Preparation Nanocomposites 5ASA–GG-MMT nanocomposites had been synthesized in

Preparation of 5 aminosalicylic acid –
Loaded GG-MMT Nanocomposites

5ASA–GG-MMT nanocomposites had
been synthesized in two steps. Firstly, Guar gum was  to dissolve in water and then to emulsify in
the solution of acetone/ethanol containing 5-ASA with magnetic stirring
followed by sonication. In the second step, the primary w/o emulsion had been
emulsified in the external aqueous phase of MMT and Pluronic F68 (1%, w/v) to
form a w/o/w-emulsion. The intermediate organic phase was separated the
internal water droplets from each other as well as from the external aqueous
continuous phase. After solvent evaporation the 5ASA–GG-MMT nanocomposites were
collected by centrifugation and washed with double distilled water before
freeze-drying. 5ASA-GG-MMT NC’s formulations were labeled s code (NC1, NC2, NC3
& NC4) (Table 2) 28.

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Morphological Studies

The
shape and surface morphology of 5ASS-GG NP’s (NP4) and MMT-5ASA-GG NC’s (NC4) were
observed by Scanning electron microscopy (SEM) (JELO 5400, USA)

 

Particle
Size, Polydispersity Index (PDI) and Zeta Potential

Evenly diluted
suspension of 5ASA-GG nanoparticles (NP4) and MMT-5ASA-GG (NC4)
nanocomposites had been crammed in
the chamber of a laser diffraction particle size analyzer (DTS Ver. 4.10,
Malvern Instruments, Malvern, UK) for examination of PDI and average particle
size. Zeta potential of 5ASA-GG NP’s and MMT-5ASA-GG NC’s were also obtained by using  Malvern Zetasizer (DTS Ver. 4.10, Malvern
Instruments, Malvern, UK) 29.

Differential
Scanning Calorimetry (DSC)

Differential scanning calorimetery (DSC) of  pure 5ASA, MMT, GG, 5ASA-GG NP’s (NP4)
and MMT-5ASA-GG NC’s (NC4) were estimated by heating the
sample from 30 to 400ºC at the
heating rate of 10ºC/min, in a
nitrogen environment (nitrogen gas flow rate of 60.0ml/min). DSC studies had
been conducted on NETZSCH DSC 200F3­­ 240-20-427-L Proteus Software 30.

 

Powder
X-ray Diffractometry (PXRD)

Powder X-ray diffractometry (PXRD)
diffraction of formulations (NP4) & (NC4) analyses had been carried out in
order to find out crystalline nature of polymer and drug. Powder X-ray
diffraction patterns of samples were completed using power X-ray diffractometer
(Bruker, Munich, Germany). X-ray diffraction analysis was observed using a
nickel-filtered Cu-Ka radiation (a voltage of 40 kV and a current of 20 mA).
The scanning rate  2/min over a 2y range
of 0–40° and with an interval of 0.02? was set 31.

 

Determination
of Drug Entrapment Efficiency

50
mg of 5ASA-GG NP’s and MMT-5ASA-GG NC’s were
dissolved in acetone and ethanol (10 ml). Further suspension was centrifuged,
and supernatant was decanted than GG was precipitated by adding 10 ml of
ethanol. Percentage entrapment of 5ASA was determined using HPLC system
(Agilant Technoloogies, 1220 infinity LS, UK) the mobile phase was made of  2 parts i.e. part A phosphate buffer (pH 6.6)
and part B (acetonitrile) (77:23 v/v) delivered at a flow rate of 1 ml/min at
25ºc, wavelength detector, and a zorbax 5? C18 column (250 × 4.60 mm) at 329
nm. The concentration of free drug in the supernatant was taken by comparing
the absorption of the supernatant with standard curve 32. The amount of drug
entrapped into nanocomposites had been calculated as the difference between the
amount of drug used for the formulation and the amount of drug in the
supernatant and present as encapsulation efficiency. Encapsulation efficiencies
had been calculated by using formula :-  

                         Encapsulation
efficiency (%) =   total
drug?free
drug/ total drug ×100

 

                      

Drug
Release Studies

 In-vitro drug release of both the formulations
(NP4 & NC4) were observed in simulated gastric fluid (SGF) with pH 1.2 and
simulated intestinal fluid (SIF) with pH 7.5. These pH values were identified
based upon the normal variation of gastrointestinal tract (GIT) in the stomach
(pH 1.5), to the colon (pH 7 to 7.8) 33. 10 mg of 5ASA-GG NP’s and
MMT-5ASA-GG NC’s were dispersed into 2ml of PBS (pH 1.2) for 2 hrs and it was put
into a dialysis tube (MWCO 2000 Da). Then pH was managed to 7.5 and the release
profile studies were continued for 24 hrs. The dialysis tube was put into 50 ml
of aqueous recipient PBS medium (pH 1.2) and (pH 7.4) with continuous stirring
at 100 rpm and 37±2 ºC, for the total separation of drug in PBS. At a proper
time intervals, the whole medium (50 ml) were replaced with the same volume of
fresh PBS (pH 1.2 and 7.4) (50 ml) and samples were processed for analysis by
HPLC.

 In vivo Study

 These studies were planned according to the
guidelines of the CPCSEA, Ministry of Social Justice and Empowerment,
Government of India and prior approval from the Institutional Animal Ethical
Committee (Reg. No. 147/PO/a/11/CPCSEA) of Guru Ramdas Khalsa Institute of Science and Technology, Pharmacy,
Jabalpur, MP, India.

 

 

Organ
Distribution Study

 Male albino rats (150-200 g) were selected throughout
the experiment. Animals were categorized into 4 groups of 6 rats in each. The
group I served as control, group II received 5ASA suspension (30 mg/kg), group
III were introduced 5ASA-GG NP’s (0.5 mg/kg body weight related to 5ASA content)
and group IV was given with 5ASA-GG-MMT NC’s (0.5 mg/kg body weight related to
5ASA content) . The formulations were orally administered in suspension form
followed by enough volume of intake water. Three animals from each group were made
unconscious till their death by deep chloroform anesthesia at 1, 2, 3, 4, 6, 8,
10, 12, 14, 16, 20 and 24 hrs after drug administration. The GI tract was
eleminated; Stomach, small intestine, and colon were obtained. The luminal
contents were eleminated by applying mild pressure with wet scissors to the
tissues. Luminal and organs contents were processed to weigh. The organs were
incised open longitudinally and washed with saline solution (0.9% NaCl) to
detach any remaining luminal contents. The organs (small pieces) were taken for
homogenization by Micro Tissue Homogenizer (Mac, Mumbai, India) at 4 ºC along
with a small quantity of HPLC grade water. These organs were selected for
homogenization along with a small amount of PBS (pH 7.4); Acetonitrile (1 ml)
was cautiously used and added to homogenate and it was kept for 30 minutes.
Then it was centrifuged at 10,000 rpm for 5min and supernatant was filtered.
This filtrate was used for assay for the drug content by measuring the
absorbance HPLC method. The drug content at different part of GI tract at different
time period was determined.

 

Therapeutic
Activity

Male albino rats (avarage
weight 150 g) were utilized for the inflammation model.
Animals were treated with customary
existing conditions such as maintenance of 12h light and 12h dark timings,
controlled temperature and humidity (19 ± 29 °C; 35–60% humidity), standard
pellet diet, water and beddings in polypropylene caging.

Study protocol consisted of division of 24 animals
in four groups each group having 6 animals. details of which are as follows:-

Group 1: Animals were kept
untreated and given salines only. 

Group 2, 3 & 4: Animals were
treated with trinitrobenzenesulfonic
acid (TNBS) for induction of inflammation after the following process: firstly
catheterization of animals was done through 4cm intrarectally followed by light
narcotizing with ether. TNBS (100 ?l) was then introduced in ethanol and
applied in a dose of 160 mg/kg.  

Drug treatment

Group 2: Animals were processed for treatment
with 5ASA solution (at a dose of 30 or
100 mg/kg body weight)

Group 3:  Animals were processed for treatment with
5ASA-GG NP suspension (0.5 mg/kg body
weight related to 5ASA content)

Group 4: Animals
were processed for treatment with 5ASA-GG-MMT NC suspension (0.5 mg/kg body weight related to 5ASA
content)

All
administrations were given once daily for six days. Further animals were used
to sacrify 24h after the last drug/NP/NC administration and their colons were reselected.

 

 

 Pathophysiological Parameters

Inflammation
intensity was estimated with a medical examination consisting weight loss,
stool consistency and rectal bleeding as previously intimated 34, 35.
Longitudinal opening of rectal colon tissue samples was performed and luminal
content was removed after rinsing with iced phosphate buffer. Further wet
weight of tissue and colon was estimated and expressed as colon weight/length
quotient. Myeloperoxidase activity measurement was done in order to examine the
severity of the colitis since it is a trustworthy index of severity of
inflammation caused by infiltration of activated neutrophils into inflamed
tissue. Enzymatic activity was performed to analyse according to a standard
method 36.

 

Statistical
Analysis

The results
were outlined as mean values±S.D. For the analysis of statistical significance
ANOVA on ranks was used followed by Dunn’s test for all pairwise comparison. In
all cases, P

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