| Objective1.To prepare "Wu Bai Powder" anti-blemish gel paste and conduct quality standard and preclinical pharmacodynamic study research to lay the foundation for the subsequent research.2.To evaluate the clinical efficacy of "Wu Bai Powder" traditional powder acupoint paste for the treatment of melasma,and provide a practical method for the clinical treatment of melasma.To optimize the treatment protocol of melasma with acupoint paste,evaluate the clinical efficacy of "Wu Bai Powder" anti-blemish gel paste for melasma treatment,and provide a new option for the clinical application of melasma treatment with acupoint paste.3.To predict the mechanism of "Wu Bai Powder" anti-blemish gel paste in the treatment of melasma based on network pharmacology and conduct relevant molecular biology experiments,providing scientific basis for the treatment of melasma with "Wu Bai Powder" anti-blemish gel paste.Method1.The active ingredients is determinated by high performance liquid chromatography(HPLC).1,1-diphenyl-2-trinitrophenylhydrazine(DPPH)free radical scavenging and tyrosinase inhibition experiments were performed to optimize the extraction process of "Wu Bai Powder".The optimum nitrogen ketone content of "Wu Bai Powder" anti-blemish gel paste was investigated by transdermal test in vitro,and "Wu Bai Powder" anti-blemish gel paste was prepared.The quality standard of "Wu Bai Powder" anti-blemish gel paste was evaluated through the characterization,thin layer identification,fugacity,paste content,viscosity determination,content determination and so on.Skin irritation test and skin sensitivity test were also conducted to complete the safety of "Wu Bai Powder" anti-blemish gel paste.The clean-grade tricolor guinea pigs were randomly divided into normal group,model group,"Wu Bai Powder" anti-blemish gel paste group,"Wu Bai Powder" traditional powder paste group,blank gel matrix paste group and positive drug control group.After successful modeling,the normal group and the model group were only treated with blank dressing,while the rest of the groups were treated with the corresponding patch/drug for 30 days.After the treatment,immunohistochemical staining was performed to observe the melanin in each group,and the total superoxide dismutase(T-SOD)activity and malondialdehyde(MDA)content of guinea pig skin tissue homogenate were measured by hydroxylamine method and thiobarbituric acid method.2.90 cases of chloasma patients who met the inclusion conditions were randomly divided into "Wu Bai Powder" traditional powder paste group,"Wu Bai Powder" anti-blemish gel paste groupand positive drug control group.Patients in the 3 groups were given corresponding application/smear intervention,once every other day,1 month as a course of treatment,a total of 3 courses of treatment.The skin area and skin color were scored before and after each course of treatment,and the serum T-SOD activity and MDA content were detected before and after the third course of treatment.The patients were followed up one month after the end of treatment,and the skin lesion area and skin lesion color were scored again to comprehensively evaluate the clinical efficacy of acupoint paste in the treatment of chloasma.3.Network Pharmacology Study: BATMAN database was used to screen the compound active ingredients and potential targets of "Wu Bai Powder",and Drugbank,OMIM,Dis Ge NET,CTD and Gene Cards databases were used to obtain melasma related targets.Biso Genet,a plug-in in Cytoscape 3.7.2,was used to expand the potential targets of "Wu Bai Powder" compound and chloasma respectively,and then the intersection was obtained to obtain the core targets,and then the core targets were imported into DAVID database for KEGG pathway enrichment analysis.Animal experiments: Western blot and real-time quantitative PCR were used to detect melanin production and EGFR/PI3K/AKT/GSK3β signaling pathway in guinea pig skin of normal group,model group and "Wu Bai Powder" anti-blemish gel paste group.Result1.Preparation,quality standard and Preclinical efficacy studyThe results of comprehensive weighting of all indicators showed that the best extraction method for the "Wu Bai Powder" herbal compound was alcohol extraction followed by water extraction;the orthogonal test results showed that the extraction process of the "Wu Bai Powder" herbal compound was A2B3C1,i.e.,the first extraction was done by adding 8 times the amount of 70% ethanol,and then the second extraction was done by adding 10 times the amount of pure water.The results of the transdermal experiments showed that the penetration effect of "Wu Bai Powder" anti-blemish gel paste with 1% azone content is the best.The relevant preparation examination of "Wu Bai Powder" anti-blemish gel paste was completed;the thin layer identification method of the two components called imperatorin and 1,4-bis[4-(glucoseoxy)benzyl]-2-isobutyl malate(MLTR),and the HPLC determination method of the two components were established,and the tentative paste content of "Wu Bai Powder" anti-blemish gel paste was determined to be not less than 15.8168g/100cm2.The safety evaluation,including skin irritation and skin allergy test,showed that no erythema,edema and other abnormalities on the experimental site within 72 hours.Immunohistochemical results showed that: comparing with the model group,blank gel matrix paste group,"Wu Bai Powder" traditional powder paste group positive drug control group,"Wu Bai Powder" anti-blemish gel paste group was lower in positive cells rate,positive cell density,average optical density value,H-Score,and the difference was statistically significant(P<0.01).T-SOD activity assay results showed that the "Wu Bai Powder" anti-blemish gel paste group can significantly incrase the T-SOD activity than other four group,with significant difference statistically significant(P<0.01);MDA content determination results showed that the "Wu Bai Powder" anti-blemish gel paste group can significantly reduce the MDA content than other four group,with significant difference statistically significant(P<0.01).2.Clinical studyThe total score of clinical skin lesions showed that the total score of skin lesions in the three groups decreased after the third course of treatment with that before treatment,the first course of treatment and the second course of treatment,respectively,with statistically significant difference(P<0.01).Comparison between the three groups after the treatment,the "Wu Bai Powder" traditional powder paste group and positive drug control group have no statistical significance(P>0.05)in skin total score of skin lesions,the "Wu Bai Powder" anti-blemish gel paste group and the "Wu Bai Powder" traditional powder paste group have no statistical significance(P>0.05)in skin total score of skin lesions.Compared with the positive drug control group,the total score of skin lesions of the "Wu Bai Powder" anti-blemish gel paste group was lower than that of the positive drug control group,and the difference was statistically significant(P<0.05).In the follow-up period,compared with the third course of the treatment period,the total skin score of the "Wu Bai Powder" anti-blemish gel paste group decreased,the difference was statistically significant(P<0.05);the total skin score of the "Wu Bai Powder" traditional powder patch paste group showed no significant difference(P>0.05);the total skin score of the positive drug control group increased,with the difference was statistically significant(P<0.05).The results of T-SOD activity measurement showed that the T-SOD activity of the three groups was lower than that of the healthy control group after the clinical study,and the differences were statistically significant(P<0.01).Compared with the positive drug control group,there was no statistical significance in "Wu Bai Powder" traditional powder patch paste group(P>0.05).Compared with the traditional powder paste group,the T-SOD activity of the "Wu Bai Powder" anti-blemish gel paste group increased,and the difference was statistically significant(P<0.01).Compared with the positive drug control group,the T-SOD activity of "Wu Bai Powder" anti-blemish gel paste group increased,and the difference was statistically significant(P<0.05).After rank sum test,the T-SOD activity of the three groups increased compared with that before treatment,and the differences were statistically significant(P<0.01).The content of MDA in the three groups was significantly higher than that in the healthy control group after the clinical study(P<0.01).Compared with the positive drug control group,there was no statistical significance(P>0.05).MDA content in the "Wu Bai Powder" anti-blemish gel paste group was decreased compared with the "Wu Bai Powder" traditional powder paste group and the positive drug control group,and the difference was statistically significant(P<0.01).After rank sum test,MDA content in the three groups decreased compared with that before treatment,and the differences were statistically significant(P<0.01).Clinical efficacy: the total effective rate of "Wu Bai Powder" anti-blemish gel paste group was 93.3%,the total effective rate of "Wu Bai Powder" traditional powder paste group was 83.3%,and the total effective rate of positive drug control group was 80.0%.In terms of clinical efficacy,"Wu Bai Powder" anti-blemish gel paste group was superior to "Wu Bai Powder" traditional powder paste group and positive drug control group,the difference was statistically significant(P<0.05).Compared with the positive drug control group,the efficacy of "Wu Bai Powder" traditional powder paste group was similar,and the difference was not statistically significant(P>0.05).3.Mechanism researchNetwork pharmacology research: After searching BATMAN database and deleting duplicate values,113 components and 248 targets of "Wu Bai Powder" compound were collected.Based on Drugbank,OMIM,Dis Ge NET,CTD and Gene Cards data,156 potential targets for the treatment of chloasma were predicted.The potential targets of "Wu Bai Powder" compound and chloasma were respectively expanded by using the plug-in Biso Genet in Cytoscape 3.7.2 software,and the intersection was taken.The targets with Degree value greater than or equal to 210 were selected based on the literature.Among them,SOD1 and TYR were closely related to melanin generation,so they were also retained.KEGG pathway enrichment analysis in DAVID database showed that potential targets were involved in a total of 20 signaling pathways(P < 0.05),including thyroid hormone signaling pathway,PI3K-Akt signaling pathway,Hippo signaling pathway,etc.In animal experiments,Western blot results showed that: "Wu Bai Powder" anti-blemish gel paste could significantly reduce the relative protein expression levels of MITF,TYR,TRP-1 and TRP-2 in skin tissue of guinea pig with chloasma model(P<0.01).The relative protein expression levels of EGFR,Pi3 k,P-PI3 K,AKT1,P-AKT1 and P-AKT1 /AKT1 in guinea pig skin tissue of chloasma model were significantly increased(P<0.01),and the relative protein expression levels of P-PI3 K /Pi3 k in guinea pig skin tissue of chloasma model were increased(P<0.05).QPCR results showed that: "Wu Bai Powder" anti-blemish gel paste could significantly reduce the m RNA relative expression levels of MITF,TYR,TRP-1 and TRP-2 in the skin tissue of guinea pig with chloasma model(P<0.01),and significantly increase the m RNA relative expression levels of EGFR,Pi3 k and AKT1 in the skin tissue of guinea pig with chloasma model(P<0.01).Conclusion1.The optimal extraction process of "Wu Bai Powder" Chinese herbal compound is to add 8 times the amount of 70% ethanol for the first extraction,and then add 10 times the amount of water for the second extraction,each 1h.The optimum azone content of "Wu Bai Powder" anti-blemish gel paste is 1%.The "Wu Bai Powder" anti-blemish gel paste prepared in this study has reached the quality standards required by The Chinese Pharmacopoeia 2020 edition,with reliable quality and no irritation or sensitization within 72 h,which can be used for subsequent related tests."Wu Bai Powder" anti-blemish gel paste can reduce melanin in melasma guinea pig model,improve T-SOD activity in guinea pig skin tissue,reduce MDA content in guinea pig skin tissue,achieve the therapeutic purpose by inhibiting melanin generation and antioxidant,and the curative effect is better than "Wu Bai Powder" traditional powder and positive drugs.2.Compared with the positive drugs,the traditional powder acupoint paste of "Wu Bai Powder" has the same effect as the positive drugs in effectively improving the skin lesions of patients with chloasma and anti-oxidation,but it has the characteristics of lasting and stable curative effect in the treatment of chloasma."Wu Bai Powder" anti-blemish gel paste group can effectively improve the skin lesions of patients with chloasma,increase the serum T-SOD activity of patients with chloasma,reduce MDA content,curative effect and long-term curative effect are better than "Wu Bai Powder" traditional powder paste group,positive drug control group,and clinical use is more standardized and convenient.3.Network pharmacology studies show that "Wu Bai Powder" TCM compound treatment of chloasma has the characteristics of multi-component,multi-target and multi-pathway synergistic effect.KEGG pathway enrichment analysis shows that EGFR/PI3K/AKT/GSK3β signaling pathway has the most prominent effect.Based on this,further animal experiments demonstrated that "Wu Bai Powder" anti-blemish gel paste may regulate EGFR/PI3K/AKT/GSK3β signal transduction,inhibit melanin production and resist oxidation to achieve the effect of chloasma treatment. |
PDF Full Text Request