| Persicae Semen, dry mature seed of Prunus persica (L.) Batsch or Prunus davidiana (Carr.) Franch, which is root in the family of Rosaceae. Mature fruit is harvested, pulp and nucleocapsid is removed, Taking out seeds and drying.The nature is calm and the taste is bitter and sweet which is ascriptioned the meridian of heart, liver and colon.It has many effects, such as removaling the stasis of blood, lubricating intestinal, relieving constipation, suppressing cough and relieving constipation. Pregnant using it shoud with caution. This product is contained in the"Shen Nong's Materia Medica", Modern research shows that Persicae Semen effects on the cardio-cerebral vascular disease significantly, which is received extensive attention in recent years. But systematic research on Active ingredients of Persicae Semen is penurious, and there is no specific quality evaluation system, which leding the circulation of Persicae Semen medicine market in disarray. Because the main ingredients of Persicae Semen is amygdalin, this study choose amygdalin as the major components, Respectively using TLC, HPLC as well as the fingerprint method, the content of amygdalin of the 15 samples which is taken from Shandong, Shanxi, and Hebei Chengde, anguo different origin and different varieties of Persicae Semen is qualitative and quantitative Studied, providing a scientific basis for evaluating the quality of Persicae Semen and rational drug using.Objective: To establish a method of TLC, a method to detect the content of amygdalin in Persicae Semen and a method of fingerprint through experiments on different batches of Persicae Semen,Which providing a basis for scientific evaluation and effective control of Persicae Semen quality.Methods: 1 The establishment of TLC: (1) Extracted conditions: Investigating the effects of different extraction methods and extraction solvents on the results, and selecting the best appropriate extraction method and extraction solvent. (2) The selection of development conditions: According to the chemistry of main indicators composition from Persicae Semen, determing the appropriate stationary phase, expand System, color systems.2 The establishment of content determination by HPLC: (1) Extraction: to examine the influence of different extractions and different extraction solvents on distilling the effective ingredient from Persicae Semen by optimizing the beforehand handling procedure of quality control through experiment which focuses 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: Choose appropriate fixed phase, adjust different formulation and proportion of mobile phase , regulate flow rate,the injection volume and column temperature in order to separate the peak of amygdalin from others well. (3) System suitability test: In this chromatographic condition, calculate the resolution and theoretical plate of amygdalin peak. (4) Preparation of standard curve: Prepare a series of the reference solution, examine peak area, then the regress equation was obtained with the content of amygdalin as abscissa, the relevant peak area as ordinate. (5) In the experiment of precision, take the same test solution, inject to the apparatus for six times, determine the relevant peak area of amygdalin, and then calculate the precision. (6) In the experiment of recovery, transfer 6 shares of Persicae Semen and add the reference solutions of amygdalin on same levels, detect the relevant peak area, then calculate the content of amygdalin and the RSD. (7) In the experiment of stability, transfer the sample solution from Semen Armeniacae Amarum to determine the relevant peak area at 0, 4, 8, 12, 24 and 48 h, then calculate the content and RSD. (8) In the experiment of recovery, transfer 9 shares of Persicae Semen and add the reference solutions of amygdalin on three levels and every level repeat three times, detect the relevant peak area , then calculate the content of amygdalin. (9) Determination of the lowest detection limitation: Dilute the reference solution of amygdalin until the value of S/N was more than or equal to 3. The relevant concentration was the lowest detection limitation. (10) Assay: Under above-mentioned conditions, determine the content of amygdalin in different batches of Persicae Semen.3 Establishment of fingerprint: (1) Extraction: An optimal extracting condition was chose by comparing the experimental results. (2) Chromatographic condition: Choose appropriate column and adjust different formulation and proportion of mobile phase and column temperature in order to establish a better fingerprint chromatogram. (3) System suitability test: In this chromatographic condition, calculate the resolution and theoretical plate of amygdalin peak. (4) Precision test: In the experiment of precision, take the same test solution, inject to the apparatus for six times, determine the relevant retention time and peak area, respectively. (5) Reproducibility test: In the experiment of reproducibility, prepare one concentration sample of Persicae Semen, repeat each concentration solution for six times in the same way, determine the relevant retention time and peak area, respectively. (6) Specificity test: In the experiment of stability, transfer the sample solution from Persicae Semen to determine the relevant retention time and peak area at 0, 4, 8, 12, 24 and 48 h, respectively. (7) Development of fingerprints: the test solution was prepared from different producing areas and different varieties and then their relevant fingerprint chromatograms were obtained.Results: 1 The method of TLC: (1) Extraction: put 10ml of methanol in to the sample, and ultrasonic treated 30 min, take supernatant solution as for the solution for the test product. (2) Development condition: Take the lower solution of chloroform-ethyl-methanol-water (15:40:22:10) which is placed 12 hours on 5-10℃as for development solvent, developing,taking it out and drying, putting it in to the solution of 0.8% phosphomolybdate acid of 15% sulfate ethanol, heating and waiting the spot clearly on 105℃, 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 The method of content determination by HPLC: (1) Extraction: The experiment ascertains that the beforehand handling procedure of quality analysis of Persicae Semen is the ultrasonic extraction of the mixture of fifty portion of methanol and one portion of Persicae Semen, which are performed once and lasts for 30 minutes. (2) Chromatogram conditions: The HPLC system was performed on a C18 analytical column gradient eluted with a mixture consisting of methanol-water (15:85) at a flow rate of 1.0 ml/min.The temperature of column was 30℃. The UV detection wavelength was set at 210 nm. 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) The draw 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 1.22%. (5) Reproducibility: The RSD value of repeatability was 0.53%. (6) Stability: The control solution and test solution were sTable in 48 h and the RSD values of amygdalin were 1.44% and 0.96%. (7) Recovery: The average recoveries of amygdalin was 99.57% and the RSD value was 2.50%. (8) The lowest detection limitation was 40.1032 ng. (9) The results showed the contents of amygdalin in Persicae Semen of different sources were between 2.332%-3.532%.3 The method of fingerprint chromatography: (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.The temperature of column was 30℃. The UV detection wavelength was set at 225 nm. 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 0.024%-0.137% and between0.278%-1.621%, respectively. (5) The reproducibility of sample was good and the RSD values of relative retention time and relative area were between0.069%-0.573% and between1.075%-2.269 %, respectively. (6) The test solution was sTable in 48 h and the relevant RSD values of relative retention time and relative area were between 0.004%-0.245% and between1.075%-1.951%, respectively. (7) Development of relevant fingerprint chromatograms: Fingerprint chromatograms from 15 batches of Persicae Semen were got. In addition, the fingerprint chromatograms of different varieties were also obtained. (8) Data analysis: similarity clustering analysis was performed and the result can significantly reflect the quality of Persicae Semen from different producing areas and different varieties.Conclusion:1 TLC: This method revealed a clear spots, repeatability and stability is well, It is the better qualitative identification method to be used to differentiate Persicae Semen.2 Content Determination: this study choose the content of amygdalin as the quality Index, the HPLC method was established to determine concentration of amygdalin in Persicae Semen. The method was found to be accurate, sensitive, quick and sTable It provides a scientific basis for scientific evaluation and effective control of the quality of Persicae Semen.3 FingerprintChromatogram: Getting feature comparison Chromatogram though the establishment of the HPLC fingerprint of Persicae Semen.then calculation the similarity. The method was found to be accurate, repetitive.It contributes to the quality control of Persicae Semen, And provides a new basis to the identification of Persicae Semen at the same time. |
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