| Background: Bupleurum chinense DC,a traditional chinese medicine for relieving exterior syndrome,is the dried root of Umbelliferae plant Bupleurum chinese DC or Bupleurum scorzonerifolium Willd.Bupleurum have the effects of reducing fever,relieving exterior syndrome,promoting yang qi,soothing liver and relieving depression.Modern pharmacological studies show that the main effective components of Bupleurum include Bupleurum volatile oil and Bupleurum saponin,Bupleurum polysaccharide,Bupleurum volatile oil is the main active component with antipyretic effect.In recent years,the preparations developed with volatile oil of Bupleurum as the main ingredient mainly include Bupleurum injection,Bupleurum choral liquid,Bupleurum nasal spray,Bupleurum transdermal patch,etc.Which are used to treat influenza,relapsing fever,fever in the development stage of tuberculosis and other symptoms.It is an adaptive response of the host to the damage of inflammatory mediators in the co-regulation of febrile immune system and nervous system,characterized by elevating body temperature,headache and fatigue.Commonly used antipyretic drugs in clinic mainly include ibuprofen,lysine aspirin,analgin,etc,its efficacy is intense,which is not suitable for patients with low fever.Bupleurum injection was once known as the "star variety" of fever-reducing,but because of its complex components and incompatibility with many drugs,the incidence of adverse reactions is high,which limits its clinical application.Therefore,in order to meet the demand of clinical medication,this study takes Bupleurum as the research object to prepare pediatric Bupleurum antipyretic suppository.In this study,the volatile oil of Bupleurum was extracted by steam distillation,and the volatile oil of Bupleurum was included by saturated aqueous solution method.Finally,the pediatric Bupleurum antipyretic suppository was prepared by melting method.Refering to the 2020 edition of the standard of “Chinese Pharmacopoeia”,to study the quality standard of antipyretic suppository of Bupleurum for children,and its quality control and pharmacodynamic research were carried out,to provide theoretical basis for clinical application and pharmacological research.Objective: To prepare Bupleurum antipyretic suppository for children,formulate its quality standard,the pharmacodynamics and mechanism were preliminarily studied.Methods: 1.Study on the preparation of Bupleurum antipyretic suppository for children The extraction process of volatile oil from Bupleurum was optimized by orthogonal experiment with the ratio of solid to liquid,soaking time and extraction time as influencing factors.The inclusion rate of Bupleurum volatile oil was used as the evaluation index,and β-CD dosage,inclusion temperature and inclusion time were used as the influencing factors to optimize the inclusion process of Bupleurum volatile oil by orthogonal experiment.The preparation technology of Bupleurum antipyretic suppository for children was optimized by single factor experiment with suppository properties and melting time limit as evaluation indexes.To optimize the preparation process of Bupleurum antipyretic suppository with the content.2.To study the quality standard of antipyretic suppository of Bupleurum for children Refering to the 2020 edition of the standard of “Chinese Pharmacopoeia”,to study the quality standard of antipyretic suppository of Bupleurum for children.The characteristics,weight difference,melting time and microbial limit of Bupleurum antipyretic suppository for children were examined respectively.Taking hexanal and heptanal as its quality indicators,and the content of volatile oil in Bupleurum chinense DC was determined by GC-MS.3.To study the pharmacodynamic of antipyretic suppository of Bupleurum for children New Zealand rabbits were divided into groups,New Zealand rabbits were divided into blank group,model group,antipyretic suppository of Bupleurum for children group,Bupleurum injection group and ibuprofen suppository for children group.And fever model was established by intravenous injection of endotoxin in ear margin.Each group was given blank suppository,antipyretic suppository of Bupleurum for children（0.8 g/kg）,Bupleurum injection（0.6m L/kg）and ibuprofen suppository（25mg/kg）for children.The changes of behavior and temperature of rabbits in each group were observed 1 hour before modeling,1.5 h after modeling and after administration.The levels of interleukin-1β（IL-1β）,interleukin-6（IL-6）and tumor necrosis factor-α（TNF-α）in plasma and the levels of c AMP,arginine vasopressin（AVP）and prostaglandin E2（PGE2）in hypothalamus of rabbits in each group were detected by ELISA.4.To study the antipyretic mechanism of antipyretic suppository of Bupleurum for children THP-1 cells were divided into groups,THP-1 cells were divided into blank group,LPS group,CH group（8 mg/m L）,LPS+CH group（2 mg/m L）,LPS+CH group（4 mg/m L）and LPS+CH group（8 mg/m L）.And the inflammatory model was established by stimulating THP-1 cells with LPS,LPS（10 μg/ m L）,CH（8 mg/m L）,LPS+ CH（2 mg/ml）,LPS+ CH（4 mg/ml）and LPS+ CH（8 mg/ml）were given to each group respectively.The protein expressions of COX-2 and IL-1β in each group were detected by Western blot and gene expression of TNF-α,IL-1β,IL-6,PGE2 in each group were detected by PCR.Results: 1.Study on the preparation of Bupleurum antipyretic suppository for children The optimum extraction conditions of Bupleurum volatile oil were as follows: the ratio of material to liquid was 1:10,soaking time was 8 h,and extraction time was 2 h;And the optimal inclusion process conditions were 30 g of β-CD,50℃ of inclusion temperature and 2 h of inclusion time.60 tablets of Bupleurum antipyretic suppository for children were prepared from 300 g Bupleurum decoction pieces.2.Study the quality standard of antipyretic suppository of Bupleurum for children Antipyretic suppository of Bupleurum for children is milky white bulletshaped,with uniform color,smooth,complete and good formability at room temperature and no softening or deformation.The overall average weight of 3 batches of antipyretic suppository of Bupleurum for children were（1.136±0.034）g,and the overall average melting time were（18.20±0.47）min.No pathogenic bacteria such as staphylococcus aureus and pseudomonas aeruginosa were detected.GC-MS experimental mass spectrometry show that the matrix and impurities have no interference,the specificity was good and the mass spectrometry conditions meet the requirements.Hexaldehyde and heptanal showed a good linear relationship at 6.19~99.00 μg/m L and 4.90~78.40 μg/m L,with correlation coefficients of 0.998 and 0.998 respectively.The precision RSD were 1.26% and 0.78% respectively.The repeatability RSD were 1.35% and 2.32% respectively.The stability RSD were 2.82% and 1.58% respectively.The recovery rates were 97.4% and 98.9% respectively,RSD were 1.84% and 1.28% respectively.The lowest average contents of hexanal and heptanal in three batches of antipyretic suppository of Bupleurum for children determined by GC-MS were 1.015 mg/10 capsule and 1.145 mg/10 capsule,respectively.Therefore,the content of volatile oil from Bupleurum should not be less than 0.173 mg/capsule.3.Study the pharmacodynamic of antipyretic suppository of Bupleurum for children Compared with the model group,the antipyretic suppository of Bupleurum for children group can significantly reduce the temperature of febrile rabbits to the normal level within 1~2.5 h after administration（P<0.01）,with a decrease of 0.82±0.07 ℃.Furthermore,the antipyretic suppository of Bupleurum for children can significantly down-regulate the levels of IL-1β,IL-6 and TNF-α in the plasma of febrile rabbits（P <0.05）,down-regulated by 19.13±1.57,33.76±5.82,25.48±9.07 pg/m L respectively.antipyretic suppository of Bupleurum for children can obviously down-regulate the levels of c AMP（P <0.05）,PGE2（P <0.01）in hypothalamus of febrile rabbits,down by 1.70±0.19 nmo L/L,79.65±14.99 pg/m L respectively,and up-regulate AVP（P <0.01）and up by 0.93±0.18 nmo L/L.4.Study the antipyretic mechanism of antipyretic suppository of Bupleurum for children Compared with the blank group,the expression of IL-1β and COX-2 protein in LPS group increased significantly（P <0.001,P <0.01）,up-regulated to 0.22±0.02,0.12±0.04 respectively.Compared with LPS group,the expression of IL-1β and COX-2 protein in LPS+CH group（8 mg/m L）decreased significantly（P <0.01,P <0.05）,down-regulated to 0.08±0.01,0.04±0.02 respectively.Compared with the blank group,the gene expression of IL-1β,TNF-α,COX-2,PGE2（P <0.01,P <0.001,P<0.0001）in LPS group were increased significantly,up-regulated by 10.90±0.14,8.17±0.05,3.90±0.23,2.20±0.10 respectively.Compared with LPS group,the expression of IL-1β,TNF-α,COX-2 and PGE2（P <0.01,P <0.001,P <0.0001）genes in LPS+CH group（8mg/m L）were decreased significantly,down-regulated by 10.10±0.15,5.89±0.03,4.10±0.04,2.05±0.14 respectively.Conclusion: 1.Antipyretic suppository of Bupleurum for children is reasonable and feasible,and the prepared suppository meets the requirements of quality standards.GC-MS can be used for the content of volatile oil of Bupleurum chinense DC.2.The antipyretic effect of antipyretic suppository of Bupleurum for children is obvious,and the mechanism may be related to the regulation of the expression of inflammatory factors and heat medium such as IL-1β,IL-6,TNF-α,COX-2 and PGE2. |
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