INTRODUCTION:

Oral controlled release (CR) dosage forms (DFs) have been developed over the past three decades due to their considerable therapeutic advantages such as ease of administration, patient compliance and flexibility in formulation.

Recently, dosage forms that can precisely control the release rates and target drugs to a specific body site have made an enormous impact in the formulation and development of novel drug delivery systems but the problem frequently encountered with controlled release dosage forms is the inability to increase the residence time of the dosage form in the stomach and proximal portion of the small intestine, due to the rapid gastrointestinal transit phenomenon of the stomach which may consequently diminish the extent of absorption of many drugs since almost most of the drug entities are mostly absorbed from the upper part of the intestine, therefore it would be beneficial to develop a sustained release formulation which remain at the absorption site for an extended period of time¹.

Furazolidone is a nitrofuran antimicrobial specialist utilized in the treatment of looseness of the bowels or enteritis brought about by microscopic organisms or protozoan diseases. Furazolidone is additionally dynamic in treating typhoid fever, cholera and salmonella diseases. Furazolidone is an individual from the class of oxazolidines that is 1,3-oxazolidin-2-one in which the hydrogen connected to the nitrogen is supplanted by a N-{[(5-nitro-2-furyl)methylene]amino} bunch. It has antibacterial and antiprotozoal properties, and is utilized in the treatment of giardiasis and cholera. It has a job as an EC 1.4.3.4 (monoamine oxidase) inhibitor, an antitrichomonal drug, an antiinfective specialist and an antibacterial medication. Furazolidone ties bacterial DNA which prompts the steady restraint of monoamine oxidase ^2-3.

MATERIAL AND METHODS:

Furazolidone was procured from Alkem India. Eudragit RSPO was obtained from EvonikIndia, Mumbai while Eudragit RLPO was obtained from Alkem India. HPMC K4M was being provided by Colorcon India, Goa. Chitosan was obtained from Central Marine Fisheries, Kochi, India. All other Chemicals and Reagent were used as obtained and were of Analyticle grade.

Drug Excipient Interaction Study:

FTIR Spectroscopy:

The FTIR spectrum of Furazolidone was recorded using FTIR spectrophotometer (Paragon 500 Perkin Elmer) using KBr pellet technique⁴.

Differential Scanning Calorimeter Studies:

Thermal analysis was performed using (DSC6220 Shimadzu) system with a differential scanning calorimeter equipped with a computerized data station. All samples were weighed and heated at a scanning rate of 10°C/min between 30 and 400°C and 40 ml/min of nitrogen flow. The differential scanning calorimetry analysis gives an idea about the interaction of various materials at different temperature. It also allows us to study the possible degradation pathway of the materials⁵.

Calibration Curve for Furazolidone:

10 mg Furazolidone was accurately weighted and transferred to 100ml volumetric flask. It was then dissolved in 0.1N HCl (pH 1.2) and sonicated for 10 min and diluted to volume with 0.1N HCl to give stock solution containing 100µg/ml. This solution was appropriately diluted with 0.1N HCl to obtain a concentration of 5, 10, 15, 20, 25, and 30µg/ml. The above solutions were analyzed by U.V. Spectrophotometer at 365 nm. All the dilutions were made using 0.1N HCL and 0.1N HCl was used as a blank during spectrometric analysis^6-7.

Preparation of Mucoadhesive Microspheres of Furazolidone:

Microspheres were prepared by a solvent evaporation method. The solvent system acetone/liquid paraffin was used. Agglomeration of microspheres was prevented by using 1% w/v Span 80. Eudragit RSPO was used to form a matrix of microspheres and mucoadhesive polymer were chosen to produce mucoadhesion is Chitosan and Hydroxypropyl methyl cellulose K4M. Eudragit RSPO and Furazolidone were dissolved in acetone and weighed quantity of Chitosan and Hydroxypropyl methyl cellulose K4M were dispersed it. The total volume of acetone was 12 ml. This homogeneous final dispersion was cooled to 5 °C and poured slowly with stirring (700 rpm) into 80 ml of liquid paraffin containing 1% w/v span 80, which was previously also cooled to 5 °C. The obtained emulsion was stirred at 40 °C for 40 min. The suspension of microspheres in liquid paraffin was filtered and microspheres were washed by petroleum ether and dried in vacuum at room temperature overnight⁸.

Table No.1: Table showing the Batch codes and polymer content

Batch	Drug	Eudragit RSPO	Eudragit RLPO	Chitosan	HPMC K4M
1	1	1	1	2	^-
2	1	1.5	0.5	2	-
3	1	0.5	1.5	1.5	0.5
4	1	2	-	1	1
5	1	-	2	1	1
6	1	1	1	1.5	0.5
7	1	2	-	1.5	0.5
8	1	1.5	0.5	1.5	0.5
9	1	0.5	1.5	-	2

Evaluations of Mucoadhesive Microspheres ^9-10

The Flow Properties:

Flow properties of microspheres were characterized in terms of angle of repose, Carr’s index and Hausner's ratio. The bulk density and tapped density were determined and from this data Carr's index and Hausner's ratio were calculated.

(a) Bulk Density and Tapped Density:

Both bulk density, ρ_b(often called loose or aerated bulk density) and tapped density, ρ_t were determined. Amount of microspheres was introduced in a 10 ml measuring cylinder up to 9 ml volume. Then the weight of Microspheres was determined by substracting the weight of empty measuring cylinder from final weight of measuring cylinder. The cylinder was allowed to fall onto a hard surface from a height of 2 cm at 2 sec intervals. The tapping was continued till no volume change was noted. ρ_b and ρ_t were determined by following formulas,

ρ_b=M / V_b

ρ_t = M / V_t

b) Carr’s Compressibility Index:

An important measure that can be obtained from bulk density determinations is the percent compressibility C, which is defined as follows

I_c= (ρt-ρb)/ρb×100

c) Hausner Ratio: A similar index has been defined by Hausner.

H_R=ρt / ρb

d) Angle Of Repose:

The angle of repose of the microspheres was determined by using funnel method. The accurately weighed powder were taken in a funnel. The height of the funnel was adjusted in such a way that the tip of the funnel just touched the apex of the heap of the microspheres. The diameter of the microspheres cone was measured and angle of repose was calculated by using the equation.

Ѳ =tan^-1h/r

where h and r are the height and radius of the microspheres cone.

Percentage Yield:

The prepared microspheres of all batches were accurately weighed. The weight quantity of prepared microspheres was divided by the total amount of all the excipients and drug used in the preparation of the microspheres, which give the total percentage yield of mucoadesive microspheres. It was calculated by using following equation.

Actual weight of microspheres

Percentage yield = -------------------------------------_X 100

Total weight of drug and excipients

Particle Size Determination:

Microsphere size was determined by using an optical microscope under regular polarized light, and the mean microsphere size was calculated by measuring 100 particles with the help of a calibrated ocular micrometer.

(9n_i× logX_i)

X_{g =}-------------------------×10

Where X_g is geometric mean diameter, n_iis number of particle in range, X_ithemidpoint of range and N is the total number of particles.

Determination of Drug Content:

To determine the total drug content of microspheres a known amount of microspheres wereground to fine powder. Accurately weighed (50mg) grounded powder of microspheres weresoaked in 50 ml of distilled water and sonicated using probe sonicator for 2 h. The whole solution was centrifuged using a tabletop centrifuge to remove the polymeric debris. Then the polymeric debris was washed twice with fresh solvent (water) to extract any adhered drug. The clear supernatant solution was filtrated through a 0.45 μm whatman filter paper then analyzed for Furazolidone content by UV-Vis spectrophotometer at 365 nm¹¹.

Determination of Drug Entrapment:

A known amount of microspheres wereground to fine powder. Accurately weighed (50mg) grounded powder of microspheres weresoaked in 50 ml of distilled water and sonicated using probe sonicator for 2 h. The whole solution was centrifuged using a tabletop centrifuge to remove the polymeric debris. Then the polymeric debris was washed twice with fresh solvent (water) to extract any adhered drug. The clear supernatant solution was filtrated through a 0.45 μm whatman filter paper then analyzed for Furazolidone content by UV/Vis spectrophotometer at 365 nm against blank¹¹. The amount of drug entrapped in the microspheres was calculated.

The drug entrapment was calculated by the equation

Practical drug content

DEE = -------------------------------------------- X 100

Theoretical drug content

Swelling Study:

Accurately weighed 10 mg of dried microspheres were incubated in HCl solution (pH 1.2) for 3 h. At predetermined times the microspheres were removed from the swelling media, blotted with a piece of tissue paper to absorb excess surface water and weighed¹¹.The swelling index(I) of the microspheres were calculated by the following equation:

I = (w2‐ w1)/w1

Where w1 is the weight of the dried sample and

w2 is the weight of the swollen sample.

The swelling ratio was plotted as a function of time.

In vitro Evaluation of Mucoadhesiveness:

For the evaluation of mucoadesiveness of furazolidone microspheres perfusion techniques was used, in which falling liquid film method was used. A strip of goat intestinal mucosa was mounted on a glass slide and accurately weighed mucoadhesive microspheres in dispersion form was placed on the mucosa of the intestine. This glass slide was incubated for 15 min in a desiccator at 90% relative humidity to allow the polymer to interact with the membrane and finally placed in the cell that was attached to the outer assembly at an angle 45⁰. Phosphate buffer saline (pH 6.4), previously warmed to 37 ± 0.50C, was circulated to the cell over the microspheres and membrane at the rate of 1 ml/min with the help of pump. Washings were collected at different time intervals and microspheres were separated by centrifugation followed by drying at 50⁰C.¹²The weight of microspheres washed out was taken and percentage mucoadhesion was calculated by

Percentage Mucoadhesion = Wa-Wl ×100 / Wa

Where Wa = weight of microspheres applied;

W1 = weight of microspheres leached out.

In vitro Drug Release Studies:

Release of Furazolidone from the microspheres was studied in 0.1N HCl (900 ml) using a USP dissolution testing apparatus II (Paddle type) with a rotating paddle stirrer at 50 rpm and 37° ± 1°C. A 5 ml sample solution was withdrawn from the dissolution apparatus for an every hour for 12 hrs. Samples were replaced by its equivalent volume of dissolution medium. The samples were filtered through Whatman filter paper and solutions were analyzed at 365 nm by UV Spectrophotometer (Libindia Disso-2000). Cumulative percentage drug release was calculated^13-15.

Drug release kinetics:

Dissolution data of above methods was fitted in Zero order, First order and Higuchi eqations.

The mechanism of drug release was determined by using Korsmayer Peppas equation^16-18.

RESULT:

FTIR:

FTIR is called as fingerprint technique and is widely used in identification of drugs and excipients. The FTIR spectra of drug were compared with standard spectra of Furazolidone and showed all corresponding peaks as per standard spectra.

FTIR Spectroscopy:

FTIR spectrum of Furazolidone showed all the peaks corresponding to the functional groups present in the structure of Furazolidone.

Figure No.1: FTIR spectrum of Furazolidone

Calibration Curve for Furazolidone:

The calibration curve for Furazolidone in 0.1N HCl. The graph of absorbance vs. concentration for Furazolidone was found to be linear in the concentration range of 5-30 μg/ml at 365 nm. The r²of the calibration curve was found to be 0.9955.

Figure No.2: Calibration curve for Furazolidone

Differential Scanning Calorimeter Studies:

Differential Scanning Calorimetry studies indicated a sharp endothermic peak at 260.2°C for pure Furazolidone. There was no significant change in the position of this peak in the thermograms of drug and excipients mixture. So, it can be concluded that the excipients and drug do not interact with each other.

Differential Scanning Calorimetry (DSC):

DSC thermograms of drug and formulation batch showed the peaks at 260.2⁰C and 258.8⁰C respectively. These are the temperatures corresponding to the melting points of Furazolidone and B7 formulation batc.

Figure No.3: DSC graph of Furazolidone

Figure No.4: DSC of Optimized Batch of Furazolidone

Evaluation of Mucoadhesive Microspheres:

Flow Properties of Microspheres:

Size of prepared mucoadhesive microspheres was found between 421mm to 517mm. range. Microspheres size of formulation batch B1-B2 was increased as chitosan polymer concentration increases than other batches.

The prepared mucoadhesive microspheres were characterized with respective bulk density, Tapped Density, Carr’s index, Hausner ratio, and angle of repose. From the above value, bulk density value ranged between 0.302-0.584, where tapped density ranged between 0.344-0.638. Carr’s index in between 8-17 % and Hausner ratio within 1.08-1.16. The angle of repose was less than 30º for all the batches indicating good flow properties.

Table no.4: Evaluation of flow properties of microspheres

Batch	Bulk Density (g/cc)	Tapped Density (g/cc)	Carr’s compressibility index	Hausner’s ratio	Angle of repose (degree)
B1	0.302	0.352	16.88	1.16	29⁰16’
B2	0.309	0.344	11.32	1.11	30⁰82’
B3	0.389	0.425	8.47	1.09	30⁰82’
B4	0.467	0.528	11.55	1.13	28⁰24
B5	0.481	0.525	9.14	1.09	30⁰82’
B6	0.386	0.442	12.66	1.14	29^O72’
B7	0.489	0.547	10.60	1.11	30⁰82’
B8	0.584	0.638	8.46	1.09	30⁰82’
B9	0.504	0.549	8.19	1.08	27⁰45

Evaluations of Mucoadhesive Microspheres:

The percentage yield of all the formulation B1 to B9 in the range of 65.23-79.45. From this data, it shows percentage yield above 65% that means all formulations having good percentage yields. The percentage yield of mucoadhesive microsphere determined by weighing after drying. Formulations B4 and B7 containing mucoadhesive microsphere gives higher percentage yield i.e. 74.63% to 79.45% respectively.

Table no.5: Evaluations of Mucoadhesive microspheres

Batch	% Yield	Drug content	% DEE	Particle Size (µm)
B1	65.23	54.15	64.21	509
B2	72.41	59.21	72.16	517
B3	68.57	55.64	66.43	421
B4	78.59	62.79	68.38	463
B5	74.63	60.42	71.06	409
B6	72.98	56.83	63.78	486
B7	79.45	64.83	73.45	426
B8	67.51	51.13	61.37	461
B9	73.42	57.94	63.52	487

SEM:

Scanning electron microscopy (SEM) was utilized to observe the surface and inner part of the mucoadhesive microspheres of optimized formulation B7. It was found that even surface morphology at 100x was in the range 430-490 µm.

Figure No.5 SEM image of formulation batch B7

Swelling Index:

Swelling:

Swelling studies of mucoadhesive microsphere done on the basis swelling ratio. Swelling ratio increased with weight gain by microsphere was increased proportionally with the rate of hydration. Microsphere is influenced by the environmental pH, being generally greater at lower rather than higher pH value.

From the given value of swelling index, it was founded that swelling ratio of the formulation B9> B5> B4 containing HPMC K4M was more as compared to the remaining batches due to high concentration and swelling property of HPMC K4M in acidic media. The gelling of chitosan did not have significant effect on the swelling property it was observed that as we enhance the concentration of HPMC K4M swelling index increases due to swelling property of it.

Figure no.6: Swelling behaviour of Furazolidone Batches B1-B9

In Vitro Mucoadhesion:

Microspheres consisting of HPMC K4M and Chitosan in different concentrations exhibited good mucoadhesive properties in the in-vitro falling liquid film test up to 12 hr.

The order of bioadhesion in the media based on percentage was as follows: B7> B8> B6> B5> B4> B3> B9> B1>B2. The results of the falling liquid film test indicated that the microspheres had fairly good mucoadhesive properties in the gastric conditions.

Figure no.7 In Vitro Mucoadhesion behavior of Batch B1-B9

In Vitro Dissolution study:

In vitro Dissolution Test Drug Release

Order of drug release from batches was found to be B7> B8> B5> B9 due to gelling nature of chitosan in combination with swelling of HPMC K4M. B1 to B3 batches shows low drug release due to gelling of chitosan with sustained release polymers. To develop sustained drug release combination of Mucoadhesive polymers i.e. chitosan and HPMC K4M with sustained release polymer is required. However all batches shows drug release shows release above than 76%.

Figure no.8: In Vitro Drug Release of Batch B1-B9

Kinetic Study:

B7 Batch:

Zero order > Korsmeyer Peppas > Higuchi > First order

Drug Release Mechanism:

For all the formulation Korsmeyer’s Peppas exponent (n) was found to be greater than 0.85(>0.85). This indicated that mechanism of drug release controlled from the all formulations was based on Case-II Transport or typical zero-order release.The in vitro release kinetics exhibited a Case-II Transport model. This kind of diffusion corresponds to a more predictable type of sustained drug delivery system.

Data Comparison:

Effect of concentration polymers: of mucoadhesive:

To determine this effect, dissolution data of formulations B2 and B3 containing Chitosan and Chitosan+HPMC K4M respectively were used, as these polymers showed drug release for longer period in all the cases as mentioned above.

Effect of polymers (Eudragit) On Drug Release:

To determine this effect, dissolution data of formulations B6, B7 and B8 containing Eudragit RSPO+Eudragit RLPO (1:1), Eudragit RSPO and Eudragit RSPO+RLPO (1_1/2:1/2) respectively were used, as these polymers showed drug release for longer period in all the cases as mentioned above.

Table No.9: ANOVA of Formulation B6, B7 and B8

Source of variation	Degree of freedom	Sum of Squares	Mean square	F ratio
Between Columns Formulations	2	94.54	47.27	24.38
Between Rows Time	11	25235	2294	1183
Residual	22	42.65	1.939
Source of Variation	P value summary		Significant?
Formulations	****		Yes
Time	****		Yes

ANOVA results clearly showed that the effect of different type of eudragit i.e RSPO and RLPO affects drug release determined by paddle method significantly (P<0.0001). Drug release mechanism of batch B6, B7, and B8 was found Case II transport i.e typical zero order release and it is clearly indicating from the experimental results of formulations, which shows complete release more than 12 hrs. The experimental results support this fact.

Figure No. 9: Effect of Eudragit (RSPO and RLPO)

CONCLUSION:

Mucoadhesive microspheres of Furazolidone prepared by solvent evaporation method by using eudragit RSPO, eudragit RLPO, chitosan and HPMC K4M. The proportion of drug to polymer (Furazolidone: Eudragit RSPO+ Eudragit RLPO and chitosan: HPMC K4M) influence the physical characteristic as well as mucoadhesive behaviour of the microspheres. The drug release was sufficiently sustained and Case-II Transport or typical zero-order release of the drug from mucoadhesive microspheres was confirmed. ANOVA result shows significant effect of polymers and concentration of polymers on formulation. The optimized formulation batch B7 shows high percentage yield, entrapment efficiency, percent cumulative release and mucoadhesiveness.

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Received on 23.07.2021 Modified on 27.11.2021

Asian J. Res. Pharm. Sci. 2022; 12(1):1-7.

DOI: 10.52711/2231-5659.2022.00001