| Objective:Under the guidance of the theory of Mongolian medicine,the traditional Mongolian medicine Ulan-3 decoction was transformed into Ulan-3 ultramicro powder granules with modern technology.The safety and side effects of Ulan-3 were verified by oral acute toxicity test,and the anti-inflammatory effects on mice induced by Streptococcus pneumoniae were discussed.Methods:(1)The particle size,Angle of reactivity,fluidity,hygroscopicity,water soluble extract,alcohol soluble extract and so on were compared between Ulan-3 conventional powder and ultrafine powder.The content of total flavonoids was determined at 510nm by UV spectrophotometry with sodium nitrite,aluminum nitrate and sodium hydroxide colorimetry.The contents of rubiin and hydroxyl rubiin were determined by HPLC.The chromatographic conditions were Agilent Eclipse XDB-C18 column(4.6mm×250mm,5μm).The mobile phase was 0.2%phosphoric acid solution:methanol,0-6min 84-90(B),6-15min90-99(B);Detection wavelength 250nm;Column temperature 30℃;Volume flow 1ml/min;The sample size was 10μl.(2)The dry granulation method was used to carry out the granulation orthogonal test with the single molding rate as the index.According to the Chinese Pharmacopoeia(2020 Edition),the obtained ultrafine powder particles were checked for character,particle size and moisture,and the stability of the granules was determined at60℃and 92.5%±5%high humidity.(3)40 SPF Kunming mice were divided into blank control group and drug administration group(half male and half female).The administration group was given Ulan-3 ultra-fine powder particles(0.25g/ml)twice within 24h,and the blank group was given the corresponding amount of normal saline(1ml/time).The toxic reaction of mice was observed at 14 days,and the body weight and food intake were recorded.(4)84 SPF BALB/c mice were divided into 7 groups,including blank control group,model group,low-dose Ulan-3 decoction group,high-dose ulan-3 decoction group,low-dose ulan-3ultramicro powder granule group,high-dose ulan-3 ultramicro powder granule group and amoxicillin positive control group.During modeling,mice were anesthetized with isoflurane,kept in a vertical position,and received nasal drops of Streptococcus pneumoniae with a concentration of 50μl(1×108 cfu/ml).The blank group was given the corresponding amount of normal saline.After successful modeling,the drug was administered once a day,continuously for 7 days,and blood was collected from the eyeball 2h after the last administration.The expressions of IL-1,IL-6 and TNF-αin serum were determined by enzyme-linked immunosorbent assay.HE staining was used to observe the pathological changes of lung tissue in mice.Results:(1)It was found that the fluidity of Ulan-3 ultrafine powder was worse than that of Ulan-3 conventional powder,and the moisture absorption was stronger,with more water-soluble and alcohol-soluble extracts.The linear regression equation y=0.0849x+0.0031,R~2=0.9993 was obtained.The results showed that the concentration of rutin had a good linear relationship between 0.019894 and 0.079579mg/g.RSDS of precision,stability and reproducibility tests were 0.53%,0.73%and 1.77%,respectively.The average recovery was 100.32%with RSDS of 1.69%.The average content of total flavonoids in Wulan-3 conventional powder,ultra-fine powder and ultra-fine powder granules was39.36mg/g,43.33mg/g and 51.98mg/g,respectively.The results obtained by HPLC were in the concentration ranges of 119.997-1199.97μg/ml and 56.9088-569.088μg/ml,respectively.There was a good linear relationship between the injection size and the peak area.The regression equation of alizarin was y=0.2165x-0.6073.The regression equation of hydroxy-alizarin was y=0.2376x-0.3477,R~2was 1 and 0.9999,respectively.Methods The precision,stability,repeatability and recovery RSDS of rubitin and hydroxyl rubitin were0.18%and 0.73%,0.41%and 1.44%,1.20%and 1.39%,1.41%and 1.92%,respectively.The average contents of rubitin and hydroxyl rubitin in Ulan-3 conventional powder,ultramicro powder and ultramicro powder granules were 1.9mg/g and 2.49mg/g,2.84mg/g and 2.79mg/g,2.96mg/g and 2.57mg/g,respectively.(2)On the basis that Ulan-3 ultrafine powder is superior to conventional powder,orthogonal experiment using dry granulation method was carried out with single molding rate as the index.The optimal granulation conditions were finally selected as rotary pressure 4,press wheel speed 7,feeding speed 20,and the highest single molding rate of particles was obtained under these conditions.The average molding rate is80.47%by parallel experiments with optimized process parameters.In the test of influencing factors,the content loss rate of each component of Ulan-3 ultrafine powder was less than 10%when it was placed at 60℃for 10 days.Under the condition of relative humidity(RH=92.5%±5%)for 10 days,the loss rates of rubiin and hydroxyl rubiin were both greater than 10%,and the particle increase was greater than 10%.(3)The maximum dosage was0.25g/ml and the final dosage reached 15.98g/kg(the dosage was 186 times of the clinical human dose).During the observation period of 14 days,no abnormality was observed in activities,behavior,diet,hair color and other general states of the mice in each group,no death or poisoning symptoms,and no obvious abnormality in the appearance of major organs.Compared with before administration,the body weight of male and female mice in blank group and administration group increased significantly on 2d,7d and 14d after administration.Compared with the female mice in the blank group,the female mice in the administration group had slower weight gain after 2d(P<0.05).Overall,both male and female mice in the blank control group and the administration group gained weight naturally.(4)HE staining of lung tissue showed that the lung structure of mice in the model group was incomplete,alveolar parenchyma was observed,accompanied by a large number of neutrophil infiltration,bronchial structure was eroded and destroyed,more inflammatory cells were observed in the alveolar cavity,and circular infiltration of lymphocytes was observed around blood vessels.A small amount of inflammatory cell infiltration and red blood cells were observed around the blood vessels of lung tissue in Ulan-3 decoction,Ulan-3 ultramicron powder granule group and amoxicillin positive control group,and the inflammation was significantly reduced compared with model group.ELISA results showed that compared with blank control group,serum levels of IL-1,IL-6 and TNF-αin model group were significantly increased,and the differences were statistically significant(P<0.01).Compared with model group,serum levels of IL-1,IL-6 and TNF-αin all administration groups were significantly decreased,and the difference was statistically significant(P<0.01).Conclusion:(1)The ultra-fine powder particles of Ulan-3 obtained by dry granulation can withstand high temperature and high humidity conditions,and the experimental results can provide reference data for the formulation of quality standards of Mongolian medicine Ulan-3.(2)Based on acute toxicity test,the oral toxicity of Ulan-3 ultramicro powder granules was low and the safety range was wide.(3)The two forms of ulan-3 had certain anti-inflammatory effects on pneumococcal pneumonia model. |
PDF Full Text Request