Quality Evaluation Of Acorus Calamus And Research On Transmembrane Transport Component

Purpose:To analyze the quality of Acorus calamus from different markets,determine the main index components and analyze the content of the main components,and provide support for the quality evaluation.To establish the determination method of Acorus acorus and used in the analysis of Rhizoma anemones altaicae.The transmembrane components of Acorus calamus that can be absorbed into the brain via olfactory nerve through nasal mucosa were screened by Calu-3 monolayer cell membrane to provide experimental data for the pharmacodynamic material basis of aromatic drugs of Acorus acorus delivered through nose.Materials and Methods:1.The mechanism of Acorus calamus and Poria in the treatment of Alzheimer’s disease was analyzed through network pharmacology.2.To evaluate the quality of Acorus calamus and local customs Rhizoma anemones altaicae,through the analysis of character,identification,extract,heavy metal,pesticide residue,fingerprint and content determination,to comprehensively evaluate the quality of Acorus calamus from different markets,and to provide data reference for its quality control.3.The 12-well Transwell culture plate was used to establish the Calu-3single-layer cell membrane model.The safe concentration of Acorus calamus was screened by MTT method.The integrity and permeability of monolayers were determined by transmembrane analysis with sodium fluorescein and immunofluorescence staining with TJ connexin.The transmembrane components of Acorus calamus were qualitatively analyzed by GC-MS to determine the components passing through Calu-3 monolayer cell membrane.Results:1.According to network pharmacology,19 compounds were identified in Acorus calamus and poria mainly acting on AD,and the main information pathways were PI3K-Akt and Ca²⁺,and there were 7 potential gene targets for jointly acting on AD disease.2.the characters of medicinal materials and microscopic results showed,there were significant differences between the two varieties of Acorus acorus and Rhizoma anemones altaicae.Acorus acorus has the characteristics of air aroma,hard quality and fibrous cross-section,while Rhizoma anemones altaicae has the characteristics of light air,easy to break,powder cross-section,etc.The results of thin layer identification showed that there wereβ-asarin spots in R_f 0.5 of Acorus acorus and Rhizoma anemones altaicae,but there were no characteristic spots in Rhizoma anemones altaicae.According to the relevant provisions of the 2020 edition of Chinese Pharmacopoeia and Shaanxi Provincial Standards for Traditional Chinese Medicine Decoction Pieces,the water content,total ash content and extract of the two medicinal materials were tested,and all of them were in line with the relevant provisions.It was found that the content of 5 kinds of heavy metals in medicinal materials from different producing areas had significant difference.The harmful elements such as cadmium and arsenic in Acorus mongolicum produced in a Hubei province sample、Zhejiang sample were relatively low and the quality was relatively good.The pesticide residues of 12 batches of medicinal materials were measured in the range of 3.72E^-04,with very low pesticide residue content.The GC fingerprints of Acorus acorus and Rhizoma anemones altaicae were analyzed,and it was found that there were 4 common peaks in 12 batches.Through stoichiometric statistical analysis,12 batches of samples were found to be divided into two categories,including Rhizoma anemones altaicae one category and Acorus acorus the other,Acorus acorus can be divided into three subgroups.The extraction methods and chromatographic optimization were investigated.It was found that Agela DA-5 was the best capillary column,andα-naphthol was the internal standard substance.25 ml ethanol was used as the solvent with heat reflux once as the best sample pretreatment conditions.The content ofα-asarum andβ-asarum was higher in an Anhui province sample,followed by a Hubei Province sample.The content of eugenol methyl ether and isoeugenol was the highest in a Hubei province sample,a small amount in an Anhui province,but not detected in Rhizoma anemones altaicae.3.Investigate the transport of Acorus acorus components in the cell monolayers,and determine the concentration range of cytotoxicity of Acorus tatarinowii samples by MTT method.It was found that the safe dosage of samples obtained by different extraction methods was different,The mass concentrations of volatile oil from steam distillation and supercritical CO₂ extraction were below 20 ug/ml,themassconcentration of lyophilized powder from deoiling water was below 6mg/ml,and the mass concentration of lyophilized powder from alcohol extraction was below2mg/ml,all of which had no toxic effect on cells.The integrity and permeability of monolayer membrane were investigated.The Papp of sodium fluorescein was less than 5×10^-7 cm/s.The tight junction protein fluorescence staining showed significant difference between the experimental group and the control group.It was found that theβ-asarine could pass through Calu-3 cell monolayers.Conclusion:1.To explore the mechanism of action of Acorus tatarinowii-Poria cocos in the treatment of AD disease by using network pharmacological method,It provides information and knowledge support for the study of material basis and mechanism of action of Acorus acorus in the treatment of AD,and also provides new research data for the treatment of AD based on the theory of"brain-gut axis".2.Quality evaluation and analysis were conducted from the aspects of characters,identification,heavy metals and harmful substances of medicinal materials,and it was found that the test results were in line with the relevant provisions of the 2022 edition of Chinese Pharmacopoeia.The results of thin layer identification indicated that the quality of Acorus tatarinowii were good.The pesticide residue content and heavy metal content of medicinal materials from some producing areas were in line with the limits stipulated in the Green Industry Standard for Foreign Trade of Medicinal Plants and Preparations（WM/T2-2004）,suggesting that attention should be paid to the monitoring of heavy metal and pesticide residues in medicinal materials.The subsequent studies of Acorus tatarinowii and Rhizoma anemones altaicae should not only focus on the main components of volatile oil,but also focus on the trace elements.The content of asarum in Acorus calamus was high,and the quality of two batches of Acorus calamus produced in Hubei was the best.3.After establishing the study model of transmembrane transport and absorption of Acorus calamus components by Calu-3 cell monolayers,the established Calu-3 cell monolayers can be used to simulate the absorption and transport of Acorus calamus components by nasal administration.The cytotoxicity of acorus calamus samples to Calu-3 cells was investigated to determine the safe administration dose of the cell model.Under gas-liquid conditions,the established monolayer cell membrane has certain integrity and permeability,which can be used for subsequent transmembrane transport research.To investigate the transport of Acorus calamus in the monolayers of cells and confirm that asarum can be transported through the membrane,indicating thatβ-asarine,the main component of Acorus calamus,could be absorbed through nasal mucosa,which might be the main active component of its aroma induction.