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AIM To investigate the effects of aqueous extraction at normal temperature,40 ℃,and water boiling on total coumarins extract of Angelicae dahuricae Radix.METHODS For the extracts Ⅰ-Ⅳ obtained by ethanol reflux extraction and three aqueous extraction preprocessing methods,respectively,UV and HPLC were adopted in the content determination of total coumarins and imperatorin,then the powder properties and dissolution rates were compared.RESULTS The contents of total coumarins in four extracts were 3.98％,6.03％,6.81％ and 4.46％,while those of imperatorin were 0.633％,0.540％,0.465％ and 0.155％,respectively.The angles of repose were 48.455°,42.587°,42.689° and 42.024° with the bulk densities of 0.214,0.324,0.316 and 0.354 g/cm3,respectively.Within 100 min,extract Ⅰ demonstrated higher moisture adsorption rate and equilibrium moisture absorption content than the other three extracts.The accumulative dissolution rates of various extracts were much higher than that of medicinal material fine powder within 120 min.CONCLUSION All the three aqueous extraction preprocessing methods can obviously improve the powder properties of total coumarins extract of A.dahuricae Radix and increase the dissolution rate of medicinal material fine powder.
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AIM To establish the quality standard for Weigela japonica Thunb.var.sinica (Rehd.) Bailey (W.j.).METHODS TLC was adopted in this medicinal material's qualitative identification after morphological identification and microscopic identification.The contents of water,total ash,acid-insoluble ash and extracts were detected according to Chinese Pharmacopoeia methods.Then the contents of scopoletin and total coumarins were determined by HPLC and UV,respectively.RESULTS The morphologies and microscopic characters of W.j.could be distinguished from other same generic plants.The clear TLC spot displayed good resolution.The contents of water,total ash,acid-insoluble ash,water-soluble extract and acid-soluble extract were no more than 12.0％,no more than 2.0％,no more than 0.5％,no less than 5.0％ and no less than 4.5％,respectively.Scopoletin and total coumarins showed good linear relationships within the ranges of 1.25-40.0 μg/mL(r =0.999 7)and 2.0-64.0 μg/mL (r =0.999 9),whose average recoveries were 98.19％ (RSD =0.90％) and 99.21％ (RSD =2.5％),respectively.CONCLUSION This accurate and reliable method can be used for the quality control of W.j..
ABSTRACT
OBJECTIVE: To explore the effect of total coumarins isolated from Hedyotis diffusa (total coumarins from Hedyotis diffusa, TCHD) on proliferation inhibition of leukemia cells, and to explore its related mechanism. METHODS: The purity of TCHD prepared by ethanol reflux extraction was tested by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) system. The cells (KG-1 Kasumi-1, THP 1 cells, U937 cells and K562 cells) were treated with TCHD(0.02, 0.04, 0.06, 0.08, 0.10 mg·mL-1) for 24 or 48 h, the inhibitive effect of TCHD on cells growth were determined by MTT method. After Kasumi-1 cells were incubated with TCHD for 24 h, the apoptosis of cells were analyzed by flow cytometry stained with Annexin V/PI. The expression levels of caspase-3, caspase-8, caspase-9, PARP and Bcl-2 family protein were assayed by Western blot. RESULTS: TCHD in certain concentration range could markedly inhibit the proliferation of AML cells, their IC50 on Kasumi-1, THP-1 KG-1, U937 and K562 cells were 0.077, 0.083, 0.096, 0.087, 0.096 mg·mL-1 for 24 h, and 0.059, 0.067, 0.072, 0.064, 0.068 mg·mL-1 for 48 h. TCHD has significant inhibitory effect on Kasumi-1, which was stronger than those on other cell lines, and showed a dose- and time-dependent manner(r=0.357, P<0.05). The apoptotic proportion of Kasumi-1cells in 0, 0.02, 0.04, 0.06, 0.08, 0.10 mg·mL-1 TCHD treatment groups for 24 h were (5.33±0.41)%, (7.99±0.45)%, (10.22±0.32)%, (20.10±1.99)%, (28.66±0.67)% and (33.24±2.12)%, respectively. After treated with TCHD(0.02-0.06 mg·mL-1) for 24 h, G0/G1 phase ratio of Kasumi-1 detected by flow cytometry were (51.43±3.21)%, (62.91±2.35)% and (76.42±4.14)%, respectively, which were significantly higher than that of the control group (35.8±5.25)% (P<0.05).Western blot results showed that different concentrations of TCHD could activate caspase-8, caspase-9, caspase-3 and PARP, promote the expression of cyto-C, down-regulate the cyclin E and CDK6, CDK2, p-CDK2 and cyclin D1 protein, and up-regulate the expression of p21 proteinin concentration- dependent manner(P<0.01). CONCLUSION: TCHD can obviously inhibit the proliferation of Kasumi-1 in a dose- and time-dependent manner, which may relate to the apoptosis of Kasumi 1 induced by activating caspase-3, 9, PARP protein through the mitochondrial pathways and Kasumi-1 cell block in G0/G1 phase through the influence of CDK2, p-CDK2, CDK4/6, cyclin E, cyclin D1 and p21.