CN105153324A - Golden camellia polysaccharide extracting method - Google Patents
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Abstract
The invention provides a golden camellia polysaccharide extracting method. The golden camellia polysaccharide extracting method golden camellia polysaccharide extracting method comprises the following steps that 1, golden camellia leaves are subjected to extraction with water to obtain a water extract solution; 2, after the water extract solution is concentrated, ethanol is added to perform precipitation, and a precipitate is obtained for standby application; 3, the precipitate obtained in the step 2 is washed by using absolute ethyl alcohol and acetone sequentially, then distilled water is added for dissolution, and then protease is added to perform enzymolysis so as to obtain enzymatic hydrolysate; 4, the enzymatic hydrolysate is processed under frequency ultrasonic waves, dialysis is performed by using a dialysis bag capable of cutting off 18000-28000 molecular weight, the retained liquid is subjected to dialysis by using a dialysis bag capable of cutting off 400000-500000 molecular weight, the obtained permeated liquid is concentrated, then ethanol is added for precipitation, and a precipitate is obtained for standby application; 5, the precipitate obtained in the step 4 is washed by using absolute ethyl alcohol and acetone sequentially, then distilled water is added for dissolution, and activated carbon decoloration, filtration and drying are performed to obtain golden camellia polysaccharide. The golden camellia polysaccharide prepared by means of the golden camellia polysaccharide extracting method has higher immunological competence.
Description
Technical field
The invention belongs to technical field of biological extraction, be specifically related to the extracting method of camellia chrysantha polysaccharide.
Background technology
Camellia nitidissima Chi (Camelliachrysantha (Hu) Tuyama) belongs to Theaceae Camellia Camellia Section Chrysantha, is described as " tea race queen " and " vegitabilia giant panda ".Camellia nitidissima Chi leaf, as a kind of herbal medicine of Zhuang nationality in Guangxi folk tradition, has hypoglycemic, reducing blood-fat, Tumor suppression growth, improves the different physiological roles such as body immunity, and wherein, camellia chrysantha polysaccharide is considered to one of important functional component." research of camellia chrysantha polysaccharide anti-mouse immunological liver injury effect " (is published in West China pharmaceutical journal, 2014, 9 (5): 525 ~ 527) extracting method disclosing camellia chrysantha polysaccharide is: the Camellia nitidissima Chi leaf meal 1kg of drying is added 10L sherwood oil backflow degreasing and extract 2 times, each extraction 1.5h, after waving the sherwood oil in the most dregs of a decoction, boiling water extraction 2 times, each 2h, united extraction liquid, be concentrated into 150ml, repeatedly extract with chloroform-propyl carbinol (3:1), be removed the supernatant liquor of protein, add 95% alcohol settling to spend the night, suction filtration, abandoning supernatant, filter residue uses ether successively, dehydrated alcohol, washing with acetone, obtain Camellia nitidissima Chi Crude polysaccharides.Through DE52 type resin chromatography post wash-out, vacuum-drying obtains Powdered camellia chrysantha polysaccharide, adopts phend-sulphuric acid colour developing, UV-vis spectroscopy measurement is fixed and calculated purity is 69.16%.The document further discloses BCG combined LPS inducing mouse and occurs immunological liver injury, when Mice Body inner cell receives immune attack, show as internal organ to damage, the index rising caused as hepatosplenomegaly and the Thymus atrophy caused by immunocompromised, this shows that BCG combines LPS and has the special aggressiveness of target organ.After camellia chrysantha polysaccharide treatment, mice organs degree of injury obviously alleviates, consistent with the dye result of pathological examination of HE, and this shows that camellia chrysantha polysaccharide passes through recovery body internal organs activity and strengthening immunity, thus suppresses hepatocellular immunotoxicity.Although above-mentioned document discloses camellia chrysantha polysaccharide have certain immunocompetence, immunocompetence is not high.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of extracting method of camellia chrysantha polysaccharide, and the immunomodulatory effect that the method extracts the camellia chrysantha polysaccharide obtained significantly improves than existing camellia chrysantha polysaccharide.
Technical scheme provided by the invention is the extracting method of camellia chrysantha polysaccharide, comprises the following steps:
1) by Camellia nitidissima Chi leaf extracting in water, Aqueous extracts is obtained;
2), after Aqueous extracts is concentrated, add alcohol settling, taking precipitate is for subsequent use;
3) by step 2) throw out use dehydrated alcohol, washing with acetone successively, then adding distil water dissolves, then adds protease hydrolyzed, obtains enzymolysis solution;
4) enzymolysis solution being placed in frequency is process 20 ~ 80s under 1000 ~ 5000kHz ultrasonic wave, then be the dialysis tubing dialysis 24 ~ 72h of 18000 ~ 28000 through molecular weight cut-off; go bail for and stay liquid to be the dialysis tubing dialysis 24 ~ 72h of 400000 ~ 500000 through molecular weight cut-off; get permeate and concentrate; add alcohol settling again, taking precipitate is for subsequent use;
5) by step 4) described in throw out use dehydrated alcohol, washing with acetone successively, then adding distil water dissolves, activated carbon decolorizing, filters, dry, is camellia chrysantha polysaccharide.
Step 1) in, extracting in water refers to the water adding its weight 20 ~ 30 times in Camellia nitidissima Chi leaf, and refluxing extraction 2 ~ 3 times, extracts 1 ~ 3h at every turn, united extraction liquid, and filter, filtrate is Aqueous extracts.
Step 2) in, Aqueous extracts being concentrated into relative density at 60 DEG C is the medicinal extract of 1.1 ~ 1.3.
Step 2) in, described in add alcohol settling and refer to that ethanol to the alcohol content adding 90 ~ 95v% in concentrated solution is 80 ~ 85v%.
Step 3) in, the consumption of dehydrated alcohol, acetone is 2 ~ 4 times of weight of precipitate, and the consumption of distilled water is 2 ~ 4 times of the Sediment weight after washing.
Step 3) in, the consumption of proteolytic enzyme is 0.3 ~ 3% of Camellia nitidissima Chi leaf dry weight, and hydrolysis temperature is 40 ~ 60 DEG C, and enzymolysis time is 1 ~ 3h.Macro-molecular protein enzymolysis in extract can be polypeptide and amino acid by proteolytic enzyme.
Step 4) in, enzymolysis solution being placed in frequency is process 20 ~ 80s under 1000 ~ 5000kHz ultrasonic wave, the part sugar chain of great for molecular weight polysaccharide (pectin) can be disconnected, reduce molecular weight, reduce viscosity, improve water-soluble, strengthen immunocompetence.To be 20 ~ 80s be ultrasonic treatment time is greater than the glycosides key of the polysaccharide of 500,000 in order to ensure only broken molecule amount, and the breaking degree of polysaccharide molecule is less, does not destroy activity unit, to reach reduction molecular weight, improve water-soluble, and strengthen immunocompetent object.
The dialysis tubing that enzymolysis solution after ultrasonication is first 18000 ~ 28000 through molecular weight cut-off carries out first time dialysis, and small molecules pigment, polypeptide and amino acid and proteolytic enzyme through dialysis tubing filtering, will improve the purity retaining liquid greatly.Be the dialysis tubing dialysis 24 ~ 72h of 400000 ~ 500000 again through molecular weight cut-off by reservation liquid, obtain the effluent liquid of molecular weight at the camellia chrysantha polysaccharide in 18000 ~ 28000 to 400000 ~ 500000 intervals, described permeate (effluent liquid) is concentrated refers to that being concentrated into relative density at 60 DEG C is the medicinal extract of 1.1 ~ 1.3.The described alcohol settling that adds refers to that ethanol to the alcohol content adding 90 ~ 95v% in concentrated solution is 80 ~ 85v%.
Step 5) in, the consumption of dehydrated alcohol, acetone is 2 ~ 4 times of weight of precipitate, and the consumption of distilled water is 2 ~ 4 times of the Sediment weight after washing.
The present invention adopts ultra-high frequency ultrasonic wave by the glycosides bond rupture in great for molecular weight polysaccharide molecule (molecular weight is more than 500000), reduces its molecular weight, reduces viscosity, improves water-soluble, greatly strengthen the immunocompetence of end product.After testing, the purity of end product is more than 98.8%.
Embodiment
The present invention is further elaborated for following specific embodiment, but not as a limitation of the invention.
Embodiment 1
1) pulverized by Camellia nitidissima Chi cured leaf, add the water of its weight 20 times, refluxing extraction 2 times, extracts 1h at every turn, united extraction liquid, and filter, filtrate is Aqueous extracts and obtains Aqueous extracts;
2) Aqueous extracts being concentrated into relative density at 60 DEG C is the medicinal extract of 1.1, and the ethanol adding 90v% is 80v% to alcohol content, and staticly settle, suction filtration, taking precipitate is for subsequent use;
3) by step 2) throw out use dehydrated alcohol, washing with acetone successively, the consumption of dehydrated alcohol, acetone is 2 times of weight of precipitate, then adding distil water dissolves, the consumption of distilled water is 2 times of the Sediment weight after washing, add the proteolytic enzyme of Camellia nitidissima Chi leaf dry weight 0.3% again, at 40 DEG C, enzymolysis time is 1 ~ 3h, and enzyme-deactivating, obtains enzymolysis solution;
4) enzymolysis solution being placed in frequency is process 20s under 1000kHz ultrasonic wave, then be the dialysis tubing dialysis 24h of 18000 ~ 28000 through molecular weight cut-off; go bail for and stay liquid to be the dialysis tubing dialysis 24h of 400000 ~ 500000 through molecular weight cut-off; getting permeate, to be concentrated into relative density at 60 DEG C be the medicinal extract of 1.1; ethanol to the alcohol content adding 90v% is 80v%; staticly settle, suction filtration, taking precipitate is for subsequent use;
5) by step 4) described in throw out use dehydrated alcohol, washing with acetone successively, the consumption of dehydrated alcohol, acetone is 2 times of weight of precipitate, then adding distil water dissolves, the consumption of distilled water is 2 times of the Sediment weight after washing, activated carbon decolorizing, filter, dry, be camellia chrysantha polysaccharide.After testing, purity is 98.8%.
Embodiment 2
1) pulverized by Camellia nitidissima Chi cured leaf, add the water of its weight 30 times, refluxing extraction 3 times, extracts 3h at every turn, united extraction liquid, and filter, filtrate is Aqueous extracts and obtains Aqueous extracts;
2) Aqueous extracts being concentrated into relative density at 60 DEG C is the medicinal extract of 1.3, and the ethanol adding 95v% is 85v% to alcohol content in mixed solution, and staticly settle, suction filtration, taking precipitate is for subsequent use;
3) by step 2) throw out use dehydrated alcohol, washing with acetone successively, the consumption of dehydrated alcohol, acetone is 4 times of weight of precipitate, then adding distil water dissolves, the consumption of distilled water is 4 times of the Sediment weight after washing, add the proteolytic enzyme of Camellia nitidissima Chi leaf dry weight 3% again, at 60 DEG C, enzymolysis time is 3h, and enzyme-deactivating, obtains enzymolysis solution;
4) enzymolysis solution being placed in frequency is process 80s under 5000kHz ultrasonic wave, then be the dialysis tubing dialysis 72h of 18000 ~ 28000 through molecular weight cut-off; go bail for and stay liquid to be the dialysis tubing dialysis 72h of 400000 ~ 500000 through molecular weight cut-off; getting permeate, to be concentrated into relative density at 60 DEG C be the medicinal extract of 1.3; ethanol to the alcohol content adding 95v% is 85v%; staticly settle, suction filtration, taking precipitate is for subsequent use;
5) by step 4) described in throw out use dehydrated alcohol, washing with acetone successively, the consumption of dehydrated alcohol, acetone is 4 times of weight of precipitate, then adding distil water dissolves, the consumption of distilled water is 4 times of the Sediment weight after washing, activated carbon decolorizing, filter, dry, be camellia chrysantha polysaccharide.After testing, purity is 99.0%.
Embodiment 3
1) pulverized by Camellia nitidissima Chi leaf, add the water of its weight 25 times, refluxing extraction 2 times, extracts 2h at every turn, united extraction liquid, and filter, filtrate is Aqueous extracts and obtains Aqueous extracts;
2) Aqueous extracts being concentrated into relative density at 60 DEG C is the medicinal extract of 1.2, and the ethanol adding 90v% is 80v% to alcohol content in mixed solution, and staticly settle, suction filtration, taking precipitate is for subsequent use;
3) by step 2) throw out use dehydrated alcohol, washing with acetone successively, the consumption of dehydrated alcohol, acetone is 3 times of weight of precipitate, then adding distil water dissolves, the consumption of distilled water is 3 times of the Sediment weight after washing, add the proteolytic enzyme of Camellia nitidissima Chi leaf dry weight 1.5% again, at 50 DEG C, enzymolysis time is 2h, and enzyme-deactivating, obtains enzymolysis solution;
4) enzymolysis solution being placed in frequency is process 40s under 4000kHz ultrasonic wave, then be the dialysis tubing dialysis 48h of 18000 ~ 28000 through molecular weight cut-off; go bail for and stay liquid to be the dialysis tubing dialysis 48h of 400000 ~ 500000 through molecular weight cut-off; getting permeate, to be concentrated into relative density at 60 DEG C be the medicinal extract of 1.2; ethanol to the alcohol content adding 90v% is 80v%; staticly settle, suction filtration, taking precipitate is for subsequent use;
5) by step 4) described in throw out use dehydrated alcohol, washing with acetone successively, the consumption of dehydrated alcohol, acetone is 3 times of weight of precipitate, then adding distil water dissolves, the consumption of distilled water is 3 times of the Sediment weight after washing, activated carbon decolorizing, filter, dry, be camellia chrysantha polysaccharide.After testing, purity is 98.9%.
Embodiment 4
1) pulverized by Camellia nitidissima Chi leaf, add the water of its weight 20 times, refluxing extraction 3 times, extracts 1h at every turn, united extraction liquid, and filter, filtrate is Aqueous extracts and obtains Aqueous extracts;
2) Aqueous extracts being concentrated into relative density at 60 DEG C is the medicinal extract of 1.3, and the ethanol adding 90v% is 85v% to alcohol content in mixed solution, and staticly settle, suction filtration, taking precipitate is for subsequent use;
3) by step 2) throw out use dehydrated alcohol, washing with acetone successively, the consumption of dehydrated alcohol, acetone is 2 times of weight of precipitate, then adding distil water dissolves, the consumption of distilled water is 4 times of the Sediment weight after washing, add the proteolytic enzyme of Camellia nitidissima Chi leaf dry weight 0.3% again, at 60 DEG C, enzymolysis time is 1h, and enzyme-deactivating, obtains enzymolysis solution;
4) enzymolysis solution being placed in frequency is process 80s under 1000kHz ultrasonic wave, then be the dialysis tubing dialysis 24h of 18000 ~ 28000 through molecular weight cut-off; go bail for and stay liquid to be the dialysis tubing dialysis 72h of 400000 ~ 500000 through molecular weight cut-off; getting permeate, to be concentrated into relative density at 60 DEG C be the medicinal extract of 1.1; ethanol to the alcohol content adding 95v% is 80v%; staticly settle, suction filtration, taking precipitate is for subsequent use;
5) by step 4) described in throw out use dehydrated alcohol, washing with acetone successively, the consumption of dehydrated alcohol, acetone is 4 times of weight of precipitate, then adding distil water dissolves, the consumption of distilled water is 2 times of the Sediment weight after washing, activated carbon decolorizing, filter, dry, be camellia chrysantha polysaccharide.After testing, purity is 99.1%.
Experimental example
1, camellia chrysantha polysaccharide is on the impact of experimental immune liver injury
Choose 70 Kunming mouses, body weight 20 ± 2g, male and female half and half, be divided into 7 groups at random, be respectively blank group, model group, positive controls and experimental group 1 ~ 4, laboratory animal drinking public water supply, put room temperature 20 ~ 25 DEG C, after adapting to 1 week in the laboratory environment of humidity 60 ~ 75%, start experiment.Experiment first day is except blank group, and all the other respectively organize rat equal abdominal injection BCG (bacille Calmette-Guerin vaccine) 8mg (about 5 × 10
7individual viable bacteria).Within 2nd day, rise, except blank group, each group rat starts administration, model group gastric infusion physiological saline, and consumption is 0.2ml/kg; The camellia chrysantha polysaccharide of positive controls gavage background technology, consumption is 0.2g/kg; The medicine of experimental group 1 ~ 4 difference gastric infusion embodiment 1 ~ 4, consumption is 0.1g/kg.Successive administration 12d, after last administration last administration 16h, blank group tail vein injection saline, all the other respectively organize mouse peritoneal injection LPS normal saline solution 30 μ g/kg body weight, eyeball blood sampling after 8h.Calculate liver, Thymus and spleen index, and adopt HE dyeing to carry out liver histopathology inspection, adopt Elisa method to detect the content of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and microsome nitrogen protoxide (NO) in hepatic tissue.
Table 1
Note: compared with blank group, * P < 0.05, * * P < 0.01; Compared with model group, △ P < 0.05, △ △ P < 0.01.
As shown in Table 1, the liver in model group mice plasma and Spleen coefficient significantly rise, and thymus index reduces.Liver coefficient, the Spleen coefficient of experimental mice reduce, and thymus index increases, and compared with model group, has significant difference.TNF-α, IL-6 and NO level in model group murine liver tissue obviously increases, and there were significant differences compared with blank group, and modeling success is described.In the hepatic tissue of experimental mice, the level of TNF-α, IL-6 and NO reduces, and compare with model group that there were significant differences, the liver of polyoses extract to immunological liver injury in mice of visible experimental group has certain provide protection.Experimental group is compared with positive controls; liver index, index and spleen index significantly reduce; thymus index significantly increases, and TNF-α, IL-6 and NO level obviously reduces, and illustrates that polysaccharide of the present invention has better provide protection than the liver of polysaccharide to immunological liver injury in mice of background technology.
2, camellia chrysantha polysaccharide is on the impact of mouse phagocytic cell and index
Get the Kunming mouse 70 that body weight is 20g ± 2g, male and female half and half, be divided into 7 groups at random, be respectively blank group, model group (physiological saline is by 0.2ml/kg body weight), experimental group 1 ~ 4 (camellia chrysantha polysaccharide of administration embodiment successively 1 ~ 4, by the administration of 0.1g/kg body weight) and positive controls (camellia chrysantha polysaccharide of background technology presses the administration of 0.2g/kg body weight).Gastric infusion, every day 1 time, successive administration 7d.Administration the 2nd day and the 5th day are except normal group, and all the other respectively organize lesser sac injection endoxan 80mg/kg, make immunosuppressed mice model.Administration 5d, except normal group, each group of mouse peritoneal injects 2% starch normal saline solution 1.5ml, after last administration 24h, the CRBC suspension 1ml of each group mouse peritoneal injection 2% (CRBC:NS, V/V), after 2h, cervical dislocation is put to death, gently rub belly, extract peritoneal fluid 0.5ml, calculate phagocytic percentage and phagocytic index.
Table 2
| Group | Phagocytic percentage | Phagocytic index |
| Blank group | 34.6±3.91 | 0.57±0.13 |
| Model group | 20.22±3.12** | 0.32±0.09* |
| Positive controls | 35.53±4.24△ | 0.59±0.15△△ |
| Experimental group 1 | 56.82±5.33△△ | 0.79±0.18△△ |
| Experimental group 2 | 58.13±3.29△ | 0.80±0.16△△ |
| Experimental group 3 | 59.42±5.34△△ | 0.81±0.20△ |
| Experimental group 4 | 57.16±6.10△ | 0.79±0.07△△ |
Note: compared with blank group, * P < 0.05, * * P < 0.01; Compared with model group, △ P < 0.05, △ △ P < 0.01.
As shown in Table 2, model group phagocytic percentage and phagocytic index, significantly lower than blank group, illustrate immunosuppressed mice model success.Wherein between experimental group and model group, there were significant differences, shows that camellia chrysantha polysaccharide can significantly improve phagocytic percentage and the phagocytic index of immunosuppressed mice peritoneal macrophage caused by Cy.Experimental group is compared with positive controls, and phagocytic percentage and phagocytic index significantly improve, known, and camellia chrysantha polysaccharide of the present invention has better non-specific immune function compared with the camellia chrysantha polysaccharide of background technology to immunosuppressed mice.
3, camellia chrysantha polysaccharide is on the impact of mice serum hemolysin level
Get body weight 20g ± 2g Kunming mouse 70, male and female half and half, be divided into 7 groups at random, often organize 10, be respectively blank group, model group (physiological saline is by 0.2ml/kg body weight), positive controls (camellia chrysantha polysaccharide of background technology is by the administration of 0.2g/kg body weight) and experimental group 1 ~ 4 (camellia chrysantha polysaccharide of administration embodiment successively 1 ~ 4, by the administration of 0.1g/kg body weight).Gastric infusion, every day 1 time, successive administration 7d.Administration the 2nd day and the 5th day are except normal group, and all the other respectively organize lesser sac injection endoxan 80mg/kg, make immunosuppressed mice model.Respectively organize mouse peritoneal in the 7d of first administration and inject 20% sheep red blood cell (SRBC) (SRBC) 0.2ml, 4d after immunity, goes eyeball to get blood, separation of serum, is supplied afterwards to measure by serum with normal saline dilution 500 times.
Diluted mice serum 1ml, 5%SRBC0.5ml is added successively, 10% complement 1ml in reaction tubes, put in 37 DEG C of waters bath with thermostatic control and be incubated 30min, then move to termination reaction in ice bath, the centrifugal 5min of 1500r/min, gets supernatant liquor 1ml, add Dou Shi reagent 3ml, shake up and place 10min, in 540nm wavelength working sample absorbance, with not increase serum dual-purpose physiological saline replacement, but add equally sheep red blood cell (SRBC) and complement as sample blank control tube, measure absorbance.
Get 5%SRBC0.25ml, add Dou Shi reagent to 4ml, shake up and place 10min in 540nm wavelength colorimetric reading absorbancy, be absorbance (HC during SRBC HD50
50).
Table 3
| Group | Half hemolysis value (HC 50) |
| Normal group | 18.62±4.35 |
| Model group | 10.55±2.32* |
| Positive controls | 19.23±4.16△ |
| Experimental group 1 | 32.87±5.53△△ |
| Experimental group 2 | 33.12±5.10△△ |
| Experimental group 3 | 34.01±4.33△ |
| Experimental group 4 | 33.10±6.03△ |
Note: compared with blank group, * P < 0.05, * * P < 0.01; Compared with model group, △ P < 0.05, △ △ P < 0.01.
As shown in Table 3, the camellia chrysantha polysaccharide of experimental group can suppress the serum hemolysin of mouse to generate by Promote immunity, on promotion Hemolysin formation, there is significant difference with model group, illustrate that camellia chrysantha polysaccharide significantly can resist the restraining effect of CY induced mice serum hemolysin formation.Experimental group is compared with positive controls, and the camellia chrysantha polysaccharide of experimental group is formed immune response later stage antibody secreting cell has better enhancement.
Claims (9)
1. the extracting method of camellia chrysantha polysaccharide, is characterized in that: comprise the following steps:
1) by Camellia nitidissima Chi leaf extracting in water, Aqueous extracts is obtained;
2), after Aqueous extracts is concentrated, add alcohol settling, taking precipitate is for subsequent use;
3) by step 2) throw out use dehydrated alcohol, washing with acetone successively, then adding distil water dissolves, then adds protease hydrolyzed, obtains enzymolysis solution;
4) enzymolysis solution being placed in frequency is process 20 ~ 80s under 1000 ~ 5000kHz ultrasonic wave, then be the dialysis tubing dialysis 24 ~ 72h of 18000 ~ 28000 through molecular weight cut-off; go bail for and stay liquid to be the dialysis tubing dialysis 24 ~ 72h of 400000 ~ 500000 through molecular weight cut-off; get permeate and concentrate; add alcohol settling again, taking precipitate is for subsequent use;
5) by step 4) described in throw out use dehydrated alcohol, washing with acetone successively, then adding distil water dissolves, activated carbon decolorizing, filters, dry, is camellia chrysantha polysaccharide.
2. the extracting method of camellia chrysantha polysaccharide according to claim 1, is characterized in that: step 1) in, extracting in water refers to the water adding its weight 20 ~ 30 times in Camellia nitidissima Chi leaf, refluxing extraction 2 ~ 3 times, extracts 1 ~ 3h at every turn, united extraction liquid, filter, filtrate is Aqueous extracts.
3. the extracting method of camellia chrysantha polysaccharide according to claim 1, is characterized in that: step 2) in, Aqueous extracts being concentrated into relative density at 60 DEG C is the medicinal extract of 1.1 ~ 1.3.
4. the extracting method of the camellia chrysantha polysaccharide according to claim 1 or 3, is characterized in that: step 2) in, described in add alcohol settling and refer to that ethanol to the alcohol content adding 90 ~ 95v% in concentrated solution is 80 ~ 85v%.
5. the extracting method of camellia chrysantha polysaccharide according to claim 1, is characterized in that: step 3) in, the consumption of dehydrated alcohol, acetone is 2 ~ 4 times of weight of precipitate, and the consumption of distilled water is 2 ~ 4 times of the Sediment weight after washing.
6. the extracting method of camellia chrysantha polysaccharide according to claim 1, is characterized in that: step 3) in, the consumption of proteolytic enzyme is 0.3 ~ 3% of Camellia nitidissima Chi leaf dry weight, and hydrolysis temperature is 40 ~ 60 DEG C, and enzymolysis time is 1 ~ 3h.
7. the extracting method of camellia chrysantha polysaccharide according to claim 1, is characterized in that: step 4) in, described permeate concentrates and refers to that being concentrated into relative density at 60 DEG C is the medicinal extract of 1.1 ~ 1.3.
8. the extracting method of the camellia chrysantha polysaccharide according to claim 1 or 7, is characterized in that: step 4) in, described in add alcohol settling and refer to that ethanol to the alcohol content adding 90 ~ 95v% in concentrated solution is 80 ~ 85v%.
9. the extracting method of camellia chrysantha polysaccharide according to claim 1, is characterized in that: step 5) in, the consumption of dehydrated alcohol, acetone is 2 ~ 4 times of weight of precipitate, and the consumption of distilled water is 2 ~ 4 times of the Sediment weight after washing.
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