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Drug Deliv, 2014; 21(6): 487–494! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10717544.2014.886640

ORIGINAL ARTICLE

Neuroprotective study of Nigella sativa-loaded oral provesicular lipidformulation: in vitro and ex vivo study

Mohd. Akhtar1, Syed Sarim Imam2, Mohd. Afroz Ahmad1, Abul Kalam Najmi1, Mohd. Mujeeb3, and Mohd. Aqil2

1Department of Pharmacology, 2Department of Pharmaceutics, and 3Department of Pharmacognosy, Faculty of Pharmacy, Jamia Hamdard,

New Delhi, India

Abstract

Aim: The aim of this research was to develop proniosome (niosomes) of Nigella sativa (NS) toimprove its drug release, gastrointestinal (GI) permeation and neuroprotective activity.Materials and methods: Proniosomes were prepared by thin film method using variouscompositions of nonionic surfactants, cholesterol, and phosphatidylcholine. The optimuminfluence of different formulation variables of NS such as surfactant type, phosphatidylcholineand cholesterol concentration were optimized for size and entrapment efficiency.Results and discussion: Results indicated that prepared niosome showed smaller size withhigh entrapment efficiency. The permeation enhancement ratio was found to be 2.16 incomparison to control with maximum flux value obtained was 7.23 mg/cm2/h for formulationNS6. The in vivo study revealed that the niosomal dispersion significantly improvedneuroprotective activity in comparison to standard and control formulation.Conclusion: In conclusion, developed proniosomal formulation could be one of the promisingdelivery system for NS with better drug release and GI permeation profiles and improvedneuroprotective activity and merits for further study.

Keywords

Lipid formulation, neuroprotective, NigellaSativa, oral

History

Received 8 January 2014Accepted 20 January 2014

Introduction

The majority of the drugs administered by oral route

frequently encounter bioavailability problems due to several

reasons like poor dissolution, poor permeation across the

gastrointestinal (GI) barrier, unpredictable absorption, inter-

and intrasubject variability and lack of dose proportionality

(Lobenberg & Amidon, 2000; Gurrapu et al., 2012). It has

been conceptualized to develop the formulation of colloidal

lipid carrier systems as a means to improve the drug

solubilization and permeation across the GI barrier (Porter

et al., 2007; Janga et al., 2012). Among different colloidal

particulate drug delivery systems, liposomes are very distinct

when compared with conventional dosage forms. Liposomes

have shown limited success in oral delivery, can be explicit in

terms of physicochemical stability issues such as aggregation,

fusion, phospholipid hydrolysis and/or oxidation. The pronio-

some formulation ameliorates these problems by using dry,

free-flowing product, which is more stable during storage

(Hu & Rhodes, 1999; Gurrapu et al., 2012). Proniosomes are

dry powder formulations containing water-soluble carrier

particles coated with surfactant and can be hydrated to form

niosomal dispersion on brief agitation in aqueous media.

Nigella sativa (NS) is an annual herbaceous plant with

black seeds, commonly known as black cumin, black caraway

seed, and Habbatul barakat, belongs to Ranunculaceae

family. Numerous studies have shown that seeds and oil

from this plant are characterized by a very low degree of

toxicity (Ali & Blunden, 2003). The major biologically

active compound of NS is thymoquinone. Various pharma-

cological properties have been reported in literature

(Babazadeh et al., 2012). It also contain fixed and essential

oils, proteins, alkaloids and saponins. Henceforth, this study

was designed to develop and optimize NS-loaded pronioso-

mal formulation. The optimized formulation was further

evaluated ex vivo permeation study to assess the permeation

for drug, in vitro drug release to check release kinetics

and in vivo pharmacodynamics study to ascertain

its neuroprotective activity.

Materials and methods

Material

NS was procured locally; seeds were identified, authenticated

and standardized by Department of Pharmacognosy and

Phytochemistry, Jamia Hamdard, New Delhi. Surfactant (span

20, span40, span60, tween20, tween40 and tween80) and

cholesterol were purchased from SD fine company, Mumbai.

All chemicals and reagents used were of analytical grade, and

solvents were of HPLC grade. Freshly collected double-

distilled water was used all throughout the experiments.

Address for correspondence: Dr. Mohd. Aqil, Senior Assistant Professor,Department of Pharmaceutics, Faculty of Pharmacy, HamdardUniversity, MB Road, New Delhi-110 062, India. Email: aqilmalik@yahoo.com

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Animals

The study was carried out under controlled conditions using

adult Wistar albino rats of 150–250 g, procured from Central

Animal House Facility of Hamdard University, New Delhi,

selected and acclimatized accordingly. All animals were

housed in cages kept with a natural light–dark cycle. They had

free access to standard pellet diet (Amrut Laboratory rat

and mice feed, Nav Maharastra Chakan oil mills Ltd., Pune)

and water. The experimental protocol was approved by the

Institutional Animal Ethics Committee, Jamia Hamdard and

CPCSEA, New Delhi (173/CPCSEA 28 January 2008, Project

no. 575, dated 26 November 2009). Ethical norms were

strictly followed during all experimental procedures.

Preparation of NS proniosomes

Nigella sativa proniosomes were prepared by film hydration

method (Balakrishnan et al., 2009). Cholesterol with different

type of nonionic surfactant, and phosphatidylcholine were

dissolved in 5 ml chloroform–methanol mixture (1:2).

Separately calculated amount of NS (equivalent to 4 mg/ml)

was dissolved in above solvent. The lipid mixture and drug

solution were transferred to round bottom flask, and solvent

was evaporated under reduced pressure at a temperature of

60 �C by a rotary evaporator (Model-HS-2005V-N; Hahnshin

Scientific Co., Korea) until a thin lipid film was deposited on

the wall of the flask. The excess organic solvent was removed

by keeping the flask under vacuum overnight in desiccator.

After ensuring the complete removal of solvent, the resultant

dried free-flowing powders were stored in a tightly closed

container for further evaluation. Proniosomes were trans-

formed to niosomes by hydrating with 10 ml distilled water and

agitation for 2 min. The resulting niosomes dispersion and

proniosomal powder were further used for the quality

evaluation.

Quality evaluation

Micromeritic properties

The flow properties of the proniosomal powder were studied

through measuring the Angle of repose, Carr’s compressibil-

ity index and Hausner’s ratio. The angle of repose of

proniosome was determined by using conventional fixed

funnel method (Staniforth, 1988). The Carr’s compressibility

index and Hausner’s ratio were calculated from the bulk and

tapped density of the powders (Carr, 1965).

Number of vesicles per cubic millimeter

The number of vesicles formed after hydration of the

proniosomal powder is an important parameter in formulation

development. The proniosome powder was subjected to

hydration with distilled water and the formed niosomes

were counted by optical microscope using a hemocytometer

(Jukanti et al., 2011). The niosomes in 80 small squares were

counted and calculated by using the following formula.

Number of niosomes per cubic mm

¼

Total number of niosomes counted

�Dilution factor� 4000

� �

Total number of squares counted

Vesicles size and size distribution

The size distribution of all the proniosomal formulations

were measured by dynamic light scattering technique

using a computerized instrument (Zetasizer, HAS 3000,

Malvern, UK). The measurement of vesicle is done

diluting with appropriate medium and the measurements

were taken in triplicate. The polydispersity index (PDI)

was determined as a measure of homogenecity (Sentjurc

et al., 1999).

Vesicle morphology

The prepared niosomal formulation was characterized for

their morphology using transmission electron microscopy

(TEM; MORGANI 268D, The Netherland). The sample

was prepared by taking small quantity of formulation, adding

1% phosphotungstic acid and mixing gently. One drop of

the above mixture was placed on the carbon-coated grid

and excess sample was drawn out by filter paper. The grid

was allowed to dry for 2 min to absorb the sample, and

it was observed under TEM by using soft imaging viewer

software.

Entrapment efficiency

The entrapment efficiency of the proniosomal formulation

was determined by measuring the concentration of free

drug in the dispersion medium using centrifugation

method (Alsarra et al., 2005). Five milliliter of niosomal

suspension was taken in a tube and sonicated in a bath

sonicator for 30 min. The untrapped drug was separated

by centrifuging the sample at 14 000 rpm at 4 �C for 30 min

(REMI Cooling centrifuge; C-24, Mumbai, India). The

supernatant was taken and diluted with phosphate buffer,

and assayed (Aboul-Enein & Abou-Basha, 1995). The

experiment was performed in triplicate, and percentage

entrapment of NS in proniosome was calculated from the

following equation:

% Entrapment efficiency

¼ Total amout of added� Free Drug

Total amount of Drug added� 100

In vitro release

The drug release of NS-loaded proniosomes was performed

for 12 h and was compared with pure drug. The study was

performed using USP-type II apparatus (Veego, VDA-8DR,

Mumbai, India). Calculated amount of proniosome (equiva-

lent to 10 mg of NS) was placed in dialysis bag. The release

medium was 500 ml phosphate buffer (pH 6.8) with stirring

speed 100 rpm, and the temperature was maintained at

37 ± 1 �C (Nasr, 2010). The samples (5 ml) were withdrawn

at various time intervals and filtered through 0.2-mm mem-

brane filter, and samples were replaced by fresh medium. The

amount of drug released from the formulation was assayed

and mean cumulative amount of drug released was plotted

against time (Aboul-Enein & Abou-Basha, 1995). The

obtained data was fitted into various kinetic data to explain

kinetics and mechanism of drug release from formulations

(Szuts et al., 2010).

488 M. Akhtar et al. Drug Deliv, 2014; 21(6): 487–494

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Ex vivo permeation studies

The ex vivo permeation study was carried out using the rat

intestinal segment, since majority of drugs get absorbed

from small intestine. The abdomen was opened and a

segment of the intestine was removed and flushed with

Krebs–Ringer solution to remove the mucus and adhered

intestinal contents. One end of the intestine segment was

tied and niosomal dispersion (equivalent to 10 mg of NS)

was filled into the lumen and was tightly closed. The tissue

was placed in an organ bath with continuous aeration and

maintained at a temperature of 37 ± 1 �C. The receptor

compartment contained 10 ml of phosphate buffer. At

predetermined time intervals, an aliquot of 0.5 ml was

collected and replenished with equal volume of fresh

medium. The samples were centrifuged and the supernatant

was quantified(Aboul-Enein & Abou-Basha, 1995). The

cumulative amount of drug permeated (Q) was plotted

against time. The steady state flux (Jss) was calculated

from the slope of linear portion of the cumulative

amount permeated per unit area versus time plot. The

permeability coefficient (Kp) of the drug through intestine

was calculated by dividing steady state flux with initial

concentration of NS in donor compartment. The enhance-

ment ratio (ER) was calculated by using the following

equation:

ER ¼ Jss of proniosome formulation

Jss of control:

Assessment of physical stability

The fusion of the vesicles as a function of temperature was

determined as the change in entrapment efficiency after

storage. The prepared vesicles were stored in glass vials at

different temperature like (room temperature or refrigerated

temperature 4 �C) for 3 months. The initial retention of

entrapped drug was measured after preparation and then after

1, 2 and 3 months of storage. The stability for formulation was

defined in terms of change in vesicle size and entrapment

efficiency. Stable formulations were defined as those showing

not much variation in size and entrapment efficiency at each

time interval.

In vivo activity

Drugs and administration

Animals were divided into five groups, each group consisting

of six rats each receiving different treatments orally for dose

administration. Group I – normal control, Group II – sham

operated, Group III – middle cerebral artery occluded

(MCAO) only, Group IV – aspirin + MCAO, Group V –

proniosomal formulation + MCAO. The proniosomal formu-

lation (NS6) was made as previously described. All the drugs

were administered before 3 h of inducing cerebral ischemia in

rats. Normal control group and sham-operated animals

received distilled water. Ischemia was induced for 2 h

followed by reperfusion for 22 h. After 24 h of ischemia,

behavioral parameters were assessed and the animals were

then immediately sacrificed for infarct volume and oxidative

stress parameters in brains.

Induction of cerebral ischemia

Rats were anesthetized with chloral hydrate (dissolved in

distilled water) at a dose of 400 mg/kg i.p. A middle incision

was performed on right common carotid artery, external

carotid artery and internal carotid artery and was exposed

under an operative magnifying glass. A 4.0 monofilament

Nylon thread (40–3033 Pk 10; Doccol Corporation

Pennsylvania Ave, Red Lands, CA) with its tip rounded by

heating quickly near a flame was advanced from the external

carotid artery into the lumen of the internal carotid artery

until resistance was felt, which ensures the occlusion of the

origin of the middle cerebral artery. The nylon filament was

allowed to remain in place for 2 h. After 2 h, the filament was

retracted so as to allow the reperfusion of ischemic region

(Longa et al., 1989). Sham-operated rats had the same

surgical procedures except that the occluding monofilament

was not inserted. After 24 h, the animals were studied for

locomotor activity and grip strength test. The animals were

sacrificed immediately after behavior study and their brain

was removed to measure infarct volume and biochemical

estimations.

Behavioral tests

Locomotor activity (closed field activity monitoring)

Spontaneous locomotor activity was assessed using a digital

photoactometer (Lannert & Hcfyer, 1998). Each animal was

observed for a period of 10 min in a square closed arena

equipped with infrared light sensitive photocells. The appar-

atus was housed in a darkened light and sound attenuated

ventilated testing room. During activity testing, only one

animal was tested at a time.

Grip test

Grip strength meter was used for recording the grip strength

of the animal. The animal’s front paws were placed on the

grid of grip strength meter and was moved down until its front

paws grasping the grid was released. The force achieved by

animal was displayed on the screen and was recorded as

kilogram unit (Ali et al., 2004).

Estimation of oxidative stress markers

The animals were decapitated, brains were quickly removed

and necrotic parts of the brains were taken for estimation. The

collected samples were weighed and homogenized in ice-cold

KCI phosphate buffer (0.1 M, pH 7.4) and centrifuged at

2000 rpm for 5 min at 4 �C. The supernatant containing crude

membrane was used for the estimation of thiobarbituric

acid reactive substance (TBARS) and reduced glutathione

(GSH). The remaining supernatant was again centrifuged at

10 000 rpm at 4 �C for 20 min. The post mitochondrial

supernatant was used for the study of antioxidant enzyme

activities and protein estimation. Catalase and super oxide

dismutase activities were determined immediately after

sample preparation. Protein concentrations were determined

DOI: 10.3109/10717544.2014.886640 NS-loaded oral provesicular lipid formulation 489

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using purified bovine serum albumin as standard (Lowry

et al., 1951).

Measurement of lipid peroxidation

Lipid peroxidation test was measured as reported

(Ohkawa et al., 1979). Briefly, 1 ml of suspension medium

was taken from the 10% tissue homogenate. About 0.5 ml

of 30% TCA was added to it, followed by 0.5 ml of 0.8%

TBA reagent. The tubes were covered and kept in shaking

water bath for 30 min at 80 �C. After 30 min, tubes were taken

out and kept in ice-cold water for 30 min. These were

centrifuged at 3000 rpm for 15 min. The absorbance of the

supernatant was read at 540 nm at room temperature against

appropriate blank. Blank consist of 1 ml distilled water, 0.5 ml

of 30% TCA and 0.5 ml of 0.8% TBA. TBARS values were

expressed as n moles MDA/mg protein.

Measurement of reduced glutathione

Glutathione was measured by taking equal quantity of

homogenate (w/v) and 10% trichloroacetic acid and centri-

fuged to separate the proteins. To 0.01 ml of this supernatant,

2 ml of phosphate buffer (pH 7.4), 0.5 ml 5,50-dithiobisnitro

benzoic acid and 0.4 ml of double-distilled water were added.

The mixture was vortexed and absorbance was read at 412 nm

within 15 min. GSH values were expressed as micromoles

GSH milligram protein (Ellman, 1959).

Measurement of catalase

Catalase activity was measured as per reported (Claiborne &

Greenworld, 1985). A total of 0.1 ml of supernatant was added

to cuvette containing 1.9 ml of 50 mM phosphate buffer

(pH 7). The reaction was started by the addition of 1 ml

freshly prepared 30 mM H2O2. The rate of decomposition of

H2O2 was measured spectrophotometrically at 240 nm.

Catalase values were expressed as n moles H2O2 consumed/

min/mg protein.

Measurement of superoxide dismutase

Superoxide dismutase (SOD) activity was measured by the

reported method (Kagiyama et al., 2003). The supernatant

was assayed for SOD activity by following the inhibition

of pyrogallol auto-oxidation. Hundred microliters of cyto-

solic supernatant was added to Tris HCI buffer (pH 8.5).

The final volume of 3 ml was adjusted with the same

buffer. At least 25 ml of pyrogallol was added and changes

in absorbance at 420 nm were recorded at 1 min interval

for 3 min. The increase in absorbance at 420 nm after

the addition of pyrogallol was inhibited by the presence

of SOD.

Statistical analysis

All the data were expressed as the mean ± SEM. For statistical

analysis of the data, group means were compared by one-way

analysis of variance (ANOVA) followed by Dunnett’s ‘‘t’’

test. The p value 50.05 was considered significant. It was

carried out with graph pad in Stat 3 software.

Results and discussion

In this study, the NS proniosomes were optimized and

evaluated for their quality in improving the oral delivery.

The formulations were developed by film hydration technique

using cholesterol, surfactant and phospholipid for use

of oral delivery (Table 1). The formation of niosomes

after reconstitution of proniosome depends on the ease of

dispersibility of the carrier in aqueous fluids. The optimum

NS proniosome formulation was selected based on the criteria

of attaining the optimum vesicles size and maximum encap-

sulation efficiency and permeability. The solid state and

high-phase transition temperature render more stability in GI

fluids and augment the flow characteristics of the pronio-

somes, respectively, which is an important prerequisite for

solid dosage forms.

Quality evaluation

Micromeritics study

The micromeritics properties of the proniosome powders are

important in handling and processing operations because the

dose uniformity and ease of filling into container are dictated

by the powder flow properties. The value of angle of repose

will be small for noncohesive particles, whereas in case of

cohesive particles, the value is increased due to high internal

friction between particles. Our results (Table 2) indicate small

angle of repose (530�) assuring good flow properties for

proniosome powder formulations. In addition to angle of

repose, Carr’s index and Hausner’s ratio were also less than

21 and 1.25, respectively, ensuring acceptable flow for

proniosome powder formulations (Staniforth, 2002).

Number of vesicles per cubic millimeter

The maximum benefit from the proniosome formulations can

be speculated when abundant numbers of vesicles are formed

after hydration in the GI tract. All the formulations have

Table 1. Composition of Nigella sativa-loaded proniosome formulation.

Code S20:CH:PC S40:CH:PC S60:CH:PC T20:CH:PC T40:CH:PC T80:CH:PC

NS1 9:1:1 – – – – –NS2 – 9:1:1 – – – –NS3 – – 9:1:1 – – –NS4 – – – 9:1:1 – –NS5 – – – – 9:1:1 –NS6 – – – – – 9:1:1

S20 – Span20; S40 – Span40; S60 – Span60; T20 – Tween 20; T40 – Tween 40; T80 – Tween 80; CH – cholesterol;PC – phosphatidylcholine. Each formulation contains 4 mg/ml Nigella sativa.

490 M. Akhtar et al. Drug Deliv, 2014; 21(6): 487–494

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exhibited good number of vesicles that is in well correlation

with the size and entrapment efficiency results (Table 2).

Vesicles size and size distribution

Vesicle size and size distribution is an important parameter

for the vesicular systems (Plessis et al., 1991). The mean

size of the vesicles were in the range of 378.54–435.43 nm

(Figure 1). The difference in the mean vesicle size could be

explained, span-based niosomes showed smaller vesicle

size than tween-based niosomes that are more hydrophilic.

Yet, within each class, the surfactant of higher chain length

produced a larger vesicle than those of smaller chain length.

The PDI used as a measure of a unimodal size distribution

was within the acceptable limits for all the formulations.

Small value of PDI (50.1) indicates a homogenous popula-

tion, while a PDI (40.3) indicates a higher heterogeneity

(Table 3).

Vesicle morphology

To confirm the formation of vesicle structures from the

proniosome powder, the morphology of the reconstituted

dispersion was examined using negative stain TEM and

the obtained photomicrographs are presented in Figure 2.

The reconstituted dispersion revealed well-identified vesicles

present in a nearly perfect spherical shape. They show the

outline and core of the well-identified spherical vesicles,

displaying the retention of sealed vesicular structure.

Entrapment efficiency

The entrapment was expressed as a percentage of the total

amount of NS incorporated in proniosomes. The entrapment

efficiency relies on the stability of the vesicle that is highly

dependent on amount of surfactant concentration and chol-

esterol amount shown in Table 3. The mean entrapment

efficiency of proniosome-derived niosomes was found in the

range of 77.71–89.89%. The maximum entrapment was found

in NS6 formulation due to their high HLB and its longer alkyl

chain.

In vitro release

The release rates of NS from the developed niosomes

formulations were significantly slower compared to free

drug (p50.05). The optimized formulation NS6 showed

typical biphasic release pattern with an initial rapid phase

followed by a slow phase for a period of 12 h (Figure 3). The

initial rapid phase in the release as expected could be due to

presence of untrapped drug in the outer region of proniosome.

The maximum release of NS after 12 h was found to be

94.98% from formulation (NS6), respectively. NS molecules

could be entrapped in the internal aqueous core or intercalated

Figure 2. TEM image of niosome dispersion from reconstitutedproniosomal formulation.

Figure 3. In vitro release of Nigella sativa from proniosome.

Figure 1. Size distribution of optimized NS-loaded niosome.

Table 3. Quality evaluation parameters.

Code Size (nm)Entrapment

efficiency (%)Flux

(mg/cm2/h)Enhancement

ratio

NS1 378.54 ± 8.65 77.71 ± 3.56 6.11 ± 5.43 1.82NS2 391.22 ± 8.38 79.23 ± 5.24 6.87 ± 6.36 2.05NS3 417.98 ± 9.12 89.15 ± 4.98 7.11 ± 4.64 2.12NS4 388.95 ± 7.65 81.54 ± 6.65 5.38 ± 7.54 1.61NS5 435.43 ± 5.72 85.59 ± 5.71 6.21 ± 6.12 1.72NS6 406.76 ± 6.48 86.89 ± 6.22 7.23 ± 7.08 2.16Control 3.34 ± 4.17

Table 2. Micromeritic and physicochemical evaluation.

FormulationAngle of

repose (h)aCarr’sindexa

Hausner’sratioa PDI

No. of vesiclesPer mm3� 103

NS1 29.1 13.9 1.13 0.215 2.92NS2 27.5 11.1 1.12 0.243 2.71NS3 26.9 10.0 1.11 0.225 3.21NS4 24.8 10.9 1.12 0.196 2.98NS5 25.4 8.20 1.12 0.226 3.01NS6 27.1 7.51 1.13 0.274 2.88

DOI: 10.3109/10717544.2014.886640 NS-loaded oral provesicular lipid formulation 491

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within the bilayer structure of niosomal membrane. These

release experimental results clearly show that the release

of NS was greatly retarded in niosomes. The release data

have been fitted into various kinetic equations to know the

release order and mechanism. The correlation coefficient

of release profiles showed Fickian diffusion transport mech-

anism (n¼ 0.37). If n lies in the range 0–0.5, the system

follows a Fickian diffusion transport mechanism, while

if n lies in the range 0.5–1, convective mass transfer is

the pertinent mechanism (anomalous transport) (Pando

et al., 2013).

Ex vivo permeation study

The ex vivo permeation study facilitates to ascertain the

potential of proniosomes for improved absorption of NS

across GI tract. The study was performed by using rat

intestine to assess the potential of formulations for improv-

ing the permeation of NS across the intestinal barrier.

The maximum flux of NS6 (7.23 ± 7.08 mg/cm2/h) across rat

intestine was found to be with 2.16 times permeation

enhancement over control formulation (3.34 ± 4.17 mg/cm2/

h), which produced the least flux (p50.01) (Table 3).

The significant enhancement in NS from NS6 (p50.01)

with respect to control reveals that proniosomes obviate

the barrier properties of the GI tract thus favoring the

absorption. The ER well above 1 indicates improved perme-

ation and in our findings we could notice an ER greater

than 1 for all developed proniosome compared to control.

To summarize, the results reveal the potential of

proniosome carriers for improved absorption of NS across

the biological membrane. The optimized formulation (NS6)

has been further selected for in vivo neuroprotective activity

on rats.

Assessment of physical stability

The results of entrapment efficiency showed that there was no

appreciable change in the percent retention of NS at

refrigerated temperature. But in case of formulation stored

at room temperature, the entrapment varied from the initial

value. The higher amount of drug leakage at elevated

temperature may be due to the degradation of lipids

constituting bilayers resulting in defects in membrane

packing and loss of overall rigidity that makes them leaky.

The PDI and unimodal size distribution of formulation

was also found to be low, which favors the stability of

proniosome formulation. The stability studies suggest that

the proniosome formulation was comparatively more stable

when stored at refrigerated conditions compared to room

temperature.

In vivo study

Locomotor activity

Spontaneous locomotor activity was observed over a period of

10 min for each rat in each group. In the MCA occluded rats,

significant reduction in locomotor count was observed

(p50.00 l). There was significant improvement in locomotor

counts observed with NS6 as compared to the MCAO rats

(p50.001) (Table 4).

Effect on grip strength study

MCAO group showed a significant decrease in grip strength

as compared to the normal rats (p50.01). Pretreatment of

NS6 showed improvement in grip strength when compared

with MCAO rats (p50.001) (Table 4). Our findings were in

agreement with already reported observations and results

(Abdulhakeem et al., 2006; Akhtar et al., 2008, 2012, 2013;

Pratap et al., 2011).

Effect on TBARS levels

NS proniosomal formulation (NS6) was administered in this

study, decreased TBARS levels as compared to the MCAO

rats. The TBARS levels measured after 24 h of middle

cerebral artery occlusion were found to be significantly

increased in the MCAO rats than in the normal rats. NS6

produced reduction in TBARS levels when compared to that

of MCAO (p50.001) (Table 5). This was in agreement with

our earlier reports which showed that MCA occlusion

followed by reperfusion increased TBARS formation in rats

(Pratap et al., 2011; Akhtar et al., 2012, 2013).

Effect on glutathione

The brain glutathione levels were estimated in all the groups.

Levels of reduced glutathione in MCAO rats were signifi-

cantly reduced when compared to the sham-operated rats. The

glutathione levels were elevated with NSPF when compared

with MCAO group (p50.001) (Table 5). There was a

significant decrease in GSH levels in MCA occluded rats.

Large number of reports stated that oxidative stress decreases

GSH levels (Abdulhakeem et al., 2006; Akhtar et al., 2008,

2012, 2013; Pratap et al., 2011). Pretreatment of NS6

formulation showed elevation of GSH levels as compared to

MCA occluded rats; thus, confirming its antioxidant and free

radical scavenging properties. It was already reported that NS

decreases lipid peroxidation and increases the antioxidant

defense system activity (Burits & Bucar, 2000).

Effect on SOD

The levels of SOD after 24 h in MCA occluded group were

significantly reduced as compared to the normal rats. The

levels of SOD were significantly increased with NSPF as

compared to the MCAO group (p50.001) (Table 5).

Table 4. Effect of Nigella sativa proniosome on locomotor count andgrip strength in middle cerebral artery occludes rats.

Groups(n¼ 6) Treatment

Locomotor activity(count/10 min)

Grip strength(kg/unit)

I Normal control 31.5 ± 0.62 0.62 ± 0.01II Sham operated 38.83 ± 0.71 0.52 ± 0.04III MCAO 09.50 ± 0.65a,b 0.10 ± 0.01a,b

IV Asp + MCAO 24.16 ± 0.49c 0.49 ± 0.03c

V NS6 + MCAO 15.83 ± 0.61d 0.38 ± 0.02d

MCAO – middle cerebral artery occlusion; Asp – Aspirin.Data represented as mean ± SEM.Significance by one-way ANOVA followed by Dunnett’s t test.ap50.001 versus normal control.bp50.001 versus Sham-operated.cP50.01 versus the MCAO group.dP50.05 versus the MCAO group.

492 M. Akhtar et al. Drug Deliv, 2014; 21(6): 487–494

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Effect on catalase

The levels of catalase were reduced in MCA occluded

group as compared to the normal rats. NSPF showed elevation

in the levels as compared to the MCAO group (p50.001)

(Table 5).

Effect on infarct volume

In this study, a selective neuronal damage causing more

infarction volume as evident by triphenyltetrazolium chloride

(TTC) dye staining of rats brains was observed. NS

proniosomal formulation significantly reduced the infarction

volume when compared to MCA occluded rats and thus,

showed neuroprotective effects. TTC dye staining of brains of

NSPF pretreated animals showed significant improvement in

the infarct volume. Aspirin, NSPF pretreated animals showed

a highly significant reduction in infarct volume as compared

with MCAO rats (p50.001) (Table 6).

Conclusion

The developed proniosomal formulations possess good flow

properties and prolonged drug release compared to control.

The ex vivo permeation across the rat intestine reveals the

potential of proniosome formulation for improved absorption

of NS across GI membrane. The in vivo studies showed that

NS6 reduced TBARS levels, elevated GSH, SOD and catalase

levels. Decrease in infarction volume was also observed.

Thus, the neuroprotective effects exhibited by NS6 against

ischemia in rats confirm its antioxidant, free radical scavenger

and anti-inflammatory properties reported previously. Thus, it

may be used in reducing the symptoms of cerebral ischemia.

Our results are preliminary; further research is warranted

to establish the exact role of NS as a neuroprotective agent.

Declaration of interest

All authors have approved the final manuscript and the

authors declare that they have no conflicts of interest to

disclose.

Financial support under RPS scheme from AICTE, New

Delhi is greatly acknowledged.

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Data represented as mean ± SEM.Significance by one-way ANOVA followed by Dunnett’s t test.ap50.01 versus MCAO.bp50.05 versus MCAO.

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