CN101619108B - Method for extracting radiation resistant potentilla anserine polysaccharide oxide by ultrasonic waves - Google Patents

Abstract

The invention provides a method for extracting radiation resistant potentilla anserine polysaccharide oxide by ultrasonic waves, which comprises the following steps: extracting potentilla anserine as raw material for 30 minutes-120 minutes by ultrasonic waves and filtering under the pressure condition of 0.06 MPa-0.08 Mpa; carrying out decompressing concentration, alcohol precipitation and centrifugation on filter liquor at the pressure of 0.06 Mpa-0.08 Mpa and the temperature of 55 DEG C-65 DEG C; collecting sediments at the lower layer; freezing and drying the sediments; after dissolving by nematolyt, extracting by mixture liquid of n-butyl alcohol and chloroform, decompressing, concentrating and dialyzing, obtaining natural potentilla anserine polysaccharide. The natural potentilla anserine polysaccharide has no toxic and side effect and has better repair effect on in-vivo oxidation damage caused by free radicals generated by X-ray radiation. An antioxidant ability experiment of a small mouse radiated by a 3.5Gy X-ray indicates that the SOD activity, the T-AOC vitality and the CAT vitality are respectively improved by 66.7 percent, 54.6 percent and 105.7 percent and the MDA content is reduced by 63.2 percent of an animal with radiation damage by taking the preparation for 7 days.

Description

A kind of method of extracting radiation resistant potentilla anserine polysaccharide oxide by ultrasonic waves

Technical field the present invention relates to the preparation method of natural polysaccharide, and specifically a kind of ultrasonic extraction has the method for the fern amylose of radioprotective oxygenizement.

The background technology ionizing rays also has the radioreaction by water to the direct effect of the existing transmission ofenergy of damage of organism, produces the indirect action of free radical in a large number.Base, ribose and phosphate bond all may suffer the attack of free radical in the dna molecular, cause base and ribose oxidation, splitting of chain, with polytype damage such as protein cross.The lipid peroxidation that free radical causes can cause the out of proportion of saturated and unsaturated fatty acids in the microbial film, changes the hard and soft characteristic of membrane structure, increases the permeability of film, even disintegration takes place.MDA is the degradation production of lipid peroxidation, and its content can reflect the degree of lipid peroxidation and the degree that body is attacked by free radical.SOD and CAT are catalysis O ₂ ^-And decomposing H ₂O ₂Antioxidase, they all reflect the resistance of oxidation of body.Free radical acts on peroxidatic reaction of lipid, and oxidation end product mda etc. can cause the crosslinking polymerization of life macromolecules such as protein, nucleic acid, and this phenomenon is old and feeble fundamental factor.

In order to slow down oxidation, old and feeble process, the body that repairing and treating is injured, people more edible usually contain the food of naturally occurring antioxidant, as contain the fruit, green vegetable, berry etc. of vitamin A, vitamins C, vitamin-E, selenium, zinc.Meanwhile, people are also on purpose seeking the low and active high antioxidant of toxicity.Patent CN 1143084A discloses the crosslinked polysaccharide of a kind of anti-oxidant grafted polysaccharide or antioxidant in treatment of arthritis, as the effect aspect the drug media, reduce tissue adhesion incidence, promote chronic trauma and ulcer healing.Patent CN 101353382A discloses a kind of extracting method with antioxidative active Cordyceps sinensis polysaccharide, has reported a kind of method with Chinese caterpillar fungus waste liquid extraction anti-oxidant activity polysaccharide, and the extraction yield of smart polysaccharide is about 7.8%.Patent CN 1144835A discloses and has a kind ofly repeatedly extracted the method for antioxidant the plant from the fan, and this method is before extracting antioxidant, with change essential oil in the plant of water vapor or supercritical extraction fan, extracts antioxidant wherein more earlier.Patent CN 101233950A discloses a kind of technology of extracting antioxidant from low value sea water fish and leftover protein, and its anti-oxidant activity of the antioxidant that obtains reaches 57.1～94.4% of vitamin-E; Patent CN 101099768A discloses a kind of method of extracting anti-oxidation active substance from Chinese toon old leaf.

Fern fiber crops (the piece root of Rosaceae potentilla potentilla anserina) have the insufficiency of the spleen diarrhoea of treatment, after being ill anaemia, malnutrition, invigorating spleen and reinforcing stomach, promote the production of body fluid to quench thirst, effect such as benefiting vital QI for enriching blood.Patent CN 02138522.X discloses a kind of protective foods and preparation thereof that contains the fern Nettle extract, adopt water and/or extraction using alcohol fern fiber crops, make the fern Nettle extract, add or do not make protective foods, have the effect of middleman's body immunity function, antifatigue with foodstuff additive.Patent CN 101001842A discloses the application of fern fiber crops in the preparation anti-anoxic medicine, can significantly improve survival rate and the survival time of asphyxiating anoxic and acute decompression anoxic mouse.Patent ZL200610090097.5 discloses a kind of preparation method of radiation resistant fern amylose, has obtained strengthening the polysaccharide of animal capability of resistance to radiation, and polysaccharide yield is 6.32% behind the purifying; It is the method for raw material microwave auxiliarily extracting antiradiation polysaccharide with the fern fiber crops that patent CN 101358222A discloses a kind of, and the yield of polysaccharide is 12.18% behind the purifying, is used for the treatment of the radiation injury that X ray causes.

Advantages of ultrasonic is to make the molecule in the sample produce polarization, but between solvent and sample, produce the sound wave cavatition, cause formation, growth and the explosion compression of bubble in the solution, thereby solid sample is disperseed, increase the contact area between sample and the extraction solvent, improve target compound is transferred to liquid phase from solid phase rate of mass transfer.Patent ZL200610166326.7 has authorized a kind of supersonic extracting method of rice bran polysaccharide, ultrasonic power 200～400W, and reckon without the influence of extraction conditions to sample purity, the yield of polysaccharide is lower behind the purifying, be 2.87% only, be difficult to be applied to actual production.

In the above-mentioned technology, conventional extract that there is length consuming time in the polysaccharide method, solvent-oil ratio is big, extraction efficiency low and problem such as running cost height; And microwave extraction can exert an influence to the polysaccharide molecule structure, may cause the degraded of macromolecular chain.The radioprotective oxygenizement is different from simple radiation resistance, needs antioxidant enzyme, antioxidant system comprehensive evaluation with each organ of body.And the variation meeting of polysaccharide structures produces tremendous influence to its biological activity, therefore, how to avoid above-mentioned technical problem in leaching process, overcomes the prior art defective, and obtaining the target organism active substance is problem demanding prompt solution.

Summary of the invention the object of the present invention is to provide a kind of method of extracting radiation resistant potentilla anserine polysaccharide oxide by ultrasonic waves, it uses the ultrasonic extraction method fern amylose first, extraction efficiency height, running cost are low, and can reduce to greatest extent polysaccharide molecule structure and biological activity thereof are exerted an influence.

Another object of the present invention is to provide a kind of fern amylose to have the natural product of the free-radical oxidn damage function of repairing the initiation of X-ray or the application in the protective foods in preparation.

The method of extracting radiation resistant potentilla anserine polysaccharide oxide by ultrasonic waves provided by the invention can be achieved through the following technical solutions, and the steps include:

1) takes by weighing a certain amount of fern fiber crops dry powder, place ultrasonic extraction equipment, add 20～50 times of (m/v) distilled water, stirring, mixing; At ultrasonic power is 100～180W, temperature is 40 ℃～70 ℃ and extracted 30～120 minutes down, under 0.06～0.08MPa pressure condition, filter, filtrate is 0.06～0.08MPa at pressure, temperature is to be evaporated to 1/9～1/3 of original volume under 55 ℃～65 ℃ the condition, in concentrated solution, add ethanol, described ethanol content accounts for and adds 70～80% of overall solution volume behind the ethanol, leave standstill after 24～36 hours centrifugal under 4000～5000 rev/mins rotating speed under the room temperature, collect the lower sediment thing, and at-60～-50 ℃, lyophilize is 24～30 hours under the condition of 1～13Pa, gets substance A;

2) take by weighing measured quantity of material A, dissolved in distilled water with 6～9 times of weight, the back adds 10 μ g/ml papoid liquid, the volume ratio of enzyme liquid and A liquid is 1: 100,40～50 ℃ of following enzymolysis of temperature 10～12 hours, be 1: 2～4 mixed solution again with propyl carbinol and chloroform volume ratio, extract 3～5 times, collect upper water solution, 40～50 ℃ of temperature, under pressure 0.06～0.08MPa condition, be evaporated to 1/10～1/12 of former aqueous solution volume, put into the dialysis tubing of molecular weight cut-off 6000～8000Da, flowing water dialysis 20～24 hours, used distill water dialysis again 18～36 hours, with liquid in the dialysis tubing 40～50 ℃ of temperature, be evaporated to 1/10～1/12 of former aqueous solution volume under pressure 0.06～0.08MPa condition, add ethanol, described ethanol content accounts for and adds 70～80% of overall solution volume behind the ethanol, left standstill 10～14 hours, centrifugal under 4000～5000 rev/mins rotating speed, the collecting precipitation thing is in temperature-60～-50 ℃, lyophilize is 30～36 hours under pressure 1～13Pa condition, gets the fern amylose of purifying.

Fern amylose behind the purifying is through infrared scan, and infrared spectra shows as Fig. 5: at 2829.67cm ^-1There is a characteristic peak at the place, is that the angle vibration of c h bond in the molecule causes; At 3423.56cm ^-1There is a characteristic peak at the place, is that the stretching vibration of O-H causes; 802.3cm ^-1Near absorption peak has shown the existence of α-D-galactopyranose; 862.67cm ^-1The absorption peak explanation at place contains β-D-mannopyranose, and these are the absorption peak of polysaccharide.

The method of above-mentioned extracting radiation resistant potentilla anserine polysaccharide oxide by ultrasonic waves provided by the invention, compare with microwave extraction process with the hot water lixiviate, ultrasonic extraction has following outstanding feature: need not high temperature, the extraction efficiency height, the purity height destroys little to active substance, has broad spectrum, energy consumption is less, and the technology cost is low, and overall economic efficiency is (seeing the following form) significantly.The present invention is to be raw material with the fern fiber crops, and through a kind of natural product that can repair by radiation-induced oxidative damage of ultrasonic extraction, purifying preparation, these natural product have no side effect, and the radioprotective oxidation effectiveness is good.The vivo oxidation that free radical the caused damage that X-radiation is caused has curative effect preferably.Resistance of oxidation experiment through 3.5Gy x-ray bombardment mouse shows: took said preparation and make the SOD activity of radiation injury animal raise 66.7% in 7 days; T-AOC vigor 54.6%, the CAT vigor rises 105.7%, and MDA content descends 63.2%.

Ultrasonic wave is followed the example of with traditional extraction method, microwave method and is compared

Experiment	Temperature (℃)	Time (min)	Solid-liquid ratio	Extraction yield (%)	Total sugar content (%)
						Embodiment 1	50	60	1∶20	9.8625	93.5
Embodiment 2	70	30	1∶35	10.5965	90.3
						Embodiment 3	40	120	1∶50	11.787	92.2
Tradition is extracted	70	180	1∶45	5.73	85.6
						Microwave extraction	55	45	1∶40	12.18	80.6

Fern amylose radioprotective oxidation effectiveness animal experiment of the present invention

60 of laboratory mices are divided into 5 groups at random, and are 12 every group, as follows respectively: normal control group, radiation control group, high/medium/low three the dosage groups (240mg/ (kgb.w.) of polysaccharide; 120mg/ (kgb.w.); 60mg/ (kgb.w.)), the normal control group is irritated stomach physiological saline.Administration is 15 days before the radiation, administration in the 12h after the radiation, and be administered once later every day, surveys the interior anti-oxidant index of body of mouse after 7 days.Illuminate condition is: X ray, mouse single whole body dose 3.5Gy, dose rate 1Gy/min, ource-skin Distance 1m.Except that the normal control group was not shone, other groups were all shone.

By experimental result Fig. 1 as can be known, the radiation control group is compared with the normal control group, and liver CAT vigor obviously reduces the activity of CAT of normal control group apparently higher than radiation control group (p＜0.01).Irradiation back 7d, the CAT activity is compared all with the radiation control group and is increased to some extent between each medicine feed group; Wherein low dose group CAT activation recovering reaches conspicuous level (p＜0.05).

By experimental result Fig. 2 as can be known, the MDA content of the mouse of x-ray bombardment is apparently higher than the normal control group.Irradiation back 7d, MDA content is compared with the radiation control group all and is descended to some extent between each medicine feed group, and wherein the decline of the MDA content of low dose group is the most obvious.

By experimental result Fig. 3 as can be known, the activity of the SOD of normal control group is apparently higher than the radiation control group.Irradiation back 7d, the SOD activity is compared all with the radiation control group and is increased to some extent between each medicine feed group.

By experimental result Fig. 4 as can be known, the radiation control group is compared with the normal control group, and liver T-AOC vigor obviously reduces, and reaches utmost point conspicuous level (p＜0.01).Irradiation back 7d, the T-AOC activity is compared all with model control group and is increased to some extent between each medicine feed group.

Show by above-mentioned experimentation on animals result: the fern amylose of the present invention's preparation can be applied to prepare in the natural product or protective foods with the free-radical oxidn damage function of repairing the initiation of X-ray.

Description of drawings Fig. 1 is the influence of fern amylose preparation to radiation murine liver CAT vigor

Fig. 2 is the influence of fern amylose preparation to radiation murine liver MDA content

Fig. 3 is the influence of fern amylose preparation to the radiation murine hepatic SOD

Fig. 4 is the influence of fern amylose preparation to radiation murine liver T-AOC vigor

Fig. 5 is the infrared scan figure to the fern amylose preparation

The invention will be further described below in conjunction with embodiment and comparative example for embodiment:

Embodiment 1:

1) takes by weighing a certain amount of fern fiber crops dry powder, place ultrasonic extraction equipment, add 20 times of (m/v) distilled water, stirring, mixing; At ultrasonic power is that 100W, temperature are to extract 60 minutes under 50 ℃ of conditions, under the condition of pressure 0.07MPa, filter, filtrate is to be evaporated to 1/3 of original volume under 60 ℃ of conditions in pressure 0.08MPa, temperature, in concentrated solution, add ethanol (ethanol content account for add overall solution volume behind the ethanol 75%), after leaving standstill 30 hours under the room temperature, centrifugal under 4000 rev/mins rotating speed, collect the lower sediment thing at-55 ℃, lyophilize is 28 hours under the condition of pressure 1Pa, gets substance A;

2) take by weighing measured quantity of material A, dissolved in distilled water with 6 times of weight, the back adds 10 μ g/ml papoid liquid, the ratio of enzyme liquid and A liquid is 1: 100 (v/v), 45 ℃ of following enzymolysis 11 hours, use propyl carbinol and chloroform mixed solution (1: 3 again, v/v) extraction is 4 times, collect upper water solution, under 45 ℃ of temperature, pressure 0.07MPa condition, be evaporated to 1/11 of former aqueous solution volume, put into the dialysis tubing of molecular weight cut-off 6000～8000Da, flowing water dialysis 22 hours was used distill water dialysis 24 hours again.Liquid in the dialysis tubing is evaporated to 1/11 of former aqueous solution volume under 45 ℃ of temperature, pressure 0.07MPa condition, add ethanol (ethanol content account for add overall solution volume behind the ethanol 75%), left standstill 12 hours, centrifugal under 4500 rev/mins rotating speed, the lyophilize 33 hours under temperature-55 ℃, pressure 1Pa condition of collecting precipitation thing, the fern amylose of purifying.

The yield of purified polysaccharide is 9.8625%, and detecting its total sugar content through the phenolsulfuric acid method is 93.5%.

Embodiment 2:

1) takes by weighing a certain amount of fern fiber crops dry powder, place ultrasonic extraction equipment, add 35 times of (m/v) distilled water, stirring, mixing; At ultrasonic power is that 180W, temperature are to extract 30 minutes under 70 ℃ of conditions, under the condition of pressure 0.06MPa, filter, filtrate is to be evaporated to 1/9 of original volume under 55 ℃ of conditions in pressure 0.07MPa, temperature, in concentrated solution, add ethanol (ethanol content account for add overall solution volume behind the ethanol 80%), after leaving standstill 36 hours under the room temperature, centrifugal under 5000 rev/mins rotating speed, collect the lower sediment thing at-60 ℃, lyophilize is 30 hours under the condition of 13Pa, gets substance A;

2) take by weighing measured quantity of material A, dissolved in distilled water with 9 times of weight, the back adds 10 μ g/ml papoid liquid, the ratio of enzyme liquid and A liquid is 1: 100 (v/v), 40 ℃ of following enzymolysis 12 hours, use propyl carbinol and chloroform mixed solution (1: 2 again, v/v) extraction is 5 times, collect upper water solution, under 40 ℃ of temperature, pressure 0.08MPa, be evaporated to 1/12 of former aqueous solution volume, put into the dialysis tubing of molecular weight cut-off 6000～8000Da, flowing water dialysis 24 hours was used distill water dialysis 18 hours again.Liquid in the dialysis tubing is evaporated to 1/12 of former aqueous solution volume under 50 ℃ of temperature, pressure 0.06MPa condition, add ethanol (ethanol content account for add overall solution volume behind the ethanol 80%), left standstill 10 hours, centrifugal under 4000 rev/mins rotating speed, the lyophilize 30 hours under temperature-50 ℃, pressure 13Pa condition of collecting precipitation thing, the fern amylose of purifying.

The yield of purified polysaccharide is 10.5965%, and detecting its total sugar content through the phenolsulfuric acid method is 90.3%.

Embodiment 3:

1) takes by weighing a certain amount of fern fiber crops dry powder, place ultrasonic extraction equipment, add 50 times of (m/v) distilled water, stirring, mixing; At ultrasonic power is that 150W, temperature are to extract 120 minutes under 40 ℃ of conditions, under the condition of pressure 0.08MPa, filter, filtrate is to be evaporated to 1/6 of original volume under 65 ℃ of conditions in pressure 0.06MPa, temperature, in concentrated solution, add ethanol (ethanol content account for add overall solution volume behind the ethanol 70%), leave standstill after 24 hours centrifugal under 4000 rev/mins rotating speed under the room temperature, collect the lower sediment thing at 50 ℃, lyophilize is 24 hours under the condition of pressure 8Pa, gets substance A;

2) take by weighing measured quantity of material A, dissolved in distilled water with 8 times of weight, the back adds 10 μ g/ml papoid liquid, the ratio of enzyme liquid and A liquid is 1: 100 (v/v), 50 ℃ of following enzymolysis 10 hours, use propyl carbinol and chloroform mixed solution (1: 4 again, v/v) extraction is 3 times, collect upper water solution, under 50 ℃ of temperature, pressure 0.06MPa condition, be evaporated to 1/10 of former aqueous solution volume, put into the dialysis tubing of molecular weight cut-off 6000～8000Da, flowing water dialysis 20 hours was used distill water dialysis 36 hours again.Liquid in the dialysis tubing is evaporated to 1/10 of former aqueous solution volume under 40 ℃ of temperature, pressure 0.08MPa condition, add ethanol (ethanol content account for add overall solution volume behind the ethanol 80%), left standstill 14 hours, centrifugal under 5000 rev/mins rotating speed, the lyophilize 36 hours under temperature-60 ℃, pressure 10Pa condition of collecting precipitation thing, the fern amylose of purifying.

The yield of purified polysaccharide is 11.787%, and detecting its total sugar content through the phenolsulfuric acid method is 92.2%.

Comparative example 1:

Method and hot water lixiviate with embodiment 1 experimentize.The difference of two experiments are that the hot water lixiviate adopts 70 ℃ of heating to extract to add in 180min, the fern fiber crops dry powder 45 times of distilled water to replace ultrasonic extraction in the embodiment step 1.

1) takes by weighing a certain amount of fern fiber crops dry powder, place the 2000ml beaker, add 45 times of (m/v) distilled water, stirring, mixing; Being 70 ℃ in temperature extracted 180 minutes down, under the condition of pressure 0.07MPa, filter, filtrate is evaporated to 1/3 of original volume under pressure 0.08MPa, 60 ℃ of conditions of temperature, in concentrated solution, add ethanol (ethanol content account for add overall solution volume behind the ethanol 75%), leave standstill after 30 hours centrifugal under 4000 rev/mins rotating speed under the room temperature, collect the lyophilize 28 hours under the condition of temperature-55 ℃, pressure 1Pa of lower sediment thing, substance A;

2) take by weighing measured quantity of material A, dissolved in distilled water with 6 times of weight, the back adds 10 μ g/ml papoid liquid, the ratio of enzyme liquid and A liquid is 1: 100 (v/v), 45 ℃ of following enzymolysis 11 hours, use propyl carbinol and chloroform mixed solution (1: 3 again, v/v) extraction is 4 times, collect upper water solution, under 45 ℃ of temperature, pressure 0.07MPa condition, be evaporated to 1/11 of former aqueous solution volume, put into the dialysis tubing of molecular weight cut-off 6000～8000Da, flowing water dialysis 22 hours was used distill water dialysis 24 hours again.Liquid in the dialysis tubing is evaporated to 1/11 of former aqueous solution volume under 45 ℃ of temperature, pressure 0.07MPa condition, add ethanol (ethanol content account for add overall solution volume behind the ethanol 75%), left standstill 12 hours, centrifugal under 4500 rev/mins rotating speed, the lyophilize 33 hours under the condition of temperature-55 ℃, pressure 1Pa of collecting precipitation thing, the fern amylose of purifying.

The yield of purified polysaccharide is 5.73%, and detecting its total sugar content through the phenolsulfuric acid method is 85.6%.

Comparative example 2:

Extract with the method for embodiment 2 and microwave-assisted and to experimentize.The difference of two experiments is microwave-assisted extraction employing frequency 915MHz, power 300W, and 40 times of distilled water of adding replace the ultrasonic extraction in the embodiment step 1 in 55 ℃ of heating extraction 45min of temperature and the fern fiber crops dry powder.

1) takes by weighing a certain amount of fern fiber crops dry powder, place Microwave Extraction Equipment, add 40 times of (m/v) distilled water, stirring, mixing; In frequency 915MHz, power 300W, temperature is to extract 45 minutes under 55 ℃ of conditions, under the condition of pressure 0.06MPa, filter, filtrate is to be evaporated to 1/9 of original volume under 55 ℃ of conditions in pressure 0.07MPa, temperature, in concentrated solution, add ethanol (ethanol content account for add overall solution volume behind the ethanol 80%), leave standstill after 36 hours centrifugal under 5000 rev/mins rotating speed under the room temperature, collect the lyophilize 30 hours under the condition of temperature-60 ℃, pressure 13Pa of lower sediment thing, substance A;

2) take by weighing measured quantity of material A, dissolved in distilled water with 9 times of weight, the back adds 10 μ g/ml papoid liquid, the ratio of enzyme liquid and A liquid is 1: 100 (v/v), 40 ℃ of following enzymolysis 12 hours, use propyl carbinol and chloroform mixed solution (1: 2 again, v/v) extraction is 5 times, collect upper water solution, under 40 ℃ of temperature, pressure 0.08MPa condition, be evaporated to 1/12 of former aqueous solution volume, put into the dialysis tubing of molecular weight cut-off 6000～8000Da, flowing water dialysis 24 hours was used distill water dialysis 18 hours again.Liquid in the dialysis tubing is evaporated to 1/12 of former aqueous solution volume under 50 ℃ of temperature, pressure 0.06MPa condition, add ethanol (ethanol content account for add overall solution volume behind the ethanol 80%), left standstill 10 hours, centrifugal under 4000 rev/mins rotating speed, the lyophilize 30 hours under the condition of temperature-50 ℃, pressure 13Pa of collecting precipitation thing, the fern amylose of purifying.

The yield of purified polysaccharide is 12.18%, and detecting its total sugar content through the phenolsulfuric acid method is 80.6%.

Claims (1)

1. the method for an extracting radiation resistant potentilla anserine polysaccharide oxide by ultrasonic waves is characterized in that steps of the method are:

1) takes by weighing a certain amount of fern fiber crops dry powder, place ultrasonic extraction equipment, add 20～50 times of (m/v) distilled water, stirring, mixing; At ultrasonic power is 100～180W, temperature is 40 ℃～70 ℃ and extracted 30～120 minutes down, under 0.06～0.08MPa pressure condition, filter, filtrate is at pressure 0.06～0.08MPa, temperature is to be evaporated to 1/9～1/3 of original volume under 55 ℃～65 ℃, in concentrated solution, add ethanol, described ethanol content accounts for and adds 70～80% of overall solution volume behind the ethanol, leave standstill after 24～36 hours centrifugal under 4000～5000 rev/mins rotating speed under the room temperature, collect the lower sediment thing, and at-60～-50 ℃, lyophilize is 24～30 hours under the condition of 1～13Pa, gets substance A;

2) take by weighing measured quantity of material A, dissolved in distilled water with 6～9 times of weight, the back adds 10 μ g/ml papoid liquid, the volume ratio of enzyme liquid and A liquid is 1: 100,40～50 ℃ of following enzymolysis of temperature 10～12 hours, be 1: 2～4 mixed solution again with propyl carbinol and chloroform volume ratio, extract 3～5 times, collect upper water solution, 40～50 ℃ of temperature, under pressure 0.06～0.08MPa condition, be evaporated to 1/10～1/12 of former aqueous solution volume, put into the dialysis tubing of molecular weight cut-off 6000～8000Da, flowing water dialysis 20～24 hours, used distill water dialysis again 18～36 hours, with liquid in the dialysis tubing 40～50 ℃ of temperature, be evaporated to 1/10～1/12 of former aqueous solution volume under pressure 0.06～0.08MPa condition, add ethanol, described ethanol content accounts for and adds 70～80% of overall solution volume behind the ethanol, left standstill 10～14 hours, centrifugal under 4000～5000 rev/mins rotating speed, the collecting precipitation thing is in temperature-60～-50 ℃, lyophilize is 30～36 hours under pressure 1～13Pa condition, gets the fern amylose of purifying.

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