1.Thin layer identification method
Take 0.5g of epimedium medicinal herbs powder or an appropriate amount of extract, add 10ml of ethanol, immerse for 30 minutes, filter, and evaporate the filtrate. Add 1ml of ethanol to dissolve the residue, which is the test solution. The icariin reference substance was weighed, dissolved in methanol and diluted to 0.1 mg/ml, which was the reference solution. Pipette 10 μl of each of the test solution and the reference solution, respectively, on a silica gel H thin layer plate of the same sodium carboxymethyl cellulose as a binder, with ethyl acetate-butanone-formic acid-water (10:1: 1:1) as a developing agent, unrolled, taken out, air-dried, and placed in a UV lamp (365 nm) for examination. In the chromatogram of the test sample, the same dark red spot appears on the position corresponding to the chromatogram of the reference substance; sprayed with trichlorination The aluminum test solution was re-applied with a UV lamp (365 nm) to reveal the same orange-red fluorescent spot.
2. Spectrophotometric determination of total flavonoids in Epimedium and its extracts
The reference product icariin was accurately weighed and formulated into a standard solution of 10 ug/ml with methanol. Weigh accurately the amount of this product powder (equivalent to about 1mg of icariin), sonicate with methanol 25ml for 30 minutes, dilute to a 50ml volumetric flask, filter with 0.45μm microporous membrane, that is to test Product solution. The test solution and the reference solution were taken separately, and the absorbance was measured at a wavelength of 270 nm with the reagent as a blank, and the calculation was obtained.
3. Determination of icariin in Epimedium and its extract by HPLC
Chromatographic conditions and system suitability test: ODS2 5u C18 (4mm × 150mm) column; mobile phase: acetonitrile-water (30:70); detection wavelength: 270nm; flow rate: 0.8ml / min. The number of theoretical plates is not less than 1500 based on icariin.
Preparation of the reference solution: The reference product icariin was accurately weighed, and a solution of 0.1 mg/ml was prepared with methanol.
Preparation of the test solution: accurately weigh the appropriate amount of this product powder (equivalent to about 1mg of icariin), sonicate with methanol 25ml for 30 minutes, dilute to 50ml volumetric flask, use 0.45μm microporous filter Membrane filtration is the test solution.
Assay method: 10 μl of each sample solution and reference solution were injected into the HPLC instrument, and the area was calculated by external standard method.