Jarupa Viyoch

Potential of an injectable chitosan/starch/β-glycerol phosphate hydrogel for sustaining normal chondrocyte function

An injectable hydrogel for chondrocyte delivery was developed by blending chitosan and starch derived from various sources with beta-glycerol phosphate (beta-GP) in the expectation that it would retain a liquid state at room temperature and gel at raised temperatures. Rheological investigation indicated that the system consisting of chitosan derived from crab shell and corn starch at 4:1 by weight ratio (1.53%, w/v of total polymers), and 6.0% (w/v) beta-GP (C/S/GP system) exhibited the sharpest sol-gel transition at 37+/-2 degrees C. The C/S/GP hydrogel was gradually degraded by 67% within 56 days in PBS containing 0.02 mg/ml lysozyme. The presence of starch in the system increased the water absorption of the hydrogel when compared to the system without starch. SEM observation revealed to the interior structure of the C/S/GP hydrogel having interconnected pore structure (average pore size 26.4 microm) whereas the pore size of the hydrogel without starch was 19.8 microm. The hydrogel also showed an ability to maintain chondrocyte phenotype as shown by cell morphology and expression of type II collagen mRNA and protein. In vivo study revealed that the gel was formed rapidly and localized at the injection site.

Evaluation of in vitro antimicrobial activity of Thai basil oils and their micro‐emulsion formulas against Propionibacterium acnes

The aim of this study was to evaluate the efficacy of Thai basil oils and their micro‐emulsions, on in vitro activity against Propionibacterium acnes. An agar disc diffusion method was employed for screening antimicrobial activity of the essential oils of Ocimum basilicum L. (sweet basil), Ocimum sanctum L. (holy basil) and Ocimum americanum L. (hoary basil) against P. acnes. Minimum inhibitory concentration (MIC) values of the basil oils were determined using an agar dilution assay. The obtained results indicated that the MIC values of sweet basil and holy basil oils were 2.0% and 3.0% v/v, respectively, whereas hoary basil oil did not show activity against P. acnes at the highest concentration tested (5.0% v/v). Gas chromatography‐mass spectrometry analysis revealed that methyl chavicol (93.0%) was the major compound in sweet basil oil, and eugenol (41.5%), γ‐caryophyllene (23.7%) and methyl eugenol (11.8%) were major compounds in holy basil oil. Hoary basil oil contained high amounts of geraniol (32.0%) and neral (27.2%) and small amounts of methyl chavicol (0.8%). The Oil‐in‐water (o/w) micro‐emulsions of individual basil oils with concentrations corresponding to their MIC values were formulated. The stable o/w micro‐emulsion system for basil oil consisted of 55.0% v/v water phase, 10.0% v/v oil phase (2.0 or 3.0% v/v sweet basil or 3.0% v/v holy basil oil plus 7.0% v/v isopropyl myristate), 29.2% v/v polysorbate 80 and 5.8% v/v 1,2‐propylene glycol. Hydroxyethylcellulose at a concentration of 0.5% w/v was used as thickening agent. According to the disc diffusion assay, the formulations containing sweet basil oil exhibited higher activity against P. acnes than those containing holy basil oil, and the thickened formulations tended to give a lower activity against P. acnes than the non‐thickened formulations. The prepared micro‐emulsions were stable after being tested by a heat–cool cycling method for five cycles. These findings indicate the possibility to use Thai sweet and holy basil oil in suitable formulations for acne skin care.

Effects of the blended fibroin/aloe gel film on wound healing in streptozotocin-induced diabetic rats

Delayed healing remains a major clinical problem and here we have sought to develop an improved dressing film comprising 1.95% w/v fibroin and 0.05% w/v aloe gel extract. The tensile strength of dry film was 21.1 ± 0.5 MPa and broke at 1.1 ± 0.2% elongation; corresponding values for wet film were 18.3 ± 1.3 MPa and 1.9 ± 0.1%. The film maintained its shape upon water immersion and the swelling ratio of the dry film was 0.8 ± 0.1 while the water uptake was 43.7 ± 2.6%. After 28 days of incubation in phosphate buffered saline (1 M, pH 7.4, 37 °C), the weight of film was reduced by 6.7 ± 1.1% and the tensile strength and elongation at breaking point (dry state) were 15.4 ± 0.6 MPa and 1.5 ± 0.2%, respectively. Compared to aloe-free fibroin film (2.0% fibroin extract only), the blended film enhanced the attachment and proliferation of skin fibroblasts. The bFGF immunofluorescence of fibroblasts cultured on the blended film appeared greater than those cultured on tissue culture plate or on aloe-free fibroin film while α-smooth muscle actin was maintained. In streptozotocin-induced diabetic rats, the wounds dressed with the blended film were smaller (p <0.05) by day 7 after wounding, compared to untreated diabetic wounds. Histology of repaired diabetic wounds showed the fibroblast distribution and collagen fiber organization to be similar to wounds in normal rats, and this was matched by enhanced hydroxyproline content. Thus, such accelerated wound healing by the blended fibroin/aloe gel films may find application in treatment of diabetic non-healing skin ulcers.

Properties and Biocompatibility of Chitosan and Silk Fibroin Blend Films for Application in Skin Tissue Engineering

Chitosan/silk fibroin (CS/SF) blend films were prepared and evaluated for feasibility of using the films as biomaterial for skin tissue engineering application. Fourier transform infrared spectroscopy and differential scanning calorimetry analysis indicated chemical interaction between chitosan and fibroin. Chitosan enhanced β-sheet conformation of fibroin and resulted in shifting of thermal degradation of the films. Flexibility, swelling index, and enzyme degradation were also increased by the chitosan content of the blend films. Biocompatibility of the blend films was determined by cultivation with fibroblast cells. All films showed no cytotoxicity by XTT assay. Fibroblast cells spread on CS/SF films via dendritic extensions, and cell-cell interactions were noted. Cell proliferation on CS/SF films was also demonstrated, and their phenotype was examined by the expression of collagen type I gene. These results showed possibility of using the CS/SF films as a supporting material for further study on skin tissue engineering.

Screening for phosphodiesterase inhibitory activity of Thai medicinal plants

INTRODUCTION Phosphodiesterases (PDEs) are a group of enzymes that have powerful effects on cellular signaling because they regulate the second messenger, cAMP or cGMP. PDE inhibitors have been used for treatment of many indications such as cardiovascular diseases, chronic obstructive pulmonary diseases, erectile dysfunction and pulmonary hypertension. THE AIM OF THE STUDY The aim of the study was to search for sources of PDE inhibitors from Thai biodiversity. MATERIALS AND METHODS Some Thai medicinal plants used as aphrodisiac and neurotonic agents together with plants from Leguminosae collected from the North of Thailand were screened for PDE inhibitory activity using a radioassay. RESULTS Seven from nineteen aphrodisiac and neurotonic plants as well as three from twelve Leguminosae plants showed potent PDEs inhibitory activity. The concentrations that could inhibit 50% PDE activity (IC(50)) of the active extracts were determined in comparison to the standard inhibitor, 3-isobutyl-1-methylxanthine (IBMX). Betula alnoides, Hiptage benghalensis, Leea indica and Senna surrattensis showed IC(50) values in the range of microgram per milliliter while IBMX standard showed an IC(50) value of 0.68+/-0.13 microg/ml. CONCLUSION Thai biodiversity was the great sources of PDE inhibitors.

Kaempferia parviflora, a plant used in traditional medicine to enhance sexual performance contains large amounts of low affinity PDE5 inhibitors

AIM OF THE STUDY A number of medicinal plants are used in traditional medicine to treat erectile dysfunction. Since cyclic nucleotide PDEs inhibitors underlie several current treatments for this condition, we sought to show whether these plants might contain substantial amounts of PDE5 inhibitors. MATERIALS AND METHODS Forty one plant extracts and eight 7-methoxyflavones from Kaempferia parviflora Wall. ex Baker were screened for PDE5 and PDE6 inhibitory activities using the two-step radioactive assay. The PDE5 and PDE6 were prepared from mice lung and chicken retinas, respectively. All plant extracts were tested at 50 μg/ml whereas the pure compounds were tested at 10 μM. RESULTS From forty one plant extracts tested, four showed the PDE5 inhibitory effect. The chemical constituents isolated from rhizomes of Kaempferia parviflora were further investigated on inhibitory activity against PDE5 and PDE6. The results showed that 7-methoxyflavones from this plant showed inhibition toward both enzymes. The most potent PDE5 inhibitor was 5,7-dimethoxyflavone (IC(50) = 10.64 ± 2.09 μM, selectivity on PDE5 over PDE6 = 3.71). Structure activity relationship showed that the methoxyl group at C-5 position of 7-methoxyflavones was necessary for PDE5 inhibition. CONCLUSIONS Kaempferia parviflora rhizome extract and its 7-methoxyflavone constituents had moderate inhibitory activity against PDE5. This finding provides an explanation for enhancing sexual performance in the traditional use of Kaempferia parviflora. Moreover, 5,7-dimethoxyflavones should make a useful lead compound to further develop clinically efficacious PDE5 inhibitors.

Development of curcuminoids hydrogel patch using chitosan from various sources as controlled-release matrix.

In this study, we developed the hydogel patch containing curcuminoids for application in cosmetic purpose. The powder of curcuminoids extracted from rhizome of Curcuma longa Linn. was first formulated into o/w microemulsions before incorporating into the polymer solution. The polymer solutions consisted of chitosan derived from various sources or the blended chitosan starch. We found that chitosan from squid pen gave the patch with highest strength and flexibility. After incorporation of curcuminoids microemulsion into the polymer solution of squid chitosan in ratio of 1 : 1 by weight, values of tensile strength and per cent elongation at break of the obtained patch decreased (from 4.55 ± 0.41 N mm−2 to 2.26 ± 0.01 N mm−2 for tensile strength and from 40.27 ± 1.46% to 29.65 ± 2.77% for elongation at break). The DSC thermogram of the squid patch containing curcuminoids implied non‐crystalline structure of polymeric network, corresponding to porous characteristics of the patch surface. The results showed that the curcumin content remained at a concentration of 96% and 40% of the initial content after being kept at 4°C and room temperature, respectively. When the patch was kept at 50°C, the remaining curcumin could not be detected. According to vertical diffusion cell method, we found a rapid rate of curcumin release from the patch. The curcumin release pattern, which fitted well to the Higuchi’s model, exhibited two distinct phases: the rapid phase (0–15 min), where the release rate of the curcumin averaged 0.74 μg min−1 mm−2, and the slow phase (15–120 min), where the release rate averaged 0.13 μg min−1 mm−2. Any sign of skin irritation was not observed in volunteers after single application of the curcuminoids patch in the under‐eye area for 30 min. This finding indicates mildness to skin of the developed patch.

Development of chitosan-coated liposomes for sustained delivery of tamarind fruit pulp's extract to the skin.

In this study, chitosan‐coated liposomes were developed. To entrap lyophilized tamarind extract containing alpha‐hydroxy acids (AHAs) together with tartaric acid, the reverse phase evaporation method was used to obtain well‐formed liposomes loaded with the extract. The highest entrapment efficiency of 68.3 ± 3.0% into the liposomes was obtained with liposomes consisting of phosphatidylcholine and cholesterol in a molar ratio of 2 : 1 after the extrusion process. The average particle size of the prepared liposomes was 158 ± 26 nm showing a negative zeta potential of −6 mV. For the preparation of the chitosan‐coated liposomes, two selected independent parameters were varied: chitosan concentrations of 0.1, 0.5 and 1.0% w/v and volumes of the chitosan solutions of 1, 2 and 3 mL, to study the effects of such parameters on the entrapment efficiency of the extract‐loaded liposomes. Variation in the volumes of the chitosan solution did not affect the entrapment efficiency of the liposomes. However, the entrapment efficiency of the AHAs in the chitosan‐coated liposomes significantly increased with increasing chitosan concentrations. The size of the chitosan‐coated liposomes was in the range of 200–300 nm with a positive zeta potential in the range of 6–29 mV. An in vitro release study using dialysis technique was performed to evaluate the release profile of the tartaric acid from the chitosan‐coated liposomes. The obtained results showed the effect of the chitosan‐coated liposomes on the lower release rate and on the amount of tartaric acid in comparison with that of the uncoated liposomes. The study in an in vitro skin cell model indicated that the developed system could enhance the potential of tamarind’s AHAs on the stimulation of human keratinocyte proliferation being two‐fold higher than the solution of the tamarind extract.

Formulation and development of a patch containing tamarind fruit extract by using the blended chitosan-starch as a rate-controlling matrix.

A cosmetic patch containing tamarind fruit extract was formulated and developed by blending two types of natural polymers: chitosan with molecular weight of 100 000 and starch such as corn, potato or tapioca starch. The physicochemical characteristics, i.e. flexibility, colour, transparency, integrity, gloss, water sorption and bioadhesion property and the stability of the patch without tamarind content were investigated. Stability test was performed by keeping the prepared patches at 4 degrees C, at room temperature or at 45 degrees C for 2 weeks. The results showed that the formulations composed of chitosan:corn starch ratio of 4.5 : 0.5 (CC(4.5 : 0.5)) and chitosan:tapioca starch ratios of 4.5 : 0.5 (CT(4.5 : 0.5)) and 4.0 : 1.0 (CT(4 : 1)) provide patches with favourable physical characteristics, high water sorption, good bioadhesion ability and good stability. After the lyophilized tamarind extract in an amount corresponding to 5% of tartaric acid was incorporated into the formulations of CC(4.5 : 0.5), CT(4.5 : 0.5) and CT(4 : 1), the ability of the patches to adhere to skin was improved. However, after keeping the test patches at room temperature or at 45 degrees C for 6 weeks, their colours were intensified while their flexibilities and skin adhesion properties decreased. A 12-h in vitro permeation was investigated by studying the cumulative amount of tartaric acid permeated through the Silastic membrane (Dow-Coming, Midland, MI, USA). The CC(4.5 : 0.5) patch tended to give the highest amount of tartaric acid released. The release pattern of all the blended polymeric matrices was exhibited in two distinct phases: the rapid phase, where the flux averaged 3.61 microg min(-1) mm(-2); and the slow phase, where the flux averaged 1.89 microg min(-1) mm(-2).

Development of hydrogel patch for controlled release of alpha-hydroxy acid contained in tamarind fruit pulp extract.

The aim of this study was to develop hydrogel patch using crosslinked chitosan–starch as polymeric matrix for controlling the release of the natural alpha‐hydroxy acid (AHA) contained in the extract of tamarinds fruit pulp. The chitosan (MW 100 000) was blended with corn, tapioca or rice starch in various ratios and then crosslinked with glutaraldehyde. The physical characteristics, mechanical resistance, bio‐adhesion property and surface morphology of the prepared hydrogel patches with and without the extract were investigated. The release patterns of the hydrogel patches containing the extract were investigated by measuring the amount of tartaric acid, a major AHA present in the tamarinds fruit pulp extract, accumulated in the receptor medium of the vertical diffusion cell at various time intervals over a period of 6 h. The results indicated that the formulations of chitosan : corn starch 4.5 : 0.5 with glutaraldehyde 0.02% w/w (C4.5C0.5G0.02) or 0.04% w/w (C4.5C0.5G0.04), chitosan : tapioca starch 4.5 : 0.5 with glutaraldehyde 0.04% w/w (C4.5T0.5G0.04) or 0.05% w/w (C4.5T0.5G0.05), and chitosan : rice starch 4.5 : 0.5 with glutaraldehyde 0.04% w/w (C4.5R0.5G0.04) and chitosan : rice starch 4.0 : 1.0 with glutaraldehyde 0.03% w/w (C4.0R1.0G0.03) provided the flexible and elastic patches with good bio‐adhesive property. The tensile strength values ranged from 5 to15 N mm−2 and the elasticity ranged from 30 to 60%. The addition of the extract in these formulations significantly increased the tensile strength values of the obtained patches. The patch of C4.0R1.0G0.03 formulation containing the extract showed relatively highest porosity, corresponding to its highest amount (12.02 ± 0.33 mg) and rate (0.452 ± 0.012 mg mm−2 min−1/2) of tartaric acid released. The amounts of tartaric acid released from the developed hydrogel patches were proportional to a square root of time (Higuchis model), particularly the release from C4.0R1.0G0.03 (R2, 0.9978 ± 0.0020) and C4.5R0.5G0.04 (R2, 0.9961 ± 0.0024) patches.