an optimal manner to the patient. However, novel systems like the transdermal drug delivery systems offer the potential advantage of directly delivering the drug at a controlled rate for a prolonged period ^3-4.

The disorders like eczema, where deeper skin layers are involved; there is a need for vesicular carrier system for delivery of drugs though the skin layers that are an effective barrier to drug penetration. The objective of this system is to explore skin as a site for drug administration with better patient compliance. There are numerous advantages associated with vesicular systems that are like elimination of the first-pass metabolism, steady plasma levels maintenance, reduction of dosing frequency and subsequent decrease of dose-related side effects.

Vesicular systems like ethosomes show enhanced therapeutic efficacy due to improved dissolution profile, drug retention and stability. Ethosomal systems are novel permeation enhancing lipid carriers embodying ethanol containing lipid vesicles with inter-digitated fluid bilayers. In contrast to the liposomes and deformable liposomes, ethosomes have been shown to exhibit high encapsulation efficiency, skin deposition ability, and depth of skin penetration for a wide range of molecules including lipophilic drugs and are effective at delivering molecules to and though the skin ^5-6. Ethosomes can also encapsulate herbal phytoconstituents which have been proven to be effective against several types of dermatological disorders like dermatitis, psoriasis, acne, etc.^7-9. Extracts from various herbs have been continuously developed and studied to provide relief from different types of inflammatory conditions. This is due to the presence of special active phytoconstituents. Herbal extracts or their specific active phytoconstituents lag behind due to their bioavailability. This can be combated by employing distinguishable vesicular carriers (like ethosomes itself). Thus, the well-established properties of phytoconstituents like toxicity, strengthening effects on immune system, availability in bulk, etc. can be utilized with the help of novel drug delivery systems ^{10- 11}. One of the many beneficial and constantly researched herbal phytoconstituent is quercetin.

Quercetin is a major health-benefitting, dietary flavonoid which is extensively found in food items like apples, onions, green tea, berries, red wine, buckwheat tea, etc. It is a potent pleiotropic molecule that can modulate multiple pathways. It also exhibits distinguishable biochemical and biological effects when employed as an active constituent against viral diseases, inflammatory disorder, cancer and cardiac issues ¹². Antioxidant effect of quercetin plays a very crucial role in its activity with respect to the signal transduction pathway, glutathione, environmentally induced reactive oxygen species (ROS) and enzymatic activity ¹³. Quercetin has become an actively performing anticancer drug as it has entered the clinical trial phase. In contract to the beneficial effects of quercetin, it also falls behind due to low solubility (sparingly soluble in water), limited absorption (though oral routes) and minimal bioavailability. The drug delivery system which enhances the absorption of drug in the systemic circulation by penetration though topical application, can be used to bypass the drawbacks attached with quercetin ¹⁴.

The rationale behind the current research work was to achieve improved properties of quercetin by incorporating them in nanoethosomal gel form for the possible management of dermatitis.

MATERIALS AND METHODS:

Materials:

Quercetin was obtained from- SRL, poly ethylene glycol 400, and L-α phosphatidylcholine, were obtained from Sigma Aldrich, rest of the chemicals used were of analytical grade. Wistar rats (150–250 gm) were procured from the Central Drug Research Institute, Lucknow, India. The protocols for experiment were reviewed and approved by the Institutional Animal Ethics Committee (IAEC) as followed the guidelines of the Committee for the Purpose of safety and Supervision of Experiments on Animals (CPCSEA), Government of India¹⁵. Registration number [1492/PO/Re/S/11/CPCSEA 28/3/2017 and CSIR-IITR Lucknow, 54/GO/RBi /S/99/CPCSEA].

Methods:

Preformulation studies:

The Preformulation studies for the quercetin were done. The solubility was determined by the qualitative method. The partition coefficient was determined by the conventional shake flask method. The Drug excipient interaction was performed using FTIR (Perkin Elmer Spectrum, version 10.03.06)¹⁶.

Preparation of Quercetin loaded Ethosomes:

Ethosomes were prepared by modified cold method. Weighed the quantity of L-α phosphatidylcholine and quercetin were taken in a beaker and added ethanol to it. The beaker was covered with parafilm to prevent evaporation of ethanol. The solution was stirred on a magnetic stirrer and the water was added drop by drop with the help of syringe at room temperature¹⁷. Stirring was continued for the next 15 min. Sonication was done for next 5 min to reduce the size of prepared vesicles. The dispersions were stored in refrigerator. All formulations used are shown in Table1.

Table 1. Formulation table for ethosomes preparation

Formulation Code	% L-α Phosphatidylcholine	Ethanol: Water
F1	1	10:90
F2	1	20:80
F3	1	30:70
F4	1	40:60
F5	2	10:90
F6	2	20:80
F7	2	30:70
F8	2	40:60
F9	3	10:90
10	3	20:80
F11	3	30:70
F12	3	40:60

Procedure for characterization of quercetin loaded ethosomes:

All the prepared batches were observed under phase-contrast microscopy with a magnification power of 100 X (Columbus). Photograph were taken using (Nokia 6.1 Plus). The optimized batch was observed by transmission electron microscopy (Malvern) with voltage of 200 kV for surface appearance and shape¹⁸.

PDI, Size and Zeta Potential:

The ethosomes were dispersed in distilled water and 10 µl of diluted dispersion was placed on the carbon-coated grid then zeta potential vesicle size and polydispersity index were measured by zetasizer (Nano plus- Version 5.22/3.00 from BBAU, Lucknow)

Entrapment Efficiency:

The entrapment efficiency of quercetin loaded nanoethosomal vesicles was determined by the ultra-centrifugation method in which formulation was kept in a centrifugation tube and placed in the REMI Cooling centrifugation machine. Centrifugation was carried out at 10000 RPM at 4°C for 30 min. The clear supernatant was siphoned of clearly to separate the unentrapped drug, sediment was treated with 1 ml of 0.1 % Triton X 100 to lysis the vesicles and then diluted with PBS (6.8). Entrapment efficiency was calculated using the following formula¹⁹

Amount of drug present in vesicles

% Entrapment Efficiency= -------------------------------------------× 100

Total Drug incorporated

Amount of drug present in vesicles

% Loading Efficiency=---------------------------------------------- × 100

Initial drug loaded

In vitro Studies:

In vitro study was performed using Franz diffusion cell (Ambient borosilicate fabricated) with glass, surface area available for diffusion as 2.54 cm. The dialysis membrane (50-LA, Himedia Lab. Pvt. Ltd) was placed in PBS (6.8) for 4 hours to attain saturation before starting the study. It was placed between the donor and receptor compartment and ethosomal dispersion (5ml) to be analyzed was placed into the donor cell compartment²⁰. The receptor chamber was filled with PBS (6.8) and was maintained at 37±0.5°C with continuous stirring. The donor compartment was covered with parafilm to prevent evaporation. 1 ml aliquot from receptor phase solution were withdrawn at different time intervals (5, 1, 2, 3, 4, 5, 6, 7, 8, 24 h) and exact volume of medium was readded back into the medium²¹ and quantification was done using UV Spectrophotometer (Shimadzo-1800) at 252 nm.

Ex vivo permeation and determination of the amount of remained drug in the skin:

In this study the dialysis membrane was replaced with rat skin. The skin was mounted between donor and receptor compartment with the stratum corneum side facing upwards into the donor compartment.²² Skin surface was washed first with PBS pH 6.8 and then with ethanol. The procedure was repeated twice to ensure that no traces of formulation were left onto skin surface. The permeation area of the skin was excised, weighed, and then cut into small pieces to extract the drug content present in skin with ethanol. The resulting solution was centrifuged (1500 RPM), and the quercetin concentration was measured and expressed as percent of initially added drug.

Permeation Data Analysis:

Study of release rate profile and data obtained from the in vitro drug release study are fitted in different kinetic equation: zero order as the cumulative percent drug release Vs time, first order release as the percentage release (RTR) Vs time, higuchi release kinetics as the cumulative percentage drug release Vs square root (SQRT) time, korsmeyer peppas release kinetics as the log mt/mi Vs log time.^23-24

Calculation data of skin permeation parameters:

Cumulative amount of the drug passed per unit area was plotted as a function of time. The flux was calculated from the slope of the linear portion.²⁵ The permeability coefficient (𝐾𝑝) of Quercetin though out rat skin was calculated using relation derived from Fick’s first law of diffusion, which is expressed by the following equation:

Where 𝐽 is the flux and 𝐶 is the drug concentration in donor compartment.

Confocal Laser Scanning Microscopy (CLSM):

The capability of probe loaded ethosomes to penetrate though the skin was observed by confocal laser scanning microscopy. The probe Rhodamine B (0.03%) was used in place of drug. Diffusion study of probe loaded ethosomal dispersion was performed in a similar manner as discussed in previous section²⁶. The skin was removed from the cell and thoroughly washed with distilled water at the end of the study₁₄. The treated area was cut out and sectioned using microtone. The skin specimen was observed under a confocal microscope. (Reni Shaw in via Raman Microscope Holmarc Honeycomb Tabletops (TT300-120) and penetration depth of Rhodamine B was measured²⁷.

Incorporation into a hydrophilic gel:

The pH was adjusted by adding triethanolamine dropwise. On the basis of entrapment efficiency, zeta potential, polydispersity index, and vesicle size F-6, F-11, F-1, F-8 were selected and incorporated into carbopol base gel. The gel base was prepared by dispersing the weighed quantity of Carbopol 934 P (1% w/w). The carbopol was soaked in distilled water overnight. Quercetin loaded ethosomal dispersion (5ml) was added in it with continuous stirring. ²⁸

In vitro Diffusion Study and Data analysis:

In vitro diffusion study was performed for a hydroethanolic solution (HES), ethosomal gel formulation (G F-6, GF-11, G F-1, and GF-8), conventional gel formulation and standard gel formulation.²⁹Using the same procedure as discussed in the previous section. Release data were fitted in different Kinetic equations.

For the release rate profile study, the data, collected from the in vitro drug release study were fitted in the different kinetic equations as done in the previous section.³⁰

Characterization of quercetin loaded ethosomal gel pH, Viscosity:

The pH was determined by digital pH meter (RI-152-R).³¹The viscosity was measured by using Brookfield viscometer (Model No DV-III ULTRA) using spindle no 06 at 100 RPM.³²

Texture, Spreadability:

Texture Analysis of optimized nanoethosomal gel was determined by the TAXT2 Texture Analyzer.³³Spreadability was expressed as the time required for two slides to slip off from the gel for at fixed distance by applying constant weight attached (unit gm.cm/sec). Spreadability of the gel formulations is inversely proportional to the viscosity. The lesser the time taken for the slides to slip off from the gel, the better the spreadability of the gel. A greater spreadability indicated a smaller shear is needed to spread the gel.³⁴

Drug Content:

Drug content of prepared gels were determined by dissolving an accurately weighed quantity of the gel (100mg) in 5 ml of ethanol. 1 ml was transferred to a volumetric (50ml) and appropriate dilutions were made with phosphate buffer pH 6.8.³⁵ The resulting solution was then filtered using a 0.45µm membrane filter and measured spectrophotometrically at 252 nm (Shimadzu-1800, Japan).

Stability Studies:

Drug loaded ethosomal gel was stored at 4°C and at 25°C for one month. The physical stability was examined by visual observation and pH determination. Gel formulation was assessed by change in pH.³⁶

Statistical Analysis:

Statistical analysis was done by using one-way ANOVA (Microsoft Excel). The difference was considered significant when P ≤ 0.05.³⁷

Animal study:

Wistar rats of either sex (150-200 gm) with no previous drug treatment were used for in vivo studies. The animals were fed with standard diet and water Librium. Acclimation to laboratory hygienic condition was done for seven days before the start of experiment³⁸. The experimentation on animal was performed as per Institutional Animal and Ethics Committee of Amity Institute of Pharmacy, AUUP, Lucknow.

Registration number [1492/PO/Re/S/11/CPCSEA 28/3/2017 and CSIR-IITR Lucknow, 54/GO/RBi /S/99/CPCSEA].

Dermatitis Induction:

Dinitrochlorobenzene (DNCB) induced animal model was used for the induction of Dermatitis in rat. 50 µl of 1% DNCB in acetone: olive oil (4:1) was applied on the inner and outer surface of left ear lobes of each animal from Group 2 -6 once daily for 5 days²⁷. Eight hours before the final application weighed quantity of test (G-3) and marketed preparation (Clobetasol-Zydus Pharmaceuticals) was applied on the inner and outer sides of left ear lobes to each animal of group 3 to 6 as per protocol. Animal were sacrificed and ear of both sides were cut off using sharp surgical scissor. Then skin tissue from the right and left ear of rat were excised and sent for histological examination³⁹.

Histopathology:

The tissue was fixed in 10% neutral formalin solution for 24 h, and dehydrated with a sequence of ethanol-xylene series of solutions. Materials were filtered and embedded with paraffin (40–60°C)⁴⁰. 10µ thickness was taken by microtone sections. The sections were processed in alcohol-xylene series and stained with haematoxylin-eosin dye. Histological changes were observed under a microscope. Photograph was taken from each slide⁴¹.

Skin Irritation Study:

The hair on the dorsal side of the animal were removed by clipping a day before the experiment. The animals were divided as per the protocol and treatment was given as per the protocol on the shaven back area of skin. The treated area was covered with gauge and wrapped with a bandage. The bandage and test material were removed after 24 h and after 1 h, the treated area was examined and scored for skin irritation⁴².

1. Moderate erythema (dark pink)

2. Moderate to severe erythema (light red)

3. Severe erythema (extreme redness).

Cell Viability Study:

Cell survival study using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT): The cell survival study was conducted using a MTT based assay. The cells were washed with 1mM phosphate buffer saline pH 7.2 and the 100 µl/well cells with OD 600 nm=1.0 were seeded on the 96 well plate⁴³. A 5 µl MTT solution was added to each well and incubated for 1 h at 37 ⁰C. After the incubation, the cells were centrifuged at 1000 rpm for 5 min and 150 µl of the solubilization buffer containing SDS and DMSO were added to the cell pellet⁴⁴. After 15 min of incubation, the cells were centrifuged at 1000 X g for 5 min. The absorbance of the supernatant at 570 nm was recorded with the help of a spectrophotometer⁴⁵.

Reduced Glutathione:

The measurement of GSH was done by following the protocol of Rahman et al., 2006 with some modifications. In this 40 µl of tissue, homogenate was added to 0.4 M of Ellman’s reagent and 60 mM phosphate buffer p H=7.5.⁴⁶The reaction mixture was kept in an incubator for 1 h at 37⁰C and absorbance was measured at 412 nm in a UV-Vis spectrophotometer⁴⁷

Estimation of lipid peroxidation:

The estimation of malondialdehyde (MDA) content was done using the lipid peroxidation estimation kit. A 40 µl of tissue homogenate was added to the color developing solution containing thiobarbituric acid and acid reagent. This mixture was incubated in a water bath at 60⁰C for two h to form a pink color solution of MDA-TBA adduct. The absorbance was measured at 512 nm in a UV-VIS spectrophotometer⁴⁸.

Total antioxidant activity using 2, 2-diphenyl-2-picrylhydrazyl (DPPH) assay:

The DPPH assay is a free radical scavenging method used to assess the total antioxidants present by following the protocol of Brand Williams et al., (1995) with slight modifications. A 40 µl of the tissue homogenate was added to the 0.002 ml of 0.004% methanolic DPPH solution⁴⁹. The reaction was incubated for 15 min at 37⁰C and the absorbance was measured at 520 nm in the UV-Vis spectrophotometer.

Antioxidant Enzymes:

A. Catalase:

Catalase activity was measured according to the protocol of Beers et.al., 1952 with some modifications. Briefly, 20 µl of tissue homogenate was added to 0.1% hydrogen peroxide (H₂O₂) in 2.9 ml of 1m M phosphate buffer pH 7.2. The time-dependent decomposition of H₂O₂ was monitored at 240 nm for 2 min in the UV-Vis spectrophotometer⁵⁰.

B. Superoxide Dismutase:

The estimation of superoxide dismutase (SOD) was done by following the protocol of Beauchamp and Fridovich, 1971. A 20 µl of tissue homogenate was added to the reaction mixture containing Phenazine methosulphate, 0.052 M Nitroblue tetrazolium salt, Nicotinamide adenine dinucleotide, and sodium phosphate buffer pH=8.3 and incubated for 5 min⁵¹.

Ethosomal gel formulation GF-1 to GF-4, conventional gel and HES were also evaluated for in vitro drug release study. Results showed that cumulative percentage drug release for GF-3 loaded ethosomal gel was significantly higher than quercetin loaded conventional gel at the end of 0-24 h. (p˃0.05) These results suggested that ethosomal gel can enhance the skin permeation of the drug (Figure-1). The data obtained from in vitro release study of ethosomal gel and conventional gel was fitted in various kinetic models to assess the release rate profile. Results indicated that Higuchi Kinetic model was best fitted as R² value for this was highest among all. This implies slow and steady release by the process of diffusion as proposed by Higuchi. (Table-5,6)

Table 5. Permeated drug, flux and permeability coefficient of different formulations

Formulation Code	Permeated amm at 24 h (𝜇g/cm)²	Flux (𝜇g/cm2 /h)	Permeability coefficient (𝐾𝑝) × 10−3 (cm/h)
F-1	1406.45±451.32	58.60±0.63	5.86±0.032
F-6	2454.10±293.78	102.25±3.22	10.22±0.63
F-8	2233.90±768.21	93.07±0.51	9.307±0.051
F-11	1526.19±152.18	63.59±0.57	6.359±0.043
Standard	2178.51±165.23	90.77±0.65	9.077±0.37
HES	134.75±23.66	5.61±0.37	0.561±0.011
Conventional	252.86±32.76	10.53±0.21	1.053±0.09

Table6. % Drug Residual in different formulation

S. No.	Formulation Code	Amount Used (mg)	Amount Remained (mg)	% Drug Permeated
1.	F-1	2	0.162	8.1
2.	F-6	2	0.154	7.7
3.	F-8	2	0.205	10.25
4.	F-11	2	0.179	8.95

Table 7. Kinetics assessment of release profile of Quercetin loaded ethosomal Gel by using animal skin.

Formulation Code	Zero order Model R²	First order model R²	Higuchi order model R²	Kosermeyer and Peppas model R²
F 6	0.8147	0.0375	0.9414	0.1857
F 11	0.7936	0.0376	0.9315	0.1522
F 1	0.8076	0.0372	0.9029	0.1594
F 8	0.7865	0.0377	0.9099	0.1614

The transdermal flux ranged from 102.25±3.22 (𝜇g/cm² /h) of F6 to 5.61±0.37 (𝜇g/cm² /h) of (HES) and permeability coefficient ranged from 10.22±0.63 (cm/h) of F6 to 0.561±0.011 (cm/h) (HES) respectively. The result indicated that the flux of optimized batch F6 was 18.22 time higher then hydroethanolic solution. Results obtained from skin retention study showed that 7.7 % drug retained in skin. (Table-7)

Figure 2. Confocal laser scanning graph (A- HES at the depth of 45.23 micron), (B- F-6 at the depth of 45.23 micron), (C- HES at the depth of 10 micron), (D- F-6 at the depth of 10 micron)

CLSM was used to trace the penetration depth of fluorescence marker Rhodamine B loaded across rat skin; it was observed that it reaches up to the depth of 45.23-micron CLSM into the rat skin which was significantly higher than the Rhodamine B solution at the depth of the rat skin HES solution of Rhodamine B showed negligible fluorescence. This study proves the enhanced permeation profile of Rhodamine B loaded ethosomes into a deeper layer of the skin whereas Rhodamine B hydroethanolic solution remained confined to superficial layers.

Cell Viability Assay:

The viability was conducted by the tetrazolium based colorimetric assay for the study. The mitochondrial enzyme present in the viable cell produce formazan, a blue product by the reduction of tetrazolium salt 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). The results obtained in the dermatitis induced rat showed significant decreased of 13.77% cell viability when compared with the 0 day control rat. The marketed formulation which was used as a standard and the new formulation used as a test showed insignificant decline in the cell viability when compared to the control. Upon comparison with the dermatitis induced rat results obtained demarcate increased cell viability. The data indicate that the test formulation (F-6) used in the study has the efficacy of healing the dermatitis in the defined time without itself undergoing cellular damage. While there is no significant difference between base gel and conventional gel.

The in vivo anti-oxidant enzyme (Catalase) was found to be (32.30%) less in dermatitis induced rat while the treated and the standard showed remarkable increase in catalase activity as compared to control. From the above result we can conclude that the dermatitis healing capacity of the test formulation used in the study is due to antioxidants and the enzyme involved is the Catalase. While there is no significant difference between base gel and conventional gel.

2, 2 diphenyl-1-picrylhydrazyl (DPPH) is a colorimetric estimation of the total anti-oxidant activity. The results obtained indicate that the dermatitis induced rat showed a significant low (4.25 %) antioxidant capacity. Interestingly the treated and standard showed rise in DPPH value when compared is the control rat. The rise in DPPH levels upon application of the test formulation prove to be a potent healer with immerse antioxidant properties. While there is no significant difference between base gel and conventional gel.

The results obtained in the dermatitis induced rat showed significant decreased of 13.77% cell viability when compared with the 0 day control rat. The marketed formulation which was used as a standard and the new formulation used as a test showed insignificant decline in the cell viability when compared to the control. Upon comparison with the dermatitis induced rat results obtained demarcate increased cell viability. The data indicate that the test formulation (F-6) used in the study has the efficacy of healing the dermatitis in the defined time without itself undergoing cellular damage. While there is no significant difference between base gel and conventional gel.

2, 2 diphenyl-1-picrylhydrazyl (DPPH) is a colorimetric estimation of the total anti-oxidant activity. The results obtained, indicate that the dermatitis induced rat showed a significant low (4.25 %) in the antioxidant capacity. Interestingly the treated and standard showed rise in DPPH value when compared is the control rat. The rise in DPPH levels upon application of the test formulation prove to be a potent healer with immense antioxidant properties. While there is no significant difference between base gel and conventional gel.

Figure 5. Bar graph representation of Cell viability assay

LIST OF ABBREVIATIONS

API – Active pharmaceutical ingredient

BCS –Biopharmaceutical classification system

FTIR – Fourier transform infrared spectroscopy

QC – Quercetin

CLSM - Confocal Laser Scanning Microscopy

TEM - Transmission Electron Microscopy

PDI - Polydispersity Index

MTT -3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide

SOD - Superoxide Dismutase

DPPH - 2, 2- diphenyl-1- picrylhydrazyl

GSH - Gluthiaone

DMSO- Dimethyl Sulfoxide

PBS - Phosphate Buffer Solution

ACKNOWLEDGEMENT:

The Authors would like to thank Amity Institute of Pharmacy, Amity University, Uttar Pradesh, Lucknow Campus for providing the necessary facilities for the experimental work, for providing the gift sample, CDRI-Lucknow for Transmission Electron Microscopy and Fourier Transmission Infrared Spectroscopy Facility, BBAU, Lucknow for Zeta Sizer facility and BSIP, Lucknow for confocal laser scanning microscopy facility, Anton Par for Rheological study and Shruti Pet Clinic for histopathology study.

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Received on 27.08.2020 Modified on 12.01.2021

Research J. Pharm.and Tech 2021; 14(12):6516-6526.

DOI: 10.52711/0974-360X.2021.01127

S. No.	Formulation Code	Entrapment Efficiency (%)	Vesicle Size (Nanometers)	Polydispersity Index (PDI)	Zeta Potential(mV)
1	F1	63.99±1.73	334±15	0.532±0.032	-37.2
2	F2	56.61±1.14	343±13	0.453±0.029	-38.3
3	F3	42.48±0.70	361±17	0.0342±0.054	-39.3
4	F4	23.77±2.00	359±18	0.453±0.022	-35.2
5	F5	45.29±1.11	359±12	0.327±0.037	-30.9
6	F6	94.68±1.14	324.9±19	0.241±0.11	-26.33
7	F7	52±0.83	342±14	0.354±0.019	-36.3
8	F8	57.39±2.11	341±11	0.421±0.032	-33.2
9	F9	54.2±1.83	337±14	0.387±0.029	-31.4
10	F10	57.85±0.64	339±16	0.421±0.027	-39.7
11	F11	86.36±1.21	329±17	0.114±0.031	-35.8
12	F12	50.55±0.76	332±16	0.554±0.024	-31.3

Formulation Code	Initial pH Day 0	4°C One month	25°C One month
GF-1	7.06±0.03	7.03±0.03	7.05±0.03
GF-2	7.1±0.01	7.08±0.01	7.05±0.01
GF-3	6.8±0.06	6.7±0.06	6.6±0.06
GF-4	6.9±0.07	6.8±0.07	6.6±0.07

Formulation	Colour	Appearance	Spreadability	Drug Content	Ph	Viscosity (cps)
GF-1	Yellowish	Homogeneous	34.07±0.72	96.65±1.4	7.06±0.03	5300
GF-6	Yellowish	Homogeneous	33.03±0.56	81.65±1.9	7.1±0.01	6600
GF-8	Yellowish	Homogeneous	35.1±0.71	79.47±1.4	6.8±0.07	6200