Studies On Quality Standards Of Euphorbiae Ebracteolatae Radix And Its Prepared Slices

Euphorbiae Ebracteolatae Radix (Lang Du),a traditional Chinese medicine,is the dried root of Euphoria ebracteolata Hayata or E. fischeriana Steud..But nowdays, in the clinical application and the market, Euphorbiae Ebracteolatae Radix and Stellera chamaejasme L. are often mixed because of the similar character in alias, medicinal part, product properties and the effect. Both Euphorbiae Ebracteolatae Radix and Stellera chamaejasme L. are toxical medicine which can cause serious intoxicating phenomenon, meanwhile the market of Euphorbiae Ebracteolatae Radix is confused. The Chinese pharmacopoeia (Volume 1,2010 version) have only included description, microscopic identification, moisture, total ash,yet lack of special TLC identification and HPLC determination. And the Chinese pharmacopoeia (Volume 1,2010 version) are lake of the quality santandard of its prepared slice.Considering such a mess of clinical medication, we researched the Euphorbiae Ebracteolatae Radix and its prepared slices of quality standard. Characteristic components from Euphorbiae Ebracteolatae Radix are searched to make a distinguish between the fake stamps and original species. After consulting a plenty of references, we find out that terpenes, especially diterpenes were thought to be the mainbioactive compounds in this plant with significant antitumor activity against several tumor lines. The diterpene component Jolkinolide B is the main antitumor ingredient which has a high cotent in these two original plants, so we decided to use Jolkinolide B as reference standard. By column chromatography, half preparation HPLC, we got white powder. Through IR, UV, MS, NMR, the structure is identified as Jolkinolide B.A specific TLC identification method for Euphorbiae Ebracteolatae Radix was established with Jolkinolide B to control the medicine, with cyclohyxane:ethyl-acetate= 8.5-1.5 for mobile phase, with 10% vitriol ethanol solvent as chromoginic reagent, developed at 105℃ until spots are clear. The results shows that 21 samples in 23 Euphorbiae Ebracteolatae Radix samples are real. The results show that the method is specific for identification of Euphorbiae Ebracteolatae Radix.The method of detecting Jolkinolide B in Euphorbiae Ebracteolatae Radix by HPLC-UV is developed. Chromatographic conditions for the detection of Jolkinolide B:chromatographic column:Agilent (5μm,250*4.6mm); mobile phase:Acetonitrile: water=65:35;The column temperature:30℃. There was a good linearity (R2= 0.9998)with the range of 2.125μg/ml～27.2μg/ml. The average recovery was 102.60% with RSD of 1.10%(n= 6). Acording to the experimental result, it is proposed to contain not less than 0.012% of Jolkinolide B calculated with reference to the dried drug in Euphorbiae Ebracteolatae Radix.In the Chinese pharmacopoeia (Volume Ⅰ,2010 version),there is only processing method of Euphorbiae Ebracteolatae Radix prepared slices, yet lack quality standard. The developed TLC identification and determination method of Euphorbiae Ebracteolatae Radix above is also suitable for prepared slices. It is proposed to contain not less than 0.01% of Jolkinolide B calculated with reference to the dried drug according to the experimental results.It is proposed to contain less than 13% of moisture,7.0%total ash,1.0% acid insoluble ash, no less than 20.0% of extraction with Dilute ethanol.Meanwhile, established UPLC fingerprint of Euphorbia ebracteolata Hayata and its prepared slice. Jolkinolide B was used as the internal standard substance. The reversed phase UPLC system consisted of a Waters C18 1.8μm(100mm L×2.1mm ID) column and a mixture of acetonitrile-water as the mobile phase with gradient elution mode at the flow rate of 0.4ml·min-1.The column temperature was 30℃ and the absorbance was monitored at 210nm,238nm,280 nm.1) This method for UPLC fingerprint determination of Euphorbia ebracteolata Hayata indicated 12,7,7 characteristic peaks in each absorbance, and its prepared slice indicated 11,6,6 characteristic peaks in each absorbance.2) In 210nm、238nm、280nm, the range of similarity about Euphorbia ebracteolata Hayata are 0.876～0.983,0.946～0.990 and 0.914～0.994, the prepared slice is more equal than the medicinal materials.3) Compared with Euphorbia ebracteolata Hayata.,the prepared slice has the less peaks and the main peak area are less than the medicinal materials in each determine wavelength,4)The method is simple and accuracy with good reproducibility and can be used as a quality control menthod for Euphorbia ebracteolata Hayata and its prepared slice.