Study On The Quality Standard Of Peach Blossom

The peach blossom originates from the flowers of the Rosaceae plant peach（Prunus persica（L.）Batsch）or mountain peach（Prunus davidiana（Carr.）Franch）.This product is contained in the “Shen Nong’s Materia Medica”and “Chinese Material Medica” and other books,but not included in any quality standards.Its quality is difficult to effectively control.In this study,11 batches of peach blossom were identified by TLC,and the content of rutin,hyperoside and isoquercitrin were determined.On this basis,the peach blossom were analyzed by high performance liquid chromatography（HPLC）fingerprints.Objective:The method of TLC determination,content determination and fingerprint analysis were established,to provide the basis for quality control.Methods:1 The establishment of TLCThe effects of various extraction solvents and extraction methods on the target composition spots were tested.Select the appropriate thin layer adsorbent,expansion conditions and color conditions.2 The establishment of content determination by HPLCTo examine the influence of different extraction methods、different extraction solvents and its concentration and volume、different extraction time,different granularity on extracting rutin,hyperin and isoquercitrin from peach blossom by optimizing the highest determination of the three active ingredients were selected.Choose appropriate fixed phase,adjust different formulation and proportion of mobile phase in order to separate the peak of the three active ingredients from others well.Preparation of standard curve.Verify the precision,repeatability,stability,and recovery of established assay methods.The detection limits of the three target active ingredients were determined by stepwise dilution.3 different manufacturers of chromatography column,two different manufacturers of the instrument were respectively determined the content of the 3 batches of samples,calculated the RSD respectively.In accordance with above-mentioned conditions,the content of the three active ingredients in different varieties and different collection periods of peach blossom were determined.3 Establishment of fingerprintExtraction conditions and chromatographic conditions were selected under the same content.Verify the precision,repeatability,and stability of the established assay.The test solution was prepared and then their relevant fingerprint chromatograms were obtained.Results:1 The method of TLCPut 10 mL of 70% methanol in to the sample,and ultrasonic treated 30 min,filtration,take continued filtrate.Take ethyl acetate-formic acid-water（30:3:2）as for development solvent,developing,taking it out and drying,putting it in to the solution of 5% aluminum chloride ethanol solution,heating 3 min on 105 ℃,The principal spot in the chromatogram obtained from the test solution was similar in position,colour and fluorescence intensity to the principal spot in the chromatogram obtained from the reference solution and Reference materials solution.2 The method of content determination by HPLC25 times 70% methanol ultrasonic extraction 30 min,filtration,take continued filtrate.The column was SunFire C18 column（250 mm×4.6 mm,5 μm）;the mobile phase consisted of acetonitrile（A）and 0.1% aqueous phosphoric acid（B）;then firstly kept A-B（14:86）isocratic,secondly took a linear gradient elution until isoquercitrin peak came（0⁵ min,14% A→60% A;5²0 min,60% A;20²5 min,60% A→14% A）;the flow rate was 1.0 mL·min^-1,the detected wavelength was 353 nm and column temperature was 30 ℃,and the sample size was 20 μL.Under the above condition,the number of theoretical plates were more than 5000 calculated by isoquerditrin peak.The liner range for rutin was 1.00²01.72 μg·mL^-1.The liner range for hyperin was 1.00²00.54 μg·mL^-1.The liner range for isoquercitrin was 1.00²01.72 μg·mL^-1.The precision of the method were well.The RSD were 1.9%、1.9% and 1.3% respectively.The Repeatability of the method were well.The RSD of rutin,hyperin and isoquercitrin repeatability were 1.4%、1.8% and 1.2%.The test solution were stable in 24 h.The RSD of rutin,hyperin and isoquercitrin repeatability were 1.9%、1.9% and 1.4%.The average recovery of rutin was 101.4% and the RSD was 3.8%.The average recovery of hyperin was 102.2% and the RSD was 2.8%.The average recovery of isoquercitrin was 102.7% and the RSD was 2.0%.The lowest detection limitation of the three active ingredients was 0.10、0.10、0.10μg.The durability of the instrument and method were well.（10）The results showed the contents of the three active ingredients in peach blossom were between 0.053%⁰.29% 、0.030%⁰.082% and 0.11%⁰.32% respectively.3 Establishment of finger print25 times 70% methanol ultrasonic extraction 30 min,filtration,take continued filtrate.The column was SunFire C18 column（250 mm×4.6 mm,5 μm）;the mobile phase consisted of acetonitrile（A）and 0.1% aqueous phosphoric acid（B）;then firstly kept A-B（14:86）isocratic,secondly took a linear gradient elution until isoquercitrin peak came（0-20 min,14%A→20%A;20-40 min,20%A→60%A;40-45 min,60%A→14%A）;the flow rate was 1.0 mL·min^-1,the detected wavelength was 353 nm and column temperature was 30 ℃,and the sample size was 20 μL.The precision of the method were well,the RSD of relative retention time and relative areas were between 0.08%¹.36% and between 1.43%².72%,respectively.The reproducibility of sample was good and the RSD of relative retention time and relative area were between 0.07%⁰.62% and between 1.45%².14%,respectively.The test solution was stable in 24 h and the relevant RSD of relative retention time and relative area were between 0.08%¹.36% and between 1.43%².72%,respectively.A fingerprint analysis method of peach blossom was established,and fingerprints of 11 different batches of different origin and peach blossom were tested.The data was processed and analyzed according to the State Pharmacopoeia Commission “chromatographic fingerprint similarity evaluation system version 2.0”（development of the National Pharmacopoeia Commission）,the fingerprints of P r u n u s p e r s i c a a n d P r u n u s d a v i d i a n a were analyzed and compared,and similarity calculation and deviation analysis were carried out.Conclusions:1 TLCThe chromatographic spots obtained by this method are clear and clear,the separation effect is good,the method is durable and can be effectively identified for peach blossom.2 Content DeterminationTo develop an HPLC method for simultaneous determination of rutin、hyperin and isoquercitrin in peach blossom.The method has good precision,stability and repeatability.It provides a scientific basis for scientific evaluation and effective control of the quality of peach blossom.3 Fingerprint ChromatogramGetting feature comparison Chromatogram though the establishment of the HPLC fingerprint of peach blossom.The fingerprint data of P r u n u s p e r s i c a a n d P r u n u s d a v i d i a n a were measured and the similarity was evaluated.The method could be used as the reference for the quality evaluation of peach blossom.