| Objective: To investigate whether the polysaccharides from Salvia yunnanensis and Rosa rugosa have bioactivity against pancreatic cancer cell growth,and their structures will be characterized.This study will lay a theoretical and material foundation for the biological activity against pancreatic cancer,polysaccharide quality control,and polysaccharides-base new drug development from Salvia yunnanensis and Rosa rugosa.Methods: The polysaccharides of Salvia yunnanensis and Rosa rugosa were extracted by water extraction and alcohol precipitation.The crude polysaccharides were isolated by DEAE SepharoseTM Fast Flow anion exchange column and further purified with gel filtration chromatography to obtain homopolysaccharides.The physicochemical properties of each polysaccharide were examined by phenol-sulfuric acid method,meta-hydroxydiphenyl method BCA method and high-performance gel permeation chromatography(HPGPC).The monosaccharide composition was determined using HPLC pre-column derivatization.The chemical structure of the polysaccharides was finely characterizeded using combination of GC-MS,mass spectrometry and NMR analysis.Sulfated polysaccharide SZ0P21 was obtained by sulfating Z0P21 using the pyridine sulfur trioxide method.The viability of the pancreatic cancer cells were measured by MTT assay.Interaction between Z0P21 and Galectin-3 were examined by surface plasmon resonance(SPR)technique.The antiangiogenesis bioactivity of oligosaccharide were evaluated by tube formation assay and wound healing assay using human microvascular endothelial cells(HMEC-1).Results: Crude polysaccharide ZDSW(160.3 g,yield 3.56%)was obtained from Salvia yunnanensis(4.5 kg)by water extraction.ZDSW was eluted with 0 M,0.05 M,0.1 M,0.2 M and 0.4 M Na Cl through DEAE SepharoseTM Fast Flow column,and five polysaccharides were obtained: ZDS0(6.3 g,yield 1.755%),ZDS05(141.4 mg,yield 0.003%),ZDS1(352.6 mg,yield 0.008%),ZDS2(535.6 mg,yield 0.013%),ZDS4(241.8 mg,yield 0.005%).ZDS0 was purified by Bio-Gel P2 column to obtain an oligosacc-haride Z0P21(400.28 mg,yield 0.96%).The molecular weight of Z0P21 was determined as 666 Da by ESI-MS.The glycan composition analysis showed that the oligosaccharide was mainly composed of glucose(Glc)and galactose(Gal)in a molar ratio of 31.10: 68.90,which was a glucogalectan.Methylation analysis showed that the glycosyl residues in the Z0P21 oligosaccharide were α-1,6-linked-Gal and terminal Glc in a molar ratio of 72.49: 25.88.Combining the monosaccharide composition analysis,methylation,mass spectrometry and NMR analysis,we deduced that Z0P21 was a linear tetrasaccharide which composed of three galactose and one glucose as the terminal.After Z0P21 was sulphated,the degree of sulfate substitution was measured as 0.98 by the barium chloride-gelatin method.Crude polysaccharide REPW(261.2 g yield,5.80 %)was obtained from Rosa rugosa(4.5 kg,261.2 g)by boiling water extraction.REPW was eluted with 0 M,0.05 M and 0.4 M Na Cl through DEAE SepharoseTM Fast Flow column,and three polysaccharides were obtained: REP0(4759.2 mg,yield 0.085%),REP05(1088.9 mg,yield 0.005%),REP4(8274.0 mg,yield 0.069%).REP4 was purified by Bio-Gel P2 column to achieve homogeneous polysaccharides REP4P21(60.9 mg,yield 0.96%)and polysaccharide REP4P22(59.4 mg,yield 0.96%).The result of monosaccharides composition analysis indicated that REP4P21 was consisted of mannose(Man),rhamnose(Rha),glucuronic acid(Glc A),galacturonic acid(Gal A),glucose(Glc)and arabinose(Ara)in a molar ratio of 1.81: 11.04: 3.35: 30.33: 27.94: 25.53,with a molecular weight of 117,209 Da.REP4P22 was consisted of mannose,galacturonic acid,glucose and arabinose in a molar ratio of 9.04: 79.26: 3.12: 5.22: 3.37,with a molecular weight of 55,371 Da.Bioactivity studies showed that four acidic polysaccharides ZDS05,ZDS1,ZDS2 and ZDS4 isolated from Salvia yunnanensis might inhibit the growth of pancreatic cancer SW1990 cells.The oligosaccharide Z0P21 and its sulfated one might block human microvascular endothelial cells tube formation.The targeting molecule study show Z0P21 bound to Galectin-3 with a KD value of 5.777 × 10-6.Three kinds of polysaccharides isolated from Rosa rugosa had different inhibitory effects on SW1990.Among them,the polysaccharide REP4 had the best activity in a dose dependent way,with an inhibition rate of 48.96% on pancreatic cancer cells SW1990 after the cells were treated by the polysaccharide at the concentration of 1 mg/m L for72 h.The biological activity study of a homogeneous polysaccharide REP4P22 showed that this polysaccharide might impede pancreatic cancer cells growth.The inhibition rates of As PC-1,PANC-1 and SW1990 were 31.2%,24.2% and 29.65%,respectively.However,REP4P22 had no significant toxic effects on normal pancreatic cancer cells.Conclusion: Five polysaccharide fractions(ZDS0,ZDS05,ZDS1,ZDS2,ZDS4)were extracted and isolated from Salvia yunnanensis to obtain a neutral oligosaccharide.Z0P21,was a linear tetrasaccharide which composed of one glucose and three galactoses.Three polysaccharides(REP0,REP05,REP4)were isolated and pooled from Rosa rugosa.Eventually,a homogeneous polysaccharide,REP4P21 was achieved.The bioactivity results suggested that all four polysaccharides from Salvia yunnanensis(ZDS05,ZDS1,ZDS2,ZDS4)and three polysaccharides from Rosa rugosa(REP0,REP05,REP4)showed significantinhibitory activities against pancreatic cancer cell growth.Oligosaccharide Z0P21 and its sulfate one might impede angiogenesis effects by targeting Galectin-3.Polysaccharide REP4P22 might inhibit the growth of pancreatic cancer cellswithout significant toxic effects on normal human cells.The above studies have laid a theoretical foundation for the understanding of the active substance basis of the Chinese herbal medicines Salvia yunnanensis and Rosa rugosa and their polysaccharide based innovative new drug development. |
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