research article sonication-based improvement of the...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 985259 11 pageshttpdxdoiorg1011552013985259

Research ArticleSonication-Based Improvement of the PhysicochemicalProperties of Guar Gum as a Potential Substrate for ModifiedDrug Delivery Systems

Siddique Akber Ansari1 Pietro Matricardi1 Claudia Cencetti1

Chiara Di Meo1 Maria Carafa1 Claudia Mazzuca2 Antonio Palleschi2

Donatella Capitani3 Franco Alhaique1 and Tommasina Coviello1

1 Department of Drug Chemistry and Technologies University ldquoLa Sapienzardquo 00185 Rome Italy2 Department of Sciences and Chemical Technologies University of Rome ldquoTor Vergatardquo 00133 Rome Italy3Magnetic Resonance Laboratory Annalaura Segre Institute of Chemical Methodologies CNR Research Area of RomeMonterotondo Stazione 00016 Rome Italy

Correspondence should be addressed to Tommasina Coviello tommasinacoviellouniroma1it

Received 4 April 2013 Accepted 26 June 2013

Academic Editor Rares Salomir

Copyright copy 2013 Siddique Akber Ansari et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Guar Gum is a natural polysaccharide that due to its physicochemical properties is extensively investigated for biomedicalapplications as a matrix for modified drug delivery but it is also used in the food industry as well as in cosmetics A commercialsample of Guar Gumwas sonicated for different periods of time and the reduction in the average molecular weight was monitoredby means of viscometric measurements At the same time the rheological behaviour was also followed in terms of viscoelasticityrange flow curves and mechanical spectra Sonicated samples were used for the preparation of gels in the presence of borate ionsThe effect of borax on the new samples was investigated by recordingmechanical spectra flow curves and visible absorption spectraof complexes with Congo Red The anisotropic elongation observed in previous studies with tablets of Guar Gum and borax wasremarkably reduced when the sonicated samples were used for the preparation of the gels

1 Introduction

It is well known that viscometric properties of macromole-cules are significantly affected by their molecular weight Theaim of this study was to examine the effect of the reductionof Guar Gum (GG) molecular weight for an appropriatemodulation of its flow and gelling properties accordingto specific needs and possible applications in the field ofpharmaceutics cosmetics and food industry Furthermorea reduced molecular weight should allow conjugation withhydrophobic moieties which should lead to self-assembledstructures suitable as systems for drug delivery and targetingas previously proposed with gellan gum which was sonicatedand chemically linked to prednisolone [1] Furthermorefollowing previous investigations carried out using anotherpolysaccharide (ie scleroglucan) [2] we studied the effectof borax crosslinking on the rheological properties of GG

solutions sonicated for different intervals of time and thecorresponding anisotropic swelling and elongation of tabletsobtained by compression of the GGborax system The helixconformation assumed by the GG chains in the presence ofborax [3] was tested to be stable even at high pH values bymeans of Congo Red complexation experiments The chosenpolysaccharide GG is a water-soluble galactomannan whichconsists of a linear 120573-D-(14)-mannose backbone irregularlysubstituted by uncharged 120572-D-(16)-linked galactose sidegroups [4] This seed gum generally recognised as safe(GRAS) because of its gelling viscosifying and thickeningproperties is used in pharmaceutics [5] for the stabilization ofemulsions and suspensions it is also employed in tablet man-ufacturing as a binder and disintegrating agent and for drugmicr-encapsulation Furthermore GG is suitable as a foodsupplement [6] It has been proposed in the therapy of hyper-cholesterolemia hyperglycemia and obesity [7] but also

2 BioMed Research International

in textile paper and paint industries as well as in cosmeticsAs pointed out most practical applications of GG are relatedto its rheological and gelling properties furthermore GG ispable to form hydrogels when crosslinked with glutaralde-hyde [8ndash11] and in the presence of borax [3] For the reductionof GG molecular weight the probe sonication approach wasused because in such case no chemicals are needed and suchaspect represents an important prerequisite for themarketingapproval of the obtained polymer

2 Materials and Methods

21 Materials GG was provided by CarboMer (USA) boraxwas a Carlo Erba (Italy) product and Congo Red (CR) aSigma Aldrich (USA) product For the sample preparationsdistilled water was always used except forNMR experimentswhere deuterium oxide (D

2O) (Cambridge Isotope Labora-

tories USA) was used All products and reagents were ofanalytical grade The Polysaccharide calibration kits SAC-10(Agilent Technologies UK) were used as pullulan standards

211 GG Purification A given amount of polymer was dis-solved in distilled water (polymer concentration C

119901= 05

wv) and then kept under magnetic and mechanical stirringat 60∘C for 24 h The obtained solution was exhaustivelydialysed at 7∘C against distilled water and then freeze-driedThe molecular weight cut-off of dialysis tubing was 12000ndash14000 From now on the sample purified by dialysis will becalled GG

212 Hydrogel and Tablet Preparation For the preparationof the tablets an appropriate amount of polymer (about200mg) was magnetically stirred in water for 24 h Then thecalculated amount (ie moles of borax = moles of repeatingunits of polymer) of 01M borax solution was added andthe system was left under magnetic stirring for 5min Theobtained sample (C

119901= 07 wv) was kept overnight at 7∘C

for gel setting and then freeze-dried Tablets were preparedfrom the freeze-dried sample with an IR die (Perkin Elmerhydraulic press) using a force of 50 kN for 30 s The weightof the GGborax tablets was 230 plusmn 10mg the diameter was130 plusmn 01mm and the thickness was 105 plusmn 002mm

22 Methods

221 Sonication In order to reduce the GG molecularweight a High Intensity Ultrasonic Processor (750 Wattmodel probe type sonicatormdashVibra CellmdashVC 750 Cole-Parmer USA) was used working at 20 kHz with a 65mmmicrotip applying an amplitude of 30 of the maximumpower supplied by the instrument corresponding to 20 wattsand pulser cycles of 30 s ON and 30 s OFF Aqueous solutions(50mL) of GG (05 (wv)) prepared in glass beaker werekept during sonication in an ice bath which allowed to keepan average temperature of the samples below 25∘C Theseexperimental conditions allowed to maintain the structuralcharacteristics of the polymeric chains as evidenced alsoin the case of other polysaccharides [2] All samples after

sonication were centrifuged for 20min at 5000 rpm and20∘C The centrifuge speed was selected after several testsin order to find out the minimum rpm value capable ofremoving the probe-tip residues from the polymer solutionsThe supernatant solutions were then dialysed and finallyfreeze-dried

222 Ultracentrifugation After sonication all samples werecentrifuged (Sorvall WX 80 ULTRA centrifuge (ThermoScientific USA)) for 20min at 20∘C and 5000 rpm For anappropriate comparison GG samples (C

119901= 05 wv) were

only centrifuged (without applying the sonication step) AllGG samples were then dialysed and freeze-dried

Fromnow on the dialysed sample will be calledGGwhilethe centrifuged and dialysed samples will be called accordingto the minutes of sonication GG 0 (no sonication) GG 1GG 3 GG 5 GG 10 GG 20 and GG 30

223 GPC Weight molecular weight (119872119908) and polydisper-

sity index (119872119908119872119899) were determined by GPC on a bank

of TSK gel GMPWXL columns (Tosoh Bioscience TokyoJapan) A Varian 210 HPLC system with a 356-LC refractiveindex detector was used The eluant was 55mM Na

2SO3

and 002 NaN3in double distilled water the flow rate

and temperature were maintained at 06mLmin and 40∘Crespectively All guar samples were diluted to 015 (wv) andfiltered through a 045 120583m filter prior to analysis The bank ofcolumns was calibrated using pullulan standards [12 13]

224 NMR Analysis Samples of dialysed GG and GG 30about 4mg were solubilized in 07 120583L of D

2O 1H-NMR

experiments were carried out at 45∘C on a Bruker AVANCEAQS 600 spectrometer operating at 60013MHz andequipped with a Bruker multinuclear 119911-gradient probe headA soft presaturation of the HOD residual signal was appliedbefore spectra acquisition [14]

225 Rheological Measurements The rheological character-ization of the GG and GGborax samples was performed bymeans of a controlled stress Haake Rheo-Stress RS300 rota-tional rheometer provided with aHaake DC50 thermostat Acone plate device (Haake CP60Ti diameter = 60mm cone =1∘ gap = 0053mm)was used forGG solutionswhile a grainedplate-plate device (Haake PP35 TI diameter = 35mm) wasemployed for GGborax samples in order to reduce the extentof wall slippage phenomena [15] To perform the measure-ments the samples were transferred in the rheometer andthe upper part of the selected device was then lowered until itreached the sample surface Gap-setting optimizations wereundertaken according to the procedure described elsewhere[16] Stress sweep experiments were performed (25∘C and1Hz) in the range 120574 = 0001 divide 1000 frequency sweepexperiments were carried out in the range 001 divide 10Hz inthe linear viscoelastic region (usually 120574 = 001) preliminaryassessed by stress sweep experiments flow curves wereperformed in the range 0001 divide 1000 sminus1 applying a stepwiseincrease of the stress All measurements were made at 25∘C

BioMed Research International 3

0

10

20

15 17 19 21 23 25 27Time (s)

(au

)

GGGG 0GG 1

GG 10GG 30

SampleGG

GG 0GG 1

GG 10GG 30

46558239265206

MwMn

Figure 1 Molar mass distributions of the guar samples and table of Polydispersity Index

226 Dilute Solutions Viscometry For the viscosity measure-ments an automatic viscometer (Instrument Schott AVS 370Lauda Germany) with a water bath (Lauda 015 T) allowingthe temperature control to 01∘C was used An Ubbelohdecapillary viscometer (Type no 531 01 with a capillary diameter= 054mm Schott-Gerate) for dilution sequences with aflux time for the solvent (distilled water) at 25∘C of 17828 swas used The distilled water for the sample preparationsand for the dilutions was previously filtered three times with020120583 Sartorius Biolab Products filters The flux time of GGsolutions (119905) was compared with that of the solvent (119905

0) and

the relative increase of fluxing time (1199051199050= 120578120578

0= 120578rel 120578rel minus

1 = 1199051199050minus 1 = 120578specific) was evaluated at different polymer

concentrations In the range of viscosities up to about twicethat of water (ie 120578rel = 2) the following Huggins equation isvalid

120578specific

119862= [120578] + 119896

119867[120578]2

119862 (1)

The limiting value of 120578specific119862 for 119862 rarr 0 representsthe intrinsic viscosity [120578] (cm3g) while from the slope ofthe linear trend of viscosity data it is possible to calculatethe Huggins constant 119896

119867 which gives an indication on the

aggregation state of the polymer in the tested conditionsof solvent and temperature [17] The intrinsic viscosity is ameasure of the inherent ability of the polymer to increasethe viscosity of the solvent at a given temperature and it canbe determined according toHuggins equation bymeasuringviscosity of solutions at low concentrations and extrapolatingto infinite dilution Furthermore intrinsic viscosity [120578] isrelated to the viscosity averagemolecularmass of the polymerthrough the Mark-Houwink-Sakurada relationship (MHS)to the molar mass of the sample [18] [120578] = 119870119872119886

119908 where

119870 and 119886 are constants for each polymer-solvent system at agiven temperature and are both related to the ldquostiffnessrdquo ofthe polymer For flexible polymer coils 119886 is 05 in a 120579 solventand 08 in a good solvent [12]

227 Conformational Transition Studies The capability ofsonicated GG to retain the helix conformation when linkedwith borax [3] was examined by characterising the CongoRed-GG complexes [19 20] Experimentally aliquots of aNaOH stock solution were added to GG-borax samples(1 gL) containing 10 120583M CR in order to obtain the desiredpH values (9 10 and 13)

The solutions were left to equilibrate until their UV-Visspectra did not change with time For a comparison similarexperiments were carried out on a 10120583M CR solution Theabsorption spectra were recorded with a Cary 100 spectro-photometer (Varian Palo alto CA USA) using a quartzcuvette with a path length of 1 cm

228 Water Uptake and Dimensional Increase Studies Theswelling of GGborax tablets prepared with the differenttypes of GG (ie dialysed GG centrifuged and dialysed GGand GG sonicated for different periods of time centrifugedand then dialysed) was carried out by soaking the tablets indistilled water at 37∘CAt fixed time intervals the tablets werewithdrawn the excess of water was removed with soft filterpaper for 5 s and then the corresponding weights and dimen-sional variations along the longitudinal axis were determinedby means of a screw gauge with an accuracy of plusmn01mmNo remarkable variations of cross-section dimensions weredetected during the swelling process All experiments werecarried out in triplicate and the obtained values always laywithin 10 of the mean

3 Results and Discussion

31 GPC Measurements Molecular weight distributions ofGG and sonicated GG samples by GPC are shown inFigure 1 It is apparent that depolymerization of GG by sonicirradiation causes degradation of the polysaccharide to givedifferent fractions of lower molecular weight each fractionhaving a narrow molecular weight distribution In absence


0001

001

01

1

10

100

0001 001 01 1 10 100 1000 10000

GGGG 0GG 1

GG 10GG 30

d120574dt (sminus1)

120578(P

a s)

(a)

0001

001

01

1

10

100

1000

10000

0001 001 01 1 10 100 1000 10000

GGb GGb 0GGb 1 GGb 10GGb 30 GGGG 0 GG 1GG 10 GG 30

d120574dt (sminus1)

120578(P

a s)

(b)

Figure 2 (a) Flow curves of GG samples sonicated (at C119901= 05)

for different periods of time (0 1 10 and 30min) (b) Flow curves ofGG samples with (red symbols) and without borax (black symbols)for a comparison the flow curves recorded for GG andGG 0 are alsoreported (C

119901= 07 119879 = 25∘C)

of external stimuli (GG and GG 0) the distribution curvesare very broad while by applying sonication for differentperiod of time the peaks become sharper The molecularweight distribution is in fact somehow more importantfor characterising the samples than just their 119872

119908only In

Figure 1 typical chromatograms of GG samples are showntogether with the polydispersity index (119872

119908119872119899) It should be

kept in mind that the sonicated samples are also centrifuged(and finally dialysed) and this procedure removes not only

54 52 50 48 46 44 42 40 38 36(ppm)

100

0

156

7

(a)

54 52 50 48 46 44 42 40 38 36(ppm)

100

0

156

0(b)

Figure 3 1H-NMR spectra at 60013MHz and at 45∘C (D2O) of

dialysed GG (a) and GG 30 (b)

the tip dust but also the possible aggregates present in thesamples On the other hand in the dialysed sample (GG)only the polymeric fractions of very small molecular weights(cut-off of dialysis tubing 12000ndash14000) and impuritiesif present are removed Thus the dialysed samples lackthe smallest molecular weight fractions while the sonicatedones lack also molecular aggregates if present The highpolydispersity index detected for GG 0 sample may be dueto the effect of centrifugation that breaks the aggregatesleading to the dissolution of lowermolecular weight fractions(though higher than 14000) such effect obviously doesnot occur in the sample that was only dialysed From thetable of Figure 1 it is evident how the sonication processreduces significantly the polydispersity index leading for anirradiation of 30min towards the value expected for themostprobable distribution in a random chain scission (119872

119908119872119899=

2)

32 Rheological Measurements The flow curves for the son-icated samples are shown in Figure 2 As a comparison theflow curves of GG andGG 0 are also reported It can be notedthat these two samples together with GG 1 show almostsuperimposable profiles The small differences in the threeprofiles can be attributed to the different molecular weight


00001

001

1

0001 01 10 1000

GGGGGG 0GG 0

GG 1GG 1GG 5GG 5

120574

G998400 G998400998400

(Pa)

(a)

00001

001

1

100

00001 001 1 100

GGb GGbGGb 0 GGb 0GGb 1 GGb 1GGb 5 GGb 5

GG GGGG 0 GG 0GG 1 GG 1GG 5 GG 5

120574

G998400 G998400998400

(Pa)

(b)

Figure 4 (a) Viscoelasticity plot of GG samples sonicated (at C119901= 05) for different periods of time (0 1 and 5min) (b) Viscoelasticity

curves of GG samples with (red symbols) and without borax (black symbols) for comparison the flow curves recorded for GG are alsoreported (1198661015840 = full symbols 11986610158401015840 = empty symbols C

119901= 07 119879 = 25∘C)

distributions as evidenced by the GPC measurements Thefirst Newtonian plateau is not detectable for any sample andthe drop of viscosity is quite significant until the secondNewtonian plateau is reached with values of viscosity approx-imately less than 1 Pa s By increasing the sonication timedrastic changes are observed in the flow curves A sonicationof ten min is sufficient to drastically reduce the viscosity ofthe polymer (at 119889120574119889119905 = 10 (sminus1)) which drops of one decadefrom 03 (for GG and GG 1) to 002 (Pa s) Furthermorethe interval of the Newtonian plateau is remarkably shiftedto a higher shear rates indicating that by reducing thechain length the viscosity continues to decrease while forthe native polymer or the polymer sonicated for 1min theviscosity values remain almost constant up to 119889120574119889119905 =100 (sminus1)) Thus ten minutes of sonication are sufficientto break significantly the GG chain backbones (the initialmolecular weight was reduced to one third see Section 33)Furthermore by increasing the period of sonication therange of the second Newtonian plateau is even more shiftedtowards higher shear rate values and the macroscopic effectof a sonication of 30min is that the viscosity approachesvalues only five times higher than those of water

Increasing the sonication time a further breakdown ofthe polymeric chains occurs without damaging the repeatingunit structure as evidenced by NMR analysis (Figure 3)

NMRwas carried out in D2O at 45∘C in order to separate

theHOD residual signal from the anomeric signals As shownin Figure 2 the spectra of GG andGG 30 are superimposableIn particular the ratio between the anomeric 120573 (1rarr 4)mannose protons at 473 ppm and the anomeric 120572 (1rarr 6)galactose protons at 502 ppm remains constant and equalto 156 for the tested samples This is a clear indication that

the119872119866 ratio is not affected by the sonication process that isthe repeating units of the polymer keep intact their chemicalcompositions

Thus the sonication process can be proposed as a suitablemethod for GG molecular weight reduction Actually bymeans of sonication it is possible to prepare GG within aquite wide range of molecular weights without damaging themolecular structure This is a crucial point especially withreference to possible industrial applications of GG becausethe rheological properties of the polymeric solutions pre-pared with the sonicated samples that show remarkablevariations in comparison with the native polymer can beappropriately tailored according to specific needs

The effect on the rheological properties of the addition ofborax to the various samples was also investigated (Figure 2)Addition of borate ions to the GG samples led to a dramaticchange in the flow curve trends Between the samples withoutborax and those with borax a difference in the viscosityvalues of four decades is detected A well-defined plateau isrecorded at low shears up to 119889120574119889119905 = 01 (sminus1) After thisvalue GG and GG 0 undergo a rapid decrease in viscosityfollowed by the impossibility to further proceed with themeasurements due to the brittle nature of the network Thepresence of borax leads to a better discrimination among thedifferent samples even after 1min of sonication Going from1 to 10min of sonication the viscosity is strongly reducedand the plateau is extended up to 119889120574119889119905 asymp 2 (sminus1) Thus thereduction in molecular weight induces a dramatic change inthe polymeric network When sonication is carried out for30min the sample with borax starts to show a pseudoplasticbehaviour However in no case the viscosities of the GGbsamples reach values below those recorded for the samples


001

01

1

10

001 01 1 10

GG GGGG 1 GG 1

G998400 G998400998400

(Pa)

f (Hz)

(a)

001

01

1

10

100

0001 001 01 1 10

GGb GGbGG GG

G998400 G998400998400

(Pa)

f (Hz)

(b)

1

10

100

0001 001 01 1 10

GGb GGbGGb 5 GGb 5GGb 10 GGb 10

G998400 G998400998400

(Pa)

f (Hz)

(c)

Figure 5 Mechanical spectra of GG samples with (red symbols) and without borax (black symbols) sonicated (at C119901= 05) for different

periods of time (a) Frequency sweep of GG andGG 1 (b) frequency sweep of GG andGGb (c) frequency sweep of GGb andGGb sonicatedfor 5 and 10min (c) comparison between the frequency sweep of GG and GGb (1198661015840 = full symbols 11986610158401015840 = empty symbols C

119901= 07

119879 = 25∘C)

without borax It is clear that both GG molecular weight andborax represent key parameters for the flow properties

In Figure 4 the changes of the two moduli 1198661015840 (storagemodulus) and 11986610158401015840 (loss modulus) as a function of thedeformation are shownAs expected the linear viscoelasticityinterval is reduced by lowering the sample molecular weightThe addition of borax increases the 1198661015840 values from about 1 toapproximately 100 Pa and the stressstrain response is linearup to a 100 of deformation The mechanical spectra werethen recorded at 120574 = 001

In Figure 5 the mechanical spectra of GG samples arereported It is interesting to follow the modulus variations

in the stress sweep experiments which show the significanteffect of sonication on the supramolecular structure

In Figure 5(a) the effect on GG sample of 1min sonica-tion is reported both mechanical spectra are those typical ofa solution (11986610158401015840 gt 1198661015840) and a strong dependence of the twomoduli on frequency can be detected In the case of samplessonicated for a longer period of time it was not possible tocarry out the experiment due to their predominant liquid-like characteristics Figure 5(b) compares the dynamic rheo-logical moduli prior to degradation of a GG solution with aGG-borax gel at the same polymer concentration (07 wv)There is a dramatic effect on the shape of frequency sweep


0

5

10

15

20

0 01 02 03 04

GGGG 0GG 1GG 3

GG 10GG 20GG 30

120578 spC

(dL

g)

C ( wv)

Figure 6 (a) 120578sp119862 versus polymer concentration for GG samplessonicated for different periods of time (119879 = 25∘C)

trend as a consequence of the addition of borax a well-defined transition from a liquid-like to a gel-like behaviouris detected Actually the loss modulus (11986610158401015840) dominatesthe solution response over most of the frequency domainindicating the mainly viscous nature of the GG solutionBorax crosslinking causes an increase in both moduli withthe elastic modulus (1198661015840) being more strongly affected Awell-defined plateau is observed in the elastic modulus atintermediate frequencies (005ndash10Hz) being 1198661015840 higher than11986610158401015840 within this interval These features are characteristic of anetwork structure formed by the GG-borax crosslinks Dueto the labile nature of these linkages the GGb gel displaysa terminal region similar to that of polymer solutions witha well-defined crossover between 1198661015840 and 11986610158401015840 The frequencyvalues at which the moduli inversion takes place increaseas sonication time increases Obviously by reducing the GGmolecular weight the strength of the gel formed in thepresence of borax correspondingly decreases This is clearlyevidenced by the reduction of the storage modulus values ofthe frequency interval where1198661015840 is independent of the appliedfrequencies and of the shift of the crossover towards higherfrequency values All these variations indicate the increasingweakness of the polymeric network as sonication proceedswith a widening of the mesh size (with the reduction of1198661015840 values) and a corresponding shortening of the longestrelaxation time (the crossover frequency is shifted towardshigher values)

In Figure 5(c) the effects of sonication on GGb samplesare evidenced The addition of borax to GG 5 leads to anetwork very similar to that of GGb Nevertheless it mustbe pointed out that the crossover between 1198661015840 and 11986610158401015840 isappreciably shifted to higher frequencies in comparison toGGb sample (from 0002Hz (GGb) to 0012Hz (GGb5) In the case of sonicated samples the sol-gel transitionoccurs at higher frequencies due to the reduced influence of

Table 1 Sonication times of the GG samples the correspondingintrinsic viscosity the Huggins constant and the average molecularweight calculated according to the MHS equation

Sample Sonication time (min) [120578] (dLg) 119896119867119872119908times 10minus5

GG 0 915 044 802GG 0 0 923 074 813GG 1 1 914 075 801GG 3 3 613 043 460GG 5 5 524 049 370GG 10 10 397 042 252GG 20 20 310 039 178GG 30 30 250 015 132

the longest relaxation modes as expected for a looser net-work Furthermore themaximumof11986610158401015840 does notmatchwiththe crossing point this discrepancy is reduced by increasingthe sonication time This means that by reducing the chainlength the data can be fitted by using the generalizedMaxwellmodel applying only a few or only one (in the case ofcoincidence between the 11986610158401015840 maximum and the crossingpoint) relaxation time This is even more evident when GGundergoes a 10min sonication The mechanical spectra arestill those of a gel system but both moduli are lowered by apower of ten and the crossover is further shifted to higherfrequencies (asymp03Hz) Of course in this last case a muchweaker gel is obtained being the 1198661015840 plateau significantlyreduced in comparison to that of the previous two systems

Anyhow in the case of GG one min is the maximumsonication time that can be applied in order to record themechanical spectra (a more prolonged sonication leads toliquid-like systems with moduli that are too low to beexperimentally recorded) On the other side the presenceof borax leading to the formation of a three-dimensionalnetwork allowed to acquire mechanical spectra of a GGsample sonicated up to 10min When the sonication time isfurther increased to 30min the linkages with borax are toofew due to the reduction of GG molecular weight (119872

119908=

132 times 105) for the formation of a real gel network andconsequently the modulus values become too low to berecorded

33 Reduction of Guar Gum Solution Viscosity upon Sonica-tion Aqueous solutions of sonicated samples were preparedfor the viscosity measurements carried out at 25∘C and theresults are shown in Figure 6 For an appropriate comparisonGG samples were also tested

From the intercepts that is from the intrinsic viscosities[120578] of the samples the molecular weights of the variousfractions were estimated (and reported in Table 1) accordingto the Mark-Houwink-Sakurada (MHS) equations valid forgalactomannans [21]

[120578] = 119870119872119886119908 (2)

where119870 = 513 times 10minus4 and 119886 = 072It is possible to observe that by applying the sonication

for only 5min the GG molecular weight was reduced to


0

5

10

15

20

0 5 10 15 20 25

GGGG 0GG 1

GG 10GG 30

Time (h)

(wminusw0)w0

(a)

0

10

20

30

0 5 10 15 20 25Time (h)

GGGG 0GG 1

GG 10GG 30

(hminush0)h0

(b)

0

5

10

0 1 2 3

(wminusw0)w0

Time 05 (h05)

(c)

0

10

20

0 1 2 3

(hminush0)h0

Time 05 (h05)

(d)

Figure 7 (a) Water uptake (119908 minus 1199080)1199080and (b) relative height increase (ℎ minus ℎ

0)ℎ0from tablets of GGborax (using GG samples sonicated

for different periods of time) as a function of time (c) and (d) the same data of (a) and (b) reported as a function of the square root of timeExperiments were carried out in triplicate and the obtained values always lay within 10 of the mean

more than one half The reduction was further increased byincreasing the sonication process for longer times From theslope of the linear trend of viscosity data the values of theHuggins constant 119896

119867 (see Table 1) were calculated for all

samples The Huggins coefficient is a measure of polymer-polymer interactions in solution (with values of roughly030ndash040 in good solvents and 050ndash080 in 120579 solvents)and assumes high values when intermolecular associationsexist [22] Furthermore it is known that water is a fairlygood solvent for GG However 119896

119867of GG in water is much

higher than the regular value for a good solvent (03ndash04) which indicates the importance of polymer-polymerhydrogen bonding interactions among the GG moleculesWhen sonication is applied the decrease in molecular weightreduces the number per chain of hydrogen bonding sitesleading at the same time to a reduction of the intermolecularhydrogen bonding between polymer molecules that is theattractions between guar chains [12] 119896

119867is also very sensitive

to the formation of molecular aggregates In other words theHuggins coefficient can provide additional information about

the state of guar gummacromolecules in solution and can bea measure of the goodness of the solvent [23]

34 Water Uptake and Dimensional Increase Studies In pre-vious works [24] it was reported that tablets prepared withthe GGborax freeze-dried hydrogel undergo an anomalouspeculiar swelling essentially along one direction and verysimilar to that reported for the Scleroglucanborax tablets[1 25ndash27] New tablets were then prepared with sonicatedGG samples and water uptake and elongation behaviourwere followed as a function of time As a comparison theexperiments on tablets prepared with GG (ie with thepolymer that was only dialysed) were also carried out Theresults reported in Figure 7 (in distilledwater at 37∘C) clearlyshow that both water uptake and anisotropic elongationof the tablets observed for the GGborax system weresignificantly reducedwhen the sonicated samples were tested

The tablets prepared with GG and GG 0 increased theirweight and elongated for 24 hrs those prepared with GG


GG

GG 0

GG 1

GG 10

GG 30

4 8 24

(h)

Figure 8 Pictures of swelled GGborax tablets prepared with GG sonicated for different periods of time in distilled water at 37∘C (the heightof the tablets before the swelling process was ℎ

0asymp 105mm)

sonicated for 1min follow the same trend while those pre-pared with GG 10 started to dissolve in the medium alreadyafter 8 hours This phenomenon started even earlier for thesample sonicated for 30min the tablets started to loose theirweight already after 5 hours and the anomalous elongationcould be detected only up to 5 hours After 8 hours the tabletsdissolved completely in the swellingmedium Both processesthe uptake of water and the height increase obey during thefirst hours the Fickian law (Figures 7(c) and 7(d)) Thus thereduction of the polymeric chain lengths did not change thediffusional character of watermolecules behaviour during theimbibition of the tablet matrices

In Figure 8 the pictures of swelled tablets prepared withGG sonicated for different periods of time are shown

35 Helix-Coil Transition Analysis The conformationalstructures of GG-borax and sonicated GGborax were inves-tigated by means of the formation of CR-polymer complexesat various alkali concentrations CR indeed tends to bind topolysaccharides in helical structures and as a consequenceits visible absorption maximum shifts to higher values

By following its shift in the Vis spectra of the polymer-dyecomplex it is possible to demonstrate the presence of helixconformation As reported in Figure 9 the CR absorptionmaxima of GGborax and GGborax sonicated samples arered shifted in comparison to those of the dye alone even athigh pH values indicating that the polymer at low molecularweight is still able to bind the dye keeping a helix structure

4 Conclusions

Sonication appears to be a rather easy method suitable toreduce the 119872

119908of a polysaccharide such as GG without the

use of chemicals (ie without producing side products thatcould contaminate the final product) thus allowing moreeasily acceptable applications in the field of pharmaceuticscosmetics and food industry As evidenced in the NMRspectra sonication does not destroy the structural character-istics of the polymeric chains furthermore obtained resultsshowed that depolymerization of GG by sonic irradiationleads to different fractions of lower119872

119908 each fraction having

a narrow molecular weight distribution As expected such


488

492

496

500

504

8 9 10 11 12 13 14pH

CRGGbGGb 30

120582m

ax

Figure 9 Absorption maximum shifts at different pH values forCR and GGb systems with GG sonicated 0 and 30min

119872119908reduction dramatically affects the rheological properties

of the polysaccharide leading to significantly modified flowcurves In this sense it must be pointed out that it waspossible to acquire themechanical spectra only for the samplesonicated for 1min because the moduli of the other samplesobtained after sonication was carried out for a longer periodof time were too low to be detected As in the case of otherpolysaccharides (3) the addition of borax changed com-pletely the rheological properties from liquid-like systemsto gel-like systems and this behaviour although to a lessextent was observed for all sonicated samples Only whenthe molecular weight was decreased to119872

119908= 132 times 105 the

GG chains were nomore able to build up a three-dimensionalgelled network and the rheologicalmoduli were too low to beexperimentally detected Actually the GG molecular weightas estimated by viscometric measurements decreased veryrapidly by applying ultrasounds to the polymeric solutionsleading to a reduction to one-half of the initial 119872

119908already

after a sonication of 3 minutesThe swelling behaviour of GGborax tablets was also

investigated In this respect the reduced molecular weightof the polymer led to a significant difference in water uptakeand anisotropic elongation in comparison to the starting GGsample

In conclusion the use of a simple method such as soni-cation for the reduction of GG119872

119908 represents an important

approach for an appropriate tailoring of the mechanicalproperties of GG solutions in consideration of amore suitableand rational utilization of such polysaccharide in the variousand wide fields of practical uses In particular as far asinnovative drug formulations are concerned an optimizedreduction of the GG119872

119908will allow an easy conjugation of the

obtained shorter chains with a hydrophobic drug that shouldthen lead to the formation of self-assembled structures inthe form of nanohydrogels that are suitable for modifieddrug delivery systems as already successfully carried outand reported in the case of sonicated gellan gum chemicallylinked to prednisolone [1]

Acknowledgment

SapienzaUniversityGrant no C26A119N2S is acknowledged

References

[1] G DrsquoArrigo C Di Meo E Gaucci et al ldquoSelf-assembled gellan-based nanohydrogels as a tool for prednisolone deliveryrdquo SoftMatter vol 8 pp 11557ndash11564 2012

[2] S A Ansari P Matricardi C Di Meo F Alhaique and TCoviello ldquoEvaluation of rheological properties and swellingbehaviour of sonicated scleroglucan samplesrdquoMolecules vol 17no 3 pp 2283ndash2297 2012

[3] G Bocchinfuso C Mazzuca C Sandolo et al ldquoGuar gumand scleroglucan interactions with borax experimental andtheoretical studies of an unexpected similarityrdquo Journal ofPhysical Chemistry B vol 114 no 41 pp 13059ndash13068 2010

[4] I S Dierckx and I K Dewettinck ldquoSeed gumsrdquo in Biopoly-mers Polysaccharides II S De Baets E J Vandamme and ASteinbuchel Eds pp 321ndash343 Wiley-VCH London UK 2002

[5] T Shaikh and S Sasi Kumar ldquoPharmaceutical and pharmaco-logical profile of guar gum an overviewrdquo International Journalof Pharmacy and Pharmaceutical Sciences vol 3 supplement 5pp 38ndash40 2011

[6] K T Roberts ldquoThe physiological and rheological effects offoods supplemented with guar gumrdquo Food Research Interna-tional vol 44 no 5 pp 1109ndash1114 2011

[7] M S Butt N Shahzadi M K Sharif andM Nasir ldquoGuar guma miracle therapy for hypercholesterolemia hyperglycemia andobesityrdquo Critical Reviews in Food Science and Nutrition vol 47no 4 pp 389ndash396 2007

[8] T Coviello F Alhaique A Dorigo PMatricardi andMGrassildquoTwo galactomannans and scleroglucan as matrices for drugdelivery preparation and release studiesrdquo European Journal ofPharmaceutics and Biopharmaceutics vol 66 no 2 pp 200ndash209 2007

[9] C Sandolo P Matricardi F Alhaique and T CovielloldquoDynamo-mechanical and rheological characterization of guargum hydrogelsrdquo European Polymer Journal vol 43 no 8 pp3355ndash3367 2007

[10] C Sandolo T Coviello PMatricardi and F Alhaique ldquoCharac-terization of polysaccharide hydrogels for modified drug deliv-eryrdquo European Biophysics Journal vol 36 no 7 pp 693ndash7002007

[11] C Sandolo PMatricardi F Alhaique and T Coviello ldquoEffect oftemperature and cross-linking density on rheology of chemicalcross-linked guar gum at the gel pointrdquo Food Hydrocolloids vol23 no 1 pp 210ndash220 2009

[12] Y Cheng K M Brown and R K Prudrsquohomme ldquoCharacteri-zation and intermolecular interactions of hydroxypropyl guarsolutionsrdquo Biomacromolecules vol 3 no 3 pp 456ndash461 2002

[13] A Tayal R M Kelly and S A Khan ldquoRheology and molecularweight changes during enzymatic degradation of a water-solu-ble polymerrdquoMacromolecules vol 32 no 2 pp 294ndash300 1999

[14] M Gueron P Plateau and M Decorps ldquoSolvent signal sup-pression in NMRrdquo Progress in Nuclear Magnetic ResonanceSpectroscopy vol 23 no 2 pp 135ndash209 1991

[15] R Lapasin and S Pricl Rheology of Industrial PolysaccharidesTheory and Applications Chapman amp Hall London UK 1995

[16] A J Kuijpers G H M Engbers J Feijen et al ldquoCharacteriza-tion of the network structure of carbodiimide cross-linkedgelatin gelsrdquoMacromolecules vol 32 no 10 pp 3325ndash3333 1999


[17] C Tanford ldquoTransport processes viscosityrdquo in Physical Chem-istry of Macromolecules pp 317ndash456 John Wiley amp Sons NewYork NY USA 1961

[18] C R Cantor and P R Schimmel ldquoElementary polymer-chainhydrodynamics and chain dimensionsrdquo in Biophysical Chem-istry Part IIImdashThe Behaviour of Biological Macromolecules pp1019ndash1039 W H Freeman San Francisco Calif USA 1980

[19] K Ogawa T Dohmaru and T Yui ldquoDependence of complexformation of (1ndashgt3)-beta-D-glucan with congo red on tem-perature in alkaline solutionsrdquo Bioscience Biotechnology andBiochemistry vol 58 no 10 pp 1870ndash1872 1994

[20] J I Farina F Sineriz O E Molina and N I Perotti ldquoIsolationand physicochemical characterization of soluble scleroglu-can from Sclerotium rolfsii Rheological properties molecularweight and conformational characteristicsrdquo Carbohydrate Poly-mers vol 44 no 1 pp 41ndash50 2001

[21] D R Picout and S B Ross-Murphy ldquoOn the Mark-Houwinkparameters for galactomannansrdquo Carbohydrate Polymers vol70 no 2 pp 145ndash148 2007

[22] M Bohdanecky and J Kovar Viscosity of Polymer SolutionsElsevier Scientific Publishing Company AmsterdamTheNeth-erlands 1982

[23] X Ma and M Pawlik ldquoIntrinsic viscosities and Huggins con-stants of guar gum in alkali metal chloride solutionsrdquo Carbohy-drate Polymers vol 70 no 1 pp 15ndash24 2007

[24] T Coviello L Bertolo P Matricardi et al ldquoPeculiar behavior ofpolysaccharideborax hydrogel tablets a dynamomechanicalcharacterizationrdquo Colloid and Polymer Science vol 287 no 4pp 413ndash423 2009

[25] T Coviello M Grassi A Palleschi et al ldquoA new scleroglu-canborax hydrogel swelling and drug release studiesrdquo Inter-national Journal of Pharmaceutics vol 289 no 1-2 pp 97ndash1072005

[26] A Palleschi T Coviello G Bocchinfuso and F AlhaiqueldquoInvestigation on a new scleroglucanborax hydrogel structureand drug releaserdquo International Journal of Pharmaceutics vol322 no 1-2 pp 13ndash21 2006

[27] C Di Meo T Coviello P Matricardi F Alhaique D Capitaniand R Lamanna ldquoAnisotropic enhanced water diffusion inscleroglucan gel tabletsrdquo Soft Matter vol 7 no 13 pp 6068ndash6075 2011

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


in textile paper and paint industries as well as in cosmeticsAs pointed out most practical applications of GG are relatedto its rheological and gelling properties furthermore GG ispable to form hydrogels when crosslinked with glutaralde-hyde [8ndash11] and in the presence of borax [3] For the reductionof GG molecular weight the probe sonication approach wasused because in such case no chemicals are needed and suchaspect represents an important prerequisite for themarketingapproval of the obtained polymer

2 Materials and Methods

21 Materials GG was provided by CarboMer (USA) boraxwas a Carlo Erba (Italy) product and Congo Red (CR) aSigma Aldrich (USA) product For the sample preparationsdistilled water was always used except forNMR experimentswhere deuterium oxide (D

2O) (Cambridge Isotope Labora-

tories USA) was used All products and reagents were ofanalytical grade The Polysaccharide calibration kits SAC-10(Agilent Technologies UK) were used as pullulan standards

211 GG Purification A given amount of polymer was dis-solved in distilled water (polymer concentration C

119901= 05

wv) and then kept under magnetic and mechanical stirringat 60∘C for 24 h The obtained solution was exhaustivelydialysed at 7∘C against distilled water and then freeze-driedThe molecular weight cut-off of dialysis tubing was 12000ndash14000 From now on the sample purified by dialysis will becalled GG

212 Hydrogel and Tablet Preparation For the preparationof the tablets an appropriate amount of polymer (about200mg) was magnetically stirred in water for 24 h Then thecalculated amount (ie moles of borax = moles of repeatingunits of polymer) of 01M borax solution was added andthe system was left under magnetic stirring for 5min Theobtained sample (C

119901= 07 wv) was kept overnight at 7∘C

for gel setting and then freeze-dried Tablets were preparedfrom the freeze-dried sample with an IR die (Perkin Elmerhydraulic press) using a force of 50 kN for 30 s The weightof the GGborax tablets was 230 plusmn 10mg the diameter was130 plusmn 01mm and the thickness was 105 plusmn 002mm

22 Methods

221 Sonication In order to reduce the GG molecularweight a High Intensity Ultrasonic Processor (750 Wattmodel probe type sonicatormdashVibra CellmdashVC 750 Cole-Parmer USA) was used working at 20 kHz with a 65mmmicrotip applying an amplitude of 30 of the maximumpower supplied by the instrument corresponding to 20 wattsand pulser cycles of 30 s ON and 30 s OFF Aqueous solutions(50mL) of GG (05 (wv)) prepared in glass beaker werekept during sonication in an ice bath which allowed to keepan average temperature of the samples below 25∘C Theseexperimental conditions allowed to maintain the structuralcharacteristics of the polymeric chains as evidenced alsoin the case of other polysaccharides [2] All samples after

sonication were centrifuged for 20min at 5000 rpm and20∘C The centrifuge speed was selected after several testsin order to find out the minimum rpm value capable ofremoving the probe-tip residues from the polymer solutionsThe supernatant solutions were then dialysed and finallyfreeze-dried

222 Ultracentrifugation After sonication all samples werecentrifuged (Sorvall WX 80 ULTRA centrifuge (ThermoScientific USA)) for 20min at 20∘C and 5000 rpm For anappropriate comparison GG samples (C

119901= 05 wv) were

only centrifuged (without applying the sonication step) AllGG samples were then dialysed and freeze-dried

Fromnow on the dialysed sample will be calledGGwhilethe centrifuged and dialysed samples will be called accordingto the minutes of sonication GG 0 (no sonication) GG 1GG 3 GG 5 GG 10 GG 20 and GG 30

223 GPC Weight molecular weight (119872119908) and polydisper-

sity index (119872119908119872119899) were determined by GPC on a bank

of TSK gel GMPWXL columns (Tosoh Bioscience TokyoJapan) A Varian 210 HPLC system with a 356-LC refractiveindex detector was used The eluant was 55mM Na

2SO3

and 002 NaN3in double distilled water the flow rate

and temperature were maintained at 06mLmin and 40∘Crespectively All guar samples were diluted to 015 (wv) andfiltered through a 045 120583m filter prior to analysis The bank ofcolumns was calibrated using pullulan standards [12 13]

224 NMR Analysis Samples of dialysed GG and GG 30about 4mg were solubilized in 07 120583L of D

2O 1H-NMR

experiments were carried out at 45∘C on a Bruker AVANCEAQS 600 spectrometer operating at 60013MHz andequipped with a Bruker multinuclear 119911-gradient probe headA soft presaturation of the HOD residual signal was appliedbefore spectra acquisition [14]

225 Rheological Measurements The rheological character-ization of the GG and GGborax samples was performed bymeans of a controlled stress Haake Rheo-Stress RS300 rota-tional rheometer provided with aHaake DC50 thermostat Acone plate device (Haake CP60Ti diameter = 60mm cone =1∘ gap = 0053mm)was used forGG solutionswhile a grainedplate-plate device (Haake PP35 TI diameter = 35mm) wasemployed for GGborax samples in order to reduce the extentof wall slippage phenomena [15] To perform the measure-ments the samples were transferred in the rheometer andthe upper part of the selected device was then lowered until itreached the sample surface Gap-setting optimizations wereundertaken according to the procedure described elsewhere[16] Stress sweep experiments were performed (25∘C and1Hz) in the range 120574 = 0001 divide 1000 frequency sweepexperiments were carried out in the range 001 divide 10Hz inthe linear viscoelastic region (usually 120574 = 001) preliminaryassessed by stress sweep experiments flow curves wereperformed in the range 0001 divide 1000 sminus1 applying a stepwiseincrease of the stress All measurements were made at 25∘C


0

10

20

15 17 19 21 23 25 27Time (s)

(au

)

GGGG 0GG 1

GG 10GG 30

SampleGG

GG 0GG 1

GG 10GG 30

46558239265206

MwMn



0) and


0= 120578rel 120578rel minus



120578specific

119862= [120578] + 119896

119867[120578]2

119862 (1)




119908 where








0001

001

01

1

10

100

0001 001 01 1 10 100 1000 10000

GGGG 0GG 1

GG 10GG 30

d120574dt (sminus1)

120578(P

a s)

(a)

0001

001

01

1

10

100

1000

10000

0001 001 01 1 10 100 1000 10000


d120574dt (sminus1)

120578(P

a s)

(b)



119901= 07 119879 = 25∘C)


119908only In


119908119872119899) It should be


54 52 50 48 46 44 42 40 38 36(ppm)

100

0

156

7

(a)

54 52 50 48 46 44 42 40 38 36(ppm)

100

0

156

0(b)




119908119872119899=

2)



00001

001

1

0001 01 10 1000

GGGGGG 0GG 0

GG 1GG 1GG 5GG 5

120574

G998400 G998400998400

(Pa)

(a)

00001

001

1

100

00001 001 1 100



120574

G998400 G998400998400

(Pa)

(b)



119901= 07 119879 = 25∘C)









001

01

1

10

001 01 1 10

GG GGGG 1 GG 1

G998400 G998400998400

(Pa)

f (Hz)

(a)

001

01

1

10

100

0001 001 01 1 10

GGb GGbGG GG

G998400 G998400998400

(Pa)

f (Hz)

(b)

1

10

100

0001 001 01 1 10


G998400 G998400998400

(Pa)

f (Hz)

(c)



119901= 07

119879 = 25∘C)







0

5

10

15

20

0 01 02 03 04

GGGG 0GG 1GG 3

GG 10GG 20GG 30

120578 spC

(dL

g)

C ( wv)









119908=




[120578] = 119870119872119886119908 (2)




0

5

10

15

20

0 5 10 15 20 25

GGGG 0GG 1

GG 10GG 30

Time (h)

(wminusw0)w0

(a)

0

10

20

30

0 5 10 15 20 25Time (h)

GGGG 0GG 1

GG 10GG 30

(hminush0)h0

(b)

0

5

10

0 1 2 3

(wminusw0)w0

Time 05 (h05)

(c)

0

10

20

0 1 2 3

(hminush0)h0

Time 05 (h05)

(d)















GG

GG 0

GG 1

GG 10

GG 30

4 8 24

(h)


0asymp 105mm)





4 Conclusions







488

492

496

500

504

8 9 10 11 12 13 14pH

CRGGbGGb 30

120582m

ax




119908= 132 times 105 the


119908already








Acknowledgment


References

































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0

10

20

15 17 19 21 23 25 27Time (s)

(au

)

GGGG 0GG 1

GG 10GG 30

SampleGG

GG 0GG 1

GG 10GG 30

46558239265206

MwMn



0) and


0= 120578rel 120578rel minus



120578specific

119862= [120578] + 119896

119867[120578]2

119862 (1)




119908 where








0001

001

01

1

10

100

0001 001 01 1 10 100 1000 10000

GGGG 0GG 1

GG 10GG 30

d120574dt (sminus1)

120578(P

a s)

(a)

0001

001

01

1

10

100

1000

10000

0001 001 01 1 10 100 1000 10000


d120574dt (sminus1)

120578(P

a s)

(b)



119901= 07 119879 = 25∘C)


119908only In


119908119872119899) It should be


54 52 50 48 46 44 42 40 38 36(ppm)

100

0

156

7

(a)

54 52 50 48 46 44 42 40 38 36(ppm)

100

0

156

0(b)




119908119872119899=

2)



00001

001

1

0001 01 10 1000

GGGGGG 0GG 0

GG 1GG 1GG 5GG 5

120574

G998400 G998400998400

(Pa)

(a)

00001

001

1

100

00001 001 1 100



120574

G998400 G998400998400

(Pa)

(b)



119901= 07 119879 = 25∘C)









001

01

1

10

001 01 1 10

GG GGGG 1 GG 1

G998400 G998400998400

(Pa)

f (Hz)

(a)

001

01

1

10

100

0001 001 01 1 10

GGb GGbGG GG

G998400 G998400998400

(Pa)

f (Hz)

(b)

1

10

100

0001 001 01 1 10


G998400 G998400998400

(Pa)

f (Hz)

(c)



119901= 07

119879 = 25∘C)







0

5

10

15

20

0 01 02 03 04

GGGG 0GG 1GG 3

GG 10GG 20GG 30

120578 spC

(dL

g)

C ( wv)









119908=




[120578] = 119870119872119886119908 (2)




0

5

10

15

20

0 5 10 15 20 25

GGGG 0GG 1

GG 10GG 30

Time (h)

(wminusw0)w0

(a)

0

10

20

30

0 5 10 15 20 25Time (h)

GGGG 0GG 1

GG 10GG 30

(hminush0)h0

(b)

0

5

10

0 1 2 3

(wminusw0)w0

Time 05 (h05)

(c)

0

10

20

0 1 2 3

(hminush0)h0

Time 05 (h05)

(d)















GG

GG 0

GG 1

GG 10

GG 30

4 8 24

(h)


0asymp 105mm)





4 Conclusions







488

492

496

500

504

8 9 10 11 12 13 14pH

CRGGbGGb 30

120582m

ax




119908= 132 times 105 the


119908already








Acknowledgment


References

































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0001

001

01

1

10

100

0001 001 01 1 10 100 1000 10000

GGGG 0GG 1

GG 10GG 30

d120574dt (sminus1)

120578(P

a s)

(a)

0001

001

01

1

10

100

1000

10000

0001 001 01 1 10 100 1000 10000


d120574dt (sminus1)

120578(P

a s)

(b)



119901= 07 119879 = 25∘C)


119908only In


119908119872119899) It should be


54 52 50 48 46 44 42 40 38 36(ppm)

100

0

156

7

(a)

54 52 50 48 46 44 42 40 38 36(ppm)

100

0

156

0(b)




119908119872119899=

2)



00001

001

1

0001 01 10 1000

GGGGGG 0GG 0

GG 1GG 1GG 5GG 5

120574

G998400 G998400998400

(Pa)

(a)

00001

001

1

100

00001 001 1 100



120574

G998400 G998400998400

(Pa)

(b)



119901= 07 119879 = 25∘C)









001

01

1

10

001 01 1 10

GG GGGG 1 GG 1

G998400 G998400998400

(Pa)

f (Hz)

(a)

001

01

1

10

100

0001 001 01 1 10

GGb GGbGG GG

G998400 G998400998400

(Pa)

f (Hz)

(b)

1

10

100

0001 001 01 1 10


G998400 G998400998400

(Pa)

f (Hz)

(c)



119901= 07

119879 = 25∘C)







0

5

10

15

20

0 01 02 03 04

GGGG 0GG 1GG 3

GG 10GG 20GG 30

120578 spC

(dL

g)

C ( wv)









119908=




[120578] = 119870119872119886119908 (2)




0

5

10

15

20

0 5 10 15 20 25

GGGG 0GG 1

GG 10GG 30

Time (h)

(wminusw0)w0

(a)

0

10

20

30

0 5 10 15 20 25Time (h)

GGGG 0GG 1

GG 10GG 30

(hminush0)h0

(b)

0

5

10

0 1 2 3

(wminusw0)w0

Time 05 (h05)

(c)

0

10

20

0 1 2 3

(hminush0)h0

Time 05 (h05)

(d)















GG

GG 0

GG 1

GG 10

GG 30

4 8 24

(h)


0asymp 105mm)





4 Conclusions







488

492

496

500

504

8 9 10 11 12 13 14pH

CRGGbGGb 30

120582m

ax




119908= 132 times 105 the


119908already








Acknowledgment


References

































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


00001

001

1

0001 01 10 1000

GGGGGG 0GG 0

GG 1GG 1GG 5GG 5

120574

G998400 G998400998400

(Pa)

(a)

00001

001

1

100

00001 001 1 100



120574

G998400 G998400998400

(Pa)

(b)



119901= 07 119879 = 25∘C)









001

01

1

10

001 01 1 10

GG GGGG 1 GG 1

G998400 G998400998400

(Pa)

f (Hz)

(a)

001

01

1

10

100

0001 001 01 1 10

GGb GGbGG GG

G998400 G998400998400

(Pa)

f (Hz)

(b)

1

10

100

0001 001 01 1 10


G998400 G998400998400

(Pa)

f (Hz)

(c)



119901= 07

119879 = 25∘C)







0

5

10

15

20

0 01 02 03 04

GGGG 0GG 1GG 3

GG 10GG 20GG 30

120578 spC

(dL

g)

C ( wv)









119908=




[120578] = 119870119872119886119908 (2)




0

5

10

15

20

0 5 10 15 20 25

GGGG 0GG 1

GG 10GG 30

Time (h)

(wminusw0)w0

(a)

0

10

20

30

0 5 10 15 20 25Time (h)

GGGG 0GG 1

GG 10GG 30

(hminush0)h0

(b)

0

5

10

0 1 2 3

(wminusw0)w0

Time 05 (h05)

(c)

0

10

20

0 1 2 3

(hminush0)h0

Time 05 (h05)

(d)















GG

GG 0

GG 1

GG 10

GG 30

4 8 24

(h)


0asymp 105mm)





4 Conclusions







488

492

496

500

504

8 9 10 11 12 13 14pH

CRGGbGGb 30

120582m

ax




119908= 132 times 105 the


119908already








Acknowledgment


References

































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


001

01

1

10

001 01 1 10

GG GGGG 1 GG 1

G998400 G998400998400

(Pa)

f (Hz)

(a)

001

01

1

10

100

0001 001 01 1 10

GGb GGbGG GG

G998400 G998400998400

(Pa)

f (Hz)

(b)

1

10

100

0001 001 01 1 10


G998400 G998400998400

(Pa)

f (Hz)

(c)



119901= 07

119879 = 25∘C)







0

5

10

15

20

0 01 02 03 04

GGGG 0GG 1GG 3

GG 10GG 20GG 30

120578 spC

(dL

g)

C ( wv)









119908=




[120578] = 119870119872119886119908 (2)




0

5

10

15

20

0 5 10 15 20 25

GGGG 0GG 1

GG 10GG 30

Time (h)

(wminusw0)w0

(a)

0

10

20

30

0 5 10 15 20 25Time (h)

GGGG 0GG 1

GG 10GG 30

(hminush0)h0

(b)

0

5

10

0 1 2 3

(wminusw0)w0

Time 05 (h05)

(c)

0

10

20

0 1 2 3

(hminush0)h0

Time 05 (h05)

(d)















GG

GG 0

GG 1

GG 10

GG 30

4 8 24

(h)


0asymp 105mm)





4 Conclusions







488

492

496

500

504

8 9 10 11 12 13 14pH

CRGGbGGb 30

120582m

ax




119908= 132 times 105 the


119908already








Acknowledgment


References

































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0

5

10

15

20

0 01 02 03 04

GGGG 0GG 1GG 3

GG 10GG 20GG 30

120578 spC

(dL

g)

C ( wv)









119908=




[120578] = 119870119872119886119908 (2)




0

5

10

15

20

0 5 10 15 20 25

GGGG 0GG 1

GG 10GG 30

Time (h)

(wminusw0)w0

(a)

0

10

20

30

0 5 10 15 20 25Time (h)

GGGG 0GG 1

GG 10GG 30

(hminush0)h0

(b)

0

5

10

0 1 2 3

(wminusw0)w0

Time 05 (h05)

(c)

0

10

20

0 1 2 3

(hminush0)h0

Time 05 (h05)

(d)















GG

GG 0

GG 1

GG 10

GG 30

4 8 24

(h)


0asymp 105mm)





4 Conclusions







488

492

496

500

504

8 9 10 11 12 13 14pH

CRGGbGGb 30

120582m

ax




119908= 132 times 105 the


119908already








Acknowledgment


References

































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0

5

10

15

20

0 5 10 15 20 25

GGGG 0GG 1

GG 10GG 30

Time (h)

(wminusw0)w0

(a)

0

10

20

30

0 5 10 15 20 25Time (h)

GGGG 0GG 1

GG 10GG 30

(hminush0)h0

(b)

0

5

10

0 1 2 3

(wminusw0)w0

Time 05 (h05)

(c)

0

10

20

0 1 2 3

(hminush0)h0

Time 05 (h05)

(d)















GG

GG 0

GG 1

GG 10

GG 30

4 8 24

(h)


0asymp 105mm)





4 Conclusions







488

492

496

500

504

8 9 10 11 12 13 14pH

CRGGbGGb 30

120582m

ax




119908= 132 times 105 the


119908already








Acknowledgment


References

































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


GG

GG 0

GG 1

GG 10

GG 30

4 8 24

(h)


0asymp 105mm)





4 Conclusions







488

492

496

500

504

8 9 10 11 12 13 14pH

CRGGbGGb 30

120582m

ax




119908= 132 times 105 the


119908already








Acknowledgment


References

































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


488

492

496

500

504

8 9 10 11 12 13 14pH

CRGGbGGb 30

120582m

ax




119908= 132 times 105 the


119908already








Acknowledgment


References

































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

















Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

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