金针菇多糖的制备与分析
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Experiment 2： Preparation and Generic Identification of flammulina velutipes
生物制药综合实验（二） 金针菇多糖的制备和鉴定
Purpose
(1) Learn the principle of separation, purification of fungus polysaccharides. (2) Master the quantitive determination of polysaccharides.
Principle
There are many methods for polysaccharide purification, which must correspond to the properties of polysaccharide: (1) Ethanol precipitation; (2) CTAB.
Sample acid hydrolysis
polysaccharide sample 10 mg in clean tube + 2 ml 2 mol/L trifluoroacetic acid
sealing tube (with sealing film and adhesive seal) 100℃ in boiling water 3 hours to hydrolysis
(2) Preparation of sample solution: Weigh out sample (about 10mg) accurately, put them into volumetric flask (scaled 100ml) and then add solution to 100ml. (3) Determination
Principle
flammulina velutipes
treasured medical fungus.
is a kind of traditional
Chinese
The polysaccharide of
flammulina velutipes
has an obvious
effect on immune function, antiinflammatory and antiradiation.
真空干燥 无水乙醇洗涤 真空干燥
沉淀2
(1) Standard curve of G: Weigh out G 10mg accurately, put them into volumetric flask (scaled
100ml) and then add solution to 100ml.
沉淀2 上清1 上清2
<5-15ml水 溶解 <5-15ml水
¼ 体积 of 氯仿：正丁醇(4:1)
分液漏斗振摇20min
离心
3000rpm×10min
¼ 体积 of 氯仿：正丁醇(4:1)
3000rpm×10min 3000rpm×10min 沉淀1
离心
无水乙醇洗涤
上清1 上清 2
3倍，冷无水乙醇 3倍，冷无水乙醇
Principle
Quantitive determination: After hydrolyzed in concentrated sulfuric acid, polysaccharide is further dehydrated into aldose derivation that can form colored compound with anthrone.
crude polysaccharide1 crude polysaccharide1 2
制备工艺：
金针菇子实体（200g） 去杂，切碎 1000ml水 煮 1-1.5h 上清液 上清 搅拌加无水乙醇至终浓度40% 搅拌加无水乙醇至终浓度60% 4℃， 30min 4℃，60min 离心 3000rpm×10min 离心 3000rpm×10min 沉淀1 沉淀2 沉淀1 4层纱布过滤
precipitate 2
dissolve <5-15ml water dissolve
add ¼ volume of chloroform-butanol(4:1) add ¼ volume of Shake 20min centrifugalize 3000rpm10min chloroform-butanol(4:1) Precipitate 1 Precipitate 2
Pick up 10 G tubes, number them, and add reagents according to
following table. Use solution as blank, and test the optical density at 620nm. Fill data you got in the table, and calculate the content based on
Add triple amount of alcohol Supernatant layer 1 Supernatant layer 2 Centrifugalize3000rpm×10min Wash the precipitate separately with 1v alcohol Dry Dry
0.5
0.5
0.5
0.5
Bath the tube with boiling water for 10min, cooling to room temperature and measure the OD620, determine the concentrate of sample according to the standard curve.
chromatography cylinder best to an earlier 30 min saturated exhibition layer agent with filter paper) Do not make sample spots immersion exhibition layer agent. hair dryer blow dry. Spray chromogenic agent evenly, 85℃ dry for 10 minutes, record the spot position and color.
2cm
1cm
2mm
10μl
1.5cm
ห้องสมุดไป่ตู้
Supernatant layer Add alcohol until the concentration is 60% 4℃ 60min centrifugalize Precipitate 2 3000rpm10min
Add alcohol until the
centrifugalize
precipitate 1
Operating procedures
200g flammulina velutipes
cut Water 1000ml
Filtrate(纱布）
Boiled 60-90min
Supernatant layer concentration is 40% 4℃ 30min 3000rpm10min Precipitate 1
in oven drying in vacuum
methanol washing, dissolved with distilled water
thinlayer chromatography
sample quantity control in 550 ug, wait for drying samples (hair dryer blow dry or natural drying)
standard curve.
Sample:0.1mg/mL
1 Standard G(ml) 0.1mg/ml 0
2 0.1
3 0.2
4 0.3
5 0.4
6 0.5
7
8
Sample(ml)
H2O(ml) Sulfuric acid anthrone(ml) 1 0.9 0.8 0.7 4 0.6 0.5
生物制药综合实验（二） 金针菇多糖的制备和鉴定
Purpose
(1) Learn the principle of separation, purification of fungus polysaccharides. (2) Master the quantitive determination of polysaccharides.
Principle
There are many methods for polysaccharide purification, which must correspond to the properties of polysaccharide: (1) Ethanol precipitation; (2) CTAB.
Sample acid hydrolysis
polysaccharide sample 10 mg in clean tube + 2 ml 2 mol/L trifluoroacetic acid
sealing tube (with sealing film and adhesive seal) 100℃ in boiling water 3 hours to hydrolysis
(2) Preparation of sample solution: Weigh out sample (about 10mg) accurately, put them into volumetric flask (scaled 100ml) and then add solution to 100ml. (3) Determination
Principle
flammulina velutipes
treasured medical fungus.
is a kind of traditional
Chinese
The polysaccharide of
flammulina velutipes
has an obvious
effect on immune function, antiinflammatory and antiradiation.
真空干燥 无水乙醇洗涤 真空干燥
沉淀2
(1) Standard curve of G: Weigh out G 10mg accurately, put them into volumetric flask (scaled
100ml) and then add solution to 100ml.
沉淀2 上清1 上清2
<5-15ml水 溶解 <5-15ml水
¼ 体积 of 氯仿：正丁醇(4:1)
分液漏斗振摇20min
离心
3000rpm×10min
¼ 体积 of 氯仿：正丁醇(4:1)
3000rpm×10min 3000rpm×10min 沉淀1
离心
无水乙醇洗涤
上清1 上清 2
3倍，冷无水乙醇 3倍，冷无水乙醇
Principle
Quantitive determination: After hydrolyzed in concentrated sulfuric acid, polysaccharide is further dehydrated into aldose derivation that can form colored compound with anthrone.
crude polysaccharide1 crude polysaccharide1 2
制备工艺：
金针菇子实体（200g） 去杂，切碎 1000ml水 煮 1-1.5h 上清液 上清 搅拌加无水乙醇至终浓度40% 搅拌加无水乙醇至终浓度60% 4℃， 30min 4℃，60min 离心 3000rpm×10min 离心 3000rpm×10min 沉淀1 沉淀2 沉淀1 4层纱布过滤
precipitate 2
dissolve <5-15ml water dissolve
add ¼ volume of chloroform-butanol(4:1) add ¼ volume of Shake 20min centrifugalize 3000rpm10min chloroform-butanol(4:1) Precipitate 1 Precipitate 2
Pick up 10 G tubes, number them, and add reagents according to
following table. Use solution as blank, and test the optical density at 620nm. Fill data you got in the table, and calculate the content based on
Add triple amount of alcohol Supernatant layer 1 Supernatant layer 2 Centrifugalize3000rpm×10min Wash the precipitate separately with 1v alcohol Dry Dry
0.5
0.5
0.5
0.5
Bath the tube with boiling water for 10min, cooling to room temperature and measure the OD620, determine the concentrate of sample according to the standard curve.
chromatography cylinder best to an earlier 30 min saturated exhibition layer agent with filter paper) Do not make sample spots immersion exhibition layer agent. hair dryer blow dry. Spray chromogenic agent evenly, 85℃ dry for 10 minutes, record the spot position and color.
2cm
1cm
2mm
10μl
1.5cm
ห้องสมุดไป่ตู้
Supernatant layer Add alcohol until the concentration is 60% 4℃ 60min centrifugalize Precipitate 2 3000rpm10min
Add alcohol until the
centrifugalize
precipitate 1
Operating procedures
200g flammulina velutipes
cut Water 1000ml
Filtrate(纱布）
Boiled 60-90min
Supernatant layer concentration is 40% 4℃ 30min 3000rpm10min Precipitate 1
in oven drying in vacuum
methanol washing, dissolved with distilled water
thinlayer chromatography
sample quantity control in 550 ug, wait for drying samples (hair dryer blow dry or natural drying)
standard curve.
Sample:0.1mg/mL
1 Standard G(ml) 0.1mg/ml 0
2 0.1
3 0.2
4 0.3
5 0.4
6 0.5
7
8
Sample(ml)
H2O(ml) Sulfuric acid anthrone(ml) 1 0.9 0.8 0.7 4 0.6 0.5