Cell membrane chromatography for screening active ingredients of analgesic Chinese medicine
| The invention provides a cell membrane chromatography method that can simultaneously screen analgesic traditional Chinese medicine active ingredients and determine its bound opioid receptor subtypes. The method includes the following steps: establishment of fingerprints of alcohol extracts of baijiu; primary cells of vas deferens smooth muscle cells Cultivation and subculture; binding and dissociation of effector components; high-performance liquid chromatography to determine changes in the spectrum before and after binding. By making Chinese medicine act on vas deferens smooth muscle cells of different species, examining the behavior of the dissociated solution on a high-performance liquid chromatography column, and using opioid receptor agonists and opioid receptor antagonists as a reference, the active ingredients of the traditional Chinese medicine can be examined Affinity to a certain opioid receptor subtype. Using the opioid receptor on the vas deferens as a screening model for the key link of analgesics, in addition to the active ingredient screening of Chinese medicines with analgesic effects, it can also directly determine that the active ingredient has affinity with a certain opioid receptor subtype , Has a good application prospect. |
| Cell membrane chromatography for screening active ingredients of analgesic Chinese medicine |
A cell membrane chromatography method for screening active ingredients of analgesic Chinese medicines, which is characterized by the following steps: (1) establishment of fingerprints of alcohol extracts of Paeonia lactiflora: Weigh 20 g of medicinal materials of Paeonia lactiflora, add 160ml of 95% ethanol, and extract by refluxing for 1-2 hours. Dry ethanol, dissolve the residue in methanol to make up to 20ml, take 250μl of it and dilute to 7.5ml with methanol, pass through a 0.45μm microporous filter membrane, and obtain the filtrate as the alcohol extract of Baijiu; take 20μl of the filtrate and use high performance liquid chromatography The method was used to determine the fingerprint of the active ingredient group of baijiu alcohol extract and determine the 13 common fingerprint peaks. The analysis conditions of the high performance liquid chromatography were: methanol-water as mobile phase system, column temperature 30 ° C, injection volume 20μl, Lichroscher 5-C18 column with a wavelength of 254 nm, a length of 250 mm, and a diameter of 4.6 mm, mobile phase gradient: 0 to 5 min, 5% to 20% methanol; 5 to 15min, 20% methanol; 15 to 20min, 20% to 70 % Methanol; 20 to 30 min, 70% methanol; 30 to 40 min, 70 to 90% methanol; all of the above are volume percentages; the 13 common fingerprint peaks of the fingerprint of the baijiu alcohol extract are the relative retention times are: 1 Peak No .: 0.031 to 0.034; Peak No. 2: 0.086 to 0.088; Peak No. 3: 0.607 to 0.649; Peak No. 4: 0.658 to 0.665; Peak No. 5 : 0.704 to 0.708; Peak 6: 0.730 to 0.739; Peak 7: 0.749 to 0.755; Peak 8: 0.810 to 0.816; Peak 9: 0 .842 ~ 0.847; Peak No. 10: 0.856 to 0.8861; Peak No. 11: 1; Peak No. 12: 1.085 to 1.087; Peak No. 13: 1.181 to 1.186; each The relative peak area ranges of the common fingerprint peaks are: Peak No. 1: 0.05 to 0.061; Peak No. 2: 0.01 ～ 0.132; Peak No. 3: 0.0050 to 0.366; Peak No. 4: 0.0010 to 0.135; Peak No. 5: 0.03 to 0.148; Peak No. 6: 0.0006 to 0 .153; Peak No. 7: 0.086 to 0.328; Peak No. 8: 0.047 to 0.105; Peak No. 9: 0.0018 to 0.059; Peak No. 10: 0.030 to 0.113. Peak No. 11: 1; Peak No. 12: 0.251 to 0.492; Peak No. 13: 0.304 to 0.845; all above are based on peak No. 11; (2) Primary culture of vas deferens smooth muscle cells And subculture: take the vas deferens of adult male mice or adult male rabbits, strip the inner and outer membrane tissues, and culture the cells in culture flasks by enzymatic digestion; when the cells grow to cover about 80% of the bottom area of the flasks, passaging; (3 ) Binding and dissociation of effect components: cell suspension obtained by digesting cells passaged from 3 to 10 generations and alcohol extraction from paeony obtained in step (1) Mix in equal volumes, incubate at 36 to 38 ° C for 0.5 to 24 hours, centrifuge to remove the supernatant, wash the cells with DM medium for 3 to 5 times to obtain a uniform suspension, centrifuge, and discard the supernatant to obtain pelleted cells; Add PBS of pH 2 to 6 to precipitate the cells to denature the cells. Centrifuge, collect the supernatant and dry it to obtain dissociates. (4) High-performance liquid chromatography to determine the spectrum changes before and after binding: dissociate the results obtained in step (3). The product was dissolved in methanol and analyzed by high-performance liquid chromatography. The conditions of the high-performance liquid chromatography analysis were the same as those of step (1). By comparing the fingerprints of the dissociated product and the fingerprint of the alcohol extract, the peaks of the absorption peaks were compared. Changes, and opioid receptor antagonists and opioid receptor agonists were used as references to determine whether the peony binds to opioid receptors; the characteristic peaks that specifically bind to opioid receptors are peaks 7, 8 and 10 ; The active ingredients of Paeonia lactiflora screened are peaks 7, 8 and 10; the opioid receptor antagonist is naloxone hydrochloride, and the opioid receptor agonist is a salt Pethidine.