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International Journal of Biological Macromolecules 62 (2013) 137– 145

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

jo ur nal homep age: www.elsev ier .com/ locate / i jb iomac

lends of jackfruit seed starch–pectin in the development ofucoadhesive beads containing metformin HCl

mit Kumar Nayaka, Dilipkumar Palb,∗

Department of Pharmaceutics, Seemanta Institute of Pharmaceutical Sciences, Mayurbhanj 757086, Odisha, IndiaDepartment of Pharmaceutical Sciences, Guru Ghasidas Vishwavidyalaya, Koni, Bilaspur 495009, C.G., India

r t i c l e i n f o

rticle history:eceived 11 June 2013eceived in revised form 6 August 2013ccepted 17 August 2013vailable online xxx

a b s t r a c t

In this work, calcium pectinate-jackfruit (Artocarpus heterophyllus Lam.) seed starch (JFSS) mucoadhesivebeads containing metformin HCl were developed through ionotropic-gelation. Effects of pectin and JFSSamounts on drug encapsulation efficiency (DEE), and cumulative drug release after 10 h (R10 h) wereoptimized using 32 factorial design. The optimized calcium pectinate-JFSS beads containing metforminHCl showed DEE of 94.11 ± 3.92%, R10 h of 48.88 ± 2.02%, and mean diameter of 2.06 ± 0.20 mm. The in vitrodrug release from these beads was followed controlled-release (zero-order) pattern with super case-II

eywords:ectinackfruit seed starcholymer-blenducoadhesion

onotropic-gelationrug release

transport mechanism. The beads were also characterized by SEM and FTIR. The pH of test mediums wasfound critical for swelling and mucoadhesion of these beads. The optimized calcium pectinate-JFSS beadsalso exhibited good mucoadhesivity and significant hypoglycemic effect in alloxan-induced diabetic ratsover prolonged period after oral administration.

© 2013 Elsevier B.V. All rights reserved.

. Introduction

The control of small molecular drugs released from the poly-eric matrices has remained a great challenge over the years.

ractically, the drug molecules embedded in a polymeric matrixxhibits a faster rate of drug release via diffusion through theores of the matrix. Such rate of drug release is undesirable toet prolonged action in case of the drugs with low biological half-ife. Various carbohydrate polymers have been widely employedn design of drug delivery system for containment and releaseontrol of small molecular drugs. The wide applications of theseiopolymers are attributed to their biodegradability and low tox-

city [1–3]. Among various carbohydrate polymers, pectin is onef the widely used biopolymers used in biomedical applicationsue to its nontoxic, highly biocompatible, mechanically strong,nd acid-stable property [2,4]. Pectin is a water soluble naturalolysaccharide extracted industrially from citrus peels, sugar beetoots, apple pomaces, etc. and has been used as food additive,

hickener and gelling agent [4,5]. Pectin consists of linearly con-ected (1–4)-linked ∞-d-galacturonic acid residues interrupted byome rhamonogalacturonic acid residue and ∞-l-rhamnopyranose

∗ Corresponding author at: Department of Pharmaceutical Sciences, Guru Ghasi-as Vishwavidyalaya (A Central University), Koni, Bilaspur 495009, C.G.,

ndia. Tel.: +91 7389263761.E-mail addresses: drdilip2003@yahoo.co.in, drdilip71@gmail.com (D. Pal).

141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.ijbiomac.2013.08.020

by ∞-1-2 linkage. The galacturonic acid residue of the pectin-backbone is partially esterified [6,7]. Pectin can be classified aslow methoxyl (LM, with a 25–50% degree of methylation) and highmethoxyl (HM, with a 50–80% degree of methoxylation) pectins.Usually, LM pectins can form gel structures through ionotropic-gelation with various divalent cations (e.g., calcium, zinc) for the useas effective vehicles in drug delivery applications [8,9]. The inter-actions of the carboxyl groups of LM pectin backbone with divalentcross-linking cations induce the formation of the so-called “egg-box” structure, even though it slightly differs from the “egg-box”model originally defined for alginates [4]. Recent years, variousionotropically gelled pectinate beads have been widely investi-gated in the design of oral delivery carriers for many bioactiveagents [6,8–10]. Unfortunately, the solubility and swellability ofcalcium pectinate gel beads in gastrointestinal fluid experiencefrom low entrapment efficiency and premature release of incorpo-rated small molecular drugs [10]. To overcome these weaknesses,several modifications of calcium pectinate gel beads have beeninvestigated by various research groups [7,11–14].

Jackfruit (Artocarpus heterophyllus Lam., family: Moraceae) seedstarch (JFSS) is one of such natural starch with potential appli-cations in pharmaceutical and food technology [15–19]. Researchinvestigations on the physicochemical properties of JFSS indicated

several unique characteristics relating to acid resistance, ther-mal and mechanical properties compared to common starches[20,21]. JFSS has been investigated for its mucoadhesivity and con-trolled release properties [22]. In an investigation by our research

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38 A.K. Nayak, D. Pal / International Journal o

roup, we have investigated the utility of JFSS, as a mucoadhe-ive polymer-blend with sodium alginate for the developmentf mucoadhesive beads by ionotropic-gelation technique for these in oral drug delivery [23]. In the literature, it was alsoound that a few investigations had been carried out to formulatetarch-blended calcium pectinate-based beads through ionotropic-elation [10,13]. To the best of our knowledge, no report is availablen the previous literature on the development of mucoadhesiveeads composed of JFSS and pectin for controlled drug release. Inhe present study, we made an attempt to investigate the devel-pment of ionotropically gelled mucoadhesive beads composed ofackfruit seed starch (JFSS)–pectin blends for use in controlled drugelease applications.

Metformin HCl was investigated as model drug in the cur-ent study. Metformin HCl is a biguanide antihyperglycemic agent,hich is widely used in the management of non-insulin dependentiabetes mellitus (NIDDM, Type-II) [24]. It has biological half-lifef 1.5–1.6 h and daily requirement of it is 1.5–3 g/day [25]. Theain absorption site of metformin HCl is proximal small intestine

26]. Hence, this would be beneficial to develop a mucoadhe-ive polymeric beads containing metformin HCl using calciumectinate-JFSS blend through ionotropic-gelation technique. Thisype of ionotropically gelled mucoadhesive beads might facilitaten intimate contact with the absorbing surfaces of mucous mem-ranes (i.e., mucoadhesion) and thus, the gastric residence coulde prolonged to release the encapsulated drug at the drug absorb-

ng site at a controlled rate to maximize the therapeutic effect. computer-aided optimization technique using response surfaceethodology based on 32 (two factors and three levels) factorial

esign was employed to investigate the composition of polymer-lend on the properties of calcium pectinate-JFSS mucoadhesiveeads containing metformin HCl relating to drug encapsulation andrug release.

. Experimental

.1. Materials

Metformin HCl (Abhilash Chemicals Pvt. Ltd., India), lowethoxy pectin (MW ∼30,000–100,000, Loba-chemie, India), cal-

ium chloride (Merck Ltd., India) were used. JFSS was isolated fromaw seeds of jackfruit (A. heterophyllus Lam.) according to the previ-usly reported method [23]. All other chemicals and reagents wereommercially available and of analytical grade.

.2. Preparation of calcium pectinate-JFSS beads containingetformin HCl

Calcium pectinate-JFSS beads containing metformin HCl wererepared using calcium chloride (CaCl2) as cross-linking agenty ionotropic-gelation technique. Briefly, required amounts of LMectin and JFSS were dissolved in deionized water (20 ml) usingagnetic stirring for 30 min. Afterwards, metformin HCl was added

o the mixture solutions of pectin-JFSS for each formulation main-aining polymer to drug ration 2:1, and mixed thoroughly using aomogenizer (Remi Motors, India). The final pectin-JFSS mixtureolutions containing metformin HCl were ultrasonicated for 5 minor debubbling. The resulting dispersion was then added via a 21-auge needle drop wise into 100 ml of 10% (w/v) CaCl2 solution.dded droplets were retained in the CaCl2 solution for 15 min to

omplete the curing reaction. The wet beads were collected byecantation. These wet beads were washed two times with dis-illed water and dried at 37 ◦C for overnight. The dried beads weretored in a desiccator until used.

gical Macromolecules 62 (2013) 137– 145

2.3. Experimental design for optimization

The amount of pectin (X1, 650–750 mg) and JFSS (X2,150–250 mg) as polymeric blend were defined as the selected inde-pendent variables, which were varied at three levels, low level(−1), medium level (0), and high level (+1). The drug encapsulationefficiency (DEE, %), and cumulative drug release after 10 h (R10 h,%) were used as dependent variables (responses). Design-Expert®

Version 8.0.6.1 software (Stat-Ease Inc., USA) was used for the gen-eration and evaluation of the statistical experimental design. Thematrix of the design including investigated responses i.e., DEE (%),and R10 h (%) are shown in Table 1. For optimization, the effectsof independent variables upon the responses were modeled usingquadratic mathematical model generated by 32 factorial design[27,28]:

Y = b0 + b1X1 + b2X2 + b3X1X2 + b4X21 + b5X2

2 ; where Y is theresponse; b0 is the intercept, and b1, b2, b3, b4, b5 are regres-sion coefficients. X1 and X2 are individual effects; X2

1 and X22 are

quadratic effects; X1X2 is the interaction effect. One-way ANOVAwas applied to estimate the significance of the model (p < 0.05).

2.4. Determination of DEE

Hundred milligram of beads were taken and were crushed usingpestle and mortar. The crushed powders of drug containing beadswere placed in a 250 ml volumetric flask and the volume was madeup to 250 ml by phosphate buffer, pH 7.4, and kept for 24 h withoccasionally shaking at 37 ± 0.5 ◦C. After the stipulated time, themixture was stirred at 500 rpm for 20 min using a magnetic stirrer(Remi Motors, India). The polymer debris formed after disintegra-tion of bead was removed filtering through Whatman® filter paper(No. 40). The drug content in the filtrate was determined using aUV–VIS spectrophotometer (Shimadzu, Japan) at 233 nm againstappropriate blank. The DEE (%) of these prepared beads was calcu-lated by the following formula [29]:

DEE (%) = actual drug content in beadstheoretical drug content in beads

× 100

2.5. Bead size measurement

Particle size of 100 dried beads from each batch was measuredby optical microscopic method for average particle size using anoptical microscope (Olympus). The ocular micrometer was previ-ously calibrated by stage micrometer.

2.6. Scanning electron microscopy (SEM) analyses

FSM-alginate beads containing metformin HCl were gold coatedby mounted on a brass stub using double-sided adhesive tape andunder vacuum in an ion sputter with a thin layer of gold (3–5 nm)for 75 s and at 20 kV to make them electrically conductive and theirmorphology was examined by scanning electron microscope (ZEOL,JSM-5800, Japan).

2.7. Fourier transform-infrared (FTIR) spectroscopy analyses

Samples were reduced to powder and analyzed as KBr pelletsby using a Fourier transform-infrared (FTIR) spectroscope (Perkin

Elmer Spectrum RX I, USA). The pellet was placed in the sam-ple holder. Spectral scanning was taken in the wavelength regionbetween 3600 and 500 cm−1 at a resolution of 4 cm−1 with scanspeed of 1 cm/s.

A.K. Nayak, D. Pal / International Journal of Biological Macromolecules 62 (2013) 137– 145 139

Table 1Experimental plan of 32 full factorial design (coded values in bracket) with observed response values with bead size for different formulations of calcium pectinate-JFSSbeads containing metformin HCl.

Codes Factors with normalized levels Responsesa Bead diameter (mm)b

LM pectin (mg, X1) JFSS (mg, X2) DEE (%) R10 h (%)

F-1 650.00 (−1) 150.00 (−1) 66.65 ± 2.47 89.72 ± 4.03 1.52 ± 0.15F-2 650.00 (−1) 200.00 (0) 68.87 ± 2.93 82.34 ± 3.22 1.67 ± 0.18F-3 650.00 (−1) 250.00 (+1) 73.22 ± 3.02 73.99 ± 2.80 1.72 ± 0.23F-4 700.00 (0) 150.00 (−1) 67.52 ± 2.17 88.57 ± 4.15 1.68 ± 0.16F-5 700.00 (0) 200.00 (0) 70.20 ± 2.93 80.58 ± 3.17 1.75 ± 0.19F-6 700.00 (0) 250.00 (+1) 75.65 ± 3.15 70.76 ± 2.28 1.82 ± 0.24F-7 750.00 (+1) 150.00 (−1) 71.19 ± 2.42 79.05 ± 3.07 1.78 ± 0.25F-8 750.00 (+1) 200.00 (0) 74.45 ± 2.99 68.93 ± 3.16 1.85 ± 0.22F-9 750.00 (+1) 250.00 (+1) 80.32 ± 3.26 59.55 ± 2.13 1.94 ± 0.29

Observed valuesF-O 698.31 348.72 94.11 ± 3.92 48.88 ± 2.02 2.06 ± 0.20

Predicted values95.04 47.36

% Errorc −0.98 3.20

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a Mean ± S.D., n = 3.b Mean ± S.D., n = 20.c Error (%) = (observed value − predicted value)/predicted value × 100.

.8. In vitro drug release studies

The release of metformin HCl from various calcium pectinate-FSS beads was tested using dissolution apparatus USP (Campbelllectronics, India). The baskets were covered with 100-mesh nylonloth to prevent the escape of the beads. The dissolution rates wereeasured at 37 ± 1 ◦C under 50 rpm speed. Accurately weighed

uantities of beads containing metformin HCl equivalent to 100 mgere added to 900 ml of 0.1 N HCl (pH 1.2). The test was carried

ut for 2 h and then continued in phosphate buffer (pH 7.4) forext 8 h. 5 ml of aliquots was collected at regular time intervals,nd the same amount of fresh dissolution medium was replacednto dissolution vessel to maintain the sink condition throughouthe experiment. The collected aliquots were filtered, and suitablyiluted to determine the absorbance using a UV–VIS spectropho-ometer (Shimadzu, Japan) at 233 nm against appropriate blank.

.9. Analysis of in vitro drug release kinetics and mechanism

In order to predict and correlate the in vitro drug releaseehavior from formulated various calcium pectinate-JFSS beadsontaining metformin HCl it is necessary to fit into a suitableathematical model. The in vitro drug release data were evalu-

ted kinetically using various important mathematical models likeero order, first order, Hixson–Crowell, Weibull, Baker–Lonsdale,iguchi, and Korsmeyer–Peppas models [29,30].

Zero-order model: Q = kt + Q0; where Q represents the drugreleased amount in time t, and Q0 is the start value of Q; k is therate constant.First-order model: Q = Q0 ekt; where Q represents the drug releasedamount in time t, and Q0 is the start value of Q; k is the rateconstant.Hixson–Crowell model: Q 1/3 = kt + Q 1/3

0 ; where Q represents thedrug released amount in time t, and Q0 is the start value of Q; k isthe rate constant.Weibull model: m = 1 − exp [−(t)b/a]; where m represents the drugreleased amount in time t, a is the time constant and b is the shapeparameter.

Baker–Lonsdale model: 3/2 [1 − (1 − Q)2/3] − Q = kt; where Q repre-sents the drug released amount in time t, and k is the rate constant.Higuchi model: Q = kt0.5; where Q represents the drug releasedamount in time t, and k is the rate constant.

Korsmeyer–Peppas model: Q = ktn; where Q represents the drugreleased amount in time t, k is the rate constant and n is thediffusional exponent, indicative of drug release mechanism.

The accuracy and prediction ability of these models were com-pared by calculation of squared correlation coefficient (R2) usingKinetDS 3.0 Rev. 2010 software. Again, The Korsmeyer–Peppasmodel was employed in the in vitro drug release behavior anal-ysis of these formulations to distinguish between competingrelease mechanisms: Fickian release (diffusion-controlled release),non-Fickian release (anomalous transport), and case-II transport(relaxation-controlled release). When n is ≤ 0.43, it is Fickianrelease. The n value between 0.43 and 0.85 is defined as non-Fickianrelease. When, n is ≥ 0.85, it is case-II transport [31].

2.10. Evaluation of swelling behavior

Swelling behavior evaluation of optimized calcium pectinate-JFSS beads containing metformin HCl were carried out in twodifferent aqueous media: 0.1 N HCl (pH 1.2), and phosphate buffer(pH 7.4). 100 mg beads were placed in vessels of dissolution appa-ratus (Campbell Electronics, India) containing 500 ml respectivemedia. The experiment was carried out at 37 ± 1 ◦C under 50 rpmpaddle speed. The swelled beads were removed at predeterminedtime interval and weighed after drying the surface by using tissuepaper. Swelling index was determined using the following formula[31,32]:

Swelling index (%)

= weight of beads after swelling − dry weight of beadsdry weight of beads

× 100

2.11. Ex vivo mucoadhesion testing

The mucoadhesive property of optimized calcium pectinate-JFSS beads containing metformin HCl were evaluated by ex vivowash-off method [33]. Freshly excised pieces of goat intesti-nal mucosa (2 cm × 2 cm) (collected from slaughterhouse) were

mounted on glass slide (7.5 cm × 2.5 cm) using thread. About 50beads were spread onto the wet tissue specimen, and the preparedslide was hung onto a groove of disintegration test apparatus. Thetissue specimen was given a regular up and down movement in a

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40 A.K. Nayak, D. Pal / International Journal o

essel containing 900 ml of 0.1 N HCl (pH 1.2) and phosphate bufferpH 7.4), separately, at 37 ± 0.5 ◦C. After regular time intervals, the

achine was stopped and the number of beads still adhering to theissue was counted.

.12. Pharmacodynamic evaluation

In vivo pharmacodynamic studies were performed in alloxan-nduced diabetic male albino rats of either sex (weighing22–358 g) [33]. The acclimatized rats were kept fasting for 24 hith water ad libitum. All experiments were performed between

AM and 12 PM to minimize circadian influences. The experimen-al protocol was subjected to the scrutiny of the Institutional Animalthical Committee and was cleared before starting. The experimen-al animals were handled as per guidelines of committee for theurpose of control and supervision on experimental animals (CPC-EA). All efforts were made to minimize both the suffering andumber of animals used.

The male albino rats were made diabetic by intraperitonealdministration of freshly prepared alloxan solution at a dose of50 mg/kg dissolved in 2 mM citrate buffer (pH 3.0). After oneeek of alloxan administration, alloxanized rats with fasting blood

lucose of 300 mg/dl or more were considered diabetic and weremployed in the study for 10 h. After initial collection of bloodamples from the alloxan-induced diabetic rats, they were dividedandomly into 2 groups of 6 rats each and treated as follow: Group

was administered with pure metformin HCl (100 mg/kg bodyeight) in suspension form and Group B were administered with

ptimized calcium pectinate-JFSS beads containing metformin HCl,oth at a dose equivalent to 100 mg metformin HCl/kg body weightsing oral feeding needle. Blood samples were withdrawn (0.1 ml)rom tail tip of each rat at regular time intervals under mildther anesthesia, and were analyzed for blood glucose by oxidase-eroxidase method using commercial glucose kit. Comparative

n vivo blood glucose level in alloxan-induced diabetic rats afterral administration of pure metformin HCl and optimized calciumectinate-JFSS beads containing metformin HCl were evaluated.

.13. Statistical analysis

Statistical optimization was performed using Design-Expert®

ersion 8.0.6.1 software (Stat-Ease Inc., USA). The squared correla-ion coefficients (R2) of all kinetic models were determined usinginetDS 3.0 Rev. 2010 software. All measured data are expresseds mean ± standard deviation (S.D.). Each measurement was donen triplicate (n = 3). The in vivo data was tested for significant dif-erences (p < 0.05) by paired samples t-test. All other data wasnalyzed with simple statistics. The simple statistical analysis andaired samples t-test were conducted using MedCalc software, ver-ion 11.6.1.0.

. Results and discussion

.1. Preparation of calcium pectinate-JFSS beads containingetformin HCl

The interactions between the negative charged carboxylicroups of LM pectin-backbone and the positive charged divalentross-linking cations (here Ca2+) induces the formation of so-calledEgg-Box” model structure, even though it slightly differs from theEgg-Box” model originally defined for alginates [34]. Thus, variousigid and spherical calcium pectinate-JFSS beads containing met-

ormin HCl were formulated through ionotropic-gelation, whenarious mixtures of LM pectin-JFSS blends containing metforminCl were dropped into the CaCl2 solutions. In this work, polymer-lends of LM pectin and JFSS were used to formulate calcium

gical Macromolecules 62 (2013) 137– 145

pectinate-JFSS beads containing metformin HCl. Polymer-blendingis a common practice to achieve desired functional properties inboth biomedical and pharmaceutical applications [12,13,31,35,36].Blends of already known polymers represent a rational approach toobtain different and modulated properties that enable their use forspecific goals [12,13]. This approach is advantageous, since apartfrom working with well known substances, it avoids the high costof the synthesizing new materials [13]. Blending of ionic polysac-charides with other polymers improves functional properties suchas gel strength, stability, swelling, drug encapsulation, and desiredsustained drug release [31,33,36]. Blending of mucoadhesive poly-mers with other polymers can also improve mucoadhesivity ofvarious systems for the use in the development of mucoadhe-sive drug delivery [14,30,32,33,37–39]. Additionally, the changesin polymer ratio can result in wide range of physicochemical prop-erties, which could provide different drug release patterns. Theblends of pectin and other biocompatible second polymers havealso been employed in some investigations for the development ofionotropically gelled calcium pectinate-based beads to prevent pre-mature release of incorporated small molecular drugs [11–14]. Inthe present investigation, calcium pectinate-JFSS beads containingmetformin HCl were prepared by ionotropic-gelation using CaCl2as cross-linker.

3.2. Optimization

Traditional pharmaceutical development involves trial anderror method for the process and formulation optimization. Phar-maceutical development is the process of design for quality productand understanding the process that consistently bring the productfor intended performance. Design of experiment is the methodol-ogy of systemically determining the relationship between variablesaffecting the formulation and/or process and output of that for-mulation and/or process [40]. It is the statistical way of testinglarge number of formulation and process variables in a minimumnumber of experiment run. Among various statistical experimen-tal design for optimization, factorial design is a design by whichthe factors all factors involved in a process are studied in all possi-ble combinations by analyzing the influence of individual variablesand their interactions using minimum experiments [41]. Thus, theconstruction of factorial design involves the selection of factorsand the choice of responses. Based on the factorial design exper-iment, the optimization technique encompasses factorial designexperiment that will reliably measure responses, generating math-ematical model equations using experimental data, conductingappropriate statistical tests to select best possible model and deter-mining the values of independent variables to produce optimumresponses [41].

For the 32 factorial design, 9 trial formulations were suggestedby Design-Expert® Version 8.0.6.1 software (Stat-Ease Inc., USA) fortwo independent variables: amount of pectin (X1, 650–750 mg) andJFSS (X2, 150–250 mg) as polymeric-blends, which were varied atthree levels: low level (−1), medium level (0), and high level (+1).The DEE (%), and R10 h (%) were evaluated as dependent variables(responses). Design-Expert® Version 8.0.6.1 software (Stat-EaseInc., USA) was used for the generation and evaluation of the sta-tistical experimental design. According to this trial plan, calciumpectinate-JFSS beads containing metformin HCl were prepared byionotropic-gelation technique. Overview of matrix of the designincluding investigated responses (i.e., DEE and R10 h) is presentedin Table 1. The values of investigated responses measured for all

trial formulations were fitted in the 32 factorial design to get modelequations for responses analyzed in this investigation. The resultsof the ANOVA based on the two quadratic models indicated thatthese models were significant for all response parameters (Table 2).

A.K. Nayak, D. Pal / International Journal of Biological Macromolecules 62 (2013) 137– 145 141

Table 2Summary of ANOVA for the response parameters.

Source Sum of squares d.f.a Mean square f value p-Value prob > F

(a) For DEE (%)b

Model 152.36 5 30.47 1448.47 <0.0001X1 49.42 1 49.42 2349.26 <0.0001X2 94.64 1 94.64 4498.96 <0.0001X1X2 1.64 1 1.64 77.88 0.0031X2

1 3.52 1 3.52 167.33 0.0010X2

2 3.13 1 3.13 148.94 0.0012

(b) For R10 h (%)c

Model 758.21 5 151.64 780.12 <0.0001X1 247.30 1 247.30 1272.211 <0.000X2 468.87 1 468.87 2412.10 <0.0001X1X2 3.55 1 3.55 18.28 0.0235X2

1 38.25 1 38.25 196.79 0.0008X2

2 0.24 1 0.24 1.21 0.3512

a d.f., degree of freedom.

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b DEE (%), drug encapsulation efficiency (%).c R10 h (%), % drug released from calcium pectinate-JFSS beads containing metfo

uadratic effect; X1X2 is the interaction effect.

The model equation relating DEE (%) became:

EE (%) = 330.12 − 0.74X1 − 0.30X2 + 2.56 × 10−4X1X2 + 5.31

×10−4X21 + 5.01 × 10−4X2

2

× [R2 = 0.9996; F = 1448.47; p < 0.05]

The model equation relating R10 h (%) became:

10 h(%) = −710.01 + 2.40X1 + 0.14X2 − 3.77 × 10−4X1X2 − 1.75

×10−3X21 − 1.37 × 10−5X2

2

× [R2 = 0.9992; F = 780.12; p < 0.05]

Model simplification was carried out by eliminating non-ignificant terms (p > 0.05) in model equations resulting from theultiple regression analysis [31], giving:

DEE (%) = 330.12 − 0.74X1 − 0.30X2 + 2.56 × 10−4X1X2 + 5.31×10−4X2

1 + 5.01 × 10−4X22

R10 h(%) = −710.01 + 2.40X1 + 0.14X2 − 3.77×10−4X1X2 − 1.75 × 10−3X2

1

The influences of main effects (factors) on responses (here, DEE,nd R10 h) were further elucidated by response surface methodol-gy. Response surface methodology is a widely proficient approachn the development and optimization of drug delivery devices10,28,31,36,40]. The response surface methodology is used forptimizing a formula (i.e. maximizing one or more of the responses,eeping the formulation variable setting within a satisfactoryange), carrying out simulations with the model equations and plot-ing the responses. The three-dimensional response surface graphs very useful in learning about the main and interaction effectsf the independent variables (factors), whereas two-dimensionalontour plot gives a visual representation of values of the response41]. The three-dimensional response surface plots (Fig. 1a and b)nd corresponding contour plots (Fig. 1c and d) were presentedo estimate the effects of the independent variables (factors) onach response investigated. The three-dimensional response sur-ace graph relating DEE (Fig. 1a) indicates the increment in both thealues with the increasing of pectin amount (X1), and JFSS amount

X2) in the formulated calcium pectinate-JFSS beads containing

etformin HCl prepared by ionotropic gelation technique. How-ver, a decrease in R10 h values with the increasing pectin amountX1), and JFSS amount (X2) is indicated by the three-dimensional

HCl after 10 h; X1 and X2 represent the main effects (factors); X21 and X2

2 are the

response surface graph relating R10 h (Fig. 1b). All the contour plotsrelating measured responses (Fig. 1c and d) indicate nonlinearrelationships between two independable variables (here, pectinamount, X1 and JFSS amount, X2) on all measured responses, inves-tigated in this study.

A numerical optimization technique using the desirabilityapproach was employed to develop new formulations with desiredresponse (optimum quality). The desirable ranges of the undepend-able variables (factors) were restricted to 650 ≤ X1 ≤ 750 mg, and150 ≤ X2 ≤ 250 mg; whereas the desirable ranges of responses wererestricted to 95 ≤ DEE ≤ 100%, and 45 ≤ R10 h ≤ 50%. The optimal val-ues of responses were obtained by numerical analysis using theDesign-Expert 8.0.6.1 software based on the criterion of desirabil-ity. The desirability plot indicating desirable regression ranges foroptimal process variable settings and overlay plot indicating theregion of optimal process variable settings was presented in Fig. 1eand f, respectively.

In order to evaluate the optimization capability of the mathe-matical models generated according to the results of 32 factorialdesign, optimized calcium pectinate-JFSS beads containing met-formin HCl were prepared using one of the optimal process variablesettings proposed by the design (R2 = 1). The selected optimalprocess variable setting used for the formulation of optimizedformulation was X1 = 715.38 mg and X2 = 349.87 mg. Optimized cal-cium pectinate-JFSS beads containing metformin HCl (F-O) wereevaluated for DEE (%) and R10 h (%). The optimized beads contain-ing metformin HCl (F-O) showed DEE of 94.11 ± 3.92% and R10 h of48.88 ± 2.02% with small percentage error-values (−0.98 and 3.20,respectively) (Table 1). Percentage error evaluation is helpful inestablishing the validity of generated model equations to describethe domain of applicability of optimization model. The percentageerror values indicate that mathematical models obtained from thefull 32 factorial design were well fitted.

3.3. DEE

The DEE (%) of these newly formulated ionotropically gelledcalcium pectinate-JFSS beads containing metformin HCl ranged66.65 ± 2.47 to 94.11 ± 3.92% (Table 1). It was observed that the DEEin these beads containing metformin HCl was increased with theincreasing of both pectin and JFSS amount as polymer-blend. The

increased DEE with the increasing amount of both pectin and JFSSin these newly developed beads could be due to the increase in vis-cosity of the polymer-blend solutions with the increasing amountof polymer addition. This might have been prevented drug leaching

142 A.K. Nayak, D. Pal / International Journal of Biological Macromolecules 62 (2013) 137– 145

Fig. 1. Three-dimensional response surface plots showing the effects of amount of pectin (mg) and JFSS (mg) on (a) DEE (%) and (b) R10 h (%); Two-dimensional correspondingc ) ando

fthmc

3

cfsmasrt

ontour plots showing the effects of amount of pectin (mg) and JFSS (mg) (c) DEE (%verlay plot (d) indicating the region of optimal process variable settings

rom the prepared beads to the cross-linking solution. In addition,he increasing amount of pectin in polymer-blend solution mightave been elevated the cross-linking by CaCl2 through availingore numbers anionic of sites of pectin molecules for ionotropi-

ally cross-linking by calcium ions.

.4. Bead size

The average bead diameters of these dried ionotropically gelledalcium pectinate-JFSS beads containing metformin HCl rangedrom 1.52 ± 0.15 to 2.06 ± 0.20 mm (Table 1). Increasing the beadize was found with the increasing amount of both the poly-ers used in the polymer-blends (pectin and JFSS). This could be

ttributed due to the increase in viscosity of the polymer-blendolutions with incorporation of both the polymers in increasingatio, which in turn increased the droplet size during addition ofhe polymer-blend solutions to the cross-linking solutions.

(d) R6 h (%); The desirability plot (c) indicating desirable regression ranges and the

3.5. SEM

The morphological analysis of the optimized ionotropicallygelled calcium pectinate-JFSS beads containing metformin HCl(F-O) was visualized by SEM and is presented in Fig. 2. Themicrophotograph of these beads showed almost spherical shapeand free from agglomeration. The surface topography of thesebeads revealed rough surfaces with wrinkles, which might becaused by partly collapsing the polymeric gel network during dry-ing. However, polymeric derbies were seen on the bead surface,which could be due to the method of preparation (i.e., simultaneousgel bead preparation and formation of the polymer-blend matrix).

3.6. FTIR

The FTIR of pectin, JFSS, calcium pectinate-JFSS blank beads,optimized calcium pectinate-JFSS beads containing metformin HCl(F-O), and metformin HCl are shown in Fig. 3. In Fig. 3, both the FTIR

A.K. Nayak, D. Pal / International Journal of Biological Macromolecules 62 (2013) 137– 145 143

Fgi

stadcp1aopIttsioHwst

FpJ

Fig. 4. In vitro drug release from various ionotropically gelled calcium pectinate-JFSS

ig. 2. Scanning electron microphotograph of the optimized ionotropicallyelled calcium pectinate-JFSS beads containing metformin HCl prepared throughonotropic gelation (F-O)

pectra of pectin (LM) and JFSS showed strong and broad absorp-ion band peaks between 3600 and 3200 cm−1 due to OH stretcheslong with some complex bands in the region of 1200–1030 cm−1

ue to C O and C O C stretching vibrations, which are theharacteristic of the natural polysaccharides. In addition, princi-al absorption peaks were observed in the FTIR spectra of pectin at459.27 cm−1 and 1356.45 cm−1, which could be assigned to CH2nd OH bending vibration peaks respectively. The FTIR spectrumf calcium pectinate-JFSS blank beads showed all characteristiceaks of both pectin and JFSS without any significant interaction.

n the FTIR spectrum of metformin HCl, the principal absorp-ion peaks appeared at 3169 cm−1 due to the N H stretching ofhe primary amine group ( NH2) and at 1063 cm−1 due to C Ntretching, and a peak at 1584 cm−1 occurs due to N H bend-ng vibrations of the primary amine group. In the FTIR spectrumf optimized calcium pectinate-JFSS beads containing metformin

Cl, various characteristic peaks of pectin, JFSS, and metformin HClere appeared without any significant shifting of these peaks. In

hort, the ionotropically gelled calcium pectinate-JFSS beads con-aining metformin HCl prepared with pectin-JFSS polymer-blend

ig. 3. FTIR spectra of (a) pectin, (b) isolated JFSS, (c) ionotropically gelled calciumectinate-JFSS blank bead, (d) optimized ionotropically gelled calcium pectinate-

FSS beads containing metformin HCl (F-O), and (e) pure metformin HCl

beads containing metformin HCl prepared through ionotropic gelation [Mean ± S.D.,n = 3]

had significant characters of metformin HCl in the FTIR spectrum,suggesting absence of any interaction between the drug, metforminHCl and the polymer-blend used (pectin and JFSS).

3.7. In vitro drug release

The newly developed ionotropically gelled calcium pectinate-JFSS beads containing metformin HCl showed prolonged metforminHCl release over 10 h, in vitro (Fig. 4). Metformin HCl release fromthese beads in the gastric pH (1.2) was found slow (less than 15%after 2 h). This could be due to the shrinkage of pectinate gel at acidicpH. The trace amount of metformin HCl release could probably bedue to the presence of drug crystals onto bead surface. After that,faster metformin HCl release was observed in alkaline intestinal pH(7.4) comparatively, probably due to the higher swelling of thesebeads in alkaline pH. The cumulative percentage metformin HClreleased from calcium pectinate-JFSS beads containing metforminHCl after 10 h (R10 h, %) was within the range of 48.88 ± 2.02 to89.72 ± 4.03%. Nevertheless, the metformin HCl release from thesebeads was also found to be delayed with the increasing of polymeramounts (both pectin and JFSS). In case of beads containing higherpolymer contents, the more hydrophilic property of the polymerscould probably binds better with water to form viscous gel struc-ture, which might blockade the pores on the surface of beads andcould delayed the drug release from these formulated beads. Theanother reasonable explanation of the delayed drug release can beattributed to increasing coating efficiency of the drug particles withthe increasing polymer content employed in the formulation.

The in vitro drug release data from various calcium pectinate-JFSS beads containing metformin HCl were evaluated kineti-cally using various mathematical models like zero-order, first-order, Hixson–Crowell, Weibull, Baker–Lonsdale, Higuchi, andKorsmeyer–Peppas models. The result of the curve fitting intovarious mathematical models is given in Table 3. When respec-tive correlation coefficients of these beads were compared, themetformin HCl release from these beads was found to followzero-order model (R2 = 0.9927–0.9981) over a period of 10 h. How-ever, Korsmeyer–Peppas model (R2 = 0.9860–0.9940) and Weibullmodel (R2 = 0.9856–0.9939) were found to be closer to the best-fit zero-order model. The best fit of zero-order model indicatedthat the drug release from these beads followed controlled-releasepattern. The values of diffusional exponent (n) determined fromKorsmeyer–Peppas model ranged from 1.04 to 1.12, indicating the

drug release from these beads followed the super case-II trans-port mechanism controlled by swelling and relaxation of calciumpectinate-JFSS matrix. This could be attributed due to polymer dis-solution and enlargement or relaxation of polymeric chain.

144 A.K. Nayak, D. Pal / International Journal of Biological Macromolecules 62 (2013) 137– 145

Table 3Results of curve fitting of the in vitro metformin HCl release data from various calcium pectinate-JFSS beads containing metformin HCl.

Models Formulation codes

F-1 F-2 F-3 F-4 F-5 F-6 F-7 F-8 F-9 F-O

Zero order R2 0.9927 0.9958 0.9932 0.9952 0.9955 0.9927 0.9975 0.9953 0.9981 0.9941First order R2 0.8593 0.8883 0.8931 0.8674 0.8937 0.9010 0.9034 0.8827 0.9007 0.8949Hixson–Crowell R2 0.9248 0.9449 0.9449 0.9324 0.9469 0.9480 0.9539 0.9391 0.9541 0.9476Weibull R2 0.9856 0.9893 0.9898 0.9863 0.9874 0.9887 0.9862 0.9916 0.9923 0.9939Baker–Lonsdale R2 0.8976 0.9138 0.9327 0.8911 0.9186 0.9389 0.9110 0.9315 0.9223 0.9447Higuchi R2 0.5494 0.5432 0.5257 0.5480 0.5554 0.5751 0.5694 0.5925 0.5319 0.5280Korsmeyer–Peppas R2 0.9860 0.9940 0.9926 0.9893 0.9927 0.9927 0.9937 0.9916 0.9944 0.9940

1.08 1.05 1.05 1.04 1.10 1.11

3

piiailptpiJtimti

3

pniwpntogafi

Fb(

ered rapidly toward the normal level. In case of the group (Group

na 1.12 1.10 1.12 1.11

a n, diffusional exponent.

.8. Swelling behavior

The swelling index of optimized ionotropically gelled calciumectinate-JFSS beads containing metformin HCl was found lower

n gsatric pH (1.2) in comparison with that of in intestinal pH (7.4),nitially (Fig. 5). This was occurred due to shrinkage of pectinate atcidic pH. Maximum swelling of these beads was noticed at 2–3 hn alkaline intestinal pH (7.4) and after which, erosion and disso-ution took place. The swelling behavior of these beads in alkalineH could be explained by the ion exchanging between Ca2+ ions ofhe ionotropically cross-linked beads and the Na+ ions present inhosphate buffer with the influence of Ca2+-sequestrant phosphate

ons. This could result in disaggregation in the calcium pectinate-FSS matrix structure leading to matrix erosion and dissolution ofhe swollen beads. This phenomenon suggests that the ionotrop-cally gelled optimized calcium pectinate-JFSS beads containing

etformin HCl are able to swell slightly in the stomach and begino swell more when these beads subsequently move to the upperntestine, where the metformin HCl is to be absorbed.

.9. Mucoadhesivity

The results of ex vivo wash-off test of optimized calciumectinate-JFSS beads containing metformin HCl using goat intesti-al mucosa in gastric pH (1.2) and intestinal pH (7.4) are presented

n Fig. 6. The ex vivo wash off of these newly developed beadsas found faster in intestinal pH than at gastric pH. In gastricH (1.2), the percentage of beads adhering to the goat intesti-al mucosal tissue varied from 70.05 ± 4.22% over 8 h; whereas,his was 36.64 ± 3.45% in intestinal pH (7.4). The rapid wash-offbserved at intestinal pH could be due to ionization of functional

roups of polymers, which increased their solubility with reduceddhesive strength. Therefore, the results of the wash-off test con-rmed that the newly developed optimized ionotropically gelled

ig. 5. Swelling behavior of optimized ionotropically gelled calcium pectinate-JFSSeads containing metformin HCl in acidic pH (0.1 N HCl, pH 1.2), and in alkaline pHphosphate buffer, pH 7.4) [mean ± SD, n = 3]

Fig. 6. Mucoadhesive behavior of optimized ionotropically gelled calciumpectinate-JFSS containing metformin HCl in acidic pH (0.1 N HCl, pH 1.2), and inalkaline pH (phosphate buffer, pH 7.4) [mean ± SD, n = 3]

calcium pectinate-JFSS beads containing metformin HCl had goodmucoadhesivity.

3.10. Pharmacodynamic evaluation

In alloxan-induced diabetic rats, the comparative in vivo bloodglucose level and the mean percentage reduction in blood glucoselevel after oral administration of pure metformin HCl and optimizedcalcium pectinate-JFSS beads containing metformin HCl (F-O) arepresented in Figs. 7 and 8, respectively. In case of the group treatedwith pure metformin HCl (Group A), a rapid reduction in blood glu-cose level in alloxan-induced diabetic rats was observed within 3 hof oral administration, and after that, the blood glucose level recov-

B) treated with calcium pectinate-JFSS beads containing metforminHCl (F-O), the reduction in blood glucose level was found slowerthan that of the group treated with pure metformin HCl (Group

Fig. 7. Comparative in vivo blood glucose level in alloxan-induced diabetic ratsafter oral administration of pure metformin HCl and optimized ionotropicallygelled calcium pectinate-JFSS mucoadhesive beads containing metformin HCl (F-O)[Mean ± S.D., n = 6]. The data were analyzed for significant differences (*p < 0.05) bypaired samples t-test. The statistical analysis was conducted using MedCalc softwareversion 11.6.1.0

A.K. Nayak, D. Pal / International Journal of Biolo

Fig. 8. Comparative in vivo mean percentage reduction in blood glucose level inaot

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[[[[38] D. Pal, A.K. Nayak, Drug Deliv. 19 (2012) 123–131.

lloxan-induced diabetic rats after oral administration of pure metformin HCl andptimized ionotropically gelled calcium pectinate-JFSS mucoadhesive beads con-aining metformin HCl (F-O)

) up to 3 h. Significant differences (p < 0.05) were found betweenhe blood glucose level after administration of pure metformin HClnd optimized calcium pectinate-JFSS beads containing metforminCl (F-O) at each time-point measured. However, the reductions

n glucose level were increased gradually with the increment ofime in case of Group B (treated with optimized calcium pectinate-FSS beads containing metformin HCl), and were sustained over0 h. A 25% reduction in glucose level is considered a significantypoglycemic effect [38]. Therefore, the significant hypoglycemicffect by the calcium pectinate-JFSS beads containing metforminCl (F-O) was observed over 10 h. Thus, the results of this studylearly suggested that these mucoadhesive beads containing met-ormin HCl swelled slowly in the stomach and accordingly adheredo the stomach mucosa. This might allow more amount of drug to bebsorbed minimizing the diffusion barriers to increase the absorp-ion period by prolonging the gastric residence time. Then, theseeads were subsequently move to the upper intestine, where theywelled more and released drug through the polymeric gel layer,ormed at the matrix-periphery. Thus, the ability of these devel-ped mucoadhesive system was found suitable to maintain tightlood glucose level and improved patient compliance by enhanc-

ng, controlling and prolonging systemic absorption of metforminCl.

. Conclusion

Mucoadhesive beads containing metformin HCl made ofectin-JFSS polymer-blends was successfully developed through

onotropic-gelation technique and optimized using 32 factorialesign. The method for the preparation of these mucoadhesiveeads was found to be simple, easily controllable, economicalnd consistent. In addition, the excipients used for the formula-ion of ionotropically gelled mucoadhesive beads were cheap andasily available. These newly developed ionotropically gelled cal-

ium pectinate-JFSS mucoadhesive beads containing metforminCl displayed high drug encapsulation, good mucoadhesivity with

he biological membrane (excised goat intestinal mucosa), suit-ble controlled in vitro drug release pattern and also significant

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gical Macromolecules 62 (2013) 137– 145 145

hypoglycemic activity in alloxan-induced diabetic rats over pro-longed period after oral administration, which could possibly belucrative in terms of prolonged systemic absorption of metforminHCl maintaining tight blood glucose level and advanced patientcompliance. This type of mucoadhesive beads can also be formu-lated to load other drugs demanding sustained release in controlledmanner over a longer period to improve their bioavailability andtherapeutic efficacy.

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