| Herba Lysimachiae, the dry whole plant of Lysimachia christinae Hance, is a common used traditional Chinese medicine, which has long been widely used as conservative treatment for hepatolithiasis. biliary calculus and urinary calculus. Besides used as decoction in traditional way, Herba Lysimachiae is prepared as preparations alone or together with other medicinal materials together, among which Jinqiancao Granules is the most common one. In traditional production process of Jinqiancao Granules, a lot of sugar is used to granulate and to correct the flavour. However, with the increasing incidence of diabetes and obesity, the Sugar-free Jinqiancao Granules is anticipated by a lot of patients. So, Chongqing Heping pharmaceutical Co., Ltd. is trying to develop Sugar-free Jinqiancao Granules and authorized us to establish quality standards for Sugar-free Jinqiancao Granules.To prepare reference substances for quantifying ingredients of Herb Lysimachiae, the n-BuOH extraction of the material was studied on its chemical composition. Three flavonoids were isolated and their structures were identified as: quercetion-3-O-α-L-rhamnosyl-(1→2)-β-D-galactoside (1), myricetin-3,3’-di-O-a-L-rhamnoside (2), quercetion-3,3’-di-O-a-L-rhamnooside (3). Quercetion-3-O-α-L-rhamnosyl-(1→2)-β-D-galactoside was used as component marker to evaluate the quality of Sugar-free Jinqiancao Granules.A quantitative method of quercetion-3-O-a-L-rhamnosyl-(1→2)-β-D-galactoside by HPLC was established and was methodologically validated. The preparation method of sample solution is determined as follows:Extract 1 g of sample by ultrasonic for 30 minutes with 30 mL of methanol at room temperature. Then,15 mL of subsequentfiltrate is evaporated to dryness and the residue is dissolved with 1 mL of methanol. The HPLC conditions were optimized as follows:phenomenex C18 column (4.6 mm×150 mm,5μm), acetonitrile-0.5% acetic acid (A) and H2O-0.5% acetic acid (B) as mobile phases and the gradient program was as follows:0-25 min,15% A; 25-25.05 min,15% A→100%A; 25.05-30 min,100%A; 30-30.05 min, 100%A→15%A; 30.05-40 min,15% A. The flow rate was 1.0 mL·min-1, the column temperature was 35℃and the detection wavelength was 255 nm. The methodological validation showed that the peak area was linearly correlated to the quantity of quercetion-3-O-α-L-rhamnosyl-(1→2)-β-D-galactoside within the range of 0.2μg-20μg, the sample solution was stable within 5 days, the intra-day and inter-day precision were good, and that the recovery of spiked analyte was around 95%.The quantitative method of total flavonoids in Sugar-free Jinqiancao Granules was established too. Sample solution preparation method was determined as follows:Extract 2 g of sample by ultrasonic for 30 minutes with 20 mL of methanol at room temperature and take the subsequentfiltrate as sample solution. The quantity of total flavonoids in Sugar-free Jinqiancao Granules was determined with spectrometry with rutin as reference substance, with AlCl3-methanol solution as chromogenic reagent, and with O.D. measured at 410 nm. The methodological tests showed that there was a good linear correlation between O.D. and the concentration of flavonoids in the color reaction solution within the range of 0.012 mg·mL-1-0.040 mg·mL-1 of rutin, the intra-day and inter-day precision were good, the sample solution was stable within 4 days, and that the recovery of spiked analyte is 96.63%-97.59%. The quantitative method of total flavones in Sugar-free Jinqiancao Granules shows feasible and credible.A method was established to assay the fingerprint of Sugar-free Jinqiancao Granules by HPLC. Sample solution should be prepared as follows:Extract 5 g of sample by ultrasonic for 30 minutes with 50 mL of methanol at room temperature,15 mL of subsequentfiltrate is evaporated and the residue is dissolved with 1 mL of methanol to give sample solution. The HPLC conditions were optimized as follows: phenomenex C18 column (4.6 mm×150 mm,5μm), acetonitrile-0.5% acetic acid (A) and H2O-0.5% acetic acid (B) as mobile phases and the gradient program was as follows:0-10 min,5% A; 10-10.5 min,5% A→10%A; 10.5-30 min,10%A→15%A; 30-50 min,15% A→18% A; 50-60 min,18% A→20%A; 60-60.5 min,20% A→100% A; 60.5-70 min,100% A. The flow rate was 1.0 mL·min-1, the column temperature was 30℃and the detection wavelength was 255 nm. Similarity evaluation system for chromatographic fingerprint of TCM (Edition A) issued by State Pharmcopoeia Committee was used to analyze the HPLC fingerprints of Sugar-free Jinqiancao Granules. The HPLC fingerprints of 6 batches of Sugar-free Jinqiancao Granules samples were compared and analyzed to give the standard HPLC fingerprint of Sugar-free Jinqiancao Granules which contains 19 common peaks.The quantitative methods of quercetion-3-O-α-L-rhamnosyl-(1→-2)-β-D-galacto-side and total flavonoids in Sugar-free Jinqiancao Granules and the assay method of fingerprint of Sugar-free Jinqiancao Granules by HPLC were sensitive and accurate, easy to operate, and can be used for the routine quality control of Sugar-free Jinqiancao Granules.A series of physical and chemical properties of Sugar-free Jinqiancao Granules were measured on six batches of Sugar-free Jinqiancao Granules samples, including granularity, bulk density and tapped density, weight loss on drying, content of water insoluble substance, residue of ignition, contents of heavy metals. The results showed that Sugar-free Jinqiancao Granules produced by Chongqing Heping pharmaceutical Co., Ltd was in line with SFDA’s requirements. |
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