Abstract
In the present study, moxa oil was encapsulated into the gelatin-arabic gum microcapsules using a complex coacervation technique in order to improve the antibacterial property of moxa oil. The physical properties including surface morphology, particle size, drug loading as well as release profile and anti-Staphylococcus aureus activity of moxa oil containing microcapsules were investigated. The results showed that the moxa oil containing microcapsules had relatively spherical appearance with the average particle size (6.42 µm) and the average oil loading (0.20 ± 0.01 mg/ml). The moxa oil was demonstrated to be gradually released from the microcapsules. The antibacterial study revealed that the moxa oil microcapsule-treated cotton fabrics showed a significantly stronger growth inhibition towards Staphylococcus aureus (S. aureus).
In recent years, people have focused more attention on functional textiles used in hygienic and medical applications. Researchers have focused on exploiting the technologies to develop the healthcare textile materials in order to fulfill the requirements of customers. Microencapsulation technology is one of the effective methods to achieve these ideas by introducing active substances into wearable textiles. Microencapsulation is actually a micro-packaging technique for the production of microcapsules or microspheres by surrounding the small particles of solids, droplets of liquids or dispersions of solids in liquids within polymer coatings to give small capsules helpful for various applications. Encapsulated reservoirs of core active agents under controlled release can offer useful functions for specific end uses.1–9 With reference to various microencapsulation techniques, coacervation is one of the simplest and cheapest ways to form microcapsules. 7 Gelatin and arabic gum have been used as the wall materials to form the microcapsules for various applications10–13 because of their abundance and their degradability.
Moxa (Artemisia argyi) leaf is one of the most used herbs in Chinese traditional medicine which is commonly applied to heal eczema, inflammation, hemostasis and tuberculosis.14–16 It is also recommended to help improve blood circulation, discomfort during menstrual flow and itching. 10 In the present study, we demonstrated the controlled release profile and anti-Staphylococcus aureus activity of moxa oil containing gelatin-arabic gum microcapsule-treated cotton fabrics.
Experimentation and research and development
Materials
Moxa oil (Green Ying Artemisia Company, Henan Province, China) was used as a core agent while gelatin (Shanghai Chemical Reagent Co., Ltd., China) and arabic gum (Guangzhou Southern-based Company, China) were used as the wall materials of microcapsules. Span 80 (Tianjin Kermel Chemical Reagent Co., Ltd.) and formaldehyde (Tianjin Ke Meng Chemistry Co., Ltd.) were applied as a nonionic surface-active agent and a curing agent, respectively, during the microcapsule formation. Acetic acid solution and sodium hydroxide were obtained from Tianjin Damao Chemical Reagent Factory, China and Xilong Chemical Industry Incorporated Co. Ltd., China, respectively. 3D resin as a cross-linker and MgCl2 as a catalyst (Dymatic Chemicals, Inc., China) and fatty alcohol polyoxyethylene ether (JFC) (Guangzhou Chemical City, China) as a penetrant were used in the process of microcapsule fabrication. Agar and Luria broth (LB) were purchased from Sigma-Aldrich, Germany.
Preparation of microcapsules
Moxa oil microcapsules were prepared by a complex coacervation method. 1.5 g of gelatin and 1.5 g of arabic gum were hydrated in 40 g of deionized water respectively which were then heated at 50℃. Afterwards, 3 g of moxa oil, 1.5 g of span 80 and 2 g of a nonionic surfactant agent were dissolved in 20 g of deionized water and they were stirred at 2000 rpm for 15 minutes using a magnetic stirrer. Subsequently, the oil mixture was added to a completely dissolved arabic gum solution and they were emulsified at 1200 rpm for five minutes using a mechanical mixer. The stirring speed was then adjusted to 400 rpm and afterwards a completely dissolved gelatin solution was poured into the emulsion. Simultaneously, a 10% acetic acid solution was dropped until the pH value of the emulsion reached approximately 4. The reaction lasted for 15 minutes. The reaction system was subject to the ice bath and it was allowed to cool down to 5–10℃. 6 ml of 25% formaldehyde was then added into the reaction system and it was stirred for 30 minutes in the ice bath. Afterwards, a 10% sodium hydroxide solution was added to adjust the pH to 8–9. Finally, the reaction system was heated at 50℃ using the water bath. Moxa oil containing microcapsules was obtained after two hours of continuous stirring.
Sample preparation
Pure woven cotton fabrics were supplied by Esquel Group, China. The cotton fabrics were first soaked in a 10% acetone solution for 30 minutes and then they were rinsed with water and dried at room temperature. The cotton fabrics were cut into 10 × 10 cm2 sections. The microcapsule-finishing liquid was composed of 200 g/l of moxa oil microcapsule emulsion, 100 g/l of 3D resin, 2 g/l of catalyst MgCl2 and JFC. The cotton fabrics were immersed into the microcapsule-finishing liquid at the bath ratio of 1:30 for 30 minutes. A small padder (Model, LZ, Supplier: Widerly Development Limited, USA) with the pressure of 0.3 MPa was used to squeeze out the extra water of the microcapsule-treated samples evenly. The wet pick up of the finished specimens was 100%. Afterwards, the finished fabrics were preliminary dried at 80–90℃ for three minutes and afterwards baked at 110–120℃ for 1.5 minutes, and then cooled at room temperature. Consequently, microcapsule-treated cotton samples were obtained after rinsing and drying.
Scanning electron microscopy (SEM) and determination of particle size
Moxa oil containing microcapsules were dropped on the surface of a mica piece and they were allowed to dry at room temperature. Microcapsule-treated cotton samples were coated with gold prior to evaluation using an ion sputtering device (JEOL JFC 1100, Tokyo, Japan). The surface morphology of the test samples was evaluated using a scanning electron microscope (Model: TM-3000, Supplier: TECHCOMP LIMITED, China) with 20 kV accelerating voltage. The particle size of the microcapsules was examined according to the captured scanning electron microscopy (SEM) images. A total number of 200 microcapsules were counted manually.
Determination of encapsulated oil in microcapsules
A given amount of microcapsules was dispensed in 1 ml of HCl in order to dissolve the wall materials of the microcapsules. The amount of moxa oil entrapped in the microcapsules was then quantified after all the oil was removed by extraction from the microcapsules with hexane, which was repeated three times. The absorbance was determined at 280 nm using an ultraviolet visible spectrophotometer (Lambda 18, Perkin Elmer). Three measurements of each sample were taken in order to obtain the average amount of oil existing in the microcapsules.
Release test
1 g of moxa oil microcapsules and cotton fabric treated with 1 g of moxa oil microcapsules were placed into a medium containing 100 ml of a 80% phosphate buffer saline (pH 7.4) and 24% ethanol and incubated at 50 rpm and 37℃, respectively. Aliquots were withdrawn at the desired time intervals and the medium was then replaced in order to ensure the constant amount of release medium. The absorbance was measured at 280 nm using the ultraviolet visible spectrophotometer. 17
Antibacterial study of microcapsule-treated cotton fabrics
Staphylococcus aureus (S. aureus) was used to study the antibacterial activity of moxa oil containing microcapsule-treated cotton fabrics. S. aureus was firstly diluted with LB medium and plated on a sterilized agar plate. The fabric samples were cut into circular shape 1 cm in diameter. Cotton fabrics containing microcapsules with 25 µg/cm2 and 50 µg/cm2 of moxa oil as well as cotton fabrics containing 25 µg/cm2 and 50 µg/cm2 of moxa oil were prepared by adding the required amount of moxa oil containing microcapsules and moxa oil into the finishing liquid as previously mentioned in the Sample Preparation Section, respectively, and then applied to the cotton fabric samples. Afterwards, cotton fabrics containing microcapsules with 25 µg/cm2 and 50 µg/cm2 of moxa oil, cotton fabrics containing 25 µg/cm2 and 50 µg/cm2 of moxa oil and control cotton samples were placed on the agar surface. The plates were incubated at 37℃ for 24 hours and the growth inhibition of S. aureus on agar plates was recorded.8,18
Results and discussion
SEM of moxa oil containing microcapsules
The SEM image of a moxa oil containing microcapsule is shown in Figure 1. It was observed that moxa oil containing microcapsules showed relatively spherical appearance and good dispersion.
Scanning electron microscopy micrographs of moxa oil containing microcapsules.
SEM of microcapsule-treated cotton fabrics
Figure 2(a) shows the SEM image of the control sample at a magnification of 500 × with normal fibrillose being apparently found on the fabric surface. From Figure 2(b), it is obvious that the fiber surface was evenly adhered to with many tiny microcapsules. Microcapsules penetrated and attached into the gaps among the fibers. This behavior could result in the prevention of broken microcapsules which might be caused by friction during the padding process.
(a) Scanning electron microscopyimage of control cotton fabric; (b) scanning electron microscopy image of microcapsule-treated cotton fabric.
Particle size
Figure 3 shows the particle size distribution of moxa oil containing microcapsules. The developed microcapsules ranged from 2.3 to 9.8 µm, and the mean particle size of microcapsules was examined to be 6.4 µm. It was noticed that a majority of microcapsules (75%) were in a range of 6.0 to 8.0 µm.
Particle size distribution of moxa oil containing microcapsules.
Amount of oil encapsulated in the microcapsules
The oil was extracted from the microcapsules using hexane so that the gelatin and arabic gum did not interfere with the measurement process, since both substances are insoluble in hexane. The moxa oil loading of microcapsules was estimated to be 0.20 ± 0.01 mg/ml.
Release of moxa oil from the microcapsules and microcapsule-treated cotton fabric
Figure 4 shows the release profile of moxa oil containing microcapsules. It was illustrated that approximately 30% of the moxa oil was released after the first 6 hours and about 25% of the oil was further released from the microcapsules after 24 hours. Afterwards, the moxa oil was released at a slower rate for the next 24 hours. The oil was then continuously released and about 90% of oil was released after 96 hours. Figure 5 illustrates the moxa oil release from microcapsule-treated cotton fabric. The release rate of moxa oil from the fabric sample was relatively slower than that of the microcapsules. This reduced rate might be associated with the fact that some microcapsules penetrated and attached into gaps among the fibers which resulted in a reduced surface area for oil release.
Release profile of moxa oil containing microcapsules. Release profile of moxa oil from microcapsule-treated cotton fabric.
Antibacterial study
From Figure 6, it was demonstrated that moxa oil containing microcapsules significantly improved the antibacterial activity when compared with that of the free moxa oil since the microcapsule-treated fabrics had a larger inhibition zone than that of the moxa oil-treated fabrics. This result might be due to the controlled release of moxa oil from microcapsules which resulted in sustained growth inhibition towards S. aureus.8,18 Microcapsules containing 50 µg/cm2 of moxa oil (inhibition zone = 7.5 mm ± 0.5) showed stronger growth inhibition towards S. aureus than that of 25 µg/cm2 of moxa oil (inhibition zone = 5 mm ± 0.5). Control cotton as a negative control did not show any antibacterial activity.
Growth inhibition of moxa oil containing microcapsules towards Staphylococcus aureus. Three independent experiments were performed for each sample in order to obtain the average inhibition zones of each sample. Shown is one of the representative results.
Conclusion
In conclusion, encapsulation of moxa oil in gelatin-arabic gum microcapsules by a complex coacervation is an effective method to protect the oil from the external environment and to enhance the antibacterial property of moxa oil. The characterization of moxa oil containing gelatin-arabic gum microcapsules was established in terms of surface morphology, particle size, drug loading and release rate as well as antibacterial activity. Lee and Vairappan 19 in 2011 reported that there was no obvious antibacterial activity of free moxa (Artemisia argyi) oil. However, our results revealed that the controlled release of moxa oil from microcapsules could promote a prolonged and stronger antibacterial activity for textile materials. The moxa oil containing microcapsule-treated cotton fabric is believed to provide a potential application for the textile industry since it is user-friendly when compared to the free oil form. It is proposed that moxa oil containing microcapsules could be applied to medical textiles in order to produce the hygienic and antibacterial conditions required for contact with human beings.
Footnotes
Acknowledgement
We would like to thank Dr. C.H. Chui (Department of Medicine and Therapeutics, Prince of Wales Hospital, The Chinese University of Hong Kong) and Dr. S.Y. Cheng (Institute of Textiles and Clothing, The Hong Kong Polytechnic University) for their technical advice in antibacterial study.
Funding
This research is supported by RGC Early Career Scheme (F-PP0A, Design of smart functional apparel products for moxa moxibustion).