Chiang Mai J. Sci. 2013; 40(3) 517

Chiang Mai J. Sci. 2013; 40(3) : 517-533http://it.science.cmu.ac.th/ejournal/Contributed Paper

The Potential of Electrospun Poly(L-lactic Acid) FiberMats Containing a Crude Garcinia dulcis Extract forUse as Wound DressingsOrawan Suwantong*[a], Porntipa Pankongadisak [a], Suwanna Deachathai [a] andPitt Supaphol [b][a] School of Science, Mae Fah Luang University, Tasud, Muang, Chiang Rai 57100, Thailand.[b] The Petroleum and Petrochemical College and The Center for Petroleum, Petrochemicals, and

Advanced Materials, Chulalongkorn University, Pathumwan, Bangkok 10330, Thailand.*Author for correspondence; e-mail: o.suwantong@gmail.com

Received: 12 March 2012Accepted: 9 November 2012

ABSTRACTElectrospun poly(L-lactic acid) (PLLA) fiber mats containing an acetone crude extract

of Garcinia dulcis Roxb. (GD) at either amount of 30% or 50% (based on the weight ofPLLA) were successfully prepared by electrospinning. Both the neat and the GD-loadedPLLA fibers were smooth, with the average diameters of the fibers ranged between 0.60 and1.13 μm. The release characteristics of GD from the GD-loaded PLLA fiber mats werecarried out by total immersion method in acetate or phosphate buffer solution that contained0.5% v/v Tween 80 and 3% v/v methanol (hereafter, A/T/M or P/T/M medium) at either32 or 37°C, respectively. The cumulative amounts of the released GD from the GD-loadedPLLA fiber mats in both types of the releasing medium gradually increased with increase insubmersion time and then reached a plateau value at the longest submersion investigated time.The antioxidant activity based on the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay of theGD-loaded PLLA fiber mats remained active even after it had been subjected to a highelectrical potential during electrospinning process. The antimicrobial activity of the GD-loadedPLLA fiber mats was greatest against Streptococcus pyogenes. Lastly, the GD-loaded PLLA fibermats were proven non-toxic to normal human dermal fibroblasts, making their potential foruse as wound dressings.

Keywords: poly(L-lactic acid), Garcinia dulcis extract, electrospinning, antioxidants,wound dressing

1. INTRODUCTIONControlled drug delivery systems are

used to improve therapeutic effect of theconventional treatment, reduce toxicity, andenhance compliance of the patients [1].Controlling drug release at predefined andreproducible rate for a prolonged period of

time can be accomplished by incorporationdrug or therapeutic agent into polymericmaterials [2]. In particular biodegradableand biocompatible materials can be used tocontrol drug release occurs through diffusionand degradation [3]. Recently, the extensive

518 Chiang Mai J. Sci. 2013; 40(3)

research activities on electrospun polymernanofibers are encouraged by the greatpotential applications, especially in biomedicalapplications such as drug delivery system[4-7], wound dressing [8-11], and tissueengineering [12, 13]. Since the electrospunfibers have very high specific surface area,small diameter and large porosity [14].Electrospinning is become a promisingprocess for producing ultra-fine fibers withdiameters in the sub-micrometer down tonanometer range [15]. This process involvesapplying a strong electric field across aconductive capillary tip attaching to apolymer liquid-containing reservoir and acollector [16]. When electric field reaches acritical value where the Coulombic of thecharges overcomes the surface tension of thepolymer solution droplet at the tip of thecapillary, a charged jet is ejected. During thecharged jet is accelerated to the collector,the charged jet thins down and solidifies.Finally, the ultra-fine fibers are collected onthe collector [17]. However, the morphologyand the diameters of the electrospun fibersdepend on the concentration of polymers inthe solvent, the applied voltage, the distancebetween the electrodes and the flow rate ofsolution [18].

Poly(L-lactic acid) (PLLA) is a major kindof biodegradable poly(α-hydroxy acids) andthe product resulting from polymerization ofL-lactide [19]. PLLA has been widely studiedfor use in biomedical applications includingtissue engineering, wound dressing anddrug delivery system because of its goodprocess-ability, good biocompatibility, goodbiodegradability and suitable mechanicalproperties [20, 21]. PLLA fibers can beprepared by various processing such as meltspinning, dry spinning, wet spinning, dry-jet-wet spinning, and electrospinning [22].However, the fabrication of drug-loadedPLLA electrospun fibers via electrospinning

for drug delivery system has been studiedby many researchers [23]. Zeng et al. [4]prepared electrospun PLLA fibers containingvarious amounts (5-100 wt.%) of rifampinfrom 3.9 wt.% PLLA solution in 2:1 v/v ofchloroform/acetone. The drug release ratewas constant if proteinase K or enzyme wasadded in buffer solution and burst release ofrifampin was not observed indicating thatthe perfect incorporation of drug in thefibers. Thakur et al. [9] fabricated electrospunPLLA fiber mats containing two modeldrugs (i.e., lidocaine and mupirocin) withdifferent lipophilicities by the dual spinneretelectrospinning technique. The release profilesof the two drugs from the fiber mats showeddifferent profiles. Lidocaine showed an initialburst release (80% release in PBS) within firsthour followed by a plateau after the first fewhours, while mupirocin showed only a 5%release within the first hour. Chuysinuan et al.[24] prepared PLLA fibers containing gallicacid via electrospinning using dichloromethane(DCM)/N,N-dimethylformamide (DMF)(7:3, v/v) as solvent and also studied thecumulative amount of gallic acid releasedfrom the gallic acid-loaded PLLA fiber matsinto three types of medium (i.e., the acetatebuffer, the citrate-phosphate buffer, and thenormal saline). The results showed that thecumulative amount of gallic acid releasedfrom these fiber mats increased rapidlyduring the initial submersion times, graduallyincreased with further increases in thesubmersion times, and finally reachedplateau levels at longer submersion times [24].

Garcinia dulcis Roxb. (Guttiferae) is knownas Asian medical plant. It is a plant belongingto the Guttferae family. It is a native tropicalplant found in Indonesia, now widespread inwarm countries such as Philipines, Jawa,Borneo and also in south of Thailand.In Indonesia, the leaves and seeds of thisplant have been used for the treatment of

Chiang Mai J. Sci. 2013; 40(3) 519

lymphatitis, parotitis and struma [25].In Thailand, it is known as Ma-Phut whichis a native herb. Garcinia dulcis is one of theimportant species of genus Garcinia whichare a rich source of aromatic metabolites, e.g.,flavonoids, benzophenones and xanthones[26-31]. These compounds are well knownto exhibit a variety of biological activities, suchas antimicrobial, antioxidant, anti-malarial,and anti-inflammatory properties [31-34].

In this study, the electrospinning processof PLLA solution (10% w/v in 7:3 v/vDCM/DMF) was used to fabricate the PLLAfiber mats as carriers for delivery of a crudeacetone extract of the flowers of Garcinia dulcis(GD). A crude GD extract was incorporatedin PLLA solution (either 30 or 50% w/wbased on the weight of PLLA powder).Various properties (i.e., morphological, waterretention, mass loss, and cytotoxicityproperties) of both the neat and the GD-loaded PLLA fiber mats were investigated.The release characteristics of GD from thePLLA fiber mats were also investigated bythe total immersion method in the A/T/Mor the P/T/M medium. Lastly, the GD andthe GD-loaded PLLA fiber mats were testedfor the antioxidant activity via DPPH assay,as well as for their antimicrobial activity againstsome common pathogenic microorganismsfound on burn wounds.

2. EXPERIMENTAL DETAILS2.1 Materials

Poly(L-lactic acid) (PLLA; intrinsicviscosity = 1.28 dL⋅g-1) was obtained fromNature Works (USA). Dichloromethane(DCM) and dimethylformamide (DMF) werepurchased from Labscan (Asia) (Thailand).Sodium acetate, sodium chloride, anhydrousdisodium hydrogen orthophosphate, sodiumdihydrogen orthophosphate (Ajax Chemicals,Australia), glacial acetic acid (Carlo Erba, Italy),and all other chemicals were of analytical

reagent grades and used without furtherpurification.

2.2 Extraction of Garcinia dulcis (GD)The flowers of G. dulcis were collected

from Songkhla province in the southern partof Thailand. The dried flowers (1.2 kg) weregrinded and extracted with 5.0 L of acetonefor 5 days. The acetone extracts had beenfiltered through No. 4 Whatman filterpapers and concentrated with a rotaryevaporator to remove the solvent, before abrown viscous liquid extract was obtained[33]. The crude acetone extract (81.5 g) wasfurther separated by silica gel quick columnchromatography and eluted with hexane,dichloromethane and acetone in a polarity-gradient system. The eluted fractions werecombined into 18 fractions on the basis oftheir chromatographic characteristics.Finally, the eighteenth fraction (12.4582 g),eluted with 100% acetone, was used in thisstudy.

2.3 Preparation and Electrospinning ofNeat and GD-containing PLLA Solutions

A weighed amount of PLLA powderwas dissolved in 7:3 v/v DCM/DMF toprepare the base PLLA solution at a fixedconcentration of 10% w/v. The GD-containing PLLA solutions were preparedby dissolving the same amount of PLLApowder and GD in the amount of either 30or 50 wt% based on the weight of PLLApowder in the DCM/DMF mixture. Priorto electrospinning, the as-prepared solutionswere characterized for their viscosity andconductivity, at room temperature (26 ± 1°C),using a Brookfield/RVDV-II+P viscometerand a CyberScan con 100 conductivity meter,respectively. These solutions were thenelectrospun under a fixed electrostaticfield strength (EFS) of 20 kV/18 cm.The collection time was ~12 h resulting

520 Chiang Mai J. Sci. 2013; 40(3)

in the fiber mats of 70 ± 10 mm inthickness.

2.4 Characterization of Neat andGD-loaded PLLA Fiber Mats

Morphological appearance of both theneat and the GD-loaded electrospun PLLAfiber mats was observed by a LEO 1450 VPscanning electron microscope (SEM). Eachsample was coated with a thin layer of goldusing a Polaron SC-7620 sputtering deviceprior to observation under SEM. Diametersof the fibers were measured directly fromSEM images using a SemAphore 4.0 software.

The water retention and the mass lossbehavior of both the neat and the GD-loadedPLLA fiber mats were measured in an acetateor a phosphate buffer solution (Procedure forpreparation of acetate and phosphate buffersolutions is available as SupplementaryInformation) containing 0.5% v/v Tween 80and 3% v/v methanol (hereafter, the A/T/M medium and the P/T/M medium,respectively) at the skin and the physiologicaltemperatures of 32 and 37°C, respectively,for 24 and 48 h according to the following

equations:where M is the weight of each sample

after submersion in the buffer solution fora certain period of time (24 and 48 h), Md isthe weight of the sample after submersion inthe buffer solution for a certain period oftime (24 and 48 h) in its dry state, and Mi isthe initial weight of the sample in its dry state.

2.5 Release of GD from GD-loadedPLLA Fiber Mats2.5.1 Actual GD content

The actual amounts of GD in the GD-

loaded PLLA fiber mats were firstdetermined. Each sample (circular disc;~2.8 cm in diameter) was dissolved in 10mL of 7:3 v/v DCM/DMF. After that, 1.0mL of the solution was measured by aPerkin-Elmer Lambda 35 UV-visspectrophotometer at the wavelength of650 nm. The actual amounts of GD in theGD-loaded PLLA fiber mats were thenback-calculated from the obtained dataagainst a predetermined calibration curvefor GD.

2.5.2 GD release assayThe release characteristics of GD from

the GD-loaded PLLA fiber mats wereinvestigated by total immersion methodin either of the A/T/M (pH 5.5) or theP/T/M (pH 7.4) medium. Each sample(circular disc; ~2.8 cm in diameter) wasimmersed in 20 mL of the A/T/M mediumat the skin temperature of 32°C or ofthe P/T/M medium at the physiologicaltemperature of 37°C. At a specifiedimmersion time period ranging between 0 and48 h (2880 min), 1.0 mL of a sample solutionwas withdrawn and an equal amount of thefresh medium was refilled. The amounts ofGD in the sample solutions were determinedusing a Perkin-Elmer Lambda 35 UV-visspectrophotometer at the wave length of650 nm. The obtained data were carefullycalculated to determine the cumulativeamounts of the released GD. The experimentswere carried out in triplicate and the resultswere reported as average values.

2.6 Antioxidant ActivityThe antioxidant activity of the as-

extracted and the as-loaded GD wasdetermined using DPPH assay. For the as-extracted GD, the stock solution of GD inacetone was serially diluted to obtain GDsolutions with the final concentrations of

Water retention(%) = × 100, (1)

And Mass loss(%) = × 100, (2)

M-MdMd

Mi-MdMi

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9.75, 4.875, 2.438, 1.219, 0.304, 0.038,and 0.019 μg⋅mL-1. Exactly 1.0 mL of amethanolic solution of DPPH (100 μM)was added to 1.0 mL of each of the GDdilution and the obtained mixtures wereincubated for 30 min at the physiologicaltemperature of 37°C. The free radicalscavenging activity was determinedspectrophotometrically at the wavelengthof 517 nm. As for the as-loaded GD, themethod was a slight modification fromthat utilized by Re et al. [35] Specifically,each sample (circular disc; ~2.8 cm indiameter) was first dissolved in 10 mL of7:3 v/v DCM/DMF and subsequentlytreated with a methanolic solution of DPPH(100 μM) for 30 min (i.e., 1.0 mL of the as-loaded GD solution against 1.0 mL ofthe DPPH solution) at 37°C. The freeradical scavenging activity was determinedspectrophotometrically at the wavelengthof 517 nm. The antioxidant activity (%AA)of either of the as-extracted or the as-loadedGD was expressed as the percentage ofDPPH that was decreased in comparisonwith that of the control condition (i.e., the

testing solution without the presence ofeither type of GD), according to thefollowing equation:where Acontrol and Asample are the absorbancevalues of the testing solution without andwith the presence of either type of GD.

2.7 Antimicrobial EvaluationThe antimicrobial activity of both the neat

and the GD-loaded PLLA fiber mats wastested against some common pathogenicmicroorganisms, e.g., Acinetobacter calcoaceticus,Escherichia coli, Pseudomonas aeruginosa,

Staphylococcus aureus ATCC 25923,Staphylococcus aureus DMST 20654 ,Staphylococcus epidermidis, Streptococcusagalactiae, Streptococcus pyogenes andCandida albican, by the disc diffusion method.The suspensions of microorganisms in theCriterion™ Nutrient Broth (NB) were spreadas thin layers on the Criterion™ Mueller-Hinton (MH) agar in Petri dishes. After that,each of the neat and the GD-loaded PLLAfiber mat specimens (13 mm in diameter) wasplaced on top of the smeared agar andthe plate were incubated at 37°C for 24 h.The neat PLLA fiber mats were used ascontrol. If inhibitory concentrations werereached, there would be no growth of themicrobes, which could be seen as clear orinhibition zones around the disc specimens.

2.8 Indirect Cytotoxicity EvaluationThe indirect cytotoxicity evaluation of

both the neat and the GD-loaded PLLA fibermats was conducted in adaptation from theISO 10993-5 standard test method in a96-well tissue-culture polystyrene plate(TCPS; Corning Costar®, USA) using normalhuman dermal fibroblasts (NHDF; 24th

passage) as reference. The cells werecultured in Dulbecco’s modified Eagle’smedium (DMEM; Sigma-Aldrich, USA),supplemented by 10% fetal bovine serum(FBS; Invitrogen Corp., USA), 1%L-glutamine (Invitrogen Corp., USA) and 1%antibiotic and antimycotic formulation[containing penicillin G sodium, streptomycinsulfate, and amphotericin B (Invitrogen Corp.,USA)]. The samples cut from both the neatand the GD-loaded PLLA fiber mat werefirst sterilized by UV radiation for ~1 h andthen immersed in serum-free medium (SFM;containing DMEM, 1% L-glutamine and 1%antibiotic and antimycotic formulation) for24 h in incubation to produce extractionmedia at various extraction ratios (i.e., 10, 5,

%AA = × 100, (3)Acontrol-Asample

Acontrol

522 Chiang Mai J. Sci. 2013; 40(3)

and 0.5 mg⋅mL-1). NHDF cells wereseparately cultured in wells of TCPS at 8,000cells/well in serum-containing DMEM for24 h to allow cell attachment. The cells werethen starved with SFM for 12 h. After that,the medium was replaced with an extractionmedium and the cells were re-incubated for24 h. The viability of the cells cultured byeach of the extraction media was finallydetermined with 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT)assay (see Supplementary Information). Theviability of the cells cultured by the freshSFM was used as control.

2.9. Statistical AnalysisData were presented as means ± standard

errors of means. Statistical analysis wascarried out by the one-way analysis ofvariance (one-way ANOVA) and Scheffe’spost hoc test in SPSS (IBM SPSS, USA).The statistical significance was accepted atp < 0.5.

3. RESULTS AND DISCUSSION3.1 Electrospinning of Neat andGD-containing PLLA Solutions

Prior to electrospinning, the shearviscosity and electrical conductivity of theneat and the GD-loaded PLLA solutionswere characterized.The results are summarizedin Table 1. The presence of GD in the basePLLA solution increased both the shearviscosity and the electrical conductivity ofthe GD-containing PLLA solutions.While, Chuysinuan et al. [24] reported theconductivity values of the neat and the gallicacid-loaded PLLA solutions were 2.8 ± 2.2and 14.6 ± 1.3 μS⋅cm-1, respectively. Theincrease in the conductivity should be duethe dissociation of gallic acid into ionicspecies [24]. Moreover, Suwantong et al. [36]reported the conductivity values of theneat and the GC-loaded PLLA solutions

were ranging from 1.56 to 3.32 μS⋅cm-1.Electrospinning of these solutions was carriedout at fixed electric field of 20 kV/18 cm.Representative SEM images of the neat andthe GD-loaded PLLA fiber mats are alsoshown in Table 1. Evidently, cross-sectionallyround fibers with smooth surface andwithout the presence of beads wereobtained indicating that the as-loaded GDwas incorporated well within the fibers.The diameters of these fibers were measuredand the results are also summarized inTable 1. The diameters of the neat PLLAfibers were 1.13 ± 0.22 μm, while those ofthe 30%GD- and 50%GD-loaded PLLAfibers were 0.60 ± 0.20 and 0.96 ± 0.20 μm,respectively. Comparatively, Chuysinuan et al.[24] reported that the diameters of the neatand the gallic acid-loaded PLLA fibers were965 and 843 nm, respectively. While,Suwantong et al. [36] reported that thediameters of the neat and the GC-loadedPLLA fibers were ranging from 0.80 to1.13 μm. The increased conductivity of thespinning solution was due to the presence ofGD, possibly a main cause for the reduceddiameters of the GD-loaded PLLA fibersas compared to those of the neat ones.The increase in the electrical conductivity ofthe GD-containing PLLA solutions was adirect result of the increase in the number ofcharges carried within the GD-containingPLLA solutions, hence the increase in thenumber of charge carriers within a jetsegment. The increase in the charge carrierscaused both the electrostatic and theCoulombic repulsive forces to increase,hence the further thinning down of theobtained GD-loaded PLLA fibers incomparison with the neat PLLA counterparts.Moreover, the observed increase in thediameters of the 50%GD-loaded PLLAfibers in comparison with 30%GD-loadedPLLA fibers should be a result of the

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greater viscosity of the more GD-containing PLLA solutions.

3.2 Water Retention and Mass LossBehavior of Neat and GD-loaded PLLAFiber Mats

The water retention and the mass lossbehavior of both the neat and the GD-loadedPLLA fiber mats after submersion in eitherthe A/T/M (at 32°C) or the P/T/M (at37°C) medium for 24 h and 48 h wereinvestigated and the results are shown inFigure 1. It should be noted that the fibermats from the PLLA solutions that contained30 or 50% of GD were hereafter denoted as

30% and 50%GD-loaded PLLA fiber mats.The water retention of the neat, the 30%GD-loaded and the 50%GD-loaded PLLAfiber mats after submersion in A/T/M (at32°C) for 24 h was ~397, ~533 and ~536%,respectively, while that for 48 h was ~481,~586 and ~644%, respectively (see Figure 1a).At 24 h after submersion in the P/T/Mmedium, the water retention of the neat,the 30%GD-loaded and the 50%GD-loaded PLLA fiber mats was ~486, ~584and ~656%, respectively, while, at 48 h, itwas ~487, ~616 and ~714%, respectively(see Figure 1a). According to the results, thewater retention of both the neat and the

Table 1. Shear viscosity and electrical conductivity of neat and GD-containing PLLAsolution (n = 3), representative SEM images of the corresponding fiber mats and diametersof the individual fibers (n ≈ 100).

Type of PLLAsolution

Shearviscosity

(Pa s)

Electricalconductivity

(μS cm-1)

Representative SEMimages of fiber mat

Fiber diameters(μm)

Neat

with 30% GD(based onweight ofPLLA)

with 50% GD(based onweight ofPLLA)

73.3 ± 0.3

85.8 ± 0.2

88.3 ± 0.2

1.56 ± 0.02

38.73 ± 0.23

42.33 ± 0.67

1.13 ± 0.22

0.60 ± 0.20

0.96 ± 0.20

524 Chiang Mai J. Sci. 2013; 40(3)

GD-loaded PLLA fiber mats in both typesof the releasing medium increased with anincrease in the submersion time, except forthe neat PLLA fiber mats in the P/T/M

medium that showed equivalent values atboth time points. In comparison between twotypes of the releasing medium, the waterretention of both the neat and the GD-

Figure 1. (a) Water retention and (b) mass loss behavior of neat and GD-loaded PLLAfiber mats in two types of medium, i.e., acetate or phosphate buffer solution containing3% v/v methanol and 0.5% v/v Tween 80 (i.e., A/T/M or P/T/M medium) (n = 3). *p< 0.05 compared between A/T/M and P/T/M at any given type of samples andsubmersion time point and #p < 0.05 compared with PLLA fiber mats at any givensubmersion time point and type of medium.

Submersion time(h)

Wat

er re

tent

ion(

%)

Submersion time(h)

Mas

s los

s(%

)

(a)

(b)

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loaded PLLA fiber mats after submersionin the P/T/M medium was greater thanthat after submersion in the A/T/Mmedium.

The mass loss of the neat and the GD-loaded PLLA fiber mats after submersionin either the A/T/M (at 32°C) or the P/T/M (at 37°C) medium for 24 h and 48 his shown in Figure 1b. The mass loss of theneat, the 30%GD-loaded and 50%GD-loaded PLLA fiber mats after submersionin the A/T/M medium for 24 h was ~8,~19 and ~23%, respectively, while thatfor 48 h was ~8, ~31 and ~32%,respectively. At 24 h after submersion inthe P/T/M medium, the mass loss of theneat, 30%GD-loaded and 50%GD-loadedPLLA fiber mats was ~16, ~23 and~34%, respectively, while, at 48 h,it was ~21, ~25 and ~42%, respectively.Similar to the water retention behavior,the mass loss of both the neat and theGD-loaded PLLA fiber mats in both typesof the releasing medium increased with anincrease in the submersion time, except forthe neat PLLA fibers mats in the A/T/Mmedium that showed equivalent values atboth time points. Also similar to the waterretention behavior, the mass loss of all of thefibrous matrices, at any given time point, wasgreater in the P/T/M medium than in theA/T/M medium, except for the 30%GD-loaded PLLA fiber mats.

For the neat PLLA fiber mats, their lossin the masses in the A/T/M medium wasequivalent at both time points. In the P/T/Mmedium, the mass loss of the neat PLLA fibermats was greater and also increased with anincrease in the submersion time. In the morebasic pH of the P/T/M medium as opposedto the A/T/M counterpart, partial hydrolysisof the ester bonds on the surface of the fibersoccurred more readily. The partial hydrolysisnot only resulted in the observed increased

in the mass loss, but also in the increasedhydrophillicity, hence the slight increase inthe water retention behavior, of the neatPLLA fiber mats upon their submersion inthe P/T/M medium [36]. When GD wasincorporated within the fibers, marginalincrease in the loss in the masses of theGD-loaded PLLA fiber mats was observedupon their submersion in a given medium,which was expected to be a result of the releaseof GD from the fibers. The release of GDfrom the fibers led to their increasedaccessibility to water molecules, resulting inthe observed increase in the water retentionof the GD-loaded PLLA fiber mats.

3.3 Release of GD from GD-loadedPLLA Fiber Mats

The actual amount of GD in the GD-loaded PLLA fiber mats needed to bedetermined prior to investigating the releasecharacteristics of GD from these samples.It was found that the actual amounts of GDin the 30%GD- and the 50%GD-loadedPLLA fiber mats were 96.3 ± 2.4 and 98.4 ±1.0 (base on the amounts of GD initiallycontained within spinning solutions),respectively. These values were later used tocalculate the cumulative amount of GDreleased from these GD-loaded samples.

The release characteristics of GD fromthe GD-loaded PLLA fiber mats were carriedout by the total immersion method in twotypes of the releasing medium, i.e., the A/T/M (at 32°C) or the P/T/M (at 37°C) mediumover a period of 2880 min. The cumulativerelease profiles of GD from all of theGD-loaded PLLA fiber mats were reportedas the percentage of the weights of GDreleased divided by the actual weights of GDin the samples are shown in Figure 2. In allcases, the cumulative amounts of GD releasedin any type of the medium increased quiterapidly with an initial increase in the

526 Chiang Mai J. Sci. 2013; 40(3)

submersion time and increased moregradually afterwards. In the A/T/Mmedium, the cumulative released amountsGD from both the 30%GD- and the50%GD-loaded PLLA fiber mats wereincreased quite rapidly with an initialsubmersion time and then reacheda plateau value at the longest submersioninvestigated time. The cumulative releasedamounts of GD from the 50%GD-loadedPLLA fiber mats were greater than thosefrom the 30%GD-loaded counterparts atmost submersion investigated time, exceptwithin 10 min submersion time. On the otherhand, the cumulative released amounts ofGD from the 50%GD-loaded PLLA fibermats in the P/T/M medium were slightly

Submersion time (min)(a)

Cum

ulat

ive

rele

ase

of G

D(%

bas

ed o

n ac

tual

am

ount

s of

GD

)

greater than those from the 30%GD-loadedcounterparts at most submersion investigatedtime. The cumulative released amounts GDfrom both the 30%GD- and the 50%GD-loaded PLLA fiber mats increased with anincrease in submersion time and then reacheda plateau value at the longest submersioninvestigated time. Additionally, incomparison between the A/T/M mediumand the P/T/M medium, the cumulativeamounts of GD released from both the30%GD- and the 50%GD-loaded PLLAfiber mats in the P/T/M medium weregreater than those in the A/T/M medium.Specifically, the maximum cumulativeamounts of GD released from the 30%GD-and the 50%GD-loaded PLLA fiber mat

Figure 2. Cumulative release profiles of GD from GD-loaded PLLA fiber mats, reportedas the percentage of the weights of GD released divided by the actual weights of GD inthe samples, by total immersion method in (a) A/T/M medium at the skin temperatureof 32°C or (b) P/T/M medium at the physiological temperature of 37°C.

Submersion time (min)

Cum

ulat

ive

rele

ase

of G

D(%

bas

ed o

n ac

tual

am

ount

s of

GD

)

(b)

Chiang Mai J. Sci. 2013; 40(3) 527

upon their submersion in the A/T/Mmedium were ~66 and ~83%,respectively. On the other hand, themaximum cumulative amounts of GDreleased from the 30%GD- and the

50%GD-loaded PLLA fiber mat upontheir submersion in the P/T/M mediumwere ~86 and ~88%, respectively.

3.4 Release Kinetics of GD fromGD-loaded PLLA Fiber Mats

The release kinetics of GD from acarrier can be characterized using asequation of the following form [37, 38]:Where Mt is the cumulative amount of GDreleased at an arbitrary time t, M∞ is thecumulative amount of the GD released in aninfinite time, n is an exponent characterizingthe mechanism with which the releasekinetics can be described and k is the rate ofrelease of the GD that incorporates physicalcharacteristics of the matrix/drug system aswell as some physical contributions from themeasurement methods.

For n = 0.5, the release mechanism canbe described as Fickian diffusion [39]. For thismechanism, a straight line is expected whenthe fractional accumulative amounts of GDreleased (i.e. Mt/M”) are plotted as a functionof t0.5. Here, the release characteristics ofGD from all of the GD-loaded PLLA fibermats were analyzed further by means of theFickian diffusion type of the releasingmechanism. The average values of k (alongwith the values of r2, signifying the goodness

of the curve-fitting, reported inparentheses) associated with the releasekinetics of GD from the 30%GD-loadedPLLA fiber mats in the A/T/M and theP/T/M media were determined to be0.0034 (0.98) and 0.0033 (0.98) s -0.5,respectively, while those associated with therelease kinetics of GD from the 50%GD-loaded PLLA fiber counterparts were0.0034 (0.94) and 0.0039 (0.99) s -0.5,respectively.

3.5 Antioxidant Activity of As-extractedand As-loaded GD

Many phenolic compounds are knownto be antioxidants, exhibiting good hydrogenand/or electron donor ability. DPPH⋅ is astable free radical and is capable of acceptingan electron or a hydrogen radical to revert toa stable molecule. When a DPPH solution ismixed with a substrate that acts as a protondonor, a stable non-radical form of DPPHis obtained. This is coupled with a change ofcolor from violet to pale yellow. Here, theDPPH radical scavenging assay was used toquantify the antioxidant activity of bothof the as-extracted and the as-loaded GD.Table 2 shows the antioxidant activity ofthe as-extracted GD as a function of itsconcentration, using acetone as the solvent.As expected, the antioxidant activity of theas-extract GD was an increasing functionwith its concentration. Specifically, as theconcentration of the as-extract GD solutiondecreased from 9.75 to 0.019 μg⋅mL-1, theantioxidant activity was found to decreasefrom ~76 to ~2.8%. Moreover, the halfmaximal inhibitory concentration (IC50) ofthe as-extracted GD was interpolated to be

MtM∞

= ktn ,for < 0.6 (4)MtM∞

528 Chiang Mai J. Sci. 2013; 40(3)

Table 2. Antioxidant activity of crude acetone extract of Garcinia dulcis (GD) (n = 3).

Concentration of the as-extracted GD in acetone(μg⋅mL-1)

Antioxidant activity(%)

9.754.8752.4381.2190.3040.0380.019

76.5 ± 2.143.2 ± 0.225.8 ± 1.614.0 ± 1.78.8 ± 0.84.4 ± 3.42.8 ± 2.6

about 6.0 μg⋅mL-1. Similarly, theantioxidant activity of the as-loaded GDwas also determined and it was found tobe 87.6 ± 1.1 and 85.3 ± 0.5% for the GDthat had been loaded in the 30%GD- andthe 50%GD-loaded PLLA fiber mats,respectively. These results confirm that theantioxidant activity of the as-loaded GDwas still retained, even after it had beensubjected to a high electrical potentialduring electrospinning.

3.6 Antimicrobial Activity of GD-loadedPLLA Fiber Mats

The potential for use of the GD-loadedPLLA fiber mats as a wound dressing wasevaluated by observing their antimicrobialactivity (base on the disc diffusion method)against some common pathogenicmicroorganisms found on burn wounds, e.g.,Acinetobacter calcoaceticus, Escherichiacoli, Pseudomonas aeruginosa, Staphylococcusaureus ATCC 25923, Staphylococcus aureusDMST 20654, Staphylococcus epidermidis,Streptococcus agalactiae, Streptococcuspyogenes and Candida albican and the resultsare summarized in Table 3. The activity ofthe neat PLLA fiber mats against thesemicrobes was used as a control and the

initial diameter of all of the samples wasfixed at 1.3 cm. As expected, the neat PLLAfiber mats showed no activity against thetested microbes. After 24 h of incubation,the inhibition zones against almost all ofthe tested microbes were observed aroundthe edges of the 30%GD- and the 50%GD-loaded PLLA fiber mat samples. This is inexception to the 30%GD-loaded PLLAfiber mat sample that showed no activitytowards C. albican. In addition, both the30%GD- and 50%GD-loaded PLLA fibermats showed no activity towards E. coli.However, both the 30%GD- and 50%GD-loaded PLLA fiber mats showed highpotential of the antimicrobial activityagainst S. pyogenes. Apparently, the50%GD-loaded PLLA fiber mats showedhigh potential of the antimicrobial activityagainst A. calcoaceticus, P. aeruginosa, S.aereus strains (i.e., ATCC 25923 andDMST 20654), S. epidermidis, and S.pyogenes, with the inhibition zones in therange of 1.76-2.15 cm. Moreover, the50%GD-loaded PLLA fiber mats showedmoderate activity against the growth of S.agalactiae, with the inhibition zones in therange of 1.51-1.75 cm. While theantimicrobial activity of the 50%GD-

Chiang Mai J. Sci. 2013; 40(3) 529

Extraction ratio

Cel

l via

bilit

y(%

)

loaded PLLA fiber mats was low againstthe growth of C. albican. On the otherhand, the 30%GD-loaded PLLA fiber matsshowed only high potential of theantimicrobial activity against S. pyogenes,with the inhibition zones in the range of1.76-2.15 cm. Furthermore, the 30%GD-loaded PLLA fiber mats showed moderateactivity against the growth of P. aeruginosa,S. aereus ATCC 25923, and S. epidermidis, withthe inhibition zones in the range of 1.51-1.75cm. While the antimicrobial activity of the30%GD-loaded PLLA fiber mats was lowagainst the growth of A. calcoaceticus, S. aureusDMST 2064, and S. agalactiae.

3.7 Indirect Cytotoxicity EvaluationThe potential for use of the GD-loaded

PLLA fiber mats as wound dressings wasassessed by investigating the cytotoxicityof these samples, using the neat PLLA fibermats as an internal control group. Theviability of the normal human dermalfibroblasts (NHDF) that had been culturedwith the extraction media from thesesamples in comparison with that of the cellsthat had been cultured with the freshculture medium is shown in Figure 3.Three extraction ratio of the extractionmedium (i.e., 10, 5, and 0.5 mg⋅mL-1) wereinvestigated. Obviously, the relative

Figure 3. Indirect cytotoxicity evaluation of neat and GD-loaded PLLA fiber mats interms of the viability of normal human dermal fibroblasts (NHDF) that had been culturedwith the extraction media from the fibrous materials in comparison with the viability ofthe cells that had been cultured with fresh culture medium (n = 3).

Table 3. The antimicrobial activity of both the neat and the GD-loaded PLLA fiber mats.Materials Antimicrobial activity in terms of disc diffusion method reported as inhibition zone diameter (cm)

A.calcoaceticus E.coli P.aeruginosa S.aureusATCC25923

S.aureusDMST20654

S.epidermidis

Neat30%GD-loadedPLLA fiber mats50%GD-loadedPLLA fiber mats

S.agalactiae S.pyogenes C.albican

-+

+++

--

-

-++

+++

-++

+++

-+

+++

-++

+++

-+

++

-+++

+++

--

+

Note: (-) : No inhibition zone (1.3 cm) No antimicrobial activity(+) : Inhibition zone (1.35-1.50 cm) Low potential of antimicrobial activity(++) : Inhibition zone (1.51-1.75 cm) Moderate potential of antimicrobial activity(+++) : Inhibition zone (1.76-2.15 cm) High potential of antimicrobial activity

530 Chiang Mai J. Sci. 2013; 40(3)

viability of the cells that had been culturedwith all extraction ratio from theneat PLLA fiber mats ranged between ~98and ~102% (relative to the viability of thecells that had been culture with freshculture medium) indicating that the neatPLLA fiber mats released no substance thatwas harmful to the cells. At the lowestconcentration of the extraction medium(i.e., 0.5 mg⋅mL-1), all of the GD-loadedPLLA fiber mats were non-toxic to thecells, with the viability of the cells ~109%.At the greater concentration of theextraction medium (i.e., 5 and 10 mg⋅mL-1), both the 30%GD- and 50%GD-loadedPLLA fiber mats still posed no threat tothe cells, as the relative viability of the cellsstill exceeded the threshold value of at least~80%. The obtained results indicated thatthese samples released no substances in thelevels that were harmful to the cells.

4. CONCLUSIONSIn present contribution, both the neat

and the GD-containing PLLA solutions weresuccessfully fabricated into fibers byelectrospinning under fixed electrostatic fieldstrength of 20 kV/18 cm. The incorporationof GD in the neat PLLA solution did notaffect the morphology of fibers. The smoothsurfaces with rather cross-sectionally roundfibers were obtained. The average diametersof both the neat and the GD-loaded PLLAfiber mats ranged between ~0.60 and ~1.13μm. The water retention and mass lossbehavior of the GD-loaded PLLA fiber matsin both types of the releasing medium (i.e.,A/T/M or P/T/M medium) increased withan increase in the submersion time. Moreover,the water retention and mass loss behaviorof these samples after submersion in theP/T/M medium were greater than those inthe A/T/M medium. The cumulative releasedamounts of GD from the GD-loaded

PLLA fiber mats in the A/T/M mediumgradually increased with increase insubmersion time and then reached a plateauvalue at the longest submersion investigatedtime, while that in the P/T/M mediummore gradually increased over the testingperiod and reached a plateau value at longestsubmersion investigated time. The observedgreater cumulative amounts of the releasedGD from the GD-loaded PLLA fibers inthe P/T/M medium should be due to theobserved greater values of both the waterretention and the mass loss of these samplesafter submersion in the P/T/M medium.The half maximal inhibitory concentration(IC50) of GD was ~6.0 μg⋅mL

-1. Moreover,the antioxidant activity, based on DPPHassay, of the GD-loaded PLLA fiber matsremained active even after it had beensubjected to a high electrical potential duringelectrospinning process. The antimicrobialactivity of the GD-loaded PLLA fiber matswas greatest against S. pyogenes, followedby P. aeruginosa, S. aureus ATCC 25923,S. epidermidis, A. calcoaceticus, S. aureus DMST20654, S. agalactiae, and C. albican, respectively.Lastly, the potential for use of the GD-loadedPLLA fiber mats as wound dressings wasassessed by the indirect cytotoxicity test.The results showed that these samples werenon-toxic to the normal human dermalfibroblasts.

ACKNOWLEDGEMENTSThis work was supported by the Thailand

Research Fund (grant number: MRG5380120).We are grateful to Mae Fah Luang Universityfor partial financial support and laboratoryfacilities.

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