Study On Preparation Technology Of Effective Parts Of Frankincense-myrrha And Its Protective Effect On Ischemic Stroke

This study was completed with the support of the national natural science foundation of China?No.30973885?and the key project of jiangsu collaborative innovation center for the industrialization of traditional Chinese medicine resources?No.Fjgjs-2015-12?,mainly around the effective parts of frankincense and myrrh of preparation technology optimization,effective parts to improve the effect of the compatibility of ischemic stroke and mechanism research,etc,so as to lay a foundation for the further development and utilization of this drug.The main research contents and results are summarized as follows:Chapter one,Literature reviewThis chapter systematically summarized the main chemical constituents of frankincense and myrrh.Among them,the main compositions of boswellic acids,including pentacyclic triterpenes and macrocyclic diterpenes,and the main compositions of myrrh have single terpenoids,sesquiterpenoids,two terpenoids,triterpene.Modem pharmacological activity studies show that frankincense and myrrh have anti-inflammatory,analgesic,anti-tumor and other biological activities.It provides scientific basis for further open utilization of frankincense and myrrh.Based on the understanding of Tranditional Chinese medicine,etiology and pathogenesis of ischemic stroke and the mechanism of stroke injury,it is found that the disease was closely related to depolarization,calcium overload,?xidative stress,apoptosis and inflammatory response.Chapter two,Study on the preparation of the effective parts of frankincense and myrrhSection 1,Data analysis of the clinical application of frankincense and myrrh in traditional Chinese medicineThrough data mining analysis of frankincense,myrrh in prescription compatibility application rules,read out 2236 prescriptions of frankincense,myrrh in traditional Chinese medicine prescription information database.the statistics of frankincense and myrrh compatibility proportion has 50 kinds,including 1:1,2:1,1:2,2:3,3:2,5:3,3:4,were higher,and in the form of 1:1 ratio as high as 1863 prescriptions.Therefore,in this section,analysis 1:1 formulations for the treatment of the disease families,genera,formulations and dosage forms and its correlation.Results show that frankincense-myrrh are mainly used for surgical diseases,the main effects are to promote blood circulation and remove stasis,promoting wound healing sore muscle,relieving pain,dispelling wind,resolving carbuncle and expulsing boil,detoxification and reunion of bone.the main dosage form are powder,pill,paste.Section 2,Dissolution analysis of chemical components of frankincense-myrrh with different compatibility ratiosThe UPLC-TQ/MS was used to analyze the chemical composition dissolution of frankincense and myrrh at different compatibility ratios?1:1,1:2,2:1,2:3,3:2,3:4,5:3?.The results show that the chemical components of the different ratios of frankincense and myrrhare different,and the most soluble chemical components are the highest when the compatibility ratio are 1:1 and 2:1Section 3,Optimization of extraction and purification process effective components of boswellic triterpenoid acids from frankincenseThe extraction amount and extract yield of 13 boswellic triterpenoid acids?3-oxotirucall-8,24-dien-21-oic acid,3?-acetoxy-tirucall-7,24-dien-21-oic acid,3-hydroxytirucall-8,24-dien-21-oic acid,acetyl 11?-methoxy-?-boswellic acid,3?-hydroxy tirucall-7,24-dien-21-oic acid,11-keto-boswellic acid,3-O-acetyl-?-boswellic acid.3?-acetyloxylanosta-8,24-dien-21-oic acid,3?-aeetoxy-5?-lanosta-8,24-dien-21-oic acid,3-acetyl-11-keto-?-boswellic acid,3-acetyloxy-tirucall-8,24-dien-21-oic acid,?-boswellic acid.?-boswellic acid?in frankincense were detected by UPLC-TQ/MS,the extraction method was investigated by single factor and response surface,the extraction solvent,ratio of solid to liquid,extraction time and extraction times were investigated on the extraction process,and the extract was purified by alkali dissolving acid precipitation method and the purification process parameters were investigated by single factor and orthogonal test to determine the best purification process.The results show that optimum purification process was as follows:reflux extraction with 95%alcohol for 62 min,extraction 4 times,ratio of material to liquid was 1:20,alkali pH was pH12-13,acid pH<2,acid precipitation condition was to set it at 0-4? for 30 min.The purity of boswellic acids was 73.87%Section 4,Optimization of extraction and purification of sesquiterpenoids from myrrhThe extraction amount and extract yield of two sesquiterpenes in myrrha were detected by UPLC,and the effects of extraction solvent,ratio of material to liquid,extraction time and extraction times on the extraction process of the myrrha were investigated by single factor and response surface;and the myrrh extract was purified by silica gel column chromatography and the purification process parameters were investigated to determine the best purification process.The results show that the optimum purification process was as follows:reflux extraction with 92%alcohol for 2.5 h every time,extract twice,ratio of material to liquid was 1:7.The ethyl acetate was purified with 300-400 mesh silica gel,loading quantity of sample was 1:40,the impurity was removed with petroleum ether?PE?,and then eluted with petroleum ether:ethyl acetate?PE:EA?20:1 as eluent for 3-6 BY.The purity of sesquiterpenoids could reach 61.43%.Chapter three,Study on the effect and mechanism of MCAO model improved by effective part compatibility proportion of frankincense and myrrh in SD ratsSection 1,Evaluation of the effect of effective part compatibility proportion of frankincense and myrrh on the improvement on MCAO model in SD ratsIn this section,the ischemic stroke model of SD rats was established by means of middle brain artery occlusion?MCAO?,and the success of the model was determined by neurological function score.In other words,the injured contralateral forelimb could not be extended when the tail was lifted.Rotating to the damage side;To tip to the opposite side;Cannot walk spontaneously,unconsciousness.Different compatibility ratios of boswellic total triterpenoid acids and myrrh sesquiterpenes?BA-MS,10:1,5:1 and 20:1 were converted from the compatibility ratios of 1:1,1:2 and 2:1?were administered twice daily,for three days later,collect urine and plasma brain/lung samples,respectively through determination of biochemical indicators cerebral infarction area,brain lung tissue pathology analysis and Western blotting analysis in brain tissue Ang?/Ang?/Tie2 signaling pathways and TLR4/NF-?B signaling pathways,and immunohistochemical analysis of TDP-43,NeuN,TGF-?1,VWF and bFGF protein expression.The results showed that BA-MS could improve the neurological function score,improve the area of cerebral infarction,reduce the morphological edema of brain tissue,neuron loss,the degree of nuclear shrinkage,and improve the alveolar structure.Regulating the TLR4/NF-?B signaling pathway and the Angl/Angll/Tie signaling pathway,thereby reducing the expression of related neuroinflammatory factors?IL-1?,nNOS?,neurotrophicfactors?BDNF,NGF?and promoting the expression of related angiogenic growth factors?ET-1,VEGF,PDGF,Angll,PAI-1?,plays a protective role in ischemic stroke,among which H10:1 and H20:1 have the best effect.Section 2,Metabolomics was used to study the regulation effect and mechanism of MCAO model in SD rats by the ratio of frankincense and myrrhThis section based on metabonomics to study the intervention effect of different compatibility ratios of boswellic triterpenoid acids and myrrh sesquiterpenes?BA-MS?on ischemic stroke.By analyzing the plasma and urine samples of rats,it was found that the compatibility ratios of H10:1 and H5:1 were the best,and identified 21 kinds of endogenous metabolites?plasma of 9 and urine of 12?,and thus build 14 related metabolic pathways?impact value is greater than 0.1?5 namely the retinol metabolism,pentose and glucuronic acid,linoleic acid metabolism,glycerol phospholipid metabolism,biotin metabolism,ascorbate and aldarate metabolism,amino acid metabolism?phenylalanine metabolism,pyruvate metabolism,cysteine and methionine metabolism?,terpenoid backbone biosynthesis,pantothenate and CoA biosynthesis,ether lipid metabolism,sphingolipid metabolism,galactose metabolism,arachidonic acid metabolism,steroid hormone biosynthesis.Among them,the metabolic pathway of linoleic acid was the most likely?impact=1?and the correlation analysis suggested that after drug administration intervention,the expression of IL-1? and VWF could be reduced by regulating the metabolism of linoleic acid,so as to play an improving role in ischemic stroke Section 3,Effects of effective part ratio of frankincense-myrrh ratio on the regulation of intestinal microorganisms in MCAO model of SD ratsTo evaluate the regulatory effect of different proportions of boswellic triterpenoid acids and myrrh sesquiterpenes?BA-MS?on the diversity of intestinal flora in MCAO rats after ischemia-reperfusion by 16S rDNA sequencing.Compared with the Sham group,the relative abundance of intestinal flora of rats in the ischemic stroke model group changed from phylum classification level to genus classification level.At the phylum level,H5:1 significantly reduced Actinobacteria and Tenericutes;At the genus level,H2O:1 was significantly reduced Dubosiella,Odoribacter:H5:1 significant increase in Clostridium_sensu_stricto₁,Collinsella,enterorhabdus,Lactobacillus,norank_fLachnospiraceae,g_norank_oMollicutes_RF39:H10:1,L10:1,H5:1 and H20:1 are significantly reduced Parasutterella;YXY is significantly increase Pygwiaiobacter.It can be seen that BA-MS can improve intestinal flora imbalance in ischemic stroke rats.In general,H5:1 has the best effect.Six short chain fatty acids?SCFAs?in the cecum contents of rats in each group were determined.Compared with the Sham group,the model group decreased significantly.After administration,the H5:1 group had a significant effect on reversing the contents of acetic acid and propionic acid in the intestinal contents of ischemic stroke rats,suggesting that the effect might be achieved by increasing the contents of acetic acid and propionic acid in the intestinal contents of ischemic stroke rats.The correlation analysis between intestinal microorganisms and SCFAs showed that IVA was positively correlated with norank_fLachnospiraceae,Clostridium_sensu_surico₁?r? 0.4?;AA was positively correlated with Lactobacillus?r? 0.4?,and AA,PA and BA were negatively correlated with Parasutterella?r ?-0.4?,suggesting that H5:1 might reduce the relative abundance of Parasutterella by increasing the relative abundance of lactobacillus and norank_fLachnospiraceae,thus achieving the improvement effect on intestinal injury in ischemic stroke.Chapter four,Study on biological effects and mechanism of effective part ratio of frankincense-myrrh based on cell modelSection 1,Protective effects of different ratios of effective part of frankincense-myrrh on LPS-induced PC12 cellsMTT assay was used to detect the protective effects of 7 different ratios of extracts?1:1,2:1,1:2,2:3,3:2,5:3,3:4?and different ratios of effective parts?10:1,20:1,5:1,20:3,15:1,50:3,15:2?on PC12 cells in LPS environment.The inflammatory factors such as TNF-?,IL-1?,IL-6,IL-18 and pain indicators ENK and SP were tested by ELISA kit to evaluate 7 different ratios of extracts anti-inflammatory and analgesic activities.The results showed that 7 extracts and effective compounds of different proportions had certain protective effects on PC 12 cells in the LPS environment,and showed different degrees of anti-inflammatory and analgesic activities.Compared with the model group,the administration group could inhibit the secretion of inflammatory cytokines?TNF-?,IL-1?,IL-6,IL-18?and reduce the secretion of the pain indicator?ENK and substance P?,and especially for IL-6?P<0.001?,in which the compatibility ratio of 1:1 and 5:3 are better.Section 2,Protective effects and mechanism of different ratios of effective part of frankincense-myrrh on LPS-induced BV2 cellsThrough the establishment of LPS induced nerve inflammation model of BV2 cells.MTT analysis different ratios of boswellic triterpenoid acids and myrrh sesiterpenoids compatibility?BA-MS?of BV2 cell toxicity.IL-1?,IL-6,INOS mRNA expression by q-PCR analysed.Western blotting analysis TLR4/NF-?B/PI3K/AKT signaling pathway.Results show that when the concentration is greater than 12.5 ?g·mL-1,BV2 cell growth by different degree inhibition,inhibition rate was positively correlated with concentration.In addition,BA-MS have different degrees of inhibition of IL-1?,IL-6,INOS mRNA expression and abate the p-NF-?B/NF-?B,p-AKT/AKT,TLR4 protein expression,and the promotion of p-PI3K protein expression,with the optimal ratio of 5:1.Section 3,Network pharmacology was used to study the neuroprotective effect and mechanism of different ratios of effective parts of frankincense and myrrhThe contents of boswellic triterpenoid acids and myrrh sesquiterpene?BA-MS?at different ratios were determined by UPLC-TQ/MS.The Pearson correlation analysis of those contents and biochemical indicators?Chapter four section 2?was constructed to obtain the important contributing components for the treatment of neuroinflammation,and the network pharmacological analysis of the obtained contribution components.The results show that 2-methoxy-5-acetoxy-fruranogermacr-1?10?-en-6-one,3?-acetyloxylanosta-8,24-dien-21-oic acid,11-keto-boswellicacid,3-acetyl-11-keto-?-boswellic acid are contribution components,and acts on 31 neuroinflammation-related targets,among which PI3K,AKT and IL-6 have been confirmed in the second section of chapter 4;32 signaling pathways are involved,including the toll-like signaling pathway in the second section of chapter 4.