Polym. Bull. DOI 10.1007/s00289-014-1102-9 ORIGINAL PAPER
Electrospun poly(L-lactic acid) fiber mats containing crude Garcinia mangostana extracts for use as wound dressings Orawan Suwantong • Porntipa Pankongadisak Suwanna Deachathai • Pitt Supaphol
•
Received: 29 September 2013 / Revised: 12 December 2013 / Accepted: 22 January 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Poly(L-lactic acid) (PLLA) fiber mats containing two types of crude Garcinia mangostana Linn. (GM) extract [i.e., dichloromethane extract (dGM) and acetone extract (aGM)] were successfully prepared by electrospinning process. Both the neat and the GM-loaded PLLA fibers were smooth, with the average diameters ranging between 0.77 and 1.14 lm. The release characteristics of GM from the GMloaded PLLA fiber mats were carried out by total immersion method in acetate buffer or simulated body fluid that contained 0.5 % v/v Tween 80 and 3 % v/v methanol (hereafter, A/T/M or S/T/M medium) at either 32 or 37 °C, respectively. The maximum cumulative amounts of GM released from the GM-loaded PLLA fiber mats in the S/T/M medium were greater than those in the A/T/M medium. Moreover, the cumulative amounts of GM released from the aGM-loaded PLLA fiber mats were greater than those from the dGM-loaded PLLA fiber mats in both types of medium. The antibacterial activity of the dGM-loaded PLLA fiber mats was greatest against Staphylococcus aureus DMST 20654, while that of the aGMloaded PLLA fiber mats was greatest against S. aureus ATCC 25923 and S. epidermidis. Lastly, only the dGM-loaded PLLA fiber mats at extraction ratio of 10 mg mL-1 were toxic to the human dermal fibroblasts. Keywords Poly(L-lactic acid) Electrospinning Drug delivery systems Garcinia mangostana extract Wound dressings Electronic supplementary material The online version of this article (doi:10.1007/s00289-014-1102-9) contains supplementary material, which is available to authorized users. O. Suwantong (&) P. Pankongadisak S. Deachathai School of Science, Mae Fah Luang University, Tasud, Muang, Chiang Rai 57100, Thailand e-mail:
[email protected] P. Supaphol The Petroleum and Petrochemical College, The Center of Excellence on Petrochemical and Materials Technology, Chulalongkorn University, Pathumwan, Bangkok 10330, Thailand
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Introduction Many fabrication techniques have been used to prepare the fibrous structures [1–7]. Among those techniques, electrospinning technique has named as the simplest technique which can produce continuous ultrafine fibers with diameters in the submicrometer down to nanometer range from various types of polymers [8]. Since these ultrafine fibers have unique characteristics, for examples, high surface area to mass/volume ratio, small diameters and high porosity with interconnected porous structure [9], thus these characteristics make them suitable for various applications, especially biomedical applications such as tissue engineering [10–12], drug delivery system [13–16], and wound dressing [17–20]. Electrospinning process involves an application of a strong electric field across a conductive capillary tip attaching to a polymer solution and a collector [21]. When the electric field exceeds a critical value, charges on the surface of a pendant drop overcome the surface tension of the polymer solution droplet leading to the ejecting of the charged jet. During the jet traveling, the charged jet thins down and solidifies to finally achieve the ultra-fine fiber on the collector [22]. Poly(L-lactic acid) (PLLA) is biodegradable aliphatic polyester produced from renewable resources. Its properties are good biocompatibility, good biodegradability, and good mechanical properties which are suitable for use in biomedical applications [18, 23–26]. Electrospun PLLA fibers containing various amounts (5–100 wt %) of rifampin from 3.9 wt % PLLA solution in 2:1 v/v of chloroform/ acetone were fabricated by Zeng et al. [27]. The effect of addition of surfactants (anionic, cationic, and nonionic) on the diameter and uniformity of the electrospun PLLA fibers was investigated. The results showed that all three types of surfactants can reduce the diameters and size distribution of the electrospun PLLA fibers. In addition, the burst release of rifampin into buffer solution containing proteinase K was not observed [27]. The electrospun PLLA fiber mats containing two model drugs (i.e., lidocaine and mupirocin) with different lipophilicities by the dual spinneret electrospinning technique were fabricated [28]. The release profiles of the two drugs from the fiber mats showed different profiles. An initial burst release (80 % release in PBS) of lidocaine within first hour was observed, while mupirocin showed only a 5 % release within the first hour. From these results, the presence of two types of drug in the same polymer can alter the release kinetics of at least one drug [28]. Chuysinuan et al. [29] studied release characteristics of gallic acid from the electrospun PLLA fibers containing gallic acid into three types of medium (i.e., the acetate buffer, the citrate–phosphate buffer, and the normal saline). The cumulative amount of gallic acid released from these fiber mats was the greatest in the normal saline, followed by those in the citrate–phosphate buffer and the acetate buffer, respectively [29]. Recently, Suwantong et al. [18] prepared PLLA fiber mats containing a crude extract of Garcinia cowa Roxb. (GC) by electrospinning. The release characteristics of GC from the GC-loaded PLLA fiber mats were investigated by the total immersion method in acetate or phosphate buffer solution that contained 0.5 % v/v Tween 80 and 3 % v/v methanol (A/T/M or P/T/M medium). The results showed that the maximum amounts of GC released from these fiber mats after submersion in the P/T/M medium was greater than that released in
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the A/T/M medium [18]. Moreover, the PLLA fiber mats containing an acetone crude extract of G. dulcis Roxb. (GD) were fabricated by Suwantong et al. [30]. The release characteristics of GD from these fiber mats into the A/T/M or P/T/M medium were also investigated by a total immersion method. The results showed that the greater cumulative amounts of the GD released from the GD-loaded PLLA fiber mats were observed in the P/T/M medium [30]. Mangosteen [G. mangostana Linn. (GM)], named as ‘‘the queen of fruit’’, is a tropical plant found in Thailand. The mangosteen-fruit is dark purple or reddish with white, soft and juicy with a sweet and slightly acid edible pulp [31]. The fruit hulls of mangosteen fruit have been used as a medicinal agent for the treatment of diarrhea, dysentery, fever, skin infections and wounds [32]. The major bioactive compounds of xanthones found in fruit hull of mangosteen are a- and c-mangostin [33–35]. The biological activity of xanthones has been reported including antiinflammatory [36, 37], antiulcer [38], antimalarial [39], antiacne [40], antibacterial [41, 42], antioxidant [43–45], antitumor activities [46, 47]. Since the variation of xanthones distribution in different solvent extracts caused the variation of the antioxidant activities of GM, hence, in this study, two types of crude extract [i.e., dichloromethane extract (dGM) and acetone extract (aGM)] of G. mangostana Linn. (GM) were incorporated into a 10 % w/v PLLA solution in 7:3 v/v DCM/DMF at either 30 or 50 % (based on the weight of PLLA), and the resulting solutions were further fabricated into ultrafine fibers by electrospinning. Various properties (i.e., morphological, water retention, weight loss, and cytotoxicity properties) of both the neat and the GM-loaded PLLA fiber mats were investigated. The release characteristics of GM from the PLLA fiber mats were investigated by the total immersion method in the A/T/M or S/T/M medium. The GM and the GM-loaded PLLA fiber mats were investigated for the antioxidant activity against DPPH assay as well as for their antibacterial activity against some bacterial pathogens found on burn wounds.
Experimental Materials Poly(L-lactic acid) (PLLA; intrinsic viscosity = 1.28 dL g-1) was obtained from Nature Works (USA). Dichloromethane (DCM) and dimethylformamide (DMF) were purchased from Labscan (Asia) (Thailand). Sodium acetate, sodium chloride, potassium chloride, calcium chloride dehydrate, sodium hydrogen carbonate (Ajax Chemicals, Australia), potassium phosphate dibasic anhydrous (Rankem, India), sodium sulfate anhydrous (QRe¨CTM, New Zealand), tris-(hydroxymethyl)-aminomethane (Fisher Scientific, UK), Magnesium chloride (Aldrich, USA), hydrochloric acid (Merck, Germany), glacial acetic acid (Carlo Erba, Italy), and all other chemicals were analytical reagent grades and used without further purification.
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Extraction of G. mangostana (GM) The hulls of GM were bought from the fruit market in Chiang Rai province (the northern part of Thailand). The dried hulls (*5.3 kg) were ground and extracted with 18.6 L of dichloromethane for 10 days. The dichloromethane extracts were filtered through No. 4 Whatman filter papers and concentrated with an evaporator to remove the solvent before a black-brown viscous liquid extract was obtained. The crude dichloromethane extract (*22 g) was further separated by silica gel quick column chromatography and eluted with hexane and dichloromethane in a polaritygradient system. The eluted fractions were combined into 8 fractions on the basis of their chromatographic characteristics. Finally, the third fraction (9.1502 g), eluted with 100 % dichloromethane, was used in this study. This crude dichloromethane extract was hereafter defined as dGM. While the acetone extract was extracted from the dried hulls of GM (*4.6 kg). The dried hulls were ground and extracted with 5.4 L of acetone for 3 weeks. The acetone extracts were filtered through No. 4 Whatman filter papers and concentrated with an evaporator to remove the solvent before a brown viscous liquid was obtained. The crude acetone extract (*62 g) was further separated by silica gel quick column chromatography and eluted with dichloromethane and acetone in a polarity-gradient system. The eluted fractions were combined into 8 fractions on the basis of their chromatographic characteristics. Finally, the eighth fraction (5.9538 g), eluted with 100 % acetone, was used in this study. Preparation and electrospinning of neat and GM-containing PLLA solutions The base PLLA solution in 7:3 v/v DCM/DMF was first prepared at a fixed concentration of 10 % w/v. The GM-containing PLLA solutions were prepared by dissolving the same amount of PLLA powder and GM in the amount of either 30 or 50 % (based on the weight of PLLA powder) in the DCM/DMF mixture. Prior to electrospinning, the solutions were characterized for their viscosity and conductivity, at room temperature (26 ± 1 °C), using a Brookfield/RVDV-II ? P viscometer and a CyberScan con 100 conductivity meter, respectively. The solutions were then electrospun under a fixed electric field of 20 kV/18 cm. The collection time was *12 h, resulting in the fiber mats of 70 ± 10 lm in thickness. Characterization of neat and GM-loaded PLLA fiber mats Morphological appearance of both the neat and the GM-loaded PLLA fiber mats was observed by a LEO 1450 VP scanning electron microscope (SEM). Each sample, prior to the observation under SEM, was coated with a thin layer of gold using a Polaron SC-7620 sputtering device. Diameters of the fibers were directly measured from SEM images using a SemAphore 4.0 software. The water retention and the mass loss behaviors of both the neat and the GMloaded PLLA fiber mats were measured in an acetate buffer solution or a simulated body fluid (procedure for preparation of acetate buffer solution and simulated body fluid is available as Supplementary information) containing 0.5 % v/v Tween 80 and
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3 % v/v methanol (hereafter, the A/T/M medium and the S/T/M medium, respectively) at the skin or the physiological temperature of 32 and 37 °C, respectively, for 24 and 48 h according to the following equations: Water retention ð%Þ ¼
MMd 100; Md
ð1Þ
And Mass loss ð%Þ ¼
Mi Md 100; Mi
ð2Þ
where M is the weight of each sample after submersion in a buffer solution for a certain period of time (24 or 48 h), Md is the weight of the sample after submersion in the buffer solution for a certain period of time (24 or 48 h) in its dry state, and Mi is the initial weight of the sample in its dry state. Release of GM from GM-loaded PLLA fiber mats Actual GM content The actual amounts of GM in the GM-loaded PLLA fiber mats were first determined. Each sample (circular disc; *2.8 cm in diameter) was dissolved in 10 mL of 7:3 v/v DCM/DMF. After that, 1.0 mL of the solution was measured by a Perkin-Elmer Lambda 35 UV–vis spectrophotometer at the wavelength of 450 and 442 nm (for dGM and aGM, respectively). The actual amounts of GM in the GMloaded PLLA fiber mats were then back-calculated from the obtained data against a predetermined calibration curve for GM. GM release assay The release characteristics of GM from the GM-loaded PLLA fiber mats were investigated by the total immersion method in either of the A/T/M (pH 5.5) or the S/T/M (pH 7.4) medium. Each sample (circular disc; *2.8 cm in diameter) was immersed in 20 mL of the A/T/M medium at the skin temperature of 32 °C or 20 mL of the S/T/M medium at the physiological temperature of 37 °C. At a specified immersion time period ranging between 0 and 48 h (2,880 min), 1.0 mL of the sample solution was withdrawn and an equal amount of the fresh medium was refilled. The amounts of GM in the sample solutions were determined spectrophotometrically at the wavelength of 450 and 442 nm (for dGM and aGM, respectively). The obtained data were carefully calculated to determine the cumulative amounts of the released GM. The experiments were carried out in triplicate and the results were reported as average values. Antioxidant activity The antioxidant activity of the as-extracted and the as-loaded GM was determined using the DPPH assay. For the as-extracted dGM, the stock solution of dGM in DCM was serially diluted to obtain dGM solutions with the final concentrations of
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0.075, 0.050, 0.025, 0.0125, 0.00625, and 0.003125 mg mL-1 while the stock solution of aGM in acetone was serially diluted to obtain aGM solutions with the final concentrations of 0.01875, 0.01250, 0.00625, 0.003125, 0.001563, and 0.000781 mg mL-1. Exactly 1.0 mL of a methanolic solution of DPPH (100 lM) was added to 1.0 mL of each GM dilution, and the obtained mixtures were incubated for 30 min at the physiological temperature of 37 °C. Free radical scavenging activity of the as-extracted GM was determined spectrophotometrically at the wavelength of 517 nm. As for the as-loaded dGM and aGM, the method was a slight modification from that utilized by Re et al. [48]. Specifically, each sample (circular disc; *2.8 cm in diameter) was first dissolved in 10 mL of 7:3 v/v DCM/ DMF and subsequently treated with a methanolic solution of DPPH (100 lM) for 30 min (i.e., 1.0 mL of the as-loaded GM solution against 1.0 mL of the DPPH solution) at the physiological temperature of 37 °C. The free radical scavenging activity of the as-loaded GM was determined spectrophotometrically at the wavelength of 517 nm. The antioxidant activity (%AA) of either the as-extracted or the as-loaded GM was expressed as the percentage of DPPH decreased in comparison with that of the control condition (i.e., the testing solution without the presence of either type of GM), according to the following equation: %AA ¼
Acontrol Asample 100; Acontrol
ð3Þ
where Acontrol and Asample are the absorbance values of the testing solution without and with the presence of either type of GM. Antibacterial evaluation The antibacterial activity of both the neat and the GM-loaded PLLA fiber mats was tested against some common pathogenic bacteria, e.g., Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus ATCC 25923, S. aureus DMST 20654, and S. epidermidis, by the disc diffusion method. The suspensions of microorganisms in the CriterionTM Nutrient Broth (NB) were spread as thin layers on the CriterionTM Mueller–Hinton (MH) agar in Petri dishes. After that, each of the neat and the GM-loaded PLLA fiber mat specimens (13 mm in diameter) was placed on top of the smeared agar and then the plate was incubated at 37 °C for 24 h. The neat PLLA fiber mats were used as control. If inhibitory concentrations were reached, there would be no growth of the bacteria, which could be seen as clear or inhibition zones around the disc specimens. These were photographed for further evaluation. Indirect cytotoxicity evaluation The indirect cytotoxicity evaluation of both the neat and the GM-loaded PLLA fiber mats was conducted in adaptation from the ISO 10993-5 standard test method in a 96-well tissue-culture polystyrene plate (TCPS; Corning CostarÒ, USA) using normal human dermal fibroblasts (NHDF; 22nd passage) as reference. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, USA), supplemented by 10 % fetal bovine serum (FBS; Invitrogen Corp., USA),
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1 % L-glutamine (Invitrogen Corp., USA) and 1 % antibiotic and antimycotic formulation [containing penicillin G sodium, streptomycin sulfate, and amphotericin B (Invitrogen Corp., USA)]. The samples cut from both the neat and the GMloaded PLLA fiber mat were first sterilized by UV radiation for *1 h and then immersed in serum-free medium (SFM; containing DMEM, 1 % L-glutamine and 1 % antibiotic and antimycotic formulation) for 24 h in incubation to produce extraction media at various extraction ratios (i.e., 10, 5, and 0.5 mg mL-1). NHDF cells were separately cultured in wells of TCPS at 8,000 cells/well in serumcontaining DMEM for 24 h to allow cell attachment. The cells were then starved with SFM for 12 h. After that, the medium was replaced with an extraction medium and the cells were re-incubated for 24 h. The viability of the cells cultured by each of the extraction media was finally determined with 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay. The viability of the cells cultured by the fresh SFM was used as control. The MTT assay is based on the reduction of the yellow tetrazolium salt to purple formazan crystals by dehydrogenase enzymes secreted from the mitochondria of metabolically active cells. The amount of the purple formazan crystals is proportional to the number of viable cells. First, the culture medium in each plate was aspirated and replaced with 25 lL/well of MTT solution at 5 mg mL-1. The plate was further incubated for 4 h at 37 °C. The solution was then aspirated and 100 lL/well of dimethylsulfoxide (DMSO; Sigma-Aldrich, USA) was added to dissolve the formazan crystals. After 3 min of rotary agitation, the absorbance at the wavelength of 570 nm representing the viability of the cells was measured using a SpectraMax M2 Microplate Reader. Statistical analysis Data were presented as means ± SD of means. Statistical analysis was carried out by the one-way analysis of variance (one-way ANOVA) and Scheffe’s post hoc test in SPSS (IBM SPSS, USA). The statistical significance was accepted at p \ 0.5.
Results and discussion Electrospinning of neat and GM-containing PLLA solutions Both the neat and the GM-containing PLLA solutions were characterized for shear viscosity and electrical conductivity prior to the electrospinning and the results are summarized in Table 1. The presence of dGM in the neat PLLA solution did not affect both the shear viscosity and the electrical conductivity of the resulting solutions, except for the 50 % dGM-containing PLLA solutions, while the presence of aGM in the neat PLLA solution caused both the shear viscosity and the electrical conductivity of the resulting solutions to increase marginally. From previous work, Suwantong et al. [18] reported that the presence of G. cowa (GC) in the neat PLLA solution did not affect the shear viscosity of the resulting solutions but caused the electrical conductivity to increase significantly. For another work, the addition of G.
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Shear viscosity (Pa s)
73.3 ± 0.3
68.9 ± 0.4
Type of PLLA solution
Neat
123
With 30 % dGM (based on weight of PLLA)
1.40 ± 0.05
1.56 ± 0.02
Electrical conductivity (lS cm-1) Representative SEM images of electrospun fiber mat
1.10 ± 0.28
1.13 ± 0.22
Fiber diameters (lm)
Table 1 Shear viscosity and electrical conductivity of neat and GM-containing PLLA solutions (n = 3) as well as representative SEM images of the corresponding electrospun fiber mats including diameters of the individual fibers (n & 100)
Polym. Bull.
Shear viscosity (Pa s)
74.1 ± 0.3
77.3 ± 0.3
Type of PLLA solution
With 50 % dGM (based on weight of PLLA)
With 30 % aGM (based on weight of PLLA)
Table 1 continued
6.49 ± 0.11
1.95 ± 0.10
Electrical conductivity (lS cm-1) Representative SEM images of electrospun fiber mat
0.92 ± 0.23
1.14 ± 0.26
Fiber diameters (lm)
Polym. Bull.
123
Shear viscosity (Pa s)
79.5 ± 0.2
Type of PLLA solution
With 50 % aGM (based on weight of PLLA)
Table 1 continued
123 11.33 ± 0.11
Electrical conductivity (lS cm-1) Representative SEM images of electrospun fiber mat
0.77 ± 0.13
Fiber diameters (lm)
Polym. Bull.
Polym. Bull.
dulcis (GD) in the neat PLLA solution caused both the shear viscosity and the electrical conductivity to increase [30]. In addition, Chuysinuan et al. [29] reported that a dramatic increase in the electrical conductivity of the PLLA solution was observed when gallic acid (at 40 % based on the weight of PLLA) was incorporated into the neat PLLA solution. Hence the increase in the electrical conductivity of the PLLA solution might be a result of the dissociation of herbal substances into ionic species upon being subjected to a high electric field. Electrospinning of both the neat and the GM-containing PLLA solutions was carried out at the fixed electric field of 20 kV/18 cm. Representative SEM images of both the neat and the GM-loaded PLLA fiber mats are also shown in Table 1. Crosssectionally round fibers with smooth surface were obtained indicating that GM was incorporated well within the fibers. Moreover, the diameters of these fibers were measured and the results are also summarized in Table 1. It should be noted that the fiber mats from the PLLA solutions contained 30 and 50 % of dGM were hereafter denoted as 30 % dGM-loaded and 50 % dGM-loaded PLLA fiber mats, respectively. While the fiber mats from the PLLA solutions contained 30 and 50 % of aGM were hereafter denoted as 30 % aGM-loaded and 50 % aGM-loaded PLLA fiber mats, respectively. The diameters of the neat PLLA fibers were 1.13 ± 0.22 lm, while those of the 30 % dGM-loaded, 50 % dGM-loaded, 30 % aGM-loaded, and 50 % aGM-loaded PLLA fiber mats were 1.10 ± 0.28, 1.14 ± 0.26, 0.92 ± 0.23, and 0.77 ± 0.13, respectively. From these results, the diameters of the 30 % dGM-loaded and the 50 % dGM-loaded PLLA fibers were not much different from those of the neat PLLA fibers. Since the presence of dGM in the neat PLLA solutions did not affect the shear viscosity and the electrical conductivity of the resulting solutions. While the diameters of the 30 % aGMloaded and the 50 % aGM-loaded PLLA fibers were smaller than those of the neat PLLA fibers. Since the marginal increase in the electrical conductivity of the aGMcontaining PLLA solutions from that of the neat PLLA solution should be a direct result of the increase in the number of charges carried within the aGM-containing PLLA solutions. The increase in the charge carriers within a jet segment caused both the electrostatic and the Coulombic repulsive forces to increase, leading to further thinning down of the obtained aGM-loaded PLLA fibers compared to their neat PLLA counterparts. Comparatively, Chuysinuan et al. [29] reported that the diameters of the neat and the gallic acid-loaded PLLA fibers were 965 and 843 nm, respectively, while Suwantong et al. [18, 30] reported that the diameters of the 30 % GC, 50 % GC, 30 % GD and 50 % GD-loaded PLLA fibers were 0.80 ± 0.16, 1.04 ± 0.24, 0.60 ± 0.20, 0.96 ± 0.20 lm, respectively. Physicochemical properties of neat and GM-loaded PLLA fiber mats Physicochemical properties, in terms of water retention and mass loss, of both the neat and the GM-loaded PLLA fiber mats after submersion in either the A/T/M (at 32 °C) or the S/T/M (at 37 °C) medium for 24 or 48 h were characterized, and the results are shown in Fig. 1. At 24 h after submersion in the A/T/M medium, the water retention of the neat, the 30 % dGM-loaded, the 50 % dGM-loaded, the 30 % aGM-loaded, and the 50 % aGM-loaded PLLA fiber mats was 447, 455, 464, 535,
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(a)
700
#
Water retention (%)
600 500 400 300 200 100
# **
# # *
# **
#
#
# #
# #
*
PLLA fiber mats in A/T/M 30%dGM-loaded PLLA fiber mats in A/T/M 50%dGM-loaded PLLA fiber mats in A/T/M 30%aGM-loaded PLLA fiber mats in A/T/M 50%aGM-loaded PLLA fiber mats in A/T/M PLLA fiber mats in S/T/M 30%dGM-loaded PLLA fiber mats in S/T/M 50%dGM-loaded PLLA fiber mats in S/T/M 30%aGM-loaded PLLA fiber mats in S/T/M 50%aGM-loaded PLLA fiber mats in S/T/M
0 24
48
Submersion time (h)
(b) 60 55 50
Mass loss (%)
45 40 35 30
PLLA fiber mats in A/T/M 30%dGM-loaded PLLA fiber mats in A/T/M 50%dGM-loaded PLLA fiber mats in A/T/M 30%aGM-loaded PLLA fiber mats in A/T/M 50%aGM-loaded PLLA fiber mats in A/T/M PLLA fiber mats in S/T/M 30%dGM-loaded PLLA fiber mats in S/T/M 50%dGM-loaded PLLA fiber mats in S/T/M 30%aGM-loaded PLLA fiber mats in S/T/M 50%aGM-loaded PLLA fiber mats in S/T/M
# #
#
25 20
#
#
15 10 5 0 24
48
Submersion time (h) Fig. 1 a Water retention and b mass loss behavior of neat and GM-loaded PLLA fiber mats in two types of medium, i.e., acetate buffer or simulated body fluid containing 3 % v/v methanol and 0.5 % v/v Tween 80 (i.e., A/T/M or S/T/M medium) (n = 3). *p \ 0.05 compared between A/T/M and S/T/M at any given type of samples and submersion time point and #p \ 0.05 compared with PLLA fiber mats at any given submersion time point and type of medium
and 542 % on average, respectively (see Fig. 1a). At 48 h, the values increased to 481, 544, 579, 657, and 675 % on average, respectively. In the S/T/M medium, such values at 24 h after submersion were 522, 535, 571, 589, and 599 % on average, respectively, while, at 48 h, they increased to 528, 561, 611, 657, and 688 % on average, respectively. Apparently, the water retention of both the neat and the GMloaded PLLA fiber mats in both types of the releasing medium increased with an increase in the submersion time. Between the two media, the water retention of both the neat and the GM-loaded PLLA fiber mats in the S/T/M medium, at any given time point, was greater than that of the materials in the A/T/M counterpart, except for the water retention of the 30 % aGM-loaded PLLA fiber mats at 48 h which showed equivalent values in both types of medium.
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The mass loss of the neat and the GM-loaded PLLA fiber mats upon submersion in either the A/T/M (at 32 °C) or the S/T/M (at 37 °C) medium is shown in Fig. 1b. At 24 h after submersion in the A/T/M medium, the mass loss of the neat, the 30 % dGM-loaded, the 50 % dGM-loaded, the 30 % aGM-loaded, and the 50 % aGM-loaded PLLA fiber mats was 7, 8, 11, 15, and 17 % on average, respectively, while, at 48 h, the values increased to 11, 13, 17, 20, and 23 % on average, respectively. In the S/T/M medium, such values at 24 h after submersion were 8, 11, 15, 16, and 17 % on average, respectively, while, at 48 h, they increased to 13, 16, 20, 23, and 28 % on average, respectively. Similar to the water retention behavior, the mass loss of both the neat and the GM-loaded PLLA fiber mats in both types of the releasing medium increased with an increase in the submersion time. Also similar to the water retention behavior, the mass loss of both the neat and the GMloaded PLLA fiber mats in the S/T/M medium, at any given time point, was greater than that of the materials in the A/T/M medium, except for the mass loss of the 50 % aGM-loaded PLLA at 24 h showed equivalent values in both types of medium. Comparison between two types of medium (i.e., the A/T/M and the S/T/M), the mass loss of the neat PLLA fiber mats in the S/T/M medium was greater than that in the A/T/M medium, and also increased with an increase in the submersion time. In the more basic pH of the S/T/M medium as opposed to the A/T/M counterpart, partial hydrolysis of the ester bonds on the surface of the fibers occurred more readily. The partial hydrolysis not only resulted in the observed increase in the mass loss, but also in the increased hydrophilicity, hence the increase in the water retention behavior of the neat PLLA fiber mats upon their submersion in the S/T/M medium was observed. When GM was incorporated within the fibers, the increase in the loss in the masses of the GM-loaded PLLA fiber mats was observed upon their submersion in a given medium, which was expected to be a result of the release of GM from the fibers. The release of GM from the fibers led to their increased accessibility to water molecules, resulting in the observed increase in the water retention of the GM-loaded PLLA fiber mats. Moreover, the water retention and mass loss of the aGM-loaded PLLA fiber mats were greater than those of the dGMloaded PLLA fiber mats. This result could be a greater solubility of acetone extract that led to increased accessibility of aGM molecules to diffuse out of materials. Due to the highly porous structure of the fiber mats, the fiber mats absorbed and retained water within their porous structure by the capillary action. These actions might contribute to the higher water retention of the neat and the GM-loaded PLLA fiber mats [29]. Chuysinuan et al. [29] studied the water retention of the gallic acidloaded PLLA fiber mats in the acetate buffer (pH 5.5), the citrate–phosphate buffer (pH 5.5) and the normal saline (pH 7.0) at 32 °C. They reported that the water retention of the materials in the normal saline was the greatest, followed by that in the citrate–phosphate buffer and the acetate buffer, respectively. Moreover, the water retention of the materials that had been submerged in the normal saline, the citrate–phosphate buffer, and the acetate buffer for 48 h had 348, 347, and 74 %, respectively [29].
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Release of GM from GM-loaded PLLA fiber mats The actual amounts of GM in the GM-loaded PLLA fiber mats were determined prior to investigating the release characteristics of GM from these samples and it was found that the actual amounts of GM in the 30 % dGM-loaded, the 50 % dGMloaded, the 30 % aGM-loaded, and the 50 % aGM-loaded PLLA fiber mats were 98.4 ± 2.9, 99.2 ± 0.9, 96.8 ± 1.8, and 97.5 ± 2.1 % (based on the amounts of GM initially contained within the spinning solutions), respectively. These values were later used to calculate the cumulative amounts of GM released from these GMloaded materials. The release characteristics of GM from the GM-loaded PLLA fiber mats were carried out by the total immersion method over a period of 2,880 min in either the A/T/M (at 32 °C) or the S/T/M (at 37 °C) medium. The cumulative release profiles of GM from all of the GM-loaded PLLA fiber mats were reported as the percentage of the weight of GM released divided by the actual weight of GM in the samples (see Fig. 2 and Figure I in the Supplementary information). In all cases, the cumulative amounts of GM released into any type of the medium increased quite rapidly with increasing submersion time, increased more gradually afterwards, and then reached a plateau value at the longest submersion investigated time. Moreover, in both types of medium, the cumulative amounts of GM released from both the 50 % dGM-loaded and the 50 % aGM-loaded PLLA fiber mats were greater than those from both the 30 % dGM-loaded and the 30 % aGM-loaded PLLA fiber mats at most submersion investigated time. In addition, in comparison between the dGM-loaded PLLA fiber mats and the aGM-loaded PLLA fiber mats, the cumulative amounts of GM released from the aGM-loaded PLLA fiber mats in both types of medium were greater than those from the dGM-loaded PLLA fiber mats at all submersion investigated time due to the greater solubility of aGM leading to the increased accessibility of aGM molecules to diffuse out of materials. Additionally, in comparison between the A/T/M and the S/T/M medium, the cumulative amounts of GM released from all the GM-loaded PLLA fiber mats in the S/T/M medium were greater than those in the A/T/M medium due to the more basic pH of the S/T/M medium. The partial hydrolysis of the ester bonds on the surface of the fiber mats occurred more readily in the S/T/M medium because of the more basic pH of the S/T/M medium. Thus the hydrophilicity of the fiber mats increased and led to the increased cumulative amounts of GM released from the fiber mats. These results corresponded with the water retention and the mass loss results of all the GMloaded PLLA fiber mats after submersion in the S/T/M medium. Specifically, the maximum cumulative amounts of GM released from the 30 % dGM-loaded, the 50 % dGM-loaded, the 30 % aGM-loaded, and the 50 % aGM-loaded PLLA fiber mats upon submersion in the A/T/M medium were *56, *62, *65, and *70 %, on average, respectively. On the other hand, the maximum cumulative amounts of GM released from the 30 % dGM-loaded, the 50 % dGM-loaded, the 30 % aGM-loaded, and the 50 % aGM-loaded PLLA fiber mats upon their submersion in the S/T/M medium were *63, *67, *73, and *78 %, on average, respectively.
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Cumulative release of GM (%, based on actual amounts of GM)
(a)
80 70 60 50 40 30 20
30%dGM-loaded PLLA fiber mats 50%dGM-loaded PLLA fiber mats 30%aGM-loaded PLLA fiber mats 50%aGM-loaded PLLA fiber mats
10 0 0
500
1000
1500
2000
2500
3000
3500
Submersion time (min)
Cumulative release of GM (%, based on actual amounts of GM)
(b)
80 70 60 50 40 30 20
30%dGM-loaded PLLA fiber mats 50%dGM-loaded PLLA fiber mats 30%aGM-loaded PLLA fiber mats 50%aGM-loaded PLLA fiber mats
10 0 0
500
1000
1500
2000
2500
3000
3500
Submersion time (min)
Fig. 2 Cumulative release profiles of GM from GM-loaded PLLA fiber mats, reported as the percentage of the weights of GM released divided by the actual weights of GM in the samples, by total immersion method in a A/T/M medium at the skin temperature of 32 °C or b S/T/M medium at the physiological temperature of 37 °C
Release kinetics of GM from GM-loaded PLLA fiber mats The release kinetics of a therapeutic agent from a carrier can be characterized further using an equation of the following form [49, 50]: Mt Mt ¼ ktn for \ 0:6; M1 M1
ð4Þ
where Mt is the cumulative amount of the drug released at an arbitrary time t, M? is the cumulative amount of the drug released at an infinite time, n is an exponent characterizing the mechanism with which the release kinetics can be described, and k is the rate of release of the drug that incorporates the physical characteristics of the matrix/ drug system as well as some physical contributions from the measurement method.
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Polym. Bull. Table 2 Analyses of the release kinetics of GM from dGM-loaded and aGM-loaded PLLA fiber mats based on the Fickian diffusion type of the release mechanism (n = 3) Type of sample
Rate parameter, k (s-0.5)
r2
30 % dGM-loaded PLLA fiber mats In A/T/M medium
0.0033
0.95
In S/T/M medium
0.0037
0.96
50 % dGM-loaded PLLA fiber mats In A/T/M medium
0.0037
0.95
In S/T/M medium
0.0039
0.98
30 % aGM-loaded PLLA fiber mats In A/T/M medium
0.0037
0.95
In S/T/M medium
0.0038
0.97
50 % aGM-loaded PLLA fiber mats In A/T/M medium
0.0037
0.97
In S/T/M medium
0.0037
0.97
When n equals 0.5, the release mechanism can be described as Fickian diffusion [51]. For this, a straight line is expected when the fractional, cumulative amounts of the drug released (i.e., Mt/M?) are plotted versus the square root of the submersion time (i.e., t0.5). Here, the release characteristics of GM from all of the GM-loaded PLLA fiber mats as determined by the total immersion method in the A/T/M and the S/T/M media were analyzed, and the results are summarized in Table 2. The average values of k (along with the values of r2, signifying the goodness of the curve-fitting, reported in parentheses) associated with the release kinetics of GM from the 30 % dGM-loaded PLLA fiber mats in the A/T/M and the S/T/M media were determined to be 0.0033 (0.95) and 0.0037 (0.96) s-0.5, respectively, while those associated with the release kinetics of GM from the 50 % dGM-loaded PLLA fiber mats were 0.0037 (0.95) and 0.0039 (0.98) s-0.5, respectively. On the other hand, the average values of k (along with the values of r2) associated with the release kinetics of GM from the 30 % aGM-loaded PLLA fiber mats in the A/T/M and the S/T/M media were determined to be 0.0037 (0.95) and 0.0038 (0.97) s-0.5, respectively, while those associated with the release kinetics of GM from the 50 % aGM-loaded PLLA fiber mats were 0.0037 (0.97) and 0.0037 (0.97) s-0.5, respectively. Suwantong et al. [18, 30] reported that the k value for the release of GC from the 30 % GC-loaded and the 50 % GC-loaded PLLA fiber mats in the A/T/ M medium was 0.0039 and 0.0064 s-0.5, respectively, while that for the release of GD from the 30 % GD-loaded and the 50 % GD-loaded PLLA fiber mats in the A/T/M medium was 0.0034 and 0.0034 s-0.5, respectively. Antioxidant activity of as-extracted and as-loaded GM There were many reports that G. mangostana Linn. has rich phenolic compounds known as antioxidants, exhibiting good hydrogen and/or electron donor ability [43– 45]. DPPH is a stable free radical, which is capable of accepting an electron or a
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Polym. Bull. Table 3 Antioxidant activity of crude DCM extract and acetone extract of Garcinia mangostana (GM) (n = 3) Concentration of the asextracted GM (dGM) in DCM (mg mL-1)
Antioxidant activity of dGM (%)
Concentration of the as-extracted GM (aGM) in acetone (mg mL-1)
Antioxidant activity of aGM (%)
0.075
81.3 ± 0.3
0.01875
81.4 ± 0.5
0.050
55.4 ± 3.2
0.01250
51.5 ± 1.4
0.025
29.5 ± 0.8
0.00625
27.8 ± 1.8
0.0125
15.0 ± 0.6
0.003125
11.9 ± 0.4
0.00625
6.7 ± 0.7
0.001563
8.6 ± 1.2
0.003125
4.1 ± 0.8
0.000781
4.3 ± 1.4
hydrogen radical to revert to a stable molecule. When a DPPH solution is mixed with a substrate which acts as a proton donor, a stable non-radical form of DPPH is obtained. This is coupled with a change of color from violet to pale yellow. Here, the DPPH radical scavenging assay was used to quantify the antioxidant activity of both the as-extracted and the as-loaded GM. Table 3 shows the antioxidant activity of the as-extracted dGM and the as-extracted aGM as a function of their concentration, using DCM and acetone as the solvent, respectively. As expected, the antioxidant activity of the as-extract GM was an increasing function with its concentration. Specifically, as the concentration of the as-extract dGM solution decreased from 0.075 to 0.003125 mg mL-1, the antioxidant activity was decreased from *81 to *4 %. Moreover, the half maximal inhibitory concentration (IC50) of the as-extracted dGM was interpolated to be approximately 0.045 mg mL-1. On the other hand, as the concentration of the as-extract aGM solution decreased from 0.01875 to 0.000391 mg mL-1, the antioxidant activity was decreased from *81 to *2 %. The half maximal inhibitory concentration (IC50) of the as-extracted aGM was interpolated to be approximately 0.012 mg mL-1. In 2009, Zarena and Sankar [52] studied the radical scavenging and antioxidant properties of the G. mangostana extracts obtained by various solvents [i.e., ethyl acetate, hexane, acetone, acetone/ water (80/20), methanol, and ethanol] due to the variation of distribution of xanthone in different extracts. The results showed that ethyl acetate and acetone were suitable solvents to extract the antioxidant compounds from mangosteen hull. Since the antioxidant activity of the compounds in the extract is strongly dependent on the solvent polarity [53], it could be concluded that the antioxidant activity of the dGM extracts was lower than that of the aGM extracts due to its low polarity. Additionally, the antioxidant activity of the as-loaded dGM was also determined and it was found to be 41.3 ± 1.2 and 89.9 ± 0.3 % for the dGM that had been loaded in the 30 % dGM-loaded and the 50 % dGM-loaded PLLA fiber mats, respectively, while the antioxidant activity of the 30 % aGM-loaded and the 50 % aGM-loaded PLLA fiber mats was 89.6 ± 1.0 and 89.7 ± 0.1 %, respectively. These results confirm that the antioxidant activity of the as-loaded GM was still retained, even after it had been subjected to a high electrical potential during electrospinning. Previously, Suwantong et al. [18, 30] reported that the antioxidant activity of the 30 % GC-loaded, the 50 % GC-loaded, the 30 % GD-loaded, and the
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50 % GD-loaded PLLA fiber mats was 8.7 ± 0.4, 11.8 ± 0.5, 87.6 ± 1.1, and 85.3 ± 0.5 %, respectively. Antibacterial activity of GM-loaded PLLA fiber mats The antibacterial activity of the GM-loaded PLLA fiber mats against some common pathogenic bacteria was evaluated by disc diffusion method, and the results are summarized in Table 4. Photographic images showing the antibacterial activity of the GM-loaded PLLA fiber mats against E. coli, P. aeruginosa, S. aureus strains (i.e., ATCC 25923 and DMST 20654), and S. epidermidis are shown in Fig. 3. The activity of the neat PLLA fiber mats against these bacteria was used as a control. The initial diameter of all samples was fixed at 1.3 cm. From Table 4, the neat PLLA fiber mats showed no activity against the tested bacteria. After 24 h of incubation, the inhibition zones against almost all of the tested bacteria were observed around the edges of both the dGM-loaded and the aGM-loaded PLLA fiber mats, except for the 30 % aGM-loaded PLLA fiber mats which showed no activity toward E. coli. In case of the dGM-loaded PLLA fiber mats, the 50 % dGM-loaded PLLA fiber mats showed high potential for antibacterial activity against both S. aureus DMST 20654 and S. epidermidis, with the inhibition zone in the range of 1.76–2.15 cm, while they showed moderate potential for antibacterial activity against E. coli, P. aeruginosa, and S. aureus ATCC 25923, with the inhibition zone in the range of 1.51–1.75 cm. In addition, the 30 % dGM-loaded PLLA fiber mats also showed moderate potential for antibacterial activity against S. aureus DMST 20654. The antibacterial activity of the 30 % dGM-loaded PLLA fiber mats was low against E. coli, P. aeruginosa, S. aureus ATCC 25923, and S. epidermidis, with the
Table 4 Antibacterial activity of neat and GM-loaded PLLA fiber mats Materials
Qualitative analysis for antibacterial activity, based on the inhibition zone diameters E. coli
P. aeruginosa
S. aureus ATCC 25923
S. aureus DMST 20654
S. epidermidis
Neat
-
-
-
-
-
30 % dGM-loaded PLLA fiber mats
?
?
?
??
?
50 % dGM-loaded PLLA fiber mats
??
??
??
???
???
30 % aGM-loaded PLLA fiber mats
-
?
???
??
???
50 % aGM-loaded PLLA fiber mats
?
??
???
???
???
(-) No inhibition zone (1.3 cm), no antibacterial activity (?) Inhibition zone (1.35–1.50 cm), low potential of antibacterial activity (??) Inhibition zone (1.51–1.75 cm), moderate potential of antibacterial activity (???) Inhibition zone (1.76–2.15 cm), high potential of antibacterial activity
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inhibition zone in the range of 1.35–1.50 cm. For the aGM-loaded PLLA fiber mats, both the 30 % aGM-loaded and the 50 % aGM-loaded PLLA fiber mats showed high potential for antibacterial activity against S. aureus strains (i.e., ATCC 25923 and DMST 20654), and S. epidermidis, except for the 30 % aGM-loaded PLLA fiber mats which showed moderate potential for antibacterial activity against S. aureus DMST 20654. The antibacterial activity of the 50 % aGM-loaded PLLA fiber mats was moderate against P. aeruginosa, while the antibacterial activity of the 30 % aGM-loaded PLLA fiber mats was low against P. aeruginosa. In addition, the 50 % aGM-loaded PLLA fiber mats showed low potential for antibacterial activity against E. coli. A variety of xanthones found in the fruit hull of mangosteen, including a-mangostin, b-mangostin, and c-mangostin, have the biological acitivity. Among these, a-mangostin has the most antibacterial acitivity [41]. Since the chemical compounds of the GM extracts depend on the extraction method, the dGM and the aGM extracts might have different chemical compounds. From the results in Table 4, the higher antibacterial activity was observed with most of the aGM-loaded PLLA fiber mats compared to the dGM-loaded PLLA fiber mats. Since a-mangostin is a polar compound, the acetone extracts have more polar than the dichloromethane extracts, which might have more amounts of a-mangostin leading to the higher antibacterial activity. Indirect cytotoxicity evaluation The potential use of the GM-loaded PLLA fiber mats as wound dressings was assessed by investigating the cytotoxicity of these samples and using the neat PLLA fiber mats as an internal control group. The viability of the normal human dermal fibroblasts (NHDF) that had been cultured with the extraction media from these samples in comparison with that of the cells that had been cultured with the fresh culture medium is shown in Fig. 4. The extraction media prepared at three different extraction ratios (i.e., 10, 5, and 0.5 mg mL-1) were investigated. Obviously, the relative viability of the cells that had been cultured with all of the extraction media from the neat PLLA fiber mats ranged between *105 and *112 %, indicating that the neat PLLA fiber mats released no substance in the level that was harmful to the cells. At the lowest extraction ratio investigated (i.e., 0.5 mg mL-1), all the GMloaded PLLA fiber mats were non-toxic to the cells, with the viability of the cells ranged between *104 and *115 %. At the greater concentration of the extraction medium (i.e., 5 and 10 mg mL-1), all the GM-loaded PLLA fiber mats still posed no threat to the cells, as the relative viability of the cells still exceeded the threshold value of at least *80 %, except for both the 30 % dGM-loaded and the 50 % dGMloaded PLLA fiber mats at the extraction ratio of 10 mg mL-1. The relative viability of the cells that had been cultured with the extraction ratio of 10 mg mL-1 from the 30 % dGM-loaded and the 50 % dGM-loaded PLLA fiber mats was lower than the threshold value, indicating that there must be some substances in the crude extract that had been leached off from the fibers in the levels that were harmful to the cells.
123
123
PLLA
50aGM
50dGM
30aGM
30dGM PLLA
50aGM
50dGM
P. aeruginosa
30aGM
30dGM PLLA 50aGM
50dGM
S. aureus ATCC 25923
30aGM
30dGM
PLLA
50aGM
50dGM
S. aureus DMST 20654
Fig. 3 Representative zone of inhibition or clear zone of neat and GM-loaded PLLA fiber mats against some common bacteria
30aGM
30dGM
E. coli
30aGM
30dGM PLLA
50aGM
50dGM
S. epidermidis
Polym. Bull.
Polym. Bull.
*
120
Cell viability (%)
100 80
* *
60 40 20
Control PLLA fiber mats 30%dGM-loaded PLLA fiber mats 50%dGM-loaded PLLA fiber mats 30%aGM-loaded PLLA fiber mats 50%aGM-loaded PLLA fiber mats
0 10 mg/mL
5 mg/mL
0.5 mg/mL
Extraction ratio Fig. 4 Indirect cytotoxicity evaluation of neat and GM-loaded PLLA fiber mats in terms of the viability of normal human dermal fibroblasts (NHDF) that had been cultured with the extraction media from the fibrous materials in comparison with the viability of the cells that had been cultured with fresh culture medium (n = 3). *p \ 0.05 compared with fresh culture medium at any given extraction ratio
Conclusions In present contribution, G. mangostana extract from the fruit hull of G. mangostana Linn. (Guttiferae) (GM) in two types of solvent extraction (i.e., dichloromethane and acetone) was added to the neat PLLA solution [10 % w/v in 7:3 v/v dichloromethane (DCM)/dimethylformamide (DMF)] in various amounts (i.e., 30 and 50 wt % based on the weight of PLLA powder). Both the neat and the GMloaded PLLA fiber mats were successfully prepared by electrospinning process. The cross-sectionally round fibers with smooth surface were obtained. The average diameters of both the neat and the GM-loaded PLLA fiber mats ranged between *0.77 and *1.14 lm. The water retention and mass loss behavior of the GMloaded PLLA fiber mats in both two types of the medium (i.e., A/T/M or S/T/M medium) increased with an increase in the submersion time. Moreover, the water retention and mass loss behavior of these samples after submersion in the S/T/M medium were greater than those in the A/T/M medium. The cumulative amounts of GM released from the GM-loaded PLLA fiber mats in both types of medium increased quite rapidly with increasing submersion time, increased more gradually afterwards, and then reached a plateau value at the longest submersion time investigated. The cumulative amounts of GM released from all the GM-loaded PLLA fiber mats in the S/T/M medium were greater than those in the A/T/M medium. The maximum amounts of GM released from the 30 % dGM-loaded, the 50 % dGM-loaded, the 30 % aGM-loaded, and the 50 % aGM-loaded PLLA fiber mats after submersion in the A/T/M medium were *56, *62, *65, and *70 %, respectively, while those in the S/T/M medium were *63, *67, *73, and *78 %, respectively. The greater cumulative amounts of GM released from the GM-loaded PLLA fiber mats into the S/T/M medium should be due to the water retention and
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Polym. Bull.
the mass loss behaviors of these samples after submersion in the S/T/M medium. Moreover, the cumulative amounts of GM released from the aGM-loaded PLLA fiber mats were greater than those from the dGM-loaded PLLA fiber mats due to the greater solubility of aGM in the medium. The half maximal inhibitory concentrations (IC50) of dGM and aGM were 0.045 and 0.012 mg mL-1, respectively. Moreover, the antioxidant activity based on DPPH assay, of the GM-loaded PLLA fibers remained active even after it had been subjected to a high electrical potential during electrospinning process. The antibacterial activity of the dGM-loaded PLLA fiber mats was greatest against S. aureus DMST 20654, followed by S. epidermidis, E. coli, P. aeruginosa, and S. aureus ATCC 25923, respectively, while that of the aGM-loaded PLLA fiber mats was greatest against S aureus ATCC 25923 and S. epidermidis, followed by S. aureus DMST 20654, P. aeruginosa, and E. coli, respectively. Lastly, from the indirect cytotoxicity results, only the 30 % dGMloaded and the 50 % dGM-loaded PLLA fiber mats at extraction ratio of 10 mg mL-1 were toxic to the normal human dermal fibroblast. Acknowledgments This work was supported by the Thailand Research Fund (Grant Number: MRG5380120). We are grateful to Mae Fah Luang University for partial financial support and laboratory facilities.
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