Accepted Manuscript

Title: Locust bean gum in the development of sustained

release mucoadhesive macromolecules of aceclofenac

Author: Vipul D. Prajapati Girish K. Jani Naresh G. Moradiya

Narayan P. Randeria Pankaj M. Maheriya Bhanu J. Nagar

PII: S0144-8617(14)00637-7
DOI: http://dx.doi.org/doi:10.1016/j.carbpol.2014.06.061
Reference: CARP 9026

To appear in:

Received date: 20-10-2013

Revised date: 19-5-2014
Accepted date: 18-6-2014

Please cite this article as: Prajapati, V. D., Jani, G. K., Moradiya, N. G., Randeria, N.
P., Maheriya, P. M., and Nagar, B. J.,Locust bean gum in the development of sustained
release mucoadhesive macromolecules of aceclofenac, Carbohydrate Polymers (2014),
http://dx.doi.org/10.1016/j.carbpol.2014.06.061

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Locust bean gum in the development of sustained release mucoadhesive

macromolecules of aceclofenac

Author names: Vipul D. Prajapatia,*, Girish K. Jania, Naresh G. Moradiyaa, Narayan P. Randeriaa,

Pankaj M. Maheriyaa and Bhanu J. Nagarb

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Authors Affiliation:
a
Department of Pharmaceutics, S.S.R. College of Pharmacy, Saily-Silvassa Road, Saily, Silvassa,

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U.T. of D.N.H.-396230, India.
b

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Department of Pharmaceutics, Rofel Shri G. M. Bilakhia College of Pharmacy, Namdha Campus,

Namdha Road, Vapi – 396191, Gujarat, India.

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*Corresponding author: Prof. Vipulkumar D. Prajapati

Mailing Address: Department of Pharmaceutics and Pharmaceutical Technology, SSR College of

Pharmacy, Sayli-Silvassa Road, Sayli, Silvassa, U.T. of Dadra and Nagar Haveli- 396 230, India.

Tel: +91-09824284159; Fax: +91-0260-2681104

E-mail: vippra2000@yahoo.com

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 8 Abstract

9 The study shows the development and optimization of Locust bean gum (LBG)-alginate

10 mucoadhesive macromolecules containing aceclofenac through ionotropic-gelation using 32

11 factorial design. The effect of amount of LBG and sodium alginate on drug entrapment

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12 efficiency (% DEE), % mucoadhesion at 8h (M8) and % in vitro drug release at 10h (% Q10h)

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13 were optimized. The percentage yield, average size and DEE of macromolecules were found

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14 within the range of 93.19 to 96.65%, 1.328±0.11 to 1.428±0.13µm, and 56.37 to 68.54%

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15 respectively. The macromolecules were also characterized by SEM, FTIR and DSC. The in

16 vitro drug release from these macromolecules (84.95±2.02 to 95.33±1.56% at 10h) exhibited

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17 sustained release (first-order) pattern with super case-II transport mechanism. The swelling

18 and mucoadhesivity of these macromolecules were affected by pH of the medium. The design
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19 established the role of derived polynomial equations and plots in predicting the values of

20 dependent variables for the preparation and optimization.

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21 Key words: Locust bean gum; Sodium alginate; Mucoadhesive macromolecules;

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22 Aceclofenac.
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33 1. Introduction

34 The goal of any drug delivery system is to provide a therapeutic amount of drug to the

35 proper site in the body to achieve promptly & then maintain the desired drug concentration. In

36 the last two decades, mucoadhesion has shown renewed interest for prolonging the residence

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37 time of mucoadhesive dosage forms through various mucosal routes in drug delivery

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38 applications. Mucoadhesive-based topical and local systems have shown enhanced

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39 bioavailability. Mucoadhesive drug delivery system (MDDS) gives rapid absorption and good

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40 bioavailability due to its considerable surface area and high blood flow. Drug delivery across

41 the mucosa bypasses the first-pass hepatic metabolism and avoiding the degradation of

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42 gastrointestinal enzymes (Ahuja, 1997). Adhesion can be defined as the bond produced by

43 contact between a pressure sensitive adhesive and a surface. The American Society of Testing
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44 and Materials (ASTM) has defined it as the state in which two surfaces are held together by

45 interfacial forces, which may consist of valence forces, interlocking action or both.
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46 Mucoadhesive drug delivery systems prolong the residence time of the dosage form at the site
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47 of application or absorption. They facilitate an intimate contact of the dosage form with the
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48 underlying absorption surface and thus improve the therapeutic performance of the drug. In
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49 recent years, many such MDDS have been developed for oral, buccal, nasal, rectal and

50 vaginal routes for both systemic and local effects (Prajapati, et al. 2011).
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51 Dosage forms designed for MDDS should be small and flexible enough to be acceptable

52 for patients and should not cause irritation. Other desired characteristics of a mucoadhesive

53 dosage form include high drug loading capacity, controlled drug release (preferably

54 unidirectional release), good mucoadhesive properties, smooth surface, tastelessness, and

55 convenient application. Erodible formulations can be beneficial because they do not require

56 system retrieval at the end of desired dosing interval. A number of relevant mucoadhesive

57 dosage forms have been developed for a variety of drugs (Asane, 2008).

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58 Natural biodegradable polysaccharides like various gums and mucilage like guar gum,

59 karaya gum, tara gum, cassia tora gum, locust bean gum, fenugreek seed mucilage; pectin,

60 chitosan, carrageenans and sodium alginate have been used in controlled drug delivery

61 (Kulkarni, et al. 2001), (Soppimth, et al. 2000), (Hwagno, et al. 1998), (Aydin, & Akbuga,

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62 1996), (Kedziereuciz, & Lemory, 1999). Multiparticulate systems obtained by ionotropic

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63 crosslinking of these polymers have been used to develop mucoadhesive and floating drug

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64 delivery system of various drugs.

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65 Mucoadhesive drug delivery is one of the approaches to design a formulation, which can

66 adhere to the lining of the stomach, thus retaining the drug at the absorption site for a

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67 prolonged period. This concept utilizes the phenomenon of bioadhesion - an adhesive

68 interaction between the dosage form and the biological surface. It has been suggested that a
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69 delay in GI transit, brought about by an intimate and extended contact between bioadhesive

70 systems and mucus/mucosal lining, will improve drug bioavailability and duration of action.
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71 A prolonged retention at the mucosal surface provides intimate contact between the dosage
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72 form and absorbing tissues which results in a prolonged period of drug exposure to the
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73 region. Therefore, an increased retention time is a desirable property of bioadhesive drug

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74 delivery systems (Lehr, 1991).

75 In the present study a non-steroidal anti-inflammatory drug [NSAID], aceclofenac was

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76 selected as a model drug. Aceclofenac has been indicated for various conditions like post-

77 traumatic pain, rheumatoid arthritis, ankylosing spondylitis. It is one of the emerging NSAID

78 molecules for arthritis treatment (Yesmin, et al. 2008). The molecule is practically insoluble

79 in water, but almost totally absorbed from gastrointestinal tract, its biological half-life is 4hrs

80 and administered twice daily with single dose of 100mg. To overcome the side effects

81 associated with conventional administration of NSAIDs and increase the patient compliance,

82 controlled release dosage forms have been formulated in the form of single unit and multiunit

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 83 dosage forms. Compared to single unit dosage forms, multi unit drug delivery system avoid

84 the variations in gastric emptying and different transit rates through the gastrointestinal tract

85 (Beckett, 1980), release drugs in a more predictable manner (Follonier, & Doelkar, 1992), and

86 spread over a large area preventing exposure of the absorbing site to high drug concentration

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87 on chronic dosing (Davis, et al. 1986). The successful treatment of arthritis depends on the

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88 maintenance of effective drug concentration level in the body for which a constant and

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89 uniform supply of drug is desired. Sustained release dosage forms deliver the drug at a slow

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90 rate over an extended period of time and achieve this object. Short biological half life (about

91 4 h) and dosing frequency more than one per day makes aceclofenac an ideal candidate for

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92 sustain release (Parfitt, 1999). Several synthetic polymers have been used to formulate

93 multiunit dosage forms. Recently, much research efforts have been concentrated to develop
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94 drug-loaded mucoadhesive macromolecules using a natural polymer. However, its adverse

95 effects are seen in patients with active or suspected peptic or duodenal ulcer or history of
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96 recurrent peptic or duodenal ulcer or who have gastrointestinal bleeding or other active
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97 bleedings or bleeding disorders. These concerns led to the objective of this study. The aim of
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98 the present work was to formulate aceclofenac entrapped LBG-alginate mucoadhesive

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99 macromolecules (beads) for the oral sustained release application using natural polymers

100 (Locust bean gum and sodium alginate) which could overcome the problems associated with
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101 oral sustained release systems like drug loaded calcium-alginate macromolecules,

102 polyelectrolytes coated drug loaded alginate macromolecules.

103 Locust bean gum (LBG) has been utilized in the formulation of mucoadhesive

104 macromolecules as fewer research works has been done on it (Vineet, & Sokindra, 2012).

105 Secondly, it exhibits an effective mucoadhesive property require for the desired formulation

106 of mucoadhesive macromolecules. LBG is a high molecular weight branch polysaccharide

107 and is extracted from the seeds of carob tree Ceratonia siliqua. It is a non-starch

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108 polysaccharides consisting of galactose and mannose in the ratio of 1:4 and hence they are

109 known as galactomannan. It consists of a (1, 4)-linked β-D mannopyranose backbone with

110 branch points from their 6-positions linked to α-D-galactose. The mannose elements from a

111 linear chain linked with branched galactopyranosyl residues at varying distance of parent

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112 chain as a function of the plant origin (Sharma, Dhuldhoya, & Merchant, 2008). The

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113 molecular weight of LBG ranges between 300,000 and 12,00,000 Da. It is less soluble in

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114 water and needs heating to dissolve. Being non-ionic, its aqueous solubility is not affected by

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115 pH or ionic strength of the liquid medium. LBG is approved for food uses by the US Food

116 and Drug Administration (FDA). Sodium alginate is chosen as an ion source (anionic) and it

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117 facilitates the mucoadhesive property of other non-ionic natural polymer. It is one of the most

118 extensively studied natural, hydrophilic polysaccharide composed of D-mannuronic acid and
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119 L-guluronic acid residues. The interpenetrating polymeric networks (IPNs) formation between

120 naturally occurring polysaccharides are reported in the literature for better mechanical
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121 strength and to obtained needful release behavior of entrapped drug. These include gellan
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122 gum-alginate (Rao, et al., 2007), carrageenan-alginate (Mohamadnia, et al., 2007), guar gum-
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123 alginate (George, & Abraham, 2007), carboxymethyl xanthan-alginate (Ray, Maity, Mandal,
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124 Chatterjee, & Sa, 2010) etc.

125 Hence, in present investigation LBG (non-ionic, natural mucoadhesive substance) and
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126 sodium alginate (anionic, release retardant) were used in different ratio to formulate and

127 optimize lab-scale sustained release mucoadhesive macromolecules of aceclofenac by

128 ionotropic gelation method using CaCl2 as a crosslinker in order to extend the application of

129 locust bean gum in mucoadhesive macromolecule formulation.

130

131 2. Experimental

132 2.1Materials

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133 Aceclofenac was received as a kind gift sample from Torrent Pharmaceuticals Ltd.,

134 Ahmedabad-Gandhinagar, Gujarat, India. Locust bean gum powder (mean arithmetic particle

135 size (davg) 255 m, M/G ratio 4:1 and molecular weight 320,000 Da, specifications received

136 from supplier) was received as a gift sample from Triveni Chemicals, Vapi, Gujarat, India.

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137 Sodium alginate (molecular weight 242 kDa, 80 cP viscosity of 0.1 % (w/v) aqueous solution,

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138 particle size <375) and calcium chloride were procured from Ozone Pvt. Ltd., India and

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139 Chemdyes Pvt. Ltd., India respectively. All other chemicals and reagents were of analytical

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140 grade purchased from commercial supplier and used as received.

141 2.2 Characterization of powder of locust bean gum and sodium alginate

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142 The needful physical characteristics like pH of aqueous solution, viscosity, swelling index

143 (%w/w) and moisture content (%w/w) of both powder samples were performed individually
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144 according to following methods in order to explore their effect on desire goal of study. Both

145 are nontoxic and safe to use for needful formulation of drugs according reports of supplier.
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146 The experiential results are shown in table 1.

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147 2.2.1 Determination of pH

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148 The pH of 0.1%w/v aqueous solution of LBG and sodium alginate were noted by pH
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149 meter (EUTECH Instruments, Singapore).

150 2.2.2 Measurement of viscosity

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151 0.1, 0.2 and 0.3 % w/v aqueous solution of LBG (prepared by applying heat at

152 temperature less than 50C and then cooled at 25C) and sodium alginate were prepared

153 separately and their viscosity was determined at 25C, 10 RPM using TL6 spindle small

154 sample adapter (EXPERT L, Fungilab rotational viscometer, Barcelona, Spain). The viscosity

155 in cPs was recorded in triplicates of each sample.

156 2.2.3 Measurement of swelling index (%w/w)

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157 Equilibrium swelling measurements of both the powder samples were performed in

158 triplicate in two different media (0.1N HCl, pH 1.2 and phosphate buffer, pH 6.8) to exhibit

159 the effect of pH for 2 h at ambient temperature. Accurately 0.5 g of dry samples was

160 immersed in 10 ml of each media containing measuring cylinder, mixed thoroughly for 5

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161 minute and then left to swell for 2 h at ambient temperature. After 2 h, the supernatant media

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162 was removed from each cylinder and the swollen mass was recovered carefully. The excess

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163 media from swollen was removed using tissue paper and reweighed using a single pan

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164 electronic balance (XB 220A, Precisa Instruments Ltd., Switzerland) having accuracy up to

165 fifth decimal. Percentage weight gained by the swollen mass was calculated using the formula

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166 (Yeole, et al. 2006);

Wt - W0
167 Swelling index (%w/w) = x 100 (1)
W0
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168 Where, Wt = weight of swollen mass at time‘t’ and W0 = initial dry weight of powder mass.
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169 2.2.4 Measurement of moisture content (%w/w)

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170 Moisture content was expressed as percentage weight loss on drying (%LOD). Two grams

171 of powder sample (LBG & sodium alginate) was weighed and oven dried at 105 C for 4 h to
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172 a constant weight. The experiment was done in triplicates and mean of triplicates was taken.
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173 The percentage loss on drying was then calculated using the formula;
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Weight of water in sample

174 % loss on drying = x 100 (2)
Total weight of wet sample
175

176 Table 1 here.

177 2.3 Preparation of aceclofenac loaded LBG-alginate mucoadhesive macromolecules

178 The macromolecules were prepared by ionotropic gelation technique (Yesmin, et al. 2008).

179 According to described proportion as shown in table 2, sodium alginate and LBG

180 (mucoadhesive polymer) were dissolved in purified water (20 ml) to form a colloidal

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181 dispersive solution. Based on preliminary batches of drug loaded LBG-alginate

182 macromolecules, fixed quantity of aceclofenac (0.8 g) was dispersed in above polymer

183 dispersion and mixed thoroughly to form a smooth viscous dispersion in order to entrap

184 added quantity of drug in polymeric macromolecules. Variables such as concentration and

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185 volume of CaCl2 solution (5% w/v, 100 mL), stirring speed (50 rpm), and flow rate of

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186 addition of dispersed phase into dispersion medium (8 –10 drops/min), and dropping distance

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187 from the tip of a needle to the hardening dispersion medium (3.5 cm) were fixed based on

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188 preliminary trials of batches in order to get desired strength, size and shape macromolecules.

189 The resultant bubble free dispersion was dropped through 18G syringe needle into calcium

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190 chloride solution (5% w/v, 100 ml), which was kept under stirring (50 rpm) to improve the

191 mechanical strength of the macromolecules and also to prevent aggregation of the formed
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192 macromolecules. Immediate formations of LBG-alginate mucoadhesive macromolecules were

193 developed. The developed macromolecules were retained in the calcium chloride solution for
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194 30 minutes to complete the curing reaction. The formed macromolecules were collected by
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195 filtration and dried under sunlight for 1 day. The dried macromolecules were stored in tightly
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196 closed glass vials for further studies. Table 2 shows formulation composition of batches of
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197 mucoadhesive macromolecules.

198 2.4 Experimental design for optimization

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199 In order to obtain “best” or an “optimized product” nine different formulations were

200 generated using 32 factorial design. Amount of Locust bean gum (X1) and amount of Sodium

201 alginate (X2) were taken as independent formulation variables while % drug entrapment

202 efficiency (Y1), % mucoadhesion at 8 hrs in phosphate buffer (pH 6.8) (M8) (Y2) and % drug

203 release at 10 h (Q10h) (Y3) were considered as dependent or response variables. A statistical

204 model incorporating interactive and polynomial terms was used to evaluate the responses.

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205 Design Expert® (Version 8.0.7.1) software was used for the generation and evaluation of the

206 statistical experimental design.

207 The matrix of the design including investigated responses i.e., % DEE, M8 (%) and Q10h

208 (%) are shown in Table 2. The effects of independent variables were modeled using a

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209 quadratic mathematical equation generated by a 32 factorial design such as;

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210 Y  b 0  b1X1  b 2 X 2  b12 X1X 2  b11 X12  b 22 X 2 2 (3)

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211 Where, Y is the response; b0 is the intercept, and b1, b2, b12, b11, b22 are regression

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212 coefficients. X1 and X2 are individual effects; X12 and X22 are quadratic effects; X1X2 is the

213 interaction effect. One-way ANOVA was applied to estimate the significance of models (p <

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214 0.05). Individual response parameters were evaluated using the F-test. The surface response

215 plots, and contour plots were analyzed to reveal the effect of independent factors (amount of
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216 LBG and sodium alginate) on the measured responses (% DEE, M8 and Q10h).

217 Table 2 here.

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218 2.5 Percentage yield (%w/w)

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219 The percentage yield of mucoadhesive macromolecules of each batch was calculated
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220 using the following formula;

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Weight of dried beads

221 % yield = x 100 (4)
Weight of drug + Weight of polymer
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223 2.6 Morphology and Particle size analysis

224 Particle size of the prepared macromolecules was determined using a digital calibrated

225 vernier caliper (For-bro engineers, Mumbai, India). Size of 20 dried macromolecules from

226 each batches were measured for calculating the mean diameter of macromolecules (n=3).

227 Shape and surface morphological examination of the surface structure of dried

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228 macromolecules was carried out by scanning electron microscopy (JEOL, JSM 5160, New

229 Delhi, India).

230 2.7 Drug entrapment efficiency (%DEE or PDEE)

231 Accurately weighed 100 mg drug loaded macromolecules were crushed in glass mortar

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232 and pestle and the powdered mass was transferred in a volumetric flask (50 ml) containing

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233 few ml of methanol. It was diluted to the mark using methanol and kept it for 10 mins. It was

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234 allowed to stir for 2 h at 100 rpm on magnetic stirrer (5 MLH Plus, Remi laboratory

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235 instruments, India), then it was filtered using a 0.45 micron filter paper (MF-Millipore

236 membrane filter). Drug content in the filtrate was determined by spectrophotometrically.

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237 %DEE was calculated using the formula;

Actual drug content (%)

238 Entrapment efficiency = x 100 (5)
Theoretical drug content (%)
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239 2.8 Drug excipients compatibility study
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240 Drug excipient compatibility study was carried out by FTIR and differential scanning
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241 calorimetry studies.

242 2.8.1 Fourier transform infrared (FTIR) spectroscopy

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243 Drug excipients interaction was checked out by comparing the IR spectra of pure drug
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244 aceclofenac, polymers (LBG and sodium alginate) and IR spectra of the formulation. All

245 samples in powders were triturated in a small size mortar and pestle until the powder became
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246 fine and uniform. The spectral scanning was carried out by ATR (Bruker Opus, Germany) in

247 the wavelength region between 4000 and 600 cm-1 and at a resolution 4 cm-1 by keeping dried

248 sample on Zinc selenide crystal. Stack all the IR spectra using Opus software (Vineet, &

249 Sokindra, 2012).

250 2.8.2 Differential scanning calorimetry (DSC) study

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251 Differential scanning calorimetry (DSC) was performed on pure drug, locust bean gum,

252 sodium alginate, placebo mucoadhesive macromolecules (beads) and drug-loaded LBG-

253 alginate mucoadhesive macromolecules (beads). DSC measurements were done on modulated

254 DSC (Shimadzu, Japan). About 3.0 mg of sample was placed in an aluminium pan and then

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255 hermetically sealed with an aluminium lid. The thermograms were obtained at a scanning rate

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256 of 5°C min–1 over a temperature range of 0 to 300°C under an inert atmosphere flushed with

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257 nitrogen at a rate of 20 mL min–1 (Yassin, et al. 2006).

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258 2.9 Swelling study

259 Swelling extent was measured in terms of percent (%) weight gain by the macromolecules

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260 in triplicate as a function of pH. 20 mg macromolecules of each batch were immersed in petri

261 plates containing 4 ml of 0.1 N HCl (pH 1.2) triplicately. At the end of 1h, macromolecules
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262 were withdrawn from respective petri plates and reweighed after removing the excess water

263 using blotting papers. The weight measurements of swollen macromolecules was determined
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264 using a single pan electronic balance (XB 220A, Precisa Instruments Ltd., Switzerland)
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265 having accuracy up to fifth decimal. For every 1h weight of macromolecules was noted, and
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266 process was continued till the end of 10h. The macromolecules were handled carefully in
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267 order to avoid any mass loss due to breaking or erosion. Percentage weight gained by the

268 macromolecules was calculated using Eq. (1) (Yeole, et al. 2006). Same procedure was
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269 carried out in triplicate using phosphate buffer (pH 6.8) and percentage weight gained by

270 macromolecules was calculated.

271 2.10 In-vitro wash off test

272 Mucoadhesive property of various formulations of aceclofenac loaded macromolecules

273 was evaluated by the in vitro wash-off method. A freshly excised piece of goat intestinal

274 mucosa (3X3 cm2) was mounted on a glass slide using thread. About 50 macromolecules

275 were spread out on each piece of mucosa and then it was hanged from the arm of the tablet

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276 disintegration test apparatus. A regular up and down movement was give to the tissue

277 specimen in a vessel containing 900 ml of 0.1 N HCl (pH 1.2) maintained at 37 ± 0.5°C. The

278 adherence of macromolecules was regularly observed. The macromolecules that remained

279 adhered to the mucosa was counted at regular intervals for up to 10 h. Same procedure was

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280 followed by replacing 0.1 N HCl with phosphate buffer of pH 6.8. Disintegration test

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281 apparatus was stopped at different time intervals up to 10 h and the number of

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282 macromolecules still adhering to the tissue was counted and % mucoadhesion was calculated

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283 (Ritesh, et al. 2012).

284 2.11In-vitro drug release study

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285 In-vitro release of aceclofenac from the macromolecules of each batch was carried out in

286 the simulated gastrointestinal condition by the pH change method at 37 ± 2 °C (Yesmin, et al.
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287 2008). 0.1 N HCl (0.1 % SLS, pH 1.2) was used to represent the gastric condition; pH 6.8

288 (phosphate buffer) is a compromise condition between the pH of the gastric and small
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289 intestine. Dissolution process was carried out in USP apparatus-I (basket method) at 50 rpm
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290 by taking macromolecules equivalent to 200 mg aceclofenac in 900 ml of 0.1 N HCl

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291 containing 0.1% SLS media for first 2h, followed by 900 ml of pH 6.8 phosphate buffer for
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292 10h. Total process was continued for 12h. Aliquots of 5 ml was withdraw every half an hour

293 and replaced an equivalent amount of fresh dissolution media equilibrated at the same
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294 temperature. Aliquots solution was diluted suitably, filtered and analyzed at 274nm using UV

295 visible spectrophotometer. All the release studies were conducted in triplicate (n=3).

296 2.12 Analysis of in vitro drug release kinetics and mechanism

297 In order to investigate the mechanism of drug release, the data were fitted to various

298 release kinetic model equations such as zero order (cumulative % aceclofenac release vs.

299 time), first order (Log cumulative of % drug retaining vs. time), Higuchi’s square root of time

300 model (cumulative % aceclofenac release vs. square root of time), Hixson Crowell cube root

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301 plot (cube root of drug % remaining in matrix vs. time) and Korsmeyer-Peppas kinetic plot

302 (fraction release of drug vs. time).

303 The zero order rate Eq. (6) describes the system where the drug release rate is independent of

304 its concentration. The first order Eq. (7) describes the release from system where release rate

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305 is concentration dependent. Higuchi described the release of drugs from insoluble matrix as a

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306 square root of time dependent process based on Fickian diffusion Eq. (8). The Hixson-

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307 Crowell cube root law Eq. (9) describes the release from systems where there is a change in

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308 surface area and diameter of particles. The Korsmeyer-Peppas exponential model Eq. (10)

309 describes the drug transport mechanism.

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310 C= K 0 t (6)

311 Where, K0 is zero-order rate constant expressed in units of concentration/time and t is the
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312 time.

Log C0 - Kt
313 Log C = (7)
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2.303
314 Where, C0 is the initial concentration of drug and K is the first order rate constant.
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315 Q = Kt1/2 (8)

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316 Where Q is the amount of drug released at time t and K is the diffusion rate constant.
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317 Q01/3 - Q t1/3 = K HC t (9)

318 Where, Qt is the amount of drug released in time t, Q0 is the initial amount of the drug in the
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319 bead, and KHC is the rate constant for Hixson-Crowell rate equation.

Mt
320 =Kt n (10)
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321 Where Mt/M8 is the fractional release of the drug, t is the release time, K is a constant

322 incorporating structural and geometric characteristic of the release device and the diffusional

323 exponent ‘n’ is dependent on the geometry of the device as well as the physical mechanism

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324 for release. The values of n for a cylindrical shaped device are = 0.43 for Fickian release. For

325 non-Fickian (anomalous) release, ‘n’ value is 0.5 to 1.0; for quasi-Fickian diffusion, n < 0.5;

326 for Fickian diffusion (square root of time kinetics), n = 0.5; for zero order release case II

327 transport (relaxation controlled), n = 1.0; for super case transport II, n > 1.0.

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328

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329 3. Results and discussion

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330 3.1 Characterization of powder of locust bean gum and sodium alginate

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331 The pH of 0.1%w/v aqueous solution of LBG powder and sodium alginate were found to

332 be 5.9  0.08 and 9.1  0.22 respectively as shown in table 1 indicating LBG as slightly acidic

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333 and sodium alginate as alkaline in nature.

334 According to table 1, the viscosity of aqueous solution of LBG as well as sodium alginate
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335 increased gradually with their proportion at low RPM indicating their nature as highly

336 branched random coils in composition and also due to water uptake capacity of their
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337 polysaccharide units. LBG is composed of galactose and mannose units (mannose to
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338 galactose content 4:1) and the longer the galactose side chain greater will be the viscosity.
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339 The viscosity of sodium alginate solution was dependent on its concentration and length of
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340 alginate molecules or the number of monomer units in the chain. The characteristic of

341 viscosity was helped in the preparation of aqueous dispersion containing LBG and sodium
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342 alginate, selection of their proportion with respect to needful quantity of addition of drug as

343 well as its effect on selection of needle size for extruding them in continuous phase without

344 blockage. Based on viscosity characteristics, 18G needle and proportion of LBG to sodium

345 alginate were fixed keeping fixed amount of aceclofenac (0.8 g) in optimization batch

346 preparation.

347 From the swelling study (Table 1), it was observed that LBG powder showed least

348 swelling in both pH 1.2 and pH 6.8 media (16.50  1.45 %w/w and 17.65  0.82 %w/w,

15
Page 15 of 43
349 respectively). In both the media, LBG powder did not any significant difference in swelling

350 behavior. This was due to its non ionic nature. Natural gums generally on hydration swell

351 rapidly forming the viscous gel layer on the surface, which increases the diffusion path length

352 and slows down the solvent imbibition. Moreover, as it is a galactomannan, it has lesser

t
353 tendency to interact with water molecule due to its steric bulkiness and random coil nature

ip
354 (Lundin et al., 1995). On the other hand, sodium alginate in powder form exhibited higher

cr
355 swelling in both pH 1.2 and pH 6.8 media (41.45  1.62 %w/w and 52.50  1.74 %w/w,

us
356 respectively) in comparison to LBG powder. Being polyelectrolyte, sodium alginate was

357 exhibited higher swelling in acidic media as compared to LBG powder. The higher swelling

an
358 of sodium alginate powder in pH 6.8 with compare to pH 1.2 might be due to its acid resistant

359 nature towards acidic pH. The swelling extent of sodium alginate will be affected during
M
360 blending with LBG and crosslinking with CaCl2 following their concentration. This study was

361 helped in selection of ratio of LBG to sodium alginate with respect to CaCl2 as cross-linker to
d

362 get desired oral sustained release mucoadhesive macromolecules of aceclofenac.

te

363 The moisture content was calculated as percentage loss on drying to determine purity of
p

364 both polymers. It was found to be 9.4  0.17 % for LBG and 8.8  0.51 % for sodium alginate
ce

365 (Table 2). Pharmacopoeial lime for moisture content of natural gums has been set at  15.0

366 %. The moisture content of the LBG and sodium alginate was within the limit set for natural
Ac

367 gums. If natural materials like gums contain excess moisture it is therefore, important to

368 assess the moisture content of natural gums which indicate to certain extent its stability.

369 3.2 Optimization

370 A 32 factorial experimental design technique was employed to investigate the effect of

371 independent variable on dependent variables like % drug entrapment efficiency, %

372 mucoadhesion at 8 h and % drug release at 10 h using the Design Expert® Software (Version

373 8.0.7.1).

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Page 16 of 43
374 3.2.1 Effect of formulation variable on % Drug entrapment efficiency (Y1)

375 The R2 (0.9896) was high indicating the adequate fitting of the quadratic model. The

376 polynomial equations can also be used to draw conclusions considering the magnitude of co-

377 efficient and the mathematical sign it carries; i.e. positive or negative. The % drug entrapment

t
378 efficiency of all batches was within 56.37 to 68.54%. Among the nine batches F6 showed

ip
379 highest % drug entrapment efficiency, this may be due to a high level of sodium alginate

cr
380 which allows the drug to get entrap more as compared to formulation containing a low level of

us
381 sodium alginate. Locust bean gum does not affect the % drug entrapment efficiency as it is

382 neutral polymer and only shows mucoadhesive property. F3, F6 and F9 contain the highest

an
383 concentration of sodium alginate so they showed highest % drug entrapment efficiency than

384 other batches. The % drug entrapment efficiency increases with increase in the concentration
M
385 of sodium alginate. The effect of formulation variable on % DEE is presented by response

386 surface plot and contour plots in Fig. 1(a) and 2(d) respectively. The polynomial equation for
d

387 % drug entrapment efficiency was calculated using the equation;

te

388 Y1  8.51  95.15 X1  63.30 X 2 – 83.50 X1X 2 – 73.50 X12  2.0 X 2 2 (11)
p

389 Figure 1 and 2 here.

ce

390 3.2.2 Effect of formulation variables on % mucoadhesion at 8 hours in phosphate buffer pH

391 6.8 (M8) (Y2)

Ac

392 The R2 (0.9756) was high indicating the adequate fitting of the quadratic model. The

393 polynomial equations can also be used to draw conclusions considering the magnitude of co-

394 efficient and the mathematical sign it carries; i.e. positive or negative. The % mucoadhesion

395 at 8 h of all batches was within 60 ± 1.62 to 85 ± 1.83%. Among the nine batches F9 showed

396 highest % mucoadhesion at 8 h in phosphate buffer pH 6.8, this may be due to a high level of

397 locust bean gum. The batches containing higher level of locust bean gum showed

17
Page 17 of 43
398 significantly greater mucoadhesive property in phosphate buffer (pH 6.8) may be due to the

399 presence of a certain amount of unionized hydroxyl groups within Locust bean gum which

400 forms a strong gel network with the mucus glycoprotein network of the intestinal mucosa,

401 which results in formation of stable mucoadhesive joint and explains the large force required

t
402 to detach the mucoadhesive dosage form from the mucosal surface. Macromolecules of batch

ip
403 F6 showed 80 ±1.59 % mucoadhesion at 8 h (M8) although containing medium level of locust

cr
404 bean gum because of it contains the highest level of sodium alginate as it shows little

us
405 mucoadhesive property. The effect of formulation variable on M8 (%) is presented by

406 response surface plot and contour plots in Fig. 1(b) and 2(e) respectively. The polynomial

an
407 equation for % mucoadhesion at 8 h in phosphate buffer (pH 6.8) was calculated using the

408 equation;
M
409 Y2  113.88  200.0 X1 – 216.66 X 2 – 3.77 X1X 2 – 333.33 X12  166.66 X 2 2 (12)

410 3.2.3 Effect of formulation variables on % drug release at 10 hours (Q10h) (Y3)
d

411 The R2 (0.7227) was high indicating the adequate fitting of the quadratic model. The
te

412 polynomial equations can also be used to draw conclusions considering the magnitude of co-
p

413 efficient and the mathematical sign it carries; i.e. positive or negative. The % drug release at
ce

414 10 h of all batches was within 84.95 ±2.02 to 95.33 ±1.56%. Among the nine batches F5

415 showed highest % drug release at 10 h, this may be due to a entrapment of drug occurs in an
Ac

416 ideal way and medium concentration of sodium alginate cannot get entrapped whole amount

417 of drug and so some particles of the drug remained on the surface which releases first while

418 dissolution. In case of batches containing highest concentration of sodium alginate entrapped

419 whole amount of drug and releases drug slowly than other batches and whole amount of drug

420 available lately. The effect of formulation variable on Q10h (%) is presented by response

18
Page 18 of 43
421 surface plot and contour plots in Fig. 1(c) and 3(f) respectively. The polynomial equation for

422 % drug release at 10 h was calculated using the equation;

423 Y3  – 207.10  72.50 X1  711.71 X 2 – 19.25 X1X 2 – 159.66 X12 – 428.66 X 2 2 (13)

424 Validation of factorial design was carried out using Design Expert® software (Version

t
ip
425 8.0.7.1) and check point was generated. Amount of two variables were found and new batch

426 of LBG-alginate mucoadhesive macromolecules was prepared by using that amount of two

cr
427 variables. Batch F6 showed actual value nearby to new generated batch and so batch F6 was

us
428 confirmed as optimized batch (Table 3).

429 Table 3 here.

an
430 3.3 FTIR study

431 The characteristics absorption peaks of aceclofenac were obtained at 1566.92, 3314.98,
M
432 1247.11, 1442.41, 1766.70, 1710.17, 1340.95, 742.91 and 664.28. The principle peaks

433 obtained for the combination were almost similar to that of the drug. The possibility of
d

434 interaction was ruled out as there was no major shift in the absorption bands of drug and the
te

435 formulation. The frequencies of functional groups of drug aceclofenac remained intact in IR
p

436 spectra of formulation so it was concluded that there was no interaction occurred between the
ce

437 drug and excipients used in the study (Fig. 3).

438 Figure 3 here.

Ac

439 3.4 DSC study

440 In an effort to investigate the possible physical and chemical interactions between drug

441 and polymer, DSC study was carried out of pure aceclofenac, locust bean gum, sodium

442 alginate, placebo LBG-alginate mucoadhesive macromolecules (beads) and drug loaded LBG-

443 alginate mucoadhesive macromolecules (beads). The results are displayed in Fig. 4. The DSC

444 thermogram showed a sharp endothermic peak at 149.32°C for pure aceclofenac as the

19
Page 19 of 43
445 melting point of the drug. The DSC thermogram showed a endothermic peak at 197.12°C and

446 119.45°C for locust bean gum and sodium alginate respectively.

447 Figure 4 here.

448 3.5 Percentage yield

t
449 The production yields of prepared formulations were in the range of 93.19 to 96.65%.

ip
450 This high yield of production is because all amount of the polymer is available for gelation

cr
451 into cross linking agent.

us
452 3.6 Morphology and Particle size analysis

453 Particle size of the 20 macromolecules was measured and average was calculated. The

an
454 particle size of prepared formulations was in the range of 1.328 ± 0.11 to 1.428 ± 0.13 mm.

455 The macromolecules were found to be discrete, uniform in size, spherical and free flowing.
M
456 The SEM photographs (Fig. 5) indicated that the macromolecules were spherical and

457 completely covered with the coat polymer, with a size range of 1000-1100 μm. The increase
d

458 in polymer (sodium alginate) concentration showed increase in particle size and spherical
te

459 nature of macromolecules might be due to more viscous nature of polymer solution. Also, the
p

460 difference in the shape of macromolecules was observed, as the macromolecules containing
ce

461 higher amount of sodium alginate (Batch F6-F9) were more spherical and regular as

462 compared to that of macromolecules having lower percent of sodium alginate (Batch F1-F3).
Ac

463 Thus, sodium alginate has a great influence on the characteristics of macromolecules. The

464 drug-loaded macromolecules were spherical and brownish in appearance.

465 Figure 5 here.

466 3.7 Drug entrapment efficiency

467 Encapsulation efficiency of the macromolecules was dependent mainly on the

468 concentration of sodium alginate; it was found that by increasing the concentration of sodium

469 alginate, the encapsulation efficiency of the microcapsules also increases. It was found from

20
Page 20 of 43
470 literature that 5% CaCl2 concentration was suitable for preparing macromolecules to provide

471 proper hardness. Thus, increasing alginate concentration and taking 5% CaCl2 concentration,

472 increased microencapsulation efficiency of negatively charged drug. It was also assumed that

473 gelation proceeds radially from the surface of the microcapsule to its centre. As gelation

t
474 proceeds, water is expelled due to cross-links formed by the cations and the contraction of the

ip
475 gel volume. The entrapment efficiency was in the range of 56.37 to 68.54%.

cr
476 3.8 Swelling study

us
477 The results of swelling studies, shown in Fig. 6a and 6b, indicate that swelling varied

478 with the pH of the medium. The macromolecules did not show any sign of disintegration in

an
479 both the media at pH 1.2 and 6.8 over a period of 10 h. However the swelling ratio of the

480 macromolecules at pH 6.8 was considerably greater than that at pH 1.2. The swelling ratio of
M
481 the formulations ranged from 175.65 to 244.85 % in pH 1.2 and 848.70% to 935.55% in pH

482 6.8. The formulation F9 exhibited the highest swelling index in both the media. From this
d

483 result it was observed that the swelling ratio was enhanced due to increased in polymer
te

484 concentration in the formulations. Locust bean gum readily hydrates; absorb water with good
p

485 degree of swelling. Diffusion of the drug significantly depends on the water content of
ce

486 macromolecules. As polymer chain becomes more hydrated and gel becomes more diluted,

487 the disentanglement concentration may be reached (i.e. the critical polymer concentration
Ac

488 below which the polymer chain disentangles and detaches from the gelled matrix) which

489 result in swelling. Consequently, faster and greater swelling of macromolecules might lead to

490 increased dimension of macromolecules resulting to an increasing diffusion pathway and

491 thus, a reduction in diffusion rate. So, the drug release was found to be high initially and then

492 gradually decreased.

493 Figure 6 here.

494 3.9 In-vitro wash off test

21
Page 21 of 43
495 The in-vitro wash off test using goat intestinal mucosa for assessing mucoadhesivity of

496 LBG-alginate macromolecules containing aceclofenac was performed at both gastric pH (0.1

497 N HCl, pH 1.2) and intestinal pH (phosphate buffer, pH 6.8) for 10 h. In 0.1N HCl, the

498 percentage of macromolecules adhering to the goat intestinal mucosal tissue varied from 30 ±

t
499 1.29 to 45 ± 1.76% (Fig. 7a) over 8 h. Whereas, this was varied from 60 ± 1.62 to 85 ± 1.83%

ip
500 in phosphate buffer (Fig. 7b). The less mucoadhesion of Locust bean gum-alginate

cr
501 macromolecules containing aceclofenac in 0.1 N HCl (pH 1.2) may be due to the erosion of

us
502 calcium ion. The significantly greater mucoadhesive property of Locust bean gum-alginate

503 macromolecules in phosphate buffer (pH 6.8) may be due to the presence of a certain amount

an
504 of unionized hydroxyl groups within Locust bean gum which forms a strong gel network with

505 the mucus glycoprotein network of the intestinal mucosa, which results in formation of stable
M
506 mucoadhesive joint and explains the large force required to detach the mucoadhesive dosage

507 form from the mucosal surface. Hence mucoadhesive property depends on concentration of
d

508 polymers and polymer type. Therefore, the results of the wash off test indicated that the
te

509 Locust bean gum-alginate macromolecules containing aceclofenac possessed good

p

510 mucoadhesivity in intestinal pH.

ce

511 Figure 7 here.

512 3.10In-vitro drug release study

Ac

513 The in-vitro drug release from the macromolecules in 900 ml 0.1 N HCl (0.1% SLS) for 2

514 h, followed by 900 ml phosphate buffer pH 6.8 for 10 h was performed using the USP I

515 Dissolution test apparatus. Aceclofenac release from the LBG-alginate mucoadhesive

516 macromolecules (beads) was slow and depended on the composition of the coat. The

517 differences in the drug release characteristics of various macromolecules are due to the

518 differences in the porosity of the coat formed and its solubility in the dissolution fluid. It was

519 observed that with the increase in the concentration of sodium alginate, the release of drug

22
Page 22 of 43
520 from macromolecules also increases as sodium alginate act as a release rate retardant. At pH

521 1.2, there was no discernible release of aceclofenac from macromolecules and but maximum

522 drug releases in pH 6.8.

523 The in-vitro dissolution results showed that macromolecules were sustaining the drug release.

t
524 The macromolecules were able to achieve the maximum solubility of the encapsulated drug.

ip
525 This may be due to the crosslinking. The reason for the sustained release might be due to the

cr
526 entanglement of polymer chains in the macromolecules because of strong ionic interactions.

us
527 Thus, the penetration of the dissolution medium into the hydrogel macromolecules was made

528 difficult. The drug release from these macromolecules was characterized by an initial phase of

an
529 low release (burst effect) due to poor solubility of aceclofenac in pH 1.2. However, as

530 gelation proceeded, the remaining drug was released at a slower rate followed by a phase of
M
531 moderate release. This bi-phasic pattern of release is a characteristic feature of matrix

532 diffusion kinetics. Comparison of CCPR of all batches is illustrated in Fig. 8. The cumulative
d

533 percentage drug release from the macromolecules of the all formulations ranged from 88.05
te

534 to 96.99% at 12 h time period. The formulation F5 exhibited the highest drug release at 12 h.
p

535 Figure 8 here.

ce

536 3.10.1Kinetics and Mechanism of drug release

537 In order to investigate the mechanism of drug release, the data were fitted to models
Ac

538 representing zero-order, first order, Higuchi’s square root of time, Korsmeyer-Peppas kinetic

539 plot and Hixson-Crowell cube root plot. The examination of the coefficient of determination

540 (R2) indicated that drug release from the prepared macromolecules followed a diffusion

541 controlled mechanism, since the R2 values for first order (from 0.942 to 0.987) was always

542 higher compared to zero-order (from 0.852 to 0.935), Higuchi model (from0.893 to 0.927) and

543 to the Hixson-Crowell ones (from 0.916 to 0.983). Most conventional dosage forms exhibits

544 first order dissolution mechanism. Some modified release preparation, particularly prolonged

23
Page 23 of 43
545 release formulations, adheres to this type of dissolution pattern. Since the release from the

546 prepared macromolecules followed a biphasic profile, it was decided to use a more stringent

547 test in order to distinguish between the mechanisms of drug release. The release data were

548 fitted to the Peppas exponential model which characterizes the drug transport mechanism. The

t
549 n values were in the range of 1.206 to 1.303, indicating that all the prepared formulations

ip
550 followed the first-order with Super case II transport release mechanism of aceclofenac (n >

cr
551 1.0) (Table 4).

us
552 Table 4 Here.

553

an
554 4 Conclusion

555 LBG-alginate mucoadhesive macromolecules (beads) containing aceclofenac were

M
556 successfully prepared by ionotropic gelation method. This type of system shows a sustained

557 drug release behavior due to the diffusion, swelling and mucoadhesive properties. By the
d

558 result of optimization study the desired amount of natural polymer was obtained to make the
te

559 mucoadhesive macromolecules which have good mucoadhesive property and shows better
p

560 sustain release of drug. The particle size analysis revealed that the size of the macromolecules
ce

561 was found to be increase with increase in the concentration of polymers. SEM showed that

562 the macromolecules were almost spherical and had rough surface. FTIR studies showed the
Ac

563 presence of functional groups. No significant chemical interaction was observed. The in-vitro

564 drug release study showed a slow release profile for LBG-alginate mucoadhesive

565 macromolecules. From the in-vitro drug release study it was observed that the drug release

566 decreased with increase in the sodium alginate concentration at some level. Drug release was

567 diffusion controlled and followed first order kinetics. There was a significant difference in the

568 release could be seen in 0.1 N HCL (0.1% SLS) and phosphate buffer (pH 6.8). The release

569 rate was slower in acidic media than buffer media was observed. The macromolecules

24
Page 24 of 43
570 showed considerable slow swelling behavior in phosphate buffer (pH 6.8), which helped to

571 release drug slowly and gave sustained drug release behavior.

572 Finally this study demonstrated the observations of spherical to oval shaped particles with

573 high % DEE, strong mucoadhesive property and a possible sustained drug release profile

t
574 from the LBG-alginate mucoadhesive macromolecules (beads). Thus, the prepared drug

ip
575 delivery system could be utilized for a possible sustained release product of aceclofenac.

cr
576 Acknowledgement

us
577 The authors are highly thankful to SSR College of Pharmacy, Silvassa for providing all

578 the necessary support and the essential library information resources. The authors are also

an
579 thankful to Torrent Pharmaceuticals Ltd., Torrent Research Centre, Ahmedabad-Gandhinagar,

580 Gujarat, India for proving gift sample of aceclofenac and Triveni Chemicals, Vapi, Gujarat,
M
581 India for providing gift sample of Locust bean gum.

582
d

583
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584 References
p

585 Ahuja, A. (1997). Mucoadhesive drug delivery systems. Drug Development and
ce

586 Industrial Pharmacy, 23(5), 489–515.

587 Asane, G. S. (2008). Polymers for Mucoadhesive Drug Delivery System: A Current
Ac

588 Status. Drug Development and Industrial Pharmacy, 34, 1246–1266.

589 Aydin, Z., & Akbuga, J. (1996). Preparation and evaluation of pectin beads. International

590 Journal of Pharmaceutics, 137, 133–136.

591 Beckett, A. H. (1980). Alternative routes of drug administration and new drug delivery

592 systems. In: Breimer, D. D. (Ed.), Towards Better Safety of Drug and Pharmaceutical

593 Products. North Holland: Elsevier Biomedical Press, 247-263.

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594 Davis, S. S., Stockwell, A. F., Taylor, M. J., Hardy, J. G., Whalley, D. R., Wilson, C. G.,

595 Bechgaard, H., & Christensen, F. N. (1986). The effect of density on the gastric emptying

596 of single- and multiple-unit dosage forms. Pharmaceutical Research, 3(4), 208-213.

597 Follonier, N., & Doelkar, E. (1992). Biopharmaceutical Comparison of Oral Multiple-

t
598 unit and Single-unit Sustained Release Dosage Forms. STP Pharma Sciences, 2, 141-158.

ip
599 George, M., & Abraham, T.E. (2007). pH-Sensitive alginate-guar gum hydrogel for the

cr
600 controlled delivery of protein drugs. International Journal of Pharmaceutics, 335, 123-129.

us
601 Hwagno, J., Skinner, G. W., Harcu, W. W., & Barnum, P. E. (1998). Pharmaceutical

602 application of naturally occurring water soluble polymer. Pharmaceutical Science and

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603 Technology Today, 1, 254–261.

604 Kedziereuciz, K., & Lemory, C. (1999). Effect of the formulation on the in vitro release
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605 of propranolol from gellan beads, International Journal of Pharmaceutics, 178, 129–136.

606 Kulkarni, A. R, Soppimath, K. S., Aminabhavi, T. M., & Rudzinski, W. E. (2001). In

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607 vitro release kinetics of cefadroxil, loaded sodium alginate interpenetrating network beads.
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608 European Journal of Pharmaceutics & Biopharmaceutics, 51, 127–133.

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609 Lehr, C. M. (1991). An estimate of turnover time of intestinal mucus gel layer in the rat in
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610 situ loop. International Journal of Pharmaceutics. 70, 235-240.

611 Lundin, L., Hermansson, A. M. (1995). Supermolecular aspects of xanthan-locust bean

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612 gum gels based on rheology and electron Microscopy. Carbohydrate Polymers, 26, 129.

613 Mohamadnia, Z., Zohuriaan-Mehr, M.J., Kabiri, K., Jamshidi, A., Mobedi, H. (2007). pH-

614 Sensitive IPN hydrogel beads of carrageenan-alginate for controlled drug delivery. Journal of

615 Bioactive and Compatible Polymers, 22, 342-356.

616 Parfitt, K. (1999). Analgesics Anti-inflammatory and antipyretics, In: Reynolds JEF. ed.

617 Martindale: The Complete Drug Reference (Massachusetts). 32nd edition, 2-12.

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618 Prajapati, R. K., Mahajan, H. S., & Surana, S. J. (2011). PLGA Based Mucoadhesive

619 Microspheres for Nasal Delivery: In Vitro / Ex Vivo Studies. Indian Journal of Novel Drug

620 delivery. 3(1), 9-16.

621 Rao, V.U., Vasudha, M., Bindu, K., Samanta, S., Rajinikanth, P.S., Mishra, B., & et al.

t
622 (2007). Formulation and In Vitro Characterization of Sodium Alginate- Gellan Beads of

ip
623 Glipizide. Acta Pharmaceutica Sciencia, 49, 13-28.

cr
624 Ray, R., Maity, S., Mandal, S., Chatterjee, T.K., Sa, B. (2010). Development and

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625 evaluation of a new interpenetrating network bead of sodium carboxymethyl xanthan and

626 sodium alginate for ibuprofen release. Pharmacology and Pharmacy, 1, 9-17.

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627 Ritesh, S., Anuratha, P., & Anil, J. (2012). Development and evaluation of Mucoadhesive

628 algino-HPMC microparticulate system of Aceclofenac for oral sustained drug delivery. Indian
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629 Journal of Pharmaceutical Education and Research, 46(2), 120-128.

630 Sharma, B. R., Dhuldhoya, N.C., & Merchant, S. N. (2008). Glimpses of galactomannans.
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631 Science Tech Entrepreneur, 3, 1-10.

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632 Soppimth, K. S., Kulkarni, A. R., & Aminabhavi, T. M. (2000). Controlled release of
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633 antihypertensive drug from the interpenetrating network poly (vinyl) alcohol, guar gum
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634 hydrogel microspheres. Journal of Biomaterial Science, Polymer Edition, 11, 27–43.

635 Vineet, B., & Sokindra, K. (2012). Design and Characterization of Novel Interpenetrating
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636 Polymer Network Mucoadhesive Microspheres of Locust Bean Gum and PVA for Controlled

637 Release of Metformin HCl. Internationale Pharmaceutica Sciencia, 2(2), 115-121.

638 Yassin, A. B., Alsarra, I. A., & Al-Mohizea, A. M. (2006). Chitosan beads as a new

639 gastroretentive system of verapamil, Scientia Pharmaceutica, 74, 175-188.

640 Yeole, P. G., Galgatee, U. C., Babla, I. B., & Nakhat, P. D. (2006). Design and evaluation

641 of Xanthan gum based sustained release matrix tablets of Diclofenac Na. Indian Journal of

642 Pharmaceutical Sciences. 68 (2), 185-189.

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643 Yesmin, F., Mesbah, U. T., Muhammad, S. I., Susmita, L., & Tasnuva, H. (2008).

644 Evaluation of Aceclofenac Loaded Agarose Beads Prepared by Ionotropic Gelation Method.

645 Stamford Journal of Pharmaceutical Sciences, 1(1&2), 10-17.

646

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646 Figure Captions

647 Figure 1 Three-dimensional response surface plots showing the effect of amount of LBG (g)

648 ‘X1’ and sodium alginate (g) ‘X2’on responses (a) DEE (%), (b) mucoadhesion at 8 h ‘M8’

649 (%), and (c) Q10h (%).

t
ip
650 Figure 2 Contour plots showing the effect of amount of LBG (g) ‘X1’ and sodium alginate (g)

651 ‘X2’on responses (d) DEE (%), (e) mucoadhesion at 8 h ‘M8’ (%), and (f) Q10h (%).

cr
652 Figure 3 FTIR Spectrum of (a) aceclofenac and (b) aceclofenac loaded LBG-alginate

us
653 mucoadhesive macromolecules.

654 Figure 4 DSC thermograms of (a) aceclofenac, (b) LBG, (c) sodium alginate, (d) Placebo

an
655 LBG-alginate mucoadhesive macromolecules and (e) aceclofenac loaded LBG-alginate

656 mucoadhesive macromolecules.

M
657 Figure 5 SEM microphotographs of (a) placebo LBG-alginate muacoadhesive

658 macromolecules, (b) drug loaded LBG-alginate mucoadhesive macromolecules and (c)
d

659 surface of drug loaded LBG-alginate macromolecules.

te

660 Figure 6 Swelling behavior of LBG-alginate mucoadhesive macromolecules of all batches in

p

661 0.1 N HCl, pH 1.2 (a) and in phosphate buffer, pH 6.8 (b).
ce

662 Figure 7 Results of in-vitro wash off test of LBG-alginate mucoadhesive macromolecules of

663 all batches in 0.1 N HCl, pH 1.2 (a) in phosphate buffer, pH 6.8 (b).
Ac

664 Figure 8 Comparative in vitro dissolution profile of aceclofenac from LBG-alginate

665 mucoadhesive macromolecules of all batches.

666

29
Page 29 of 43
666 Table Captions

667 Table 1 Observed needful characteristics like pH, swelling index (%w/w), moisture content

668 (%w/w) and viscosity of powdered locust bean gum and sodium alginate.

669 Table 2 Experimental plan of 32 factorial design (coded values in bracket) with observed

t
ip
670 response values for different formulations.

671 Table 3 Results of experiments for conforming optimization capability.

cr
672 Table 4 Results of curve fitting of the in vitro aceclofenac release data from LBG-alginate

us
673 mucoadhesive beads.

674

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d
p te
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30
Page 30 of 43
Table 1

655 Table 1

656 Observed needful characteristics like pH, swelling index (%w/w), moisture content (%w/w)

657 and viscosity of powdered locust bean gum and sodium alginate.

Characteristics Powdered locust bean gum Powdered sodium alginate

pH (0.1 % w/v aqueous solution at 25C) 5.9 ± 0.08 9.1 ± 0.22

t
ip
0.1 % w/v aqueous solution 70.56 ± 1.12 82.25 ± 1.34
Viscosity (cP)

at 25C

0.2 % w/v aqueous solution 127.24 ± 2.50 139.50 ± 1.52

cr
0.3 % w/v aqueous solution 168.30 ± 1.05 189.65 ± 2.32

us
Swelling index (%w/w) pH 1.2 16.50 ± 1.45 41.45 ± 1.62

pH 6.8 17.65 ± 0.82 52.50 ± 1.74

Moisture content (%w/w)

an
9.4 ± 0.17 8.8 ± 0.51

658 All values indicate mean ± S.D., n = 3.

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Table 2

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659 Table 2

660 Experimental plan of 32 factorial design (coded values in bracket) with observed response values for different formulations.

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Normalized levels of factors Responses (Y1, Y2, Y3)
Experimental

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Amount of LBG Amount of sodium alginate M8 (%) Q10h (%)
formulations % DEE
(g) (g) (Mean ± S.D., n = 3) (Mean ± S.D., n = 3)
batch code (Y1)

M
(X1) (X2) (Y2) (Y3)

F1 0.1 (-1) 0.7 (-1) 56.37 60 ± 1.62 84.95 ± 2.02

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F2 0.1 (-1) 0.8 (0) 63.33 65 ± 1.32 93.12 ± 1.99

F3
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0.1 (-1) 0.9 (+1) 67.95 70 ± 1.66 89.47 ± 1.54
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F4 0.2 (0) 0.7 (-1) 58.48 70 ± 1.57 85.02 ± 1.89

F5 0.2 (0) 0.8 (0) 62.52 75 ± 1.32 95.33 ± 1.56

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F6 0.2 (0) 0.9 (+1) 68.54 80 ± 1.59 89.95 ± 2.11

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F7 0.3 (+1) 0.7 (-1) 58.30 75 ± 1.62 86.25 ± 1.99

F8 0.3 (+1) 0.8 (0) 62.18 75 ± 1.58 87.23 ± 2.21

F9 0.3 (+1) 0.9 (+1) 66.45 85 ± 1.83 90 ± 1.54

661 DEE = drug encapsulation efficiency; M8 = percentage mucoadhesion at 8 h in phosphate buffer (pH 6.8); Q10h = percentage cumulative drug

662 release from LBG-alginate beads at 10 h.

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Table 3

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663 Table 3

664 Results of experiments for conforming optimization capability.

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Experimental Factors Responses (Y1, Y2, Y3)

an
formulation Amount of LBG Amount of sodium % DEE M8 (%) Q10 (%)

batch code (g) alginate (g) (Y1) (Y2) (Y3)

M
(X1) (X2) PV AV Error (%) PV AV Error (%) PV AV Error (%)

F10 0.25 0.9 67.33 66.85 -0.712 83.01 82.67 ± 3.21 -0.409 90.06 89.39 ± 1.25 -0.743

d
665 DEE = drug encapsulation efficiency; M8 = percentage mucoadhesion at 8 h in phosphate buffer (pH 6.8); Q10h = percentage cumulative drug

666
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release from LBG-alginate beads at 10 h; LBG = Locust bean gum; SA = sodium alginate; PV = predicted value; AV = actual value.
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Table 4

667 Table 4

668 Results of curve fitting of the in vitro aceclofenac release data from LBG-alginate

669 mucoadhesive beads.

Experimental Regression coefficient (R2) Release

formulations Zero First Higuchi Hixson Korsmeyer- exponent (n)

t
batch code order order Crowell Peppas

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F1 0.910 0.978 0.921 0.965 0.813 1.303

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F2 0.902 0.983 0.912 0.976 0.795 1.275

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F3 0.895 0.972 0.919 0.955 0.721 1.134

F4 0.915 0.982 0.914 0.970 0.814 1.293

F5 0.879 0.982 0.909

an0.968 0.783 1.280

F6 0.895 0.977 0.914 0.963 0.762 1.206

M
F7 0.935 0.987 0.927 0.983 0.788 1.224

F8 0.884 0.971 0.913 0.951 0.759 1.204

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F9 0.852 0.942 0.893 0.916 0.748 1.200

pt

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*Highlights

1 Highlights

2  Sustained release mucoadhesive macromolecules of aceclofenac were developed.

3  Locust bean gum (mucoadhesive agent), sodium alginate (release retardant) were

4 utilized.

5  Variables affected on macromolecules were optimized using 32 facorial design.

t
 These macromolecules showed pH-sensitive swelling, good mucoadhesive property.

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6

7  The in vitro drug release followed first-order with super case-II transport mechanism.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Figure 7

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Figure 8

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