Preliminary Study On The Mechanism Of "Significant Attenuation And Syneristic Effect" And Processing Technology Of Momordicae Semen Cream

ObjectiveMomordica cochinensis（Lour.）Spreng.is a dry mature seed of the cucurbitaceae Momordicae,which is a toxic Chinese medicine and its clinical application must be made into a cream.This project aims to optimize the best processing technology of Momordicae Semen cream,and analyze the differences in chemical composition,toxicity and anti-tumor efficacy of Momordicae Semen before and after frosting,so as to explore the mechanism of significant attenuation and synergistic effect of Momordicae Semen cream.Method1 The best process study of Momordicae Semen creamThe OD is the comprehensive evaluation index of the index of the saponin3-O-β-D-glucuronide methyl ester,β-sitosterol,oleanolic acid content and fatty oil content,using three factors three.The three-factor and three-level Box-Behnken response surface method was used to design the experiment,and the effects of baking time,baking temperature and pressing time on the processing technology of Momordicae Semen cream were investigated.2 Study on chemical composition difference of Momordicae Semen before and after frostingUltra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-QTOF-MS）was used to acquire high samples of 9 batches of Momordicae Semen and Momordicae Semen cream by time-dependent mass spectrometry data scanning mode（MS^E）,combined with Unifil software and Progenesis QI V2.0 software analysis data,based on principal component analysis（PCA）and orthogonal partial least squares discriminant analysis（OPLS-DA）to compare the chemical composition of Momordicae Semen before and after frosting difference.3 Comparative study on acute toxicity and anti-tumor effect of H₂₂ before and after frostingAccording to the acute toxicity test method of mice,the LD₅₀ of the alcohol extract was orally administered by the probabilistic unit method（Bliss method）to calculate the LD₅₀ of the alcohol extract and the Momordicae Semen cream.After 14 days,the spontaneous activity,mental status,toxicity and death of each group of mice were observed and recorded.The mice were sacrificed at the end of the experiment,and the liver and kidney were taken for pathological examination.The Kunming mice were inoculated with H₂₂ liver cancer ascites to establish a transplanted tumor model.H₂₂ tumor-bearing mice were divided into model group（sterile saline）,positive CTX group（20mg/kg）,and positive compound Shougong powder group（1.95g/kg）,Momordicae Semen group（1.65g/kg,0.55g/kg）,Momordicae Semen cream administration group（1.65g/kg,0.55g/kg）,and normal group,continuous intragastric administration for 10 days.The living state and body mass of the tumor-bearing mice were observed,and the tumor inhibition rate and organ index were measured.The levels of IL-2,IL-6,IL-12 and TNF-αin serum were determined by ELISA.The tumor and liver were observed by HE staining.The pathological changes of the tissues were detected by immunohistochemistry to detect the expression levels of Bax and Bcl-2 proteins in tumor tissues.Results1 Study on the best technology of Momordicae Semen creamAccording to the BBD-RSM method,it is preferred that the optimum processing conditions of the Momordicae Semen cream are a baking time of 1.7 h,a baking temperature of 80℃,and a pressing time of 30 min.2 Study on chemical composition difference of Momordicae Semen before and after frostingMomordicae Semen identified 32 components,and Momordicae Semen cream added 9ingredients,a total of 41 compounds.1 Add 9 ingredients,namely oleragenin;Goyaglycoside f;Charantosides A;momordicoside U;3-O-β-D-glucofuranosidurono-6,3-lactone-gypsogenin;Karavilagenins D;daucosterol;Kuguacins K;Kuguacins O.2content is significantly different in 6 components,Momordins III,Neokuguaglucoside,nonadec-10-enoic acid,oleanolic acid Momordicae Semen cream is higher;（9Z,11Z,13E）-octadeca-9,11,13-trienoic acid,Karavilosides I Momordicae Semen is higher.3 Study on acute toxicity and anti-tumor effect of H₂₂ before and after frosting3.1 Study on acute toxicity of Momordicae Semen before and after frostingThe mice in the group of Momordicae Semen and Momordicae Semen cream showed different degrees of spontaneous activity reduction,drowsiness,eyelid hemiplegia,severe tonic convulsions,and death.According to Bliss method,the LD₅₀=16.777g/kg of Momordicae Semen and the LD₅₀=28.560g/kg of Momordicae Semen cream were calculated,which was significantly lower than that of Momordicae Semen cream.The degree of liver and kidney tissue in the Momordicae Semen cream group was reduced to some extent compared with the Momordicae Semen group.3.2 Anti-tumor effect of H₂₂ tumor-bearing mice before and after frostingThe mice in the group of Momordicae Semen and Momordicae Semen cream improved the general condition of H₂₂ tumor-bearing mice,increased the spleen and thymus index,and inhibited the growth of the tumor,but there was no significant difference between the two;The levels of IL-6,IL-12 and TNF-α,and the level of IL-2 and TNF-αof Momordicae Semen cream was significantly higher than that of Momordicae Semen（1.65g/kg）.The IL-12 level of the sub-（0.55g/kg）was significantly decreased;both increased the expression of bax in tumor tissues,inhibited the expression of bcl-2 and increased the ratio of bax/bcl-2,and the Momordicae Semen cream was significantly higher than that of Momordicae Semen;relieves pathological changes in tumor and liver tissue.ConclusionThis project is stable and feasible with BBD-RSM’s preferred Momordicae Semen cream processing method,and the model predictability is good.There are differences in the chemical and chemical components of Momordicae Semen and Momordicae Semen cream.The acute toxicity of Momordicae Semen cream is significantly lower than that of Momordicae Semen;while Momordicae Semen cream may increase IL-2,IL-6 and TNF.The level ofα-factor and the regulation of apoptotic genes bcl-2 and bax enhance the anti-tumor effect,and preliminarily clarify the mechanism of“attenuation and synergy”of Momordicae Semen cream.