Preparation And Characterization Of Perilla Leaf Superfine Powder And Its Therapeutic Effect On Ulcerative Colitis In Mice

Enteritis is a common disease in veterinary clinical practice,which seriously affects the growth of livestock and poultry,and reduces the income of farmers.Among them,ulcerative colitis（UC）,as the main type of enteritis,has characteristics such as chronic,recurrent,and refractory.At present,the treatment of UC is still mainly anti-inflammatory,commonly used drugs such as aminosalicylic acid and glucocorticoids.However,these drugs have unsatisfactory clinical efficacy and have serious adverse reactions.Therefore,it is urgent to find a more reasonable and effective alternative therapy.Chinese veterinary medicine has a long history of treating enteritis and has great development value.Perilla leaf has a long history of clinical application in China,and is one of the first batch of 60Chinese medicinal and food homologous herbs issued by the Ministry of Health of the People’s Republic of China.According to traditional Chinese medicine theory,Perilla frutescens has the effects of relieving exterior and dispersing cold,promoting lung and resolving phlegm,promoting qi,and stomach.In this study,Perilla leaf superfine powder was prepared using superfine grinding technology,determine the best preparation process,characterize and validate the Perilla leaf superfine powder.The antioxidant and anti-inflammatory effects of Perilla leaf superfine powder were confirmed through in vitro antioxidant and anti-inflammatory activity tests.And on this basis,a mouse model of ulcerative colitis was established to evaluate the therapeutic effect of Perilla leaf superfine powder on ulcerative mice.Finally,the pharmacodynamic material basis of Perilla leaf superfine powder was determined using UPLC-Q-TOF-MS.The specific research results are as follows:（1）Through the combination of single factor test and principal component analysis,the preparation process of Perilla leaf superfine powder was optimized.The best preparation process was as follows:80mesh of coarse powder,20 g of sample volume,and 90 s of crushing time.Under this preparation process,the content of effective components in the Perilla leaf superfine powder is the highest,in which the content of rosmarinic acid is 7.45±0.07 mg/g;the content of total phenols is 14.33±0.57 mg/g;and the content of total flavonoids is 11.60±0.48 mg/g.The best physical characteristics parameters of Perilla leaf superfine powder are:bulk density is 0.20±0.01 g/m L;compaction density is 0.42±0.01 g/m L;compressibility is 57.56±0.97%;angle of repose is 46.78±0.06°;sliding angle is 46.78±0.06°;the water holding capacity is 2.59±0.04 g/g;the oil holding capacity is 1.62±0.01 g/g;the expansion force is 1.81±0.04 m L/g;and the wetting settling time is 49.66±1.17 s.（2）The antioxidant activity of Perilla leaf superfine powder in vitro was investigated by ABTS+·free radical scavenging test.The test results showed that its IC₅₀ was 5.67 mg/m L,and at 20 mg/m L,the scavenging rate was 91.99±1.37%.The effect of Perilla leaf superfine powder on RAW264.7 cell viability was measured using the CCK8 method.The results showed that the cell survival rate was above90%at concentrations below 8 mg/m L,indicating that it had no cytotoxicity.Using LPS to induce RAW264.7 to establish inflammatory cell model,the results showed that Perilla leaf superfine powder could significantly reduce NO and Tumor necrosis factor-α（TNF-α）,Interleukin-6（IL-6）,Interleukin-1β（IL-1β）in cell supernatantand The content of Perilla leaf superfine powder showed good anti-inflammatory activity in vitro.（3）Perilla leaf superfine powder was characterized by optical microscope and scanning electron microscope.The results showed that Perilla leaf superfine powder achieved cell-level pulverization and the powder uniformity was significantly improved.The chemical structure changes before and after ultrafine grinding were analyzed by infrared spectrum.The results showed that the peak positions of infrared spectrum were basically the same,that is,no new substances were produced.（4）The solubility of Perilla leaf superfine powder in different dissolution media was evaluated by paddle method.The results showed that the maximum dissolution rate of total phenols was 98.34±1.79%in Perilla leaf superfine powder after 20 minutes in artificial gastric juice,and the maximum dissolution rate of total flavonoids was 98.07±1.91%after 30 minutes.The maximum dissolution rates of total phenols and total flavonoids were 98.38±1.63%and 98.51±1.13%,respectively,when Perilla leaf superfine powder was immersed in artificial intestinal fluid for 20 minutes.Compared with Perilla leaf coarse powder,the effective component dissolution rate of Perilla leaf superfine powder in different dissolution media is better than that of Perilla leaf coarse powder.（5）The therapeutic effect of Perilla leaf superfine powder on ulcerative colitis was evaluated by establishing a mice model of ulcerative colitis.The results showed that Perilla leaf superfine powder could significantly improve the clinical symptoms of mice with ulcerative colitis,slow down the decline of body weight,reduce the Disease activity index（DAI）score,reduce the degree of colon shortening and spleen index.Reduce the level of White blood cell（WBC）,increased the level of Red blood cell（RBC）,Hemoglobin（HGB）and Platelet（PLT）in mice.Not only that,Perilla leaf superfine powder can also significantly improve the morphology of mouse colon mucosa,alleviate tissue ulcer and edema,significantly reduce the activities of Myeloperoxidase（MPO）and Malondialdehyde（MDA）in mouse colon tissue,increase the activity of Superoxide dismutase（SOD）（p<0.01）.,and significantly reduce TNF-α、IL-6、IL-1βand TNF-α、IL-6、IL-1βexpression level of m RNA in mouse colon（p<0.01）.,significantly increased the protein content of occulin and ZO-1 in mouse colon tissue（p<0.05）.（6）UPLC-Q-TOF-MS technology was used to detect the blood inflow components of Perilla leaf superfine powder,and a total of 8 effective components were identified:glycyrrhetinic acid、α-Linolenic acid、aesculetin、asiaticic acid、panaxadiol、ethyl linolenic acid、Lingerythrin A,and gingerone.Indicate that the efficacy of Perilla leaf superfine powder is the result of the combined action of various components.In summary,this study successfully prepared Perilla leaf superfine powder,completed process optimization and characterization.It evaluated the anti-inflammatory and antioxidant activities of Perilla leaf superfine powder in vitro,clarified the therapeutic effect of Perilla leaf superfine powder on mice with ulcerative colitis,and clarified the material basis of efficacy of Perilla leaf superfine powder.This study provides a new idea for the development and clinical application of Perilla leaf,and provides a theoretical basis for the development of related functional products.