| Semen Armeniacae Amarum, the semen of Prunus armeniaca L. Var. ansu. Maxim., Prunus. sibirica. L., Prunus.mandshurica (Maxim.) Koehne, or Prunus armeniaca L., is multi-origin variety in the family of Rosaceae. It is reported that amygdalin and fatty acid are the main bioactive components of this drug, as a result, many effects, such as repessing upward perversion of Qi, suppressing cough, relieving dyspnea and relieving constipation with laxatives, have been proved thoutsand years ago. Their complex origin, wide distribution, unstandadd cultivation and elaboration lead to its unsTable quality, and there is no effective method for its quality evaluation, for this reason, it is difficult for people to control its quality. According to the researches, besides the ones which have been recorded in the Chinese Pharmacopoeia (2005 Edition) , there are also some varieties used as Semen Armeniacae Amarum in the market, like Prunus armeniaca dasycarpa (Ehrh.) Borkh and Prunus armeniaca (L.) Lam. var. pubescens Kost. In this study, the methods of thin-layer chromatography, content determination of Amygdalin and HPLC fingerprint were used to study the variety and quality control of Semen Armeniacae Amarum, and its quality evaluation method was also established.Objective:To establish a TLC method for qualitative identification, and a method to determine the contents of amygdalin in Semen Armeniacae Amarum, which can provide fast and efficient guideline to identify Semen Armeniacae Amarum. Besides, to establish HPLC fingerprints of Semen Armeniacae Amarum and get reference fingerprint, to compare the fingerprints of Semen Armeniacae Amarum collected from different varieties, so as to establish a scientific method to control the quality of Semen Armeniacae Amarum.Methods:1 Studies on TLC of Semen Armeniacae Amarum: By selecting suiTable extraction solvent, extraction approach, eligible fixed phase, development system, and colouration system, Semen Armeniacae Amarum was identified with TLC.2 Determination of amygdalin in Semen Armeniacae Amarum by HPLC: (1) Extraction: To examine the influence of different extractions and different extraction solvents on distilling the effective ingredient from Semen Armeniacae Amarum, and to optimize the beforehand handling procedure of quality control, the orthogonal experiment was performed, which focused on three major factors affecting the efficiency of extraction, including the rates of solvent and medicine material, extraction time and extraction times. (2) Chromatogram conditions: The appropriate fixed phase, the different formulation and proportion of mobile phase, the flow rate and the column temperature were studied to make the separation of the peak for amygdalin better. (3) Preparation of standard curve: Different concentrations of reference solution were prepared, and the peak area was examined, then the regress equation was obtained with the content of amygdalin as abscissa, the relevant peak area as ordinate. (4) Precision: The same test solution was determined for six times, and the relevant peak area of amygdalin was recorded to calculate the RSD value. (5) Reproducibility: The test samples of Semen Armeniacae Amarum were prepeared for six times used the same group of this drugs in the same way, and the relevant peak area of amygdalin was recorded to calculate the RSD value. (6) Stability: The same test solution was determined at 0, 4, 8, 12, 24 and 48 h, and the relevant peak area of amygdalin was recorded to calculate the RSD value. (7) Recovery: Six shares of Semen Armeniacae Amarum were took, and the same amount of reference substance was added respectively, then the test samples were prepeared in the same way, and the relevant peak areas of amygdalin was recorded to calculate the RSD value. (8) Determination of the lowest detection limitation: The reference solution of amygdalin was diluted until the value of S/N was more than or equal to 3. The relevant concentration was the lowest detection limitation. (9) Assay: Under above-mentioned conditions, the content of amygdalin in different batches of Semen Armeniacae Amarum was determined.3 Establishment of fingerprint: (1) Extraction: An optimal extracting condition was chose by comparing the experimental results. (2) Chromatographic condition: The appropriate column, the different formulation and proportion of mobile phase, the flow rate and the column temperature were studied to make the separation of the peak for amygdalin better. (3) System suitability test: The separation and theoretical plate of amygdalin peak were calculated in the chromatographic condition. (4) Precision: The same test solution was determined for six times, and the relevant peak area of amygdalin was recorded to calculate the RSD value. (5) Reproducibility: The test samples of Semen Armeniacae Amarum were prepeared for six times used the same group of this drugs in the same way, and the relevant peak area of amygdalin was recorded to calculate the RSD value. (6) Stability: The same test solution was determined at 0, 4, 8, 12, 24 and 48 h, and the relevant peak area of amygdalin was recorded to calculate the RSD value. (7) Development of fingerprints: The samples of different producing areas and different varieties were prepared in the same way, and then their relevant fingerprint chromatograms were obtained.Results:1 Studies on TLC of Semen Armeniacae Amarum: TLC methods were established for the identification of Semen Armeniacae Amarum, the principal spot in the chromatogram obtained from the test solution was similar in position, colour and intensity to the principal spot in the chromatogram obtained from the reference solution.2 Determination of amygdalin in Semen Armeniacae Amarum by HPLC: (1) Extraction: The experiment ascertained that the beforehand handling procedure of quality analysis of Semen Armeniacae Amarum was the ultrasonic extraction of the mixture of fifty portion of methanol and one portion of Semen Armeniacae Amarum, which were performed once and lasted for 30 minutes. (2) Chromatogram conditions: The HPLC system was performed on a C18 analytical column eluted with a mixture consisting of methanol-water (15:85) at a flow rate of 1.0 ml·min-1, the temperature of column was 30℃, the UV detection wavelength was 210 nm, and the injection volume was 10μl. Under the above condition, the peaks of samples were separated well with the resolution of not less than 1.5. The retention time is about 12 min. The theoretical plates were more than 4000. (3) Preparation of standard curve: The liner range for amygdalin was 0.138-13.8μg. Regression equation was Y=513.57X+20.704, r=0.9999 (n=6). (4) Precision: The precision of instrument and method were good and the RSD values of amygdalin were 0.71% and 0.78% respectively. (5) Reproducibility: The RSD value of repeatability was 0.76%. (6) Stability: The control solution and test solution were sTable in 48 h and the RSD values of amygdalin were 1.44% and 1.25% respectively. (7) Recovery: The average recovery of amygdalin was 99.51% and the RSD value was 1.81%. (8) The lowest detection limitation was 40.1032 ng. (9) The results showed the contents of amygdalin in Semen Armeniacae Amarum of different sources were between 0.0724%-5.668%.3 Establishment of fingerprint: (1) Extraction: The method of supersonic wave-extraction with 70% methanol for 60 min was simple, quick and sTable. (2) The HPLC system was performed on a Waters Symmetry-C18 analytical column gradient eluted with a mixture consisting of acetonitrile, water at a flow rate of 1.0 ml·min-1, the temperature of column was 30℃, the UV detection wavelength was set at 225 nm, and the injection volume was 10μl. (3) System suitability test: Under the above condition, the peak corresponding to amygdalin of the test solution was separated well with the resolution of more than 1.0 and about 50000 of theoretical plate. (4) The precision of sample was good, amygdalin peak as reference peak, the RSD values of relative retention time and relative area were between 0.009%-0.26% and between 1.01%-2.43%, respectively. (5) The reproducibility of sample was good and the RSD values of relative retention time and relative area were between 0.009%-1.04% and between 1.76%-2.59%, respectively. (6) The test solution was sTable in 48 h and the relevant RSD values of relative retention time and relative area were between 0.009%-1.04% and between 1.07%-2.59%, respectively. (7) Development of relevant fingerprint chromatograms: Fingerprint chromatograms from 10 batches of Semen Armeniacae Amarum were got. In addition, the fingerprint chromatograms of different varieties were also obtained. (8) Data analysis: Similarity clustering analysis was performed and the results could significantly reflect the qualities of Semen Armeniacae Amarum from different producing areas and different varieties.Conclusion:1 In identification, the relevant spots were clear and characteristic and could be used to differentiate Semen Armeniacae Amarum.2 The HPLC method was established to determine concentration of amygdalin in Semen Armeniacae Amarum. The method was found to be accurate, sensitive, quick and sTable.3 It was the first time to establish the HPLC fingerprint chromatogram of Semen Armeniacae Amarum, get reference fingerprint chromatogram. We compared the fingerprints of 8 samples which were from different varieties and studied their difference with similarity. This method provides a scientific basis for the scientific evaluation and utility control of the quality of Semen Armeniacae Amarum.
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