Chiang Mai J. Sci. 2021; 48(4) : 952-968http://epg.science.cmu.ac.th/ejournal/Contributed Paper

Bacterial Cellulose and Bacterial Cellulose/Chitosan Films Containing Mangosteen Pericarp Extract for Wound DressingsPronpatsorn Moonsungnoen [a,b], Duangjai Ochaikul*[a] and Pathavuth Monvisade [c][a] Department of Biology, Faculty of Science, King Mongkut's Institute of Technology Ladkrabang, Bangkok 10520,

Thailand.[b] Department of Animal Husbandry, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330,

Thailand.[c] Polymer Synthesis and Functional Materials Research Unit, Department of Chemistry, Faculty of Science, King

Mongkut's Institute of Technology Ladkrabang, Bangkok 10520, Thailand.

*Author for correspondence; e-mail: daungjai.oc@kmitl.ac.thReceived: 19 October 2020

Revised: 11 January 2021 Accepted: 14 January 2021

ABSTRACT Appropriate wound dressings for maintaining a moist wound environment, inducing

re-epithelialization and protection from infection have been widely developed. Recently, many biocompatible polymers and bioactive substances have been extensively used for wound healing applications. In this study, bacterial cellulose film (BC-MPE film) and bacterial cellulose/chitosan film (BC/CH-MPE film) containing 1.56 mg/mL of mangosteen pericarp extract (MPE) were prepared. Antibacterial activity, cytotoxicity, physical and mechanical properties of the dry films were investigated. The BC film with MPE presented non-cytotoxic effect after 20 h of exposure to mouse fibroblast cell line (L929). The prepared films performed carrier of bioactive compounds that exhibited antibacterial activity against bacterial infection in burn wounds. The morphology of the films showed the characteristic of ultra-fine network structures with the entrapment of compounds. In addition, the compact structure was observed due to the rapid moisture loss during vacuum drying process. SEM images also showed that BC/CH-MPE film formed layers with chitosan entrapment causing the film to become thicker than BC-MPE film. The formation of MPE and chitosan in modified films was also confirmed by FTIR. The compact structure of BC/CH-MPE film led to the decrease in cumulative release of xanthone, WVTR and WAC. Moreover, the existence of chitosan in BC layers provided more flexible properties than the non-chitosan film. The chitosan addition demonstrated an influence of barrier film to protect the wound. Therefore, BC and chitosan were considered as suitable candidates for wound dressing material and xanthone content of MPE promoted wound healing as an effective therapeutic agent.

Keywords: bacterial cellulose, chitosan, mangosteen pericarp, wound dressings

1. INTRODUCTION Cellulose is the most abundant biopolymer on the earth. It has been mostly focused on cellulose

from plants as the main producers of cellulose [1]. However, several species of bacteria have been

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reported to produce extracellular cellulose such as Acetobacter, Agrobacterium, Pseudomonas, Rhizobium and Sarcina [2]. Among the cellulose producing bacteria, the most efficient cellulose producer is Gluconacetobacter (formerly Acetobacter), which forms membranes on the media surface. Bacterial cellulose (BC) is an attractive candidate in various fields, especially for biomedical applications in terms of high chemical purity, lack of cytotoxicity, biocompatibility, high water holding capacity and fluid systems in the human body cannot digest cellulose [3,4]. Moreover, the properties of polymer can be changed by combining them or adding a second phase to alter the molecule structures [5]. BC-based materials are also easily improved by association with collagen, gelatin, fibroin, chitosan, silver and alginate [6].

In particularly, chitosan, deacetylated product of chitin with alkaline treatment, is the second most abundant biopolymer in nature. The existence of chitosan in nature is discovered by isolation from yeasts and a major component of the fungal cell wall. However, chitosan has been produced on an industrial scale by alkaline deacetylation of crustaceans, mainly shrimps and crab shells [7]. Chitosan is also considered as a good biological application owing to its non-toxicity, non-immunogenicity, biocompatibility, biodegradation and antibacterial activity [8,9]. The properties of chitosan have been studied in variety of biomedical applications such as drug delivery, tissue engineering and wound healing. Furthermore, chitosan can be formed in various forms such as membranes, sponges, gels, microparticles and nanoparticles [10].

Mangosteen (Garcinia mangostana Linn.) is a tropical fruit belonging to the Guttiferae family and known as the queen of fruits. It is widespread cultivated in Southeast Asia, especially Indonesia, Malaysia and Thailand, including other tropical areas. Mangosteen pericarps contain variety of secondary metabolites such as xanthones, tannins, flavonoids, and other bioactive compounds. Among these metabolites, major bioactive

compound is xanthone, which exhibits anti-inflammatory, antioxidant and antimicrobial activity [11,12]. The most abundant xanthone derivatives in the pericarp of mangosteen are α-mangostin and β-mangostin [13]. Thus, several studies have attempted to isolate antimicrobial compounds from plant materials for utilization in pharmaceutical industries to be used as an alternative to antibiotics.

Recently, burn wound infection has become a major concern. However, the use of antibiotics in patients with burn wound infection is more complicated than in other wound types [14]. It is found that about 75% of the cause of death relate to infections rather than osmotic shock and hypovolemia. The most common bacterial isolate from burn wounds in the first 24 h is Staphylococcus epidermidis and presence of Staphylococcus aureus within the first week [15,16], including Pseudomonas aeruginosa, Klebsiella spp. and Escherichia coli [17].

The aim of this study was to develop the properties of BC film by chitosan penetration method with alkali solution and evaluate potential of modified BC film for controlled release of MPE, which was non-toxic to mammalian cells (L929 cells) and presented antibacterial activity.

2. MATERIALS AND METHODS2.1 Materials

Mangosteens and mature coconut water were purchased from local market in Bangkok, Thailand. Xanthone (purity 97%) and calcium chloride were purchased from Sigma-Aldrich Pte. Ltd., Singapore. Chitosan (CS) with degree of acetylation and molecular weight of 85% and 270 kDa, respectively, was purchased from Eland Co., Ltd., Thailand. 95% ethanol was purchased from Liquor Distillery Organization, Thailand. Nutrient agar (NA) and Mueller-Hinton broth (MHB) were purchased from Sisco Research Laboratories Pvt. Ltd., India. Phosphate buffer saline (PBS) powder was purchased from Vivantis Technologies Sdn. Bhd., Malaysia. Resazurin

Chiang Mai J. Sci. 2021; 48(4)954

tablets were purchased from VWR International LLC., USA. Sodium hydroxide was purchased form Ajax Finechem, Australia.

2.2 Mangosteen Pericarp Extraction The extraction was carried out by adapting

from the literature [18]. In detail, the pericarps of mangosteen (G. mangostana L.) were collected and washed several times. After cutting into small pieces, mangosteen pericarps were subjected to heat using hot air-dryer (INB 500, Memmert, Germany) at 40 °C for 3 days before grinding to fine powder. The powder (100 g) and 95% ethanol (500 mL) were mixed under agitation at room temperature. After 7 days, the supernatant segment was filtrated. The extraction was repeated by adding 95% ethanol into residue powder from filtration, according to the previous condition. A mixture of two portions of supernatant was evaporated (Heidolph, Germany) to yield the mangosteen pericarp extract (MPE). The MPE was stored to dryness in a desiccator (%RH = 0). The amount of xanthone in MPE was determined by dissolving MPE in 95% ethanol and then measured at the wavelength of 254 nm using UV-vis spectrophotometer (UV-1800, Shimadzu, Japan) with using the calibration curve of xanthone.

2.3 Determination of the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC)2.3.1 Preparation of inoculum suspension

The test microorganisms in this study were Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228, Bacillus cereus DMST 5040, Bacillus subtilis TISTR 1248, Micrococcus luteus ATCC 9431, Salmonella Typhimurium TISTR 1469, Pseudomonas aeruginosa ATCC 27859, Serratia marcescens TISTR 1354, Klebsiella pneumoniae ATCC 13883 and Escherichia coli ATCC 25922. All strains were cultured on Nutrient agar (NA) and incubated at 37 °C for 24 h. The colonies of each strain were transferred into 0.85% sterile saline to

achieve the turbidity of a 0.5 McFarland standard. Inoculum was then diluted with Mueller-Hinton broth (MHB) 1:200 (approximately 7.5 x 105 CFU/mL).

2.3.2 MIC test MIC values were determined by broth

micro dilution assay [19]. Fifty microliters of MPE solution, two-fold dilution with MHB, was added into 96-well plate. Then, 50 µL of adjusted microorganism was added into each well with the final concentration of MPE ranging from 0.001-100 mg/mL. The panel was placed at 37 °C for 24 h. Then, 10 µL of resazurin indicator was added and incubated at 37 °C for 3 h. Clindamycin and gentamicin (50 µg/mL) were used as positive control to determine the sensitivity of gram-positive and gram-negative bacteria, respectively. MIC was defined as the lowest concentration of MPE, which prevented the color changing of resazurin after incubation.

2.3.3 MBC testEach well which exhibited no bacterial

growth or no color changing was selected to inoculate on Mueller-Hinton agar (MHA) surface by using the streak plate technique, according to MIC results. An inoculated plate was incubated at 37 °C overnight. The lowest MPE concentration with no colonies growth was considered as MBC.

2.4 Preparation of Bacterial Cellulose Membranes

Bacterial cellulose (BC) membranes were obtained from the cultivation of Acetobacter xylinum TISTR 976 on glucose-yeast extract-calcium carbonate agar (GYC) seed medium for 3 days. Two loopfuls of colonies were transferred into 50 mL of coconut water medium containing 5% (w/v) sucrose, 0.1% (w/v) ammonium sulfate and 1% (v/v) acetic acid, the volume of medium was adjusted with mature coconut water. The starter culture was carried out under static condition at 30 °C for 2 days. BC production

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was performed in 250 mL Erlenmeyer flasks containing 45 mL of coconut water medium. Each flask was inoculated with 10% of starter, and then statically incubated at 30 °C for 5 days. After incubation, BC membranes were rinsed with running water and pressed with the press machine into sheets. BC membranes were subsequently treated with 1% (w/v) NaOH solution for 24 h to eliminate microbial cells. BC membranes were further treated in boiling water for 30 min and washed with distilled water until the pH became neutral. The membranes were pressed to squeeze excess water out and sterilized (Autoclave ES-315, Tomy Seiko, Japan) at 121 °C for 15 min. 2.5 Preparation of MPE Loaded BC Film

BC membrane was immersed into 100 mL of ethanolic MPE solution at room temperature for 24 h. The ethanolic MPE solution was prepared by using the MBC value and other 5 concentrations, which were provided by two-fold increase from the MBC value. Afterward, the membranes were dried under vacuum drying oven (VD 53, Binder, Germany) at 35 °C for 3 h. These films were stored in dry place and protected from light.

2.6 Antibacterial Activity of BC-MPE FilmColony suspension of the test microorganisms

was cultured on NA and incubated at 37 °C for 24 h. The colonies were directly transferred into 0.85% sterile saline and adjusted to match a 0.5 McFarland standard. A sterile cotton swab was dipped into the inoculum suspension, slightly pressed on the wall tube to remove excess inoculum, and inoculated on the surface of MHA. The entire plate was then swabbed from edge to edge along with rotating the plate 60° and repeated swabbing for 3 times. Dry films were punched into a circular shape with 6 mm diameter. Then, the film specimens were placed on the inoculated MHA plate and incubated at 37 °C for 24 h. BC film with clindamycin at concentration of 50 µg/mL was used as positive

control. After incubation, the inhibition zones were measured from the diameter of clear zone surrounding the films in mm.

2.7 MTT AssayThe evaluation of MTT assay was performed

in accordance with ISO 10993-5: 2009 Biological evaluation of medical devices - Part 5: Tests for in vitro cytotoxicity. L929 cells were seeded into 96-well plate to achieve 1x105 cells/100 µL of Dulbecco’s Modified Eagle Medium (DMEM) per well. The plate was incubated at 37 °C for 24 h with 5% CO2. Culture medium was discarded and then treated with 100 µL of test sample extract per well at 37 °C for 20 h. The sample extraction was prepared by macerating 0.01 g of each film in 10 mL of DMEM medium with 5% FBS for 24 h, then the sample was filtrated through sterilized syringe filter with 0.45 µm pore size. After incubation, 10 µL of MTT solution was added into each well. The plate was incubated at 37 °C for 4 h with 5% CO2. MTT solution was discarded, then 100 µL of 100% DMSO : 10% SDS was added to dissolve formazan. The cell viability was determined by microplate reader (Anthos Multiread 400, Biochrom, UK) equipped with 570 nm. If the cell viability of the test sample is less than 70% of the blank, the sample is considered as toxic. Cytotoxicity was calculated using the following equation (1).

Cytotoxicity (%) =

7

with 570 nm. If the cell viability of the test sample is less than 70% of the blank, the sample is 168

considered as toxic. Cytotoxicity was calculated using the following equation (1). 169

Cytotoxicity (%) = A-BA

× 100 (1) 170

Where A is the mean value of the measured optical density of the blanks and B is the mean 171

value of the measured optical density of the test samples. 172

173

2.8 Preparation of Modified BC Film 174

Bacterial cellulose/chitosan film (BC/CH-MPE film) was prepared by immersing purified BC 175

membrane into 1% (w/v) chitosan in 1% (v/v) acetic acid solution for 24 h. After that, BC membrane 176

was treated with 1% (w/v) NaOH solution for 24 h at room temperature and washed with running water 177

until pH became neutral. Each of BC membranes was immersed into 100 mL of ethanolic MPE solution 178

at room temperature for 24 h and dried under vacuum condition at 35 °C. BC-MPE film was prepared 179

similarly to the method described above without using chitosan/alkali treatment. The BC film without 180

the extract was served as a control. To prepare chitosan control film (CH film), chitosan powder (1% 181

w/v) was dissolved in 1% (v/v) acetic acid aqueous solution. The solution was degassed by 182

ultrasonicator (Tru-Sweep 2800HT, Crest Ultrasonics, USA) for an hour before casting. The sample 183

was dried at 50 °C for 24 h. CH film was then peeled off from the dish and conditioned in a desiccator. 184

185

2.9 SEM Analysis 186

The surface and cross-sectional morphology of the films were analyzed by Scanning electron 187

microscope (Quanta 250, FEI, USA). The dry specimens, a size of approximately 10 x 10 mm, were 188

mounted on a specimen stab with carbon tape and coated with gold before the examination on a 189

microscope. The cross-section specimens were prepared by fracturing the sample in liquid nitrogen. 190

The samples were then mounted onto the holder for cross-sectional observation. 191

192

2.10 FTIR Analysis 193

(1)

Where A is the mean value of the measured optical density of the blanks and B is the mean value of the measured optical density of the test samples.

2.8 Preparation of Modified BC FilmBacterial cellulose/chitosan film (BC/CH-

MPE film) was prepared by immersing purified BC membrane into 1% (w/v) chitosan in 1% (v/v) acetic acid solution for 24 h. After that, BC membrane was treated with 1% (w/v) NaOH

Chiang Mai J. Sci. 2021; 48(4)956

solution for 24 h at room temperature and washed with running water until pH became neutral. Each of BC membranes was immersed into 100 mL of ethanolic MPE solution at room temperature for 24 h and dried under vacuum condition at 35 °C. BC-MPE film was prepared similarly to the method described above without using chitosan/alkali treatment. The BC film without the extract was served as a control. To prepare chitosan control film (CH film), chitosan powder (1% w/v) was dissolved in 1% (v/v) acetic acid aqueous solution. The solution was degassed by ultrasonicator (Tru-Sweep 2800HT, Crest Ultrasonics, USA) for an hour before casting. The sample was dried at 50 °C for 24 h. CH film was then peeled off from the dish and conditioned in a desiccator.

2.9 SEM AnalysisThe surface and cross-sectional morphology

of the films were analyzed by Scanning electron microscope (Quanta 250, FEI, USA). The dry specimens, a size of approximately 10 x 10 mm, were mounted on a specimen stab with carbon tape and coated with gold before the examination on a microscope. The cross-section specimens were prepared by fracturing the sample in liquid nitrogen. The samples were then mounted onto the holder for cross-sectional observation.

2.10 FTIR AnalysisFTIR spectra of films were recorded

with Fourier transform infrared spectrometer (IRTracer-100, Shimadzu, Japan) at room temperature. All film samples were scanned from 700 to 4000 cm-1 with 4.0 cm-1 resolution.

2.11 In vitro Xanthone Release Study2.11.1 Actual xanthone content

The films were cut into a size of 8 x 4 cm. Each film was shaken along with 50 mL of 95% ethanol under speed of 200 rpm at 30 °C for 24 h. Xanthone release was quantified by measuring the absorption at the wavelength of 254 nm using

UV-vis spectrophotometer and evaluated by using the calibration curve of xanthone.

2.11.2 In vitro xanthone release In vitro xanthone release was investigated

by immersing each specimen into 50 mL of phosphate buffer saline (PBS), pH 7.4, under 50 rpm agitation at 37 °C for 120 h. The sample was withdrawn with periodic sampling. After each sampling, an equal amount of fresh PBS was added. The amount of xanthone in the sample solution was measured at the wavelength of 254 nm.

2.11.3 Determination of xanthone release efficiency

To determine the efficiency of bioactive compound release from BC-MPE and BC/CH-MPE films. The film specimens were applied on an inoculated MHA plate and then incubated at 37 °C for 24 h. The diameter of clear zones was recorded in mm.

2.12 Water Vapor Transmission Rate (WVTR)The test was performed in accordance with

the ASTM E 96-95 standard test methods for water vapor transmission of materials. The film was mounted on the cup with a diameter of 6.70 cm containing 10 g of calcium chloride. The relative humidities of the desiccant and the chamber were maintained at %RH = 0 and %RH = 75, respectively and temperature of 30 °C was used. The movement of water vapor through the film into the desiccant was determined by periodic weighing for 7 days. A graph was plotted at least 6 points of weight against elapsed time, the slope of a straight line was the rate of water vapor transmission. WVTR was calculated using the following equation (2). The results were expressed as the mean and standard deviation of triplicate data.

WVTR (g/m2·day) =

9

rate of water vapor transmission. WVTR was calculated using the following equation (2). The results 221

were expressed as the mean and standard deviation of triplicate data. 222

WVTR (g/m2·day) = G/tA

(2) 223

Where G is the weight change (g), t is time (day), G/t is the slope of the straight line (g/day) 224

and A is the cup mouth area (m2) 225

226

2.13 Water Absorption Capacity (WAC) 227

WAC test was performed by immersing the pre-weighted dry film (20 x 20 mm) into 20 mL of 228

PBS solution (pH 7.4) at room temperature until the film reached an equilibrium point. The film was 229

then moved out from the solution. After wiping with a filter paper (Whatman qualitative filter paper, 230

grade 4, GE Healthcare, UK) to remove any excess liquid on the surface of the film, swollen film was 231

weighed. The percentage of WAC was calculated using the following equation (3). 232

WAC (%) = W-WdWd

× 100 (3) 233

Where W is the weight of swollen film and Wd is the weight of dry film. 234

235

2.14 Mechanical Properties 236

Mechanical properties were performed in accordance with the ASTM D 882-02 standard test 237

methods for tensile properties of thin plastic sheeting. Specimens were measured by a universal testing 238

machine (LR5K, Lloyd instruments, UK). Films were run on dumbbell-shaped and gauge length of the 239

specimens was 26 mm. Materials were tested with 7 specimens in each examination. A preload force 240

of 0.5 N was used and the speed of testing was 50 mm/min. This test method covered the determination 241

of tensile strength, young’s modulus and elongation at break. 242

243

2.15 Statistical Analysis 244

All Data were presented as the mean values with error by standard deviation. The experiments 245

were analyzed with one-way analysis of variance (One-way ANOVA) using IBM SPSS Statistics 246

(2)

Chiang Mai J. Sci. 2021; 48(4) 957

Where G is the weight change (g), t is time (day), G/t is the slope of the straight line (g/day) and A is the cup mouth area (m2)

2.13 Water Absorption Capacity (WAC)WAC test was performed by immersing the

pre-weighted dry film (20 x 20 mm) into 20 mL of PBS solution (pH 7.4) at room temperature until the film reached an equilibrium point. The film was then moved out from the solution. After wiping with a filter paper (Whatman qualitative filter paper, grade 4, GE Healthcare, UK) to remove any excess liquid on the surface of the film, swollen film was weighed. The percentage of WAC was calculated using the following equation (3).

WAC (%) =

9

rate of water vapor transmission. WVTR was calculated using the following equation (2). The results 221

were expressed as the mean and standard deviation of triplicate data. 222

WVTR (g/m2·day) = G/tA

(2) 223

Where G is the weight change (g), t is time (day), G/t is the slope of the straight line (g/day) 224

and A is the cup mouth area (m2) 225

226

2.13 Water Absorption Capacity (WAC) 227

WAC test was performed by immersing the pre-weighted dry film (20 x 20 mm) into 20 mL of 228

PBS solution (pH 7.4) at room temperature until the film reached an equilibrium point. The film was 229

then moved out from the solution. After wiping with a filter paper (Whatman qualitative filter paper, 230

grade 4, GE Healthcare, UK) to remove any excess liquid on the surface of the film, swollen film was 231

weighed. The percentage of WAC was calculated using the following equation (3). 232

WAC (%) = W-WdWd

× 100 (3) 233

Where W is the weight of swollen film and Wd is the weight of dry film. 234

235

2.14 Mechanical Properties 236

Mechanical properties were performed in accordance with the ASTM D 882-02 standard test 237

methods for tensile properties of thin plastic sheeting. Specimens were measured by a universal testing 238

machine (LR5K, Lloyd instruments, UK). Films were run on dumbbell-shaped and gauge length of the 239

specimens was 26 mm. Materials were tested with 7 specimens in each examination. A preload force 240

of 0.5 N was used and the speed of testing was 50 mm/min. This test method covered the determination 241

of tensile strength, young’s modulus and elongation at break. 242

243

2.15 Statistical Analysis 244

All Data were presented as the mean values with error by standard deviation. The experiments 245

were analyzed with one-way analysis of variance (One-way ANOVA) using IBM SPSS Statistics 246

(3)

Where W is the weight of swollen film and Wd is the weight of dry film. 2.14 Mechanical Properties

Mechanical properties were performed in accordance with the ASTM D 882-02 standard test methods for tensile properties of thin plastic sheeting. Specimens were measured by a universal testing machine (LR5K, Lloyd instruments, UK). Films were run on dumbbell-shaped and gauge length of the specimens was 26 mm. Materials were tested with 7 specimens in each examination. A preload force of 0.5 N was used and the speed of testing was 50 mm/min. This test method covered the determination of tensile strength, young’s modulus and elongation at break.

2.15 Statistical AnalysisAll Data were presented as the mean values

with error by standard deviation. The experiments were analyzed with one-way analysis of variance (One-way ANOVA) using IBM SPSS Statistics version 22. The comparison of mean values was analyzed by Duncan’s Multiple Range Test (DMRT) at 95% confidence interval (p<0.05).

3. RESULTS AND DISCUSSION3.1 Extraction Yield of MPE and Amount of Xanthone

The appearance of the MPE was red-brown color and highly sticky texture. The extraction yield of MPE was 21.69±0.96% (w/w of dry powder) and the amount of total xanthone in MPE was 56.30±0.01% (w/w of extraction yield).

3.2 MIC and MBC MIC was defined as the lowest concentration

of MPE active against M. luteus ATCC 9431, B. cereus DMST 5040, B. subtilis TISTR 1248, S. aureus ATCC 25923 and S. epidermidis ATCC 12228 at concentrations of 0.05, 0.05, 0.10, 0.10 and 0.20 mg/mL, respectively. The MBC value, the lowest concentration of MPE which resulted in killing bacteria, of MPE against gram-positive bacteria was found to be 0.39 mg/mL. In addition, all bacterial strains were highly susceptible to low dose of antibiotics. However, the extract did not show any activity against gram-negative bacteria. The bioactive efficiency of MPE was previously reported that MPE had an activity against gram-positive bacteria, but no inhibitory effect on gram-negative bacteria [20]. This might have been due to lack of specificity of active functional groups. Narasimhan et al. [21] informed that the synthetic derivatives of α-mangostin from mangosteen pericarp, which the core structure was altered through semi-synthetic modification, showed more bioactive properties and antimicrobial activity than the primary natural molecule. The report indicated that modified α-mangostin exhibited antibacterial activity against both gram-positive and gram-negative bacteria and antifungal activity. Therefore, the functional groups and purity of xanthone derivatives were considered as essential for biological activity enhancement.

3.3 Antibacterial Activity of BC-MPE Films According to the MBC result, the values

of 0.39-12.50 mg/mL of MPE were carried

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out for BC preparation. As shown in Table 1, BC film without MPE exhibited no zone of inhibition, which BC generally had no impact on the antibacterial activity. Whereas BC-MPE films had the potential of bioactive release and antibacterial activities. BC-MPE film with loading of MPE at concentration of 12.50 mg/mL promoted the maximum resistance to gram-positive bacteria i.e., M. luteus ATCC 9431, B. cereus DMST 5040, B. subtilis TISTR 1248, S. aureus ATCC 25923 and S. epidermidis ATCC 12228. Their zones of inhibition were 12.12±0.67, 10.80±0.27, 9.85±0.44, 11.65±0.79 and 12.80±0.39 mm, respectively. Whereas the reduction of the MPE concentrations resulted in the decrease in size of the inhibition zones. BC film with 0.39 mg/mL of the MPE, the MBC value, showed inhibition on M. luteus ATCC 9431, B. cereus DMST 5040, S. aureus ATCC 25923 and S. epidermidis ATCC 12228, except for B. subtilis TISTR 1248. For inhibition of sporulation and cell growth in B. subtilis TISTR 1248, it needed at least 0.78 mg/mL of the MPE loading in BC film. Generally, the spores of Bacillus species are

highly resistant to environmental stresses such as wet and dry heat, radiation, antibiotic, dehydration and disinfectants. Sporulation in Bacillus subtilis is a massively complex process [22,23]. The multiple membrane layers of the spore are spore coating, spore shell, outer membrane, cortex, cell wall and inner membrane, which the nucleus is surrounded [24].

3.4 CytotoxicityBC-MPE films at various MPE concentrations

of 0.78-12.50 mg/mL were tested for cytotoxicity assessment. The cytotoxicity was determined for the biological response on L929 cells by an indirect MTT test. As shown in Figure 1, the MPE concentrations ranging from 3.13-12.50 mg/mL had a cytotoxic potency since the viabilities of L929 cells were reduced over 70% of the blank, according to the international standard for biological evaluation of medical devices (ISO 10993-5). Hence, this study was focused on the films with an acceptable level of toxicity to mammalian cells and providing good inhibition activity. The films with MPE at the concentrations

Table 1. Antibacterial activity of the BC-MPE films with various concentrations of MPE.

Concentrations of MPE

(mg/mL)

Zone of inhibition in mm

M. l

uteu

s A

TC

C 9

431

B. c

ereu

s D

MST

504

0

B. su

btili

s T

IST

R 1

248

S. a

ureu

s A

TC

C 2

5923

S. e

pide

rmid

isA

TC

C 1

2228

12.50 12.12±0.67b 10.80±0.27b 9.85±0.44b 11.65±0.79b 12.80±0.39b

6.25 11.20±0.36c 10.18±0.33b 9.51±0.34b 10.77±0.35c 11.79±0.44c

3.13 10.23±0.39d 9.10±0.92c 8.61±0.36c 9.63±0.71d 10.81±0.72d

1.56 10.11±0.05d 8.88±0.53c 8.08±0.42c 9.46±0.21de 10.47±0.23de

0.78 9.47±0.41e 7.73±0.48d 7.34±0.32d 8.70±0.48e 9.73±0.51e

0.39 8.40±0.08f 6.81±0.18e 6.00±0.00e 7.92±0.24f 8.85±0.61f

0 6.00±0.00g 6.00±0.00f 6.00±0.00e 6.00±0.00g 6.00±0.00g

Positive control 22.45±0.38a 18.34±0.19a 16.64±0.27a 20.45±0.13a 21.21±0.18a

Note: Diameter of film = 6 mm, Different letters in the same column indicate significant difference using one way ANOVA (p<0.05, n=3, Data are mean±SD).

Chiang Mai J. Sci. 2021; 48(4) 959

of 1.56 mg/mL (13.60±2.54 %cytotoxicity) and 0.78 mg/mL (1.00±0.73 %cytotoxicity) were antibacterial films and non-toxic. Thus, the film with 1.56 mg/mL of MPE was selected for the further testing.

The results were consistent with the previous studies showing that mangosteen extract coated polypropylene filter for face mask provided to be non-toxic to L929 mouse fibroblast cells at the concentrations of 0.01, 0.1 and 1 mg/mL and exhibited bacterial reduction [25]. Boonmak et al. [26] also informed that bioactive wound dressing in the type of PVAc spray-on dressing containing different concentrations of mangosteen extract (1%, 2% and 3% w/v) was non-toxic to L929 cells, HaCaT cells and NHFs cells. It should be noted that, in this study, cytotoxic effects of the films have come from in vitro experiment using L929 cells. Although the film with 1.56 mg/mL of MPE has been proved to be suitable for medical device through in vitro testing, further clinical studies are needed to evaluate the properties of ideal wound dressings.

L929 or mouse fibroblast cell line is a common cell of connective tissue with spindle

shaped morphology that strongly adheres to the surface of culture well [27,28]. As usual, the adhesion and proliferation of L929 cells are observed during incubation with culture medium [29]. Figure 2 shows the morphology of L929 cells after exposure to various concentrations of MPE. The morphology of the untreated cells revealed the spindle shaped cells. When the treatment medium with small amount of sample extract exposed to the cells, the morphology of L929 cells was relatively similar to the untreated cells. However, the appearance of the cells which were treated with high concentration of the extract did not preserve its normal morphology but exhibited a spherical shape. Moreover, floating cells were observed due to cytotoxic effect of the test sample extract.

3.5 SEM Analysis To provide an appropriate wound dressing

material for the particular wound type, natural polymers were suggested in combination. BC/CH-MPE film and BC-MPE film were prepared, both of films were carried out with 1.56 mg/mL of MPE. The surface morphologies

13

the morphology of L929 cells after exposure to various concentrations of MPE. The morphology of the 316

untreated cells revealed the spindle shaped cells. When the treatment medium with small amount of 317

sample extract exposed to the cells, the morphology of L929 cells was relatively similar to the untreated 318

cells. However, the appearance of the cells which were treated with high concentration of the extract 319

did not preserve its normal morphology but exhibited a spherical shape. Moreover, floating cells were 320

observed due to cytotoxic effect of the test sample extract. 321

322

Figure 1. Cytotoxicity of BC-MPE films on L929 mouse fibroblast cells after exposure to the film 323

extract for 20 h (The data are expressed as the mean±SD, n=3). 324

325

326

0

20

40

60

80

100

0.78 1.56 3.13 6.25 12.50

% C

ytot

oxic

ity

Concentrations of film extract (mg/mL)

aab

c

d

A B C

D E F

Figure 1. Cytotoxicity of BC-MPE films on L929 mouse fibroblast cells after exposure to the film extract for 20 h (The data are expressed as the mean±SD, n=3).

Chiang Mai J. Sci. 2021; 48(4)960

and cross-sectional images are shown in Figure 3. Ultra-fine network structure and tight network of BC fibers generated a dense mesh with small pore size and compact layer. The apparent particles of MPE were observed on the surface of BC-MPE film, which also loosened the packed layers of BC in cross-sectional image. A previous study also reported that the extract penetration was the cause of the loosening of the packed structures and relaxation of polymer chain [30]. In general, bacterial cellulose exhibits high water absorption capacity in the wet state [7]. Thus, the extract particles were entrapped not only on the surface of films but also between the fiber layers. BC/CH-MPE film also showed that the network structure and the fibers became thicker than BC film and BC-MPE film due to chitosan penetration and coating on BC fibers. Normally, chitosan is completely soluble in aqueous acid solution. Additionally, the acidic chitosan solution can be precipitated by adding an alkaline solution, which releases the hydroxide

ion into solution so that protonated amino group (-NH3

+) of chitosan solution is deprotonated to form a chitosan precipitate [31]. Cross-sectional image of BC/CH-MPE showed the densified layers by insertion of chitosan on the cellulose fiber layers. Unlike others, CH film was smooth, homogeneous and compact structure with neither cracks nor pores, while the cross-sectional image did not show the layer by layer assembly.

3.6 FTIR AnalysisThe spectra of films are presented in

Figure 4. The FTIR spectra of BC film showed characteristic peak in the region of 3400-3300 cm-1 for O-H stretching of hydroxyl group, peak at 2895 cm-1 for C-H stretching and peak near 1640 cm-1 for carbonyl group [32-34]. The addition of the MPE in the films also showed additional peaks at 1624 cm-1 for C=C stretching of aromatic ring and 1442 cm-1 for the presence of O-H bending [35]. For BC/CH-MPE film, the broad peak from 3450-3200 cm-1 was assigned to

13

the morphology of L929 cells after exposure to various concentrations of MPE. The morphology of the 316

untreated cells revealed the spindle shaped cells. When the treatment medium with small amount of 317

sample extract exposed to the cells, the morphology of L929 cells was relatively similar to the untreated 318

cells. However, the appearance of the cells which were treated with high concentration of the extract 319

did not preserve its normal morphology but exhibited a spherical shape. Moreover, floating cells were 320

observed due to cytotoxic effect of the test sample extract. 321

322

Figure 1. Cytotoxicity of BC-MPE films on L929 mouse fibroblast cells after exposure to the film 323

extract for 20 h (The data are expressed as the mean±SD, n=3). 324

325

326

0

20

40

60

80

100

0.78 1.56 3.13 6.25 12.50

% C

ytot

oxic

ity

Concentrations of film extract (mg/mL)

aab

c

d

A B C

D E F

Figure 2. Morphology of L929 mouse fibroblast cells under microscopic investigation after 20 h of exposure to film extract at the concentrations of 0.78 mg/mL (A), 1.56 mg/mL (B), 3.13 mg/mL (C), 6.25 mg/mL (D), 12.50 mg/mL (E) and untreated cells (F).

Chiang Mai J. Sci. 2021; 48(4) 961

15

351

Figure 3. SEM images of surface and cross section of (A) BC film, (B) BC-MPE film, (C) BC/CH-352

MPE film and (D) CH film at a magnification of 20,000x. 353

354

3.6 FTIR Analysis 355

The spectra of films are presented in Figure 4. The FTIR spectra of BC film showed 356

characteristic peak in the region of 3400-3300 cm-1 for O-H stretching of hydroxyl group, peak at 2895 357

cm-1 for C-H stretching and peak near 1640 cm-1 for carbonyl group [32-34]. The addition of the MPE 358

A

B

C

D

Figure 3. SEM images of surface and cross section of BC film (A), BC-MPE film (B), BC/CH-MPE film (C) and CH film (D) at a magnification of 20,000x.

Chiang Mai J. Sci. 2021; 48(4)962

O-H and N-H stretching vibrations and peaks of the characteristic functional groups of chitosan shifted to wavenumber at 1641 cm-1 for amide I and 1581 cm-1 for amide II [36]. However, the characteristic peak of MPE was not clearly observed owing to overlapping peaks of chitosan and extract. Therefore, SEM images and film appearance were required for the observation of the characteristic of MPE on BC/CH-MPE film.

3.7 In vitro Release In this study, the actual amount of xanthone

in each film was investigated by immersion the film specimen in 95% ethanol to dissolve the extract. The actual amounts of xanthone in BC-

MPE and BC/CH-MPE films were 94.84 and 86.23 µg/cm2, respectively. The periodic releases of xanthone from the films were investigated in PBS solution (pH 7.4). The cumulative releases of xanthone are shown in Figure 5. The results presented that both BC-MPE and BC/CH-MPE films revealed an initial burst-release in the first 8 h particularly at 15 min after immersion. Both films continuously released throughout 120 h period. The total xanthone release from BC-MPE and BC/CH-MPE films were 18.72±0.53 and 14.51±0.58 µg/cm2 at 120 h, respectively. The results showed that in vitro release of BC-MPE film was slightly higher than that of BC/CH-MPE film with a statistically significant difference

16

in the films also showed additional peaks at 1624 cm-1 for C=C stretching of aromatic ring and 1442 359

cm-1 for the presence of O-H bending [35]. For BC/CH-MPE film, the broad peak from 3450-3200 cm-360

1 was assigned to O-H and N-H stretching vibrations and peaks of the characteristic functional groups 361

of chitosan shifted to wavenumber at 1641 cm-1 for amide I and 1581 cm-1 for amide II [36]. However, 362

the characteristic peak of MPE was not clearly observed owing to overlapping peaks of chitosan and 363

extract. Therefore, SEM images and film appearance were required for the observation of the 364

characteristic of MPE on BC/CH-MPE film. 365

366

Figure 4. FTIR spectra of BC film, BC-MPE film, BC/CH-MPE film and CH film. 367

368

3.7 In Vitro Release 369

In this study, the actual amount of xanthone in each film was investigated by immersion the 370

film specimen in 95% ethanol to dissolve the extract. The actual amounts of xanthone in BC-MPE and 371

BC/CH-MPE films were 94.84 and 86.23 µg/cm2, respectively. The periodic releases of xanthone from 372

the films were investigated in PBS solution (pH 7.4). The cumulative releases of xanthone are shown 373

0

4

8

12

16

20

700120017002200270032003700

3344

3344

3342

3354

2895

2897

2916

2918

1641

1624

1641

1631

1442

1581

1543

1421

BC film

BC-MPE film

BC/CH-MPE film

CH film

Wavenumber (cm-1)Figure 4. FTIR spectra of BC film, BC-MPE film, BC/CH-MPE film and CH film.

Chiang Mai J. Sci. 2021; 48(4) 963

(p<0.05). A previous report found that a major problem of drug delivery system was excessive drug release in the initial release may be toxic and wasteful. On the other hand, a loading dose or high burst release was required for some drugs [37]. However, drug release systems frequently had the initial burst release phase due to rapid diffusion mechanisms of surface associated drug molecules [38].

The xanthone release efficiency of BC-MPE film was compared with that of BC/CH-MPE film. Antibacterial activities of both films are presented in Table 2. Those films showed antibacterial activity against M. luteus ATCC 9431, B. cereus DMST 5040, B. subtilis TISTR 1248, S. aureus ATCC 25923 and S. epidermidis ATCC 12228. However, neither BC film nor CH film showed inhibition zones surrounding the films, but CH film demonstrated inhibitory effect on the contact surface only.

3.8 Water Vapor Transmission Rate (WVTR)WVTR analysis was assessed with 35.27 cm2

of the mount area, which was defined as the testing area. The WVTR of each film is shown in Table 3.

The WVTR of CH film was found to be higher than those of the test films due to the hydrogen bonding in chitosan molecules, homogeneous structure and hydrophilic character, that led to high moisture absorption and an increase of the WVTR [39,40]. Moreover, The WVTR was dependent on several factors. The most obvious factor was the thickness of polymer layers. In particular, as the thickness of polymer layers was increased, there was the reduction in WVTR [41]. The results indicated that the WVTR of BC-MPE film was higher than that of BC/CH-MPE film with a statistically significant difference (p<0.05). The film specimens appeared to have a compact form, which was the result of rapid moisture loss during vacuum drying process, thus water vapor passing through the film was slightly low [42]. For BC-MPE film, the formation of MPE aggregates was found to prevent the vapor movement through the films. When adding chitosan, BC/CH-MPE film revealed more compact and thicker layer than those of test films. It could be attributed to the penetration of chitosan onto the porous structures of BC causing an obstruction of water vapor transmission.

17

in Figure 5. The results presented that both BC-MPE and BC/CH-MPE films revealed an initial burst-374

release in the first 8 h particularly at 15 min after immersion. Both films continuously released 375

throughout 120 h period. The total xanthone release from BC-MPE and BC/CH-MPE films were 376

18.72±0.53 and 14.51±0.58 µg/cm2 at 120 h, respectively. The results showed that in vitro release of 377

BC-MPE film was slightly higher than that of BC/CH-MPE film with a statistically significant 378

difference (p<0.05). A previous report found that a major problem of drug delivery system was 379

excessive drug release in the initial release may be toxic and wasteful. On the other hand, a loading 380

dose or high burst release was required for some drugs [37]. However, drug release systems frequently 381

had the initial burst release phase due to rapid diffusion mechanisms of surface associated drug 382

molecules [38]. 383

The xanthone release efficiency of BC-MPE film was compared with that of BC/CH-MPE film. 384

Antibacterial activities of both films are presented in Table 2. Those films showed antibacterial activity 385

against M. luteus ATCC 9431, B. cereus DMST 5040, B. subtilis TISTR 1248, S. aureus ATCC 25923 386

and S. epidermidis ATCC 12228. However, neither BC film nor CH film showed inhibition zones 387

surrounding the films, but CH film demonstrated inhibitory effect on the contact surface only. 388

389

Figure 5. Cumulative release profile of xanthone from the films in PBS solution; (■) BC-MPE film, 390

(▲) BC/CH-MPE film, () BC film and () CH film (The data are expressed as the mean±SD, n=3). 391

392

02468

101214161820

0 12 24 36 48 60 72 84 96 108 120

Cum

ulat

ive

rele

ase

(µg/

cm2 )

Time (h)

Figure 5. Cumulative release profile of xanthone from the films in PBS solution; (■) BC-MPE film, (▲) BC/CH-MPE film, (♦) BC film and (●) CH film (The data are expressed as the mean±SD, n=3).

Chiang Mai J. Sci. 2021; 48(4)964

Table 3. WVTR assessment of different wound dressings.

Sample Manufacturer Thickness (mm)

WVTR(g/m2·day) Reference

Comfeel® Coloplast A/S 1.27±0.11 285±8 [44]

Dermiflex® Johnson & Johnson 2.41±0.05 76±5 [44]

Duoderm CGF® ConvaTec Ltd. 2.27±0.08 120±19 [44]

Granuflex E® Extra Thin ConvaTec Ltd. 0.50±0.05 216±6 [44]

Restore Cx® Hollister Inc. 1.19±0.08 476±18 [44]

Tegasorb® ConvaTec Ltd. 2.10±0.15 136±15 [44]

Bioclusive® Johnson & Johnson 0.077±0.01 394±12 [44]

Askina® Braun Co. 3.32 586 [45]

Suprasorb® Lohmann & Rauscher Co. Ltd. 4.82 724 [45]

Polymem® Ferris Manufacturing Co. 2.74 550 [45]

BC-MPE film - 0.025±0.001 152.68±5.61 This study

BC/CH-MPE film - 0.115±0.012 115.51±12.53 This study

BC film - 0.022±0.002 161.84±14.23 This study

CH film - 0.019±0.003 183.74±8.92 This study

Table 2. Antibacterial activity of BC-MPE film, BC/CH-MPE film, BC film and CH film.

Sample

Zone of inhibition in mm

M. l

uteu

s A

TC

C 9

431

B. c

ereu

s D

MST

504

0

B. su

btili

s T

IST

R 1

248

S. a

ureu

s A

TC

C 2

5923

S.

epid

erm

idis

AT

CC

122

28

BC-MPE film 9.72±0.14b 8.98±0.13b 8.58±0.19b 9.41±0.13b 10.46±0.36b

BC/CH-MPE film 8.81±0.05c 8.80±0.46c 7.86±0.15c 8.47±0.03c 9.34±0.16c

BC film 6.00±0.00d 6.00±0.00d 6.00±0.00d 6.00±0.00d 6.00±0.00d

CH film 6.00±0.00d 6.00±0.00d 6.00±0.00d 6.00±0.00d 6.00±0.00d

Positive control 22.12±0.13a 18.69±0.16a 16.22±0.09a 20.36±0.12a 21.25±0.06a

Note: Diameter of film = 6 mm, Different letters in the same column indicate significant difference using one way ANOVA (p<0.05, n=3, Data are mean±SD).

The WVTR is one of the most important characteristics of wound dressings. Generally, the rate of water loss for normal skin is 204±12 g/m2·day, while that for injured skin can range from 279±26 g/m2·day for a first degree burn to 5138±202 g/m2·day for a granulating wound. Thus, water losses from injured skin are

greater than normal skin [43]. BC-MPE and BC/CH-MPE films showed WVTRs within the range of 116-153 g/m2·day, which were found to be similar to the occlusive dressing products such as Duoderm CGF® and Tegasorb®, as shown in Table 3. The occlusive dressings showed inhibition of transmission of water vapor from the wound

Chiang Mai J. Sci. 2021; 48(4) 965

surface to the atmosphere and maintaining a moist wound environment to provide rapid epithelialization [46]. Film thicknesses were also found to be smaller than those of commercial dressings that provided comfort to patient and highly adaptable to wound and body contours.

The WVTR of BC/CH-MPE film was found to be suitable for burnt skin. However, the performance characteristics of dressings need to be adjusted for specific wound types since some types of dressings might not be suitable for all wounds.

3.9 Water Absorption Capacity (WAC)The water absorption capacity was the

estimation of the water retention in the films. The results are revealed in Figure 6. All of the films had extremely fast absorption in the first 5 min of immersion and reached the saturation point at 4 h of immersion. A large amount of water absorption capacity of CH film was represented at 679.96±77.80%, followed by BC-MPE film (334.70±25.5%), BC film (258.00±23.56%) and BC/CH-MPE film (140.26±9.65%) with a significant reduction (p<0.05). Polymer chains

of BC generally form closely linked structure by intramolecular and intermolecular hydrogen bonding. When BC is exposed to water, the expansion of polymer chain, water molecules can penetrate into BC through diffusion and interaction with hydroxyl groups of BC [47]. The results presented that MPE particles in BC-MPE film were diffused into PBS solution when the film was exposed to PBS solution. That also increased the free hydroxyl groups and the relaxed layer structure of BC-MPE film, which allowed water to be absorbed. On the other hand, BC film had tightly packed structure between cellulose fibers and did not recover swelling well. Moreover, addition of chitosan to form BC/CH-MPE film also showed high effectiveness of blocking the diffusion of water and a tendency to lose more absorption capacity.

3.10 Mechanical PropertiesThe mechanical properties of the films were

analyzed in terms of tensile strength, young’s modulus and elongation at break. Tensile strength and young’s modulus of the films significantly decreased with the addition of chitosan and 21

443

Figure 6. Water absorption capacity of the films in PBS solution; (■) BC-MPE film, (▲) BC/CH-MPE 444

film, () BC film and () CH film (The data are expressed as the mean±SD, n=3). 445

446

3.10 Mechanical Properties 447

The mechanical properties of the films were analyzed in terms of tensile strength, young's 448

modulus and elongation at break. Tensile strength and young’s modulus of the films significantly 449

decreased with the addition of chitosan and MPE. As shown in Table 4, tensile strength and young’s 450

modulus of BC-MPE film were 34.75±7.11 MPa and 3,378.90±361.21 MPa, respectively, greater than 451

those of BC/CH-MPE film which were 21.63±3.84 MPa and 458.12±86.95 MPa, respectively. 452

Elongation at break values of BC-MPE and BC/CH-MPE films were 3.34±0.59% and 7.35±2.38%, 453

respectively, which obviously increased with addition of MPE and chitosan. It is based on the fact that 454

BC exhibits a great mechanical strength with high intermolecular reaction among its hydroxyl groups 455

[48]. However, the penetration of MPE and chitosan into the layer of BC affects the flexibility by 456

reducing the intermolecular cohesive forces between the polymer chains. 457

458

Table 4. Mechanical properties of BC-MPE film, BC/CH-MPE film, BC film and CH film. 459

0

100

200

300

400

500

600

700

800

0 8 16 24 32 40 48

Wat

er a

bsor

ptio

n ca

paci

ty (%

)

Time (h)

Figure 6. Water absorption capacity of the films in PBS solution; (■) BC-MPE film, (▲) BC/CH-MPE film, (♦) BC film and (●) CH film (The data are expressed as the mean±SD, n=3).

Chiang Mai J. Sci. 2021; 48(4)966

MPE. As shown in Table 4, tensile strength and young’s modulus of BC-MPE film were 34.75±7.11 MPa and 3,378.90±361.21 MPa, respectively, greater than those of BC/CH-MPE film which were 21.63±3.84 MPa and 458.12±86.95 MPa, respectively. Elongation at break values of BC-MPE and BC/CH-MPE films were 3.34±0.59% and 7.35±2.38%, respectively, which obviously increased with addition of MPE and chitosan. It is based on the fact that BC exhibits a great mechanical strength with high intermolecular reaction among its hydroxyl groups [48]. However, the penetration of MPE and chitosan into the layer of BC affects the flexibility by reducing the intermolecular cohesive forces between the polymer chains.

4. CONCLUSIONSThe unique properties and antibacterial

potency of BC film were enhanced by loading of MPE and chitosan precipitation. MPE entrapment in BC films at the concentration of 1.56 mg/mL exhibited to be non-toxic to L929 cells and could provide antibacterial activity. The morphology of the test film was analyzed by SEM that revealed the penetration of extract particles and precipitated chitosan between the fiber layers of modified BC film. The formation of BC, chitosan and MPE was also proved by FTIR analysis. In vitro release of BC-MPE film was higher than that of BC/CH-MPE film with xanthone release of 18.72±0.53 and 14.51±0.58 µg/cm2, respectively. The effect

of chitosan addition, which revealed a compact arrangement, was the cause of decrease in the WVTR and WAC of the film. According to the compact structure of BC film that combined with chitosan, BC/CH-MPE film also exhibited more flexible properties than BC-MPE film. The combination of chitosan and BC was found to be suitable for occlusive dressings production. However, there was no suitable material or wound healing product for all types of wounds. Therefore, wound care dressing should be selected based on wound types.

ACKNOWLEDGEMENTSThis work was supported by Faculty of

Science, King Mongkut’s Institute of Technology Ladkrabang.

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CH film 28.30±3.05c 606.53±172.77c 19.11±3.89a

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