| Pumila Flos originates from the dried flowers of Malus pumila Mill which is root in the family of Rosaceae.Picking in April to May,dried,store in dry place.Pumila Flos is calm which is ascriptioned the meridian of spleen,stomach and kidney.It has many effects,such as treatment of neuralgia,detoxifcation,nourish the blood,bright eye,Acne treatment and whitening.After checking the relevant information,no literature on Pumila Flos was found.Currently,Pumila Flos on the market In the form of sales of Pumila Flos tea,but not included in any quality standards,its quality is difficult to effectively control.Therefore,this research has carried on character identification,microscopic identification,TLC identification,determination of the impurity,moisture,total ash content,acid insoluble ash content,alcohol soluble extract(Hot dipping method)and the specific effective composition content of Phloretin.On the basis of the above,we established the method of HPLC fingerprint analysis of 12 batches of Pumila Flos from 8 provinces,it may provide the basis for species identification and the establishment of quality standards of Pumila Flos.Objective:Research on each project In accordance with the requirements of the quality standards.It provides the basis for establishment of quality standards of Pumila Flos.Methods:1 Character identification and microscopic identification were established for 12 batches of samples from 8 provinces,determination of the impurity,moisture,total ash content,acid insoluble ash content and alcohol soluble extract(Hot dipping method)content of 12 batches of samples.2 The establishment of TLC(1)Extracted conditions: Investigating the effects of different extraction solvents and extraction methods on the results,and selecting the best appropriate extraction solvent and extraction method.(2)The selection of development conditions: According to the main chemistry composition from Pumila Flos,determing the appropriate expand system,stationary phase,color system etc.3 The establishment of content determination by HPLC(1)Extraction: to Investigate the influence of different extraction methods,different extraction solvents and its concentration,different extraction time,different concentrations of hydrochloric acid hydrolysate,different granularity on extracting specific effective composition Phloretin from Pumila Flos.We selected the extraction conditions with the highest extraction efficiency.(2)Chromatogram conditions: Selecting the best detection wavelength,select appropriate stationary phase,adjust different formulation and proportion of mobile phase in order to separate the peak of quercetin,Phloretin and kaempferol from others well.(3)System suitability test: Under the established chromatographic conditions,calculate the resolution and the number of theoretical plates of the Phloretin peak.(4)Preparation of standard curve: Prepare a series of the reference solution of Phloretin,Separate sampling and the peak areas was recorded.Then the standard curves were obtained.The concentration of Phloretin were abscissa,and the relevant peak areas were ordinate.(5)Precision: The same reference solution and test solution were separate determined for six times,and the peak areas of Phloretin were recorded to calculate the RSD.(6)Reproducibility: The test samples of Pumila Flos were prepeared for six times which used the same sample in the same way,and the peak areas of Phloretin were recorded to calculate the RSD.(7)Stability: The same reference solution and test solution were determined at 0,2,4,6,12,24 and 48 h,and the peak areas of Phloretin were recorded to calculate the RSD.(8)Recovery: The amount of Precision known content of Pumila Flos was took,and the amount of reference substance was added which equivalent the content of Pumila Flos 80%,100%,120% respectively,then the test samples were prepeared in the same way,and the peak areas of Phloretin were recorded to calculate recovery and the RSD.(9)Determination of the detection limitation and the quantitation limitation: The reference solution of Phloretin was diluted until the values of S/N was more than or equal to 10,the concentration was the quantitation limitation;when the values of S/N was more than or equal to 3,the concentration was the detection limitation.(10)Durability : Three different manufacturers of chromatography column and two different manufacturers of the instrument were respectively determined the content of one batche of sample,calculated the RSD and the RD respectively.(11)Assay: Under the above-mentioned conditions,the content of Phloretin of 12 batchs of Pumila Flos from different areas were determined.4 Establishment of fingerprint(1)The extraction conditions and chromatographic conditions were the same to the content in addition to the detection wavelength was 260 nm.(2)Precision test: In the experiment of precision,take the same test solution,inject to the apparatus for six times,determine the retention time and the peak area respectively to Investigate the precision of the test method.(3)Reproducibility test: In the experiment of reproducibility,prepare one concentration sample of Pumila Flos,repeat the solution for six times in the same way,determine the retention time and the peak area respectively to Investigate the reproducibility of the test method.(4)Stability test: In the experiment of stability,The same test solution was determined at 0,2,4,6,12,24 and 48 h respectively,and record the retention time and the peak areas of Phloretin to Investigate the stability of the test method.(5)Development of fingerprints: Prepare the test solution of 12 batches of Pumila Flos from different provinces.Then we carried out the fingerprint analysis and similarity evaluation.Results:1 The character of Pumila Flos and microscopic identification features of powder of Pumila Flos were summarized;the results of impurity determination is 1%~3%;the results of moisture determination is 5.9%~7.9%;the results of total ash content determination is 6.2%~8.5%;the results of acid insoluble ash content determination is 0.6%~2.0%;the results of alcohol soluble extract content determination(Hot dipping method)is 34.9%~41.7%.2 The method of TLC(1)Extraction: Take this powder 0.5g,put in the Erlenmeyer flask with stopper,add ethanol 40 mL,Plug,ultrasonic treatment for 20 minutes,cool,shake,filtration.Take the filtrate 20 mL,put in the 250 mL Erlenmeyer flask with stopper,add athanol 20 mL,25% hydrochloric acid 10 m L,shake,85? water bath heating reflux for 30 minutes,evaporate to dryness,The residue was dissolved in ethanol 4mL as the sample solution.(2)Development condition: Using Silica gel G plate and take the upper solution of methylbenzene-ethylacetate-methane acid(7:2:1)as for development solvent,developing,taking it out and drying,spray with 3% ethanol solution of aluminum chloride,heating 10 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 sample solution in 365 nm under UV lamp.3 The method of content determination by HPLC(1)Extraction: Take this powder 0.2g precisely(over four sieve),put in the Erlenmeyer flask with stopper,add ethanol 20 mL precisely,Plug,weigh precisely,ultrasonic treatment for 20 minutes,cool,weigh precisely again,The loss weight complement with ethanol,shake,filtration.Take the filtrate 10 mL precisely,put in the 100 mL Erlenmeyer flask with stopper,add athanol 10 mL,25% hydrochloric acid 5mL,shake,85?water bath heating reflux for 1 hour,cool to the room temperature,transfer to 50 mL volumetric flask,dilute with ethanol to the scale,shake,filtration.(2)Chromatogram conditions: The column was YMC ODS-A C18 column(250 mm×4.6 mm,5 ?m);the mobile phase was methanol-0.4 %phosphoric acid(50:50);the detected wavelength was 286 nm;the flow rate was 1.0 mLˇmin-1;column temperature was 30? and the injection volume was 10 ?L.(3)System suitability test: Under the established chromatographic conditions,the number of theoretical plates were more than or equal to 5000 calculated by Phloretin peak.the resolution is more than 1.5.(4)Drawing of standard curve: The liner range for Phloretin was 1.23137 ~ 246.274?g/mL,had a good linear relationship,regression equation was y=40466x-6026,r=1.0000(n=6).(5)Precision: The precision of instrument and method were well.The RSD values were all 0.1%.(6)Reproducibility: The repeatability was well and the RSD value was 0.2%.(7)Stability: The reference solution and the test solution were stable in 48 h.The RSD values were 0.2% and 0.1% respectively.(8)Recovery: The average recovery of Phloretin was 104% and the RSD value was 1.7%.(9)The detection limitation was 0.4ng and the quantitation limitation was 1.2ng.(10)Durability: The durability of chromatographic column and instrument were well.(11)The results showed the contents of Phloretin in Pumila Flos was between 2.3%~4.2%.4 Establishment of fingerprint(1)The extraction conditions and chromatographic conditions were the same to the content in addition to the detection wavelength was 260 nm.(2)Precision test: Phloretin peak as reference peak,the RSD values of relative retention time and relative peak areas were between 0.03%~0.11% and between 0.25%~0.98% respectively.The precision of sample was good.(3)Reproducibility test: The RSD values of relative retention time and relative peak areas were between 0.02% ~ 0.10% and between 0.19% ~ 1.36% respectively.The reproducibility of sample was good.(4)Stability test: The test solution was stable in 48 h and the RSD values of relative retention time and relative peak areas were between 0.04%~0.19% and between 0.10%~1.04% respectively.The stability of sample was good.(5)Development of relevant fingerprint chromatograms: Fingerprint chromatograms from 12 batches of Pumila Flos were got and 12 common peaks were also obtained.(6)Data analysis: The Chromatographic Fingerprint Similarity Evaluation software 2004 A edition(Pharmacopoeia Committee of china developed)was used to analyse the data and evaluate the the HPLC-fingerprint establishment.The result can provide the basis for the species identification and the establishment of quality standards of Pumila Flos from different areas.Conclusions:1 The method of character identification,microscopic identification,inspection and alcohol soluble extract content(Hot dipping method)all have good stability.2 TLCThe method revealed a clear spots,resolution,repeatability and stability are well,It is the better qualitative identification method to be used to differentiate Pumila Flos.3 Content DeterminationTo develop an HPLC method for determination of specific effective composition Phloretin in Pumila Flos.The method has good precision,repeatability and stability.It can evaluate the quality of Pumila Flos and provide basis for the establishment of quality standards of Pumila Flos.4 Fingerprint ChromatogramGetting comparison Chromatogram through the establishment of the HPLC fingerprint of Pumila Flos.Then calculation the similarity.The method was found to be accurate and repetitive.It contributes to the quality control of Pumila Flos,and provides data support to the identification of Pumila Flos at the same time. |
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