Asian Journal of Pharmaceutical Sciences
July 2015, Vol.10(4)
* Jirapornchai SuksaereeLaksana CharoenchaiFameera MadakaChaowalit MontonApirak SakunpakTossaton CharoonratanaWiwat Pichayakorn
Our work was to study the preparation, physicochemical characterization, and in vitro characteristic of Zingiber cassumunar blended patches. The Z. cassumunar blended patches incorporating Z. cassumunar Roxb. also known as Plai were prepared from chitosan and polyvinyl alcohol with glycerin as plasticizer. They were prepared by adding all ingredients in a beaker and homogeneously mixing them. Then, they were transferred into Petri-dish and dried in hot air oven. The hydrophilic nature of the Z. cassumunar blended patches was confirmed by the moisture uptake, swelling ratio, erosion, and porosity values. The FTIR, DSC, XRD, and SEM studies showed revealed blended patches with amorphous region that was homogeneously smooth and compact in both surface and cross section dimensions. They exhibited controlled the release behavior of (E)-4-(3′,4′-dimethoxyphenyl) but-3-en-l-ol (compound D) that is the main active compound in Z. cassumunar for anti-inflammation activity. However, in in vitro skin permeation study, the compound D was accumulated in newborn pig skin more than in the receptor medium. Thus, the blended patches showed the suitable entrapment and controlled release of compound D. Accordingly, we have demonstrated that such chitosan and polyvinyl alcohol formulated patches might be developed for medical use.
Topical and transdermal drug delivery systems are intended for external use. They are often dermatologic products such as sunscreens, local anesthetics, antiseptics and anti-inflammatory agents intended for localized action on one or more layers of the skin. Conversely, some transdermal drug delivery systems are designed for percutaneous route of drug delivery in which case skin is not the target. In such case, the drug must be absorbed across the skin which is made up of dermis and epidermis, especially the stratum corneum barrier including sweat glands, sebaceous glands, and hair follicles , and pass into deeper dermal layers to reach the systemic blood circulation. Generally, substances intended for transdermal delivery systems are low molecular weight (100-500 Da), potent non-irritation and non-allergenic ,  and . The delivery system can be categorized as either i) drug in adhesive or ii) drug in matrix systems. The drug is dispersed or dissolved in a polymer matrix and attached to an adhesive layer that contacts the skin. In some cases, the polymer matrix can act as the adhesive layer. Polymer matrix layers and/or the added adhesive layer act as a control of the rate of delivery  and .
Thai traditional medicines (herbal medicines) are popular for the treatment of various symptoms and diseases and to promote good health. Although the Western modern medicines are increasingly popular, Thai traditional medicines are still widely used especially among the rural Thais. Herbal medicines may contain variations of active ingredients parts of plants, other plant materials, or combinations that included herbs, herbal materials, herbal preparations, and finished herbal products. Zingiber cassumunar Roxb., also known as Thai name “Plai”, is a medicinal plant widely cultivated in Thailand and tropical Asia. It is frequently used as an ingredient in marketed phytomedicines  and . The rhizome of Z. cassumunar Roxb. has an anti-inflammatory activity. It has been the source of Thai traditional herbal remedies and extracts for topical application to alleviate inflammation ,  and . The chemical composition of the rhizome oils of Z. cassumunar Roxb. has been previously reported , , , , ,  and . The major constituents of the crude oils are terpinen-4-ol, α- and β-pinene, sabinene, myrcene, α- and γ-terpinene, limonene, terpinolene, sabmene, and monoterpenes  and . (E)-4-(3′,4′-dimethoxyphenyl) but-3-en-l-ol (compound D) is the main active compound in Z. cassumunar that exhibits anti-inflammatory ,  and , analgesic and antipyrectic ,  and  activity in experimental models. It is also used as topical treatment for sprains, contusions, joint inflammations, muscular pain, abscesses, and similar inflammation-related disorders ,  and . Thus, this work used the compound D as the marker compound for in vitro study.
Herbal patches are adhesive patches that incorporate the herbal medicines or extracted herb. When applied to the skin the active compound is released at a constant rate. Such patches are recommended for smoking cessation, herbal body detox foot patch, relief of stress, to increase sexuality, as insect repellants, as male energizer, to improve sleep, to postpone menopause, for rheumatoid arthritis, as herbal plasters patches, etc. .
The aim of the current study was to prepare a Z. cassumunar containing product incorporating the crude Z. cassumunar oil in blended patches that consisted of chitosan and polyvinyl alcohol (PVA) polymer matrix combination using glycerin as plasticizer. Similarly prepared blended patches without crude Z. cassumunar oil served as control. The patches were evaluated with regard to the physicochemical properties as moisture uptake, swelling ratio, erosion, porosity, Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), X-ray diffraction (XRD), scanning electron microscope (SEM), and in vitro release and skin permeation studies.
2. Materials and methods
The Z. cassumunar rhizome powder was purchased from Charoensuk Osod, Thailand. The Z. cassumunar powder was extracted in 95% ethanol and filtered through a 0.45 μm of polyamide membrane to obtain crude Z. cassumunar oil. Chitosan (degree of deacetylation = 85%, mesh size 30) was purchased from Seafresh Industry Public Co., Ltd, Thailand. PVA and glycerin were purchased from Sigma–Aldrich, USA. All organic solvents were analytical grade obtained from Merck KGaA, Germany.
2.2 Analytical method
An Agilent 1260 Infinity system (Agilent Technologies, USA.) was used for this experiment with detection at 260 nm, a 4.6 mm × 250 mm diameter, 5 μm particle size C18 column (ACE 5, DV12-7219, USA.), a flow rate of 1 ml/min, and injection volume of 10 μL. The mobile phase was a gradient elution of 2% acetic acid in ultrapure water (A) and methanol (B) of 60 to 50% of A, 50 to 30% of A, 30 to 20% of A, 20 to 50% of A, 50 to 60% of A, and 60% of A for 0–5 min, 5–15 min, 15–25 min, 25–30 min, 30–32 min, and 32–40 min, respectively . The HPLC validation method of compound D provided a limit of detection of 0.20 μg/ml, the limit of quantification of 0.80 μg/ml, good accuracy (95.38–104.76%), precision (less than 2%CV), and linearity with good correlation coefficient (r2) > 0.9999 in the required concentration range of 2–40 μg/ml. The separation method and validation method of compound D from crude Z. cassumunar oil was described in previous publication  and .
2.3 Z. cassumunar blended patches preparation
The chitosan was dissolved in 1% acetic acid in distilled water in concentration of 3.5%w/v. The PVA was dissolved in distilled water in concentration of 20%w/v. The blank blended patches were prepared by 2 g of 3.5%w/v chitosan were mixed together with 5 g of 20%w/v of PVA, and homogeneously mixed with 2 g of glycerin as plasticizer to obtain clear polymer blended solution. The Z. cassumunar blended patches were prepared as 3 g of the crude Z. cassumunar oil completely dissolved in absolute ethanol and continuously mixed in polymer blend solution. They were transferred into Petri-dish and dried in hot air oven at 70 ± 2 °C for 5 h. Finally, they were peeled from Petri-dish and kept in desiccator until used.
2.4 Evaluation of blank and Z. cassumunar blended patches
2.4.1 SEM photography
The surface and cross section of blank blended patches and Z. cassumunar blended patches were placed onto copper stub and then coated with gold in a sputter coater. They were photographed under SEM equipment (model: Quanta 400, FEI, Czech Republic) with high vacuum and high voltage of 20 kV condition, with Everhart Thornley detector (ETD).
2.4.2 FTIR study
The FTIR study employed the Attenuated Total Reflectance – FTIR (ATR-FTIR) technique for the chitosan film, PVA film, crude Z. cassumunar oil, blank blended patches, and Z. cassumunar blended patches. They were scanned at a resolution of 4 cm−1 with 16 scans over a wavenumber region of 400 – 4000 cm−1. The FTIR spectrometer (model: Nicolet 6700, DLaTGS detector, Thermo Scientific, USA.) was used to determine IR transmission spectra and record the characteristic peaks.
2.4.3 DSC study
A DSC instrument (model: DSC7, Perkin Elmer, USA) was used to investigate the endothermic transition of the substances that also confirmed the compatibility of each ingredient. The 1 – 10 mg of sample was weighted in DSC pan, hermetically sealed, and run in the DSC instrument at the heating rate of 10 °C/min under a liquid nitrogen atmosphere from 20 °C to 350 °C.
2.4.4 XRD study
The XRD (model: X'Pert MPD, PHILIPS, Netherlands) was employed to study the compatibility of the chitosan, PVA, blank blended patches, and Z. cassumunar blended patches. The generator operating voltage and current of X-ray source were 40 kV and 45 mA, respectively, with an angular of 5 – 40° (2θ), and a stepped angle of 0.02° (2θ)/s.
2.4.5 Moisture uptake, swelling ratio, and erosion studies
For determination of moisture uptake, swelling ratio and erosion, 1 cm × 1 cm patch specimens were used. For moisture uptake determination, the patch specimens were weighed for their initial value (W0), then moved to a stability chamber (model: Climate Chamber ICH/ICH L, Memmert GmbH + Co. KG, Germany) which controlled the temperature at 25 ± 2 °C and 75% relative humidity environment. The specimens were removed and weighed until constant (Wu). The percentage of moisture uptake was calculated by Equation (1) 
The swelling ratio and erosion study were also determined by drying patch specimens at 60 ± 2 °C overnight. Then, they were weighed (W0) and immersed in 5 ml of distilled water and moved to stability chamber (model: Climate Chamber ICH/ICH L, Memmert GmbH + Co. KG, Germany) which controlled the temperature at 25 ± 2 °C and 75% relative humidity environment for 48 h. After removal of excess water, the hydrated patches were weighed (Ws). They were then dried again at 60 ± 2 °C overnight, and weighed again (Wd). The percentage of swelling ratio and the percentage of erosion were calculated by (2) and (3), respectively.
2.4.6 Porosity determination
After the patch specimens were equilibrated in water, the volume occupied by the water and the volume of the membrane in the wet state were determined. The porosity of patch specimens was obtained by Equation (4).
where W1 and W2 = the weights of the membranes in the wet and dry states (g), respectively, dwater = the density of pure water at 20 °C, and w, l, t = the width (cm), length (cm), and thickness (cm) of the membrane in the wet state, respectively  and .
2.5 The determination of compound D in patches
The blended patches were cut into 2 cm × 2 cm specimens from different sites. Each Plai patch sample was soaked with ethanol in 10 ml volumetric flask, and sonicated at 25 °C for 30 min. Then, the solution was sampled for 0.5 ml and transferred into 100 ml volumetric flask and adjusted to volume of 100 ml with ethanol. The solution was filtered through a 0.45 μm filter and analyzed with HPLC method.
2.6 In vitro release study of compound D
The modified Franz-type diffusion cell having effective diffusion area of 1.77 cm2 was used for in vitro release and skin permeation study of compound D from the Z. cassumunar blended patches. The receptor medium was 12 ml of isotonic phosphate buffer solution pH 7.4: ethanol = 80:20, thermoregulated with a water jacket at 37 ± 0.5 °C and stirred constantly at 600 rpm with a magnetic stirrer. The crude Z. cassumunar oil was applied on the cellulose membrane (MWCO: 3500 Da, CelluSep® T4, Membrane Filtration Product, Inc., USA) which was used as a barrier between the donor compartment and the receptor compartment. The Z. cassumunar blended patch preparations were cut and placed directly on the donor cells. The 1 ml of receptor solution was withdrawn at 0, 0.5, 1, 2, 3, 4, 6, and 24 h intervals, and immediately replaced with an equal volume of fresh receptor medium. The compound D content in these samples was determined by an HPLC method.
2.7 In vitro skin permeation study of compound D
The in vitro skin permeation of the compound D from the Z. cassumunar blended patches was also carried out using a modified Franz-type diffusion cell , and pig skin with hair removed was an applied partitioning membrane  and . The newborn pigs of 1.4–1.8 kg weight that had died by natural causes shortly after birth were freshly purchased from a local pig farm in Chachoengsao Province, Thailand. The full thickness of flank pig skin was excised, hair was surgically removed, and the subcutaneous fat and other extraneous tissues were trimmed with a scalpel, cleaned with isotonic phosphate buffer solution pH 7.4, blotted dry, wrapped with aluminum foil and stored frozen. Before permeation experiments, this isolated skin was soaked overnight in isotonic phosphate buffer solution pH 7.4, and mounted on the modified Franz-type diffusion cell with the stratum corneum facing upward on the donor compartment. The crude Z. cassumunar oil and Z. cassumunar blended patches were laid onto the isolated skin in the same way as for the release study. The receptor compartment was 12 ml of isotonic phosphate buffer solution pH 7.4: ethanol = 80:20 and stirred constantly at 600 rpm by a magnetic stirrer, at a constant temperature of 37 ± 0.5 °C. A 1 ml of the receptor solution was withdrawn at 0, 0.5, 1, 2, 3, 4, 6, and 24 h intervals and an equal volume of fresh receptor medium was immediately replaced. The compound D content in these samples was determined by the HPLC method.
All in vitro release and skin permeation studies were performed in triplicate and the means of all measurements calculated. The results were presented in terms of cumulative percentage release or skin permeation as a function of time using the following formula:
where Dt was the amount of compound D released or permeated from the Z. cassumunar blended patches at time t and Dl was the amount of compound D loaded into the Z. cassumunar blended patches.
3. Results and discussion
3.1 Evaluation of blank and Z. cassumunar blended patches
Generally, the Z. cassumunar rhizomes were of deep yellow color possessing a strong camphoraceous smell, warm, spicy, and bitter taste ,  and . The extraction of the Z. cassumunar rhizome powder yielded a clear, high viscosity, yellow-orange crude Z. cassumunar oil. The solvent extraction of plant materials likely produced oleoresin, which contained not only the volatile compounds but also waxes and color pigments . In addition, Sukatta et al. 2009 reported two pathways for Z. cassumunar rhizome extraction-hydro distillation and hexane extraction. They confirmed that hydro distillation produced the yellowish, low viscosity crude Z. cassumunar oil, while the crude Z. cassumunar oil from the hexane extraction was yellow-orange in color and had high viscosity. Commonly, our work could confirm from its appearance that crude Z. cassumunar oil was obtained. Therefore, when crude Z. cassumunar oil was added in blank blended patches, it produced the dark yellow patches referred to as Z. cassumunar blended patches. The photographs of blank blended and Z. cassumunarblended patches were shown in previous reports by our research group .
The SEM technique was used to photograph the high resolution morphology of the surface and cross section of blank blended patches and Z. cassumunar blended patches (Fig. 1). The surface of blank blended patches was homogeneously smooth and dense with no visual pores (Fig. 1A). The surface of Z. cassumunar blended patches became rough and uneven as a result of widely distributed conglomeration and aggregation in the matrix of Z. cassumunar blended patches (photographed by digital camera and presented in previous publication ) (Fig. 1B).
Fig. 1. Surface (×500 (A), ×1000 (B), and ×1500 (C)) and cross section morphology (×1000 (D), ×1500 (E), and ×5000 (F)) of blank blended patches (upper) and Z. cassumunar blended patches (bottom) under SEM technique.
Recorded spectra are shown in Fig. 2. For the chitosan film, the absorption peaks of stretching vibrations of –OH groups broadly overlapped the stretching vibration of N–H ranging from 3750 to 3000 cm−1. The broad stretching vibrations of C–H bond were observed at 2920–2875 cm−1. The bending vibrations of methylene and methyl groups were also absorbed at 1375 cm−1 and 1426 cm−1, respectively. The spectrum bands in the range of 1680–1480 cm−1 were identified as vibrations of carbonyl bonds of the amide group and vibrations of protonated amine group. The vibrations of CO group occurred in the range from 1160 cm−1–1000 cm−1. In addition, the spectrum band located at around 1150 cm−1 related to asymmetric vibrations of CO in the oxygen bridge resulting from deacetylation of chitosan. The spectrum bands at 1080–1025 cm−1 were attributed to –CO of the ring COH, COC, and CH2OH. Finally, the small spectrum peak at ∼890 cm−1 corresponded to wagging of the saccharide structure of chitosan . Furthermore, the spectrum of acetic acid were found at 3050, 1720, and 1432 related to–OH bond in carboxylic acid, C–O bond, and C–O bond, respectively. In addition, the PVA spectrum showed both O–H stretching and C–O stretching at 3449 and 1637 cm−1, respectively  and .
Fig. 2. FTIR spectra of chitosan, PVA, blank blended patches, and Z. cassumunar blended patches.
The chitosan film, blank blended, and Z. cassumunar blended patches weighed 1.662, 8.747, and 8.821 mg, respectively. They were run with DSC instrument to study the thermal behavior. The thermogram of chitosan film, blank blended, and Z. cassumunar blended patches showed an initial broad peak at 70.33 °C with 231.35 J/g of enthalpy of peak (ΔH), 99.34 °C with 188.307 J/g of ΔH, and 92.37 °C with 78.76 J/g of ΔH, respectively, which was attributed to evaporation of moisture and represented the required energy to vaporize water present in their samples. Moreover, the degradation DSC peak of chitosan film broadly occurred at 323.67 °C with 127.30 J/g of ΔH. In addition, the blank blended patches and Z. cassumunar blended patches revealed high broad endothermic peaks at 257.00 °C with 363.24 J/g of ΔH and 261.00 °C with 606.41 J/g of ΔH, respectively. Although the observed endothermic peaks in blank blended patches and Z. cassumunar blended patches were slightly changed, there were no new exo- or endo-thermic peaks in any experimental ranges indicating compatibility of all ingredients (Fig. 3).
Fig. 3. DSC thermograms of chitosan, blank blended patches, and Z. cassumunar blended patches.
The XRD technique was used to identify and characterize crystalline and amorphous form of chitosan film, PVA film, blank blended patches, and Z. cassumunar blended patches that had been studied in range of 5–40° (2θ values) (Fig. 4). The X-ray diffraction profile of chitosan film showed peaks at ∼10° and ∼23° (2θ). The intensity result of PVA film was 19.69° representing their semi-crystalline characters because of the strong intermolecular interaction between PVA chains through intermolecular hydrogen bonding . Thus, the chitosan and PVA film exhibited the semi-crystalline characteristics, but the XRD patterns of blank blended patches and Z. cassumunarblended patches had broad diffraction halo of amorphous region.
Fig. 4. XRD patterns of chitosan, PVA, blank blended patches, and Z. cassumunar blended patches.
From above experimentals, the FTIR, DSC and XRD results showed that there were no chemical interactions between any components in blank blended patches or Z. cassumunar blended patches.
Limpongsa and Umprayn (2008) reported that moisture uptake, swelling ratio, erosion, and porosity values play important roles for the release behavior of active compound in matrix type patches . Thus, this research evaluated these variables as show in Fig. 5. We found that the moisture uptake, swelling ratio, erosion, and porosity of blank blended patches were 28.85 ± 4.17, 21.01 ± 5.38, 2.39 ± 0.41, and 1.92 ± 0.22%, respectively. When crude Z. cassumunar oil was added in blank blended patches, the moisture uptake, swelling ratio, erosion, and porosity of blank blended patches were 28.51 ± 0.78, 20.93 ± 5.88, 2.42 ± 0.98, 1.86 ± 0.24%, respectively, which were not significantly different from blank blended patches. These results are due to the fact that hydrophilic parts of ingredients could be dissolved and eroded from the blended patches. The chitosan and PVA could swell and immediately had the hydrated blended patches contents. The chains mobility of chitosan and PVA increased, therefore, increasing the hydrodynamic volume of the polymer compact.
Fig. 5. The moisture uptake, swelling ratio, erosion, and porosity values of blank blended patches and Z. cassumunar blended patches.
3.2 In vitro release study of compound D
In vitro release of the crude Z. cassumunar oil released compound D calculated as cumulative percentage release 90.43 ± 19.28% after 24 h (Fig. 6). The almost 100% release of compound D in 24 h might be due to rapid diffusion in the receptor medium as a fast, initial burst during the first 6 h.
Fig. 6. In vitro release of compound D content from crude Z. cassumunar oil and Z. cassumunar blended patches and in vitrorelease kinetics of zero order model (A), first order model (B), and Higuchi's model (C).
The amount of compound D in the Z. cassumunar blended patches was 2.19 ± 0.16 mg/cm2. When the Z. cassumunar blended patches were studied in in vitro, the cumulative percentage release of compound D was 81.49 ± 10.92% after 24 h (Fig. 6). The release behavior was similar to the compound D release behavior from crude Z. cassumunar oil that had a fast initial burst release during the first 6 h. This behavior was likely due to the compound D on the surface of patches might be rapid diffusion. However, the effect may be attributed to the moisture uptake, swelling ratio, erosion, and porosity whereby the patch could absorb the moisture, and create a space and a large free volume within the blended patches that enhanced compound D diffusion . Moreover, Guo et al. 2011 reported enhanced drug diffusion with amorphous matrix type patches  which supports our results in in vitro study. The in vitro release kinetics model of compound D provided a better fit to first-order model than to the zero-order and Higuchi's model (Fig. 6).
3.3 In vitro skin permeation study of compound D
The in vitro skin permeation study was carried out in a modified Franz-type diffusion cell using newborn pig skin as a partition membrane. The mean cumulative amount of compound D permeated from crude Z. cassumunar oil and Z. cassumunar blended patches were 38.55 ± 18.48% and 36.72 ± 11.29% after 24 h, respectively (Fig. 7). Although another publication reported that glycerine could enhance drug permeability  and , the Z. cassumunar blended patches contained only a small amount of glycerin as plasticizer which was unlikely to affect drug permeation. Moreover, compound D was only slightly detected in the receptor medium. Because of its structure, compound D exhibits less hydrophilicity than hydrophobicity ,  and . The in vitro skin permeation kinetics model of compound D provided a better fit to a first-order model than zero-order and Higuchi's model (Fig. 7).
Fig. 7. In vitro skin permeation of compound D content from crude Z. cassumunar oil and Z. cassumunar blended patches and in vitro skin permeation kinetics of zero order model (A), first order model (B), and Higuchi's model (C).
Thus, the newborn pig skins were removed from modified Franz-type diffusion cell apparatus. They were cut into small pieces and homogenized, and then were extracted in absolute ethanol. These solutions were analyzed for the remaining compound D content by HPLC method. They contained 60.54 ± 39.55% and 46.77 ± 17.93% compound D content in crude Z. cassumunar oil and Z. cassumunar blended patches, respectively. Thus, the compound D was highly accumulated in newborn pig skin layer minimum permeation into receptor medium. However, the underlying mechanisms for this effect was never reported and will be further studied.
In the current work prepared the Z. cassumunar blended patches made from chitosan and PVA polymer blends incorporating the crude Z. cassumunar oil. The surface and cross section were photographed for morphology study under SEM technique and the physicochemical properties evaluated by FTIR, DSC, XRD, moisture uptake, swelling ratio, erosion, and porosity. The results revealed compatible, homogeneous, smooth, and compact blended ingredients. The blended patches could absorb the moisture that resulted in swelling of blended patches. They were eroded which increased the number of porous channels homogenously to pass compound D from Z. cassumunar blended patches. The blended patches provided a controlled release and skin permeation of compound D when studied by modified Franz-type diffusion cell apparatus. Thus, the blended patches could be suitably used for herbal medicine application.
The authors reported no declaration of interests. The authors are thankful to the Faculty of Pharmacy and the Research Institute of Rangsit University (Grant No.74/2555) for financial supports.