| Retained placenta is a common the reproduction disorder of dairy cows,which seriously affects the health of the cows and the economic benefits of the dairy farmers.Semen plantaginis by stir-frying with wine as a traditional folk Chinese medicine proved recipe cure retained placenta of cows,it has a remarkable clinical effect.The separation system of active ingredients of Semen plantaginis has been established,and active components III and VI has been also determined in our laboratory,but the chemical constituents of active components have not been reported and be worth further studying.Therefore,the purpose of current study is to analyze the chemical components of the active components that semen plantaginis by stir-frying with wine in the treatment of dairy cows with retained placenta.Experiment 1: The active components III and VI were separated according to the established separation system in our laboratory,and ODS reverse-phase column with a diameter-to-height ratio of 1:10 was used,the methanol-water was used as the mobile phase to perform purification by gradient elution on its active components III and VI,and then to compute separation and purification rate.Experiment 2: Fifteen cows with retained placenta were randomly divided into negative control group(physiological saline),active components III group(purified active components III)and active components VI group(purified active components VI).The expulsion time and cure rate of placentae were recorded and counted during the 48 hours after administration.Experiment 3: Chromatograpic separation was performed,Hyper gold C18 column(100 x 2.1 mm;1.9 μm)was used as chromatographic column,acetonitrile(containing 0.1% formic acid)-water(containing 0.1% formic acid,5% acetonitrile)was used as mobile phase for gradient elution,the flow rate was 0.3 mL/min,the column temperature was 40 °C.The ESI ion source of LC-MS / MS was used to collect the primary and secondary mass spectra data of active components III and VI in the positive-ion and negative-ion modes.The results of Experiment 1 showed that the recovery rate of separation of active component III was 28.00%,and the recovery rate of purification was 88.65% under the eluent of methanol(70%)-water(30%);the recovery rate of separation of active component VI was 25.02%,and the recovery rate of purification was 72.88% under the eluent of methanol(60%)-water(40%).The results of Experiment 2 indicated that the average expulsion time of placentae was 11.90 h±3.92 h,and the cure rate was 60% in active III group,while the average expulsion time of placentae was 15.67 h±9.27 h,and cure rate was 80% in active VI group.But in the negative control groups,the cure rate was 0%.The results of Experiment 3 suggested that according to the accurate charge-mass ratio and the MS /MS data,Metlin,HMDB,MassBank and PubChem databases,the fragmentation regularities of mass spectra.In positive-ion and negative-ion modes,the thirty-eight chemical components were found in active components III,mainly including lipids,flavonoids,amine,organoheterocyclic compound,vitamins,iridoides and organic acid etc.The thirty-six chemical components were found in active components VI,mainly including lipids,flavonoids,organoheterocyclic compound,phenols and terpenes etc.The above results suggested that 38 compounds in active ingredient III and 36 compounds in active ingredient VI analyzed and identified by LC-MS /MS technology are the active ingredients of semen plantaginis by stir-frying with wine on cure of retained placenta in dairy cows,and this is the theory base for the research and development of traditional Chinese medicine of preventing and controlling the retained placenta in the dairy cows. |
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