Formulation development of antibacterial films containing mangosteen peel extract

 

Thanaporn Amnuaikit1, Turawat Phadungkarn1, Chatchai Wattanapiromsakul2, Prapaporn Boonme1*

1Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences,

Prince of Songkla University, Hat-Yai, Songkhla 90112, Thailand

2Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences,

Prince of Songkla University, Hat-Yai, Songkhla 90112, Thailand

*Corresponding Author E-mail: prapaporn.b@psu.ac.th

ABSTRACT:

The aims of this study were to formulate films containing mangosteen peel extract (MPE) and to investigate their physicochemical characteristics, mechanical properties, as well as antibacterial activity. The film backbone composed of natural biodegradable polymers, i.e. agar, sodium alginate and pectin. Propylene glycol and glycerin were also used as plasticizers. All film components used have been in a GRAS status. The films were prepared by casting and drying method. The in-house prepared MPE by column chromatography technique possessed 77.861.13% α-mangostin, an antibacterial marker. The optimal film formulation with area of 25x30 cm2 was composed of 1-g agar, 1-g sodium alginate, 1.5-g pectin, 20-ml propylene glycol and 0.9-ml glycerin. It was selected to incorporate with MPE to obtain 160 mg/film or 0.213 mg/cm2 antibacterial films. The obtained films were clear with yellow color and had smooth surface. Their physicomechanical characteristics, i.e. appearance, thickness, moisture uptake, tensile strength and elongation break were acceptable for application on the skin. X-ray diffraction patterns exhibited that the form of α-mangostin was not changed after film incorporation. The active entrapment, assayed by HPLC using standard α-mangostin as a marker, was higher than 90%. The antibacterial films had satisfied stability, active release via dissolution of the polymers, and antibacterial activity against harmful skin-infectious bacteria, i.e. S. aureus, S. epidermidis and P. acnes. It was concluded that MPE could be incorporated in films for treatment of skin infection.

 

KEYWORDS: antibacterial, a-mangostin, films, mangosteen peel extract

 


INTRODUCTION:

Several plants have been reported that they can offer antibacterial potential.1 Fruit peel of Mangosteen (Garcinia mangostana L.), a tropical plant in Guttiferae family, has been commonly used in Thai traditional medicine as an antibacterial ingredient in both oral and topical administrations.2 Moreover, the mangosteen peel extract (MPE) has been proved for activities of antioxidant, cytoprotective and antibacterial against several bacteria such as Staphylococcus aureus, Helicobacter pyroli, Propionibacterium acnes as well as Staphylococcus epidermidis.3-8

 

The compounds discovered in MPE belong to a group of xanthones such as α-mangostin, β-mangostin, γ-mangostin, and gartanin.9-11 Among these chemicals, α-mangostin is recognized as the major substance.12-16 It has been reported for multiple biological activities such as anti-inflammatory, histamine H1 receptor antagonist, and antibacterial against H. pylori, methicillin-resistant S. aureus (MRSA) as well as vancomycin resistant Enterococci (VRE).7,17,18 Therefore, it is interesting to prepare MPE, which has α-mangostin as the marker, in a suitable dosage form for skin application.

 

Many dosage forms can be used for topical administration of antibacterial agents, such as lotion, cream, gel and film. Among these dosage forms, film can provide specific dose, ease of application and good adherence on the skin, resulting in reaching the potential of the actives and increasing patient compliance. Therefore, natural polymers such as sodium alginate, pectin and agar were selected for film formation in this study due to their sustainability and biodegradability. Propylene glycol and glycerin were added in film formulations as plasticizers. All components have been classified in a generally recognized as safe (GRAS) status

 

The aims of this study were to prepare films containing MPE and to investigate their physicochemical characteristics, mechanical properties and antibacterial activity.

 

MATERIALS AND METHODS:

Materials

Mangosteen peels used in this experiment were obtained from the fruits of G. mangostana which collected from Pha Toh District, Chumphon Province in August 2007. The voucher specimen (number: SKP 083071301) was kept by the Herbarium of the Faculty of Pharmaceutical Sciences, Prince of Songkla University.

 

The α-mangostin standard with purity of 98.5% was purchased from Chromadex Inc., USA. Pectin from citrus peel and agar were purchased from Sigma-Aldrich Co., Denmark. Sodium alginate was purchased from Merck, Germany. Propylene glycol and glycerin were purchased from S. Tong Chemicals Co., Ltd., Thailand. Acetonitrile, methanol, 37% hydrochloric acid and sodium chloride were purchased from Lab Scan Asia Co., Ltd., Thailand. Formic acid was purchased from May & Baker Ltd., UK. Petroleum ether, dichloromethane, 95% ethanol and ethyl acetate were purchased from High Science Distributor, Thailand. Di-methyl sulfoxide (DMSO) was purchased from Riedel-de Han, Germany. Polyamide membrane filter was purchases from Sartorius AG, Germany.

 

The tested bacteria, i.e. S. aureus ATCC 25923, S. epidermidis TISTR 517 and P. acnes DMST 14916, were obtained from the Department of Pathology, Faculty of Medicine, Prince of Songkla University, the Thailand Institute of Scientific and Technology Research and the Department of Medical Science, Ministry of Public Health of Thailand, respectively. Mueller-Hinton Agar (MHA) and Brain Heart Infusion (BHI) were purchased from Merck, Germany. Standard neomycin and standard neomycin paper disc were purchased from Oxoid Ltd., UK.

 

Preparation of MPE

The 2 kg dried mangosteen peel were grinded and then macerated in 3 liters ethanol for 7 days. Afterwards, the macerated substance was evaporated by rotary evaporator. The process was repeated 3 times to get a total crude extract. Each portion (around 30 g) of the crude extract was dissolved in enough amount of ethanol and then mixed with 400 g silica gel until the dry mixture was obtained. This mixture was then spread on the surface of the stationary phase (silica gel which was previously mixed with the eluting solution of 7:3 petroleum ether and ethyl acetate) in a column (5 cm diameter and 30 cm height). Afterwards, it was eluted with 50 ml eluting solution. The elution was repeated for 42 fractions. Each gathered fraction was evaporated using rotary evaporator and then analyzed by thin layer chromatography (TLC) comparing to α-mangostin standard. The fractions of the extract with high α-mangostin content were combined together to obtain MPE and then determined for the yield of total α-mangostin content by high performance liquid chromatography (HPLC) method.

 

Validation of HPLC for α-mangostin assay

The quantitative amount of α-mangostin was analyzed by HPLC modifying from the method mentioned in previous report.19 A reversed phase Hypersil BDS C18 column (5 mm particle size, 125x4 mm) was used as the stationary phase. The HPLC system consisted of an auto injector, a pump and a UV-visible detector (Agilent 1100 series, USA). A mixture of acetonitrile and 0.2% formic acid (70:30 v/v) was used as mobile phase with a flow rate of 1.0 ml/min. The mobile phase was filtered through 0.45 mm polyamide membrane filter (Sartorius AG, Germany) and degassed by sonication before use. The injection volume was 20 ml. The samples were detected at 240 nm. The peak area was integrated with a G2220AA2D-Value Solution ChemStation software program.

 

Standard solutions of α-mangostin were prepared at the concentrations of 1.0, 5.0, 10.0, 15.0 and 20.0 g/ml using methanol as a solvent. These standard solutions were analyzed by HPLC in triplicate. The standard curve of α-mangostin was prepared by plotting the determined peak area versus the α-mangostin concentration. Linearity of the standard curve was calculated using linear regression analysis. The accuracy and precision were also validated.

 

Investigation of MPE properties

For chemical analysis, MPE was dissolved in methanol at the appropriate concentration. Subsequently, they were analyzed by HPLC in triplicate.

 

Antibacterial activity of MPE was determined by minimum inhibitory concentration (MIC) obtained from broth micro-dilution assay. S. epidermidis and S. aureus were cultured in MHA while P. acnes was cultured in BHI. A sequential two-fold dilution method was used in MIC test. The MPE was diluted in DMSO to the concentration of 1 mg/ml and then diluted with the medium to the concentration of 64 g/ml. The obtained mixture was then filtered through 0.45 m membrane filter. Neomycin, a positive control, was diluted in sterile water to a concentration of 64 g/ml and filtered through 0.45 m membrane filter. The test was performed in 96-well plate. The stock solution of neomycin was serially two-fold diluted with the medium to give the final concentrations of 0.125 to 32 g/ml. The stock solution of the MPE was serially two-fold diluted with the medium to give the final concentrations of 0.25 to 64 g/ml. The inoculum containing a tested microorganism of 106 CFU/ml was diluted from that of 108 CFU/ml, having identical turbidity to the McFarland 0.5 standard, by mixing with MHB at the ratio of 1:100. The 2 l of the inoculum was added to each well. The samples with S. epidermidis and S. aureus were then incubated at 37 C under aerobic conditions for 24 hr and those with P. acnes were incubated at 37 C under anaerobe conditions for 48 hr The MIC was calculated as the highest dilution showing complete inhibition of the test strains.20 Furthermore, the minimum bactericidal concentration (MBC) was measured as the lowest concentration of the compound to kill microorganisms. The incubation mixtures of the studied aerobic and anaerobic bacteria which showed positive result of inhibitory effect were streaked on MHA and BHI plates, respectively. Afterwards, they were incubated in previous mentioned conditions. The lowest concentration of the compound that the bacteria did not show any growth was taken as the MBC.20

 

Preparation of blank films

The 13 formulations of blank films as exhibited in Table 1 were prepared and evaluated. The film base was prepared by dissolving agar in boiling water at 1:10 ratio. Sodium alginate, pectin, propylene glycol, and glycerin were added in the agar solution and homogeneously mixed. Finally, the film base was casted into a plate (25x30 cm2) and followed by drying at 50 C in a hot-air oven for 24 hr, resulting in film formation. After physicomechanical characterization, the film formulations which provided good characteristics were selected for incorporation with suitable amount of MPE.

 

Physicomechanical characterization of blank films

The films were characterized for the following physicomechnical properties.21,22 The color and texture of the films was optically observed. The thickness of each film was measured at 3 different positions using a micrometer (Series 102-139, Mitutoyo, Japan). In order to measure the moisture uptake, each film was cut into 1x1 cm2 size. It was subsequently placed in a desiccator containing silica gel for 24 hr and weighed (Ws). Afterwards, the film was put in a desiccator containing saturated sodium chloride solution which had 75% relative humidity (RH) at room temperature until the film was fully saturated. It was then weighed (Wm). Moisture uptake capacity of the film was calculated using Eq. 1.

...Eq. 1

For evaluation of mechanical properties, each film was fixed the two clamps of universal testing machine (LR10K, Lloyd Instrument Limited., UK). After that, the film was slowly pulled by 5 kg of load cell at the speed of 30 mm/min. The mechanical properties of the films, i.e., tensile strength (TS) and elongation break (EB) were determined by Eq. 2 and Eq. 3, respectively.

...Eq. 2

 

...Eq. 3

 

 

Finding suitable amount of MPE for antibacterial film formulations

Antibacterial films were prepared with different concentrations of MPE, i.e. 160, 200, 240, 280 and 320 mg/film or 0.213, 0.267, 0.320, 0.373 and 0.427 mg/cm2, respectively. The antibacterial activity experiment was performed by the disc diffusion method with some modifications. Microorganisms tested, S. aureus, S. epidermidis and P. acnes, were adjusted the turbidity to yield approximately 108 CFU/ml with 0.85% sodium chloride compared to turbidity of McFarland No. 0.5. Each obtained inoculum was streaked on the surface of agar medium with cotton stick. The film was pressed down with slight pressure on the agar. The negative and positive controls were the blank film and standard 30-g neomycin paper disc, respectively. For S. aureus and S. epidermidis, the agar plates were incubated at 37 C under aerobic conditions for 24 hr. For P. acnes, the agar plates were incubated at 37 C under anaerobic conditions for 48 hr. The antibacterial activity was expressed as the mean of inhibition diameter (including the diameter of disc).20 After the suitable concentration of MPE was optimized, antibacterial films were prepared by dissolving MPE in propylene glycol before mixing with the other ingredients and then the films were formed via casting and drying technique.

 

Property and stability determination of antibacterial films

The antibacterial films or MPE loaded films were determined of the nature of the crystalline or amorphous form comparing with the form of MPE and blank counterparts by X-ray diffraction technique.22 Briefly, the samples were placed into the glass sheet and analyzed using X-ray Diffractrometer (X'Pert MPD, Philips, Netherland) at 40 kV and 50 mA during 2q of 5-40.

 


Table 1. Film formulations for the area of 25x30 cm2

Formulation

Sodium alginate (g)

Pectin (g)

Agar (g)

Propylene glycol (ml)

Glycerin (g)

1

1

1

0.5

20

0

2

1

1

1

20

0

3

1

1

1

20

0.03

4

1

1

1

20

0.3

5

1

1

1

20

0.6

6

1

1

1

20

0.9

7

1

1

1

20

1.2

8

1

1

1.5

20

0

9

1

1

1.5

20

0.03

10

1

1

1.5

20

0.3

11

1

1

1.5

20

0.6

12

1

1

1.5

20

0.9

13

1

1

1.5

20

1.2


The antibacterial films were investigated for physical and chemical stabilities at ambient condition (300C) and at stress condition (450C and 75% RH) for 120 days comparing to those after preparation. The samples were kept in plastic bags wrapped with aluminum foil for light protection. The physicomechnical changes of the films were observed at the end of storage period. For analysis of α-mangostin in the films, the 2x1 cm2 piece of each film was cut into small pieces and mixed with 5 ml methanol under vigorous agitation. Afterwards, the mixture was centrifuged at 4,000 rpm for 5 min. The extraction was performed again. The supernatants obtained from both extractions were pooled and subsequently diluted with methanol to obtain 25 ml solution. The resulted solution was further analyzed by HPLC. At least three replicates from different points of each film were performed in triplicate for consistency evaluation. The obtained amount of α-mangostin in a film after preparation was calculated and referred to entrapment of MPE in that film. The α-mangostin was also extracted from the samples and analyzed by HPLC after storage at tested conditions for 30, 60, and 120 days. The remained amounts of α-mangostin at various times were compared to those at the initial for stability determination.

 

In vitro release study

The release of the α-mangostin from the films was studied via modified Franz diffusion cells (Hanson Research Corporation, California, USA) which the temperature was constantly controlled at 370C by a circulating water bath. Each film was cut into 3.14 cm2 piece and was placed between donor and receptor compartments of the diffusion cells. Each receptor compartment contained 11 ml isotonic phosphate buffer solution pH 7.4 (IPB) as the receptor fluid which was continuously stirred at 200 rpm by a magnetic stirrer. The diffusion area was 1.77 cm2. The sample was collected for 0.5 ml at 0.25, 0.5, 1, 2, 3, 4, 6, 8 and 12 hr. Equal volume of fresh IPB was replaced immediately after sampling in order to maintain sink condition. After that, the samples were filtered through 0.45 m membrane filter and then analyzed for α-mangostin content by HPLC. The cumulative α-mangostin release (Qt) was calculated by Eq. 4.

...Eq. 4

 

Where, Ct is the drug concentration of the receptor fluid at each sampling time, Ci is the α-mangostin concentration of the ith sample, and Vr and Vs are the volumes of the receptor fluid and the sample, respectively.

 

Moreover, each film sample was crossed by cutting and then placed on the grid with adhesive tape for fixing. Then, it was coated with gold and subsequently observed feature of the cross section under scanning electron microscope (JSM-5200, JEOL, Japan). The observation was performed before and after in vitro release study.

 

 

Statistical Analysis

Each experiment was repeated at least three times. The results were reported as meanSD. One-way analysis of variance (ANOVA) and Tukeys multiple comparison test were used to compare the difference and a P value of 0.05 was considered to be significant.

 

RESULTS AND DISCUSSION:

Validation of HPLC method

It was found that the standard curve plotted from peak areas versus α-mangostin concentrations was linear with the r2 > 0.99. The validation results showed that the bias was 11.82% (less than 15%) and the relative standard deviation (RSD) was in the range of 0.09-0.28% (less than 5%). Therefore, this analysis method was appropriate for α-mangostin assay due to high linearity, accuracy and precision. The retention time was approximate 5.1 min.

 

Characteristics of MPE

It was found that 2 kg dried mangosteen peels provided 181 g crude extract, resulting in the yield of 9.05% w/w. In pre-purifying step with silica gel column, high content of α-mangostin was observed in fractions 27-42 by TLC. Therefore, these fractions were pooled together to obtain yellow MPE. The yield value was 15.40% w/w when compared with total crude extract which equalized to 1.39% w/w when compared with total dried mangosteen peel. The collected MPE had 77.861.13% w/w α-mangostin when analyzed with HPLC.

 

In addition, MIC values against S. aureus, S. epidermidis, and P. acnes of the MPE were 4.00, 2.00 and 4.00 g/ml, respectively. Those of neomycin were 0.50, 0.50, and 1.00 g/ml, respectively. Although the antibacterial growth inhibition against S. aureus, S. epidermidis, and P. acnes of MPE with high content of α-mangostin was lower than that of neomycin, the results were still satisfied. Also, bactericidal activity of MPE against the tested bacteria was found when concentration of the extract was 32 g/ml or higher.

 

Properties of blank films

According to optical observation, the blank films from Formulation 1 were easily tattered. Those from Formulations 2-4 and 8-10 were crumpled with shrunk edges. Those from Formulations 5-7 and 11-13 had good appearance, i.e. clear, smooth, and pale yellow.

 

Thickness of the blank films was in the range of 0.031-0.051 mm. In previous reports23-26, thickness of the skin films was in the range of 0.035-0.070 mm. The thicknesses of the films depended on the ingredients, especially glycerin. It was seen that when glycerin was added below 20% w/w of total polymers, it did not affect the thickness of the films compared with the formulations with no glycerin. In contrast, the thickness of films containing glycerin more than 20% w/w of total polymers was significantly larger than that of the films without glycerin (P<0.05). Moisture uptake capacities, TS and EB values of the blank films were presented in Table 2.

 


Table 2. Moisture uptake capacity, TS and EB of the blank films (n = 3)

Formulation

Moisture uptake capacity (%)

TS (N/mm2)

EB (%)

1

22.153.47

25.982.70

25.863.59

2

4.251.59

49.414.54

8.711.91

3

4.090.18

49.513.27

10.881.08

4

6.222.58

42.455.84

10.311.31

5

13.540.56

35.445.51

11.081.95

6

9.370.96

39.083.33

19.993.39

7

11.85 0.41

48.503.82

25.233.90

8

9.021.57

54.046.08

14.781.33

9

7.421.20

54.706.23

16.162.96

10

12.613.16

60.003.94

17.310.75

11

14.754.11

44.524.62

13.321.53

12

13.712.27

44.434.13

22.073.39

13

13.732.09c

42.572.80

24.462.06

 

 

Table 3. Inhibition zones provided by the films containing different concentrations of MPE (n = 3)

Formulation

Amount of MPE/area(mg/cm2)

Inhibition zones (cm) for:

S. aureus

S. epidermidis

P. acnes

5

0

0

0

0

5/1

0.213

0.930.11

0.990.03

1.240.10

5/2

0.267

0.920.03

0.940.07

1.100.02

5/3

0.320

0.940.09

1.020.07

1.140.03

5/4

0.373

0.960.08

0.990.02

1.120.01

5/5

0.427

0.980.07

0.940.01

1.170.07

7

0

0

0

0

7/1

0.213

0.940.04

1.020.02

1.300.05

7/2

0.267

0.870.02

1.010.02

1.280.02

7/3

0.320

0.950.01

0.930.01

1.270.04

7/4

0.373

1.010.01

0.990.02

1.290.03

7/5

0.427

0.940.03

1.000.01

1.300.04

12

0

0

0

0

12/1

0.213

0.990.01

1.030.02

1.190.06

12/2

0.267

0.940.01

1.080.05

1.170.07

12/3

0.320

0.960.01

1.070.03

1.220.04

12/4

0.373

0.940.04

0.990.04

1.150.06

12/5

0.427

0.980.02

1.090.04

1.170.08

neomycin

30 g/disc

1.840.07

2.480.07

3.230.04

 


It could be seen that Formulation 1 provided the highest moisture uptake capacity in this experiment since high ratios of hydrophilic polymers, i.e. pectin and sodium alginate led to high moisture absorption. Glycerin at high ratios tended to increase moisture uptake capacity of the films due to its hygroscopic property. Formulation 1 had the lowest TS but the highest EB, implying the easiest torn. These results were identical to those from optical observation.

 

TS of blank patched without glycerin (Formulations 1, 2 and 8) were significantly increased when the amount of agar in film were increased (P<0.05) due to the effect of polysaccharide chain in agar.27 Glycerin at high ratios tended to decrease TS but increase EB since it augmented moisture uptake capacity of the films, leading to soft films. Additionally, glycerin could act as a plasticizer in the films by lubricating the movement of the polymer molecules and reducing their internal resistance to sliding, resulting in low flexibility.28

 

According to the physicomechanical characteristics, Formulations 5-7 and 11-13 were suitable for further preparation of the antibacterial films. These 6 formulations provided aesthetic appearance. Their moisture uptake capacity, TS and EB values were acceptable.

 

Suitable amount of MPE in film formulation

In this experiment, Formulations 5, 7 and 12 was chosen for incorporation with different concentrations of MPE in order to evaluate the antibacterial activity. The measured inhibition diameters were shown in Table 3. It was noted that the blank films did not provide inhibition zone, indicating that they had no antibacterial activity. Insignificant difference in the inhibition zones against the tested bacteria by the films containing MPE was observed (P>0.05) even if the active concentrations were varied. The diffusion from the films did not depend on concentrations of MPE since the active was eluted from the films with the equal volume of penetrated water.29 Therefore, the minimum concentration (0.213 mg/cm2) of MPE was chosen to prepare antibacterial films by incorporation in Formulations 5-7 and 11-13.

 

Properties and stability of antibacterial films

Appearance of the films containing MPE was similar to their blank counterparts but the color was slightly darker due to the color of the extract. The thickness of active-loaded films was slightly increased when compared with that of blank films. Nevertheless, the increased thickness was not more than 20% and still in the normal range of previous reports.23-26 The moisture uptake capacity, TS and EB values of the films containing MPE did not different from that of their blank counterparts (data not shown). For convenient usage, each antibacterial film can be cut into suitable size and then adhere in the middle of larger piece of a commercial adhesive tape (e.g., Fixomull), the glue at the rim of the adhesive tape will improve adherence between the film and the skin.

 

MPE was added in the films at the concentration of 0.213 mg/cm2. Table 4 showed that the films could entrap the extract in the range of 90.51-101.38% labeled amount with lower than 6% RSD. The results indicated that the films had good uniformity of MPE.30

 

The X-ray diffractograms of MPE, films of Formulation 12 without and with MPE were illustrated in Figure 1. It could be noticed that MPE was in crystalline state with many diffraction peaks during 2q of 0 to 40 while the blank film exhibited amorphous characteristics with no diffraction peaks. The diffraction peaks, especially at 2q of 2 and 12.5, were found in the film containing MPE. Since the amount of MPE in the film (0.213 mg/cm2) was very small when compared with the amount of polymer in the film, only two strong peaks of MPE could be clearly observed. The results suggested that MPE remained in the antibacterial films as crystalline form.22

 

 

Figure 1. Diffractograms of A: MPE, B: blank film of Formulation 12, and C: MPE-loaded film of Formulation 12

According to optical observation, the color of the films kept at 45 C/75% RH gradually changed from light yellow to dark yellow during the study period of 120 days while the color of those kept at 30 C did not visually alter. The reason was unclear but it was possible that high temperature catalyzed degradation of at least one inactive component in the films since it was found that temperature did not exhibit obvious effects on α-mangostin chemical stability as shown in Table 5. After the films were stored at both studied conditions for 120 days, amounts of α-mangostin significantly decreased from the initial ones due to acid hydrolysis.31 Acid functional group dissociated from sodium alginate may hydrolyze ether group of α-mangostin, leading to degrade to benzophenone derivative. Although chemical stability of α-mangostin at 30 and 45 C was not significantly different, darker color was found at high temperature. Therefore, it was recommended that the films should be stored in a condition with low temperature, low humidity and light protection. Furthermore, it could be seen that average amounts of α-mangostin in Formulations 7 and 12, which possessed high hydroxyl group from glycerin among studied formulations, were more than 80% at all studied conditions. Thus, these 2 formulations were then selected to evaluate for the active release.

 

In vitro α-mangostin release from the films

From Figure 2, no significant difference of α-mangostin release from Formulations 7 and 12 was observed (P>0.05). However, mean cumulative amount of α-mangostin released at 12 hr from Formulation 12 (18.77 g/cm2) was slightly higher than that from Formulation 7 (16.90 g/cm2). Formulation 12 could absorb moisture higher that Formulation 7, leading to slightly higher dissolution of the films or diffusion of the α-mangostin. It has been known that the mechanism of the active release from the films might be dissolution of the films or diffusion of the α-mangostin or combination of both.29

 

Figure 2. The in vitro release profiles of α-mangostin from antibacterial films, Formulations 7 and 12 (n = 3)


Table 4. MPE content in the films (n = 3)

Formulation

Concentration of MPE in film (g/cm2)

Labeled amount (%)

RSD (%)

5

217.965.15

101.382.40

2.36

6

215.245.80

101.052.72

2.69

7

193.685.34

90.512.50

2.76

11

205.334.18

96.401.96

2.04

12

205.304.80

95.932.24

2.34

13

199.313.45

93.141.61

1.73

 

Table 5. Remaining percentages of α-mangostin in the films after stability test at various conditions (n = 3)

Formulation

% Remaining

30 C

45 C/75% RH

30 days

60 days

120 days

30 days

60 days

120 days

5

89.424.00

86.524.05

74.435.55

94.450.82

90.010.95

71.785.77

6

91.984.55

85.244.29

78.702.53

91.346.05

84.211.17

75.481.01

7

98.411.64

92.374.36

83.916.50

99.821.52

93.581.05

81.901.18

11

95.292.04

93.122.21

81.611.44

90.271.17

78.652.75

63.061.81

12

88.195.55

85.555.36

80.322.48

89.503.99

85.292.48

82.852.45

13

95.501.56

92.184.10

91.013.09

90.063.29

87.062.60

70.481.46

 

 

Figure 3. SEM micrographs of Formulation 12 at different conditions, i.e. A: without MPE; B: with MPE before release study, and C: with MPE after release study

 

 


The SEM micrographs of Formulation 12 without and with MPE at before and after release test were illustrated in Figure 3. The cross-sectional morphology of blank films and of the films containing MPE before release study was similarly homogeneous (Figure 3A, 3B) due to compatibility of all components. Nevertheless, pores were seen in the cross-sectional morphology of the films containing MPE after release study (Figure 3C).

 

The α-mangostin is hydrophobic, resulting low diffusion to aqueous medium. From the release profile in Figure 2, α-mangostin was detected in the receptor fluid. Hence, it could be explained that the mechanism of the active release from the investigated films was dissolution of the polymers in the films.29

 

CONCLUSIONS:

MPE was found to possess high content of α-mangostin, resulting in antibacterial activity against skin infective bacteria. It could be incorporated in biodegradable film bases to form antibacterial films, resulting in ease of dose specification and use. The ratios of the components in film formulations influenced on properties of the resulted films. It was found the formulation composed of 1-g agar, 1-g sodium alginate, 1.5-g pectin, 20-ml propylene glycol and 0.9-ml glycerin (Formulation 12) provided the optimal films with area of 25x30 cm2. The suitable concentration of MPE in the antibacterial films was 0.213 mg/cm2. The stability of α-mangostin in the antibacterial films depended on acidic reaction rather that temperature. However, the products should be kept in a condition with low temperature, low humidity and light protection. The data obtained from this work showed that an herb used in traditional medicine could be proved for it activity and developed into usable formulation.

 

ACKNOWLEDGEMENTS:

This research is financially supported by Graduate School and Faculty of Pharmaceutical Sciences, Prince of Songkla University, and the National Research Council of Thailand. Department of Pathology, Faculty of Medicine, Prince of Songkla University is thanked for supporting S. aureus ATCC 25923.

 

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Received on 22.06.2012 Modified on 12.07.2012

Accepted on 29.07.2012 RJPT All right reserved

Research J. Pharm. and Tech. 5(8): August 2012; Page 1058-1065