ABSTRACT
The present work is to establish an HPLC characteristic chromatograms of Asarum heterotropoides var. mandshuricum(AH) and A. sieboldii(AS), combined with cluster analysis for the identification of the two species, and predict their potential anti-inflammatory related targets by network pharmacological method. Eighty-nine samples(12 batches of AS and 77 batches of AH) were analyzed, and 11 characteristic peaks were identified by reference substances, UV spectrum and LC-MS. Cluster analysis showed that AS and AH were divided into two groups, and the ratio of characteristic peak areas can be used to distinguish them. When the ratio of characteristic peak sarisan to kakuol was greater than 5, it was AS, and when the ratio was less than 2, it was AH. The network pharmacological analysis of 119 constituents of Asari Radix et Rhizoma suggested that the anti-inflammatory effect of Asari Radix et Rhizoma might be related to COX-2, COX-1, iNOS, MAPK14, NR3 C1, PPARG and TNF. Among them, COX-2 is a relatively key target, which interacted with the characteristic constituents, asarinin, sesamin, safrole, methyleugenol and sarisan. The characteristic constituents asarinin and sesamin also interacted with the iNOS and MAPK14. Safrole and sarisan can also interact with iNOS, COX-1 and LAT4 H. Methyleugenol also showed interaction with COX-1 and LAT4 H. Since asarinin and sesamin interacted with three targets, COX-2, iNOS and MAPK14, it implied that they were the main active constituents for the anti-inflammatory activity of Asari Radix et Rhizoma. The COX-2 inhibitory activities of asarinin and sesamin were further studied by molecular docking and bioassay. The HPLC method established was simple, feasible and reliable, with predicted anti-inflammatory targets and anti-inflammatory constituents, which could provide a reference for improving the quality evaluation system of Asari Radix et Rhizoma.
Subject(s)
Anti-Inflammatory Agents/isolation & purification , Asarum/chemistry , Chromatography, High Pressure Liquid , Molecular Docking Simulation , Phytochemicals/isolation & purification , Rhizome/chemistryABSTRACT
A headspace-solid phase microextraction-gas chromatography-mass spectrometry method（HS-SPME-GC-MS） was adopted for the quantitative study of 4-allylanisole, methyl eugenol, 2,3,5-trimethoxytoluene, 3,4,5-trimethoxytoluene, sarisan, 3,5-dimethoxytoluene and safrole in mice brain, liver tissues and blood after intragastric administration of Asari Radix et Rhizoma. A VF-WAXms (30 m×0.25 mm, 0.25 μm film thickness) capillary column and SPME fiber coated with 65 μm polydimethylsiloxane/divinylbenzene (PDMS/DVB) were used. The calibration curves of seven volatile constituents were established to validate the method's stability (RSD<15%), repeatability (RSD<9.5%), accuracy (RSD<22%), relative recovery (87.0%-108%) and extraction recovery (74.9%-102%). The validated HS-SPME-GC-MS assay was applied to determine the concentrations of seven constituents in liver, brain and blood. The detected contents were 0.22,0.14 μg•g⁻¹,0.25 mg•L⁻¹ (4-allylanisole), 1.1, 0.39 μg•g⁻¹, 0.69 mg•L⁻¹ (methyl eugenol), 0.45, 0.13 μg•g⁻¹, 0.54 mg•L⁻¹ (2,3,5-trimethoxytoluene), 0.51, 0.15 μg•g⁻¹, 0.45 mg•L⁻¹ (3,4,5-trimethoxytoluene), 0.48, 0.039 μg•g⁻¹, 0.69 mg•L ⁻¹ (sarisan), 2.2, 1.2 μg•g⁻¹, 1.5 mg•L⁻¹ (3,5-dimethoxytoluene) and 1.3, 0.67 μg•g⁻¹, 1.1 mg•L⁻¹ (safrole) respectively. This HS-SPME-GC-MS method is rapid and convenient, with a small sample size, and applicable for the analysis and determination of volatile constituents in traditional Chinese medicines, which provides scientific data for further studies on effective substances and toxic substances in Asari Radix et Rhizoma.
ABSTRACT
To develop an analytic method for qualitative and quantitative analysis of dodecatetraenamides A, B in 42 samples of two official species of Asari Radix et Rhizoma( ARR) (37 samples of Asarum heterotropoides var. mandshuricum with different collection time and 5 samples of Asarum sieboldiivar. seoulense). The HPLC-IT-TOF-MS/MS methods for the qualitative and UPLC-PDA methods for the quantitative analysis were established. Dodecatetraenamides A, B were identified by comparing the retention time, UV absorption spectrum and quasi-molecular ion peak [ M + H]+ with the reference compound using HPLC-IT-TOF-MS/MS. The content of dodecatetraenamides A and B in ARR were determined by UPLC-PDA. The separation was successfully carried out on a ACQUITY UPLC BEH C18 (2.1 mm x 100 mm, 1.7 µm) column eluted with mobile phases of water (A) and acetonitrile (B) in gradient program (0-3 min, 35% B; 3-5 min, 35%-36% B; 5-6 min, 36%-43% B; 6 min-11 min 43% B; 11-12 min, 43%-100% B). The column temperature was 45 °C, and the detection wavelength was set at 254 nm. The flow rate was 0.6 mL · min(-1). On one level mass spectrometry scanning, the results showed that the quasi-molecular ion [M + H] + of both dodecatetraenamides A and B were m/z 248.20. The quantitative method with UPLC-PDA has made the baseline separation of the constituents, which were reported as mixtures in the most literatures. The average recovery of dodecatetraenamides A and B were 97.90% and 99.86%, the relative standard deviation were 0.4% and 1.1%, respectively. The contents of dodecatetraenamides A, B in all ARR samples was in the range of 0.11-3.89 and 0.24-6.65 mg · g(-1). Their contents reduced with the extension of storage time. Compared with the samples of 2013, the average content of the two constituents in the samples collected in year 2002-2003 reduced 34% and 36%, respectively (P < 0.05). Compared the A. sieboldii var. seoulense and A. heterotropoides var. mandshuricum with the same collective time and production area, the average contents of the two constituents in latter were up to (1.59 ± 0.75) mg · g(-1) and (2.90 ± 1.17) mg · g(-1), respectively, significantly higher than that in A. sieboldii var. seoulense (dodecatetraenamide A were (0.78 ± 0.52) mg · g(-1), dodecatetraenamide B were (1.69 ± 0.83) mg · g(-1)) (P < 0.05). The content of the dodecatetraenamide A in overground part was in the range of 0.11-0.33 mg · g(-1), dodecatetraenamide B was 0. 24-0.60 mg · g(-1), which were much lower than that of the underground part of ARR (dodecatetraenamide A was in the range of 0.73-3.89 mg · g(-1), dodecatetraenamide B was 2.11-6.24 mg · g(-1)). The method was certified to be simple, accurate and reliable and could be used for qualitative and quantitative analysis of dodecatetraenamide A and B in different species of ARR, also can be used for the comprehensive quality control of traditional Chinese medicine, Asari Radix et Rhizoma.