The Preparation Of Angelica Keiskei Koidzumi Chalcone And Its Inhibition On Mice H₂₂ Hepatoma Angiogenesis

Object To research and build a process for the preparation of Angelica keiskei Koidzumi chalcoe(AC) and prepare the sample for in vivo experiment. To determine the content of AC and its structural characteristics by ultraviolet spectrum(UV), intrared spectrum(IR) and high performance liquid chromatography(HPLC). To investigate the inhibition of AC on H22 hepatoma angiogenesis.Method Including the preparation and analysis of AC and its inhibition on H22 hepatoma angiogenesis:1 The extraction, purification and analysis of AC1.1 The extraction of AC:Extraction process: fresh ACâ†'homogenizationâ†'extraction with different solventâ†'collectionâ†'detectionâ†'AC crude extract. To select the optimum process for the AC extraction by comparing the influence factors including solvent, ratio of material to solvent, extracting time, temperature and times of extracting.1.2 The purification of ACProcess: AC crude extract dissolved in 65% ethanolâ†'filtrationâ†'absorption in columnâ†'washingâ†'elutionâ†'testingâ†'concentrationâ†'dryingâ†'sample of AC preparation1.3 The analysis of prepared AC sample1.3.1 Determination of the AC content:The absorbance of AC sample was determined by aluminium nitrate color-test. The content, extraction rate and purity of AC sample was calculated based on the standard rutin equation of linear regression as below:CX =(Y+0.0002)/0.484CX —the concentration of AC in the sample(mg/m L)Y—the absorbance of AC sampleCalculation of AC extraction rate:crude extract(%) = crude extract weight/material weight×100%prepared sample(%) = prepared sample weight/material weight×100%Calculation of prepared AC sample purity:crude extract(%) = AC content/crude extract weight×100%prepared sample(%) = AC content/prepared AC sample weight×100%1.3.2 Structural analysis of AC:The skeletal structure and specific absorption peak was detected by UV and IR respectively, and the composition of AC sample was detected by HPLC.2 Study on AC inhibition on mice H22 hepatoma angiogenesis2.1 MethodsHepatocarcinoma H22 cell resuspensions(0.2 ml, 1×107 cell/ml) were transplanted into the armpits of test mice subcutaneously as an experimental model. Ten days after H22 cell transplantation, 50 mice bearing tumors were selected and ready for grouping and testing. The selected 50 mice were divided into five groups, and orally administered 5 mg/kg(low dose), 25 mg/kg(middle dose) or 50 mg/kg(high dose) of AC or 4mg/kg of Endostar by intraperitoneal injection once a day for 10 days. The control group was treated with normal saline. Then the mice were sacrificed. Tumor samples were fixed in a 10% formaldehyde solution and the slides were prepared for hematoxylin and eosin staining and microscopy.2.2 Mesurement index2.2.1 Tumor weight and inhibition rate: the tumors were collected and weighed by electronic balance. The tumor inhibition rate was calculated as follows:inhibition rate(%) =(1-the tumor weight in treatment group/the tumor weight in control group) × 100%.2.2.2 Tumor cell proliferation: tested by the MTT assay.2.2.3 Analysis of MVD and VEGF expression in tumor tissue: measured by immunohistochemistry method.2.2.4 Analysis of HIF-1α concentration in tumor tissue: measured by enzyme linked immunosorbent assay(ELISA).Results The results of our study are as follows:1 The extraction, purification and analysis of AC1.1 The preparation of AC crude extractThe optimum process condition of AC crude extract preparation: water:ethanol(v/v) =35:65, ratio of material to solvent: 1:10, extract 2h at 60℃ for only once. And the extraction rate is 3.38%.1.2 The purification of AC crude extractThe purity of AC after our purification process impoves over 12 times. The purity is higher when using polyamide column chromatography for purification compared with AB-8 macroporous resin column chromatography.1.3 Analysis of AC in prepared sample1.3.1 Detection of AC contentThe content of prepared AC crude extract and purified sample were 0.570 mg and 7.250 mg respectively, while their purity was 7.71% and 96.6% respectively.1.3.2 UV spectrum analysis of AC prepared sampleAt 380 nm the AC sample appears maximum absorption, which is in accordance with the standard chalcone’s.1.3.3 IR spectrum analysis of AC prepared sampleThe IR spectrum of AC sample shows specific absorption of phenolic hydroxyl group, carbonyl, methyl, methylene, C-O bond and benzene ring, proves the characteristic structure of chalcone derivatives.1.3.4 HPLC analysis of ACThe results of HPLC show the AC sample contains 8 chalcone derivatives. Our process didn’t obtain single component of chalcone derivative.2 Study on the AC inhibition of H22 hepatoma angiogenesis2.1 Tumor weight and inhibition rate: The average tumor weight in tumor control group and AC high dose group were 0.548±0.021 g and 0.332±0.032 g respectively, while their inhibition rate were 4.20% and 12.20% respectively. The differences were statistically significant(p<0.05).2.2 Tumor cell proliferation: The proliferation activity of AC middle dose and high dose group were 0.657±0.073 and 0.506±0.032 respectively, which significantly lower than the tumor control group(p<0.05).2.3 Analysis of MVD and VEGF expression in tumor tissue: Compared with tumor control group, the MVD count of AC middle dose and high dose group were 7.2±1.135 and 4.5±1.286 respectively, which is significantly lower(p<0.05). The VEGF positive expression of AC middle dose and high dose group were 26.12 % and 12.22 % respectively, which the differences were statistically significant comparing to the tumor control group(p<0.05).2.4 Analysis of HIF-1α concentration in tumor tissue: The HIF-1α concentration of AC middle dose and high dose group were 81.16±10.31ng/L and 77.34±11.39ng/L respectively, while the tumor control group was 88.92±11.73ng/L. The differences were statistically significant comparatively(p<0.05).Conclusion1. Our study compares four different extraction solvent systems to optimize the extraction process of AC from fresh Angelica keiskei Koidzumi. We establish the optimal process to prepare the AC crude extract as follows: water:ethanol(v/v) =35:65, 60℃, ratio of material to solvent: 1:10, extract 2h for only once. And the extraction rate is 3.38%.2. The purity of prepared AC purified sample is improved by over 12 times after the purification process mentioned before. And polyamide column proves better than AB-8 macroporous resin for AC purification.3. The UV spectrum shows the AC purified sample appears maximum absorption at 380 nm while the IR spectrum shows specific absorption of phenolic hydroxyl group, carbonyl, methyl, methylene, C-O bond and benzene ring. The HPLC results show the AC purified sample contains 8 chalcone derivatives.4. AC can inhibit the proliferation of hepatoma cell. It can also downregulate the expression of MVD and VEGF in the tumor tissue and reduce the HIF-1α concentration. The tumor weight was significantly reduced after AC treatment. These results imply AC can inhibit the tumor angiogenesis.5. The innovation points of our study:(1) A direct extraction process from fresh Angelica keiskei Koidzumi has been established. The process is rapid, efficient, green, energy saving, and easy to operate, which is suitable for industrial application. The process is applied for national invention patent and has been authorized.(2) A whole process of AC extraction, purification and analysis from fresh Angelica keiskei Koidzumi has been established, while the process is systematic and operable.(3) We study the AC inhibition on the H22 hepatoma angiogenesis for the first time, and provide new experiment data for its future development of inhibition mechanism and targeted antitumor drugs.