MICROENCAPSULATION OF EUGENOL IN POLYELECTROLYTE COMPLEXES OF CHITOSAN AND ALGINATE

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1 MICROENCAPSULATION OF EUGENOL IN POLYELECTROLYTE COMPLEXES OF CHITOSAN AND ALGINATE Chamaiporn Supachettapun a and Nongnuj Muangsin* b a Petrochemistry and polymer Science, Faculty of science, Chulalongkorn University, Thailand b Department of chemistry, Faculty of science, Chulalongkorn University, Thailand Keywords: Alginate, Chitosan, Eugenol ABSTRACT Eugenol is the one of essential oils which has a good antimicrobial, antifungal and antioxidant properties. Moreover, eugenol usually uses in many applications such as in food industry and cosmetic pharmaceuticals industry. However, eugenol is volatile unstable and is degraded easily by oxidation, heating and light. Alginate and chitosan also use in several applications in drug delivery, film coating or food packaging because of their biocompatibility and nontoxicity. The purpose of this study was to encapsulate eugenol by using alginate and chitosan as polyelectrolyte complex microspheres form by ionic gelation method. The particle size, shape and encapsulation efficiency (EE) were studied by scanning electron microscopy (SEM), UV-visible spectroscopy (UV-Vis), respectively. The SEM images of obtained beads were showed a regular distribution and spherical shape with the size range of 1 mm. From UV-Vis spectra showed the encapsulation efficiency between 45 and 55%. Moreover, investigated the functional group of the alginate-chitosan polyelectrolyte complexes in form of beads by using fourier transform infrared spectroscopy (FT-IR). Furthermore, studied antimicrobial properties of eugenol and their exhibited a significant reduction antimicrobial effect. *nongnuj.j@chula.ac.th INTRODUCTION Essential oils (Eos) are aromatic compound and volatile oily liquid which obtain from plants. Essential oils are extracted from plants or spices which are rich of biologically active compounds such as terpenoids and phenolic acid. Eugenol s natural phenolic compounds are primarily extracted from clove plants, it has been widely used in food, pharmaceuticals, cosmetic or active packaging materials 1. due to its antimicrobial and antioxidant properties. However, most plants-derived phenolic compound are sensitive to oxygen, light and heat. To overcome the susceptibility and improve the stability of bioactive compounds, the emerging technology of microspheres encapsulation has been recently applied in food industry. Alginate is a natural anionic polysaccharide and can be extracted from marine brown algae. Alginate is a linear binary copolymer that consists of (1 4)-linked -Dmannuronic acid (M) and -L-guluronic acid (G) residues 2. Additional, alginate is widely used in engineering and biotechnological industries, due to its biocompatibility, low cost Petrochemical and Materials Technology Tuesday May 23, 2017, Pathumwan Princess Hotel, Bangkok, Thailand Page 1

2 and non-toxic polymer. Alginate could from ionic bonds with polyvalent cation such as calcium ion, bgenerating a crosslinked structure among of G groups of difference alginate chains, forming a polymerics beads 3. Chitosan is natural cationic polysaccharide which is formed by the N-deacetylation of chitin, which is a product found in crustacean shells, Chitosan is a linear binary copolymer that consists of -(1 4) linked 2-acetoamido-2-deoxy- -D-glucopyranose (Glc- NAc; A-unit) and 2-amino-2-deoxy- -D-glucopyranose (Glc-N; D-unit) 4. The A- and D-type residues are randomly distributed along the chitosan chain. The chitosan is a nontoxic material biocompatible and biodegradable that manifests antibacterial properties. The objective of this research is to prepare the active beads bio-material based on eugenol-encapsulated alginate/chitosan microspheres (encapsulated oils) by an extrusion process. We also clarified the successful encapsulation by UV-vis spectrophotometry (UVvis), confirmed the functional group of alginates coated chitosan beads by Fourier transform infrared spectroscopy (FT-IR), determined the shape and morphology by Scanning electron Microscopy (SEM). In addition, the antimicrobial of alginate beads coated chitosan were investigated. EXPERIMENTAL A. Materials Chitosan, food grade, Mw 500 kda., Deacetylation 85%, Sodium alginate, food grade was dissolved in distilled water. Eugenol, Tween 80 and Calcium chloride were purchased from Union Chemical Acetic acid was purchased from Merck, Germany B. Encapsulation of Eos Micro-encapsulation of oils was conducted by using emulsion extrusion technique. Sodium alginate was dissolved in distilled water to produce (2 and 2.2w/v%) alginate solution, the solutions were left standing for 24 hr to disengage bubble before using. After that, mixed tween 80 (1, 2, 3, 5 and 10 % w/v) with eugenol (0.125 g) before combination of alginate solution 100 ml were homogenized into a beaker with stirring at a speed of 400 rpm for 20 mins by IKA RW20 digital. Alginate-oils emulsion was then drop into a collecting water bath containing calcium chloride solution (0.5, 1, 1.5, 2, 3, 4w/v%) for 20 min, afterwards the alginate which encapsulated with eugenol was Immersed in (1%w/v) chitosan solution. The particles were solidified for at least 30 mins before being washed with distilled water and dried in an oven of 40 C overnight. C. Encapsulation Efficiency 500 mg of encapsulated oils were dissolved in 10 ml of ethanol. Crush and stirred overnight. The solution was centrifuged at 5000 rpm for 15 min and then a eugenol content was assayed by measuring the absorbance at 282 nm after suitable dilution using a UV-vis and calculated as following equation (1) Petrochemical and Materials Technology Tuesday May 23, 2017, Pathumwan Princess Hotel, Bangkok, Thailand Page 2

3 Encapsulation efficiency = 100 (1) D. Antimicrobial Activity Profile of Encapsulated Eos To estimate the antimicrobial efficiency of control, free oils and encapsulated oils samples in chili during storage, the control sample of chili was storage and no essential oils, the free oils sample distributed onto filter paper and encapsulated oils sample were stored at room temperature for 8 days. After that the antimicrobial activity could be investigated. E. Instrumental Analyses SEM morphologies of sample were mounted on an aluminum stub using doublesided carbon adhesive tape and coated with gold-palladium. The microspheres were investigated by SEM using Philips XL30CP and performed under high vacuum and ambient temperature with beam voltage of 60 KV. FTIR-ATR spectra. FTIR-ATR spectra were obtained with a Fourier transform spectrometry, equipped with diamond crystal and measurement each sample pure alginate, pure chitosan and alginate-chitosan microspheres were recorded wave number from cm -1. RESULTS AND DISCUSSION A. Effect of Alginate Concentration The difference ratios of alginate and chitosan solution (1:1, 1.5:1, 2:1 and 2.2:1), were used to prepare alginate chitosan microspheres. (Fig.1) The morphology of the microspheres was observed by SEM. The SEM images of the alginate and coated with chitosan demonstrated spherical shape (Fig.1a), alginate and chitosan ration of 1:1, it could not be formed the spherical bead but the other ratios could be formed as and the spherical bead regular distribution size is about 1 mm. In (Fig.1c) and (Fig1d) which have more spherical and smooth bead, which alginate and chitosan ratio 2:1 and 2.2:1, respectively, it had the best morphology. (a) (b) (c) (d) Figure 1. SEM images of alginate microsphere coated 1% w/v chitosan, (a) 1%w/v alginate; (b) 1.5%w/v alginate; (c) 2%w/v alginate and (d) 2.2%w/v alginate Petrochemical and Materials Technology Tuesday May 23, 2017, Pathumwan Princess Hotel, Bangkok, Thailand Page 3

4 B. Fourier Transform Infrared Spectroscopy (FT-IR) FT-IR Spectroscopy was used to determine the chemical reaction of the sample (Fig. 2) The IR spectrum of pure alginate (Fig. 2a) showed the characteristic absorption bands at 1592, 1414and 1026 cm -1 attributed to the asymmetric and symmetric stretching vibrations of COO and the stretching of C O C, respectively. The FT-IR spectrum of chitosan (Fig. 2b) shows the characteristic peaks at 892 and 1027 cm -1 indicated that the saccharine structure and a strong characteristic amino peak appeared around 1591 cm -1. The peak at 1591 cm -1 attributed to the amide of N-acylated chitosan. The FT-IR spectrum of the alginate coated with chitosan (Fig.2c) showed a band at 1589 cm -1, which was attributed to the formation of NH 3+ and indicated to the complexation between the amino groups of chitosan and the carboxylic groups in alginates. Figure 2. Fourier transform infrared spectra: (a) alginate; (b) chitosan and (c) alginate coated chitosan C. The Concentrations of Calcium Chloride Cross-linking agent concentration on the affecting factor of encapsulation efficiency was study by using various concentrations of calcium chloride (0.5-4%w/v) with 2%w/v alginate and crosslink time for 20 mins. The results of this study were presented in (Fig.3) indicating that the increasing of calcium chloride concentration from 0.5to 4% the drug entrapment efficiency were found to increase from 8 to 15% at 2%w/v calcium is the best crosslinking. Petrochemical and Materials Technology Tuesday May 23, 2017, Pathumwan Princess Hotel, Bangkok, Thailand Page 4

5 Figure 3. Effect of calcium chloride concentration on the encapsulation efficiency D. Tween 80 Concentrations Tween 80 is the one of surfactant. The effect of tween 80 on the morphology and encapsulation efficiency prepare as the beads was using investigated by using UV-Visible spectroscopy as shown in the (Fig.4) From the results using tween 80 at 1, 2, 3, 5 and 10%w/v the drug entrapment efficiency was found that it increased from 10 to 53% and 10% of tween 80 and it can encapsulation eugenol which is the best percentage of surfactant. Figure 4. Effect of Tween 80 concentration on the encapsulation efficiency in 2%w/v alginate condition From the results of studying the effect between concentration ratio of alginate and chitosan at 2:1 condition gave the best encapsulation essential oils around 53%. To increase the encapsulation essential oils, thus we also studied the effect of tween 80 to concentration ratio of alginate and chitosan at 2.2:1 condition shown in (Fig.5). in Fig.5, it is successful to increase the percentage encapsulation essential oil to 55% Petrochemical and Materials Technology Tuesday May 23, 2017, Pathumwan Princess Hotel, Bangkok, Thailand Page 5

6 Figure 5. Effect of alginate and tween 80 concentration on the encapsulation efficiency in 2.2% w/v alginate condition E. Antimicrobial Activity Profile of Encapsulated EOs The activity profile of sample control, free oils and encapsulated tested oils with chilli in room temperature was taken study in the difference times. The results were show in Figure 6. The exhibited results after four days of the antimicrobial activity of encapsulated oils sample in chilli showed no sign of any microorganism and the colour look fresher comparing to free oils and control samples. This indicated that the encapsulated sample could be antimicrobial material. (a) (b) (c) 0 days 4 days 8 days Figure 6. Appearance changes of chilis stored at room temperature (a) control; (b) free oils sample and (c) encapsulated oils sample Petrochemical and Materials Technology Tuesday May 23, 2017, Pathumwan Princess Hotel, Bangkok, Thailand Page 6

7 CONCLUSIONS Microencapsulation of eugenol in polyelectrolyte complexes of chitosan and alginate can be synthesized by emulsion extrusion method at 2.2% (w/v) alginate, 1% (w/v) chitosan, 2% (w/v) calcium chloride, 10% tween 80 and g eugenol conditions. The encapsulated sample showed good result in antimicrobial activity after four days in chili. Moreover, it indicated that the encapsulation process of eugenol-loaded alginate coated chitosan microspheres could be considered as inexpensive and efficient technique for decreased the antimicrobial activity in agricultural crops. ACKNOWLEDGEMENTS The research was funded by the Ratchadapisek Sompoch Endoment Fund (2016), Chulalongkorn University (CU HR) REFERENCES 1 Sanla-Ead, N., et al. (2012) Packaging Technology and Science. 25 (1), Soliman, E.A., et al. (2013) Journal of Encapsulation and Adsorption Sciences. Vol.03No.01,8 3 Saether, H.V., et al. (2008) Carbohydrate Polymers. 74 (4), Hosseini, S.F., et al. (2013) Carbohydrate Polymers. 95 (1), Petrochemical and Materials Technology Tuesday May 23, 2017, Pathumwan Princess Hotel, Bangkok, Thailand Page 7