Determination Of Hyperin In Folium Rhododendri Micranthi By RP-HPLC

Folium Rhododendri Micranti is the branches and leaves of Rhododendron Micranthum Turcz.,which belongs to rhododendron section,it mainly has the kinds of flavonoid components,such as hyperin and multi-saponin,tannin and carbohydrate. Folium Rhododendri Micranthi can be mainly used to treat acute or chronic bronchitis, astbma, dysentery, dysmenorrhea and puerperal general pain in traditional Chinese medicine. It also can be used to therapy fracture and sore for external application. But so far,there are not report which related the method to determine the content of hyperin in Folium Rhododendri Micranti. Objective:To establish a method for the assay of hyperin in Folium Rhododendri Micranti in order to provide one guide line for their quality control. Methods:(1)Separate and extract hyperin from Folium Rhododendri Micranti by reflux with ethanol of 90%.(2)Choose appropriate column,different formulation and proportion of mobile phase and adjust optimal flow rate and wavelength in order to separate hyperin well. (3)Preparation of standard curve:prepare a series of the reference solutions,determine peak areas,then the regression equation was obtained with the content of hyperin as abscissa and the relevant peak area as ordinate. (4)Determination of the lowest detection limitation:dilute the reference solution until the value of S/N is more than 3. The relevant concentration is the lowest detection limitation. (5)In the experiment of stability,transfer the sample from Folium Rhododendri Micranti to determine the relevant peak area within 12 hours. (6)In the experiment of precision,take the same test solution,inject to the apparatus for six times,determine the peak areas of hyperin and calculate the concentrations of hyperin according to the same standard curve with the same apparatus in the same day,record the chromatograms and then calculate the precision.(7)In the experiment of reproducibility,prepare one concentration sample of Folium Rhododendri Micranti,repeat each concentration solution for five times in the same way,respectively. (8)In the experiment of recovery,transfer 5 shares of Folium Rhododendri Micranti and add the reference solutions,respectively. Detect the content of hyperin each.(9)Assay:under above-mentioned conditions,determine the content of hyperin in Folium Rhododendri Micranti.Results:(1)Extraction:The method of refluxing with ethanol was stable and quick.(2)The chromatographic procedure was carried out using C18 as an analytical column and a mixture of 45 volume of methanol,55 volume of 5% of phosphoric acid,0.36 volume of triethylamine as a mobile phase at a flow rate of 1.0ml·min-1. The detection wavelength was set at 360 nm. Under the above condition,the peak of the test solution was separated well with the resolution of more than 1.5. Theoretical plate was more than 6000 and the tailing factor for the peak of hyperin was between 1.02～1.14. (3)The regression equation was:Y＝8.7531X－0.0152,r＝0.9999(n＝8),the linear range of hyperin was from 0.05μg to 0.28μg. (4)The lowest detection limitation was 2.9 ng. (5)The sample was stable in 12 hours and the relevant RSD was 0.39%. (6)The precision of sample was good and the RSD was 0.38%. (7) The average content of hyperin was 0.49% and the relevant RSD was 1.1%. (8)The average recovery was 99.91% and the relevant RSD was 0.44%. (9)The result showed the content of hyperin in Folium Rhododendri Micranti was 0.49%.Conclusion:It is the first time to establish the RP-HPLC method to determine hyperin concentration in Folium Rhododendri Micranthi. This method is sensitive,quick and can be used for the quality control for Folium Rhododendri Micranthi.