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International Journal of Biological Macromolecules 66 (2014) 203–211

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

j ourna l h o mepa ge: www.elsev ier .com/ locate / i jb iomac

evelopment of pectinate-ispagula mucilage mucoadhesive beads ofetformin HCl by central composite design

mit Kumar Nayaka, Dilipkumar Palb,∗, Kousik Santraa

Department of Pharmaceutics, Seemanta Institute of Pharmaceutical Sciences, Mayurbhanj 757086, Odisha, IndiaDepartment of Pharmaceutical Sciences, Guru Ghasidas Vishwavidyalaya (A Central University), Koni, Bilaspur, 495009, Chhattisgarh(C.G.), India

r t i c l e i n f o

rticle history:eceived 5 January 2014eceived in revised form 29 January 2014ccepted 9 February 2014vailable online 18 February 2014

a b s t r a c t

Ionotropically-gelled mucoadhesive beads of metformin HCl composed of low methoxy (LM) pectin-ispaghula husk mucilage (IHM) polymer-blend was developed and optimized using central compositedesign (spherical type, single center point, and ˛ = 1.414). Effects of LM pectin and IHM amounts on drugencapsulation efficiency (DEE) and cumulative drug release at 10 h (R10h) were analysed using responsesurface methodology. The optimized beads containing metformin HCl (F-O) showed DEE of 86.98 ± 3.26%

eywords:spaghula husk mucilageonotropic-gelationontrolled drug release

and R10h of 47.20 ± 1.28%. All these beads exhibited suitable controlled in vitro sustained drug release pat-tern with super case-II transport mechanism over 10 h. These beads were also characterized by SEM andFTIR. The optimized beads also exhibited pH-dependent swelling, good mucoadhesivity with goat intesti-nal mucosa and significant hypoglycemic effect in alloxan-induced diabetic rats over prolonged periodafter oral administration, which could possibly be lucrative in terms of prolonged systemic absorption ofmetformin HCl maintaining tight blood glucose level and advanced patient compliance.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

Pectin is a branched macromolecular, water soluble, naturalolysaccharide, which is widely used as biopolymers in variousiomedical applications due to its biodegradability, biocompat-

bility and acid stable property [1,2]. It has been used as fooddditive, thickener and gelling agent in food and pharmaceuticalndustry [2,3]. It consists mainly of linearly connected (1-4)-linked-d-galacturonic acid residues interrupted by some rhamnogalac-

uronic acid residue and �-l-rhamnopyranose by �-1-2 linkage.he galacturonic acid residue of the backbone is partially esteri-ed [4]. Pectin is generally classified as low methoxyl (LM, with

25–50% degree of esterification) and high methoxyl (HM, with 50–80% degree of esterification) pectins. Usually, LM pectin canorm rigid gels through ionotropic-gelation with various divalentations (e.g., calcium, zinc, etc.) from the use as effective vehiclesn drug delivery applications [5–7]. The interaction of the car-oxyl groups of LM pectin backbone with divalent cross-linking

ations induces the so-called “egg-box” structure, even though itlightly differs from the “egg-box” model originally defined for algi-ates [2]. During last few decades’, various ionotropically-gelled

∗ Corresponding author. Tel.: +91 3244243265; fax: +91 591 2360818.E-mail addresses: drdilip2003@yahoo.co.in, drdilip71@gmail.com (D. Pal).

ttp://dx.doi.org/10.1016/j.ijbiomac.2014.02.023141-8130/© 2014 Elsevier B.V. All rights reserved.

pectinate gel beads as oral drug delivery matrices have been inves-tigated [5,7–9]. Unfortunately, the solubility and swellability ofionotropically-gelled pectinate gel beads in gastrointestinal fluidsuffer from low drug entrapment efficiency and premature releaseof incorporated small molecular drugs [8]. Various research groupshave reported several modifications of ionotropically-gelled pecti-nate beads to overcome these above said limitations [10–15].Among them, blending of pectin with a biocompatible secondpolymer is a simple and easiest way to improve desired func-tional properties like drug encapsulation, swelling, stability and toprevent premature release of incorporated small molecular drugs[12,13,15]. Blending of isolated plant polysaccharides like jack-fruit seed starch, tamarind seed polysaccharide and fenugreek seedmucilage with LM pectin in the development of ionotropically-gelled pectinate-based beads for drug delivery was also reportedby our research group [16–18]. These blends exhibited high drugencapsulation with sustained drug release over a prolonged period.Moreover, these beads showed good mucoadhesivity with the bio-logical membranes, which might lead higher bioavailability of theencapsulated drug due to increased gastric residence time with acloser contact between the absorptive membrane and these beads.

Ispaghula (Plantago ovata F.) husk mucilage (IHM) is white,

hydrophilic in nature and forms a colorless gel in presence of water[19]. It contains a high amount of highly branched arabinoxylanand non-reducing terminal sugar residues [20]. IHM is widely used

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s excipients in various drug delivery applications [20–23]. Ouresearch group has already investigated the utility of isolated IHMs mucoadhesive polymer-blend with sodium alginate in the devel-pment of ionotropically-gelled mucoadhesive beads for controlledrug release [20]. However, the use of polymer-blends of LM pectinnd IHM in the development of ionotropically-gelled mucoadhe-ive beads for controlled drug release is not reported till date.herefore, we made an attempt to develop metformin HCl-loadeducoadhesive beads for oral use using LM pectin-IHM polymer-

lend through ionotropic-gelation.Metformin HCl, a biguanide anti-hyperglycemic agent, is widely

sed in the treatment of non-insulin dependent diabetes mellitusNIDDM, Type-II) and generally, its daily requirement is 1.5–3 g/day24,25]. Its biological half-life is 1.5–1.6 h [26]. The main absorp-ion site of metformin HCl is proximal small intestine [27]. Hence,his would be beneficial to design a mucoadhesive polymeric beadsontaining metformin HCl using LM pectin-IHM blend throughonotropic-gelation technique, which might facilitate an intimateontact with the absorbing surfaces of mucous membranes (i.e.,ucoadhesion) and thus, the gastric residence time could be pro-

onged to release the encapsulated drug at the drug absorbing sitet a controlled rate to maximize the therapeutic effect.

Statistical optimization based on design of experiments (DoE)as been widely applied in the process or formulation optimiza-ion by designing a set of experiments that reliably measure theesponse variables, fitting a mathematical model to the data, con-ucting appropriate statistical test to assure that the best possibleodel is chosen and determining the values of independent for-ulation variables to produce optimum response [28–33]. Among

arious statistical optimization design, central composite design is popular response surface design has been widely used for formu-ation optimization of various drug delivery systems [34–37]. It isery efficient and flexible design providing necessary informationn experiment variable effects and overall experimental error in ainimal number of required runs [34]. In the present study, cen-

ral composite design based on response surface methodology wasmployed for the formulation optimization of ionotropically-gelledalcium pectinate-IHM mucoadhesive beads containing metforminCl.

. Experimental

.1. Materials

Metformin HCl (Abhilash Chemicals Pvt. Ltd., India), LM pectinMW ∼30,000–100,000, Loba-chemie, India), calcium chlorideMerck Ltd., India) were used. IHM was isolated from ispaghula (P.vata F.) husk (Shree Baidyanath Ayurved Bhawan Pvt. Ltd., India).he procedure of IHM isolation has been described in previouslyublished paper by our research group [20]. All other chemicalsnd reagents were commercially available and of analytical grade.

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

Calcium pectinate-IHM beads containing metformin HCl wererepared using calcium chloride (CaCl2) as cross-linker by

onotropic-gelation technique. Briefly, required amounts of LMectin and isolated IHM were dissolved in deionized water (20 ml)sing magnetic stirring for 30 min. Afterwards, metformin HCl wasdded to the mixture solutions of LM pectin-IHM for each formula-

ion maintaining polymer to drug ration 2:1 and mixed thoroughlysing a homogenizer (Remi Motors, India). The final LM pectin-IHMixture solutions containing metformin HCl were ultra-sonicated

or 5 min for debubbling. The resulting dispersion was then added

ical Macromolecules 66 (2014) 203–211

via a 21-gauge needle drop wise into 100 ml of 5% w/v CaCl2 solu-tion. Added droplets were retained in the CaCl2 solution for 15 min.The wet beads were collected through decantation, washed twotimes with distilled water and then, dried at 37 ◦C for overnight.The dried beads were stored in a desiccator until used.

2.3. Experimental design for optimization

A central composite design (spherical type, single center point,and ̨ = 1.414) was employed for the formulation optimizationof calcium pectinate-IHM beads containing metformin HCl. Theamounts of LM pectin (A) and IHM (B) as polymeric blend weredefined as the selected independent formulation variables (fac-tors); while drug encapsulation efficiency (DEE, %), and cumulativedrug release at 10 h (R10h, %) were used as dependent variables(responses). The matrix of the design including values and lev-els of investigated factors and responses is shown in Table 1. Thematrix of the design including investigated factors and responsesare also shown in Table 2. Design-Expert 8.0.6.1 software (Stat-EaseInc., USA) was used for generation and evaluation of experimentaldesign. The polynomial mathematical model generated by circum-scribed central composite design is following [34]:

Y = b0 + b1A + b2B + b3AB + b4A2 + b5B2 (1)

where, Y is the response; b0 is the intercept, and b1, b2, b3, b4, b5are regression coefficients. A and B are individual effects; A2 and B2

are quadratic effects; AB is the interaction effect. One-way ANOVAwas applied to estimate the significance of the model (p < 0.05). Thesurface response plots and contour plots were analyzed to revealthe effect of independent factors on the measured responses wereanalyzed.

2.4. Determination of DEE

Beads (100 mg) were taken and crushed using pestle and mor-tar. The crushed powders were placed in a 250 ml volumetric flask.The volume was made up to 250 ml by phosphate buffer, pH 7.4and kept for 24 h with occasionally shaking at 37 ± 0.5 ◦C. Afterthe stipulated time, the mixture was stirred at 500 rpm for 20 minusing a magnetic stirrer (Remi Motors, India). The polymer debrisformed after disintegration of bead was removed filtering throughWhatman® filter paper (no. 40). The drug content in the filtrate wasdetermined using a UV–VIS spectrophotometer (Shimadzu, Japan)at 233 nm against appropriate blank. The DEE (%) of these preparedbeads was calculated by the following formula [38]:

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

× 100 (2)

2.5. Bead size measurement

Particle size (100 dried beads from each batch) was measured byoptical microscopic method for average particle size using an opti-cal microscope (Olympus). The ocular micrometer was previouslycalibrated by stage micrometer.

2.6. Scanning electron microscopy (SEM) analyses

Beads containing metformin HCl were gold coated by mountedon a brass stub using double-sided adhesive tape and under vacuum

in an ion sputter with a thin layer of gold (3–5 nm) for 75 s and at20 kV to make them electrically conductive and their morphologywas examined by scanning electron microscope (ZEOL, JSM-5800,Japan).

A.K. Nayak et al. / International Journal of Biological Macromolecules 66 (2014) 203–211 205

Table 1Experimental plan of central composite design (coded values in bracket) with observed response values with bead size for different formulations of calcium pectinate-IHMbeads containing metformin HCl.

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

LM pectin (mg, A) IHM (mg, B) DEE (%) R10h (%)

F-1 629.29 (−1.414) 150.00 (0) 70.12 ± 2.83 79.92 ± 2.28 1.60 ± 0.08F-2 650.00 (−1) 100.00 (−1) 67.78 ± 1.87 83.66 ± 3.10 1.26 ± 0.06F-3 650.00 (−1) 200.00 (1) 76.07 ± 2.02 66.48 ± 1.82 1.54 ± 0.10F-4 700.00 (0) 79.29 (−1.414) 71.28 ± 2.77 75.05 ± 2.74 1.17 ± 0.08F-5 700.00 (0) 150.00 (0) 73.09 ± 3.03 70.27 ± 1.98 1.54 ± 0.11F-6 700.00 (0) 220.71 (1.414) 81.67 ± 3.18 57.02 ± 1.22 1.84 ± 0.15F-7 750.00 (1) 100.00 (−1) 72.28 ± 2.49 72.05 ± 2.53 1.36 ± 0.10F-8 750.00 (1) 200.00 (1) 79.56 ± 2.89 60.82 ± 2.04 1.77 ± 0.14F-9 770.71 (1.414) 150.00 (0) 76.01 ± 3.03 68.88 ± 2.18 1.38 ± 0.15F-O 679.60 263.10 Actual values 1.87 ± 0.17

86.98 ± 3.26 47.20 ± 1.28Predicted values88.59 45.55

% Errorc 1.82 3.62

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Mean ± S.D., n = 3.b Mean ± S.D., n = 20.c Error (%) = (difference between actual value and predicted value)

predicted value× 100.

.7. Fourier transform-infrared (FTIR) spectroscopy analyses

Samples were reduced to powder and analyzed as KBr pelletsy using a Fourier transform-infrared (FTIR) spectroscope (Perkinlmer Spectrum RX I, USA). The pellet was placed in the sam-le holder. Spectral scanning was taken in the wavelength regionetween 3750 and 400 cm−1 at a resolution of 4 cm−1 with scanpeed of 1 cm/s.

.8. In vitro drug release studies

The in vitro release of metformin HCl from various calciumectinate-IHM beads was tested using dissolution apparatus USPCampbell Electronics, India). The baskets were covered with 100-

esh nylon cloth to prevent the escape of the beads. The dissolutionates were measured at 37 ± 1 ◦C under 50 rpm speed. Accuratelyeighed quantities of various calcium pectinate-IHM beads con-

aining metformin HCl equivalent to 100 mg were added to 900 mlf 0.1 N HCl (pH 1.2). The test was carried out in 0.1 N HCl (pH.2) for 2 h and then continued in phosphate buffer (pH 7.4) forext 8 h. 5 ml of aliquots was collected at regular time intervalsnd the same amount of fresh dissolution medium was replaced

nto 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.

able 2ummary of ANOVA for quadratic models.

Source Sum of squares d.f.*

(a) For DEE (%)Model 61.06 5

X1 33.29 1

X2 114.49 1

X1X2 0.26 1

X12 0.15 1

X22 6.38 1

(b)For R10h (%)Model 577.29 5

X1 135.16 1

X2 363.26 1

X1X2 8.85 1

X12 14.06 1

X22 11.45 1

* d.f. indicates degree of freedom. and NS indicate significant and not significant, respectively.

2.9. Analysis of in vitro drug release kinetics and mechanism

The in vitro drug release from formulated various cal-cium pectinate-IHM beads containing metformin HCl wereevaluated kinetically using various important mathematical mod-els like zero-order, first-order, Higuchi and Korsmeyer–Peppasmodels [32].

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 = Q0ekt; where Q represents the drugreleased amount in time t, and Q0 is the start value of Q; k is therate constant.

Hixson–Crowell model: Q1/3 = kt + Q01/3; where Q represents the

drug released amount in time t, and Q0 is the start value of Q; k isthe 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 the dif-fusional exponent, indicative of drug release mechanism. When nis ≤0.43, it is Fickian release (diffusion-controlled release) if the

matrix is spherical. The n value between 0.43 and 0.85 is definedas non-Fickian release (anomalous transport). When, n is ≥0.85, itis case-II transport case-II transport (relaxation-controlled release)[32].

Mean square F value p-Value Prob > F

32.21 62.47 0.0031 (S)33.29 64.56 0.0040 (S)114.49 222.03 0.001607 (S)0.26 0.49 0.5326 (NS)0.15 0.28 0.6310 (NS)6.38 12.37 0.0390 (S)

115.46 175.34 0.0007 (S)135.16 205.26 0.0007 (S)363.26 551.66 0.0002 (S)8.85 13.44 0.0351 (S)14.06 21.36 0.0191 (S)11.45 17.39 0.0251 (S)

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.10. Evaluation of swelling behavior

Swelling evaluation of optimized calcium pectinate-IHM beadsontaining metformin HCl were carried out in two different aque-us media: 0.1 N HCl (pH 1.2) and phosphate buffer (pH 7.4). 100 mgeads were placed in vessels of dissolution apparatus (Campbelllectronics, India) containing 500 ml respective media. The exper-ment was carried out at 37 ± 1 ◦C under 50 rpm paddle speed. Thewelled beads were collected at various time intervals and weighedfter drying the surface by using tissue paper. Swelling index wasetermined using the following formula [32]:

welling index(%)

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

× 100

(3)

.11. Ex vivo mucoadhesion testing

The ex vivo mucoadhesive property of optimized calciumectinate-IHM beads containing metformin HCl were evaluatedy wash-off method [36]. Freshly excised pieces of goat intesti-al mucosa were collected from slaughterhouse. Mucosal samples2 × 2 cm) were mounted on glass slide (7.5 × 2.5 cm) using thread.bout 50 beads were spread onto the wet tissue specimen and thelide was hung onto a groove of disintegration test apparatus. Theissue specimen was given a regular up and down movement in ves-els 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, thepparatus was stopped and the number of beads still adhering tohe tissue was counted.

.12. In vivo antidiabetic evaluation

In vivo antidiabetic evaluation was performed in alloxan-nduced diabetic male albino rats of either sex (weighing22.75–346.50 g). The protocol was subjected to the scrutiny ofhe Institutional Animal Ethical Committee and was cleared beforetarting. The experimental animals were handled as per guidelinesf committee for the purpose of control and supervision on exper-mental animals (CPCSEA). All efforts were made to minimize bothhe suffering and number of animals used. The acclimatized ratsere kept fasting for 24 h with water ad libitum. All experimentsere performed between 8 AM to 12 PM to minimize circadian

nfluences.The male albino rats were made diabetic by intraperitoneal

i.p.) administration of freshly prepared alloxan solution at a dosef 150 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-IHM 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.

.13. Statistical analysis

Statistical optimization was performed using Design-Expert®

ersion 8.0.6.1 software (Stat-Ease Inc., USA). All measured data are

ical Macromolecules 66 (2014) 203–211

expressed as mean ± standard deviation (S.D.). The in vivo data wastested for significant differences (p < 0.05) by paired samples t-test.The statistical analyses were conducted using MedCalc software,version 11.6.1.0.

3. Results and discussion

3.1. Preparation of calcium pectinate-IHM beads containingmetformin HCl

In the present work, calcium pectinate-IHM beads containingmetformin HCl were prepared by ionotropic-gelation using CaCl2 ascross-linker. The ionic interactions between the carboxylic groups(negative charged) of LM pectin-backbone and the Ca2+ induces theformation of so-called “Egg-Box” model structure, even though itslightly differs from the “Egg-Box” model originally defined for cal-cium alginate [2,39]. According to the “Egg-Box” model originallyhypothesized for LM pectin, there is an initial dimerization stepof two homogeneous galacturonic chains by cooperative bridgingof parallel facing chains through Ca2+ ions in ionotropic-gelation[18]. Cooperatively, it is possible because homogalacturonic chainsare relatively rigid and binding of a first Ca2+ ion by two pectinchain facilitates their alignment with respect to each other, whichin turn allows easier binding in a next chain ion and so on along thesequence [18,40]. The anti-parallel orientation of the two chainsseems to be the most favourable arrangement and in addition toelectrostatic interactions, this initial dimer association is stronglystabilized by hydrogen bonding. When the mixtures of LM pectin-IHM blends containing metformin HCl was dropped into the CaCl2solutions, spherical ionotropically-gelled calcium pectinate-IHMbeads containing metformin HCl were formed due to electrostaticinteraction between negatively charged carboxylic groups of LMpectin and positively charged Ca2+ ions of cross-linking solutions.

3.2. Optimization

According to the trial proposal of central composite design(spherical type, single center point, and ̨ = 1.414), 9 experimentalformulations of calcium pectinate-IHM beads containing met-formin HCl were prepared using ionotropic-gelation technique.Overview of the experimental plan and observed response val-ues is presented in Table 1. The values of investigated responsesmeasured for all trial formulations were fitted in the central com-posite design to get model equations for responses analyzed in thisinvestigation. These models were evaluated statistically by apply-ing one-way ANOVA (p < 0.05) (Table 2).

The model equation relating DEE (%) as response became:DEE (%) = −8.08 + 0.18 A − 0.03 B − 1.01 × 10−4 AB − 8.76 × 10−5

A2 + 5.92 × 10−4 B2 [R2 = 0.9905; p < 0.05] The model equation relat-ing R10h (%) as response became: R10h (%) = 623.61 − 1.40 A − 0.31B + 5.95 × 10−4 AB + 8.80 × 10−4 A2 − 7.94 × 10−4 B2 [R2 = 0.9966;p < 0.05].

Model simplification was carried out by eliminating non-significant terms (p > 0.05) in model equations resulting from themultiple regression analysis [32], giving: DEE (%) = −8.08 + 0.18A − 0.03 B + 5.92 × 10−4 B2 and R10h (%) = 623.61 − 1.40 A − 0.31B + 5.95 × 10−4 AB + 8.80 × 10−4 A2 − 7.94 × 10−4 B2.

Response surface methodology is a widely used approach in thedevelopment and optimization of drug delivery devices [15,32].The purpose of the response surface methodology is to understanda model as fully as possible the effect of factors and their levels,

over the whole of the experimental domain, and to predict theresponse inside the domain. Moreover, it can be used for optimizinga formula (i.e., maximizing one or more of the responses, keep-ing the formulation variable setting within a satisfactory range),

A.K. Nayak et al. / International Journal of Biological Macromolecules 66 (2014) 203–211 207

F(

ctviptstiDimpRagsb

Fp

ig. 1. Three-dimensional response surface plot showing the effects of LM pectinmg) and IHM (mg) amounts on DEE (%).

arrying out simulations with the model equations and plottinghe responses [41]. The three-dimensional response surface plot isery useful in learning about the main and interaction effects of thendependent variables (factors), whereas two-dimensional contourlot gives a visual representation of values of the response [32]. Thehree-dimensional response surface plots (Figs. 1 and 2) and corre-ponding contour plots (Figs. 3 and 4) were presented to estimatehe effects of the independent variables (factors) on each responsenvestigated. The three-dimensional response surface plot relatingEE (Fig. 1) indicates the increment in both the values with the

ncreasing of LM pectin amount (A) and IHM amount (B) in the for-ulated calcium pectinate-IHM beads containing metformin HCl

repared by ionotropic-gelation technique. However, a decrease in10h values with the increasing LM pectin amount (A), and IHM

mount (B) is indicated by the three-dimensional response surfaceraph relating R10h (Fig. 3). All the contour plots relating mea-ured responses (Figs. 2 and 4) indicate nonlinear relationshipsetween two independable variables (here, LM pectin amount and

ig. 2. Two-dimensional corresponding contour plot showing the effects of LMectin (mg) and IHM (mg) amounts on DEE (%).

Fig. 3. Three-dimensional response surface plot showing the effects of LM pectin(mg) and IHM (mg) amounts on R10h (%).

IHM amount) on all measured responses (i.e., DEE and R10h), inves-tigated in this study.

To develop new formulations with desired response (opti-mum quality), numerical optimization technique was employed.The desirable ranges of the independable variables (factors) wererestricted to 650 ≤ A ≤ 700 mg and 250 ≤ B ≤ 300 mg; whereas thedesirable ranges of responses were restricted to 80 ≤ DEE ≤ 100%and 45 ≤ R10h ≤ 50%. The optimal values of responses were obtainedby numerical analysis using the Design-Expert 8.0.6.1 softwarebased on the criterion of desirability. The desirability plot indicat-ing desirable regression ranges for optimal process variable settingswas presented in Fig. 5 and overlay plot indicating the region ofoptimal process variable settings was presented in Fig. 6. In orderto validate the optimization capability of the mathematical mod-els generated according to the results of central composite design,

optimized calcium pectinate-IHM beads containing metformin HClwere prepared using one of the optimal process variable settingsproposed by the design (R2 = 1). The selected optimal process vari-able setting used for the formulation of optimized formulation was

Fig. 4. Two-dimensional corresponding contour plot showing the effects of LMpectin (mg) and IHM (mg) amounts on R10h (%).

208 A.K. Nayak et al. / International Journal of Biological Macromolecules 66 (2014) 203–211

Abasewg

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mTobL

F(r

Fig. 5. The desirability (R2) plot indicating desirable regression ranges.

= 679.60 mg and B = 263.10 mg. Optimized calcium pectinate-IHMeads containing metformin HCl (F-O) were evaluated for DEE (%)nd R10h (%). The optimized beads containing metformin HCl (F-O)howed DEE of 86.98 ± 3.26% and R10h of 47.20 ± 1.28%. For both thestimated responses small percentage error-values (1.82 and 3.62)ere observed (Table 1), which indicates that mathematical models

enerated from the central composite design were well fitted.

.3. DEE

The DEE (%) of all these calcium pectinate-IHM beads containingetformin HCl ranged 67.78 ± 1.87 to 86.98 ± 3.26% w/w (Table 1).

he ANOVA results of the central composite based formulationptimization showed that DEE (%) of these ionotropically-gelledeads was significantly influenced (p < 0.05) by both the factors,M pectin amount and IHM amount used as polymer-blend. It

ig. 6. The overlay plot indicating the region of optimal process variable settingsyellow area). (For interpretation of the references to color in this figure legend, theeader is referred to the web version of this article.)

Fig. 7. SEM photograph of the optimized calcium pectinate-IHM beads containingmetformin HCl prepared through ionotropic-gelation (F-O).

was also observed that the drug encapsulation in these beads con-taining metformin HCl was increased with the increasing of bothLM pectin and IHM amounts. Increasing amount of LM pectin inpolymer-blend solution might have been elevated the cross-linkingby CaCl2 through availing more numbers anionic of sites of pectinfor ionotropic cross-linking by Ca2+ ions, which might preventescape of the drug into the cross-linking solution during the prepa-ration [16]. However, the IHM contains the xylan backbone with(1 → 3) and (1 → 4) �-d linkages, which are highly substituted witharabinose or aldobiouronic acid residue. It seems that aldobiouronicacid might participate in cross-linking with CaCl2 during the beadpreparation and consequently further improving the drug encap-sulation [42]. The increased DEE with the increasing amount ofboth LM pectin and IHM in these newly developed beads couldbe due to the increase in viscosity of the polymer-blend solutionswith the increasing amount of polymer addition, which might havebeen prevented drug escape from the prepared beads to the cross-linking solution [18]. Similar results were observed in our previousworks, where we have formulated various ionotropically-gelledcalcium pectinate beads using LM pectin-other natural polysaccha-ride (fenugreek seed mucilage, tamarind seed polysaccharide andjackfruit seed starch) blends [16–18].

3.4. Bead size

The average bead diameter of these dried beads containing met-formin HCl made of LM pectin and IHM ranged from 1.17 ± 0.08to 1.87 ± 0.17 mm (Table 1). Increasing the bead size was foundwith the increasing amount of both the polymers used in thepolymer-blends (pectin and IHM), which could be explained basedon hydrodynamic viscosity concept. The viscosity increment of thepolymer-blend solution with the addition of IHM in increasing ratiomight form larger droplets of polymer-blend solutions during pass-ing through the needle to the cross-linking solution containing Ca2+

ions. This is in agreement with the earlier reports [16–18].

3.5. SEM analyses

The morphological analysis of the optimized calcium pectinate-IHM beads containing metformin HCl (F-O) was visualized by SEMand is presented in Fig. 7. The SEM photograph of these beadsshowed spherical shape with an irregular surface containing char-

acteristic large wrinkles and cracks. These cracks and wrinkles onthe bead surface might be caused by partially collapsing the poly-meric gel network during drying [17]. However, polymeric derbies

A.K. Nayak et al. / International Journal of Biological Macromolecules 66 (2014) 203–211 209

Fb(

wf

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bOL2ptsortIaopLtaaapTosHi

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cpHtiacc

Fig. 9. In vitro drug release from various calcium pectinate-IHM beads containing

ig. 8. FTIR spectra of (a) LM pectin, (b) isolated IHM, (c) calcium pectinate-IHMlank bead, (d) optimized calcium pectinate-IHM beads containing metformin HClF-O) and (e) pure metformin HCl.

ere seen on the bead surface, which could be due to simultaneousormation gel bead of the polymer-blend matrix [16].

.6. FTIR spectroscopy analyses

The FTIR spectra of LM pectin, IHM, calcium pectinate-IHM blankeads, calcium pectinate-IHM beads containing metformin HCl (F-) and metformin HCl are shown in Fig. 8. The FTIR spectra ofM pectin showed principal absorption peaks at 3401 cm−1 and933 cm−1 that were due to –OH and –CH stretching vibrationeaks. The peaks at 1459 cm−1 and 1356 cm−1 could be assignedo CH2 and –OH bending vibration peaks, respectively. In the FTIRpectrum of IHM, a strong absorption band at 3455 cm−1 wasbserved –OH stretching along with some complex bands in theegion 1200–1030 cm−1 due to –C–O and C–O–C stretching vibra-ions, which are the characteristic of the natural polysaccharides.n addition, the absorption bands in the region 930–820 cm−1

nd 785–730 cm−1 due to vibration modes of pyranose ringsf polysaccharides was observed. The FTIR spectrum of calciumectinate-IHM blank beads showed all characteristic peaks of bothM pectin and IHM without any significant change or shifting. Inhe FTIR spectrum of metformin HCl, the principal absorption peaksppeared at 3169 cm−1 due to the N–H stretching of the primarymine group (–NH2) and at 1063 cm−1 due to C–N stretching, and

peak at 1584 cm−1 occurs due to N–H bending vibrations of therimary amine group. In the FTIR spectrum of calcium pectinate-SP beads containing metformin HCl, various characteristic peaksf LM pectin, IHM and metformin HCl were appeared without anyignificant change or shifting indicating that the drug (metforminCl) maintained its identity even after the bead formation through

onotropic-gelation.

.7. In vitro drug release

The in vitro drug release behavior of these ionotropically-gelledalcium pectinate-IHM beads containing metformin HCl showedrolonged metformin HCl release over 10 h (Fig. 9). MetforminCl release from these beads in the acidic medium was slow (less

han 15% after 2 h). After that, faster drug release was observed

n phosphate buffer (pH, 7.4) comparatively. However, there wasn increase in the amount of drug release with time. When cal-ium pectinate-based beads were exposed to acidic condition, theross-linking Ca2+ ions might be displaced from the gel-network

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

and the carboxyl residues of LM pectin might be protonized to formwater insoluble pectinic acid, which could lead to the disruptionof ionic linkages with little electrostatic repulsion, resulting in theshrinkage of the calcium pectinate-based bead matrix [8]. The ini-tial sudden release and subsequent release rate observed in theacidic pH medium might be because the calcium pectinate-basedbeads became more compact after the initial shrinkage. The higherdrug release rate in alkaline pH compared to acidic pH might bedue to swelling and subsequent degradation of calcium pectinate-based beads. The gel structure might become loose and solublewhen exposed to alkaline pH, because Ca2+ ions involved in thecross-linked pectin network could only be displayed by Na+ ionsbut also be sequestered by phosphate present in phosphate buffer(pH, 7.4) [8]. The R10h (%) was within the range of 47.20 ± 1.28to 83.66 ± 3.10%. The results of the drug release study indicateda decrease in drug release with the increasing of polymer amounts.In case of beads containing higher polymer contents, the morehydrophilic property of the polymers could probably binds betterwith water to form viscous gel structure, which might blockade thepores on the surface of beads and could delayed the drug releasefrom these ionotropically-gelled beads [17]. The another reason-able explanation of the sustained drug release from these beadscan be attributed to the increase in densities of the polymer matrixresulting in larger beads size and this in turn increase the diffus-ional path-length [18]. Similar results were also observed in ourprevious works [16–18].

The results of the curve-fitting into various mathematical mod-els like zero order, first order, Higuchi and Korsmeyer–Peppas aregiven in Table 3. When respective correlation coefficients of thesebeads were compared, the metformin HCl release from these cal-cium pectinate-IHM beads containing metformin HCl was found tofollow zero order model (R2 = 0.9917 to 0.9976) over a period of10 h. In addition, Korsmeyer–Peppas model (R2 = 0.9892 to 0.9952)was found to be closer to the best-fit zero order model. The best-fit of zero order model indicated that the drug release from thesebeads followed controlled-release pattern. The values of releaseexponent (n) determined from in vitro drug release data of vari-ous calcium pectinate-IHM beads containing metformin HCl rangedfrom 1.03 to 1.09, indicating that the drug release from thesebeads followed the super case-II transport mechanism controlledby swelling and relaxation of polymeric-blend (calcium pectinate-IHM) matrix. This could be attributed due to polymer dissolutionand enlargement or relaxation of polymeric chain. However, therelease kinetic result is almost similar with previous reports on

release kinetics of various ionotropically-gelled natural polymeric-blend beads containing metformin HCl [16–18,20,30,32,38].

210 A.K. Nayak et al. / International Journal of Biological Macromolecules 66 (2014) 203–211

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

Formulation code Correlation coefficient (R2) Release exponent (n)

Zero-order First-order Higuchi Hixson–Crowell Korsmeyer–Peppas

F-1 0.9947 0.8472 0.6113 0.9211 0.9914 1.05F-2 0.9937 0.8971 0.5986 0.9293 0.9922 1.05F-3 0.9949 0.8610 0.6162 0.9292 0.9927 1.03F-4 0.9958 0.8449 0.5975 0.9216 0.9911 1.07F-5 0.9959 0.8542 0.5984 0.9283 0.9922 1.06F-6 0.9976 0.9035 0.5423 0.9550 0.9938 1.08F-7 0.9967 0.8808 0.5590 0.9418 0.9952 1.09F-8 0.9942 0.8836 0.5727 0.9401 0.9918 1.06F-9 0.9971 0.8551 0.6000 0.9268 0.9931 1.06F-O 0.9917 0.8968 0.5540 0.9447 0.9892 1.07

Fig. 10. Swelling behavior of optimized calcium pectinate-IHM beads contain-i[

3

c1(litmtlmiIs

3

cptoi(mosegbmbm

ferences (p < 0.05) were found between the blood glucose levelafter administration of pure metformin HCl and optimized beadscontaining metformin HCl at each time-point measured exceptstarting time-point. However, the decrease in glucose level was

Fig. 12. Comparative in vivo blood glucose level in alloxan-induced diabetic rats

ng metformin HCl (F-O) in 0.1 N HCl (pH 1.2), and phosphate buffer (pH 7.4)Mean ± S.D., n = 3].

.8. Swelling behavior

The swelling index of optimized calcium pectinate-IHM beadsontaining metformin HCl (F-O) was found lower in 0.1 N HCl, pH.2 in comparison with that of in phosphate buffer, pH 7.4, initiallyFig. 10). This was occurred due to shrinkage of pectinate gel atower pH. Maximum swelling of these beads was noticed at 2–3 hn phosphate buffer, pH 7.4 and after which, erosion and dissolu-ion took place. The swelling behavior of these beads containing

etformin HCl in phosphate buffer, pH 7.4 could be explained byhe ion exchanging between Ca2+ ions of the ionotropically cross-iked beads and the Na+ ions present in phosphate buffer. This

ight occur due to the influence of Ca2+-sequestrant phosphateons, which could result in disaggregation in the calcium pectinate-HM matrix structure leading matrix erosion and dissolution of thewollen beads [18].

.9. Ex vivo mucoadhesivity

The wash-off test of optimized calcium pectinate-IHM beadsontaining metformin HCl using goat intestinal mucosa in gastricH (pH 1.2) and intestinal pH (pH 7.4) was performed to analyzeheir ex vivo mucoadhesivity. The wash off of these newly devel-ped beads showed that the percentage of beads attached to thentestinal mucosa at gastric pH was higher than at intestinal pHFig. 11). The less percentage of beads attached to the intestinal

ucosa at intestinal pH could be due to ionization of carboxyl andther functional groups of polymers, which might increase theirolubility with reduced adhesive strength. The mucoadhesive prop-rty of these beads could be attributed to the presence of –OHroups of pectin and IHM, which have the ability to form hydrogenonds with the mucous membranes. However, hydrophilic poly-

ers like LM pectin and IHM also have ability to form non-covalent

onds like van der Waal’s forces or ionic interactions, resultingucoadhesion.

Fig. 11. Mucoadhesive behavior of optimized calcium pectinate-IHM beads con-taining metformin HCl (F-O) in 0.1 N HCl (pH 1.2), and phosphate buffer (pH 7.4)[Mean ± S.D., n = 3].

3.10. In vivo antidiabetic evaluation

The comparative in vivo blood glucose levels and the meanpercentage reduction in blood glucose level after oral administra-tion of pure metformin HCl and optimized calcium pectinate-IHMmucoadhesive beads containing metformin HCl (F-O) in alloxan-induced diabetic rats are presented in Figs. 12 and 13, respectively.A quick reduction in blood glucose level (46.18%) was observedwithin 3 h of oral administration in case of the Group A treatedwith pure metformin HCl. After that, the blood glucose levelrecovered quickly towards the normal level. In case of the GroupB treated with optimized calcium pectinate-IHM mucoadhesivebeads containing metformin HCl (F-O), the decrease in blood glu-cose level was found slower than that of the group treated withpure metformin HCl (Group A) up to 3 h (33.05%). Significant dif-

after oral administration of pure metformin HCl (standard) and optimized calciumpectinate-IHM beads containing metformin HCl (F-O) [Mean ± S.D., n = 6]. The datawere analyzed for significant differences (* p < 0.05) by paired samples t-test. Thestatistical analysis was conducted using MedCalc software version 11.6.1.0.

A.K. Nayak et al. / International Journal of Biolog

Fig. 13. Comparative in vivo mean percentage reduction in blood glucose leveli(f

iBfltphdcHt

4

nLnHawmbmiaclHc

[

[[

[[

[[[[[[[

[

[

[[

[

[[[[[[[[[

[[[

[hydr. Polym. 72 (2008) 334–341.

n alloxan-induced diabetic rats after oral administration of pure metformin HClstandard) and optimized optimized calcium pectinate-IHM beads containing met-ormin HCl (F-O).

ncreased gradually with the increment of time in case of Group (treated with optimized mucoadhesive beads containing met-

ormin HCl) and was sustained over 10 h. A 25% reduction in glucoseevel is considered a significant hypoglycemic effect [43]. Thus,he significant antidiabetic effect was observed over a prolongederiod of 10 h after oral administration of optimized mucoad-esive beads containing metformin HCl (F-O). This result clearlyemonstrates the potential of the optimized ionotropically-gelledalcium pectinate-IHM mucoadhesive beads containing metforminCl (F-O) for maintaining tight blood glucose level over a prolonged

ime.

. Conclusion

In this investigation, central composite design is used to developovel mucoadhesive beads containing metformin HCl made ofM pectin-IHM polymer-blend through ionotropic-gelation tech-ique. The optimized mucoadhesive beads containing metforminCl displayed high drug encapsulation (DEE of 86.98 ± 3.26%)nd suitable controlled in vitro sustained drug release patternith super case-II transport mechanism over 10 h. The optimizeducoadhesive beads also exhibited their pH-dependent swelling

ehavior, well ex vivo mucoadhesivity with the goat intestinalucosal membrane and significant in vivo antidiabetic activity

n alloxan-induced diabetic rats over prolonged period after oraldministration. Thus, these newly developed ionotropically-gelled

alcium pectinate-IHM mucoadhesive beads could possibly beucrative in terms of prolonged systemic absorption of metforminCl maintaining tight blood glucose level and advanced patientompliance.

[[[[

ical Macromolecules 66 (2014) 203–211 211

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