| Dendrobium densiflorum is a adiantum of the Dendrobium of the Orchidaceae. Many scholars have studied on chemical constituent and pharmacology of Dendrobium adiantums in recent decades. Numerous studies have demonstrated that Dendrobium densiflorum polysaccharides have anti-tumor, immunomodulatory and hypoglycemic effects. It seems that pharmacological activity is realtive to water-soluble polysaccharides content of Dendrobium adiantums. Acidic polysaccharides have more bioactivity than the others. But the studies on chemical constituent and pharmacology of Dendrobium densiflorum have not been reported.This article wants to screen and optimize the extraction process of crude polysaccharides in Dendrobium densiflorum by orthogonal experimental design,and to separate neureal polysaccharides and acidic polysaccharides in Dendrobium densiflorum with column chromatography on DEAE-52 cellulose.4 acidic polysaccharides (PGM1,PGM2,PGM3,PGM4) were obtained from Dendrobium densiflorum respectively eluting with 0.05mol·L-1,0.1 mol·L-1,0.2mol·L-1 and 0.3 mol·L-1 NaCl solution. It was obtained the relative molecular weight sections of 4 acidic polysaccharides by gel filtration(GF) method with Sephadex G-100 column.then we want to study whether there were some relationships on the acidity of polysaccharides and the relative molecular weight. In vitro spleen cell proliferation and tumor growth inhibition test to compare comprehensively the bioactivities of 4 acidic polysaccharides, to find the relationships on the acidity of polysaccharides and the relative molecular weight,and to determine the relative molecular weight section has the best bioactivity.Objective:1. To screen and optimize extractive technology of Dendrobium densiflorum polysaccharides. The influences of temperature, the ratio of liquor, time and material on extraction yield of polysaccharides were discussed. Orthogonal experimental design was adopted to study these four factors.2. To separate neureal polysaccharides and acidic polysaccharides of Dendrobium densiflorum. To separate acidic polysaccharides through acidic radicals.3. To obtain the relative molecular weight sections of acidic polysaccharides by gel filtration(GF) method with Sephadex G-100 column.then to study whether there were some relationships on the acidity of polysaccharides and the relative molecular weight.4. To study the effect of acidic polysaccharides (PGM1,PGM2,PGM3,PGM4) on the cell proliferations of murine splenocytes.5. To study the effect of acidic polysaccharides (PGM1,PGM2,PGM3,PGM4) on the human A549 and G2 tumor cells.6. In vitro spleen cell proliferation and tumor growth inhibition test to compare comprehensively the bioactivities of 4 acidic polysaccharides, to find the relationships on the acidity of polysaccharides and the relative molecular weight,and to determine the relative molecular weight section has the best bioactivity.Method:1.Dendrobium densiflorum powder and water heated at assigment temperature by orthogonal test design method. The filtrate was collected and diluted constant volume. Phenol-sulfuric acid method was used for the content of polysaccharides.Every test was did twice and got the average value.Then analysed the data.2. The crude polysaccharides were separated with DEAE-52 column, and were eluted by (0.05,0.1,0.2,0.3)mol·L-1 sodium chloride solution respectively. Testing by phenol-sulfuric acid method and drawing curve. Eluent of main peak was cellected dialyse(intercetion is 3500D),rotary evaporated.Then added 3 folds amount of 95% alcohol,and centrifugated, dried in oven.Then it become dry acidic polysaccharides.3. It was obtained the relative molecular weight sections of 4 acidic polysaccharides by gel filtration(GF) method with Sephadex G-100 column.The konwon molecular weight Dextran T-5,T-40 and T-70 were subjected to gel filtration successively, eluted with double dishhed water,tested with Phenol-sulfuric acid method,and recorded the eluted volume Ve respectively.Determined the volume of blank gel V0 with Dextran T-2000 as the same method above,Ve/Vo as the abscissa,the logarithm molecular weight (logM) as the ordinate,got the regression equation.Then PGM1,PGM2,PGM3, and PGM4 were subjected to gel filtration successively, Determined Ve.It substituted the equation with Ve,obtained the molecular weight of every samples,compared the relative on the different acidic polysaccharides and the relative molecular weight.4 Splenocytes from normal mouse were incubated at 37℃in the presence of acidic polysaccharides (PGM1,PGM2,PGM3,PGM4) for 44 hours. Supernatant fluid was removed. And added DMSO and shaked. Testing absorbance overλ=570nm.5. First human A549 tumor cells were digestive enzyme were incubated at 37℃for 24h, then they were incubated at 37℃in the presence of acidic polysaccharides (PGM1,PGM2,PGM3,PGM4) for 48 hours. Testing absorbance and calculating suppression ratio.Test approach of human G2 tumor cells was same as human A549 tumor cells.Results:1. Results showed the best technical condition was as follows:extraction temperature was 90℃, ratio of material and liquor was 1:20, extraction time was 2 hours and extraction number was 3,2 hours every time.The extraction yield of polysaccharides was 3.15% in the optimal technical condition.2. The crude polysaccharides were separated with DEAE-52 column, and obtained 4 acidic polysaccharides(PGM1,PGM2,PGM3,PGM4) by (0.05,0.1,0.2,0.3) mol-L"1 sodium chloride solution respectively. Neutral polysaccharides content was the most. The more concentration of sodium chloride solution, the less content of acidic polysaccharides.3. It got regression equation, y=-0.4141x+5.362, r=0.9984 through accounting three data of standard substances.It had linearity from 3.699 to 4.845. Relative molecular weight of PGM1, PGM2, PGM3, PGM4 were different, and every sample's had some, main range is 5000-70000.4. Except for PGM 1 (25μg/ml), PGM1 (100,50μg/ml), PGM2, PGM3, PGM4 (100,50,25μg/ml) have facilitated cell proliferation of murine splenocytes in a dose-dependent manner. It showed the bioactivity was PGM4>PGM2>PGM3> PGM1.5. PGM4, PGM3和PGM2 (50,100,200μg/ml),PGM1(100μg/ml) could inhibite the growth of human A549 tumor cells (P<0.05). PGM4,PGM3 (50,100,200μg/ml) and PGM2(200μg/ml) could inhibite the growth of human G2 tumor cells. It showed the bioactivity was PGM4> (PGM2,PGM3)> PGM1.Conclusion:1. The best technical condition was as follows:extraction temperature was 90℃, material and liquor was 1:20,extraction time was 2 and 3 hours each time.This process can guide the scale in industry.2.Neutral polysaccharides content was the most, while the content of acidic polysaccharides was very little.The more the acidic group in the polysaccharides,the less polysaccharides in Dendrobium densiflorum3. It showed the acidity was PGM4>PGM3>PGM2>PGM1. Relative molecular weight of every acidic polysaccharide was different. Accordingly it had not affinity between acidity and relative molecular weight.4. The bioactive polysaccharide was concentrated in (70000-50000) D, (50000-28000) D and (10000-5000) D, especially in (10000-5000) D. |
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