| Objective:In order to explore the differences of Acorus calamus L.medicinal materials in different habitats and to provide a theoretical basis for the development and utilization of Acorus calamus L.resources in different origins and clinical rational drug use,this paper conducted a series of studies from the optimization of extraction process,component analysis,bacteriostatic activity,antioxidant activity and antitumor activity of Acorus calamus L..Method:1.Taking the medicinal materials of calamus from the production area of Nyingchi as the research object,the essential oil was extracted by steam distillation,and on the basis of the single-factor experiment,3 factors of immersion time,extraction time and solvent multiple were selected as independent variables,and the essential oil yield was taken as the dependent variable,and the Box-Behnken response surface method was used to optimize the extraction process of Acorus calamus L.essential oil.The components of essential oil were analyzed and studied by gas chromatography-quadrupole time-of-flight mass spectrometry(GC/Q-TOF MS),and the composition was analyzed and studied through NIST 11.L.The standard spectrum library was compared and combined with the literature for compound identification,and the relative percentage content of each component was calculated by peak area normalization.2.By using headspace-solid phase microextraction-gas chromatography-mass spectrometry(HS-SPME-GC-MS)the volatile compounds were determined for 8 different habitats of Acorus calamus L.in 5 provinces of Tibet,Sichuan,Hubei,3.Jiangxi and Anhui.And orthogonal partial quadratic multiplication-square discriminant analysis(OPLS-DA)was used to analyze the common and different components for explore the influence of different environments on the composition of volatile components.3、Three gram-positive bacteria and four gram-negative bacteria were used as test strains.The antibacterial activity of essential oil from 3 different areas in Tibet was analyzed by filter paper method,to investigate the antibacterial ability of essential oil from different habitats.The antioxidant activity of essential oil from 3 different habitats was investigated by the scavenging ability of DPPH radicals and hydroxyl radicals.4、CCK-8 method was used to analysis the effects of essential oil and ethanol extract from different habitats and concentrations on the viability of breast cancer MDA-MB-231 cells;Flow cytometry was used to determine the DNA cycle and apoptosis rate of each cell;Transwell chamber assays on the effect of cell migration;Western Blot measures mitochondrial pathway apoptotic protein expression to explore possible mechanisms and pathways for apoptosis in breast cancer cells.Result:1、After optimization by the response surface method,the optimal extraction process was determined to be as follows:soaking time was 2.4 h,extraction time was 6.7 h,and solvent multiple was 10.6 times.Under these conditions,the measured yield of essential oils in three replicates was 1.476%,and the RSD was 0.4%,which was close to the predicted value of 1.482%and the error was 0.41%.A total of 58 compounds were identified from calamus essential oil,mainly terpenoids,and 11 compounds with a relative percentage content of more than 3%.2、A total of 96 volatile components were identified in 8 different habitats,including 47 terpenes,17 alcohols,14 ketones,5 ethers,3 phenylpropanoids,4 olefins and 6 other compounds,and there are 46 components of Lhasa,43 components of Nyingchi,44 components of Qamdo,35 components of Anqing,38 components of Bozhou,38 components of Chengdu,38 components of Yichang,44 components of Yichun,of which 17 components were same to 8 samples.OPLS-DA screened out 34 chemical components with VIP value>1 as the main difference components of different samples.3、The essential oil from 3 different habitats had different degrees of inhibitory effect on the remaining six bacteria except Pseudomonas aeruginosa,and the essential oil from Nyingchi had the strongest inhibitory activity against the six test bacteria.The essential oils of 3 different habitats showed obvious antibacterial effect on Staphylococcus aureus,but the effect on Escherichia coli was not good.Essential oils of different habitats have certain antioxidant activity,and within a certain concentration range,the size of antioxidant capacity is concentration-dependent.The IC50 value of essential oils to remove DPPH radicals from Lhasa,Nyingchi and Qamdo were 118.73,114.91 and 165.01(μL/mL).The IC50 value of essential oils to scavenge hydroxyl radicals from Lhasa,Nyingchi and Qamdo were 179.59,175.10 and 216.00(μL/mL).4、The essential oil from 3 different habitats could reduce the viability of MDA-MB-231 cells in time and concentration-dependent.The ethanol extract of calamus from Lhasa and Nyingchi had a stronger inhibitory effect on cell proliferation than that from Qamdo,among which the ethanol extract from Nyingchi was the strongest,and its strength was positively correlated with the treatment time and ethanol extract concentration.The results of Transwell experiments showed that both essential oil and ethanol extract of Linzhi calamus significantly reduced the number of migrating MDA-MB-231 cells,indicating that the migration ability of MDA-MB-231 cells was inhibited.Essential oil from Nyingchi significantly reduced the ratio of G1 phase cells and significantly increased the proportion of S phase cells,resulting in S-phase arrest of MDA-MB-231 cells,which in turn inhibited cell proliferation.After the ethanol extract of Calamus calamus from Nyingchi was applied to MDA-MB-231 cells,the cell ratio of G1 phase decreased and the cell ratio of G2 phase increased,but there was no significant change in S phase.Both could significantly induce apoptosis of MDA-MB-231 cells to varying degrees,and both were dominated by early apoptosis.Both significantly upregulated the expression level of pro-apoptotic protein Bax,while significantly inhibiting the expression level of anti-apoptotic protein Bcl-2,the ratio of Bax to Bcl-2(Bax/Bcl-2)was significantly upregulated,and the expression level of Caspase-9 was also significantly increased.Conclusion:1、The mathematical model established by the Box-Behnken response surface method has good predictability,and the selected process conditions are stable and reproducible.The 3 factors had significant effects on the yield of essential oils,and the interaction between soaking time and extraction time,extraction time and solvent multiple was obvious.GC/Q-TOF-MS can perform rapid analysis of essential oil components,which has high sensitivity,good accuracy and simple operation,and is suitable for the analysis of Acorus calamus L.essential oil.2、Using HS-SPME-GC-MS combination technology,the rapid detection and visual analysis of volatile components of calamus medicinal materials are realized.There were great differences in the composition and content of volatile components of Acorus calamus L.in 8 different habitats,and there were 17 kinds of components in each area,but there were obvious differences in content,indicating that the resource quality of Calamus was uneven,affected by geographical factors such as soil,water and light.The 3 habitats of Tibet are rich in types and contents,which may be due to their growth in the unique plateau extreme environment,and the rich and diverse secondary metabolites,which can be in-depth research in future scientific research and production.This study provides basic research data for the analysis of volatile substances of calamus from different origins,and can provide a theoretical basis for the identification and quality evaluation of calamus medicinal materials from different origins.3、The essential oil of Acorns calamus L.from Nyingchi are superior to other habitats in terms of bacteriostatic and antioxidant capacity.There are differences in bacteriostatic and antioxidant activity of essential oils from different origins,which may be due to differences in growth environment,storage conditions and harvest season,which make them different in essential oil content and composition.Essential oils from Nyingchi habitat can be further studied in depth to provide a theoretical reference for the development of new bacteriostatic agents and antioxidants.4、The essential oil and ethanol extract of Acorus calamus L.can inhibit the proliferation and migration of MDA-MB-231 cells to varying degrees,block the cell cycle,and induce apoptosis of MDA-MB-231 cells by activating endogenous mitochondria-mediated apoptosis pathway. |
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