Study On Relationship Of ’Composition-Toxicity/Efficacy’ And Aging Detoxification Of Asarum Essential Oil

The volatile oil of traditional Chinese medicine is the material basis for the prevention and treatment of diseases with aromatic drugs.It has unique physical and chemical properties,significant biological activity,which is prone to spontaneous aging over time,changing the composition of substances,and affecting quality.Clarifying its aging relationship and mechanisms is the key to scientific understanding,effective application,and systematic development of volatile oils.After aging,some volatile oils of traditional Chinese medicine can play a role in reducing toxicity,preserving efficacy,and slowly increasing fragrance,which is of great research value.Due to the unclear correlation between the"dose-effect"and"dose-toxicity"of Asarum essentia oil（AEO）,there is a lack of methods for"reducing toxicity and retaining efficacy",which affects clinical safe medication.This project takes AEO as the research object,studies the relevant laws of its composition toxicity/effectiveness,establishes safety evaluation standards for AEO,so sa to determine the safe treatment window for"reducing toxicity and retaining effectiveness"of AEO,and confirms the objective effectiveness of"reducing toxicity and retaining effectiveness".Studying the suitability and feasibility of different forms of aging methods,providing technical references for the rapid preparation of high-quality volatile oils.The main research content of this project are as follows:1.Prediction of the Related Targets and Pathways of the AEO in Analgesia and NeurotoxicityNetwork pharmacology and network toxicology were used to perform data mining on Pub Chem,NCBI,GEO,Gene Cards,Swiss Target Prediction and other databases with the keywords of’Asarum and toothache’and’Asarum and neurotoxicity’.The network relationship between AEO and toothache,AEO and neurotoxicity was established to reveal the related targets and pathways of AEO in the treatment of toothache and neurotoxicity.Through network diagram analysis,it was found that AEO affected PPARA,ACHE,SLC6A4,NOS3 and HTR2A targets through key components such as myristicin,γ-asarone and methyl eugenol,thereby affecting HIF-1 signaling pathway,calcium signaling pathway and 5-hydroxytryptamine synapse,so as to achieve the purpose of treating toothache.Through network diagram analysis,it was found that the AEO affected the targets of ACHE,ESR1 and PARP1 through key components such as elemicin,γ-asarone and myristin,and then affected the prolactin signaling pathway,resulting in neurotoxicity.2.Study on anti-inflammatory and analgesic effects and mechanism of AEOThe analgesic experiment of AEO was carried out by acetic acid writhing model in mice,and the number of writhing in mice was used as the evaluation index.The experimental results showed that the low,medium and high doses（107.92,161.88,215.84 mg/kg）of AEO effectively reduced the writhing phenomenon caused by acetic acid in mice,from 44 times to 23 times,which proved that AEO did have obvious analgesic effect.The anti-inflammatory activity of AEO was studied by LPS-induced RAW264.7 cell inflammation model,and the inflammatory factors IL-6 and TNF-αwere used as anti-inflammatory level evaluation indicators.The results showed that the expression of TNF-αand IL-6 in RAW 264.7 cells was significantly increased after LPS induction.After AEO intervention,the increase of inflammatory factors TNF-αand IL-6 was reduced（P<0.01）,which proved that the AEO did have anti-inflammatory activity.The anti-inflammatory activity and analgesic effect of the AEO confirmed that AEO is the main pharmacological active ingredient of Asarum.3.Study on the toxicity and mechanism of AEOThe acute toxicity experiment of AEO on AB zebrafish model was carried out,and the embryo morphology and survival rate were observed after administration.The results showed that the median lethal concentration of AEO to zebrafish was 0.174μL/mL（equivalent to 170.346 mg/L）.According to the acute toxicity grading standard of fish（GB/T21281-2207）,LC₅₀>100 mg/L was low toxicity,indicating that the AEO has low toxicity,and it can be observed that it has teratogenicity at high dose.The acute toxicity test of AEO on KM mice was carried out,and the LD₅₀was1.9116 g/kg.According to the oral acute toxicity grading standard,the AEO had low toxicity.After administration,the mice were observed to walk and shake,drunken.Results of body weight and organ ratio of mice showed that there was a significant difference between the high-dose administration group and the blank group（P<0.05）.The biochemical indexes of serum and tissue of mice were detected and histopathological observation was carried out.The results of biochemical indexes of serum and tissue of mice showed that the contents of AKP,ALT,AST and MDA in liver tissue of AEO group were significantly higher than those in blank group,and the content of GSH in heart tissue of AEO group was significantly lower（P<0.05）.The histopathological results showed that different degrees of damage were observed in all tissues after administration,with disordered tissue structure,disordered arrangement and direction of nucleus,and inflammatory infiltration.The transcriptome detection and analysis of mice brain tissue were carried out to predict the related proteins and pathways of the toxicity of AEO.The functional enrichment analysis of GO and KEGG showed that they were mainly enriched in oxidoreductase activity,apoptosis process,inner mitochondrial membrane protein complex,calcium signaling pathway and Oxytocin signaling pathway.It is speculated that AEO neurotoxicity is related to apoptosis and oxidative stress.4.Study on separation and toxicity of AEOThe above experimental results show that the AEO has good anti-inflammatory activity and analgesic effect,but it has low toxicity in mice and zebrafish,and the toxicity affects its clinical application.Therefore,it is very important to achieve attenuation.The essence of attenuation is the change of material basis.It is necessary to first understand the related components affecting the toxic effect in the AEO.Under the guidance of the holistic view of traditional Chinese medicine theory,molecular distillation technology was used to separate and enrich the components of AEO,and the chemical composition,anti-inflammatory activity and toxicity of different fractions were studied.Crude oil of AEO（C）was separated into three fractions by molecular distillation:Heavy Fraction（H）,Medium Fraction（M）and Light Fraction（L）.H achieved the enrichment of methyl eugenol and ethers.Methyl eugenol increased from 24.0203%to 31.3644%,and ethers increased from 69.4369%to 86.2078%.The monoterpenes in M and L were enriched.The relative percentage ofα-pinene andβ-pinene in L was 39.5302%,which was 5 times that of crude oil.The ether component contained only 5.8280%,which was 0.08 times that of crude oil.The results of LPS-induced RAW264.7 cell inflammation model showed that compared with the model group,the expressions of AEO and three fractions of TNF-αand IL-6 were significantly decreased（L>M>H/C）.According to the correlation analysis of component-anti-inflammatory activity,the substances that affect the anti-inflammatory activity of AEO mainly include:methyleugenol,myristicin,α-pinene andβ-pinene.The results of acute toxicity experiment in mice showed that the LD₅₀of C,H and M were 1.8852,1.9566 and 3.6741 g/kg,respectively.When the dose of L was increased to 5.1349 g/kg,the mortality rate of mice was 0.The results showed that the toxicity of L was much lower than that of C,H and M,and the toxicity of M was significantly lower than that of C（P<0.01）.According to the composition-toxicity correlation analysis,the main chemical components related to the toxicity of AEO include ethers such as safrole,methyleugenol and myristicin.PC12 cells were used to analyze the neurotoxicity of AEO.The results showed that AEO induced oxidative damage and dysfunction of mitochondria in PC12 cells,obvious changes in cell morphology,abnormal nucleus and increased apoptotic cells.The apoptosis rate of PC12 cells treated with AEO was detected by Annexin V-FITC double staining.The results showed that the apoptosis rate of PC12 cells increased after AEO treatment,indicating that AEO could induce apoptosis of PC12 cells.These results showed that AEO neurotoxicity was related to apoptosis and oxidative stress.Methyl eugenol and myristicin ether in AEO are common components that exert efficacy and toxicity.Correctly grasping their proportional changes is the key to achieving’attenuation and preservation’.5.To clarify the relationship between the’composition-toxicity/efficacy’of the AEO,and to establish the safety evaluation standard of’ether/olefin ratio’.According to the results of correlation analysis,methyleugenol,safrole and myristicin are not only the effective components in the AEO,but also the main toxic components.Therefore,it is necessary to control the content of AEO toxicity-effect related components and establish AEO safety evaluation standards,so as to achieve the goal of’reducing toxicity and keeping effect’.Eight fractions（A-H）containing AEO characteristic components were prepared by molecular distillation.GC-MS was used for component analysis,zebrafish acute toxicity was used to investigate the toxicity of volatile oil,and LPS-induced RAW264.7 cell inflammation model was used to study the anti-inflammatory activity of volatile oil.The results of GC-MS showed that the relative percentages of ethers in the eight groups were 61.11,67.11,71.36,37.03,68.25,70.63,68.59 and 53.79%,respectively.The relative contents of monoterpenes were 16.12,4.25,13.91,52.45,6.10,5.50,9.46and 33.49%,respectively.The results of IL-6 and TNF-αcontent showed that compared with the LPS-induced model group,the release of three concentrations（0.5,0.25 and 0.125μL/mL）in the dexamethasone group and the A-H group showed a downward trend,with significant differences（F/G>E>B>C>A>H>D）.The correlation between the toxicity results of zebrafish and the six components was analyzed:the percentage of ethers;the relative percentage content of monoterpenes;the ratio of relative percentage of ethers and monoterpenes;the sum of relative percentages of methyl eugenol,safrole,3,5-dimethoxytoluene,3,4,5-trimethoxytoluene and myristicin;the sum of relative percentage content of methyl eugenol and safrole;the relative percentage content ratio of methyleugenol and safrole.Finding the’safe and effective’volatile oil components of Asarum provides the target components for subsequent attenuation experiments.The results showed that the toxicity was significantly correlated with the evaluation indexes of six components（P<0.05）.The efficacy of AEO was positively correlated with the content of monoterpenes and methyl eugenol,and the toxicity was positively correlated with the content of ether components safrole and methyl eugenol.Therefore,in order to take into account the toxicity and pharmacological activity of AEO and avoid the phenomenon of toxicity reduction,the ratio of ethers to monoterpenes was selected as the quality evaluation index of AEO.The ratio decreases,that is,the toxicity decreases,and the ideal ratio range is 1.61～3.79.6.Study on aging law and mechanism of AEOThe methods of natural aging,physical aging and processing aging were used to study the attenuation of AEO.GC-MS was used to detect the components.Electronic nose,polarimeter and refractometer were used to determine the aroma,rotation and refraction of AEO.The effects of natural aging on the physical and chemical properties of AEO:The extracted AEO was placed in a shady cabinet（2-8℃）and stored in a refrigerator at-20℃ on days 0,30,60,90,180 and 360.GC-MS component analysis showed that the relative content of each component in the natural aging process of AEO did not change significantly,while the absolute content decreased.The acute toxicity experiment of zebrafish showed that the LD₅₀of AEO was 0.195μL/mL after 360 days of aging,and the toxicity was reduced compared with the LD₅₀of AEO on the day of extraction（0 day,0.174μL/mL）.The results of acute toxicity test in mice showed that there was no significant difference in LD₅₀of volatile oil between 360 days of aging and the same day of extraction.The effect of physical aging on the physical and chemical properties of AEO:The aging of AEO was intervened by infrared light,ultraviolet light,incandescent light,high temperature and microwave.The physical and chemical properties of volatile oil under different aging conditions were detected.The effect of aging on the physical and chemical properties of AEO was discussed,and the aging process to achieve’toxicity reduction and efficiency enhancement’was found.The results showed that microwave aging had an effect on the physical and chemical properties of AEO.After 900 W airtight aging,the monoterpene components of AEO decreased,while the ether components increased.The ratio of ethers and monoterpenes in AEO increased after microwave aging,which was opposite to the target trend.The density and refractive index also change with the composition.The physicochemical properties of AEO did not change significantly under UV,high temperature and incandescent light conditions.Under the irradiation of infrared light,two new substances,β-ocimene and sabinene,were generated.At the same time,the composition and structure of AEO were changed fromL-α-pinene to R-α-pinene,2-methyl-5-（1-methylethyl）-1,3-cyclohexadiene to 1-methyl-4-（1-methylethyl）-1,4-cyclohexadiene,and 1,8-terpinene to 1-methylene-4-（1-methylvinyl）cyclohexane.The effects of processing and aging on the physical and chemical properties of AEO:The traditional processing methods such as salt processing,vinegar processing,honey processing,alkali processing,alkali vinegar processing,honey processing,stir-frying and microwave processing were used to process AEO.The volatile oil of AEO was extracted,the physical and chemical properties were determined,the differences of components were compared,and the feasibility of processing and attenuation was explored.The results showed that the processing would affect the physical and chemical properties of the volatile oil.The ratio of ethers to monoterpenes in volatile oil extracted directly without processing was 5.66,and various processing techniques could reduce the ratio,indicating that the toxicity was reduced.Among them,wine stir-frying（3.37）and salt stir-frying（4.03）can significantly reduce the toxicity of AEO.The effects these two processing techniques on the changes of AEO components were studied,and the feasibility of using them as a detoxification method was investigated.It lays an experimental foundation for the safe and effective use and development and utilization of AEO.To further prove the significance and value of Chinese medicine processing,it is speculated that the ancients’use of Asarum processing to achieve attenuation mechanism may be related to the reduction of’ether-alkene ratio’.In summary,the AEO safety evaluation standard with’ether/olefin ratio’as the index was established by studying the correlation between volatile oil components of Asarum and toxicity/efficacy.To explore the feasibility analysis of aging method on’attenuation and preservation effect’of the AEO,to clarify the mechanism of aging and attenuation,and to provide new ideas for the attenuation of toxic aromatic Chinese medicine.