Research On Prescription Fuse-1Aqueous Extract’s Effect And Its Preliminary Study Of External Preparation

Objective:1. To research the mechanism of action of Prescription Fuse-1curing for vitiligo.2. To investigate technological process of Prescription Fuse-l’s tincture and gelata. And estimate the quality of Prescription Fuse-l’s tincture and gelata。3. To estimate the safety of Prescription Fuse-l’s tincture and gelata applied on skins.4. To estimate the clinical efficalcy of Prescription Fuse-l’s tincture and gelata on skins.Methods:1. Adopt MTT method to measure absorbance value(value A) of cultured B16melanoma cells of the five different density of Prescription Fuse-1aqueous extrcts including0.001g/1,0.005g/l,0.01g/l,0.05g/l and0.1g/1. Suppose the pure culture medium as contrast group.To observe the effect of Prescription Fuse-1on B16melanoma cells proliferation.According to the experimental results of MTT, select the medicine density and adopt the oxidation rate of DL dopa method to measure the effect of three different density Prescription Fuse-1aqueous extracts including0.001g/1.0.005g/l and0.01g/l on tyrosinase activity of cells. Suppose the pure culture medium as contrast group.measure the value A of the four groups by using the MicroplateReader after24h、48h and72h later.2. The75%ethanol as dissolvant is used to soak Prescription Fuse-1, and then made for tinctures. Prescription Fuse-1aqueous exact as medicine. carbomer940as main matrix, PEG-4000and glycerine as hygroscopic agent and thickening agent, triethanolamine as carbomer’s neutralizer, laurocapram as transdermal agent, Kathon as antiputrefactive, we make the gelata by its method. Adopt the naked eye view and centrifugalization method to evaluate the surface and clarity of tinctures; use retention samples stored at room temperature method to observe the stability of tinctures; adopt self coating, naked eye view and microscope observation methods to evaluate the surface and exquisite degree of gelata; use constant temperature incubator,refrigerator, centrifuges and room temperature storage methods to evaluate the stability of gelata; and adopt pH meter to evaluate the pH value of Prescription Fuse-l’s tinctures and gelata.3.Select4cavies and40rats.①Cutaneous stimulation test. Fristly, partition the back skin of4cavies whose back hair is shaved into four areae by latin square design, each area is multiplying2.0by2.0cm2. Secondly,embrocate the tincture group, gelata group, tinctures contrast(75%ethanol and0.16%laurocapram) and gelata excipient contrast to the skin by the design method. Tinctures that damp one layer gauze.and gelata that embrocates0.1g per area are appropriate, carefully and slowly coating and shall not be loss of the medicine. Thirdly, use one layer preservative film covered to moisturize, add gauze covered to protect, and adopt non-irritating rubberized fabric and bandages to properly fix for last, preventing adjunct from falling off. Fourthly everyday tested medicine and contrast coating simultaneously once, continuing14days, and6hours after each coating, wash the residual smear with warm water Fifthly,1hour after eliminating smear every time and before coating again, observe and record the situation of erythema and oedema, pigmentation, bleeding point, pachulosis or thin skin on the coating area, especially the occuring and disappearing time, and then grade the erythema and oedema. Sixthly,1hour,24hours,48hours and72hours after coating for last time and eliminating the smear, observe whether erythema and oedema occur on the coating area.②Skin allergies test:Divide40rats into5groups randomly,8for each group. The first group is compound tincture group, the second group is gelata group, the third group is positive control group (2,4-Dinitrobenzoyl chloride), the fourth group is tincture’s blank contrast group (tincture matrix), the fifth group is gelata’s blank contrast group (gelata diluents bases). Each coating quantity is0.1g(tincture that damp one layer gauze is appropriate). Sensitized dose is on the left of the rats’ skin, exciting dose is on the right. Sensitized test:coating the tested medicine on the left unhairing area of all groups of rats, fixation as the cutaneous stimulation test, after6hours wash the tested medicine with warm water, repeat it in the same way on7th day and14th day. Positive control group uses the1%2,4-dinitrobenzoyl chloride solution. Exciting test:14days after sensitizing tested medicine for the last time, coating the tested medicine and contrast medicine on the right unhairing area of the rats’ back. Positive control group uses the1%2,4-dinitrobenzoyl chloride solution. After6hours wash the tested medicine with warm water, observe immediately, and observe the anaphylactic reaction of erythema and oedema, and grade the erythema and oedema by the standards after24hours,48hours and72hours.4.45patients with vitiligo were test with Prescription Fuse-1’s tincture and gelata to observe these two dosage forms’ irritation and sensitization. Two patients with vitiligo in progression stage were selected to treat with Prescription Fuse-l’s tincture and gelata combined with oral medications, and then observe the curative effect. The degree of recovery of hickie’s pigment is as the curative index.5.Statistical method:SPSS13.0software for statistical analysis of experimental data, x土S representing the Measurement data, Independent samples T-test for the significance test between the two groups, One-way Annova analysis for the significance test among the multiple groups, General-Linear-Model-Univariate analysis for the significance test among the multiple factor and multilevel groups. Bonferroni test analysis for comparing in pairwise comparison among the multiple groups, when P<0.05, differences are statistically significant.Results:1. Compared to the contrast group, Prescription Fuse-l’s effect on B16melanoma cells proliferation(value A) within the density between0.001g/1to0.01g/l is no statistical significance (P>0.05), but between0.05g/l to0.1g/1is significant (P<0.001), presenting a certain inhibiting effect.Compared to the contrast group, Prescription Fuse-l’s effect on B16melanoma cells’tyrosinase activity within the density between0.001g/1to0.1g/l is significant (F=2059.17, P<0.001), and as the drug concentration increased, the activity of tyrosinase improve. There is significance (F=351.76, P<0.001) of value A(tyrosinase activity in B16melanoma cells curtured by Prescription Fuse-1water extract)after24h、48h and72h later, and yrosinase activity gradually increased with time prolonged.2. The tinctures prepared are clear, purple-brown, spicy,stimulating smell of alcohol. The gelata prepared are brown translucent cream, uniform color and texture, exquisite appearance. The tinctures samples after centrifuging appear no obvious precipitation, pH values are6.15±0.005measured by pH meter. The tinctures samples’package and appearance color clarity have no marked change in the room temperature by laying up for1month and3months. PH values after1month are6.14±0.009, and fter3months are6.12±0.013.The gelata samples are dispersing uniform,exquisite texture, suitable spissitude, and easily coated. The gelata appearance color and spissitude are no marked change,non-hierarchical and discoloration after the room temperature storage and cold heat-resisting experiment. And the gelata have no bold, layered and precipitation phenomena after centrifuging experiment, the average pH values are5.96±0.012. However, the tested average pH values are5.94±0.011、5.93±0.013after3months laying in the4℃refrigerator and room temperature.3. According to the grade standards of cutaneous stimulation reaction and stimulus intensity, Prescription Fuse-l’s tinctures and gelata have no stimulation reaction such as erythema, oedema and furfuration on the cavies’skin. According to the grade standards of skin allergies and sensitizing rate, Prescription Fuse-l’s tinctures and gelata have no anaphylactic reaction of erythema,oedema on the rats’ skin, and serious systemic anaphylactic reaction such as asthma、shock. However, the positive control group sensitized and excited by2,4-Dinitrobenzoyl chloride have anaphylactic reaction of erythema,oedema. The cutaneous stimulation reaction points are>1, and sensitizing rates are100%.4.There were no obvious adverse reactions in the course of treatment.Two cases of vitiligo patients got satisfactory curative effect.Conclusion:1. The study results show that Prescription Fuse-1water extract can promot the B16melanoma cells’tyrosinase activity. Promoting tyrosinase activity is a mechanism of action of Prescription Fuse-1curing for vitiligo. Prescription Fuse-1water extract can’t promot the B16melanoma cells’proliferation, So cells’ proliferation may not be the mechanism of action of Prescription Fuse-1curing for vitiligo.2. The Prescription Fuse-1tinctures prepared by traditional method are purple-brown, clear liquid. And the Prescription Fuse-1gelata prepared by the prescription we designed are light brown, dispersing uniform, exquisite texture, suitable spissitude, and easily coated.The stability tests show that the characters of tincture and gelata are stable. The2forms are in line with the provisions of tincture and gelata of "Chinese Pharmacopoeia"3. The cavies’cutaneous stimulation tests results show that Prescription Fuse-1tinctures and gelata have no stimulation reaction on the cavies’skin. The skin allergies tests results show that Prescription Fuse-1tinctures and gelata have no anaphylactic reaction on the rats’skin. The skin medicine are safe.4. Prescription Fuse-l’s tinctures and gelata are safe to vitiligo.