http://informahealthcare.com/drd ISSN: 1071-7544 (print), 1521-0464 (electronic) 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 lipid formulation: in vitro and ex vivo study Mohd. Akhtar1, Syed Sarim Imam2, Mohd. Afroz Ahmad1, Abul Kalam Najmi1, Mohd. Mujeeb3, and Mohd. Aqil2 Department of Pharmacology, 2Department of Pharmaceutics, and 3Department of Pharmacognosy, Faculty of Pharmacy, Jamia Hamdard, New Delhi, India

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Abstract

Keywords

Aim: The aim of this research was to develop proniosome (niosomes) of Nigella sativa (NS) to improve its drug release, gastrointestinal (GI) permeation and neuroprotective activity. Materials and methods: Proniosomes were prepared by thin film method using various compositions of nonionic surfactants, cholesterol, and phosphatidylcholine. The optimum influence of different formulation variables of NS such as surfactant type, phosphatidylcholine and cholesterol concentration were optimized for size and entrapment efficiency. Results and discussion: Results indicated that prepared niosome showed smaller size with high entrapment efficiency. The permeation enhancement ratio was found to be 2.16 in comparison to control with maximum flux value obtained was 7.23 mg/cm2/h for formulation NS6. The in vivo study revealed that the niosomal dispersion significantly improved neuroprotective activity in comparison to standard and control formulation. Conclusion: In conclusion, developed proniosomal formulation could be one of the promising delivery system for NS with better drug release and GI permeation profiles and improved neuroprotective activity and merits for further study.

Lipid formulation, neuroprotective, Nigella Sativa, oral

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, interand 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 proniosome 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.

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

History Received 8 January 2014 Accepted 20 January 2014

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 pharmacological 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 proniosomal 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 doubledistilled water was used all throughout the experiments.

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Animals

Vesicles size and size distribution

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.

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).

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Drug Deliv, 2014; 21(6): 487–494

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 transformed 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 compressibility 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   Total number of niosomes counted  Dilution factor  4000 ¼ Total number of squares counted

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 Total amout of added  Free Drug ¼  100 Total amount of Drug added 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 (equivalent 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 membrane 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).

NS-loaded oral provesicular lipid formulation

DOI: 10.3109/10717544.2014.886640

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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 enhancement 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 formulation (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

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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 apparatus 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

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Drug Deliv, 2014; 21(6): 487–494

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 NS2 NS3 NS4 NS5 NS6

9:1:1 – – – – –

– 9:1:1 – – – –

– – 9:1:1 – – –

– – – 9:1:1 – –

– – – – 9:1:1 –

– – – – – 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.

using purified bovine serum albumin as standard (Lowry et al., 1951).

for 3 min. The increase in absorbance at 420 nm after the addition of pyrogallol was inhibited by the presence of SOD.

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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 centrifuged 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 cytosolic 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

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 encapsulation efficiency and permeability. The solid state and high-phase transition temperature render more stability in GI fluids and augment the flow characteristics of the proniosomes, 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

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Table 2. Micromeritic and physicochemical evaluation. Angle of Carr’s Hausner’s Formulation repose (h)a indexa ratioa NS1 NS2 NS3 NS4 NS5 NS6

29.1 27.5 26.9 24.8 25.4 27.1

13.9 11.1 10.0 10.9 8.20 7.51

1.13 1.12 1.11 1.12 1.12 1.13

PDI

No. of vesicles Per mm3  103

0.215 0.243 0.225 0.196 0.226 0.274

2.92 2.71 3.21 2.98 3.01 2.88

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Figure 2. TEM image of niosome dispersion from reconstituted proniosomal formulation.

Figure 1. Size distribution of optimized NS-loaded niosome. Table 3. Quality evaluation parameters.

Code NS1 NS2 NS3 NS4 NS5 NS6 Control

Size (nm)

Entrapment efficiency (%)

Flux (mg/cm2/h)

Enhancement ratio

378.54 ± 8.65 391.22 ± 8.38 417.98 ± 9.12 388.95 ± 7.65 435.43 ± 5.72 406.76 ± 6.48

77.71 ± 3.56 79.23 ± 5.24 89.15 ± 4.98 81.54 ± 6.65 85.59 ± 5.71 86.89 ± 6.22

6.11 ± 5.43 6.87 ± 6.36 7.11 ± 4.64 5.38 ± 7.54 6.21 ± 6.12 7.23 ± 7.08 3.34 ± 4.17

1.82 2.05 2.12 1.61 1.72 2.16 Figure 3. In vitro release of Nigella sativa from proniosome.

exhibited good number of vesicles that is in well correlation with the size and entrapment efficiency results (Table 2).

outline and core of the well-identified spherical vesicles, displaying the retention of sealed vesicular structure.

Vesicles size and size distribution

Entrapment efficiency

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 population, while a PDI (40.3) indicates a higher heterogeneity (Table 3).

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 cholesterol 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.

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

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

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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 mechanism (n ¼ 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).

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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 improving 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 permeation 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).

Drug Deliv, 2014; 21(6): 487–494

Table 4. Effect of Nigella sativa proniosome on locomotor count and grip strength in middle cerebral artery occludes rats. Groups (n ¼ 6) I II III IV V

Treatment

Locomotor activity (count/10 min)

Grip strength (kg/unit)

Normal control Sham operated MCAO Asp + MCAO NS6 + MCAO

31.5 ± 0.62 38.83 ± 0.71 09.50 ± 0.65a,b 24.16 ± 0.49c 15.83 ± 0.61d

0.62 ± 0.01 0.52 ± 0.04 0.10 ± 0.01a,b 0.49 ± 0.03c 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. a p50.001 versus normal control. b p50.001 versus Sham-operated. c P50.01 versus the MCAO group. d P50.05 versus the MCAO group.

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 significantly 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).

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Table 5. Effect of Nigella sativa proniosome on TBARS, GSH, SOD and catalase in middle cerebral artery occludes rats. Groups (n ¼ 6) I II III IV V

Treatment

TBARS (nmoles MDA formed/mg protein)

GSH (mmol/mg protein)

SOD (U/mg protein)

Catalase (nmoles of H2O2/ min/mg protein)

Normal Control Sham Operated MCAO Asp + MCAO NS6 + MCAO

4.32 ± 0.051 3.98 ± 0.35 7.11 ± 0.11a,b 3.21 ± 0.14e 3.01 ± 0.11e

2.15 ± 0.023 3.08 ± 0.059 0.89 ± 0.01c,d 1.29 ± 0.03e 1.10 ± 0.01e

10.21 ± 0.51 12.01 ± 0.32 3.93 ± 0.41c,d 8.18 ± 0.43e 7.18 ± 0.62e

12.21 ± 0.10 15.50 ± 0.31 5.12 ± 0.10c,d 8.40 ± 0.08e 8.01 ± 0.1e

Significance by one-way ANOVA followed by Dunnett’s t test. a p50.05 versus control. b p50.05 versus Sham. c p50.01 versus control. d p50.01 versus Sham. e p50.05 versus MCAO.

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Table 6. Effects of Nigella sativa proniosome on infarct volume in middle cerebral artery occlusion occluded rats. Groups (n ¼ 6) I II III

Treatment

Infarction volume (mm3)

MCAO Aspirin + MCAO NS6 + MCAO

65.21 ± 0.01 39.70 ± 0.03a 48.01 ± 0.05b

Data represented as mean ± SEM. Significance by one-way ANOVA followed by Dunnett’s t test. a p50.01 versus MCAO. b p50.05 versus MCAO.

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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Neuroprotective study of Nigella sativa-loaded oral provesicular lipid formulation: in vitro and ex vivo study.

The aim of this research was to develop proniosome (niosomes) of Nigella sativa (NS) to improve its drug release, gastrointestinal (GI) permeation and...
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