| Aim The root of Angelica sinensis(Oliv.)Diels has beenused to treat deseases for thousands of years. Polysaccharide is one of its mainly effective compositions. Angelicapolysaccharide has lots of activities, such as hematopoiesis, immunomodulation, anti-tumor, etc. In addition, it can inhibit the proliferation and induce thedifferentiation of leukemia cells. Recently, some scholars also found that the acidic angelica polysaccharide was bioactive.Because there is short of quick and effective method to study their structures, the structures of acidic polysaccharide are rarely reported. Furthermore, because of their complex structures and pharmacological mechanisms, the relationship between structure and bioactivity remains unknown, which hampered the development and application of this polysaccharide as medicine. In this research, we attempted to optimize the extraction and separation process of acidicpolysaccharide, establish a new method to simultaneously determine netrual,acidic and aminosugars, study the complete structures of acidic homogeneous polysaccharides from Angelica sinensis, compare the inhibition effects on leukemic cells of homogeneous acidic angelicapolysaccharides, and further explore their mechanism of inducing apoptosis in leukemic cells. These results were helpful in understanding the pharmacological mechanism of Angelicapolysaccharides, and developingnovel anti-tumor agents.Methods(1) The acidic polysaccharide was isolated by the use of anion-exchange and size exclusion chromatography, then ten purified acidic polysaccharide were obtained. The homogeneity and molecular weight, contents of carbohydrates, proteins and galacturonic acids were determined using HPSEC, the phenol-sulfuric acid assay, Bradford method and carbazole method, respectively.(2) Pre-column HPLC was used to establish a new method to simultaneously determine Man, Gal N, Rha, Gal N, Glc A, Gal A, Glc, Gal, Ara and Fuc.(3) The structures of three main acidic homogeneous polysaccharides were determined by the chemical and instrumental analysis, including total acidic hydrolysis, methylation analysis, reduction of uronic acid, IR, HPLC, GC-MS and NMR(1H, 13 C, 1H/1H-COSY, HMQC, HMBC and NOESY).(4) The effects of six acidic homogeneous polysaccharides and the total sugar on the proliferation of human leukemic cells were investigated by the use of CCK-8 assay.(5) The morphological assessment of apoptosis staining with Hochest33342 was conducted with fluorescence microscope. The amount of apoptotic cells was measured by flow cytometry. The levels of Caspase-3, Caspase-9 and Gal-3 were demonstrated with western blotting.Result(1) AAPS-1I, AAPS-1II, AAPS-1â…¢, AAPS-2â… , AAPS-2â…¡, AAPS-3I, AAPS-3II, AAPS-4I, AAPS-4II and AAPS-5 were obtained from Angelica sinensis. The productions of AAPS-1II, AAPS-2â… and AAPS-2â…¡were more than other 6 acidic polysaccharides. AAPS-1I, AAPS-1II, AAPS-2I, AAPS-2II, AAPS-3I and AAPS-4I were homogeneous polysaccharides, and the molecular weight were 1.7×104, 2.3×103, 7.2×105, 1.0×104, 5.9×105 and 8.1×103 Da respectively. The physical and chemical properties differed from each other.(2) A pre-column HPLC method to determine the uronic acid, amino and neutral sugars was established. The optimal condition was the use of BDS C18(4.6 mm i.d.×250 mm, 5 μm) column. The mobile phase was composed of aqueous ammonium acetate buffer(100mmol/L, p H 5.0), acetonitrile(Me CN) and tetrahydrofuran in the ratio of 81∶17∶2. Analytes were performed with a flow rate of 1.0 m L/min.(3) The monosaccharide composition of AAPS-1II were Man, Glc N, Rha, Gal N and Gal A with the molar ratio of 8.3∶1.6∶1.0∶3.1∶2.5; the linkage compositions were T-Manp, 1,6-Manp, T-Rha, 1,4-Gal A and 1,2-Manp, in the molar ratio of 1.2∶2.5∶ 1.4∶1.1∶1.0; further analysis showed that AAPS-1II contained[Rha-(1â†'4)-Gal A-(1â†'2)-Manp-(1â†'6)-Manp-(1â†']n and Manp-[(1â†'6)-Manp-(1â†']ntwo chains. The monosaccharide composition of AAPS-2I were Gan, Glc N, Glc A, Gal A, Glc and Gal with the molar ratio of 1.;1∶4.3∶3.5∶1.1∶1.0∶1.1; AAPS-2â… was mainly composed of T-Manp, 1,4-Glcp, 1,3- Gal A, T-Galp and 1,2-Manpin the molar ratio of 1.3∶1.0∶ 1.3∶1.6∶1.9; further analysis showed that AAPS-2I contained [â†'3)-Gal A-(1â†'2)-Manp-(1â†'4)-Glcp-(1â†'2)-Manp-(1â†']n, Manp-[(1â†'2)-Manp-(1â†']n and Galp-[(1â†'4)-Glcp-(1â†']n three chains. The monosaccharide composition of AAPS-2IIwas Man, Glc N, Rha, Gal N and Gal with the molar ratio of 15.3∶5.1∶1.0∶15.3∶1.1; AAPS-2II contained T-Glcp, 1,6-Rha, 1,2-Manp and 1,3,6-Manp four residues, in the molar ratio of 1.0∶1.8∶2.8∶1.3. The monosaccharide composition of AAPS-1I was Man, Rha, Gal N, Glc A, Glc and Ara with the molar ratio of 3.0∶2.5∶2.9∶2.9 ∶ 1.5∶1.0. The monosaccharide composition of AAPS-3I was Man, Glc N, Gal N, Gal A, Glc and Gal in the molar ratio of 1.0∶3.9∶1.3∶2.1∶1.4∶1.4. The monosaccharide composition of AAPS-4I was Man, Glc N, Rha, Gal A and Ara with the molar ratio of 1.2∶3.9∶ 2.9∶1.0∶1.1.(4) The effects of six acidic polysaccharides on the proliferation of K562 cells differed from each other. The effect of AAPS-1II, AAPS-2I and AAPS-2II were in a concentration-dependent manner.AAPS-2I showed the most significant anti-tumor activities(IC50=0.056 mg/L).(4) Typical morphological changes of apoptotic K562 cells were found after treated with AAPS-2I for 72 h. The apoptosis-inducing effect was in a concentration-dependent manner. The expression of pro-caspase 3 and pro-caspase 9 was reduced, while the expression of activated-caspase-3 and caspase-9was increased. Furthermore, the expression of Gal-3 was inhibited by AAPS-2I obviously, which meant that Gal-3 may be the upstream target for AAPS-2I inducing the apoptosis of K562 cells.Conclusion(1)It was the first time to set up a new HPLC method which could simultaneously determine the composition and content of uronic acid, amino and neutral sugars. The accuracy and precision of this method was well.(2) The extraction method of acidic angelica polysaccharide was optimized. The acidic angelica polysaccharide was separated systematically and ten polysaccharides were obtained, among which six were homogenous polysaccharides. All of the six homogenous polysaccharides were analyzed with amino sugars, which was the first time to find amino sugars in umbelliferae. The structures of three main polysaccharides were analyzed and the primary structures of two were got.(3) The six acidic polysaccharides displayed different inhibition effects on K562 cells, and AAPS-2I had the most significant anti-tumor effect.(4) AAPS-2I induced the apoptosis of K562 cells in a concentration-dependent manner, and the mitochondria associated apoptotic pathway and Gal-3 were likely involved in the effects. |
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