Research On The Anti-Asthmatic Ingredients And Mechanisms Of Herba Ephedrae & Pheretima Drug Partnership

To research on the anti-asthmatic ingredients and mechanisms of the combination of Herbal Ephedrae & Pheretima, firstly, we evaluated the dosage ratio and anti-asthmatic property. Methods: To observe the relaxant effect of E1L1,E1L3,E1L9(the dosage ratio between Ephedra and Pheretima is 1:1, 1:3, 1:9 respectively)on precontracted isolated tracheal rings by 10μmol/L Carbachol (CCh) and 10μmol/L Histamine (His); To study inhibitory effect of tracheal epithelium secretion by observation of the decrease effect on short- circuit current (Isc) increase induced by 10μmol/L CCh on isolated rat tracheal epithelium. Results: E1L3 was the optimized ratio for the anti-asthmatic drug partnership. The drug partnership had the relaxant effect on CCh and His precontracted isolated tracheal rings, and the inhibitory effect on CCh induced (Isc) increase on isolated rat tracheal epitheliums.Secondly, we compared methods of extracting together or separately of the combination, using both fingerprint spectrum and quantification of index chemical components. The fingerprint spectrum indicated that the similarity of whole spectrum was more than 0.9, peak area with less than 0.9, there were 22 common peaks. Spectrophotometric analysis revealed that extract separately produced more amino acids than extract together, however, HPLC analysis revealed that more ephedrine alkaloids were obtained by extracting together.Two routes of exploring drug partnership are developed. One is"integration route", regarding the mixture of the two drugs as a whole, the other is"fraction combination route", extracting individual drug separately, and then the combination of their fractions.As an example of"integration route", we evaluated the different fractions with various polarity separated from the mixture of Herbal Ephedrae & Pheretima (mass ratio1:3), namely, 0%E(water eluant), 50 %E (50% ethanol eluant) and 95% E (95% ethanol eluant ) over macrophorous resin. All fractions showed relaxant effects on CCh and His precontracted isolated tracheal rings, but only water eluant displayed decreasing Isc effects on CCh pretreated epitheliums. It was concluded that the anti-asthmatic constituents had a broad range of polarity, but large polar part was compromised to be the best.As an example of"fraction combination route", firstly,the alkaloid extract from Herbal Ephedrae (M) was obtained with alkaloid content up to 80%. Then the water extract of Pheretima was separated into three fractions of the ethanolic precipitate (D), the alkaline fraction (J) and the acidic fraction (S). S was screened to be the anti-asthmatic fraction in vitro using the aforementioned models. Further in vivo studies also displayed that the acidic fraction could protect experimental asthma model induced by the combination of His and acetylcholine chloride in guinea pigs to prolong the latent periods of asthma (P < 0.05) and significantly decrease the cough frequency caused by ammonia water in mice (P < 0.001). Furthermore, S was separated into two fractions of high polar fraction (S30) and low polar fraction (S95) over large pore resin to find the more active ingredients by observing the anti-inflammation and immune-regulation effects on ovalbumin (OVA)-sensitized BALB/c mice. The result indicated that S inhibited eosinophilia (EOS) secretion in BALF more efficiently than S30 and S95, and for the level of IGE, IL-4, IL-5, IL-13, S30 and S95 did not show better decreasing effect. So S was screened to be the final active fraction.Furthermore, we combined M with D, J and S respectively, then evaluated their anti-asthmatic effects. MS was significantly more effective than MJ and MD in vitro; in vivo, MS displayed better protective effect than M alone against bronchospasm in guinea pigs challenged by mixture spray of His and acetylcholine chloride, and better antitussive effect than S alone in ammonia-induced mice. In conclusion: Combined Herbal Ephedrae and Pheretima was advantages over single one for asthma therapy; MS was the active constituent combination. M and S produced a synergistic anti-asthmatic effect, justifying the traditional usage of Herbal Ephedrae & Pheretima on the management of asthma.More than 80% constituents in the effective fraction of Herbae Ephedrae were determined, including ephedrine and pseudoephedrine; 80% constituents in the effective fraction of Pheretima were identified, specifically, including 16 amino acids, 5 purines and pyrimidines, 4 multi-hydroxy compounds, 17 fatty acids using HPLC, GC-MS and UPLC-MS.`Total 44 chemical ingredients in the combination were identified...