Study On Evaluation And Mechanism Of Anti-inflammatory And Analgesic Activity Of KTDA And FSA Compatibility In Frankincense-Myrrh

I.Literature research.(1)The research background of inflammatory painBy consulting the literature,the occurrence and development of inflammation were summarized,and the pathological basis of inflammatory pain was discussed.Inflammatory pain is one of the three main types of pain,which originates from tissue injury or inflammation.Macrophages,T lymphocytes and other immune cells in the damaged site release inflammatory mediators such as pro-inflammatory cytokines and chemokines,which induce the production and maintenance of pain through the activation of nociceptors.Neutralizing these cytokines relieves pain before inflammation weakens.Therefore,the drugs with anti-inflammatory activity also have a certain analgesic effect,and the treatment of inflammatory pain should also be discussed from two aspects of anti-inflammation and analgesia.(2)Research progress of anti-inflammatory and analgesic active components in frankincense myrrh.The compounds with anti-inflammatory and analgesic activities in frankincense and myrrh reported in the literature were summarized,and based on the good anti-inflammatory and analgesic activities of frankincense triterpene acid and myrrh sesquiterpene selected in previous studies,the compounds were summarized and analyzed from the point of view of functional groups.It was found that the tetracyclic triterpene acid with carbonyl and acetoxy groups in frankincense and the components with furan ring and both carbonyl and acetoxy in myrrh had good anti-inflammatory and analgesic activities,which may be the main active components in this part.Furthermore,two representative active components,KTDA(frankincense)and FSA(myrrh),were selected as the research objects of the follow-up anti-inflammatory and analgesic activity experiments.2.Preparation and chemical analysis of anti-inflammatory and analgesic active components of frankincense myrrh.(1)Preparation and chemical analysis of anti-inflammatory and analgesic components in frankincense.The frankincense powder was extracted by refluxing with 95%ethanol,and the content of KTDA in it was determined.The alcohol extract was separated by silica gel column chromatography and eluted with petroleum ether-ethyl acetate as eluent.Through the content detection,it is found that the content of KTDA in the alcohol extract of frankincense can reach 2.23%±0.12%.From the content point of view,it has high economic development value.Silica gel column chromatography gradually increased the ratio of ethyl acetate from pure petroleum ether.KTDA,was separated from petroleum ether-ethyl acetate 10:1 and was washed and purified repeatedly by petroleum ether.The target component KTDA was identified by 13C NMR and 1H NMR spectra,and its purity was 95%by UPLC analysis.(2)Preparation and chemical analysis of anti-inflammatory and analgesic components in myrrh.The myrrh powder was extracted by refluxing with 95%ethanol,and the content of FSA in it was determined.The alcohol extract was extracted with ethyl acetate,separated by silica gel column chromatography,and eluted with petroleum ether-ethyl acetate as eluent.Through the content detection,it is found that the content of FSA in the alcohol extract of frankincense can reach 1.31%±0.03%,which also has a higher content.Silica gel column chromatography gradually increased the ratio of ethyl acetate from pure petroleum ether.FSA,was isolated from petroleum ether-ethyl acetate 20:1.The purity of FSA,UPLC was identified as 95%after recrystallization and purification of petroleum ether at low temperature.The target component FSA was identified by 13C NMR and 1H NMR spectra,and its purity was 95%by UPLC analysis.The experimental results show that the above methods can be used for simple and rapid preparation of target compounds KTDA and FSA,and their high purity and yield increase their potential as medicinal compounds.3.Study on the anti-inflammatory mechanism and compatibility optimization of KTDA and FSA in vitro.(1)The effect of KTDA and FSA on the viability of RAW 264.7 cells induced by LPS and its mechanism.Based on the inflammatory model of RAW 264.7 cells induced by LPS,the anti-inflammatory activities of compounds KTDA and FSA alone and in combination were investigated,and the better compatibility ratio was selected.The results showed that KTDA had certain cytotoxicity,and when the concentration was less than 20 μM,the cytotoxicity was lower than 150 μM.Using NO as an inflammatory index,KTDA and FSA had better inhibitory effect at the ratio of 1:5.Using this ratio as the compatible ratio,the mRNA expression levels of IL-6 and IL-1β proteins,the phosphorylation levels of MAPKs family proteins and AKT proteins and the nuclear transfer of NF-κB in RAW 264.7 cells induced by LPS were used as indicators to explore the anti-inflammatory activity of KTDA and FSA alone and in combination.The results showed that KTDA could significantly reduce the expression of IL-6 and IL-1β protein mRNA at 20 uM and FSA at 75 to 100 μM,but it was not as significant as that when used alone,both KTDA and FSA had significant inhibitory effects on P38 and JNK protein phosphorylation,and the inhibitory effect was the strongest at KTDA 20 μM to FSA 100 μM,but KTDA had no significant improvement on ERK phosphorylation.High concentrations of KTDA and FSA could significantly inhibit the phosphorylated expression of AKT.For the nuclear transfer of NF-κB,KTDA showed a significant inhibitory effect at 10 to 20 μM.The results showed that both KTDA and FSA alone and in combination may inhibit the activation of NF-B and reduce the level of nuclear transfer by reducing the phosphorylation of MAPK and AKT,thus reducing the expression of inflammatory cytokines such as IL-1β,IL-6 and mRNA,so as to exert their anti-inflammatory and analgesic effects.(2)The effect of KTDA and FSA on the viability of BV2 cell inflammatory model induced by LPS and its mechanism.Based on the model of BV2 cell inflammation induced by LPS,the effect of compound KTDA and FSA on neuronal inflammation was investigated.The results showed that the cytotoxicity of KTDA was higher than that of FSA,while that of FSA was less than 150μM.the results of NO secretion of BV2 cells showed that KTDA and FSA had significant inhibitory effect at 20μM to 100μM,and the compatibility ratio needed to be further verified.the results showed that the inhibitory effect of KTDA and FSA was the most obvious at 20 μM and 100 μM,respectively,by detecting the levels of IL-6 and MCP-1 expressed by Elisa.The results suggest that KTDA and FSA may exert anti-inflammatory and analgesic effects on inflammatory diseases of CNS by reducing inflammatory factors and chemokines such as NO,IL-6,MCP-1 and so on in BV2 cells.4.Evaluation of the activity of AEA and FSA in the model of acute inflammatory pain in vivo.(1)Evaluation of the activity of KTDA and FSA in the treatment of acute inflammatory pain in mice.Based on the tail flick test and acetic acid writhing test in mice,the analgesic effects of KTDA and FSA on acute inflammatory pain were evaluated.The results showed that the maximum effect percentage of tail flick test in the compatible low dose group(KTDA 9mg/kg/d+FSA 45mg/kg/d)was higher than that in the single group,and the inhibition rate of acetic acid writhing was also better than that in other groups,which showed significant analgesic effect.The analgesic effect of FSA 45 mg/kg/d better than 90 mg/kg/d,KTDA 9 mg/kg/d and 18 mg/kg/d,low dose group was better than that of high dose group.It shows that in animal experiments,the higher the dose,the more significant the efficacy.(2)Study on the mechanism of KTDA and FSA in the treatment of acute inflammatory pain in mice.On the basis of acetic acid writhing test in mice,the serum,thymus and spleen of mice were collected after intraperitoneal injection of 0.6%acetic acid for 20 minutes.The serum was used to detect the levels of neurotransmitters and hormones such as β-EP,NE,SP and 5-HT in vivo,in order to further explore the anti-inflammatory and analgesic mechanism of KTDA and FSA.The results showed that the body weight,thymus and spleen organ index of mice did not change significantly after continuous administration for three days,but the results showed that the organ index of the administration group and the positive drug group showed an opposite trend,but the real situation remains to be further tested.The levels of SP and 5-HT in serum increased significantly after modeling,while the level of β-EP decreased.After administration,the high-dose group of KTDA and FSA had the most obvious downward trend of 5-HT and SP,while the high-dose group of KTDA and FSA had a significant increase of β-Ep.High dose of KTDA and low dose of FSA can significantly increase the levels of NE and 5-HT in cerebral cortex.The results show that KTDA and FSA may play an analgesic role by up regulating the levels of β-EP in serum and NE in cerebral cortex.(3)Study on the molecular mechanism of anti-inflammation and analgesia of KTDA and FSA based on network pharmacology.The prediction of drug-like properties of KTDA and FSA showed that KTDA had strong lipophilic solubility,while FSA showed good drug-like properties,and both of them could be used as drugs for transdermal absorption.The anti-inflammatory and analgesic targets of the two proteins were predicted and the protein interaction network was established.Nine key pivotal proteins were obtained by topological analysis,among which ERK2,AKT1 and PI3K were all involved in activating downstream NF-κB to promote the release of inflammatory factors.The previous experiments also verified that KTDA and FSA could inhibit the phosphorylation level of ERK and AKT respectively,thus reducing the level of transfer into the nucleus after activation of NF-κB,which proved the results of network pharmacological analysis.GO enrichment analysis and KEGG pathway analysis show that KTDA and FSA may participate in cellular processes and pathways such as immune system regulation,defense response,inflammatory response,cytokine signal transduction and interleukin signal transduction in the immune system,and participate in the regulation of inflammation in vivo,anti-inflammatory and analgesic effects.