Studies On Separation, Purification And Determination Of Insecticidal Active Component Angulatin A

The main insecticidal component of botanical pesticide 1% Celangulin EC having been placed in the market is angulatin A, the content of which is required higher than 0.4% in the enterprise standard. The determination method of angulatin A has not been perfect and reported in the literature, so it is important to establish a method for the determination of angulatin A in order to facilitate the enterprise to control the quality of Celangulin EC. Whereas angulatin A standard sample is unsold in the market, in this thesis the standard sample of angulatin A was firstly prepared, and the determination method of angulatin A was mainly studied.1. The preparation of angulatin A standard sample(1) In the experiment, the root bark of Celastrus angulatus from Nanyang County in Henan was extracted with 95% ethanol at 90℃, the ethanol extract was filtered and condensed until no ethanol taste, defatted with petroleum ether and extracted with ether after being dispersed with water, the ether extract was extracted with 5% Na₂CO₃ solution to remove tannins and other acidic substances, washed with distilled water to be litmusless, decolored with active carbon, and celangulin crude was obtained; (2) Celangulin crude was gradiently eluted using atmospheric silica gel column chromatography and detected by thin layer chromatography, eluate containing angulatin A was combined and angulatin A crude was obtained; (3) Angulatin A crude was further purified by preparative high performance liquid chromatography, identified by ¹³CNMR, and determined by reverse-phase high performance liquid chromatography, the result of which was that the purity of angulatin A product was 98.50%. This product met the quality requirements of standard materials, so can be used as a reference substance.2. Determination of angulatin AThe operating conditions of RP-HPLC were repeatedly explored and determined: methanol-water (70/30, V/V) at a flow-rate of 1.0 mL/min was chosen as mobile phase with UV detection at 232 nm, the sample volume was 10μL and the temperature was 25℃. Results of experiments indicated that in above conditions, the relationship between peak areas and concentrations of angulatin A in the range from 0.985 to 22.7μg/mL; The equipment had good precision, and the method had good stability and repeatability, the relative standard deviation of standard sample recoveries was small, so this method can be used for determinating angulatin A.With angulatin A having the purity of 98.50% as a reference substance, contents of angulatin A in 1% Celangulin EC, 4% Celangulin EC and 8% Celangulin mother liquor were determined using RP-HPLC; Results showed that the content of angulatin A was higher than 40% of celangulin content in each celangulin product, which met the requirements of industrial production.Effects of petroleum ether, benzene, chloroform, ethyl acetate, 95% ethanol and methanol to extraction rate of angulatin A were also discussed. Results indicated that methanol and 95% ethanol were fine distilling solvents. With methanol and 95% ethanol as solvents, contents of angulatin A in root bark of Celastrus angulatus from two producing areas were determined. Results showed that the content of angulatin A in root bark of Celastrus angulatus from Yunxian County in Hubei was higher than that from Nanyang County in Henan, which provided theoretical basis for the collection of raw materials.Determination results of angulatin A in Celangulin EC and Celastrus angulatus showed that RP-HPLC brought forward in this thesis was simple, rapid, reproducible, and provided a reliable detection method for in-depth study and industrial production.