| AimsThe aims of this study was to develop Alum-Borneol nanoemulsion based on the traditional Alum-Borneol liquid. Nano biotechnology was used to optimize the formulation and technology to improve drug loading, reduce the dose, decrease side effects and improve efficacy.Methods1. The preparation study of Alum-Borneol nanoemulsion1.1 Blank nanoemulsion formulation screening.The oil phase of the Alum-Borneol nanoemulsion has been determined on the basis of solubility of the borneol in oil phase. The oil phase has been set firstly. Tween80, RH40, OP-10, and EL were selected as surfactant, while propylene glycol, glycerin, octanol, ethylene glycol ether and polyethylene glycerol were selected as co-surfactant, and then all of those were mixed with oil phase to investigate the emulsification.1.2 The preparation of Alum-Borneol nanoemulsionThe optimized prescription was screened by pseudo-ternary phase method. The borneol and alum were added into RH40, ethyl oleate or water nanoemulsion to produce Alum-Borneol nanoemulsion. The analysis method of gas chromatography was established to detect the content of borneol in the Alum-Borneol nanoemulsion, and its drug loading and encapsulation efficiency. The content of alum (hydrated aluminum potassium sulfate) was detected by complexometric titration. The particle morphology and particle diameter were assayed by transmission electron microscopy and laser particle size distribution analyzer, respectively. Stability parameters determination, hot sterilized test, temperature test, freezing-heating cycle test were used to study its stability.1.3 In vitro release and penetration tests of Alum-Borneol nanoemulsionFranz diffusion cell was used to do percutaneous penetration test, while the gas chromatography was used to detect the drug concentration of Alum-Borneol nanoemulsion from the skin received liquid and compared with Alum-Borneol and then the transdermal properties of Alum-Borneol nanoemulsion were investigated.2. Safety evaluation of Alum-Borneol nanoemulsion2.1 Rabbit skin acute toxicity trial36 rabbits were randomly divided into 6 groups:the complete skin control group, intact skin of ice nanoemulsion low dose alum group, intact skin of ice nanoemulsion high dose alum group, skin and the control group, skin and Alum-Borneol nanoemulsion low dose group, skin and Alum-Borneol nanoemulsion high dose group. The drugs were administered 4 times a day for 14 days to observe the performance of systemic poisoning of animals and death situation of subjects on a daily basis.2.2 The long-term toxicity tests48 rabbits were randomly divided into 8 groups, including intact skin the control group (matrix group), alum skin, full of ice nanoemulsion low, medium and high dose group, skin and the control group, skin and Alum-Borneol nanoemulsion low, medium, high-dose group with 6 rabbits, respectively. Routine observations of hematology, blood biochemistry, systems autopsy and histopathologic examination were conducted after 13 weeks of continuous skin application in every group.2.3 Cytotoxicity testL-929 cells were cultivated in vitro cell as model, while MTT method was used to test the cytotoxicity of Alum-Borneol nanoemulsion to skin fibroblast cell.2.4 Skin allergy test30 white male guinea pigs were randomly divided into 3 groups, including the negative control group, positive control and test substance groups. The skin allergy was observed after the allergy exposure and stimulation in negative control group with blank nanoemulsion, positive control group with 2,4-dinitrochlorobenzene and the test substance group with Alum-Borneol nanoemulsion.2.5 Skin irritation testThere were in total 6 rabbits whose back skin hair was removed. They were divided into completed control areas of skin, completed areas of skin for drug administration, broken skin control area, and broken skin for drug administration. Those 4 areas were given Alum-Borneol nanoemulsion and blank nanoemulsion on a daily basis for 7 days and then the skin irritation was observed.3. Pharmacodynamics3.1 Deep II degree burn model in rats.140 SD rats with deep?degree burn were randomly divided into 7 groups, which were control group, matrix group, positive control group, low, medium and high dose alumina nano-ice milk group and alum icing fluid group. After appropriate medication, continuous administration was carried out in each group for 21 days. The situation of wound healing was observed everyday and the wound healing time were recorded respectively. The calculation of wound healing rate was calculated on the seventh, fourteenth and twenty-first days. The wound tissue was collected on the seventh day to do the skin hydroxyproline determination test and histopathological examination.3.2 In vitro antibacterial test Agar diffusion method, in vitro bactericidal test and tube dilution method were used to test. The effects of alumina ice nanoemulsion on clinical common pathogens in vitro antibacterial, bactericidal effects and minimum inhibitory concentration (MIC).3.3 Anti-inflammatory effect50 KM mice were randomly divided into 5 groups, including matrix group, dexamethasone acetate ointment group, low, medium and high dose Alum-Borneol nanoemulsion groups, with the xylene to induce right ear swelling, and left ear as control to observe the two swelling ears.60 SD rats were randomly divided into 6 groups, including matrix group, dexamethasone acetate group, low, medium and high Alum-Borneol nanoemulsion dose groups, alum icing fluid group. Making white rat inflamed paw swelling model using egg white. Capillary enlarge method was used to test the volume of right hind paw and the swelling was observed before and after the inflammation.3.4 Analgesic effect50 KM mice were randomly divided into 5 groups, including matrix groups, positive control group, low, medium and high Alum-Borneol nanoemulsion dose groups. Each drug was applied to each group evenly to the abdomen of mice.30 minutes after the end of administration on the fourth day, each mouse was injected with acetic acid solution, and then the mice's writhing within 15 minutes was recorded, the differences in the times of writhing between the control group and drug group were compared.50 female mice which were selected according to pre-qualified pain threshold were randomly divided into 5 groups (groups as above). The time between the mice been put in to pre-heated beaker and the time they licked the back legs were recorded before and after the treatment with drugs.3.5 Anti-itching effect60 KM mice were randomly divided into 6 groups, including matrix group, alum icing fluid group, dexamethasone acetate ointment group, Alum-Borneol nanoemulsion low, medium and high dose groups. The mice tickle model was established by injecting mice with formaldehyde. The different time when the mice licking the right hind bite or scratch and bite genitals were recorded to compare the differences among groups.60 guinea pigs were randomly divided into 6 groups described as above in order to make local itching histamine phosphate solution model. The itching threshold of each group was recorded and compared among each group.Results1.The preparation of Alum-Borneol nanoemulsionPseudo-ternary phase diagram showed the nano region formed by the RH-40 and the oil ethyl was the biggest, and the ratio of surfactant and oil phase were determined. Nanoemulsion formulation screening results showed that the surfactant of nanoemulsion was 40-polyoxyethylene castor oil, the co-surfactant was glycerol and oil phase was ethyl oleate. Blank nanoemulsion was clear, transparent, slightly opalescent pale blue liquid. The particles were spherical under electron microscope with diameter of 28.15 nm. The diffusion rate of methylene blue was significantly higher than that of Sudan III for oil in water type which is in line with the requirements of nanoemulsion.The pseudo-ternary phase diagram and the stability test were used to establish oil Alum-Borneol nanoemulsion dosage of ethyl to be 0.6% (g /g), ethyl oleate and RH-40 ratio of 1:3.5. Gas chromatography was used to detect borneol content, the standard curve for borneol had a good linear relationship between 0.1015 mg.mL-1 to 1.015 mg.mL-1. The average content of borneol was 1.48 mg·mL-1 with the average recovery rate as 100.59% and the recovery rate of RSD was 2.72%. The average concentration of aluminum potassium sulfate was 13.70 mg·mL-1 in Alum-Borneol nanoemulsion which was detected by complexometric titration. Its average recovery was 98.26%, and the recovery rate of the RSD was 0.92%. The consecutive three batches of 40-polyoxyethylene castor oil/ethyl oleate/alum borneol nanoemulsion were clear, transparent and slightly opalescent pale blue liquid. It was spherical under electron microscope with average particle diameter of 26.1 nm and zeta potential wa -0.51±1.47 mV. Its drug loading was 1.35mg·mL-1 and had more than 80% encapsulation efficiency rate. The stability parameter of Alum-Borneol nanoemulsion was 4.48±0.63.The release rate of Alum-Borneol nanoemulsion after 24 h accumulation was (2083.00±431.17)?g·cm-2, which average penetration rate 84.07?g·cm-2·h-1. The results show that the transparency of Alum-Borneol nanoemulsion was better than the control group.2. Safety evaluation2.1 There was no systematic toxicity and death in the subjects of rabbits after giving Alum-Borneol nanoemulsion. The rabbits had gained weight and there was no harm to the rabbits'biological system including respiratory, circulatory, central nervous system, limbs and other activities. There was slight erythema but no individual animal edema in the very few subjects whose symptoms disappeared after 24 h with no drug residues spots in drug area.2.2 There was no significant difference in appearance of each dose group behavior of animals, body weight, organ coefficient, hematology and hematological indices in the Alum-Borneol nanoemulsion group when compared with the control group. There were no obvious abnormalities in histopathological examination and no delayed symptoms of toxicity after stopping the drugs.2.3 The Alum-Borneol nanoemulsion had no significant toxicity on L929 cells and the cell morphology was good with first class of cell toxicity.2.4 There was no local skin erythema and no other allergic reactions in tested guinea pigs.2.5 Neither of the intact skin or damaged skin of tested rabbits showed erythema, edema or other skin irritations after single and multiple dosing.3.pharmacodynamics3.1 The wound treated with different doses of Alum-Borneol nanoemulsion were healed between 19 and 25 days. The healing time of the low and high dose groups was significantly shorten than the blank control group (P<0.05; P<0.01). The wound healing rate of the former was significantly higher (P<0.05; P<0.01) than the later and the content of hydroxyproline in each experimental groups was higher than the control group 7 days after the burn, but the difference was not significant. The pathological results suggested that Alum-Borneol nanoemulsion had significantly better healing effects on burn wounds.3.2 The effects of Alum-Borneol nanoemulsion to staphylococcus aureus, staphylococcus epidermidis, escherichia coli, pseudomonas aeruginosa, candida albicans. The killing or inhibitory activity was significantly stronger than Alum-Borneol fluid (P<0.05); the MIC90 values of Alum-Borneol nanoemulsion on staphylococcus aureus, pseudomonas aeruginosa, clinical escherichia coli were 1.02,2.04 and 2.04 mg.mL-1 which were significantly lower than the MIC90 values of Alum-Borneol fluid (P<0.05).3.3 The Alum-Borneol nanoemulsion dose groups had different degrees of inhibition for the xylene-induced ear edema in mice. There was significant differences (P<0.05) between the medium, high dose groups and the base group. There was significant different degrees of inhibition of egg white rat paw edema when compared with low, medium and high dose groups in inflammation after 1-6 h with matrix groups (P <0.05, P<0.01).3.4 The Alum-Borneol nanoemulsion could significantly inhibit the acetic acid induced writhing in mice, but there was no significant difference when compared with the control group (P> 0.05). There was a trend of prolonging the hot plate pain threshold in mice when compared with the control group, but the difference was not significant (P> 0.05).3.5 The Alum-Borneol nanoemulsion could reduce the times of licking biting or scratching biting genitals in mice when compared with the matrix group and the difference was significant (P<0.01); in medium and high dose groups it could significantly improve the itching threshold of guinea pig(P<0.05, P<0.01). Conclusions1. The proposed blank nanoemulsion can meet the requirements of nanoemulsion.2.The Alum-Borneol nanoemulsion obtained through the optimization of formulation is O/W type, it was clear, transparent and slightly opalescent pale blue liquid with average particle diameter of 26.1 nm, which meets the requirements of Chinese Pharmacopoeia (2010 edition).3.The Alum-Borneol nanoemulsion has no significant toxic effect on animals'skin, normal skin and damage after long-term administration. There was no significant toxic effect on L929 cell, or on skin allergies as well as no irritation to broken skin or normal skin.4.The Alum-Borneol nanoemulsion obviously improves the burn wound healing. It has anti-inflammatory and anti-itching effects. It may have analgesic and antibacterial effects on a broad-spectrum of bacteria and fungus. The Alum-Borneol nanoemulsion is significantly more effective than the traditional drug of Alum-Borneol fluid.5.The Alum-Borneol nanoemulsion has a good penetration capacity through skin. |
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