| PurposeAt present,in East Asia such as China,the demand for external whitening products is increasing.These whitening products mainly work by inhibiting the synthesis of skin melanin.Tyrosinase is a key enzyme for skin melanin synthesis.Therefore,according to different mechanisms,whitening products are mainly divided into two categories:tyrosinase inhibitors and tyrosinase synthesis inhibitors.The development of new inhibitors is endless.At the same time,studying the compatibility of commonly used tyrosinase inhibitors and tyrosinase synthesis inhibitors is also an efficient and practical way of developing whitening prescriptions.In this paper,we plan to select 3 commonly used tyrosinase inhibitors and 3 commonly used tyrosinase synthesis inhibitors to study the compatibility of commonly used inhibitors,and screen out the best prescription for anti-melanin that is highly effective,low-toxic and compatible with increasing efficacy and reducing toxicity.Furthermore,the best prescription is prepared as a suitable external preparation,and the preparation quality evaluation,transdermal permeability measurement and in vivo drug efficacy evaluation are carried out to provide an anti-melanin compound preparation with stable and controllable quality and obvious whitening effect.Methods1 Screening and evaluation of the compatibility of anti-melanin active substancesThis chapter aims to screen out the best anti-melanin prescription composed of 1 tyrosinase inhibitor and 1 tyrosinase synthesis inhibitor,and evaluate its activity and toxicity in vitro.First,through molecular docking and literature research,three commonly used"tyrosinase inhibitors" and three commonly used "tyrosinase synthesis inhibitors" were screened out,and they were paired to form 9 compatible combinations for prescription the study.Then,using ?-MSH-induced B16 cells as a model,the anti-melanin activity and cytotoxicity of the six inhibitors were evaluated,and the IC50 value of the inhibition of melanin content was calculated to determine the dosage range for the prescription study.Next,using ?-MSH-induced B16 cells as a model,each drug was administered at a medium dose(1/4 times IC50),and the inhibitory effects and cytotoxicity of 9 compatible combinations against melanin content were evaluated;the Coefficient of Drug Interaction(CDI)was selected as an indicator to evaluate the drug interaction properties of cell melanin content inhibition and cell viability inhibition of each combination.Among the 9 compatibility combinations,the best compatibility combination with high efficiency and low toxicity,compatibility enhancement and toxicity reduction was selected.For the best compatibility combination,the dose range of the two drugs was changed from 1/16 times IC50 to 1 times IC50 to form 25 dose combinations.Using ?-MSH-induced B16 cells as a model,25 dose combinations were used to evaluate the melanin synthesis inhibitory effect and cytotoxicity;the Coefficient of Drug Interaction(CDI)was selected as an indicator to evaluate the drug interaction properties of cell melanin content inhibition and cell viability inhibition of each combination.The best dose combination with high efficiency,low toxicity,compatibility enhancement and toxicity reduction among 25 dose combinations was screened to determine the best prescription for anti-melanin.Finally,using ?-MSH-induced B16 cells as a model,and taking IC50 doses of kojic acid as a positive control,the best prescription was tested for melanin inhibition rate and cytotoxicity,and its in vitro activity and toxicity were evaluated.2 Study on the best prescription for anti-melaninThis chapter aims to screen and prepare a preparation that takes into account the convenience,comfort and whitening effect of the best anti-melanin prescription determined in Chapter 1,and evaluate the preparation.The best prescription for anti-melanin is 1.5?g/mL A3 and ?g/mL B1.Taking into account the effectiveness and convenience,the oil-in-water cream is selected as the drug-carrying type.Under the premise of ensuring that the prescription drug is stable and the drug concentration and ratio after transdermal absorption are close to the best prescription,design and prepare 3 different prescriptions of A3-B1 cream.Choose appearance properties,particle size,pH value,uniformity,delamination phenomenon,etc.as indicators,the three prescription creams were investigated to determine the best cream prescription.Then,the A3-B1 cream was prepared according to the prescription,and the quality of the cream was evaluated,including identification,inspection,content determination,etc.to ensure that the quality of the cream was stable and controllable.Next,the back skin of SD rats was used as the material to evaluate the transdermal absorption performance of the A3-B1 cream.The 12-hour cumulative permeation amount and steady-state permeation rate were measured to determine the effective dose of the cream for transdermal administration,which was supported by data for subsequent in vivo efficacy experiments.Finally,take the colorful guinea pig induced by ultraviolet radiation as a model,take 2%kojic acid cream commonly used in clinical whitening as a positive control,and use optical density/section area as an indicator to determine the melanin content of the A3-B1 cream.The inhibition rate was measured to evaluate its anti-melanin effect in vivo.Results1 Screening and evaluation of the compatibility of anti-melanin active substances1.1 Choose compatible combinationThrough molecular docking and literature research,three tyrosinase inhibitors were identified:A1,A2,A3,and three tyrosinase synthesis inhibitors were also identified B1,B2,B3,paired together to form a compatible combination G1-G9,used for the Follow-up prescription research.1.2 Determination of anti-melanin activity and cytotoxicity of each drugThe melanin content inhibition of B16 cells of the positive control Kojic acid IC50=83.729?g/mL,and the cell viability was greater than 90%at this dose.The inhibition IC50 of melanin content in B16 cells of three tyrosinase inhibitors such as A1,A2 and A3 were all 40 times lower than that of the positive drug kojic acid.At the IC50 dose,the viability of the cells incubated with each inhibitor was similar to that of the kojic acid group.The IC50 of the three tyrosinase synthesis inhibitors including B1,B2 and B3 were 10-20 times lower than that of the positive drug kojic acid.At the IC50 dose,the viability of the cells incubated with each inhibitor was comparable to that of the kojic acid group.1.3 Screening compatibility combinationsEach drug of the compatibility combination G1-G9 was administered at a medium dose(1/4 times the IC50 dose),and its melanin content inhibitory activity and cytotoxicity on B16 cells were determined.The results showed that,compared with single administration,the compatible combination G1-G9 all increased the melanin content inhibitory potency of the drug by more than 1 time,and each combination had no obvious cytotoxicity.The best compatibility combination is G7:A3 and B1 are compatible with each other at 1/4 times the IC50 dose.The melanin content inhibition rate of G7 is as high as 56%,and the CDI value is 0.89.So,the inhibition rate and synergistic effect are significantly stronger than other combinations.The cell viability of G7 is about 87%,and the CDI value is 1.04.So the toxicity is low and the compatibility has attenuating effect on toxicity.1.4 Screening dose combinationsA3 and B1 each select 5 doses to be combined in pairs to form 25 dose combinations D1-D25.The melanin content inhibitory activity and cytotoxicity of D1-D25 on B16 cells were measured.The results showed that,compared with single administration,the compatible combination D1-D25 all increased the melanin content inhibitory potency of the drug by more than 1 time,and each combination had no obvious cytotoxicity.The best dose combination is D4:1.5?g/mL A3 and ?g/mL B1 compatibility.The melanin content inhibition rate of D4 is about 60%,and the CDI value is 0.73.So the inhibition rate is high,and the synergistic effect is significantly stronger than other combinations.The cell viability of D4 is about 93%,and the CDI value is 1.03.So the toxicity is low and the compatibility has attenuating effect on toxicity.1.5 In vitro evaluation of the best prescriptionThe best prescription(1.5?g/mL A3 in combination with ?g/mL B1)has a melanin inhibition rate of about 62%in B16 cells(84?g/mL kojic acid inhibition rate is 42%),and the CDI value of melanin inhibition is about 0.70.The cell viability of the best prescription is about 93%(the cell viability of kojic acid at 84?g/mL is 93%),and the CDI value of cell survival inhibition is about 1.03.That is,the best prescription has high in vitro anti-melanin activity and obvious synergistic effect,low cytotoxicity and compatibility with attenuating effect on cytotoxicity.2 Study on the best prescription for anti-melanin2.1 The prescription screening of A3-B1 creamThe best prescription for anti-melanin is 1.5?g/mL A3 and 1?g/mL B1.Oil-in-water cream is used as the drug-carrying type.In order to ensure the stability of the drug,the concentration and proportion of the drug in the skin after transdermal absorption,the pH value of the cream should be controlled at about 5.6,and the drug content is 0.45%of A3 and 0.55%of B1.According to the above requirements,three kinds of A3-B1 cream with different prescriptions were prepared,and the chemical properties were measured and scored,and the A3-B1 cream prescription 2 was determined as the best cream prescription.2.2 Quality evaluation of A3-B1 creamThe A3-B1 cream prepared according to prescription 2 contains the active ingredients A3 and B1,and its physical and chemical properties meet the quality standards.The content of A3 and B1 is between 90%?110%of the labeled amount.The quality of the cream is stable and controllable.2.3 Determination of the transdermal absorption performance of A3-B1 creamAfter applying A3-B1 cream at a dose of 0.75g/3.14cm2,3.539±0.391(?g·cm-2)A3 and 2.813±0.127(?g·cm-2)can be detected in the subcutaneous tissue after 1 hour.The concentration of the two drugs is similar to the concentration of best prescription.The steady-state permeation rate of A3 within 12h is JSA3=4.7358?g·cm-2·h-1,and the steady-state permeation rate of B1 is JSB1=3.1462?g·cm-2·h-1.The ratio of penetration rate JsA3:JSB1=1.505,which is close to the ratio of the drug concentration in the best prescription for anti-melanin.That is,the effective dose of the cream for transdermal administration is 0.75g/3.14cm2.2.4 In vivo efficacy evaluation of A3-B1 creamUsing ultraviolet-induced tricolor guinea pigs as a model,the in vivo anti-melanin activity of A3-B1 cream was determined.The results showed that after administering 0.45%A3-0.55%B1 cream at a dose of 0.75g/3.14cm2,once a day,for 28 consecutive days,the skin melanin inhibition rate was as high as 76%.Under the same conditions,the skin melanin inhibition rate of 2%kojic acid cream was 68%.A3-B1 cream has a good whitening effect in the body.ConclusionIn the prescription research part,this article screened and determined the best prescription for anti-melanin that is compatible with 1 tyrosinase inhibitor and 1 tyrosinase synthesis inhibitor,namely 1.5?g/mL A3 and 1?g/mL B1 Compatibility.The in vitro anti-melanin activity and cytotoxicity of the best anti-melanin prescription were determined,and the action properties of the two drugs in the prescription were evaluated,and it was determined that the anti-melanin prescription has the characteristics of high efficiency and low toxicity,effect enhancement and toxicity reduction by compatibility.In the preparation research part,this paper screened and determined the prescription of A3-B1 cream,which ensured the stability of the active ingredients in the cream and good transdermal permeability.Next,the quality evaluation of the A3-B1 cream was carried out on the identification,inspection,content determination and other items,which ensured that the quality of the cream was stable and controllable.Then,the cumulative permeation of A3-B1 cream,steady-state permeation rate and other permeation parameters were measured,and the effective dose of the cream for transdermal administration was determined.Finally,the in vivo anti-melanin activity of A3-B1 cream was measured,which proved that its anti-melanin effect was better than the commonly used 2%kojic acid cream in clinical practice.This article provides a new whitening prescription,which is made up of two anti-melanin active substances with different mechanisms,such as A3 and B1.The prescription has high-efficiency and low-toxicity anti-melanin effect,and the compatibility can increase efficacy and reduce toxicity.At the same time,this article also provides a prescription and preparation method of A3-B1 cream,which has stable and controllable quality,good transdermal absorption performance,and good whitening effect,providing a new whitening topical preparation solution. |
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