Extraction, Separation, Structural Characteristics And The Anti-tumor Effects Of Polysaccharides From Holotrichia Diomphalia Bates

Objective:Holotrichia diomphalia Bates(Qi Cao) is a species of Melolonthidae that is indigenous to eastern Asian areas and was recorded in Shennong's Herbal as traditional Chinese folk medicine. Studies have shown that Holotrichia diomphalia Bates possess many pharmacological activities, such as anti-tumor, anti-bacterial, anti-coagulation, anti-inflammatory, anti-virus, etc. In recent years, animal polysaccharides have attracted great attention in an-titumor research because of its more monosaccharide residues and more complex structure than plant polysaccharides. But so far, the preparation, structure and the activity of Qi Cao polysaccharide have been rarely reported. This study was proposed to the extraction, separation, and purification of homogeneity polysaccharides from Qi Cao, detection the structures, and study on their inhibition effects on the melanoma and relative mechanisms. These results were helpful to find active anti-tumor polysaccharides for further research of anti-tumor agents. Method:1.The alkali water extraction and alcohol precipitation methods were applied to get total polysaccharides from Qi Cao. The pigments of polysaccharides were removed by hydrogen peroxide process. The ion exchange chromatography and size exclusion chromatography were used to isolate the polysaccharides.2.The homogeneity and the molecular weight, contents of total sugar, protein, uronic acid, sulfuric acid group and amino group content of the polysaccharides were tested by high performance size exclusion chromatography, phenol-sulfuric acid method, coomassie brilliant blue method, carbazole method, barium sulfate turbidity method, cysteineSulfuric acid method, respectively. The structural characteristics of HDPS-1?, HDPS-2?, HDPS-3?and HDPS-4? were studied by the hydrolysis, methylation analysis and spectral analysis(IR?GC?GC-MS?NMR(1H, 13 C, 1H-1H COSY, HSQC, HMBC, NOESY and TOCSY).3.The xenograft tumor model was created by subcutaneous injection of B16 cells in mice to evaluate the anti-tumor activity of these homogenuous polysaccharides and total polysaccharides from Qi Cao. The tumor tissues were observed in H&E staining. The immunohistochemical assay was used to detect the tumor blood vessel density.4.The MTT method was used to detect the inhibition of human umbilical vein endothelial cells(HUVECs) proliferation. The effects of HUVECs microtubules formulation were investigated by the formation of microtubules in vitro. The expression of vascular endothelial growth factor(VEGF), P-VEGF and Gal-1 were detected by Western blotting. Results:1.HDPS-1?, HDPS-2?, HDPS-2?, HDPS-3?and HDPS-4?were obtained from Holotrichia diomphalia Bates. The yield of HDPS-2?were lowest.The protein and pigments of polysaccharide were removed using the repeated freezing and thawing method and hydrogen peroxide method, respectively.2.The homogenuous of polysaccharides(HDPS-1?, HDPS-2?, HDPS-3?and HDPS-4?) had a single symmetrical peak in HPSEC spectrum. The average molecular weight of HDPS-1?, HDPS-2?, HDPS-3?, HDPS-4? were 3.8 × 105, 8.2 × 103, 1.7 × 104 and 1.9 × 104 Da. These four kinds of polysaccharides contained sulfuric acid groups and amino groups. HDPS-1?, HDPS-2?, HDPS-3?and HDPS-4?were composed of Rha, Xyl, Glc and Gal, and their molar ratios were 1.00?1.75?4.64?6.22, 1.00?3.30?12.1? 12.7, 1.00 ? 1.76? 2.96 ? 1.53 and 3.71? 1.00 ? 1.38 ? 3.30. HDPS-1II were composed of 1,4-Rhap?1,2-Rhap?1,2,3-Rhap?1,2,4-Rhap?1,4-Glcp?1,6-Galp?T-Galp?1,2,4-Glcp?1,2,4-Xylp?T-Glcp?T-Rhap?1,4-Galp?1,3,4-Glcp and 1,3,4-Galp, and in the molar ratio of 4.03?2.54?1.00?11.0?2.82?44.7?3.61?8.92?2.51?27.6?5.03?4.15?2.92?3.69. HDPS-2II were composed of 1,4-Rhap?1,2-Rhap?1,2,4-Rhap?1,4-Glcp?1,6-Galp?1,2,4-Glcp?1,2,4-Xylp?T-Glcp?T-Rhap?1,4-Galp?1,3,4-Glcp and 1,3,4-Galp, in the molar ratio of 2.74?1.60?2.15?1.00?33.40?3.30?2.04?21.12?3.16 ? 2.31 ? 1.33 ? 2.08. HDPS-3II contained 1,4-Rhap ? 1,2,4-Rhap ? 1,6-Galp ?1,2,4-Glcp?1,2,4-Xylp?T-Glcp?T-Rhap?1,4-Galp?1,3,4-Glcp and 1,3,4-Galp, in molar ratio of 2.64?2.62?8.20?4.73?1.00?27.33?3.73?1.88?1.24?2.20. HDPS-3II were composed of 1,4-Rhap?1,2-Rhap?1,2,4-Rhap?1,6-Galp?1,2,4-Glcp?1,2,4-Xylp?T-Glcp?T-Rhap?1,4-Galp?1,3,4-Glcp and 1,3,4-Galp, and in the molar ratio of 7.47?1.69?6.66?35.50?17.45?3.12?23.20?7.64?3.98?2.06?3.13.3.Four kinds of homogeneous polysaccharide from Qi Cao had different anti-tumor activities in vivo. The tumor inhibition rates of HDPS-2?and HDPS-3?were highest among these polysaccharides. The HDPS-2?and HDPS-3?could significantly suppress tumor microvascular density.4.There were proliferation inhibition effects of HDPS-2?and HDPS-3?(100 and 300?g/m L) on HUVECs. These polysaccharides could suppress the formation of microtubules of HUVECs. Among them, HDPS–2?and HDPS-3?showed significant inhibition effects. HDPS-2II and HDPS-3II could significantly inhibit the expression of P-VEGFR2 and Gal-1 in HUVECs, whereas the VEGFR2 expressions were increased in the cells treated by HDPS–2?and HDPS-3?. Conclusions:1.The extraction and separation methods, repeated freezing and thawing method, decolorization methods of HDPS from Holotrichia diomphalia Bates were built in this study. Five kinds of polysaccharides were obtained from Qi Cao, named HDPS-1??HDPS-2??HDPS-2??HDPS-3?and HDPS-4?.2.HDPS-1?, HDPS-2?, HDPS-3?and HDPS-4?contain sulfuric acid and amino groups, and were composed of Rha, Xyl, Glc and Gal. The content of 1,6-Galp and T-Glcp are the highest among all the monosaccharide residues.3.HDPS-1?, HDPS-2?, HDPS-3?and HDPS-4? showed different anti-tumor effects and inhibition on tumor microvessel density.4. HDPS-2 ? and HDPS-3 ? could inhibit the tumor angiogenesis through the Gal-1-VEGFR2 signaling pathway.