FORMULATION AND EVALUATION OF MULTIPARTICULATE …

www.wjpr.net Vol 5, Issue 9, 2016.

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Deshpande et al. World Journal of Pharmaceutical Research

FORMULATION AND EVALUATION OF MULTIPARTICULATE

SYSTEM FOR ORAL DELIVERY OF PAPAIN

1Gaurav Laxmikant Deshpande* and 2Satish Vasudeo Shirolkar

1B. Pharm. Dr. D. Y. Patil Institute of Pharmaceutical Sciences and Research, Pimpri, Pune,

Maharashtra-411018, India.

2M. Pharm. Ph. D. Department of Pharmaceutics, Dr. D. Y. Patil Institute of Pharmaceutical

Sciences and Research, Pimpri, Pune, Maharashtra-411018, India.

ABSTRACT

Digestive enzyme supplements are used to treat enzyme deficiencies.

Plant-based enzymes, such as papain from papaya, serve as effective

digestive aids in the breakdown of proteins. The aim of this study is to

develop multi-particulate drug delivery system for digestive enzyme

papain. Multi-particulate systems offer advantage with respect to

predictable and even distribution and transportation through GI track.

Characterization of papain was carried out by physical properties,

melting point, FT-IR spectroscopy, drug qualitative stability study and

assay of papain was performed by using Hammersten-type casein as

substrate. Multi-particulate systems of papain have been attempted

using microcrystalline cellulose (MCC) and calcium hydrogen

phosphate (dihydrate) as main excipients. Dough of papain with

excipients was prepared and passed through sieve no. 16 (B.S.S.). The

extrudates were spheronised using spheronizer. Optimization of process was done by factorial

design using speed (RPM) and time (min.) for spheronisation as independent variables at

three levels. For papain containing MCC-DCP pellets effect of speed and time was studied on

percentage yield, pellet size, bulk density, tapped density, Carr‟s index, Hausner ratio, angle

of repose, hardness, disintegration time, and percentage drug content. Relationship between

dependent and independent variables was established using Design Expert® 10.

KEYWORDS: Multi-particulate systems, digestive enzyme, papain, pellets, extrusion-

spheronisation, factorial design.

World Journal of Pharmaceutical Research SJIF Impact Factor 6.805

Volume 5, Issue 9, 1153-1173. Research Article ISSN 2277– 7105

Article Received on

05 July 2016,

Revised on 26 July 2016,

Accepted on 17 Aug 2016

DOI: 10.20959/wjpr20169-6947

*Corresponding Author

Gaurav Laxmikant

Deshpande

B. Pharm. Dr. D. Y. Patil

Institute of

Pharmaceutical Sciences

and Research, Pimpri,

Pune, Maharashtra-

411018, India.


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INTRODUCTION

“Enzymes are naturally occurring protein molecules found in all living cells. They act as

catalysers, increasing the rate of biochemical reactions in the human body.” Enzymes are the

life force that activates „vitamins, minerals, proteins and other physical components‟ within

our body.[1,2]

The use of therapeutic proteins and enzymes at industrial level has increased and these

macromolecules are replacing low molecular weight chemicals due to their high specificity

and potency. However, biological limitations impose restrictions for the manufacturing and

development of protein pharmaceuticals which require controlled procedures and suitable

drug delivery systems.[3] Dietary proteins are essential for growth, repair, and regulation of

homeostasis. However, many people are intolerant of such foods, which include baked beans,

bean soup, soybean, and meat. This intolerance can lead to uncomfortable and embarrassing

symptoms, such as flatulence, belching, diarrhea/constipation, malnutrition, food allergies,

anemia, undigested food in stool, chronic intestinal parasites, and abnormal flora. These

symptoms usually occur during achlorhydria and/or pancreatin insufficiency. Therefore, the

need for a protein-digesting supplement arises to overcome the deficient manifestations.

Nowadays, the demand for digestive aids has increased, but the supply of pepsin (prepared

from hog mucosa) has decreased. Thus, plant-derived proteases like „Papain‟ can be used

because; there is no scarcity of supply.[4]

Papain is a food-grade, highly active endolytic cysteine protease (EC 3.4.22.2) derived from

Carica papaya Linne’ (Family: Caricaceae). Its active site consists of Cys-25, His-159, and

Asp-158.[5,6] Papain shows extensive proteolytic activity toward proteins, short-chain

peptides, amino acid esters, and amide links and is applied extensively in the fields of food

and medicine. Its broad substrate specificity and ability to hydrolyze small peptides as well as

large proteins make papain an ideal enzyme supplement.[4] Most marketed formulations

containing papain and other digestive enzymes need to be stored at cold (2º to 8ºC) or cool

(8º to 25ºC) temperatures conditions and still have the shelf-life of less than one year.[6] The

optimum pH for activity of papain is in the range of 5.0-9.0, varying with different

substrates.[7]

Multiple-unit dosage forms are particularly useful for the purposes of: (1) delivering highly

irritant drugs, such as nonsteroidal anti-inflammatory drugs; (2) site-specific targeting of


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acid-labile drugs within the gastrointestinal tract; and (3) the delivery of enzymes,

peptides/proteins, and vaccines.[8,9]

As papain is derived from natural source and having less or no side-effects. Multi-particulate

systems offer advantage with respect to predictable and even distribution and transportation

through GI track.[9] So, the purpose of designing this work was to develop reliable

formulation that has all the advantages of a single unit formulation and yet to devoid of the

danger of alteration in drug release profile and formulation behavior due to unit to unit

variation, change in gastro-luminal pH, and enzyme population.

The above advantages are of great commercial interest for the pharmaceutical industries;

hence, the objective of the research was to develop multi-particulate drug delivery system for

oral delivery of papain by extrusion-spheronisation technique and to optimize spheronisation

process using factorial design.

MATERIALS AND METHODS

MATERIALS

Papain (From papaya), Trichloroacetic acid (98.0%), L- Cysteine hydrochloride monohydrate

(99.0%), Citric acid (98.0%), and PVP K-30 were purchased from Himedia Laboratories Pvt.

Ltd., Mumbai. Hammersten-type casein (Bovine Grade) was purchased from Sigma Aldrich,

Mumbai. Disodium phosphate (anhydrous) (99.5%), Disodium edetate (99.5%), Toluene,

Calcium hydrogen phosphate (dihydrate), Talc, Sodium starch glycolate, and Propylene

glycol were purchased from Loba chemie, Mumbai. Cellulose Microcrystalline (MCC) and

Guar gum were purchased from Research Labs, Mumbai. Eudragit (L 100-55) was received

as gift sample from Evonik industries, Mumbai. All of the other chemicals and solvents were

of analytical grade and were used without further purification. Distilled water was used

throughout the study.

METHODS

Characterization of Papain

1. Physical properties: The enzyme powder was examined for physical properties like

appearance and odour.

2. Melting point determination: The melting point apparatus (Company: Veego; Model:

VMP-PM) was used to determine melting point of Papain.


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3. FT-IR spectroscopic study: The FT-IR spectrum was recorded by mixing dry papain and

potassium bromide. Spectral analysis was done using Fourier Transform Infra-red

Spectrophotometer (Company: Shimadzu; Model: 8400S).

4. Drug qualitative stability study: Since, papain is an enzyme, this is highly sensitive

against environmental and temperature conditions. So, the drug (papain) was studied for its

qualitative loss due to environmental degradation and temperature. Drug was kept at 40o C

temperature for 20 days in petri-dish and an IR spectrum of stored sample was then recorded

on FT-IR spectrophotometer (Company: Shimadzu; Model: 8400S).

5. Enzyme assay:[10,11] The pure drug was assayed for bioactivity determination by using

method reported in literature. The proteolytic activity of papain was estimated by modified

casein digestion method in the presence of „Phosphate cysteine disodium

ethylenediaminetetraacetate buffer.‟

a. Casein substrate: (Prepared fresh daily): 1g Hammersten-type casein substrate was

added to 50.0 mL 0.05M disodium phosphate (anhydrous) in a 250.0 mL RBF and heated

at 700 - 850 C for 25-30 min. with occasional shaking on a heating mental till bluish-white

solution was obtained. The extreme care was taken to avoid excessive bumping of

solution. Solution was immediately transferred in a beaker and cooled to room

temperature. pH was adjusted to 6.0±0.1 with 0.05M Citric acid with rapid stirring on a

magnetic stirrer (600-800 RPM). Continuous and rapid stirring had been needed to avoid

precipitation. The solution was transferred to 100.0 mL volumetric flask and diluted with

distilled water up to the mark. The finally obtained solution was filtered through sintered

glass filter assembly with vacuum pump to filter off precipitated casein if any.

b. Enzyme solution: Accurately weighed 100.0 mg of papain was added in phosphate

cysteine disodium ethylene diamine tetraacetate buffer (PCD-EDTA buffer) to make

100.0 mL. The resulting solution was sonicated for 10 min. and the 10.0 mL solution was

pipette-out and diluted to 50.0 mL with PCD-EDTA buffer. The subsequent dilutions

from 20 to 200 µg/mL were prepared as per needed.

Procedure

The 5.0 mL of buffered 1% w/v casein substrate (pH 6.0±0.1) was added to both sample and

blank test tubes. 2.0 mL of papain solution from each dilution was added to sample test tubes.


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After incubation at 40º C for 1 hour in water bath shaker (Company: Neolab), the reaction

was stopped by the addition of 3.0 mL of (30% w/v) trichloroacetic acid solution to both

sample and blank test tubes. The tubes were allowed to incubate for 10 minutes at 40º C in

water bath shaker for complete coagulation of the precipitated protein. Then, the 2.0 mL

papain solution from each dilution was added to respective blank test tubes and allowed to

incubate for 30 min. The supernatant containing digested amino acids was filtered through

Whatman filter paper number 42 by discarding first 3.0 mL filtrate. The absorbance of the

filtrate was measured at 280.0 nm against PCD-EDTA buffer using UV-VIS

Spectrophotometer (Company: Shimadzu; Model: Pharmaspec 1700).

Pellets Preparation[3,12]

The technique selected for the preparation of papain containing pellets of uniform size and

shape was „Extrusion–spheronisation.‟ The dry mixing or blending, wet massing, extrusion,

spheronisation and drying were carried out as operational stages to formulate papain

containing pellets.

1. Dry mixing: The required quantities of powered excipients i.e. Calcium hydrogen

phosphate (dihydrate), Cellulose microcrystalline PH 101, PVP K-30, Eudragit (L 100-55),

Sodium starch glycolate were weighed and passed through sieve number 60 (B.S.S.). All the

excipients were thoroughly mixed by using mortar and pestle. The accurately weighed drug

i.e. papain was added to dry mix and mixed it thoroughly for uniform mixing of drug and

excipients to form a homogenous dispersion.

2. Wet massing: The „selection of solvent‟ was very critical in papain containing pellet

formulation. As papain itself forms gel-like viscous and elastic mass with water after addition

of less than 3.0 mL of water for 15g batch, the wet massing was hardly possible. So, different

solvents like ethanol, isopropyl alcohol, propylene glycol etc. and proportional mixtures of

these solvents had been tried for wet massing. Finally mixture of water, ethanol, and

propylene glycol was selected as solvent mixture (Smix) in the proportion of 4.5: 4.5: 1.75 in

formulation.

The solvent-mixture was added to dry mixture with kneading. Successive pauses were taken

for the addition of the liquid components and proper kneading process was carried out with

pestle as well as hand mixing to obtain a damp mass of appropriate consistency for the

extrusion process.


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3. Extrusion: The damp mass was passed through sieve number 16 (B.S.S.) to get compact

cylinders – extrudates – of uniform diameter.

4. Spheronisation process: The extrudates were spheonized using lab spheronizer (Shakti

Pharmatech; Model: SSP120GMP) with a plate of 2 mm to get spherical pellets. Factorial

design was applied for optimization of process parameters i.e. speed and time of

spheronisation.

5. Drying process: The pellets were dried in the hot air oven at 40ºC and for 60 minutes.

The dried pellets were collected and sieved through sieve number 12 (B.S.S.) to separate the

fines. The final product was stored in air tight container.

Preliminary studies of papain containing pellets were carried out for selection of papain

containing batch for optimization of process parameters using factorial design. Table 1 enlists

various preliminary formulations of papain containing pellets.

Table 1: Various preliminary formulations and compositions of papain containing

pellets

Batch Code Time Papain DCP MCC PVP K-30 Eud L 100-55 SSG Smix

(min.) (g) (g) (g) (g) (g) (g) (mL)

T1 4 2 5 5 0.5 0.5 2 8

T2 4 2 5 5 0.5 0.5 2 8.5

T3 4 2 4.75 5.25 0.5 0.5 2 8.5

T4 4 2 4.5 5.5 0.5 0.5 2 8.5

T5 4 2 4.25 5.5 0.75 0.5 2 8.5

T6 2 2 4 5.5 1 0.5 2 8.5

T7 6 2 4 5.5 1 0.5 2 8

T8 9 2 4.25 5.75 0.5 0.5 2 8.5

T9 2 2 4.25 5.75 0.5 0.5 2 10

T10 2 2 4.25 5.75 0.5 0.5 2 9.5

T11 4 2 4.25 5.75 0.5 0.5 2 9 *Plate size of spheronizer was kept constant to 2 mm.

*Speed of spheronisation was kept constant to 1000 RPM.

Characterization of Pellets

Pellets were evaluated for percentage yield, pellet size analysis, bulk density, tapped density,

Carr‟s index, Hausner ratio, angle of repose, hardness, disintegration time and drug content.


1159


1. Percentage yield: The pellets were passed through sieve no. 12 (B.S.S.) to separate the

fines and total weight of pellets was taken after separation of fines. The percentage yield was

calculated by using formula.

Percentage yield =

2. Pellet size analysis: The pellet size of the prepared pellets was measured using Digital

Vernier caliper (Company: Mitutoyo). The randomly selected 20 pellets in each batch were

taken and mean diameter of pellets was measured as a pellet size.

3. Bulk density:[13,14] 10 g of pellets were weighed from each batch and filled into measuring

cylinder to note down the bulk volume of pellets. The bulk density was calculated by using

formula-

Bulk Density =

4. Tapped density:[13,14] 10 g of pellets were weighed and filled into measuring cylinder. The

measuring cylinder was fixed to an automated tap density apparatus (Company: Veego) and

tapped for 999 times the resulting volume was taken as a tapped volume. The tapped density

was calculated by using formula-

Density =

5. Carr’s index:[13,14] Compressibility index of pellets was determined for each batch by

using formula-

Carr‟s Index =

6. Hausner ratio:[13,14] Hausner ratio was calculated for each batch by using formula-

Hausner ratio =

7. Angle of repose:[13,14] Angle of repose was determined by Neumann‟s method and

calculated using the formula-

Angle of repose (θ) = Tan-1(h / r)

Where,

h = height of pile,

r = radius of the pile base.


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8. Hardness: The hardness of pellets was measured using „Digital pellet hardness tester‟

(Company: Veego). The 5 pellets were tested for hardness and average was taken as hardness

of pellets.

9. Disintegration time: Disintegration time of pellets from each batch was determined by

noting the disintegration time in distilled water. Since, there is no standard disintegration

apparatus for testing disintegration time of pellets, the pellets put in distilled water and

disintegration time was noted down by shaking them in water bath shaker till disintegration.

10. Drug content (assay of pellets): “Drug content of pellets is the amount of added drug

that is present in the formulated pellets.” The drug content of drug from pellets can be

calculated as, ratio of drug in the final formulation to the amount of drug added.

An accurately weighed crushed powder of pellets equivalent to 10 mg of papain was taken

and extracted with 100.0 mL PCD-EDTA buffer. The solution was shaked and sonicated for

15 min. The resultant solution was filtered through Whatman filter paper no. 42 and assayed

(n = 3) for enzyme content by enzyme assay method.

Drug content was calculated as-

Drug content = X 100.

Factorial Design

In this study, a 32 full-factorial design was used to determine the effect of the speed of

spheronisation (RPM) and spheronisation time (min.). Before the application of the design, a

number of preliminary trials were conducted to determine the range of independent variables.

Table 2 lists independent variables and their levels.

Table 2: Independent variables and their levels

Variables Low level (−1) Medium level (0) High level (+1)

X 1 = Speed (RPM) 800 1000 1200

X 2 = Time (min.) 2.5 4.0 5.5

Design expert 10® was used to generate the response plots and counter plots which indicated

relative effects of independent variables viz. speed of spheronisation and time allowed for

spheronisation on dependent variables viz. percentage yield (Y1), average particle size (Y2),

bulk density (Y3), tapped density (Y4), Carr‟s index (Y5), Hausner ratio (Y6), angle of

repose (Y7), hardness (Y8), and drug content (Y9).


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Drug Excipients Compatibility Study

All above formulations showed low percentage drug content. This may be because one or

more excipient/s is/are incompatible with papain. So, compatibility studies for papain were

carried out with potential formulation excipients to determine possibility of any drug-

excipient interaction/incompatibility. Drug was mixed with excipients in formulation ratio.

These samples were subjected to compatibility studies and stored for 20 days at 40o C

temperature. IR spectra of these stored samples were then obtained after 20 days.

Stability Studies of Optimized Batch

The optimized formulation was stored in airtight wide mouth glass containers at ambient

conditions (room temperature~35ºC) as well as at cool conditions in refrigerator (2º to 8º C)

for 30 days.

RESULTS AND DISCUSSION

Characterization of Digestive Enzyme Papain

Physical characterization of papain: Papain is white, hygroscopic, amorphous power

with characteristic taste and odour.

Melting point determination: The papain had started to decompose at 161.1º C and

decomposed completely at 205.1º C.

FT-IR spectroscopic study: The IR spectrum of papain obtained on a FT-IR

spectrophotometer with diffused reflectance assembly is shown in figure 1. It is similar

with one reported in literature. The interpretation of IR frequencies was done and

absorption bands are consistent that with structure of papain. Data of IR interpretation is

shown in Table 3.

5007501000125015001750200022502500275030003250350037504000

1/cm

15

30

45

60

75

90

105

120

%T

3965

.78

3553

.00

3389

.04

3344

.68

3321

.53

3282

.95

3252

.09

3012

.91

2931

.90

2727

.44

2357

.09 2322

.37

2237

.50

2125

.63

2065

.83

2046

.54

1990

.60

1936

.60

1855

.58

1822

.79

1645

.33

1454

.38

1419

.66

1346

.36

1276

.92

1236

.41

1116

.82

1062

.81

1001

.09

914.

29

858.

35

758.

05

684.

75

580.

59 526.

58 468.

72

441.

7141

0.85

papin

Fig. 1: IR spectrum of papain


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Table 3: IR spectral analysis of papain

Sr. No. IR Frequency (cm-1) Assignment Functional group

1 1645.33 O=C<NH Amide-I bond

2 3369-3352 O=C<N-H Amides

3 1666 O=C<O H Carboxylic acid

Drug qualitative stability study: The interpretation of IR frequencies of stored papain

was done and absorption bands are consistent that with structure of papain. So, there were

no such structural changes found in papain.

Assay of papain: Absorbance obtained at 280.0 nm is indicative of tyrosine production

after reaction of various concentrations of papain (enzyme) with Hammerstein type casein

(substrate).The method was found to be linear over an analytical range 20 to 200 µg/mL

with a correlation coefficient (r) of 0.9776 and regression equation were found to be y =

0.003x – 0.039.

Pellets Formulation

With regard to active substances and even more important for protein pharmaceuticals,

optimization experiments involving formulation components and process variables and the

related operational stages should provide environment to minimize protein degradation

during processing. As an attempt to provide such microenvironment, the formulation

components were carefully selected based on intrinsic properties of papain.

The selection of MCC PH 101 for the study was based on its well known applications in the

extrusion–spheronisation technique in order to allow the proper formation of pellets and

assure flexibility in formulation and processing stages. Calcium hydrogen phosphate

(dihydrate) was used as filler, which holds effects over release profile and as an advantage it

can be dried more efficiently in a short time. PVP K-30 binder appeared to be one of the most

vital formulation parameters for this method. The PVP K-30 provided strength and improves

binding capacity of dry mixture. Eudragit is well known polymer and used to give pH

dependent drug delivery in GIT. SSG was selected to provide disintegration and to improve

release profile. As solvent selection was critical issue; Ethanol was selected to avoid

excessive activity loss during drying. Propylene glycol was selected to hold moisture till end

of spheronisation procedure.


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Pellets Physical Properties

The evaluation of the physical properties of the pellets refers directly to the viability and

applicability of a formulation to an extent of the evaluation of production stages and thus

assuring the estimation of relevant quality parameters of the pharmaceutical form. The pellets

size distribution corresponds to an important parameter to be estimated considering its ability

to interfere in the release kinetics of the formulation.

By means of density measurements, the obtained values, ranging from 0.595 to 0.725 g/mL

for bulk density and from 0.663 to 0.747 g/mL for tapped density, were different for each

batch; it is relevant to point out the importance of determining such values in order to assure

subsequent procedures, such as packaging and filling during capsules manufacture. The

influence of the formulation variables on the shape and physical properties of the pellets is

reflected in results presented in Table 4, and was found to be minimum regarding the physical

properties, considering the fact that the pellets presented acceptable physical properties, even

though such values varied slightly according to each batch. This behavior indicated good

flexibility of the prototype formulations as a function of formulation variables.

Table 4: Physical characterization of papain loaded pellets

Batch

Code

%

yield

Avg.

Pellet Size

Bulk

Density

Tapped

Density

Carr’s

Index

Hausner

Ratio

Angle of

repose Hardness

(%) (mm) (g/mL) (g/mL) (%) - (º) (Kg/cm2)

T1 88.13 1.149 0.696 0.734 5.263 1.056 28º 09” 0.198

T2 88.07 1.174 0.644 0.695 7.317 1.079 30º 43” 0.277

T3 85.54 1.372 0.642 0.675 5.000 1.053 28º 36” 1.309

T4 90.37 1.163 0.646 0.695 7.142 1.077 31º 22” 0.909

T5 90.89 1.336 0.649 0.737 11.905 1.135 23º 86” 1.463

T6 97.26 1.573 0.634 0.663 4.348 1.045 30º 98” 0.984

T7 85.39 1.218 0.640 0.674 5.000 1.052 25º 93” 1.486

T8 79.77 1.183 0.725 0.747 3.030 1.031 21º 61” 1.108

T9 98.65 1.501 0.617 0.643 4.167 1.044 34º 87” 0.350

T10 91.29 1.183 0.595 0.668 10.870 1.122 35º 36” 1.758

T11 92.35 1.442 0.690 0.741 6.885 1.074 25º 87” 0.3664

Disintegration time: The pellets were swelled but not disintegrated completely even after 12

hours.

Amongst 11 formulations, batch T11 showed higher percentage yield, optimum pellet size,

enhanced sphericity, and good flow properties. Therefore, Batch T11 was selected for


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optimization of spheronisation process parameters and 32 full factorial design was applied to

optimize the process variables i.e. „Speed‟ and „Time‟ of spheronization.

Table 5 shows, results of evaluation of nine batches prepared for optimization of process

parameters.

Table 5: Effect of the independent variables on dependent variables

Batch

Code

Speed Time %

yield

Avg.

Pellet Size

Bulk

Density

Tapped

Density Hardness

Drug

content

(RPM) (Min.) (%) (mm) (gm/cc) (gm/cc) (Kg/cm2) (%)

F1 800 2.5 84.53 1.613 0.667 0.690 1.075 22.097

F2 800 4.0 88.21 1.490 0.667 0.741 0.572 19.433

F3 800 5.5 59.19 1.719 0.645 0.673 0.476 18.057

F4 1000 2.5 82.64 1.510 0.645 0.690 0.921 29.465

F5 1000 4.0 92.35 1.442 0.690 0.741 0.366 28.767

F6 1000 5.5 71.54 1.551 0.645 0.741 0.596 9.000

F7 1200 2.5 73.79 1.686 0.645 0.690 1.200 21.097

F8 1200 4.0 85.46 0.885 0.667 0.769 0.931 15.333

F9 1200 5.5 87.04 1.396 0.714 0.741 0.696 14.347

Evaluation of Factorial Batches Using Design Expert 10®

1. Effect on percentage yield

Mathematical relationship generated for percentage yield is expressed in following equation

(1).

Percentage yield (Y1) = +90.32+2.39* A-3.87* B+9.65* AB-2.47* A2-12.22* B2……. (1).

From equation (1), it can be seen that positive coefficient of A indicates increase in the Yield

(Y1) with increase in speed of spheronizer up to certain period. The negative coefficient of B

indicates decrease in yield with increase in time of spheronisation, the pellets from 2.5 to 5.5

min. The equation obtained was quadratic equation which showed the effect was nonlinear.


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(a) Counter plot showing the relationship

between various levels of two independent

variables on % Yield of pellets.

(b) Response surface plot showing the

influence of speed (RPM) and time (min.)

on the % Yield of pellets.

Fig. 2: Different plots showing effect of independent variables on % yield of pellets

The selected quadratic model showed "Prob> F" value of 0.0584 is little bit high and

indicated that there was no significant effect of process variables on percentage yield.

2. Effect on pellet size

Mathematical relationship generated for pellet size is expressed in following equation (2).

Pellet size (Y2) = +1.30-0.14* A-0.024* B-0.099* AB-0.036* A2+0.31* B2….. (2).

From equation (2), it can be seen that negative coefficient of A indicates decrease in the

pellet size (Y2) with increase in speed of spheronizer up to certain period. The negative

coefficient of B indicates decrease in pellet size with increase in time to spheronize the

pellets from 2.5 to 5.5 min. up to certain period. The equation obtained was quadratic

equation which showed the effect was nonlinear.



variables on the pellet size of pellets.



on the pellet size of pellets.

Fig. 3: Different plots showing effect of independent variables on pellet size of pellets

The selected quadratic model showed "Prob> F" value of 0.3637 which indicated that there

was no significant effect of process variables on pellet size.

3. Effect on bulk density

Mathematical relationship generated for bulk density is expressed in following equation (3).


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Bulk density (Y3) = +0.67+ (7.933E-003)* A+ (7.933E-003)* B+0.023* AB+ (7.433E-

003)* A2-0.014* B2…. (3)

From equation (3), it can be seen that positive coefficient of A indicates increase in the bulk

density (Y3) with increase in speed of spheronizer. The positive coefficient of B indicates

increase in bulk density with increase in time duration for spheronisation of pellets up to

certain period from 2.5 to 5.5 min. The equation obtained was quadratic equation which

showed the effect was nonlinear.



variables on the bulk density of pellets.



on the bulk density of pellets.

Fig. 4: Different plots showing effect of independent variables on bulk density of pellets

The selected quadratic model showed "Prob> F" value of 0.3868 which is very high and

indicated that there was no significant effect of process variables on bulk density.

4. Effect on tapped density

Mathematical relationship generated for tapped density is expressed in following equation

(4).

Tapped density (Y4) = +0.75+0.016* A+0.014* B+0.017* AB-(6.517E-003)* A2-0.046*

B2…… (4).

From equation (4), it can be seen that positive coefficient of A indicates increase in the

tapped density (Y4) with increase in speed of spheronizer. The positive coefficient of B

indicates increase in tapped density with increase in time duration up to certain period to


1167


spheronize the pellets from 2.5 to 5.5 min. The equation obtained was quadratic equation

which showed the effect was nonlinear.



variables on the tapped density of pellets.


influence of speed (RPM) and time (min.) on

the tapped density of pellets.

Fig. 5: Different plots showing effect of independent variables on tapped density of

pellets


was no significant effect of process variables on tapped density.

5. Effect on hardness

Mathematical relationship generated for hardness is expressed in following equation (5).

Hardness (Y5) = +0.76+0.12* A-0.24* B…… (5).

From equation (5), it can be seen that positive coefficient of A indicates proportional increase

in the hardness (Y5) with increase in speed of spheronizer. The negative coefficient of B

indicates decrease in hardness with proportional increase in time duration to spheronize the

pellets from 2.5 to 5.5 min. The equation obtained was linear equation which showed the

effect was linear.


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variables on the hardness of pellets.



on the hardness of pellets.

Fig. 6: Different plots showing effect of independent variables on hardness of pellets

The selected surface linear model showed "Prob> F" value of 0.0428 which indicated that

there was significant effect of process variables on hardness.

6. Effect on drug content

Mathematical relationship generated for drug content is expressed in following equation (6).

Drug content (Y6) = +23.86-1.47* A-5.21* B-0.68* AB-4.02* A2-2.17* B2….. (6).

From equation (6), it can be seen that negative coefficient of A indicates decrease in the drug

content (Y6) with increase in speed of spheronizer. The negative coefficient of B indicates

decrease in drug content with increase in time duration to spheronize the pellets from 2.5 to

5.5 min. up to certain period. The equation obtained was quadratic equation which showed

the effect was nonlinear.


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variables on the drug content of pellets.



on the drug content of pellets.

Fig. 7: Different plots showing effect of independent variables on drug content of pellets


was no significant effect of process variables on drug content.

Selection of Optimum Formulation

Table 6: Design space criteria

Sr. No. Title Lower limit Higher limit

01 Percentage yield 80 % 95 %

02 Pellet size 0.50 mm 2.00 mm

03 Bulk Density 0.65 g/mL 0.70 g/mL

04 Tapped Density 0.68 g/mL 0.75 g/mL

05 Hardness 0.30 Kg/cm2 1.00 Kg/cm2

06 Drug content 20 % 50 %

Counter plot (Overlay plot) showing the design space of percentage yield, pellet size, bulk

density, tapped density, hardness, and drug content.

800 900 1000 1100 1200

2.5

3.1

3.7

4.3

4.9

5.5Overlay Plot

A: Speed (RPM)

B: T

ime

(Min

.)

Percentage Yield : 80

Percentage Yield : 80

Bulk Density: 0.65

Bulk Density: 0.65

Tapped Density : 0.74

Hardness : 1

Drug Content : 20

Fig. 8: Counter plot (Overlay plot) showing the design space of percentage yield, pellet

size, bulk density, tapped density, hardness, and drug content

Amongst the nine formulations prepared and analyzed using ANOVA in design expert 10®,

the „Overlay plot‟ indicated that „Batch F5‟ (spheronisation speed =1000 RPM; time =4 min.)

was the batch having percentage yield, pellet size, bulk density, tapped density, hardness, and


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drug content are in the optimum range and thus, „Batch F5‟ was selected as the optimized

formulation.

Drug Excipients Compatibility Study

1. Papain + Eudragit L100-55

5007501000125015001750200022502500275030003250350037504000

1/cm

40

50

60

70

80

90

100

110

%T

3537

.57

3329

.25

3300

.31

3261

.74

3223

.16 29

16.4

7

2733

.22

2337

.80 22

39.4

3

2123

.70

2052

.33

1917

.31

1886

.44

1672

.34

1458

.23

1357

.93

1228

.70

1136

.11

1057

.03

1014

.59

922.

00

854.

49

756.

12

709.

83

590.

24 522.

73

453.

29

papin+eud l -100-55

Fig. 9: IR spectrum of Papain + Eudragit L100-55

Table 7: IR spectral analysis of papain + Eudragit L100-55

Sr. No. IR Frequency (cm-1) Assignment Interpretation

1 1672.34

1. >N-H- -O=C<OH Amide-I bond of papain and Carboxylic acid of Eudragit L 100-55 interacts by hydrogen

bonding. 2. H>N- -H-O>C=O

O=C<OH and O=C<O-C2H5 of Eudragit L 100-55 overlaps with O=C<NH of papain.

The FT-IR spectrum of drug with Eudragit L 100- 55 clearly indicates that the drug was

interacting with excipient; Eudragit L 100- 55. The papain is quite stable at 40º C and it is

also compatible with rest of the excipients used in the formulation. So, Eudragit L 100-55

needs to be replaced with some other excipient.

Stability Studies of Optimized Batch

Table 8: Stability studies of optimized batch F5

Sr. No. Test Before storage

(Day 0)

Ambient conditions

(Day 30)

Cool conditions

(Day 30)

1 Appearance White White White

2 Percentage drug content 28.77% 25.59% 26.02%


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There was not significant change observed in appearance and percentage drug content after

one month storage. Therefore prepared formulation was stable for 1 month at ambient

temperature and at ambient humidity as well as at cool temperature i.e. 2º to 8º C.

CONCLUSION

The extrusion–spheronisation technique may be applied to produce multiparticulate delivery

systems for oral delivery of therapeutic enzymes and other proteins of interest. Papain was

successfully entrapped in pellets by extrusion–spheronisation technique, optimizing the

various formulation parameters in order to attain maximum entrapment and a spherical shape,

with an almost same pellet size distribution and highest drug content. Two process variables

(speed and time of spheronization) were studied at 3 levels using 32 full factorial design. The

optimization of the process using the Design Expert® 10 resulted in 92.35% of yield, 1.442

mm average pellet size, 0.690 g/mL bulk density, 0.741 g/mL tapped density, 6.885% Carr‟s

index, 1.074 Hausner ratio, 25º 87” angle of repose, 0.366 Kg/cm2 hardness, and 28.767%

drug content for optimized formulation of papain containing pellets i.e. Batch F5. The loss of

enzyme activity is probably due to interaction between Eudragit L 100-55 and papain.

ACKNOWLEDGEMENTS

1. Authors gratefully acknowledge to Evonik Industries, Mumbai for providing gift sample

of Eudragit L 100-55.

2. Authors deeply convey their sincere thanks to Dr. R. K. Nanda for his support, valuable

suggestions and excellent guidance in all scientific endeavors.

FUNDING

1. Authors express their special thanks to BCUD, Savitribai Phule Pune University, Pune for

funding purchase of Laboratory spheronizer.

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