Biomedicine & Pharmacotherapy 112 (2019) 108622

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Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

Quercetin loaded nanoemulsion-based gel for rheumatoid arthritis: In vivo T

and in vitro studies
Jayanti P. Gokhalea, , Hitendra S. Mahajana, Sanjay J. Suranab
⁎

a
Department of Pharmaceutics, R.C. Patel Institute of Pharmaceutical Education and Research, Shirpur, Maharashtra, India
b
Department of Pharmacognosy, R.C. Patel Institute of Pharmaceutical Education and Research, Shirpur, Maharashtra, India

ARTICLE INFO ABSTRACT

Keywords: Current research reports the development, optimization and evaluation of Quercetin (QCT) loaded nanoemul-
Quercetin sion (NE)-based gel for the effective rheumatoid arthritis (RA) management. The formulation of QCT- NE was
Nanoemulsion developed using spontaneous emulsification techniques using the Box- Behnken experimental design. The cy-
Rheumatoid arthritis totoxicity study and effect on TNF-α production were evaluated respectively on HIG-82 and RAW 264.7 cells.
Gel
The study showed that QCT- NE has no toxic effect on synoviocytes and a strong inhibitory effect on LPS-induced
TNF-α production. QCT- NE gel has confirmed adequate rheological behavior with a good texture profile and
improved drug permeation compared to free QCT gel. In addition, the gel was found to be non-irritating and
showed the inhibition of paw edema in rats induced by CFA over 24 h contrary to free QCT gel. In conclusion, the
formulation of QCT- NE gel is an efficient topical treatment strategy for rheumatoid arthritis.

1. Introduction antiviral, antidiabetic, anti - inflammatory neuro-protective and anti-

Arthritic disorders are among the most common diseases and are an
important cause of joint dysfunction and disability in adults [1]. The
most prevalent type of arthritis is rheumatoid arthritis, which is an
autoimmune disorder caused by activation of pro-inflammatory med-
iators such as TNF-α, IL-1β and it is characterized by peripheral joint
pain, stiffness and swelling. Rheumatoid arthritis treatment includes
analgesics, steroidal and non-steroidal anti - inflammatory drugs, dis-
ease - modifying anti - rheumatic drugs (DMARDs), monoclonal anti-
bodies (MAbs) that only provide symptomatic relief [2]. Nevertheless,
these medications have a wide variety of harmful side effects that re-
semble stomach upset, nephrotoxicity, protein loss, toxicity, lack of
target specificity, immunosuppressive effects that ultimately lead to
poor patient consistency [3,4]. As a result, the importance of various
anti-inflammatory drugs has decreased and plant – based agents asso-
ciated with the least side effects are in the process of treating rheu-
matoid arthritis [5].
In recent years, flavonoids have attained great interest because of
the wide range of biological properties such as antioxidants, anti-pro-
liferative and anti - inflammatory activities. QCT is a leading flavonol
collectively distributed in a variety of edible plants and one of the
potent plant origin antioxidants. It is a safe and often ingested dietary
flavonoid along with numerous therapeutic advantages, such as
cancer. QCT inhibits, pro-inflammatory cytokines for example, TNF-α,
nitric oxide, IL-6, IL-1β, chemokines, etc. Consequently, it helps in the
management of chronic oxidative stress-related diseases such as ar-
thritis, inflammation and diabetes, as reported in several research stu-
dies [6]. QCT is administered orally in three daily doses of 250–500 mg;
afterwards, it is absorbed into the small intestine and colon, where QCT
and where QCT and its glycoside derivatives are combined with glu-
curonic acid. It then binds to albumin and is transported to the liver,
which means that a very small fraction of QCT is freely available in the
bloodstream and a greater proportion appears in the blood as QCT
metabolites [7]. Furthermore, QCT has poor gastrointestinal retention,
low skin penetration, insufficient water solubility and rapid excretion.
Consequently, the oral delivery of QCT is not applicable and its clinical
applications are poorly efficient. It degrades rapidly not only in alkaline
conditions but through heat exposure during product processing and
shelf life [6–13]. These obstacles regarding QCT increase the necessity
of novel formulation that stabilizes QCT and exploits the therapeutic
benefits of QCT.
In recent decades, several QCT nanoformulations, such as micro-
emulsion, microspheres, nanostructured lipid carriers, solid lipid na-
noparticles and polymer nanoparticles, have been investigated to im-
prove bioavailability by solubilization or encapsulation and to achieve
better therapeutic effects [14–18]. Despite its great biological potential,

⁎
Corresponding author.
E-mail address: jayanti.gokhale@yahoo.co.in (J.P. Gokhale).

https://doi.org/10.1016/j.biopha.2019.108622
Received 28 July 2018; Received in revised form 20 January 2019; Accepted 23 January 2019
0753-3322/ © 2019 Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
J.P. Gokhale, et al.
QCT remains unexplored with regard to the development of an in-
novative topical formulation for the treatment of rheumatism. In this
study, we hypothesized that, formulation of nanoemulsion with QCT for
topical delivery could lead to significant uptake through skin rendering
its therapeutic potential.
The colloidal delivery system has proven to be competent to solve
the practical problems associated with solubility, modified release, and
target specificity; nanoemulsion is one of them. The topical drug de-
livery based on nanoemulsion has excellence such as small droplet size
(50–1000 nm), a larger surface area and free energy, high drug loading
capacity, protection of drug candidates, controlled or sustained release
of drugs, excellent drug permeation. However, due to the low viscosity
of nanoemulsion, it has low retention at the application site and is also
inconvenient to use; it is often included in a gel system [19,21–23].
Therefore, for the treatment of RA, the nanoemulsion- based gel
system containing QCT is proposed in the present study to improve the
therapeutic efficacy of QCT which is evaluated on a CFA- induced
Wistar rat arthritis model.
2. Materials
QCT was purchased from Otto Chemie (Mumbai, India). Oleic acid,
arachis oil, castor oil, sesame oil, coconut oil, soy oil, olive oil, sun-
flower oil, isopropyl myristate (IPM), tween 20 (Polysorbate-20),
Polyethylene glycol-400 (PEG-400), propylene glycol and Transcutol P
were obtained from Loba Chemie Pvt. Ltd. (Mumbai, India). Cremophor
RH 40 was obtained from BASF India Ltd. (Mumbai, India). Capryol 90
was obtained from Gattefosse (France). Carbopol 940 was obtained
from Noveon India Ltd. (Mumbai, India). Diclofenac sodium was re-
ceived as a gift sample from Novartis Pharmaceuticals Ltd. (Mumbai,
India). All the solvents required for the experimental purpose were
received from Merck Ltd. (Mumbai, India) and were of analytical grade
and GRAS category.
3. Methods
3.1. Solubility studies
The solubility studies have been carried out to select the most ap-
propriate oil phase for the development of the formulation. The solu-
bility of QCT was determined in a variety of oils such as oleic acid,
coconut oil, castor oil, olive oil, arachis oil, sesame oil, sunflower oil,
soy oil, IPM. Similarly, surfactants (tween 20, Cremophor RH 40, and
tween 80) and co-surfactants (PEG 400, propylene glycol, and
Transcutol P) were tested. Solubility was estimated by dissolving an
excess quantity (1gm) of QCT in 2 mL of each of the selected oils,
surfactants and co-surfactants. Combinations of oils were also utilized
for the estimation of solubility. The mixtures were subjected to vortex
mixing followed by orbital shaking at 37 ± 1.0 °C for 72 h. The equi-
librated samples were centrifuged at 3000 rpm; the supernatant was
filtered, and the filtrates were diluted with appropriate solvent. The
solubility of QCT was measured at 258 nm by using validated UV
spectrophotometric method (UV 1700, Shimadzu, Japan). All estima-
tions were done in triplicates [24].
3.2. Formulation of nanoemulsion
Nanoemulsion was developed using spontaneous emulsification
technique. In brief, accurately weighed quantity of QCT (10 mg) was
added to the oil phase. Subsequently, surfactant and co-surfactant
(Smix) were then added and the mixture was vortexed for 15 min at
300 rpm. The prepared Smix then gradually added to the magnetically
stirred aqueous phase. Nanoemulsion formed rapidly. The complete
mixture was further vortex mixed for 20 min.
The resulting nanoemulsion was transparent and readily flowable
which was allowed to stand for 2 h for equilibration and characterized
2
Biomedicine & Pharmacotherapy 112 (2019) 108622
for particle size, PDI, and zeta potential [20].
For the development and optimization of the QCT loaded nanoe-
mulsion, three factors, three-level Box-Behnken experimental design
(BBD) was selected.
The Box-Behnken is an excellent design model for response surface
methodology as it allows: (i) evaluation of the quadratic model para-
meters; (ii) construction of subsequent designs; (iii) recognition of lack
of fit of the model; (iv) make use of blocks v) avoid experimentation
under extreme conditions, for which unsatisfactory results might occur;
vi) fewer number of experimental runs where number of factors are
three.
Three factors for this study were designated as A (concentration of
oil), B (concentration of surfactant), and C (concentration of co-sur-
factant) and arranged into three levels, coded as +1, 0, -1 for high,
intermediate and low values respectively. Selection of oils, surfactants,
and co-surfactants were carried out by solubility and their emulsifica-
tion capacity. Independent variables or factors selected as, (A) con-
centration of oil (10–20 %), (B) concentration of surfactant (3–9 %) and
(C) concentration of co-surfactant (3–9 %). Dependent variables or re-
sponses selected as mean globule size (nm) and (%) entrapment effi-
ciency (% EE). A total of 17 experiments with 5 center points (to allow
the estimation of pure error) were designed by the software and ex-
periments were performed in random order. Table 1 shows the in-
dependent coded variables. For the 17 batches generated, formulations
were developed and further analyzed.
3.3. Optimization of formula
The basic intension of BBD was to optimize the oil, surfactant and
co-surfactant ratio. Out of 17 runs generated from design, formulations
consisting of least amount of surfactant and co-surfactant were selected.
The selected formulations were subjected to thermodynamic stability
testing such as heating-cooling cycles, centrifugation, and freeze-thaw
cycles. During the testing, parameters such as drug precipitation, opa-
lescence, creaming, and phase separation were observed. The particular
formulations were further examined for globule size and drug entrap-
ment efficiency and optimized for further processing. The optimized
batch was selected on the basis of relative transparency, clarity, globule
size and % entrapment efficiency (% EE).
3.3.1. Thermodynamic stability studies
3.3.1.1. Heating–cooling cycles. The intension behind to perform
heating- cooling cycles was to observe the effect of temperature
variations on the nanoemulsion stability. Six cycles between 4 and
40 °C were performed with storage at not less than 48 h at each
temperature. The formulations that were found stable at these
temperatures were subjected to centrifugation test.
3.3.1.2. Centrifugation test. The formulations were centrifuged for
30 min at 2000 rpm and evaluations were carried out for phase
separation, creaming or cracking. Formulations that were found
stable were selected and subjected for freeze- thaw stress test.
3.3.1.3. Freeze–thaw cycle (accelerated ageing). The formulations were
subjected to three freeze–thaw cycles at temperatures between −21
and +25 °C and were stored at not less than 48 h at each temperature.
For further studies, formulations that survived thermodynamic stability
tests were selected [25].
3.3.2. Preparation of optimized batch of QCT-NE
Accurately weighed amount of oil phase- arachis oil and oleic acid
(15%), surfactant- tween 20 (6%) and co-surfactant- PEG-400 (6%)
were incorporated into a screw capped bottle on a vortex mixer at an
optimal speed. In this Smix, QCT (10 mg) was dissolved and gradually
added to the slightly magnetically stirred aqueous phase. Nanoemulsion
was rapidly formed. Then complete mixture was vortex mixed for
J.P. Gokhale, et al. Biomedicine & Pharmacotherapy 112 (2019) 108622

20 min. and further analysis was done.

Response Y2 (%EE) ( ± SD)

85.82 ± 0.03 3.4. Characterization of QCT loaded nanoemulsion

93.73 ± 0.31

91.12 ± 1.00
86.16 ± 0.61
91.86 ± 2.05
94.12 ± 0.87
95.76 ± 0.52
93.97 ± 0.15
87.18 ± 1.65
93.34 ± 1.83
92.55 ± 0.50
88.52 ± 1.74
90.47 ± 2.30
86.83 ± 0.39
94.65 ± 0.21
92.81 ± 0.51
3.4.1. Identification tests for optimized nanoemulsion
95.4 ± 1.09

3.4.1.1. Staining test. The oil soluble dye- Sudan Red IV, was added to
the drop of nanoemulsion, and observed under the optical microscope.

3.4.1.2. Dilution test. For the development of stable nanoemulsion,

accurate blend of surfactant and co-surfactant is essential. In order to
Response Y1 Globule size (nm) ( ± SD)

detect the stability, the nanoemulsion was diluted with 1:10 and 1: 100
ratios with double distilled water and phosphate buffer of pH 5.8 and
visually observed for any phase separation or cracking and clarity/
turbidity.

3.4.1.3. Electrical conductivity. Nanoemulsion conductivity (σ) was

1.21
0.12
1.32
0.43
0.56
1.48
2.14
0.29
0.81
1.39
1.78
2.51
0.02
0.92
0.59
0.72
1.82

estimated to confirm the type of nanoemulsion, whether it is O/W or

W/O nanoemulsion. For evaluation purposes, the conductivity meter
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
125.6
137.3
142.7
135.3
125.4
145.3
144.7
143.2
137.4
139.6
137.9
141.8
130.3
139.6
129.5
136.5
137.4

(306, Systronics, Ltd, India) consisting of Pt / platinized electrodes was

used.
All the estimates for the above mentioned parameters were per-
formed in triplicate.
Factor C Concentration of Co-surfactant (%)

3.4.2. Mean globule size, zeta potential, and morphology

The mean globule size, polydispersity index, and zeta potential were
characterized by photon correlation spectroscopy (PCS) using a
Malvern Zetasizer (Nano ZS 90, Malvern Ltd., UK). Prior to the mea-
surements, all the samples were diluted with double-distilled water,
mixed with vigorous shaking to produce appropriate scattering in-
tensity. The measurements were made at an angle of 90° at 25 °C
[26,27].
9.00 (+1)

9.00 (+1)

9.00 (+1)
9.00 (+1)
3.00 (-1)

3.00 (-1)
3.00 (-1)

3.00 (-1)
6.00 (0)

6.00(0)

6.00(0)
6.00(0)
6.00(0)

6.00(0)

6.00(0)
6.00(0)
6.00(0)

The morphology of nanoemulsion was studied by using a trans-

mission electron microscope (Jeol/JEM 2100, USA). A drop of nanoe-
mulsion was diluted with distilled water appropriately and applied to a
carbon- coated copper grid. The negative stain was used with 2%
Factor B Concentration of surfactant (%)

phosphotungstic acid and kept for 30 s. The grid was analyzed using the
transmission electron microscope with an accelerating voltage of
60–80 kV [28].

3.4.3. Viscosity, pH, refractive index

The viscosity of the nanoemulsion was estimated using Brookfield
viscometer (DV-E Brookfield Engineering Labs Inc., Middleboro, MA,
USA) with a spindle no.00 at 25 ± 0.5 °C [25]. An apparent pH of the
9.00 (+1)

9.00 (+1)

9.00 (+1)
9.00 (+1)
3.00 (-1)
3.00 (-1)

3.00 (-1)

3.00 (-1)

formulations was estimated at 25 ± 0.5 °C by a pH meter (μ pH system,

6.00(0)
6.00(0)
6.00(0)

6.00(0)

6.00(0)

6.00(0)

6.00(0)

6.00(0)
6.00(0)

362, Systronic, Ltd., India). The refractive index of the formulation was
estimated using the Abbe’s type of refractometer. All estimates for the
above parameters were made in triplicate.
Factor A Concentration of oil (%)
Factor level and response data for Box–Behnken study.

3.4.4. Degree of transparency, drug content, % EE

QCT- NE formulation was inspected by measuring percentage
Results are expressed as mean ± SD (n = 3).

transmission (% T) for optical transparency. One mL of the formulation

was diluted with methanol and spectrophotometrically analyzed at
258 nm [29].
(+1)

(+1)
(+1)
(+1)

In the formulation, QCT content was estimated using HPLC analysis

(-1)

(-1)

(-1)

(-1)
(0)

(0)

(0)
(0)
(0)
(0)

(0)

(0)
(0)

method. Briefly, 5 mL of nanoemulsion was dissolved in 10 mL of me-

10.00
15.00
20.00
15.00
10.00
20.00
20.00
20.00
15.00
15.00
15.00
15.00
10.00
15.00
10.00
15.00
15.00

thanol, centrifuged at 3500 rpm for 15 min. (REMI Instruments,

Mumbai, India) and analyzed by HPLC [30]. HPLC system (Younglin S.
K., Korea) consisted of a dual wavelength UV visible detector (UV 730 D
QCT-NE10
QCT-NE11
QCT-NE12
QCT-NE13
QCT-NE14
QCT-NE15
QCT-NE16
QCT-NE17
QCT-NE1
QCT-NE2
QCT-NE3
QCT-NE4
QCT-NE5
QCT-NE6
QCT-NE7
QCT-NE8
QCT-NE9

Shimadzu, Japan) and Autochro 3000 software (Kyungki-do, Korea)

was used. Chromolith® C18 reverse phase column (250 mm × 4.6 mm
Code

i.d., 5μ) was used with methanol: water (60:40 v/v, pH 7.0) as a mobile
phase, at a flow rate of 0.8 ml/min. All the measurements were carried
Table 1

out in triplicate.
Run

F10
F11
F12
F13
F14
F15
F16
F17
F1
F2
F3
F4
F5
F6
F7
F8
F9

Further, % EE was determined using the following formula [31].

3
J.P. Gokhale, et al.
%EE = ( Amount of drug content
Total amount of drug )× 100
(1)
3.4.5. In vitro drug release from QCT-NE
in vitro diffusion study of nanoemulsion (F16) was conducted by
Franz diffusion cell having a capacity of 10 mL. Dialysis membrane
(Avg. Mol wt. 12000 Da Himedia, India) was used as diffusion mem-
brane and was soaked in phosphate buffer saline (PBS) having pH 5.8,
for 24 h prior to experiment. Diffusion cell was filled with PBS pH 5.8
and dialysis membrane was mounted on cell. The temperature was
maintained at 37 ± 0.5 °C and magnetically stirred at 100 rpm. After a
pre-incubation time of 20 min., QCT-NE was placed in the donor
chamber. The samples (0.5 mL) were withdrawn at regular pre-
determined time intervals over a period of 4 h (0, 1, 2, 3, and 4 h) and
replaced with the same amount of fresh PBS (pH 5.8) to maintain sink
condition and analyzed by HPLC at 258 nm [7]. The similar procedure
was carried out with free QCT suspension.
3.4.6. Stability studies
The optimized batch of the nanoemulsion was prepared in tripli-
cates and kept for three months at 40 ± 2 °C/ 75 ± 5%RH. Samples
were withdrawn after specific time intervals (0, 30, 60, and 90 days)
and visually examined for any physical changes in the formulation. The
selected criteria for the stability assessment were phase separation, pH,
globular size, polydispersity index, zeta potential and drug content.
Secondary parameters were also determined, such as refractive index
and viscosity [32].
3.4.7. Cytotoxicity study
In this study, synoviocytes HIG-82 cell lines were used to investigate
the cytotoxicity of the QCT- NE. The cell growth medium comprised of
90% Ham’s F12 medium with 10% fetal bovine serum (FBS). To ex-
amine cell proliferation process, MTT [(3-(4, 5-dimethyl thiazolyl-2)-2,
5-diphenyltetrazolium bromide)] assay technique was used. The
number of viable cells present in the cell suspension was calculated by
trypan blue exclusion method using hemocytometer. For the assay,
about 5 × 104synoviocytes were seeded and loaded in each well of 6-
well culture plate and kept for 24 h in CO2 incubator at 37 °C with sa-
turated humidity 5% CO2. On a subsequent day, medium was replaced
with free QCT, QCT-NE, and DCS (diclofenac sodium) suspension pre-
pared using 10 mL Ham’s F12 medium. Each suspension was added at
various concentrations of 20,40,60,80 and 100 μM. After 48 h, the
medium containing suspensions were discarded, washed with PBS and
then MTT assay was carried out. To measure the cell viability, the ab-
sorbance was measured at the wavelength of 562 nm using ELISA mi-
croplate reader (ELx 800, Biotek). The experiments were carried out in
triplicates, and the results were expressed as mean ± SD [18,33].
3.4.8. Effect on production of TNF-α
In this study, the effect of QCT-NE on the production of TNF-α was
investigated on RAW264.7 cells (macrophage cells isolated from mouse
blood). The cells were cultured with DMEM (Dulbecco’s Modified
Eagle’s Medium - high glucose) + 2 mM Glutamine + 10% Foetal
Bovine Serum (FBS). RAW264.7 cells seeded at the density of 1 × 105/
well were preincubated with QCT, QCT-NE, and DCS for 2 h.
Lipopolysaccharide (LPS) was added to the cells for 20 h. The level of
TNF-α secreted was measured in the cell supernatants using a sandwich
ELISA kit by following the manufacturer’s experimental protocols.
Absorbance was read by an ELISA microplate reader (ELx 800, Biotek)
at 450 nm. The experiments were carried out in triplicates, and the
results were expressed as mean ± SD (n = 3) [34].
3.5. Preparation of QCT-NE based gel formulation
A gel was formulated using Carbopol 940 (1.0% w/v) as the gel
4
Biomedicine & Pharmacotherapy 112 (2019) 108622
matrix. About 0.01% w/v QCT-NE was dispersed in the gel with stirring
for 10 min at 500 rpm. The pH of the formulation was adjusted to 5–6
using triethanolamine and gel formulation was homogenized until de-
sired consistency [30].
3.6. Evaluation of QCT-NE gel formulation
3.6.1. pH and rheological measurements
The topical gel should be non-irritating and skin- pH compatible to
prevent any allergic reactions or irritation. Thus, measurement of the
pH of the gel formulation is essential. Gel pH was measured using pH
meter, (μ pH system, 362, Systronics, Ltd., India) calibrated using pH
4.0, 7.0 and 9.0 standard buffers prior to use. The rheological study was
conducted with the Brookfield Viscometer (DV-E Brookfield
Engineering Labs Inc., Middleboro, MA, USA). The viscosity of QCT-NE
gel was evaluated at rotational speeds of 5, 10, 20, 30, 60 and 100 rpm
using spindle ≠ 7.
3.6.2. Texture profile analysis (TPA)
The texture profile analysis assists to estimate the mechanical
properties of the gel such as consistency, firmness, cohesiveness, work
of adhesion, gumminess, deformation at hardness and springiness. The
measurements were carried out by using a Brookfield CT3 Texture
analyzer (Brookfield Engineering Labs Inc, Middleboro, USA). A conical
shape sample holder was filled evenly with the gel sample. The probe
(TA3/100) was programmed to move down into the sample at a speed
of 0.5 mm/s with a target value 20 mm and then go up back at the same
speed to its original position. The TPA settings were as follows: pre-test
speed- 2.0 mm/s, test speed- 0.5 mm/s, return speed- 0.5 mm/s, target
mode distance- 10 mm, trigger load- 3 g trigger type – auto, data rate-
20 points per second. The force generated by the probe to break away
from the gel when starting to ascend (the point of maximum force) was
measured [35,36]. Adhesiveness, cohesiveness, hardness, gumminess,
springiness were evaluated. The measurements were carried out in
triplicate. The calculations were performed with the software provided
along with the instrument and the results were expressed as mean ±
SD.
3.6.3. Ex vivo permeation studies
Ex vivo skin permeation studies were performed with a Franz dif-
fusion cell (cell volume 10 mL) having diffusion area of 1.00 cm2,
diameter 11.28 mm using Wistar rat abdominal skin. The skin was ex-
cised, washed and stabilized using phosphate buffer in both the com-
partments with magnetic stirring for 30 min. After 30 min, solutions
from both the compartments were withdrawn and replaced with fresh
phosphate buffer having pH 5.8. A weighed quantity of QCT-NE gel and
free QCT gel (equivalent to 10 mg of QCT) was applied to skin mem-
brane placed on donor compartment. About 0.5 mL samples were
withdrawn at predetermined time intervals over a period of 24 h (0, 2,
4, 6, 8, 10, 12 and 24 h) from receptor compartment, filtered through
0.45 μm membrane filter and analyzed for QCT content by using HPLC
at 258 nm. Replacement with fresh medium was ensured to maintain a
constant volume and sink condition [37]. The permeation profile was
constructed by plotting amount of drug permeated per unit skin surface
area (mcg/cm2) Vs. Time (h). The steady state flux (Jss, mcg/cm2h) was
calculated from the slope of the linear portion of the plot using linear
regression analysis. All measurements were carried out in triplicate.
3.6.4. Antiarthritic activity [Complete Freund’s adjuvant (CFA) model]
All animal experiments were approved and performed following the
guidelines by Institutional Animal Ethical Committee of R.C. Patel
Institute of Pharmaceutical Education and Research, Shirpur, registered
under CPCSEA, India, registration number 651/PO/ReBi/S/02/CPCSEA
and protocol number IAEC/ RCPIPER/ 2014-15/04. Male Wistar rats
(six to seven weeks old), with average body weight of 140 ± 15 g,
were randomly divided into four groups of six animals each. The
J.P. Gokhale, et al.
animals in each group were injected with 0.1 mL of CFA into the sub-
plantar region of the left hind paw. This day was considered as day ‘0′.
Group I served as untreated arthritis- CFA control, Groups II, III, and IV
animals were treated with free QCT gel, QCT-NE gel (equivalent to
10 mg of QCT), and standard diclofenac gel; respectively. Dosing was
done twice a day and continued for 28 days, including the day of in-
oculation. The animals were housed in a controlled environment (at
temperature 20 °C ± 1 °C and 52% ± 15% RH) and had free access
with standard pellet diet and water ad libitum [38,39].
3.6.5. Evaluation of the severity of Arthritis
3.6.5.1. Measurements of paw volume. The volume of the left hind paw
was measured using plethysmometer at regular interval of 0, 7th, 14th,
21st and 28th day. Changes in the paw volume were determined by
measuring the paw volume (mm) at the initial day (Vo) and a particular
day (Vt) [28].
3.6.5.2. Measurement of Arthritis index and joint stiffness. Arthritic index
(AI) scores were allotted to ankle joints of the rats of each group.
Scoring was performed on a 0–4 scale as follows: 0- no swelling or
erythema, 1- slight swelling and/or erythema, 2- low to moderate
edema, 3- pronounced edema with limited joint use and 4- excess
edema with joint stiffness.
The scoring of the joint stiffness was carried out in accordance with
the assessment scale reported by Butler et al. [41]. The body of the rats
was held from the back, bending and extension (once in each direction)
of the ankle within its limit was observed. Scoring was given as per
scale- Score 2: when there was a restriction of full range of movement of
the ankle in both bending and extension. Score 1: there was a restriction
of full range of movement of the ankle in either bending or extension.
Score 0: no restriction [39,41].
3.6.5.3. Measurement of hematological parameters. The blood markers
provide additional information for the therapeutic evaluation of drugs
in RA. Therefore, the parameters such as RBCs, WBCs, erythrocyte
sedimentation rate (ESR), hemoglobin (Hb), C- reactive protein (CRP)
and rheumatoid factor (RF) were evaluated [38].
For the estimation of RBCs, WBCs, Hb and ESR, the blood sample
was collected through vein puncture and stored in a test tube with the
appropriate anticoagulant. The cell counting was performed with the
help of Automated Hematology Analyzer (Medonic M32B cell counter).
CRP and RF were evaluated on the basis of latex agglutination method
as per the test kit manufacturer’s instructions (Microsidd India Pvt. Ltd.,
Bengaluru, India).
3.6.6. Skin irritation studies
The possibility of skin irritation on the application of QCT gel and
the QCT-NE gel was evaluated by carrying out a skin irritation test on
Wistar rats. The rats were acclimatized to the conditions for seven days
prior to the commencement of the study. The dorsal surface of the rats
was made hairless without damaging the skin surface, 4 h before the
experiment. The rats were divided into four groups each containing
three rats: Group I was treated with 0.8% v/v aqueous solution of
formalin, group II was treated with a placebo gel, group III was treated
with free QCT gel, and group IV was treated with QCT-NE gel. The
formulations (gel containing 10 mg equivalent amount of QCT) were
topically applied to the hairless skin area (1cm2) and were inspected at
24, 48 and 72 h for dermal reactions such as erythema and edema. The
mean scores for erythema and edema were recorded on the basis of
their degree of severity caused by application of formulations: 0-no
erythema/edema, 1- slight erythema/ edema, 2- moderate erythema/
edema and 3- severe erythema/ edema [42–44].
3.6.7. Data analysis
All experiments were performed in triplicate (n = 3), and the data
were expressed as mean value ± SD. Statistical data analyses were
5
Biomedicine & Pharmacotherapy 112 (2019) 108622
performed using Student’s t-test and one-way ANOVA. In all cases, a
value of p < 0.05 was considered as statistically significant.
4. Results and discussion
4.1. Solubility studies
Usually, the oil phase was selected on the basis of highest solubility
for QCT and suitability for the topical application. In the present study,
various types of oils were selected to estimate the solubility of QCT.
QCT showed the highest solubility in arachis oil (11.14 mg/mL) among
the oils tested and therefore was selected as an oil phase. Secondly, the
highest solubility was in oleic acid (9.10 mg/mL), which was selected as
the solubility and permeation enhancer for QCT in an oil phase.
Solubility efficiency and HLB value were considered for the selec-
tion of surfactant and co- surfactant. Tween 20, having HLB value 16.7
and QCT solubility 9.28 mg/mL was selected as the surfactant. PEG-
400, having HLB value 11.4 and QCT solubility 13.59 mg/mL, was se-
lected as the co-surfactant [38,45].
4.2. An experimental design using Box–Behnken model
For the development and optimization of the formulation, a three-
factor, three-level BBD was applied using Design-Expert® 7.0.0 software
(Stat-Easc, Inc., Minneapolis, MN, USA). The independent variables
(factors) were selected as - (A) concentration of oil (%), (B) con-
centration of surfactant (%), (C) concentration of co-surfactant (%). The
dependent variables (responses) were selected as- (Y1) mean globule
size (nm) and (Y2) (%) EE. Table 1 shows the factor levels and response
data for BBD.
The analysis of experimental results was carried out by using
Design-Expert® 7.0.0 software. After compilation of the data, F- values,
p-values, and model F-value for the mean globule size and % EE were
obtained from ANOVA. The value of responses Y1 (mean globule size)
and Y2 (% EE) ranges from 125.4 to 145.3 nm and 85.82 to 95.76%
respectively. The ratio of the maximum to the minimum for responses
Y1 and Y2 were 1.1586 and 1.1158 respectively. Therefore power
transformation of values is not required for mean globule size and % EE.
Transformation of response is an essential component of data analysis.
Transformation is required if the error (residuals) is a function of the
magnitude of response (predicted values). In simpler terms, power
transformation of responses is required when the ratio of maximum to
minimum response is higher than 10. For ratios less than 3, the trans-
formation has little effect. The selection of the model for analyzing the
responses was made based on the sequential model sum of squares, lack
of fit and model summary statistics. The Prob > F value of
p < 0.0001, low standard deviation, high R2 and lower predicted re-
sidual error sum of square (PRESS) value suggests to select the quad-
ratic model for both responses. ANOVA of the data confirms that the
model was significant (Model Prob > F < 0.05). The Model F value
for response Y1 and Y2 was 921.97 and 18.63 respectively, which im-
plies model is significant. ANOVA identifies the significant factors that
affect the responses. For mean globule size, oil, surfactant, and co-
surfactant were identified as significant model terms whereas, for % EE,
oil was found to be the significant model term. Lack of fit F-value for Y1
was found to be 1.65 which implies lack of fit was not significant re-
lative to the pure error, and for Y2 it was 10.64 which imply lack of fit
was significant relative to the pure error. The multiple regression terms
were also analyzed. The predicted R2 values for response Y1 and Y2
were 0.9919 and 0.9032 respectively. Adjusted R2 values for response
Y1 and Y2 were 0.9981 and 0.9084 respectively. The predicted R2 value
was found to be in reasonable agreement with adjusted R2 value, which
indicates that the model has predicted the responses well. Adeq preci-
sion for response Y1 and Y2 were 96.139 and 13.867 respectively. Adeq
precision measures the signal to noise ratio. A ratio higher than four is
desirable. The ratio of 96.139 and 13.867 indicates an adequate signal.
J.P. Gokhale, et al. Biomedicine & Pharmacotherapy 112 (2019) 108622

Fig. 1. (a) Effect of conc. of oil and conc. of surfactant on globule size, (b) effect of conc. of oil and concentration of co-surfactant on globule size, (c) effect of conc. of
surfactant and co-surfactant on globule size. In case of mean globule size, it was found to increase with increase in the (%) concentration of oil, surfactant, and co-
surfactant.

This model can be used to navigate the design space. Final equations in
term of coded factors for responses Y1 and Y2 were as follows:
(Y 1) Mean globule size = + 137.52 + 10.11 * A + 1.10 * B + 1.21 * C
0.88 * A * B 0.90 * A * C 0.52 * B * C
0.46 * A2 + 0.16 * B2 + 1.39 * C
(Y 2) % EE = + 93.47 + 3.73 * A + 0.47 * B 0.34 * C 0.58 * A * B
1.56 * A * C + 1.50 * B * C 1.34 * A 1.41 * B 2
1.73 * C 2
Where A- Concentration of oil (%), B- Concentration of surfactant (%)
and C- Concentration of co-surfactant (%)
In case of response Y1, i.e. globule size, positive coefficients of A, B,
and C indicate mean globule size was found to increase with increase in
the concentration of oil, surfactant, and co-surfactant [see
(Fig. 1(a)–(c)]. For response Y2, i.e. % EE, positive coefficients of A and
B indicates % EE was found to increase with increase in the con-
centration of oil and surfactant and the negative coefficient of C in-
dicates % EE decreases with increase in co-surfactant concentration
[see Fig. 2(a)–(c)]. Very high concentrations of oil and Smix resulted in
the formation of turbidity or microemulsion rather than nanosized
globules. The mean globule size of the nanoemulsion is a critical factor
in emulsification development as it affects not only the rate and extent
of drug release but also the absorption.
Perturbation graphs were plotted to locate the factors that affect the
response [see Fig. 3(a), (b)]. A steep slope or curvature in a factor
shows that response is sensitive to change in that factor; whereas re-
latively flat line shows insensitivity to the factor. If there are more than
(2) two factors, a perturbation plot could be beneficial to locate the factors
having maximum influence on the response. For response Y1, factor A
indicates noticeable bend or steep curvature. Factor B and C indicate a
slight bend, which implies that the concentration of oil effects mean
globule size. For response Y2, factor A, B, and C show a noticeable bend
(3) or steep curvature. It specifies that the concentration of oil, surfactant,
and co-surfactant shows significant changes in % EE.
Optimization of nanoemulsion was carried out to find the level of
factors A, B, and C, which gives Y1 in the range of 125.4–145.4 nm and
Y2 in the range of 85.82–95.76 %. The model predicted Y1 and Y2 in
the required range at A, B and C values of 12.47%, 8.79%, and 8.66%
respectively. The predicted value of responses Y1 and Y2 were
137.1 nm and 91.01%. By using these values of factors, three different
batches of nanoemulsion were prepared. The actual values of Y1 and Y2
were found to be 136.8 ± 1.2 nm and 93.7 ± 1.9%, which is in close
agreement with the predicted values.
All the batches conducted as per BBD, were subjected to thermo-
dynamic stress tests. Amongst all the batches F2, F3, F5, F7, F9, F11,
F16 and F17 passed the thermodynamic tests. It was observed that F3

Fig. 2. (a) Effect of conc. of oil and concentration of surfactant on % EE, (b) effect of conc. of oil and conc. of co-surfactant on % EE, (c) effect of conc. of surfactant
and co-surfactant on % EE. In case of %EE, it was found to increase with increase in the (%) concentration of oil and surfactant and decreases with increase in (%)
concentration of co-surfactant.

6
J.P. Gokhale, et al.
Fig. 3. Perturbation graph for effect of individual factor (a) on response Y1 (globule size) and (b) on response Y2 (% EE).
Fig. 4. Morphology of QCT loaded nanoemulsion globules visualized under
Transmission electron microscopy (TEM) voltage of 60–80 kV (Magnification
×10,000, scale of 200 nm).
had greater amount of oil and larger globule size (i.e. 142.70 nm) as
compared to F5 having lowest % EE (86.16%). Even having higher %EE
(94.12%), F7 has largest globule size (144.7 nm). So, by comparing the
selected batches F16 batch was selected for further processing which
having lower globule size (137.6 nm) and higher %EE (94.49%). Hence,
the best suited composition of the optimized formulation was selected
as oil phase (15%), surfactant (6%) and co-surfactant (6%) i.e. (F16).
Optimization was done on the basis of globule size and %EE.
The results of ANOVA for the dependent variables demonstrated
that the model was significant for the mean globule size and the %EE. In
case of mean globule size, the model F-value of 921.97 implies the
model is significant. The value p < 0.05 indicates model terms are
significant. In this case, A, B, C, AB, AC, BC, A2, B2, C2 are significant
model terms. Values higher than 0.1000 indicates the model terms are
not significant. In the case of %EE, The model F-value of 18.63 implies
the model is significant. The value p < 0.05 indicates model terms are
significant. In this case, A, AC, BC, A2, B2, C2 are significant model
terms.
4.3. Characterization of optimized QCT-NE formulation
4.3.1. Identification tests for nanoemulsion
4.3.1.1. Staining test. The optimized formulation (F16) was found to be
o/w type nanoemulsion. For staining, oil soluble dye was used. The
background was colorless and the oil globules were found red in color.
4.3.1.2. Dilution test. Visually, QCT-NE was observed clear and
transparent with bluish tinge. Furthermore, the diluted formulation
showed no sign of phase separation or cracking.
4.3.1.3. Electrical conductivity. Electrical conductivity is directly
proportional to the percentage of water. The o/w nano-emulsion
7
Biomedicine & Pharmacotherapy 112 (2019) 108622
passes the conductivity test due to water in continuous phase while,
w/o nano-emulsion vice-versa. The conductivity (σ) of QCT-NE
formulation was found to be 489.53 ± 1.08 μS/cm. This indicates
that the formulation was o/w type of nanoemulsion (n = 3).
4.3.2. Mean globule size, zeta potential, and morphology
The mean globule size of an optimized nanoemulsion was found to
be 136.8 ± 1.2 nm, and polydispersity index was 0.265 ± 0.3. The
mean globule size below 200 nm, having a polydispersity index below
one is considered ideal for topical delivery, as it would provide a large
surface area in addition, appropriate for rapid pore transport. The ne-
gative zeta potential value -25.4 ± 1.7 mV was considered satisfactory
in order to prevent coalescence and maintain the interfacial boundary
of the nanoemulsion droplets resulting in better colloidal system sta-
bility [13].
In the morphological studies (Fig. 4), the globules were found to be
distributed evenly with uniform sizes. Also, were spherical with a
smooth, flexible boundary, non-aggregated showing their stability
against Oswald ripening due to globular collapse.
4.3.3. Viscosity, pH, refractive index
The viscosity of the nanoemulsion was found to be 89.5 ± 1.41 cP.
The pH was observed to be 5.61 ± 0.07 which is entirely compatible
with the pH of the skin, which would therefore be non-irritant.
Refractive index is the net value of the components of nanoemulsion
and indicates the isotropic nature of the formulation. RI value of na-
noemulsion was found to be 1.379 ± 0.041, which reveals transpar-
ency of nanoemulsion formulation.
4.3.4. The degree of transparency, drug content, %EE
Percentage transmittance of nanoemulsion was found to be
97.68 ± 0.05%. The value closer to 100% indicates clarity and trans-
parency of formulation.
The drug content in nanoemulsion was found to be 95.65 ± 0.14%;
the batch was therefore selected for the development of gel formula-
tion.
Entrapment efficiency of the optimized nanoemulsion was found out
94.65 ± 0.14%.
4.3.5. In vitro drug release
The nanoemulsion is an excellent tool for enhancing the solubility of
hydrophobic drugs thereby the bioavailability of the drug owing to
small-scale globule size. In order to achieve better drug release and
therapeutic effect, drug was distributed into the nanoemulsion globules.
Therefore, the release of QCT from the nanoemulsion globules as
compared to free QCT suspension was observed. At the end of 4 h,
28.23 ± 1.72% of drug was released from free QCT suspension which
J.P. Gokhale, et al.
Table 2
Stability study of QCT-NE on storage.
Parameter 0 days
pH 5.81 ± 0.03
Globule size (nm) 137.6 ± 1.38
PDI 0.273 ± 1.52
Zeta potential (mV) −25.18 ± 1.97
Refractive Index 1.379 ± 0.041
Viscosity (cP) 89.5 ± 1.41
% Drug content 95.65 ± 0.14
Data is expressed as mean ± SD (n = 3), (p > 0.05).
was considerably lower (p = 0.002) than the drug was released from
QCT- NE (84.12 ± 0.51%). Higher release from nanoemulsion can be
attributed to smaller globule size, which increases the surface area for
diffusion. Hence it can be stated that, QCT-NE not only improved the
solubility of QCT but also the diffusion rate owing to its lipoidal nature
and small globule size.
4.3.6. Stability studies
The stability of QCT-NE in terms of globule size, PDI, zeta potential,
viscosity and refractive index was estimated at 0, 60, 120 and 180 days
on storage at specific temperatures. These parameters shown varying
trend with respect to time, nevertheless the changes in the studied
parameters were not statistically significant (p > 0.05). The results
were expressed as mean ± SD (n = 3) (Table 2).
4.3.7. Cytotoxicity study
The relative growth rate of cells at 48 h in free QCT and QCT-NE
was found to be 104 ± 3.2% and 130 ± 0.7%, respectively. The re-
sults indicate that the presence of QCT-NE does not inhibit the growth
of synoviocytes.
4.3.8. Effect on production of TNF-α
In the pathogenesis of RA, TNF-α plays a significant role in the
proliferation of fibroblast, stimulation of proinflammatory mediators
like PGE2, IL-1β. Thereby it promotes synovitis and stimulates cartilage
and bone breakdown [38].
LPS induced TNF-α secretion from RAW 264.7 cells was sig-
nificantly reduced post QCT-NE treatment as compared to free QCT
suspension (Fig. 5) (p = 0.041).This finding shows that the anti-ar-
thritic effects of QCT-NE complex may be mediated by the inhibition of
inflammatory cytokines like TNF- α. The inhibition of TNF-α produc-
tion is linked with suppression of inflammation and cartilage destruc-
tion which can slow down the arthritis progression.
Fig. 5. Effect of QCT concentration in formulations on TNF-α level Results are
expressed as mean ± SD (n = 3).Results were found statistically significant
(p = 0.041) as compared to free QCT suspension.
Biomedicine & Pharmacotherapy 112 (2019) 108622
60 days 120 days 180 days
5.88 ± 0.17 5.97 ± 1.20 6.08 ± 1.53
138.66 ± 0.77 142.32 ± 1.98 146.2 ± 2.12
0.286 ± 0.03 0.289 ± 1.051 0.331 ± 0.73
−25.32 ± 0.44 −26.32 ± 0.67 −27.3 ± 0.23
1.390 ± 0.007 1.461 ± 0.003 1.553 ± 0.123
86.62 ± 1.51 86.43 ± 0.96 86.97 ± 1.21
95.40 ± 0.52 94.92 ± 1.02 94.78 ± 1.36
4.4. Characterization of QCT-NE gel
4.4.1. pH and rheological measurements
The apparent pH of QCT-NE gel was found to be 5.81 ± 0.03. The
pH is slightly acidic and is physiologically compatible with the pH of
the skin, hence would be non-irritant. The viscosity of QCT-NE gel was
found to be 71,210 ± 1.12 cP at 5 rpm. Further the viscosity of QCT-
NE gel was found out decreased with the increase in rpm. In fact, with
the increase in rpm, the internal structure of the gels began to disrupt as
the contact points broke. The particles were aligned until they started to
flow and their consistency gradually decreased with rpm. The gel ex-
hibited pseudoplastic flow behavior. However, no evidence of thixo-
tropy was observed. To calculate the flow index, the power law model
was applied to the rheological properties of the gel formulation. The
flow behavior index of QCT- NE gel was found to be 0.415 ± 0.35
confirming the shear thinning behavior of the gel.
4.4.2. Texture profile analysis
Formulations intended for topical application, must show accep-
table mechanical characteristics, e.g., ease of application, stickiness,
firmness or hardness, etc. These mechanical properties of QCT-NE gel
were studied by texture profile analysis. Results of the texture profile
analysis were generated by Texture PRO CT 1.4 build 17 version soft-
ware.
Adhesiveness value of QCT- NE gel was found to be 0.3 ± 0.61 mJ.
The value near to zero implies that QCT- NE gel is more adhesive and
has a soft texture.
The cohesiveness (consistency) indicates the strength of internal
bonds that make up the gel sample and the extent to which the gel
sample can be deformed before the application. The cohesiveness of the
sample is shown by the maximum negative force. Cohesiveness value
was found to be 0.79 ± 1.98.
Gumminess is the product of hardness and cohesiveness. It is the
critical parameter of gel system with the low hardness and the high
cohesiveness extent. The higher gumminess value implies more
sample hardness. Gumminess of the QCT-NE gel was found to be
15 ± 1.34 mg.
The hardness is related to the strength of the compressed gel
structure and is the maximum force during the first compression cycle.
In sensory aspects, it is the maximum force required to apply the gel
system on the skin surface. Higher the value of hardness implies thicker
consistency of the gel system. The hardness value of QCT- NE gel was
found to be 19 ± 0.12 g.
Springiness is the rate at which a deformed sample reverses to its
undeformed state after the removal of deforming force. The springiness
value of QCT-NE gel was found to be 2.73 ± 1.32 mm. The high value
of springiness indicates that additional force is required for the appli-
cation of the gel system. [40].
4.4.3. Ex vivo permeation studies
Ex vivo permeation study showed that the drug from QCT- NE gel
permeated rapidly than free QCT gel. At the end of 24 h,
62.51 ± 0.34% and 35.87 ± 0.21% of the drug was permeated from
8
J.P. Gokhale, et al. Biomedicine & Pharmacotherapy 112 (2019) 108622

Fig. 6. Effect of QCT-NE gel on rats following CFA induced arthritis (a) effect on arthritic index, (b) effect on stiffness score (c) effect on paw circumference. [Results
were expressed as mean ± SD (n = 3). Results were found statistically significant (p < 0.05) as compared to CFA-control group].

Table 3
Alterations in hematological parameters, CRP and RF in CFA-induced arthritis in rats.
Group RBC (×106/mm3) WBC (×103/mm3) ESR (mm/h) Hb (mg%) CRP (mg/dL) RF (IU/mL)

Group I-CFA control 7.4 ± 0.5 12 ± 0.9 15 ± 0.5 11.5 ± 1.2 8.7 ± 0.2 71 ± 1.3
Group II- QCT- gel 8.2 ± 0.6 7.9 ± 0.5 10.5 ± 0.5 13.7 ± 0.2 3.6 ± 0.8 34 ± 1.9
Group III- QCT-NE gel 8.8 ± 0.4 5.6 ± 1.5 9.9 ± 1.13 15.0 ± 0.1 2.1 ± 0.3 23 ± 0.2
Group IV- Std. DCS gel 8.9 ± 1.7 5.7 ± 0.13 9.2 ± 1.4 14.3 ± 0.5 1.9 ± 0.9 22 ± 1.6

Data is expressed as (mean ± SD, n = 3) (p < 0.05) when compared to CFA- control group.

QCT- NE gel and free QCT gel respectively. Permeability coefficient of
free QCT gel and the QCT-NE gel was found to be 1.6 cm2/ min and
2.7 cm2/ min respectively. This study showed a significant improve-
ment (p = 0.04) of cumulative permeation of QCT from nanoemulsion
gels compared to the gel containing free QCT. The nanoemulsion due to
its small droplet size (< 200 nm), significantly increases the rate of
permeation since the nanosized droplets can transfer the drug through
the skin barrier and easily move into the stratum corneum. In addition,
the Smix increases the solubilization capacity and permeability coeffi-
cient of the skin as the interfacial barrier decreases and partitioning
improves due to small droplet size. Water in the gel system hydrates the
skin and causes the cells to swell in the stratum corneum, widening
drug channels, leading to improved cumulative permeation [26,28].
4.4.4. Antiarthritic activity
4.4.4.1. Measurements of paw volume. After two weeks of CFA
administration, rheumatoid arthritis was evident in rats. A significant
increase in paw circumference, erythema, swelling, joint stiffness and
hindrance in the movement was observed. Paw volume was recorded by
using digital plethysmometer on 7th, 14th, 21st and 28th day after CFA
injection. The paw circumference of CFA control group was
71.21 ± 0.33 mm, which was found to be decreased up to
51.13 ± 1.35 mm with the QCT-NE gel. The inhibition of paw
volume by QCT-NE gel was found statistically significant as compared
to CFA-control group (p = 0.006).
4.4.4.2. Arthritis index and stiffness score. The arthritic index is used to
define the severity of joint inflammation based on a calculation of
scores of the paws after arthritis induction [20]. Arthritis indices were
recorded on the 7th, 14th, 21st and 28th day after CFA injection. The
arthritic index and stiffness score of the CFA-control group were
3.7 ± 1.61 and 2.1 ± 1.53 respectively, which were reduced by
QCT-NE gel treatment i.e.1.6 ± 1.27 and 0.73 ± 0.42 respectively.
4th–14th days mark the proliferative phase which is characteristic of
inflammation, hyperplasia and macrophage activities in the synovial
membrane. The results of arthritic index and stiffness score were
found statistically significant in case of QCT-NE gel as compared to
CFA-control group (p = 0.003 and 0.02 respectively). Results of the
21st and 28th day evaluation showed that the QCT-NE gel treatment
significantly reduced CFA-induced arthritic lesions such as increased
paw circumference, erythema, swelling, joint stiffness and obstruction
in the movement, as compared to the CFA- control group. Results of
the arthritic index, stiffness score and paw circumference of group I to
IV are shown in Fig. 6.
4.4.4.3. Measurement of hematological parameters. In the CFA-control
rats, the levels of RBCs and Hb were found to be decreased initially
which is one of the clinical manifestations of RA. The QCT-NE gel
formulation treated group showed significant increase in RBCs (8.8 ±
0.4 × 106/mm3)) and Hb level (15.0 ± 0.1 mg %) as compared to
RBCs (7.4 ± 0.5 × 106/mm3) and Hb level (11.5 ± 1.2 mg %) of CFA-
control group (p = 0.007 and 0.005 respectively).
In the ESR test, the rapid sedimentation rate is an indication of the
inflammation; which was found to be decreased from 15 ± 0.5 mm/h
to 9.9 ± 1.13 mm/h with the QCT- NE gel treatment. An elevated WBC
count is also one of the characteristic arthritis diagnoses. After the
treatment with QCT-NE gel formulation, WBCs level was effectively
reduced from 12 ± 0.9×103/mm3 to 5.6 ± 1.5 × 103/mm3. Results
were found to be statistically significant (p = 0.007) as compared to
CFA-control group.

9
J.P. Gokhale, et al.
In recent years, CRP has been recognized as significant predictors for
RA diagnosis. CRP is a member of the pentraxin family of proteins, pro-
duced by the liver in response to inflammation. It plays a significant role in
bone destruction process, as it is found in lining areas of RA synovium. RF
is considered to be the main serological marker in the inflammatory ar-
thritis found in 80% of arthritis conditions. RF is an auto antibody directed
against the Fc portion of IgG antibodies. RF and IgG join in forming im-
mune complexes that contribute to the progress of RA [46]. Elevated levels
of CRP and RF show systemic inflammation and antibody production
against the injected adjuvant, which has also been found to be significantly
reduced by QCT-NE gel, as p = 0.032 and 0.004 respectively as compared
to CFA-control group. All the results are summarized in Table 3.
4.4.5. Skin irritation studies
The topical application should be free of skin irritation and er-
ythematic reactions. Conventional therapy has been linked to notice-
able skin irritation, which actively restricts its applicability and patient
acceptability. In this study, the results revealed no severe skin irritation
symptoms such as erythema (redness) and/or edema (swelling) within
72 h of QCT-NE gel application. The developed formulation can there-
fore be considered as non-sensitizing, non-irritating and safe for topical
use on the rat skin.
5. Conclusion
In conclusion, present research work emphasizes the development
of a novel formulation through topical administration in order to
overcome the drawbacks of QCT administration. QCT is extensively
used phytoconstituent having a similar activity to the NSAIDs. Even
though, it has limited applicability via topical route due to low skin
permeability. Incorporation of oleic acid: arachis oil, tween 20 and
PEG-400 (15:6:6) resulted in the spontaneous formation of nanoemul-
sion and successful carrier system for QCT. The optimized formulation
showed improved physicochemical stability, acceptable mechanical
properties and enhanced skin permeability thus increasing the ther-
apeutic efficacy, as revealed by its antiarthritic activity. Hence the
studies have confirmed that QCT-NE gel will be a promising alternative
in rheumatic complications via topical application.
Declaration of interest
The authors declare that there are no conflicts of interest.
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