Physicochemical properties and biological activities of Thai plant mucilages for artificial saliva preparation.

CONTEXT: Plant mucilages can be found in various parts of several Thai plants, which can be used as thickening, moisturizing, and lubricating agents in artificial saliva formulations. OBJECTIVE: The objective of this study was to evaluate the physicochemical properties, biological activity, and cytotoxicity of Thai plant mucilages. MATERIALS AND METHODS: The mucilages from Thai plants were extracted by various processes (temperature and pH variation, microwave oven, steam, and Tris-HCl buffer extraction). The viscosity and the rheology were evaluated using viscometer. Antioxidative activities including DPPH radical scavenging and metal chelating activities were investigated. The mucilages were determined for cytotoxicity on normal human gingival fibroblasts and anti-adherent activity of Streptococcus mutans. RESULTS: Mucilages from Ocimum citriodorum Vis. (Lamiaceae), Artocarpus heterophyllus Lam. (Moraceae), Abelmoschus esculentus (Linn.) Moench. (Malvaceae), and Basella alba Linn. (Basellaceae) exhibited pseudoplastic non-Newtonian rheology. The highest DPPH radical-scavenging and metal-chelating activities were observed in the mucilages from B. alba (microwave, 3 min) and A. esculentus (microwave, 1 min) with the SC50 and MC50 values (50% of scavenging activity and 50% of metal chelating activity, respectively) of 0.71 ± 0.32 and 1.11 ± 0.52 mg/ml, respectively. Most mucilages exhibited no cytotoxicity to normal human gingival fibroblasts. The mucilage from A. esculentus (microwave, 5 min) gave the shortest wetting time of 2.75 ± 0.51 min. The highest S. mutans adhesion inhibition was observed in A. esculentus (pH 11) of 5.39 ± 9.70%. DISCUSSION AND CONCLUSION: This study has indicated the suitable physicochemical and biological properties and the potential application of mucilages from Thai plants for artificial saliva preparation.

Biological activities of fructooligosaccharide (FOS)-containing Coix lachryma-jobi Linn. extract.

Fructooligosaccharide (FOS), a prebiotic was extracted from the grain of Coix lachryma-jobi Linn. (Job's tears) by hot water extraction at 60 °C for 1 h. The resulting dried powder extract was assayed for FOS content of 1-kestose (GF2), nystose (GF3) and 1-ß-D-fructofuranosylnystose (GF4) using HPLC equipped with RI detector. Total FOS content of the extract was 24.98 ± 7.48% (g/100 g crude extract). The biological activity including antioxidant and cytotoxicity of the FOS-containing extract was determined. The antioxidant activity by DPPH free radical scavenging of FOS-containing extract was comparable to vitamin C (0.97 fold of vitamin C) with a slight lipid peroxidation inhibition activity. The extract exhibited no cytotoxic effect on normal human skin fibroblast. These results have confirmed not only the source of FOS from Job's tears extract but also its potential application as antioxidant in food or cosmetic products.

Transdermal enhancement through rat skin of luciferase plasmid DNA loaded in elastic nanovesicles.

Transdermal absorption of luciferase plasmid (pLuc) was enhanced by loading in elastic cationic liposomes and niosomes and the application of iontophoresis or the stratum corneum (SC) stripping method. Cationic liposomes (DPPC/Chol/DDAB at a 1:1:1 molar ratio) and niosomes (Tween61/Chol/DDAB at a 1:1:0.5 molar ratio) were prepared by the freeze-dried empty liposomes method. The elastic vesicles were prepared by hydrating the lipid or surfactant film by 25% of ethanol instead of distilled water. Gel electrophoresis of all nanovesicles showed the 100% pLuc entrapment efficiency. All nanovesicles loaded with pLuc showed larger vesicular sizes than the nonloaded vesicles of about 1.4 times for liposomes and 1.7 times for niosomes. The nanovesicles loaded with pLuc demonstrated less positive zeta potential than the nonloaded vesicles. The pLuc loaded in elastic vesicles kept at 4 +/- 2 and 27 +/- 2 degrees C for 8 weeks gave the remaining pLuc of about 70 and 60% for liposomes and 85 and 73% for niosomes, respectively. For nonelastic vesicles kept at 4 +/- 2 degrees C, 56 and 61% of the remaining pLuc were observed for liposomes and niosomes, respectively, while at 27 +/- 2 degrees C, all pLuc were degraded. The deformability indices of the elastic liposomes and niosomes loaded with the pLuc were 16.64 +/- 2.92 and 20.72 +/- 0.82, whereas the nonelastic vesicles gave 9.35 +/- 0.09 and 10.08 +/- 0.12, respectively. Transdermal absorption through rat skin pretreated with SC stripping or treated with iontophoresis of pLuc loaded in nanovesicles by vertical Franz diffusion cells was investigated at 37 degrees C. The cells were stopped and the skin and the receiving solution were withdrawn at 1, 3, and 6 hours and the pLuc contents in the stripped SC, whole skin (viable epidermis and dermis; VED), and the receiving solution were assayed by the modified gel electrophoresis and gel documentation. Without the SC stripping technique or iontophoresis, the pLuc loaded and nonloaded in nonelastic cationic liposomes or niosomes were not found in SC, VED, and receiving solution. The fluxes in the whole skin of pLuc loaded in nonelastic liposomes and niosomes with SC stripping and iontophoresis at 6 hours gave 2.73 +/- 0.46 and 3.83 +/- 0.73, and 7.01 +/- 1.22 and 9.60 +/- 1.31 g/cm(2)/h, respectively, while pLuc loaded in elastic liposomes and niosomes without the SC stripping and iontophoresis at 6 hours showed 2.79 +/- 0.09 and 2.84 +/- 0.04 g/cm(2)/h, respectively. The pLuc loaded in elastic niosomes or in nonelastic niosomes with iontophoresis was found in the receiving solution with a higher amount than that loaded in elastic liposomes or nonelastic liposomes with iontophoresis. The fluxes in the receiving solution of pLuc loaded in nonelastic liposomes and niosomes with iontophoresis at 6 hours were 6.71 +/- 0.31 and 8.82 +/- 0.28 g/cm(2)/h, respectively. For elastic liposomes and niosomes, the fluxes of the loaded pLuc in the receiving solution were the same, at about 1.9 g/cm(2)/h. Although pLuc loaded in nonelastic niosomes with iontophoresis gave the highest delivery of the plasmid in VED and receiving solution, a more promising applicable approach for gene delivery has been suggested to be the elastic niosomal systems, since no equipment is required.

Potent enhancement of transdermal absorption and stability of human tyrosinase plasmid (pAH7/Tyr) by Tat peptide and an entrapment in elastic cationic niosomes.

Enhancement of transdermal absorption through rat skin and stability of the human tyrosinase plasmid (P) using Tat (T) and an entrapment in elastic cationic niosomes (E) were described. E (Tween61:cholesterol:DDAB at 1:1:0.5 molar ratio) were prepared by the freeze-dried empty liposomes (FDELs) method using 25% ethanol. TP was prepared by a simple mixing method. TPE was prepared by loading T and P in E at the T:P:E ratio of 0.5:1:160 w/w/w. For gel formulations, P, TP, PE and TPE were incorporated into Carbopol 980 gel (30 µg of plasmid per 1 g of gel). For the transdermal absorption studies, the highest cumulative amounts and fluxes of the plasmid in viable epidermis and dermis (VED) were observed from the TPE of 0.31 ± 0.04 µg/cm(2) and 1.86 ± 0.24 µg/cm(2)/h (TPE solution); and 4.29 ± 0.40 µg/cm(2) and 25.73 ± 2.40 µg/cm(2)/h (TPE gel), respectively. Only plasmid from the PE and TPE could be found in the receiving solution with the cumulative amounts and fluxes at 6 h of 0.07 ± 0.01 µg/cm(2) and 0.40 ± 0.08 µg/cm(2)/h (PE solution); 0.10 ± 0.01 µg/cm(2) and 0.60 ± 0.06 µg/cm(2)/h (TPE solution); 0.88 ± 0.03 µg/cm(2) and 5.30 ± 0.15 µg/cm(2)/h (PE gel); and 1.02 ± 0.05 µg/cm(2) and 6.13 ± 0.28 µg/cm(2)/h (TPE gel), respectively. In stability studies, the plasmid still remained at 4 ± 2 °C and 25 ± 2 °C of about 48.00-65.20% and 27.40-51.10% (solution); and 12.34-38.31% and 8.63-36.10% (gel), respectively, whereas at 45 ± 2 °C, almost all the plasmid was degraded. These studies indicated the high potential application of Tat and an entrapment in elastic cationic niosomes for the development of transdermal gene delivery system.

Enhancement of gene expression and melanin production of human tyrosinase gene loaded in elastic cationic niosomes.

OBJECTIVES: Disturbance in the synthesis of tyrosinase might be one of the major causes of vitiligo. The enhancement of tyrosinase gene expression and melanin production by loading the plasmid in elastic cationic niosomes was investigated in tyrosinase gene knocked out human melanoma (M5) cells and in tyrosine-producing mouse melanoma (B(16) F(10) ) cells. METHODS: Niosomes composed of Tween 61/cholesterol/dimethyl dioctadecyl ammonium bromide at 1:1:0.5 molar ratio were prepared by the freeze-dried empty liposomes method. The thin lipid film was redissolved in distilled water or 25% ethanol to obtain the non-elastic or elastic cationic niosomes, respectively. KEY FINDINGS: The maximum loading of the plasmid in non-elastic and elastic niosomes was 130 and 100 µg per 16 mg of the niosomal contents, respectively. The plasmid-loaded elastic cationic niosomes exhibited high specific tyrosinase activity of 1.66 and 1.50 fold in M5 cells and 6.81 and 4.37 fold in B(16) F(10) cells compared with the free plasmid and the plasmid-loaded non-elastic cationic niosomes, respectively. CONCLUSIONS: This study has demonstrated not only the enhancement of the expression of human tyrosinase gene by loading in elastic cationic niosomes, but also the potential application of this gene delivery system for the further development of vitiligo gene therapy.

Potent melanin production enhancement of human tyrosinase gene by Tat and an entrapment in elastic cationic niosomes: potential application in vitiligo gene therapy.

Potent melanin production enhancement of human tyrosinase plasmid (pAH7/Tyr, P) in mouse melanoma cells (B(16)F(10)) by Tat peptide (T) and an entrapment in elastic cationic niosomes (E) was described. The E composed of Tween 61/cholesterol/dodecyl dimethyl ammonium bromide at 1:1:0.5 molar ratio was prepared by freeze-dried emptying liposomes method. PE at P/E ratio of 1:160 w/w and TPE at T/P/E ratio of 0.125:1:160, 0.25:1:160, and 0.5:1:160 w/w/w were prepared. The final concentration of the plasmid in the study was 4 ng/µL. By sulforhodamine B assay, PE and TPE complexes showed slight or no cytotoxic effect. The cells transfected with TPE (0.5:1:160) exhibited the highest enhancement of tyrosinase enzyme activity of 11.82-, 7.67-, 5.07-, and 6.29-folds of control, P, PE, and TP (0.5:1) and melanin production of 13.03-, 8.46-, 5.36-, and 6.58-folds of control, P, PE, and TP (0.5:1), respectively. The elastic cationic niosomes demonstrated an increase in thermal stability of P at 4 ± 2, 25 ± 2, and 45 ± 2 °C. The vesicular size and the zeta potential values of PE and TPE complexes were slightly increased but still in the range of stable dispersion (out of ±30 mV). These results indicated the high potential application of the TPE complexes for further investigation for vitiligo gene therapy.

Chemical stability and cytotoxicity of human insulin loaded in cationic DPPC/CTA/DDAB liposomes.

Liposomes were prepared from DPPC (dipalmitoyl phosphatidyl choline) mixed with Chol (cholesterol) and CTA [cholest-5-en-3-ol(3beta)(trimethylammonio) acetate] or DDAB (dioctadecyl dimethyl ammonium bromide) at various molar ratios by chloroform film method with sonication. The most physical stable (no sedimentation with an average zeta potential value of 47.7+/-1.44 mV) liposomal formulation (DPPC/CTA/DDAB at 7:2:1 molar ratio) was selected to load with human insulin (0.45 mg/mL) by the freeze dried empty liposomes (FDELs) method with the entrapment efficiency of human insulin of 62.72% (determined by gel filtration). Liposomes were spherical shape with unilamellar structure and an average size of 2.26+/-0.87 microm determined by TEM. The percentages of insulin remaining in liposomes when stored at 4+/-2, 30+/-2 and 45+/-2 degrees C for 4 months were 26.21, 36.86 and 15.75% which were higher than human insulin solution of 6.13, 11.31 and 2.61 times, respectively. The percentages of entrapment of human insulin were 62.72 at initial and at 31.72, 64.10 and 8.10 when kept at 4+/-2, 30+/-2 and 45+/-2 degrees C, respectively, for 4 months. The synthesized cationic lipid, CTA, and the DPPC/Chol/CTA liposomes loaded with human insulin demonstrated no cytotoxicity on normal human skin fibroblast but some cytotoxic effects on mouth epidermal cancer cell line. This study has demonstrated the enhancement of chemical stability of human insulin with no cytotoxicity when loaded this protein in cationic DPPC/CTA/DDAB liposomes. The results indicated the potential application of this cationic liposomal formulation for topical therapeutic use.

Enhancement of transdermal absorption, gene expression and stability of tyrosinase plasmid (pMEL34)-loaded elastic cationic niosomes: potential application in vitiligo treatment.

The pMEL34 was loaded in elastic cationic niosomes (Tween61/Cholesterol/DDAB at 1:1:0.5 molar ratio) by chloroform film method with sonication and rehydrated with 25% ethanol. The amount of pMEL34 was determined by gel electrophoresis and gel documentation. The maximum loading of pMEL34 in elastic cationic niosomes was 150 microg/16 mg of the niosomal compositions. At 8 weeks, the remaining plasmid in the elastic niosomes kept at 4 +/- 2 degrees C, 27 +/- 2 degrees C were 49.75% and 38.57%, respectively, whereas at 45 +/- 2 degrees C, all plasmids were degraded. For transdermal absorption through rat skin investigated by Franz diffusion cells at 6 h, the fluxes of pMEL34 loaded in elastic and nonelastic niosomes in viable epidermis and dermis (VED) were 0.022 +/- 0.00 and 0.017 +/- 0.01 microg/cm(2)/h, respectively, whereas only pMEL34 loaded in elastic cationic noisome was observed in the receiver solution. The pMEL34 loaded in elastic cationic niosomes showed the highest tyrosinase gene expression demonstrating higher tyrosinase activity than the free and the loaded plasmid in nonelastic niosomes of about four times. This study has suggested the potential application of elastic cationic niosomes as an efficient topical delivery for tyrosinase gene in vitiligo therapy.