resveratrol-loaded calcium-pectinate beads: effects of formulation parameters on drug release and...

Resveratrol-Loaded Calcium-Pectinate Beads:Effects of Formulation Parameters onDrug Release and Bead Characteristics

SURAJIT DAS, KA-YUN NG

Faculty of Science, Department of Pharmacy, National University of Singapore, Building S4, Rm 05-02, 18 Science Drive 4,Singapore 117543, Singapore

Received 19 February 2009; revised 1 June 2009; accepted 12 June 2009

Published online 3 August 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21880

Abbreviationcalcium chlorideier transform ientrapment efficretention; T25, 2weight loss; Ca,loading; FD, fretin:resveratrol.

CorrespondenFax: 301-480-02

Journal of Pharm

� 2009 Wiley-Liss

840 JOURNA

ABSTRACT: Resveratrol has potential therapeutic efficacy on several lower gastro-intestinal (GI) diseases such as colitis and colorectal cancer. But resveratrol is quicklyabsorbed and metabolized at the upper GI tract, which renders it unsuitable for thispurpose. This study aimed at devising a delayed release formulation of resveratrol ascalcium-pectinate (Ca-pectinate) beads and investigated the impact of various formula-tion parameters on bead characteristics. Ca-pectinate beads were prepared by varyingsix formulation parameters (cross-linking pH, cross-linker concentration, cross-linkingtime, drying condition, pectin concentration, and resveratrol concentration). Theireffects were investigated on calcium entrapment, moisture content and weight lossduring drying, particle shape and size, resveratrol entrapment and loading efficiency,swelling–erosion, and resveratrol retention pattern of formulated beads. Preparativeconditions were optimized from these studies and optimized beads were further sub-jected to morphological examination, drug–polymer interaction, and enzymatic degra-dation study. Almost all prepared beads were spherical with �1 mm diameter. Swelling–erosion and drug retention pattern were changed with formulation variables. Releasedata of almost all beads showed linearity of the plots for the cumulative percentresveratrol released versus square root of time often after an initial lag period. Observa-tions from the present study revealed that optimized Ca-pectinate beads can encapsu-late a very high amount of resveratrol (>97.5%) and can be used for delayed release andsite-specific delivery to the lower GI tract. Depending on the formulation parameters,release of resveratrol after 10 h incubation in the intestinal media was 80–100%. � 2009

Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:840–860, 2010

Keywords: resveratrol; Ca-pectinate b
ead; formulation; formulation parameters;drug retention; controlled release; colonic drug delivery; stability; swelling–erosion;encapsulation
s: GI tract, gastro intestinal tract; CaCl2,; Ca-pectinate, calcium pectinate; FTIR, Four-nfra red; SER, swelling–erosion ratio; EE,iency; T75, 75% drug retention; T50, 50% drug5% drug retention; MC, moisture content; WL,calcium; ER, elongation ratio; L, resveratrol

eze-drying; RT, room temperature; P:R, pec-

ce to: Ka-Yun Ng (Telephone: 301-435-1719;87; E-mail: [email protected])

aceutical Sciences, Vol. 99, 840–860 (2010)

, Inc. and the American Pharmacists Association

L OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 2, FEBUA

INTRODUCTION

Resveratrol (trans-3,5,40-trihydroxystilbene) is apolyphenolic phytoalexin produced by a variety ofplants in response to stress.1 It displays widepharmacological activities and is widely knownfor its antioxidant, antiinflammatory, analgesic,cardioprotective, neuroprotective, chemo-preven-tive, and antiaging activities.1,2 Most recently,resveratrol has been shown to improve general

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RESVERATROL-LOADED CALCIUM-PECTINATE BEADS 841

health and survival of mice on a high-caloric diet,pointing to new approaches for treating obesity-related disorders and diseases of aging.1

As the pharmacology of resveratrol was sub-jected to extensive studies during the past decade,its pharmacokinetics has also been investigatedin preclinical models as well as in humans.1,3

Unfortunately, the pharmacokinetic properties ofresveratrol are not as favorable when comparedto its beneficial pharmacological activities invarious disease models.3 Upon oral administra-tion, resveratrol is rapidly absorbed and under-goes extensive phase II metabolism in the uppergastro-intestinal (GI) tract and liver to formglucuronide and sulfate conjugates.1,4–7 The veryshort half-life (�8–14 min)1,8 and extremely lowbioavailability of resveratrol have raised concernsregarding its systemic action.1,9 Consequently,we reason that development of a site-specific drugdelivery system that protects resveratrol duringits transit through the upper GI tract and allowsits release at the colonic region could provide apotential approach to treat lower GI diseases suchas colorectal cancer and colitis.

To achieve colon-specific drug delivery, currentapproaches use systems that (i) release the drugat a predetermined time after administration,(ii) utilize pH changes within the GI tract,(iii) make use of GI pressure differences, and(iv) exploit microbial enzymes predominantlypresent in the colonic region of the GI tract.10,11

A system relying on the transit time, pH, and/orpressure of the GI tract is expected to be pooras all these parameters are extremely variable inindividuals. In contrast, bacterial degradation ofbiopolymers in the colon to achieve colon-specificdrug delivery is a well-established phenomenonand thus could be a more realistic approach thanthe other approaches mentioned above.10,12 In thisambit, various well-known natural polysacchar-ides are currently exploited, in virtue of theirspecific degradation by colonic micro-flora whichmade them suitable carriers for colon-targeteddelivery system.11 Among such polymers, pectinsappeared to be of great practical interest dueto their low cost, biodegradability, wide variety oftypes, and flexibility in use.

Pectin is a heterogeneous polysaccharide pre-sent in the cell wall of most plants, which isconsumed as part of the human diet and used as afood additive. The influence of pectin intake on thehuman body has been well studied and regardedas safe. It is composed of partially methoxylatedpoly-a-(1–4)-D-galacturonic acids with some 1–2

DOI 10.1002/jps JOUR

linked L-rhamnose groups. It is resistant toenzymes present in stomach and intestine butcan be almost completely degraded by the colonicbacterial enzymes.11 Unfortunately, their solubi-lity and swellability in aqueous fluid prevent themfrom efficiently avoiding premature drug releasebefore reaching to the colon. However, this can bemanipulated through the choice of pectin type orthe presence of additives. The degree of methoxy-lation (DM) refers to the average number ofmethoxy groups per 100 galacturonic acid units. Itis responsible for many of the physicochemicalproperties of the pectin. High methoxy pectins(HM pectins) are poorly soluble. In contrast,pectins with low degree of methoxylation (LMpectins) are more soluble, but they can be cross-linked with divalent cations (e.g., calcium) toproduce a more water-insoluble pectate gel whichhas the potential to be an effective vehicle for drugdelivery. Various reports suggest that pectin,when cross-linked with calcium to form a multi-particulate Ca-pectinate bead system, may be aviable carrier for site-specific drug delivery to thecolon.13–15 However, to our knowledge, there is noprior study that looks at delivery of resveratrolspecifically to the colonic region. Hence in thepresent study, we attempted to prepare differentresveratrol loaded Ca-pectinate beads by varyingsix different formulation parameters (cross-linkingsolution pH, cross-linking agent concentration,cross-linking time, drying condition, pectin con-centration, and pectin to resveratrol ratio) andthoroughly studied their effect on differentphysicochemical properties and resveratrol reten-tion pattern of the formulated beads. Our presentstudy was extensive and we examined some vitalparameters which have been overlooked in pre-vious reports.13,16,17

MATERIALS AND METHODS

Materials

Amidated low methoxy (LM) pectin (GENU1

pectin type LM-104 AS-FS; degree of esterification(DE)¼ 28% and degree of amidation (DA)¼ 20%)was a generous gift from CPKelco (Lille Skensved,Denmark). Resveratrol (99.12% purity, fine crys-talline powder) and calcium chloride dihydratewere purchased from Shaanxi Sciphar Bio-technology Co., Ltd. (Xi’an, China) and Merck(Darmstadt, Germany), respectively. Sodiumhydroxide, sodium phosphate monobasic,

NAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 2, FEBUARY 2010

842 DAS AND NG

Tween1 80, and Pectinex1 Ultra SP-L (pectinase/pectinolytic enzyme from Aspergillus aculeatus)were obtained from Sigma (St. Louis, MO). HPLCgrade methanol and acetonitrile (Tedia Company,Fairfield, OH), purified water (18.2 MV cm at258C from Millipore Direct-Q1 ultra-pure watersystem, Billerica, MA) were used throughout thestudy. Disodium hydrogen phosphate anhydrousand monobasic potassium phosphate were pur-chased from Fluka (Steinheim, Germany). Allmaterials were used as received.

Formulation of Pectinate Gel BeadsContaining Resveratrol

Ca-pectinate beads were prepared using pre-viously reported iontophoretic gelation methodwith slight modification.15,18,19 Briefly, pectin wasdissolved in deionized water. Resveratrol wasthen added to the pectin solution and homoge-neously dispersed by a homogenizer. Air bubbleswere removed from the dispersion by sonicationon a bath sonicator. The pectin–resveratrolmixture was then dropped slowly (1 mL min�1)into gently agitated cross-linking solution (cal-cium chloride aqueous solution) through the bluntend of a 25-G needle from 5 cm distance at roomtemperature (RT). The beads formed were allowedto stand in the solution for specified time intervalwith gentle agitation, separated, washed withdistilled water, and dried with various conditionsmentioned for individual formulations.

The effects of different formulation variables onbead size, bead shape, moisture content, waterloss during drying, calcium content, resveratrolloading and entrapment, swelling and erosionbehavior, and resveratrol retention patterns wereevaluated by varying cross-linking solution pH,cross-linking agent concentration, cross-linkingtime, drying condition, pectin concentration, andpectin to resveratrol ratio (resveratrol concentra-tion). All batches were prepared in triplicate.

Formulation Design

Based on results from preliminary experiments,beads were prepared by varying:

(a) C

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alcium chloride concentration (2.5, 5, 10,and 20%, w/v), while keeping pectin concen-tration (5%), cross-linking time (0.5 h),cross-linking solution pH (1.5), pectin to

L OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 2, FEBUARY 2010

resveratrol ratio (3:1), and drying condition(RT) constant.

(b) C
ross-linking solution pH (1.5, 3.5, and5.5), while keeping pectin concentration(5%), calcium chloride concentration (10%),cross-linking time (0.5 h), pectin to resver-atrol ratio (3:1), and drying condition (RT)constant.
(c) C
ross-linking time (0.25, 0.5, 2, and 24 h),while keeping pectin concentration (5%),calcium chloride concentration (10%), cross-linking solution pH (1.5), pectin to resver-atrol ratio (3:1), and drying condition (RT)constant.
(d) D
rying condition (freeze-drying (FD), RT,37 and 508C), while keeping pectin concen-tration (5%), calcium chloride concentration(10%), cross-linking time (0.5 h), cross-linkingsolution pH (1.5), and pectin to resveratrolratio (3:1) constant.
(e) P
ectin concentration (3, 4, 5, and 6%, w/v),while keeping calcium chloride concentra-tion (10%), cross-linking time (0.5 h), cross-linking solution pH (1.5), pectin to resver-atrol ratio (3:1), and drying condition (RT)constant.
(f) P
ectin to resveratrol ratio (1:1, 2:1, 3:1, and4:1), while keeping pectin concentration(5%), calcium chloride concentration (10%),cross-linking time (0.5 h), cross-linkingsolution pH (1.5), and drying condition(RT) constant.
Beads were optimized based on the effects ofthese parameters on bead characteristics, entrap-ment, and retention of resveratrol in beads.

Characterization of Resveratrol-LoadedPectin Beads

The effects of formulation parameters on beadcharacteristics were investigated.

Morphological Study

Morphological examination (bead surface andcross section) of the resveratrol-free and resver-atrol-loaded beads was conducted using a JEOLscanning electron microscopy (JSM-5200) at anexcitation voltage of 20 kV. Pectinate beads werefixed on an aluminum stub and coated withplatinum (30 s) to a thickness of 2 nm (undervacuum by auto fine coater) and examined atdifferent magnifications.

DOI 10.1002/jps


Particle Size and Shape

The average diameter of 50 beads randomlyselected from each batch was determined beforeand after drying by microscopic method, usingan optical microscope (LEICA DM IL, Wetzlar,Germany). The length and breadth of each beadwere measured by precalibrated image analysisprogram (LAS EZ, version 1.4.0) after imageswere taken through a digital camera (LEICA EC3)connected with the microscope. The size of eachbead was calculated from the average of thesetwo dimensions18 and expressed as the meandiameter (mm)� standard deviation (SD). Themean diameters of beads before and after dryingwere compared.

The elongation ratio (ER) of the above-men-tioned beads was calculated to represent beadshape. ER is the quotient of length to breadth ofthe beads.18 An ER value of unity represents aperfect sphere while higher values representgreater elongation or more deviation from sphe-rical shape.

Fourier Transform Infrared Spectroscopy (FTIR)

Possible chemical interactions between resvera-trol and bead components were studied usinginfrared spectroscopy (FTIR). Infrared (IR) spec-tra of pure resveratrol, resveratrol-free pectinbead, and resveratrol-loaded pectin bead werecompared using FTIR spectrometer (PerkinElmerSpectrum 100 Series, Norwalk, CT). Pectin beadswere squashed and dried in a vacuum desiccatorfor 3 days to remove the moisture completely.Sample (�2 mg) was mixed with �198 mg dry KBr(FTIR grade, Aldrich, Steinheim, Germany). Themixture was ground into a fine powder usingmortar and pestle before compressing into a disc.Each disc was scanned at a resolution of 4 cm�1

over a wave number region of 400–4000 cm�1

using the FTIR spectrometer. The characteristicpeaks of IR transmission spectra were recorded.

Determination of Calcium Content in Beads

In order to evaluate the amount of calciumretained in the beads, �20 mg of weighed beadswas dissolved in 19 mL of 50 mM sodium phos-phate buffer (pH 7.4) containing 1% ethylenedia-minetetraacetic acid (EDTA). The solution wasacidified by adding 1 mL of concentrated hydro-chloric acid. The solution obtained was thencentrifuged at 10,000g for 10 min. Supernatantswere diluted to the calibration range and the


amount of calcium present was determined byatomic absorption spectroscopy (AAS; Perkin-Elmer AAnalyst 100 model). The beads wereanalyzed for its calcium content as a function ofthe formulation variables.

Determination of Weight Loss of Beads duringthe Drying Process

The weight loss of the beads during drying wasdetermined by gravimetric analysis. Fifty ran-domly selected beads from each batch wereweighed with an analytical balance with read-ability of 0.00001 g (Mettler Toledo, Greifensee,Switzerland) before and after drying. The meanweight loss (WL) was calculated using the fol-lowing equation:

WLð%Þ ¼ WW � WD

WW� 100% (1)

where WW is the initial weight of the beadsmeasured just after washing and WD is the weightof the beads after drying. Experiment wasperformed in triplicate for each batch.

Moisture Content

Moisture content of the beads was also deter-mined using gravimetric method. Fifty randomlyselected beads from each batch were driedcompletely at elevated temperature (608C) untilno further weight change was observed. Moisturecontent (MC) was calculated by the followingequation:

MCð%Þ ¼ WD � W

WD� 100% (2)

where WD is the weight of the beads after dryingand W is the weight of fully dried beads.Experiment was performed in triplicate for eachbatch and average was calculated.

Resveratrol Loading and Entrapment Efficiency

Resveratrol content in the beads was determinedspectrophotometrically (UV–Visible Spectrophot-ometer UV-1601, Shimadzu, Kyoto, Japan) at awavelength of 320 nm. Briefly, beads (�25 mg)were dissolved (Ca-pectinate matrix)/dispersed(resveratrol particles) in 5 mL of phosphate buffer(50 mM) by continuous stirring on a magneticstirrer. To ensure solubilization of poorly solubleresveratrol, 10 mL of methanol was added tothe system and mixed well on a magnetic stirrer.


844 DAS AND NG

The mixture was centrifuged at 10,000g toremove the pectin. The supernatant containingthe resveratrol was diluted to the calibrationrange (0.1–10mg mL�1) with methanol–watermixture (1:1). Beads prepared without resveratrolwere used as blank.

Resveratrol entrapment efficiency (EE) andresveratrol loading (L) of pectinate beads weredetermined using Eqs. (3) and (4), respectively(direct method)

EEð%Þ ¼ AQ

TQ� 100% (3)

where AQ is the actual quantity of drug present inthe beads (drug content) and TQ is the theoreticalquantity of drug (initial resveratrol loading doseduring the preparation of the beads)

Lð%Þ ¼ AQ

WP� 100% (4)

where WP was the weight of pectin used to preparethe beads.

For further confirmation, L and EE of the beadsfor resveratrol were also determined by theindirect method. In this method, an exact volume(6 mL) of pectin gel mixture containing resveratrolwas employed to prepare beads in 100 mL of cross-linking solution. The amount of resveratrol lost inthe cross-linking solution as well as washingsolution was assayed spectrophotometrically. EEand L of the beads for resveratrol were thencalculated by Eqs. (5) and (6), respectively:

EEð%Þ ¼ QT � QI

QT� 100% (5)

where QT is the amount of drug added initiallyduring the batch preparation and QI is the sum ofthe resveratrol content lost in the cross-linkingand washing solutions

Lð%Þ ¼ QT � QI

WP� 100% (6)

All experiments were performed in triplicate.

Solubility of Resveratrol in Simulated GI Fluidand in Presence of Surfactant

Resveratrol solubility was determined in simu-lated gastric fluid (SGF, pH 1.2 (USP30-NF25)),simulated intestinal fluid (SIF, pH 6.8 (USP30-NF25)), and in the presence of 0.2% (v/v) Tween-80.Excess resveratrol was added to the correspond-ing fluids; vortexed and kept on a water bath

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 2, FEBUARY 2010

shaker (100 rpm) for 1 day at 378C. The suspen-sion was filtered through a 0.22-mm membranefilter (Whatman Ltd., Kent, UK) to obtain a clearsolution. All samples were prepared in triplicate.The concentration of resveratrol in the fluids wasdetermined by HPLC assay as described in ourprevious work.8 Solubility of resveratrol in SGF wasundetectable (detection limit was 0.01mg mL�1),whereas solubility in SIF was found to be 0.11�0.01 and 0.72� 0.13mg mL�1 in the presence of0.2% (v/v) Tween-80.

Resveratrol Release Studies from Beads

As formulations in large quantity are difficult toprepare using laboratory-scale equipment andprocedures, it is inconceivable to conduct dissolu-tion experiments using the standard dissolutionapparatus mentioned in United States Pharma-copeia (USP), which requires relatively largeamounts of materials. In addition, there are otherreasons that prohibit the use of a standarddissolution apparatus for measuring drug releaseincluding: (i) shortage of ingredients, particularlypectin and resveratrol, (ii) the presence of a highvolume of the buffer which will adversely affectthe beads’ characteristics when small amount ofbeads are used (limited by factor (i)) in standarddissolution apparatus, and most importantly (iii)the marginal solubility of resveratrol in SIF/SGF,release of resveratrol (in SIF/SGF) in standarddissolution apparatus may not be observed even inthe presence of a high amount of surfactant (0.2%,v/v, Tween-80) (addition of very high amount ofsurfactant was not at all feasible as a high amountof surfactant may have a deleterious effect on therelease characteristic of the beads). Therefore, wedevised an alternative method in which only smallamounts of beads are needed to measure drugrelease and this method precludes measurementof drug released (either in dissolved or particulateform) in the release media (SIF/SGF). In thismethod, we estimated the amount of resveratrolremaining in the intact beads after each timepoint instead of measuring the drug released inthe media. This is because much of the drug is inparticulate form (rather than dissolved form) dueto the poor aqueous solubility of resveratrol andhence will impede the proper quantification.

Briefly, resveratrol-loaded beads were weighed(�25 mg) into screw cap glass test tubes andsuspended in 37� 0.28C releasing medium(10 mL). Separate tubes were used for each time

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point. The tubes were kept at 37� 0.28C withcontinuous stirring on shaking water bath(100 rpm). At each time point, 500mL releasemedia was replaced with the same volume of freshrelease media. At selected time intervals, intactbeads were separated from the specified tubes.The beads were dissolved/dispersed in 5 mL PBS(pH 7.4, 50 mM) and fully dissolved by theaddition of methanol (10 mL). The mixture wasmixed and centrifuged (10,000g) for 10 min. Thesupernatant was collected, diluted with metha-nol–water (1:1) to the calibration range. The drugcontent in the supernatant (or remaining in thebeads) was determined spectrophotometrically(UV–Visible Spectrophotometer UV-1601, Shi-madzu) at 320 nm. Beads devoid of resveratrolwere used as a control.

SIF (pH 6.8) was used as release mediathroughout the bead optimization process. Opti-mized beads were also tested in SIF (pH 6.8)containing 300 PG Pectinex1 Ultra SP-L (simu-lating the colonic environment), and in SGF(pH 1.2) to investigate resveratrol release fromthe bead formulation in colonic and gastricconditions, respectively.

Percentage of resveratrol remaining in theintact beads (in comparison to the initial resver-atrol amount in the beads) was plotted againsttime. Times corresponding to 25%, 50%, and 75%resveratrol retention in the beads (T25, T50, andT75) during release study were then determined.All experiments were performed in triplicate.

Kinetics of Drug Release

The drug release kinetics from the beads wereinvestigated by fitting the drug release data intozero order and Higuchi’s dissolution models. Thezero-order equation is expressed as

Q ¼ Q0 � k0t (7)

where Q is the percentage of drug remaining attime t (h), Q0 is the percentage of drug at t¼ 0 h,and k0 is the zero-order release rate constant.

A simplified Higuchi’s equation can be obtainedby plotting the percent drug released versussquare root of time as follows:

Qt ¼ kffiffit

p(8)

in which Qt is the percentage of drug released attime t (h) and k is the Higuchi’s release rateconstant. The original equation described by


Higuchi for drug release from an inert matrix is

Qt ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiD"ð2A � "CSÞCSt

t

r(9)

where Qt is the amount of drug released per unitarea of exposed surface of the matrix, D is thediffusion coefficient of the drug in the matrix, eis the total porosity of the matrix after the drughas been extracted, A is the amount of drug inthe matrix, CS is the solubility of the drug inthe polymeric matrix, t is the time, and t is thetortuosity.

Experimental drug release data were fitted intothe dissolution models and the appropriate modelwas selected after linear regression analysis.R2> 0.95 was set for linearity. The release rateconstants were calculated from the slope of eachstraight line of best-fit model.

Swelling–Erosion Behavior of the Beads

Preweighed (�25 mg) dry beads were placed inthe glass test tube containing enzyme-free SIF(pH 6.8) at 37� 0.28C. The test tubes were placedin the shaking water bath (37� 0.28C) at 100 rpm(exactly the same condition as in release study).One test tube was assigned to each time point. Thebeads were removed at every time point up to 6 h,blotted with filter paper to remove the excesswater, and weighed. Optimized beads were alsotested in SIF (pH 6.8) containing 300 PGPectinex1 Ultra SP-L, and in SGF (pH 1.2). Theswelling–erosion ratio (SER) was calculated bythe following equation:

SERð%Þ ¼ WT � W0

W0� 100% (10)

where WT is the weight of the beads at the giventime point, and W0 is the initial weight of the drybeads. Positive SER and upward trend denoteoverall swelling and weight gain of beads withwater absorption, while negative SER and down-ward trend demonstrate the erosion and/or drugrelease of beads. Each determination was per-formed in triplicate and the results were aver-aged. Percentage SER was plotted against time todepict SER profile of the beads in SIF.

Enzymatic Degradation

The beads prepared with optimized formulationparameters were incubated in SIF (pH 6.8) with orwithout 300 PG pectinase enzyme for 3 h. Subse-


846 DAS AND NG

quently, the beads were separated from the mediaand lyophilized. JEOL scanning electron micro-scopy (JSM-5200) was used to examine theappearance of the enzyme-treated beads andcompared with the nonenzyme-treated beads.Similar scanning electron microscopy (SEM)parameters to those of the morphological studywere used in this study.

Stability

The optimized beads (�100 mg) were stored atthree different conditions: cold (48C), RT, andaccelerated temperature (408C) for 6 months.Three samples were stored for each time point.The samples are analyzed at predetermined timeintervals (0, 3, 7, 15, 30, 90, and 180 days) by UVspectrometer (UV–Visible SpectrophotometerUV-1601, Shimadzu) using the same proceduredescribed in release study.

Statistical Analysis

Statistical analysis of drug release parameterswas performed using the software Graph-PadPrism Version 2.00 (San Diego, CA). All experi-mental data were expressed as mean�SD. One-way ANOVA with the post hoc Tukey test wereperformed for analysis except for the release studyin different release media after 6 h, where two-tailed paired t-test was performed. Statisticalsignificance was set at p< 0.05.

RESULTS AND DISCUSSION

Preparation of Ca-Pectinate Bead

In general, pectin (i.e., LM pectin) that has morefree carboxylic group (DE <50%) forms a morerigid gel structure than when most of the carboxylgroups are in esterified form (i.e., HM pectin).12

Furthermore, amidated pectins are more prone toreact with calcium ion than nonamidated pectinas hydrophilicity of pectin is reduced in thepresence of amide group which increases its gel-forming ability. In addition, hydrogen bondingbetween amide groups also leads to increased gelstrength.20 These interactions in amidated LMpectin allow consolidating cross-linking betweenpectin chains leading to the formation of a more


compact Ca-pectinate network than nonamidatedand/or HM pectin. Generally, LM pectins aresoluble at high pH; but calcium binding to pectinreduces the solubility and induces noncovalentassociations of the carbohydrate chains. Thesewere the reasons behind selecting amidated LMpectin for the formulation development.

In addition, several kinds of delivery systemshave been studied for colon-specific delivery, suchas beads, matrices, tablets, capsules, micro-, andnanoparticles. Multi-particulate systems provedto be better than single unit dosage forms due totheir reproducibility and predictable GI transittime, more reliable drug release profile, and lesslocal irritation.15 Further, multiple unit dosageforms of enzyme specificity quickly spread outupon their arrival to the colon and due to anenhanced surface area being exposed to theenzymes, rapid drug release occurs at the colon.21

Indeed, some researchers have previouslyexploited Ca-pectinate beads (multi-particulatesystem) as colon-specific drug carriers.13,15,16,22,23

In the present study, when the highly waterinsoluble resveratrol was homogeneously dis-persed into the aqueous solution of pectin andsubsequently added dropwise to a counterionsolution of calcium, gelled sphere instantaneouslyformed by ionotropic gelation method. Ionic inter-action between the negatively charged carboxylicgroups on pectin molecules and the positivelycharged divalent calcium ions led to intermole-cular cross-linking (known as ‘‘egg-box’’ conforma-tion24) and formation of gelled spheres. Theformulated beads were spherical in shape(Fig. 1A and B) and easily prepared without anysophisticated instruments.

Morphology

SEM images of both blank and resveratrol-loadedCa-pectinate beads are shown in Figure 1A–E. At75� magnification, a smooth surface topographywas observed for the blank bead (Fig. 1A). Thetopology of the resveratrol-loaded bead changed toa rough and rugged surface, which was noticedeven at lower magnification (35�) (Fig. 1B).Analyses of SEM pictures of both surface andcross-section of the drug-loaded beads taken athigher magnifications (500� and 1000�) revealedthat the drug crystals are embedded in the beadmatrix (Fig. 1C–E). Similar findings werereported for pectin bead loaded with otherdrugs.13

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Figure 1. Scanning electron micrographs of blank bead surface (75�) (A), surface ofresveratrol-loaded bead (35�) (B), surface of resveratrol-loaded bead (500�) (C), surfaceof resveratrol-loaded bead (1000�) (D), and cross-section of resveratrol-loaded bead(1000�) (E). Magnifications corresponding to each figure are presented in parentheses.


Particle Size and Shape

The mean diameter and ER of the dried andundried beads are listed in Table 1. The meandiameter ranges from 0.97 to 1.22 mm for thedried beads (except lyophilized bead), and >2 mmfor the undried beads (Tab. 1). The size of thebeads formed generally depends on the diameter(25 G) of the needle used to prepare the beads.Because a 25-G needle was used throughout the


experiment, it is obvious that other formulationvariables were affecting the particle size of thedried beads. Augmentation of particle size wasobserved with increased calcium chloride concen-tration in the cross-linking medium. This can beexplained by increased retention of calcium whichleads to higher water retention in beads (Tab. 2)due to the hygroscopic character of calciumchloride.22 However, other researchers demon-strated an inverse relationship between the


Table 1. Particle Size and Shape, and Weight of the Beads

Variables Values

Size (mm�SD),n¼ 50 Beads

Shape (ER�SD),n¼ 50 Beads

Weight of Beads(mg�SD), n¼ 50 Beads

Wet Beads Dry Beads Wet Beads Dry Beads Wet Beads Dry Beads

Calcium 2.5% 2192.82� 90.79 976.41� 74.46 1.19� 0.05 1.13� 0.08 304.50� 9.01 25.42� 1.05chloride 5% 2202.51� 83.72 997.42� 38.45 1.02� 0.02 1.12� 0.09 306.63� 9.17 28.79� 0.26concentration 10% 2211.28� 66.23 1077.77� 72.97 1.07� 0.05 1.06� 0.06 309.87� 3.93 32.62� 0.77

20% 2218.93� 42.58 1217.23� 44.03 1.03� 0.02 1.16� 0.08 315.38� 8.75 47.59� 0.67Cross-linking pH 1.5 2211.28� 66.23 1077.77� 72.97 1.07� 0.05 1.06� 0.06 309.87� 3.93 32.62� 0.77

3.5 2240.21� 53.13 1100.12� 78.14 1.05� 0.03 1.09� 0.05 314.43� 5.10 33.75� 0.415.5 2264.43� 74.28 1148.68� 83.85 1.03� 0.01 1.14� 0.13 326.00� 9.41 35.95� 0.23

Cross-linking 0.25 2198.07� 184.64 1036.84� 28.06 1.10� 0.08 1.12� 0.06 306.30� 8.01 27.13� 0.28time (h) 0.5 2211.28� 66.23 1077.77� 72.97 1.07� 0.05 1.06� 0.06 309.87� 3.93 32.62� 0.77

2 2245.01� 106.75 1078.13� 63.21 1.03� 0.02 1.12� 0.07 314.57� 2.40 35.79� 1.4224 2288.43� 94.18 1081.94� 67.88 1.17� 0.08 1.08� 0.05 328.30� 5.98 39.19� 0.92

Drying RT 2211.28� 66.23 1077.77� 72.97 1.07� 0.05 1.06� 0.06 309.87� 3.93 32.62� 0.77378C 2211.28� 66.23 1052.19� 49.14 1.07� 0.05 1.16� 0.05 309.87� 3.93 31.99� 0.32508C 2211.28� 66.23 1005.51� 30.26 1.07� 0.05 1.19� 0.11 309.87� 3.93 26.26� 0.44FD 2211.28� 66.23 1513.36� 131.24 1.07� 0.05 1.22� 0.13 309.87� 3.93 24.03� 0.10

Pectin 3% 2194.99� 142.03 1057.04� 75.52 1.13� 0.09 1.41� 0.15 315.03� 5.12 33.42� 0.87concentration 4% 2161.47� 144.16 1029.66� 84.62 1.11� 0.05 1.11� 0.10 312.47� 5.85 33.06� 0.20

5% 2211.28� 66.23 1077.77� 72.97 1.07� 0.05 1.06� 0.06 309.87� 3.93 32.62� 0.776% 2181.83� 118,47 1075.93� 105.13 1.04� 0.03 1.10� 0.08 307.57� 9.53 31.97� 0.44

Pectin:resveratrol 1:1 2231.10� 50.66 1123.50� 33.85 1.06� 0.04 1.17� 0.10 374.37� 9.25 42.54� 2.172:1 2225.82� 54.17 1099.74� 98.19 1.04� 0.03 1.10� 0.07 316.67� 1.42 35.85� 0.393:1 2211.28� 66.23 1077.77� 72.97 1.07� 0.05 1.06� 0.06 309.87� 3.93 32.62� 0.774:1 2210.37� 49.92 1020.58� 45.42 1.05� 0.03 1.06� 0.04 299.3� 5.20 31.49� 0.71

Blank — 2173.64� 68.86 971.20� 49.64 1.02� 0.01 1.09� 0.06 277.13� 7.8 28.76� 0.38

848 DAS AND NG

particle size and calcium chloride concentrationdue to pronounced gel bead shrinkage caused bysyneresis.13 Nevertheless, the cross-linking timeresulted in an insignificant change in the dia-meter of beads. An inverse relationship betweenthe particle size and drying temperature howeverwas noted. This can be explained by lowermoisture content due to higher drying tempera-ture. On the other hand, the size of the lyophilizedbeads was bigger than all other beads. It ispossible that during FD, moisture evaporatesfrom the beads without affecting the beadstructure. Slight enhancement of particle sizewas also observed with increased resveratrolconcentration. This is anticipated because of lowerpolymer to drug ratio and higher moisturecontent. Diminution of pH of the cross-linkingsolution created particles with smaller size. Aprobable reason is that at reduced pH, nonionicinteraction occurred along with ionic interactionbetween pectin chains, which led to small andcompact beads. This phenomenon will be dis-cussed later in detail. Higher moisture content at


high pH is also a probable reason behind enhancedparticle size (Tab. 2). Bead size was found to beindependent of the pectin concentration. Further,beads without drug were much smaller than drug-loaded beads. From ER, it can be seen that almostall beads (except dried beads prepared with 3%pectin) were spherical in shape (ER< 1.25)(Tab. 1).

Fourier Transform Infrared Spectroscopy (FTIR)

The FTIR spectra of pure resveratrol, blank, andresveratrol-loaded Ca-pectinate bead are depictedin Figure 2. FTIR absorption spectrum of resver-atrol showed a typical trans olefinic band at966 cm�1 and three characteristic intense bandsat 1383, 1585, and 1607 cm�1, corresponding toC–O stretching, C–C olefinic stretching, and C–Caromatic double-bond stretching. All these majorpeaks are present in the resveratrol-loaded Ca-pectinate bead, indicating that resveratrol wasnot chemically modified when formulated intoCa-pectinate beads.

DOI 10.1002/jps

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DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 2, FEBUARY 2010


Figure 2. FTIR spectra of blank Ca-pectinate bead (A), resveratrol-loaded Ca-pectinatebead (B), and pure resveratrol (C).

850 DAS AND NG

Determination of Calcium Content in Beads

Various formulation parameters have potentialimpact on the amount of calcium retained inthe beads (Tab. 2). Our findings indicated thatcalcium content per mg of beads increased withcalcium chloride concentration in cross-linkingsolution, cross-linking solution pH, and cross-linking time; on the other hand, content decreasedwith pectin concentration and resveratrol con-centration. Increase in calcium chloride concen-tration in cross-linking solution supplies morecalcium for gelation of beads that led to enhancedcalcium retention in beads. This observation is inline with a previous work.22 Similarly, increasingcross-linking time allows calcium more time toreact with the beads, which ultimately increasesits entrapment in the beads. It should be notedthat a rapid increase in calcium entrapment wasonly observed in the beginning phase (i.e., from0.25 to 0.5 h) of the process. This can be explainedby the mechanism of Ca-pectinate network for-mation, which normally occurs very quickly in atwo-step fashion: formation of dimers followedby their aggregation. Formation of dimers, whichprecedes aggregation of dimers, requires a hugeamount of calcium ions.20 By contrast, aggrega-tion of dimers occurs if more calcium is availablebut it requires less calcium ions than the initialdimer formation.22

Ionization of carboxylic group on the pectinmoiety is expected to be subdued after suppres-


sing the pH of the cross-linking medium. Thus,less calcium is bound with the pectin chain at lowcross-linking solution pH, which repressed thecalcium entrapment. As the concentration ofresveratrol increased, pectin to resveratrol ratiodecreased. So, at higher resveratrol concentra-tion, less pectin is available for cross-linking,which could probably explain lower calciumretention. More interestingly, augmentation ofcalcium entrapment was observed at lower pectinconcentration, which was probably due to easypenetration of calcium chloride into the dilutepectin drops during gelation. As a result, it can beanticipated that more calcium would be present asfree calcium chloride in the beads with low pectinconcentration. Moreover, calcium retention wasfound in the order of RT< 378C< 508C<FD.Weight of the beads decreased with dryingtemperature and was lowest in case of freeze-dried beads. So, higher amount of calciumretained per mg of beads was rational for lowerweighing beads.

Weight Loss During Drying

Weight of 50 wet as well as dry beads slightlyincreased with increasing cross-linking solutionpH, calcium chloride concentration, cross-linkingtime, resveratrol concentration, and with decreas-ing pectin concentration and drying temperature(Tab. 1). After drying, beads lost 88–92% of their

DOI 10.1002/jps


initial weight depending on the formulationconditions (Tab. 2). Weight loss during dryingslightly increased with decreasing calcium chlor-ide concentration, cross-linking solution pH, andcross-linking time; as well as with increasingpectin concentration, resveratrol concentration,and drying temperature (FD> 508C). In mostcases, this increase was not significant.

Moisture Content

The moisture contents corresponding to each typeof formulations are given in Table 2. The moisturecontent of the beads was clearly correlated withthe type of drying method undertaken. Only smallamount of moisture (<2%) was detected in freeze-dried beads and those dried at elevated tempera-ture (508C); with the lowest moisture entrapmentwitnessed in case of lyophilized beads. Very highmoisture content was observed in beads preparedwith 20% calcium chloride as cross-linking solu-tion. One earlier report already demonstratedthat only small amount of calcium is needed forCa-pectinate network formation with the majorityof calcium remaining as free calcium chloride inCa-pectinate beads. This phenomenon impartshygroscopicity to the beads that induces retentionof water.22 The moisture content of dried beadswas also found to increase linearly with increasingpH of cross-linking solution, cross-linking time,and resveratrol concentration. By contrast, decreas-ed pectin concentration causes increase in beadsmoisture content. In all cases except resveratrolconcentration and drying condition, calciumretention was related to moisture content.

Entrapment Efficiency and Loading

EE and L of each formulation type are depicted inTable 2. Instantaneous gelation of pectin allowedeasy entrapment of resveratrol in Ca-pectinatebeads. Indeed, very high resveratrol entrapmentwas found in all cases (>97.7%). We reasoned thatpoor aqueous solubility of resveratrol led to suchhigh percentage of drug entrapment in the beads.Nevertheless, a small increase in entrapmentand loading was observed in the present studywith increasing calcium chloride concentration,pectin concentration, resveratrol concentration,and with decreasing cross-linking solution pH,cross-linking time. As a matter of fact, drugconcentration increment generally leads to lowerEE due to reduced polymer to drug ratio.17 On the


other hand, EE and L slightly increased withincreasing resveratrol concentration. This isprobably due to the lower resveratrol solubilityin the cross-linking solution. At constant tem-perature, resveratrol reaches saturation in thesolution. Hence, when more resveratrol was addedinto the pectin solution, only limited resveratrolcame out from the beads to the cross-linkingsolution, which consequently led to higher entrap-ment and loading of resveratrol.

Effect of Formulation Parameters on ResveratrolRetention and SER

The following sections discuss the effects ofdifferent formulation variables on the resveratrolretention profiles, release parameters, and SERof all beads in SIF. Drug retention profiles arerepresented by plotting resveratrol retention inbead against time (Fig. 3), whereas SERs (%) areplotted against time to indicate the swelling ofbeads in SIF pH 6.8 at each time point (Fig. 4).Further, release parameters (T75, T50, T25) arecited in Table 3.

Effect of Calcium Chloride Concentration

Because calcium chloride concentration at <2.5%or >20% produced either irregular-shaped beadsor beads that adsorbed considerable amount ofmoisture, calcium concentration was kept between2.5% and 20% in the present study. As shown inFigure 3A and Table 3, the retention of resveratrolin beads within this range was clearly dependentupon the calcium chloride concentration. Beadretention of resveratrol increased with elevatedconcentration of calcium; although in case of 20%calcium, resveratrol leaked out very quickly after4 h. In particular, the release of resveratrol wassignificantly faster at 2.5% and 5% calciumchloride when compared to 10%. In addition, thekinetic profile of beads prepared in differentcounterion concentrations had similar featuresbut shifted to lower drug retention with lowcalcium chloride concentrations. This is in linewith previous works that showed drug releasesubdued at higher cross-linking agent concentra-tion.13,16,25 The effect of counterion concentrationon the release parameters (T75, T50, and T25) wasshown in Table 3. Retention of resveratrol wasmore pronounced when a higher concentration ofcalcium chloride was used for cross-linking. Anexception to this was observed for beads prepared


Figure 3. Effect of calcium chloride (CaCl2) concentration (A), cross-linkingsolution pH (B), cross-linking time (C), drying condition (D), pectin concentration (E),pectin to resveratrol ratio (P:R) (F), and release medium (G) on retention of resveratrolwithin the beads. The beads were incubated in SIF (release medium) in case of (A)–(F).Data are presented as mean�SD. p< 0.05.

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 2, FEBUARY 2010 DOI 10.1002/jps

852 DAS AND NG

Figure 4. Effect of calcium chloride concentration (A), cross-linking solution pH (B),cross-linking time (C), drying condition (D), pectin concentration (E), pectin to resver-atrol ratio (F), and release medium (G) on SER of the Ca-pectinate beads. The beads wereincubated in SIF (release medium) in case of (A)–(F). Data are presented as mean�SD.p< 0.05.

DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 2, FEBUARY 2010


Table 3. Resveratrol Retention Within the Beads

Variables Values Higuchi R2 Zero-Order R2

Timea

T75 (h) T50 (h) T25 (h)

Calcium chlorideconcentration

2.5% 0.9554 0.8849 1.76� 0.07b 3.53� 0.05b 5.90� 0.05b

5% 0.9720 0.9233 2.18� 0.17 4.27� 0.15 7.04� 0.0810% 0.9582 0.9276 2.72� 0.11 5.01� 0.09 8.00� 0.2420% 0.8887 0.9346 — — —

Cross-linking pH 1.5 0.9582 0.9276 2.72� 0.11c 5.01� 0.09c 8.00� 0.24c

3.5 0.9709 0.9312 2.28� 0.11 3.99� 0.17 6.17� 0.325.5 0.9761 0.9208 1.59� 0.16 3.09� 0.11 5.07� 0.07

Cross-linking time (h) 0.25 0.9700 0.9322 2.04� 0.06d 3.73� 0.09d 5.91� 0.14d

0.5 0.9582 0.9276 2.72� 0.11 5.01� 0.09 8.00� 0.242 0.9564 0.9190 2.73� 0.26 5.07� 0.13 8.13� 0.3124 0.9265 0.9458 — — —

Drying RT 0.9582 0.9276 2.72� 0.11 5.01� 0.09 8.00� 0.24k

378C 0.9557 0.9257 2.79� 0.16 5.13� 0.19 8.18� 0.26508C 0.9701 0.9555 3.06� 0.19 5.60� 0.29 8.89� 0.42FD 0.9648 0.8922 0.78� 0.14e 2.72� 0.20e 5.85� 0.22e

Pectin concentration 3% 0.9682 0.9028 1.89� 0.03f 3.69� 0.03f,j 6.10� 0.05f

4% 0.9861 0.9431 2.14� 0.23g 4.05� 0.20g 6.56� 0.13g

5% 0.9582 0.9276 2.72� 0.11 5.01� 0.09 8.00� 0.246% 0.9516 0.9105 2.60� 0.10 4.90� 0.13 7.93� 0.28

Pectin:resveratrol 1:1 0.9506 0.8808 1.65� 0.14h 3.04� 0.11h 4.85� 0.04h

2:1 0.9623 0.9014 2.04� 0.08 4.00� 0.09 6.63� 0.133:1 0.9582 0.9276 2.72� 0.11 5.01� 0.09 8.00� 0.244:1 0.9573 0.9349 2.86� 0.09 5.24� 0.14 8.34� 0.44

Enzyme — 0.9945 0.9898 1.75� 0.02i 3.17� 0.05i 5.01� 0.14i

aData are presented as mean�SD, N¼3.bp< 0.05 among 2.5%, 5%, and 10% calcium chloride concentration.cp< 0.05 among pH 1.5, 3.5, and 5.5.dp<0.05 between 0.25 and 0.5 h, and between 0.25 and 1 h.ep< 0.05 between RT and FD, between 378C and FD, and between 508C and FD.fp< 0.05 between 3% and 5% pectin, and between 3% and 6% pectin.gp<0.05 between 4% and 5% pectin, and between 4% and 6% pectin.hp<0.05 among 1:1, 2:1, and 3:1 pectin to resveratrol ratio.ip<0.05 between enzyme and nonenzyme treatment.jp<0.05 between 3% and 4% pectin.kp< 0.05 between RT and 508C.

854 DAS AND NG

with 20% calcium chloride which showed anopposite trend at T25. Huge difference in SERwas detected when different calcium concentra-tion was used (Fig. 4A). In general, SER decreasedwith increasing concentration of calcium. At lowconcentration, the calcium cation leads to dimerformation with pectin chain, whereas at highconcentration, the number and/or strength ofcross-links between pectin and counter ionsincreased as well. Furthermore, under suchcondition, aggregation of initial dimmers alsooccurs, which gives rise to a greater degree ofcross-linking.22 In summary, higher drug reten-tion and lower SER were observed due to highergel strength at high calcium concentrations.Based on this finding, the optimal concentration


for subsequent study was chosen to be 10%calcium chloride.

Effect of Cross-Linking Solution pH

As shown in Figure 3B, drug retention decreasedwith increasing pH. Beads formulated at pH 1.5showed more pronounced resveratrol retentionwhile T75, T50, and T25 are lower in the case of thehigher pH (Tab. 3). Chambin et al.16 demonstratedsimilar effects of cross-linking solution pH onpectinate beads prepared with either calcium orzinc ion. They noticed repression of drug release atlower pH, though the effect was more pronouncedin Zn-pectinate beads. Our data (Fig. 4B) alsoshowed SER increased with increasing pH. This is

DOI 10.1002/jps


because acidic counterion solution can lead toformation of a stronger matrix by forming a strongpectinate counterion network.26 Several authorsreported that in addition to ionic interaction withcalcium cation, polymeric chains of pectin in ‘‘egg-box’’ model of Ca-pectinate gel are also involvedwith different nonionic interactions such as hydro-phobic interaction and hydrogen bonding.27–29

Though nonionic interactions are more obviouswith high-methoxy pectin gels, Lootens et al.29

reported that stronger gels for amidated pectinwere formed at pH below 3 (lower than pectinintrinsic pKa value). According to them, low pHdecreases solubility of the pectin chain andsuppresses intermolecular electrostatic repulsionby protonation of carboxyl groups that allows thecarbonyl groups to act as hydrogen bond donors.In addition, lowering the charges of the polymerchains leads to promote conformational orderingand intermolecular association by nonionic inter-actions.29 It has been previously demonstratedthat the chain would be extended by intermole-cular charge–charge repulsion at high pH valueswhere most of the carbonyl groups are ionized,allowing conformations close to the extendedtwofold structure (two residues per turn of thehelix).28 By contrast, reduction in pH at fixedtemperature reduced charge density and pro-moted conformational transition from the twofoldto the more compact threefold helical conforma-tion of the polygalacturonate chains of pectin inthe Ca-pectinate network.28

Effect of Cross-Linking Time

The release profiles of resveratrol from beadsprepared with various cross-linking times areshown in Figure 3C. It can be seen from thesegraphs that the retention of resveratrol within thecalcium pectinate beads after incubation in SIFdepends on the cross-linking time used to preparethe beads. Drug retention within the beads andthe resveratrol release parameters (T75, T50, andT25) were lowest when the cross-linking time wasthe shortest (0.25 h) (Fig. 3C and Tab. 3, respec-tively), whereas insignificant differences in drugretention were observed between the beadsprepared at cross-linking times of 0.5 and 2 h,but more drug retentions were noticed in thesebeads than the beads prepared at 0.25 h cross-linking time. In contrast, when longer cross-linking time (24 h) was employed to prepare thebeads, drug retention was much higher in the first3 h whence drug retention dropped rapidly after-


wards (T75 was highest; T50 and T25 were higherthan the beads prepared at 0.25 h cross-linkingtime but lower than the beads prepared at 0.5 and2 h cross-linking time). Other researchers havealso reported an inverse relationship betweendrug release and cross-linking time (i.e., directrelationship between drug retention and cross-linking time).25,30,31 The observed phenomenaindicate that SER is clearly dependent on thecross-linking time (Fig. 4C). In our findings and asexpected, 24 h cross-linking time gave the lowestand 0.25 h gave the highest (only for the first 4 hwhence SER slowly dropped afterwards) SER. Asa comparison, SER was the same for the first 4 hfor beads prepared with 0.5 and 2 h cross-linkingtime. After 4 h, the trend of SER for these beadswas 0.5> 2> 0.25> 24 h. The observed SER anddelayed release pattern can be explained by thepromotion of cross-links between pectin chainsand calcium ions with respect to time. Corre-sponding to the higher calcium concentration,higher cross-linking time also probably helpedin the aggregation of primary dimmers, which,thereby, causes greater gel strength. In our study,the optimized cross-linking time was found to be0.5 h.

Effect of Drying Condition

The effect of drying condition on resveratrolrelease is illustrated in Figure 3D. Drug retentionpattern was clearly influenced by the dryingmethod used to prepare the beads. Resveratrolrelease was markedly more rapid from thelyophilized beads than beads dried by otherprocedures. FD produced spherical porous beadswhich were fragile and easily breakable uponhandling. This explains why drug retention (T75,T50, and T25) profile of freeze-dried bead was verypoor (Tab. 3). The rapid degradation and higherSER (Fig. 4D) of freeze-dried product wereprobably explained by the higher porosity of thelyophilized beads. Lyophilized beads tend to floaton the release medium while other beads stayed atthe bottom of the tubes and degraded slowly.Therefore, freeze-dried beads were unsuitable asdelivery system. A significant slow release and lowSER was observed in case of beads dried at 508C.Drying at high temperature produced dense beadswith very low moisture content. This may be thecause of slower release and lower SER for beadsdried at higher temperature. By contrast, beadsdried at RT and at 378C appear to have nosignificant difference in their release properties.


856 DAS AND NG

Sriamornsak13 also noticed similar effect of dryingcondition on the beads. In short, the rank order ofSER as a function of drying conditions wasFD>RT> 378C> 508C.

Effect of Pectin Concentration

Because of its capability of bead formation,amidated low methoxy pectin was chosen toprepare resveratrol-loaded beads. Introductionof amide group to the pectin molecule causes itshydrophilicity to decrease, which increases itstendency to form gels (compared to conventionallow methoxy pectin).32 Our studies showed thatirregular-shaped beads were produced when <3%pectin concentration was used. By contrast, whena >6% pectin concentration was used, beads witha drop-like structure were formed (data notshown). Based on these observations, a pectinconcentration between 3% and 6% was chosen forsubsequent studies. As shown in Figures 3Eand 4E and Table 3, both drug retention capabilityand SER increased with increasing pectin con-centration (3–5%). By contrast, almost similarretention and SER was observed between 5% and6%. These observations led us to decide 5% pectinas the optimum concentration for resveratrolloading.

Effect of Pectin to Resveratrol Ratio

Figures 3F and 4F demonstrated the effect ofpectin to resveratrol ratio on drug retention andSER, respectively. The retention of resveratrol(T75, T50, and T25) for beads prepared withdifferent pectin to resveratrol ratio was in thegeneral order of 1:1< 2:1< 3:1< 4:1, whereas lasttwo showed insignificant difference in resveratrolretention pattern (Tab. 3 and Fig. 3F). Thisobservation was in accordance with the previousstudies, where the authors reported enhanceddrug release for beads with high drug encapsula-tion (high drug–carrier ratio).17,25 SER decreasedwith increasing resveratrol concentration. Thedecrease in resveratrol retention and SER pre-pared with high percentage of resveratrol canprobably be explained by the decrease in thepolymer to drug ratio. Because at high resveratrolconcentration, only little pectin would be availablefor drug retention and swelling.

Effect of Release Media on ResveratrolRetention and SER

From the above-discussed observations, the fol-lowing conditions were optimized to prepare


resveratrol-loaded pectin beads: pectin concentra-tion at 5%, calcium chloride concentration at 10%,cross-linking time of 0.5 h, cross-linking solutionpH at 1.5, pectin to resveratrol ratio of 3:1, anddrying at 508C. Figures 3G and 4G demonstratedthe effect of release media on drug retentionand SER, respectively. The beads incubated inSGF showed very high resveratrol retention(>97% even after 6 h) within the beads and verylow SER. This might be because of the stability ofthe Ca-pectinate network at low pH. Enzyme-specific degradation of optimized pectin bead wasthen investigated by addition of 300 PG pectinaseto the release medium and drug retention in beadwas compared between two different releasemedia (SIF, pH 6.8 with and without pectinase).The addition of pectinase allows us to better mimicthe colonic environment and to check the suit-ability of pectin beads for colon-specific drugdelivery. Relatively faster release of resveratrolfrom pectin beads was evident in SIF withpectinase than SIF alone, although the differencewas unfortunately not huge (Fig. 3G). The releaseparameters (T75, T50, and T25) were lower in thecase of enzymatic degradation than nonenzymaticdegradation (Tab. 3). Several authors investigatedCa-pectinate beads for specific targeting of drug tothe colon.13,16 SER study revealed that erosionexceeded swelling of the beads in presence ofenzyme (Fig. 4G).

Enzymatic Degradation

After 3 h incubation in the releasing media(SIF, pH 6.8, with or without enzyme), very smallpores were found scattered all over the beads (likehoneycomb) (Fig. 5A and B). This is because thebeads absorbed water when immersed in releas-ing media and shrunk upon lyophilization.13 Thisconclusion is supported by SEM data whichindicated a more porous bead structure after 3 hincubation in SIF with enzyme than withoutenzyme (Fig. 5A and B, respectively). It is plaus-ible that enzymatic degradation and subsequenterosion of the matrix led to formation of pores.These pores allowed resveratrol particles to comeout through the partially degraded matrix. Thesemorphological studies are indicative of homoge-neous degradation of the matrix by pectinolyticenzyme.

Release Kinetics

In the present study, we noticed that the drugrelease kinetics of most beads (except those

DOI 10.1002/jps

Figure 5. Scanning electron micrographs of surfaceof resveratrol-loaded bead after 3 h incubation in SIFcontaining 300 PG pectinase (350�) (A), and surface ofresveratrol-loaded bead after 3 h incubation in SIF(350�) (B). Magnifications corresponding to each figureare presented in parentheses.

Figure 6. The release of resveratrol from Ca-pecti-nate beads after incubation in SIF with and withoutpectinolytic enzyme, plotted as the cumulative percentresveratrol released versus square root of time. Datarepresent mean�SD.


prepared in 20% calcium chloride and cross-linkedfor 24 h) fit more appropriately with the Higuchimodel (Tab. 3) with an initial lag period of <1 h(the point where the extrapolation of the linierplot intersects the x-axis is the lag time). It ispossible that a certain degree of water penetrationis needed to loosen up the gel matrix before drugrelease can occur. In most cases, zero-orderkinetics was not obeyed. Other authors haveestablished Higuchi’s diffusion model for Ca-pectinate beads.17 Figure 6 shows the linearityof plots for the cumulative percent resveratrolreleased versus square root of time. However,although resveratrol release data more appro-


priately fit with Higuchi’s square root of timeequation, release kinetics in this case might notfollow the rules of Higuchi model. As resveratrolhas very low solubility (0.11� 0.01mg mL�1) inthe release media (SIF), most of the resveratrolparticles should not be dissolved in the releasemedia that penetrated into the bead matrix. Thus,there might not be diffusion of the dissolvedresveratrol molecules, which is the basis ofHiguchi’s diffusion model. Furthermore, Higu-chi’s model is applicable to the formulations thatare nonswelling and noneroding. But, preparedCa-pectinate beads showed both swelling anderosion behavior. Thus, Higuchi’s diffusion modelis not applicable for resveratrol-loaded Ca-pecti-nate beads. In our opinion, the release of drug isdue to three phenomena: (i) hydration of thematrix followed by (ii) swelling and erosion of thematrix followed by (iii) the release of resveratrolfrom the matrix as undissolved particles ratherthan diffusion of the soluble resveratrol. As theresveratrol particles fall off from the beads, poresare created in the matrix that allow more fluidpenetration into the matrix and most likely moreresveratrol particles fall off and the bead matrixerodes. When the gel layer is thick, the length ofthe pathway required to pass through for resver-atrol particles is crucial and it slows down thedrug release. In addition, the calcium pectinate iseven more sensitive to the erosion phenomenon ifthe ionic strength of the gel is low. However, a gelresulting from low methoxy pectin cross-linked


Figure 7. Stability of resveratrol in the optimized resveratrol-loaded Ca-pectinatebeads. Data represent mean�SD. p< 0.05 for the difference between 180 days with 0, 3,7, 15, and 30 days, p< 0.05 for the difference between 30 days with 0, 3, 7, and 15 days,p< 0.05 for the difference between 90 days with 0, 3, 7, 15, and 30 days, p< 0.05 forthe difference between 180 days with 0, 3, 7, 15, 30, and 90 days, #p< 0.05 for thedifference between 40 with 48C, and RT.

858 DAS AND NG

with calcium ions expressed less tendency toerode.

Stability

The stability profile of resveratrol in the optimizedbeads is presented in Figure 7. It can be seen thateven after 6 months, stability was >99.5% in caseof storage at 48C and RT; whereas �97.5% whenstored at accelerated condition (408C). Theseresults indicate desired stability of resveratrolinside Ca-pectinate beads.

CONCLUSIONS

As resveratrol is rapidly absorbed and quicklymetabolized at the upper GI tract, it becomesindispensable that a special dosage form bedeveloped to deliver the drug to the lower GItract, for the treatment of colon-specific diseasessuch as colorectal cancer, colonic inflammation,etc. (where it is potentially active). The presentstudy on resveratrol-loaded Ca-pectinate beads forsite-specific and sustained release drug deliveryhas underlined the importance of formulationparameters and process variables to amelioratethis purpose. Calcium entrapment in the beadswas dependent on different formulation variables,and the moisture content was clearly influenced


by the calcium retention in the prepared beads.The study also unveils that some formulationparameters had a vital influence upon SER andability of the beads to resist degradation in theupper GI tract, while others had minor effect. Ingeneral, resveratrol release data more appropri-ately fit with the cumulative percent resveratrolreleased versus square root of time plots. Resver-atrol was stable and highly entrapped inside thebeads. Though optimized beads showed prolongedrelease pattern, it is anticipated that additionalmodifications of the beads such as enteric coating,use of hardening agent, and complexation withother polymers are necessary to achieve sitespecificity to lower GI tract. We are currentlycontinuing our endeavor to achieve this goal.

ACKNOWLEDGMENTS

This work was partially supported through aNational University of Singapore AcademicResearch Fund R148-050-068-101 and R148-050-068-133 (K.-Y.N.) and NIH grant R21 CA115269-02 (K.-Y.N.).

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860 DAS AND NG

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DOI 10.1002/jps

resveratrol-loaded calcium-pectinate beads: effects of formulation parameters on drug release and...

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