| Atopic dermatitis(AD)is a genetic chronic relapsing inflammatory skin condition characterized by intense itching,dry skin and rash.AD has a wide-ranging impact on a patient's quality of life and the patients suffered from physical and mental torture.Incidence of AD in China has gradually increased in recent years.The current treatments for AD are topical medication combined with systemic therapy on patient education and skin care strategies.Glucocorticoid topical drugs are used primarily for AD,but their adverse reactions are outstanding.It is an urgent need to develop a safe and effective external preparation to treat AD.Traditional Chinese medicine has advantages in the treatment of AD,therefore,the modern pharmaceutical research is important and popular in Traditional Chinese medicine.The rearch team developed an external preparation called Shenshe cream which was composed of cream base,tripterygium glycosides and traditional Chinese medicines from modified Radix Sophorae Flavescentis Decoction.The project were divided into two parts.The first part is to set up the quality standard,including a series of qualitative and quantitative examination methods,in order to ensure the quality of the Shenshe cream.The second part is the Pharmacodynamics research that used two different types of mouse model of AD to evaluate the efficacy of Shenshe cream in the treatment of AD,to understand the characteristics of the drug and explore the mechanism of drug action.Part 1 the quality standard of Shenshe creamMethods1.TLC identification method was used to identify the quality of the following 5 medicine in the cream,sophora flavescens,fructus cnidii,phellodendron amurense,fructus kochiae,smilax glabar.Matrine,osthol,berberine hydrochloride,momordin Ic and astilbin were employed as their reference substances respectively.All samples and negative reference sample were pointed on the silica gel G board,unfolded with developing agent and developed.2.With reference to the Chinese pharmacopoeia 2015 edition ? appendix creams rule,we researched Shen she cream including characteristic,particle size,content uniformity and microbial limit tests.3.To establish HPLC method for the content determination of osthole in Shenshe cream.The chromatographic separation was performed on a C18 column with the mobile phase consisting of acetonitrile-water(65:35,v/v),the flow rate was 1.0 mlˇmin-1,the wavelength detection was 277 nm,the sample size was 20?l.4.To establish LC-MS/MS method for the content determination of triptolide in Shenshe cream.The chromatographic separation was performed on an Agilent TC-C18(4.6 mm×250 mm,5 ?m)column with the mobile phase consisting of methanol-0.1% formic acid solution(73:27,v/v).Electrospray ionization(ESI)source was applied with multiple reaction monitoring(MRM)combined with positive mode.This assay method used metronidazole as internal standard.The mass transitions are 361.4?43.2 for triptolide and 172.1?128.0 for metronidazole,respectively.Results1.In the thin layer chromatography of the medicinal herbs,all samples showed the same spots at the appropriate position of the sheet,and the negative control samples were not colored.2.In 3 batches of traits,Shenshe cream were light yellow uniform semi solid,smooth,no deterioration,no separation of oil and water;Under the microscope,there is no condensation phenomenon,and not detected particles larger than 180 ?m;The average amount of each batch was more than the marked volume(15g),single volume was more than 93% marked loading capacity(13.95g);aerobic bacteria,mold and yeast count were less than 102 cfu?g-1,Staphylococcus aureus,Pseudomonas aeruginosa and living mites were not detected.3.The linear range was 5.04~80.64 ?gˇml-1 with good correlation coefficient.The method achieved satisfactory recoveries(99.36%),precision RSD was 0.21%,repeatability RSD was 0.51%,stability RSD was 0.29%.4.The linear range was 10.2 ~ 510 ngˇml-1 with good correlation coefficient(r=0.9998).The intra-and inter-day variations(RSDs)of peak area were all less than 3% and the method achieved satisfactory recoveries(96.87%)and repeatability RSD was 3.35%,stability RSD was 2.55%.Conclusions1.TLC is used to identify sophora flavescens,fructus cnidii,phellodendron amurense,fructus kochiae,smilax glabar in the cream,characterized as simple,quick separation and low cost.The selected object to identify is representative,the selected composition to identify should be specific in chemical composition,typical in pharmacological activity,and feasible in detection technology,TLC could be used as a qualitative identification method for application of cream.2.The characteristics,particle size,content uniformity and microbial limit of Shenshe cream were all up to the standard of the Chinese Pharmacopoeia,which showed that the matrix selection is reasonable and preparation process was mature.3.The established HPLC method was proved to be efficient,sensitive,accurate and repeatable for quality control of Shenshe cream by a series of experiments.4.The established LC-MS/MS method has strong specificity and good repeatability,that was proved to be simple,efficient,sensitive and accurate for quality control of Shenshe cream.Part 2 Pharmacodynamic study of Shenshe creamMotheds1.BALB/c were given OXZ and OVA,then induced to AD.We chose hydrocortisone butyrate cream as positive drug,the skin inflammation score,serum Ig E concentration as evaluation index.2.To evaluate the treat effect of Shenshe cream on AD mice model induced by OXZ.The mouse were divided randomly into four groups of 10 including control group,OXZ model group,butyrate hydrocortisone cream group and Shenshe cream group.The mouse were administered 24 hours after the first excitation,1 time a day for 15 days.24 hours after the 6th excitation,the skin condition of mice ear was observed,the inflammation score of ear was evaluated and the left ear quality diameter 6 mm was weighted.The right ear tissue was sliced and observed with HE staining.The serum Ig E concentration was detected from orbital blood.The quality of the thymus and spleen were determined to calculate the ratio of the viscera and body.3.To evaluate the treat effect of Shenshe cream on AD mice model induced by OVA.The mouse were divided randomly into four groups of 10 including control group,OVA model group,butyrate hydrocortisone cream group and Shenshe cream group.The mouse were administered 5 hours after the first excitation,1 time a day for 7 days.24 hours after the last dose,the back skin condition of mice was observed,the inflammation score of skin was evaluated and the left ear quality diameter 6 mm was weighted.The back skin tissue was sliced and observed with HE staining.The serum Ig E concentration was detected from orbital blood.The mount of the lymphocyte in blood was counted using the Ritz staining.Results1.In OXZ group,mice ears were swelling,thickening,and lichenification,vascular congestion is clearly visible,there is scaly,scratch in surface,inflammation score and ear weight increased,serum Ig E concentration increased;In OVA group,mice back skin showed erythema,edema,partial exfoliation,knot scab,desquamation,skin inf lammation score and serum Ig E concentration increased;In hydrocortisone butyrate group,mice skin inflammation score,ear weight and serum Ig E concentration decreased.2.In OXZ induced BALB/c mice of AD model,we found that when given Shenshe cream,inflammation in mice,ear weight and serum Ig E concentration decreased,spleen index also decreased to normal,did not appear ear skin thinner,smaller,thymus index,spleen index significantly reduced,which were seen in hydrocortisone butyrate group.3.In OVA induced BALB/c mice of AD model,we found that when given Shenshe cream,the mice ear inflammation scores and serum Ig E concentration decreased significantly,lymphocyte count increased significantly,in accord with hydrocortisone butyrate group.Conclusions1.When OXZ and OVA were used in BALB/c mice to induce inflammatory dermatitis,the mice showed marked inflammatory response,Ig E increased,consistent with the clinical features of AD,which suggested this model is replicable;Fourthemore,inflammation in this model could be relieved by Hydrocortisone butyrate Cream,proved that the model of pathological injury degree is moderate,with reversibility.It was concluded that OXZ and OVA induced AD models were established successfully.2.In two different allergen induced AD model,Shenshe cream has remarkable effect,and the efficacy was similar to hydrocortisone butyrate cream,but did not appear adverse reaction of glucocorticoid induced,suggesting that this drug has broad prospects in the treatment of AD.3.Shenshe cream can protect from high Ig E concentration,lymphopenia and splenomegaly induced by OXZ and OVA.It suggests that it could regulate immunity bidirectionally,maintain immune homeostasis and protect immune organs effectively,however,the active ingredient,specific mechanism and pathway is not clear,which need further study. |
PDF Full Text Request