Drug Development and Industrial Pharmacy

ISSN: 0363-9045 (Print) 1520-5762 (Online) Journal homepage: https://www.tandfonline.com/loi/iddi20

Formulation and evaluation of Thymoquinone

niosomes: Application of developed and validated
RP-HPLC method in delivery system

Sadaf Jamal Gilani, Syed Sarim Imam, Adil Ahmed, Sanjay Chauhan, Mohd.
Aamir Mirza & Mohamad Taleuzzaman

To cite this article: Sadaf Jamal Gilani, Syed Sarim Imam, Adil Ahmed, Sanjay Chauhan, Mohd.
Aamir Mirza & Mohamad Taleuzzaman (2019): Formulation and evaluation of Thymoquinone
niosomes: Application of developed and validated RP-HPLC method in delivery system, Drug
Development and Industrial Pharmacy, DOI: 10.1080/03639045.2019.1660366

To link to this article: https://doi.org/10.1080/03639045.2019.1660366

Accepted author version posted online: 26

Aug 2019.

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Formulation and evaluation of Thymoquinone niosomes: Application of developed and
validated RP-HPLC method in delivery system

Sadaf Jamal Gilani1, Syed Sarim Imam2, Adil Ahmed3, Sanjay Chauhan2, Mohd. Aamir Mirza3,
Mohamad Taleuzzaman*2

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1. College of Pharmacy, Jouf University, Aljouf, Sakaka, KSA.
2. Glocal School of Pharmacy, Glocal University, Saharanpur, Uttar Pradesh, India.

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3. School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, India.

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Corresponding Author*
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Dr. Mohamad Taleuzzaman

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Associate Professor
Department of Pharmaceutical Chemistry
Glocal School of Pharmacy
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Glocal University, Saharanpur, 247121

Uttar Pradesh, India
Email.ID- [email protected]
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M: +91-7251892850
Abstract

A rapid, accurate, and sensitive reverse phase high-performance liquid chromatographic (RP-
HPLC) method was developed and validated for estimation of Thymoquinone (TMQ) in API as
well as in noisome. The chromatograms were developed with mobile phase- water: 2-propanol:
methanol (50:45:5 v/v/v) as solvent system at 254 nm. The method was validated as per ICH
guidelines for different parameters and the recovery of TMQ was calculated in developed
niosomes. Further, TMQ niosomes (TMQNIOS) were prepared and evaluated for different
parameters. The optimized formulation TMQNIOSopt was further evaluated for surface

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morphology, in-vitro drug release, permeation study, and confocal laser scanning microscopic
(CLSM) study. The method showed linearity range between 6.25 - 100 µg/mL with low

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detection limit and quantitation limit with value of 2.08 and 6.25 μg/mL. The developed

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formulation showed the vesicle size and encapsulation efficiency in the range of 157.32 ± 3.15 to
211.44 ± 5.23 nm and 59.32 ± 4.87 to 83.21 ± 3.55 %, respectively. The drug release result
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showed the significant higher release from TMQNIOS in compare to TMQDIS, and the release
kinetics data showed Higuchi's equation with highest regression coefficient values. The
permeation study and confocal laser microscopy study further confirmed the enhancement in
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permeation of TMQ in the intestinal mucosa.
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Key Words: Thymoquinone, Niosomes, HPLC, Validation, CLSM

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Abbreviations: TMQ: Thymoquinone; TMQNIOSopt: optimized thymoquinone niosomes;

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TMQDIS: Thymoquinone dispersion; RP-HPLC: Reverse phase high pressure liquid

chromatography; CHL: Cholesterol, API: Active pharmaceutical ingredients; CLSM: Confocal
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laser scanning microscopy.

Introduction
Thymoquinone (TMQ; Figure 1) is a natural phytochemical isolated from the seed of
Nigella Sativa belongs to family Ranunculaceae [1]. It is the major bioactive compound having
IUPAC name 2-methyl-5-propane-2-yl-cyclohexane-2, 5-diene-1, 4-dione phenol and molecular
formula C10H12O2. Due to the poor aqueous solubility (550 – 650) µg/ml in different aqueous
solvents [2], its use is limited and may lead to poor dissolution and bioavailability [3]. There are
numerous pharmacological activity have been reported for TMQ as well as Nigella Sativa [4-5].
Previously many literatures reported for different TMQ loaded formulation and evaluated for

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number of pharmacological effect. The several nano formulations have been adopted to enhance
the solubility, dissolution characteristics and biological activity of TMQ by formulating it into

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chitosan vesicles [6], nanoparticles [7,8] and nano formulation [9,10].
There are numerous analytical techniques have been used for the determination of

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TMQ in black seed oil such as UV-spectrophotometer [11], TLC [12], HPLC-UV methods [2,13-
15], and HPTLC [16,17]. The reported HPLC-UV methods are primarily associated with
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multiple intricacies like use of high cost organic solvents, buffers, high flow rate and close
monitoring of controlled conditions like flow gradient, lengthy separation times, column oven
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temperature, pH, etc., for attaining consistent method performance. The literature reports suggest
that none of the available methods for TMQ is reliable enough to provide systematic and
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comprehensive understanding of the method performance and their application in vitro

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formulation evaluation. The present method designed to develop and validate new HPLC method
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utilizing simple, economical mobile phase system with low flow rate to quantify the TMQ in
delivery system.
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The majority of drugs delivered by oral route and faces bioavailability problems due to
poor solubility, poor dissolution and lack dose proportionality [18]. The colloidal delivery
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system such as liposomes [19], niosomes or proniosomes [20,21] have shown distinct advantages
over conventional delivery system. These delivery systems reported to ensure enhanced
solubility, prolonged drug release, better therapeutic effect and greater bioavailability [6,22,23].
There is no research report published with the combination of development of novel
HPLC method for TMQ and the formulation and evaluation of TMQ niosomes. The study was
designed in two steps, in first step development and validation of newer simple and sensitive RP-
HPLC method using new simple solvent system as mobile phase. In the second step TMQ loaded
nano sized niosomes was prepared and evaluated for different parameters. The developed and
validated HPLC method was further used to analyze the TMQ in niosomes for parameters like
encapsulation efficiency, ex vivo permeation study, in vitro drug release to ascertain the potential
of developed method. The proposed method has distinct advantages over previously reported
methods with its cost effectiveness, short run time and application in the estimation of TMQ in
delivery system.

Material and Methods

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Materials
Chemicals and Reagents

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TMQ (purity >99%), was obtained from Sigma Aldrich USA. Cholesterol, non ionic surfactants
(span 20, span 40, span 60, tween 20, tween 40, tween 80) and cholesterol were purchased from

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SD Fine chemicals, Mumbai, India. Methanol and chloroform (AR grade) were purchased
Thomas Baker, Mumbai India. All aqueous solutions, buffers were prepared using double
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distilled water and reagents used were of analytical grade.
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Methods
HPLC Instrumentation and chromatographic conditions
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The method development and validation was performed on HPLC system (Waters- 2695)
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connected to UV detector (Waters 2475 Multi Lambda). The LC system consist of binary pump,
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(model 1525; Milford, USA), and the column used for the study is C18 (150 mm × 3.9 mm × 5
μm). The mobile phase composition water: 2-propanol: methanol (50:45:5 v/v/v) with flow rate
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of 1 mL/min used for the study. The injection volume (20 μL) and the detection of the sample
was done at 254 nm. The data acquisition was carried out using Empower software.
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Validation
Linearity
The calibration range of TMQ was prepared by serial dilution of stock solution (1mg/mL). From
the stock solution, further dilution was done in mobile phase to get final concentration range
between 6.25 – 100 μg/mL, (Table 1). Finally, the plot between concentration vs area was drawn
and linear regression analysis was determined. The LOD and LOQ values were determined using
the slope of the calibration curve and the data was calculated as per reported procedure [24].
Accuracy
The accuracy of the developed method was performed by standard addition method using three
samples at three different concentrations. The sample TMQ (25μg/mL) was injected with the
addition of 50, 100, and 150% of the standard TMQ. The recovery (%) of sample at different
concentration was assayed and the recovered TMQ at each concentration was calculated.
Robustness
To check the robustness of the method there was small and deliberate changes in the
chromatographic condition was done to check the variation in the results. The variations were

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done in the flow rate (1.1 and 0.9 mL/min), mobile phase ratio (49:46:05 and 51:44:05v/v/v),
sample injection volume (19.5 and 20.5µL) and wavelength (249 and 259 nm). The result of the

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robustness study was observed on TMQ recovery, retention time and area (Table 2).
Formulation of TMQ niosomes

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The formulation TMQ niosomes were prepared and optimized by solvent evaporation and
hydration method with slight modification [25]. The formulations were prepared with calculated
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quantity of TMQ (25 mg) as active agent, cholesterol as vesicle membrane stabilizer, non-ionic
surfactant (span 20, span 40, span 60, tween 20, tween 40, and tween 80) and soya lecithin (10
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mg) as lipid given in Table 3. The ingredients were taken in a clean and dry round bottom flask
and dissolved in chloroform: methanol mixture (1:1) as organic solvent (10 mL). The flask was
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shaken to solubilize the ingredients and the organic solvent was evaporated to form thin lipid
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film at reduced pressure using rotary vacuum evaporator (Hahnshin Scientific Co., South Korea).
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The flask was taken from evaporator and kept in vacuum condition (24 hr) to remove the residue
of organic solvent. The dried thin lipid film was rehydrated (1 hr) above the lipid transition
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temperature using phosphate-buffer (10 mL) to get TMQ niosome. The prepared TMQNIOS was
sonicated using probe sonicator (Hielscher; Germany) in ice cold condition (4°C). The sonication
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was performed for three cycles with a gap of 5 min and one cycle run for 30 seconds to reduce
the size. Finally, the prepared formulations TMQNIOS was left overnight and stored in
refrigerator for further studies.
Vesicle size, PDI and zeta potential
The prepared formulation TMQNIOS size, PDI and zeta potential were determined by using size
analyzer (Malvern Instruments, Malvern, UK). The sample (0.1 mL) taken and diluted (100
times) with distilled water to avoid multi-scattering phenomena. The PDI value ˂0.1 indicate
homogenous population and ˃0.3 indicates high heterogenecity among the particles [26]. The
particles have a large negative or positive zeta potential then they will tend to repel each other
and there will be no tendency for the particles to come together. The general dividing line
between stable and unstable dispersions is generally taken at either +30 or -30 mV [27].
Entrapment efficiency
The entrapment efficiency (%EE) of the prepared TMQNIOS was determined by
ultracentrifugation method [25] using ultracentrifuge. The formulation TMQNIOS (2mL) was
filled in the tube and centrifuged at 15000 rpm for 30 min at 4°C to separate the untrapped TMQ.

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The collected supernatant was diluted, filtered and TMQ content was analyzed as per above
explained HPLC method. EE was calculated from the difference between the initial amount of

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TMQ added and present in unentrapped form.
Surface morphology

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The transmission electron microscopy was used to evaluate the surface morphology of
TMQNIOSopt. The sample (one drop) was taken on a carbon-coated copper grid and image
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contrasting agent phosphotungistic acid solution (2%, w/v) was added to the sample. The sample
was kept aside for some time and excess dye was removed with filter paper. The prepared
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sample was air-dried and observed for morphology using soft imaging viewer software.
In-vitro drug release
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The dialysis membrane method employed to study the comparative release behavior of
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TMQNIOS and TMQDIS (2 mL contains 5 mg of TMQ). Both the formulation were placed in
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the dialysis bag and tied from both the end to prevent the leakage. The bag was immersed in
phosphate buffer (250 mL) as release medium and the study was performed at fixed temperature
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37ºC ± 0.5°C. The sample (1 mL) was collected at predefined time intervals and replenished with
the same volume of fresh medium. The collected sample at each time point was analyzed as
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above explained HPLC method. The obtained drug release vs time data fitted to various
mathematical kinetic equations (zero order, first order, Higuchi and Korsmeyer Peppas models)
and regression analysis was analyzed [28].
Permeation study
The comparative intestinal permeation of the developed formulation TMQNIOSopt and
TMQDIS was evaluated to assess the amount of TMQ permeated. The study was performed as
per reported method in the literature and the enhancement ratio was calculated by comparing flux
of both the sample [29]. Both the formulation TMQNIOSopt and TMQDIS (each containing 5mg
TMQ) were filled in the intestinal sac at the mucosal side and tightly ligated. Both the
formulation filled sac was immersed in Krebs solution (250 mL) and the study was performed at
37 ± 0.5°C with continuous stirring. The Kreb’s solution was continuously oxygenated using an
aerator and after specified time interval the permeated samples (1mL) were collected and
replaced with fresh solution. The permeated samples were filtered through membrane filter
(0.2µ) and drug content at each time point was assayed as per the above developed HPLC
method. The graph was plotted between the cumulative amount of drug permeated (µg) vs. time

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and permeation flux (F) was calculated.
Confocal laser scanning microscopy

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The Rhodamine Red dye (0.03% w/v) loaded TMQNIOSopt and TMQDIS (2 mL) were prepared
separately to assess the permeation depth. The goat intestine was collected from slaughter house

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and washed repeatedly three times with Kreb’s solution to remove all the food content. Both the
samples filled in the sac and tied from both the ends and kept in Kreb’s solution for specific time
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intervals (6 hours). The study was performed with continuous stirring (50 rpm) and the
temperature was maintained at 37ºC. After the completion of study the intestinal sac was cut
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longitudinally and remaining formulations were removed. The intestinal sac was washed with
double distilled water to remove the excess dye. The permeation depth was evaluated using CLS
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microscope (Leica TC SPE-IIw) and image was visualized by Leica application suite Advance
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Fluorescence software.
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Results
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Optimization of chromatographic condition

The RP-HPLC method was performed with an aim to develop sensitive and robust method to
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determine TMQ in API as well as in niosomes. The preliminary investigations were performed to
optimize the final mobile phase system to get a sharp peak and better chromatographic
separation. From the results of different tried mobile phase composition the optimized mobile
phase was found to be water: 2-propanol: methanol (50:45:5 v/v/v) and shown the retention time
of 7.7 min (Figure 2).
Linearity
The concentration ranges of 6.25-100 μg/mL showed the linearity of TMQ and their linear
regression data shown in Table 1. The correlation coefficient value (R2) was found to be closer
to unity (0.998). The method showed the linear regression equation Y=204.2x+1350. The
method showed the LOD and LOQ value of TMQ was 2.08 μg/mL and 6.25 μg/mL,
respectively. The lower detection and quantification value supports the study that the developed
method was found to sensitive.
Accuracy

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The accuracy of proposed method was calculated and the method showed good TMQ recovery
between 97.36 - 98.91% at all concentration. The % RSD value was found in the range of 0.61-

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1.21 satisfying the acceptance criteria for the study as depicted in Table 1.
Precision

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The results of intra-day precision and inter day precision were found in range of 0.82-1.42 % and
0.61-1.57 % respectively as shown in Table 1. The %RSD value for both the parameters was in
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the acceptable limit.
Robustness
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The results of robustness study of the developed method showed the results within the control
limits. The influence of each of the variables was estimated as recovery (TMQ %) and retention
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time as a variables (Table 2). The method showed good TMQ recovery (97.12 – 100.23 %) and
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retention time between 7.5 – 8.1 min at all variables.

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Vesicle size and distribution

The size of all the developed TMQNIOS formulations showed in the range of 157.32 ± 3.15 to
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211.44 ± 5.23 nm with the polydispersity index of less than 0.3 (Table 4). The optimized
formulation (F3) showed the vesicle size 167.45nm as shown in Figure 3. Among six prepared
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formulation the formulation F3 has shown significantly lower vesicle size (p<.05) than other
formulations.The low value of polydispersity index indicates lesser size variation. The optimized
formulation showed the zeta potential -25.4 mV (Figure 4). The particles have a large negative
or positive zeta potential then they will tend to repel each other and there will be no tendency for
the particles to come together.
Entrapment efficiency
All prepared formulations (TMQNIOS) showed that the formulation prepared with spans have
depicted higher entrapment efficiency (%) between 67.33 ± 3.11 – 83.21 ± 3.55 than formulation
prepared with tweens 59.32 ± 4.87 – 68.66 ± 4.76 % (Table 4). Among six prepared formulation
the formulation (F3) has shown significantly (p<.001) higher encapsulation efficiency. The
formulation composition containing span 60 (F3) as surfactant showing higher encapsulation due
to the presence of greater space in the hydrophobic bilayer domain. So it may accommodate
greater amount of hydrophobic drug TMQ.

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Surface morphology
The surface morphology of the developed formulation TMQNIOSopt were evaluated by TEM as

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depicted in Figure 5. The image displayed almost spherical shape, rough surface and the
niosomes vesicles did not stick to each other. The mean diameter was found to be consistent with

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the vesicle size results.
In-vitro drug release an
The comparative drug release profile of TMQNIOS showed the biphasic release behavior and
found in the range of 59.32 ± 3.15 – 83.21 ± 2.32 %. The drug release results revealed that the
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formulation (F3) showed significant higher TMQ release profiles (p < 0.001) among prepared
formulations. There was no significant difference in TMQ release was observed between
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formulation F2 (79.32 ± 3.26) and F3 (83.21 ± 2.32). So the optimized formulation (F3) was
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selected for the further evaluation on the basis of minimum size maximum encapsulation and
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drug release. The comparative release behavior of formulation TMQNIOSopt (F3; 79.32 ± 3.26
%) and TMQDIS (36.22 ± 3.32 %) showed significant difference (p<.001) in the release pattern
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as depicted in Figure 6. The initial fast drug release was achieved (6 hrs) and further the
formulation showed prolonged release over a period of 24 h.
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Permeation study
The comparative permeation result of the prepared TMQNIOS were evaluated and compared to
select the optimized formuulation. The developed TMQNIOS showed the permeation flux
between 6.03 ± 0.19 to 8.11 ± 0.29 µg/cm2/h. The formulation F3 has shown maximum flux of
8.11 ± 0.29 µg/cm2/h and further the enhancement ratio was calculated with the control
formulation TMQDIS. The formulation TMQDIS (2.45µg/cm2/h) showed significant lower
permeation flux value (p < 0.001) than the formulation TMQNIOSopt (8.11 ± 0.29 µg/cm2/h).
The higher flux value indicates that TMQNIOSopt able to enhance the intestinal permeability.
There was significant enhancement ratio was achieved with TMQNIOSopt (3.3 times) as
compare to TMQDIS.
Confocal laser scanning microscopy
The intestinal permeation depth study was performed and the comparative image has been
depicted in Figure 7A-B. The study image revealed that RR loaded TMQNIOSopt was
distributed uniformly to a greater extent throughout the intestinal membrane. The image of
control formulation TMQDIS showed uneven distribution in the treated intestine. There was

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significant greater penetration was achieved with TMQNIOSopt vis-à-vis TMQDIS. The
formulation TMQNIOSopt showed the penetration depth of 37.61 µm (Figure 7A) as compared

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to TMQDIS (17.33µm; Figure 7B).
Discussion

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The RP-HPLC method was developed and validated to quantify the TMQ in API as well
prepared niosomes. From the different mobile phase composition and ratio, the final optimized
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mobile phase selected for the study was water: 2-propanol: methanol (50:45:5 v/v/v) get better
chromatographic separation. This composition showed the retention time of 7.7 min at 254 nm.
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The linearity range of the method showed high linearity range (6.25-100 μg/mL) with low LOD
and LOQ value of 2.08 μg/mL and 6.25 μg/mL, respectively. The lower detection and
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quantification value supports the study that the developed method was found to sensitive. The
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method showed high recovery range closer to 100 % and the % RSD value was found to be less
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than two which are under the accepted criteria for the study. The precision study showed the
results in the acceptable limit and the method was found to be precise and accurate. The change
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in variation in robustness condition of the method showed results within the control limits. The
TMQ recovery and retention time was found to be good under most conditions, and remained
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unaffected by small changes. The prepared TMQNIOS showed the nano size range of all the
formulation with the narrow polydispersity index. The low value of polydispersity index
supports the lower size variation between the vesicles. The formulations prepared with tweens
depicted larger size vesicle than the formulation prepared with the spans. The reason for this type
of property may be due to the relationship between the vesicle size and span hydrophobicity. Due
to the higher hydrophobicity of spans the reduction in surface energy takes place and forms
smaller vesicles [30]. The formulation prepared with tweens shown larger size niosomes due to it
has a much lower hydrophobicity than Span. The optimized formulation (TMQNIOSopt) showed
the vesicle size 167.45nm which is significantly lower than the size of chitosan coated vesicles
(372.8 nm) [6]. The zeta potential of the developed optimized formulation was found higher
towards negative value. The particles have a large negative or positive zeta potential then they
will tend to repel each other and there will be no tendency for the particles to come together. The
vesicles with a high ζ-potential are less likely to aggregate due to electrical repulsion [31]. The
increase in surface energy of the vesicles leads to increase the values of zeta potential towards
negative [32].

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The higher entrapment of TMQ may be due to the involvement of other reason like the highly
lipophilic part of drug is likely to be entrapped almost completely within the lipid bilayer.

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Similar observations have been previously reported by research group [33]. The surfactants used
to prepare niosomes have a low aqueous solubility and the formulation prepared with the

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addition of Tween 20, can form micelles on hydration in the presence of cholesterol [34], and it
will give lesser encapsulation than spans. The optimized formulation TMQNIOSopt showed the
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biphasic release behavior and the maximum release was found 79.32 ± 3.26%, whereas the
control formulation TMQDIS (36.22 ± 3.32 %) showed significant lower release profile. This
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type of release behavior reported due to fast release of TMQ from the niosomes surface and after
some time the slow drug release takes place. The reason of prolonged release due to release of
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TMQ from the inner core, which is responsible for the prolonged release. The release of TMQ
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from niosomes influenced by many factors, including TMQ solubility, the size of vesicle and
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surfactant nature. The release results were fitted to different release kinetics equation and the
best fit result was found to Higuchi. Among all kinetics equation the correlation coefficient was
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found to be significantly higher as shown in Table 5. The optimized formulation

(TMQNIOSopt) showed diffusion controlled release mechanism with an initial fast release phase
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followed by a slower prolonged release. This type of behavior due to TMQ desorption from
niosomes surface and at later stage the release was found to be slower which was regulated by
diffusion through the swollen niosomal bilayers [35]. This type of release behavior may be ideal
to achieve high concentration gradient required for successful drug delivery to reach the drug to
systemic circulation [36]. The permeation study results showed the significant enhancement ratio
(3.3 times) with TMQNIOSopt as compare to TMQDIS.
The enhancement in the flux achieved due to the presence of cholesterol, lipid and surfactant in
the formulation. The lipid and surfactant helps in solubility enhancement of insoluble TMQ. The
use of cholesterol in the formulation helps to disrupt the lipid bilayer and cell membranes. It may
help to open the tight junctions lead to the greater drug permeation across the mucosal surface
[37]. There are many study results matches with our results which reported the significant
enhancement in the intestinal permeation of various drugs through vesicles [38]. The intestinal
permeation was found to be higher than the previously reported by Aljoufi et al. [6]. The higher
permeation of TMQ takes place by adsorption and fusion of the formed niosomes onto the

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surface of membrane leading to a high thermodynamic activity gradient of drug at the interface,
which is thought to be the main driving force for the permeation of TMQ [39]. This effect is

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signified by the presence of lecithin which is widely known to impart high affinity between the
vesicle and skin surface layers [40]. There was significant higher drug penetration was achieved

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with TMQNIOSopt vis-à-vis TMQDIS shown by CLSM study. The presence of CHL,
soylecithin and surfactant in the formulation TMQNIOSopt may able to enhance the penetration
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to deeper layer of the membrane. There was specific darkening of the intracellular space
confirms the higher permeation. There was disruption of the mucosal membrane takes place
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which enhances the permeation deeply into mucosa.
Conclusion
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The method RP-HPLC was developed and validated using the mobile phase water: 2-propanol:
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methanol (50:45:5 v/v/v) at 254 nm. The method have shown linearity (6.25-100 μg/mL), run
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time (7.7 min), mean recovery (98.11 ± 0.57 %), to determine TMQ in API and niosomes
formulation. The developed TMQNIOS depicted the vesicle size 157.32 ± 3.15 to 211.44 ± 5.23
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nm and encapsulation efficiency in the range of 59.32 ± 4.87 to 83.21 ± 3.55 %. The developed
formulation showed significantly higher drug release and permeation (3.3 folds) in compare to
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TMQDIS. The enhanced permeation across the intestine was further confirmed by CLSM study.

Conflict of Interest
The authors declare that they have no conflicting interests.
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24. Imam SS, Aqil M, Akhtar M, Sultana Y, Ali A. Optimization of mobile phase by 32-

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43–55.
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Figure 1: Chemical structure of thymoquinone.
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Figure 2: Retention time of thymoquinone (API) using the mobile phase water:2-
propanol:methanol (50:45:5 v/v/v).
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Figure 3: Vesicle size of optimized thymoquinone niosomes formulation.

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Figure 4: Zeta potential of optimized thymoquinone niosomes formulation.

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Figure 5: Surface morphology of optimized thymoquinone niosomes formulation evaluated by
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transmission electron microscope (scale bar indicates in 200 nm).
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Figure 6: Comparative in vitro drug release profile of thymoquinone niosomes (TMQNIOSopt)
and thymoquinone dispersion (TMQDIS).
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Figure 7: Comparative confocal laser microscopy image of thymoquinone niosomes

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(TMQNIOSopt) and thymoquinone dispersion (TMQDIS).

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Table 1: Summary of TMQ method development and validation
Parameter HPLC
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Retention time (min.) 7.7
Linearity range (μg/mL) 6.25 - 100
Regression equation Y = 204.2x + 1350
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Correlation coefficient 0.998

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LOD (μg/mL) 2.08

LOQ (μg/mL) 6.25
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Accuracy ( n=3)
Mean recovery (%) 97.36 - 98.91
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% RSD 0.61 - 1.21

Precision (n=3)
Intra-day (%RSD) 0.82 - 1.42
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Inter-day (%RSD) 0.61 - 1.57

Assay of drug
TMQNIOSopt
Mean recovery (%) 98.11 ± 0.57
%RSD 0.58
 Table 2: Robustness study results with the tested parameters

Variables Range TMQ recovery Retention Area

investigated (%) time (min)
Mobile Phase 49:46:05 98.12 7.5 21900.9
50:45:05 99.76 7.7 21930.9
51:44:05 99.03 7.9 21938.2
Flow rate 0.9 98.23 8.0 21905.9
(mL / min) 1 99.11 7.7 21930.9
1.1 98.71 7.6 21928.2
Volume (μL) 20.5 99.43 7.5 21909.9

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20 100.23 7.7 21929.9

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19.5 100.11 7.9 21934.2
Wavelength 259 97.12 7.5 21904.9

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(nm) 254 98.32 7.7 21930.9
249 98.55 8.1 21932.2

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Table 3: Formulation composition of TMQ niosomes
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Formulation CHL: S20 CHL: S 40 CHL:S 60 CHL:T20 CHL:T40 CHL:T80 Soy lecithin Solvent PBS
F1 1:9 - - - - - 10 mg 10 mL 10 mL
F2 - 1:9 - - - - 10 mg 10 mL 10 mL
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F3 - - 1:9 - - - 10 mg 10 mL 10 mL
F4 - - - 1:9 - - 10 mg 10 mL 10 mL
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F5 - - - - 1:9 - 10 mg 10 mL 10 mL
F6 - - - - - 1:9 10 mg 10 mL 10 mL
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Solvent: Choloroform:methanol; S20:- span 20; S40: span 40; S60: span 60; T20: tween 20; T40:
tween 40; T80: tween 80, CHL: Cholesterol: PBS: phosphate buffer saline.
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 Table 4: Physicochemical characterization of developed TMQ niosomes.

Formulations Size (nm)a TMQ PDI TMQ release Flux

encapsulation (%)c (µg/cm2/h)d
(%)b
F1 157.32 ± 3.15 67.33 ± 3.11 0.21 65.11 ± 2.87 6.03 ± 0.19
F2 172.76 ± 4.11** 71.23 ± 2.32 0.22 83.21 ± 2.32*** 7.45 ± 0.34
F3 167.45 ± 4.28* 83.21 ± 3.55*** 0.24 79.32 ± 3.26*** 8.11 ± 0.29*

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F4 174.12 ± 3.88*** 68.66 ± 4.76 0.29 59.32 ± 3.15* 8.02 ± 0.42

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F5 191.26 ± 3.82*** 61.23 ± 5.21* 0.19 62.11 ± 3.66 7.21 ± 0.31

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F6 211.44 ± 5.23*** 59.32 ± 4.87*** 0.25 60.21 ± 3.27* 7.55 ± 0.23
All values were expressed as mean ± SD (n = 3) and data was analyzed by one way ANOVA

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followed by Tukey’s-Kramer multiple comparisons test.
a. *p<.05, vs F1; **p<.01vs. F1; ***p<.001 vs. F1); b. *p<.05, vs F4; ***p<.001 vs. F1;
c. *p<.05, vs F1; ***p<.001 vs. F1;
d. *p<.05, vs F1.
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Table 5: Drug release kinetics of TMQ niosomes with their correlation value

Release kinetics X axis Y axis Equation (R2)

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Zero order release Fraction of drug released Time Mt = M0 + k0 t 0.798

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First order release Log % drug remaining Time ln Mt = ln M0 + k1 t 0.803

Mt/M∞ = Ktn
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Korsmeyer Peppas Log fraction of drug Log time 0.891

released
Higuchi Fraction of drug released √time Mt = M0 + kH t1/2 0.911
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Notes: M0 = initial amount of drug; Mt = remaining amount of drug at time t; k0 = zero order
release constant; k1 = first order release constant; Mt/M∞ = fraction of drug released at time t;
release rate constant; K = release rate constant; n = release exponent; Higuchi dissolution
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constant