| Croton has already been used for thousands of years in the human history, and it has been recorded by Shen Nong’s Herbal Classic. Croton also has been widely used as a kind of laxative. In recent years, with the continuous development of croton medicinal materials, some researches reported that croton has the effect of anti-tumor, anti-HIV, anti-Mycobacterium tuberculosis and so on. However, the another important feature of croton is. its toxicity which has greatly restricted its clinical application. However, no certain conclusion has arrived at what kind of material does the toxic components of croton belong to.Purpose:Sieve the toxic part of the croton by the guidance of the animal toxicity, then isolate the sieved toxic parts and try to get the specific toxic ingredients of the croton. To establish a developed HPLC method for the determination of toxic components according to the results of the isolation of toxic components and to establish a HPLC fingerprint of toxic parts.Method:1. Measure the LD50of different parts of croton and croton oil by the acute toxicity test in mice respectively to sieve the toxic parts of croton.2. Isolate the sieved toxic parts by normal phase silica gel, reverse phase silica gel, Sephadex LH-20, preparative HPLC, etc. Then, combined with TLC tracking method to identify the structures of the sieved material through extensive analysis of the spectroscopic data including’H-NMR,13C-NMR.3.Take the isolated6phorbol esters as reference and establish the content detection method with six ingredients through the HPLC testing method, then examine it by methodologies. 4. Establish HPLC fingerprint of toxic parts according to the investigation result in different mobile phase, different column temperature, different columns, different flow speed and other conditions. Then choose the best wavelength and mark the calibration of the fingerprint peak according to the isolated phorbol estersResults:1. The sieved toxic parts of croton are mainly centralized in the croton oil, and the toxicity of croton oil are centralized in methanol extraction sites;2. Structures of the isolated eight phorbol ester compounds are identified, namely:4-Deoxy-4α-Phorbol-13-acetate (Number1)12-0-Tiglylphorbol-13-acetate (Number2)12-0-(2-Methyl) butyrylphorbol-13-acetate (Number3)12-0-Tiglylphorbol-13-benzoyl (Number4)4-Deoxy-4α-phorbol-12-tigliate-13-acetate (Number5)12-0-Tiglylphorbol-13-isobutyrate (Number6)12-0-(2-Methyl)butyrylphorbol-13-isobutyrate (Number7)12-0-Tiglylphorbol-13-(2-Methyl)butyrate (Number8)3. The content of esters in the six phorbol of croton oil measured by HPLC method in which formula number1, number2, number4, number5,number6, number8has a good linear relationship (r=l) respectively within the range between0.07955-0.795,0.2205-2.205,0.07955-0.795,0.0565-0.565,0.2125-2.125,0.059-0.590mg· ml-1. The average recoveries are99.83%,98.56%,98.33%,99.52%,98.44%,98.67%respectively.4.33common peaks have been identified in the HPLC fingerprint of toxic parts. The common pattern of the toxic parts has been established. The similarity of the fingerprint of toxic parts of the10batches of croton herbs are above0.85.Conclusion:The foundation of toxic substances of the croton is the phorbol esters substance in the croton oil which is centralized in methanol extraction sites of croton oil and is a type of substance with relatively great polarity. There are dozens of phorbol esters substance in the croton oil.This paper establish the content detection method of the phorbol esters substance for the first time, this method is simple, stable and reliable. And the established fingerprint of the Croton oil toxic substances indicate the basic situation. The method has high stability, good reproducibility, it can be used for the quality control of Croton. |
PDF Full Text Request