A kind of preparation method of lily polysaccharide of strengthening immunity
Technical field
The present invention relates to a kind of preparation method of lily polysaccharide of strengthening immunity.
Background technology
Immunizing power be exactly human body be basic substance with immune organ and immunoglobulin (Ig), resist the disease is kept organism physiology equilibrated ability.It can discern in the human body normal and abnormal microorganism by the human immune system, and gets rid of inside and outside pathogenic microorganism and sex change, old and feeble cell by immunne response.Immunity system is the strong guarantee of body health, but immunity system is again very fragile.Concrete as: the heredity and disease factor, as sicklemia, diabetes, AIDS, viral infection, cancer etc.; Medical treatment and medicine factor are as operation radiotherapy, chemotherapy, burn and skin excision, the external application corticoid etc. of cancer patient; Sub-health state: hypoimmunity is the key character of sub-health population performance, and this several years sub-health populations become crowds such as ascendant trend year by year, particularly intelligentsia, leading cadre, white collar, and the sub-health population number surpasses 60%; People's bad life style and usual aging can cause lower immune function.So it needs immunity system constantly to safeguard and strengthen, this is 21st century health care new ideas, and therefore seeking the medicine with good immunoregulation effect has become one of developing direction of contemporary new drug development.
Patent CN10106972 has applied for a kind of oral liquid of having following bulk drug to make: Herba Epimedii, the Radix Astragali, Chinese yam, the bark of eucommia, wolfberry fruit, hawthorn, Radix Angelicae Sinensis, Poria cocos, raspberry and Spina Date Seed have the effect of antifatigue and strengthening immunity; Patent CN1813787 has applied for a kind of composition of ginseng-astragalus polysaccharides with immuno-potentiation and preparation method thereof; Patent CN10100281 has applied for a kind of novel excessive honeybee compound Chinese herbal medicinal ingredients immunostimulant, belongs to the novel immunostimulant that strengthens immunologic function of livestock and birds; Patent CN1739790 has applied for peptidoglycan immunopotentiator and production method thereof; Patent CN1200272 has applied for that a kind of is the immunostimulant that can suppress tumor growth that main raw material is made with the worker bee larvae; Patent CN1220999 has applied for the extraction and separation method of filamentous cyanobacteria water-soluble polysaccharide and exocellular polysaccharide, has the active and very high viscosity of very strong complement activation, can be used as the thickening material in immunostimulant and the foodstuffs industry; Patent CN1099993 has applied for the biological immunopotentiator " Kangaiping " be made up of Sanguis Gallus domesticus, male Bombycis mori, pollen polysaccharide, lentinan, Spreading Hedyotis Herb, Japanese Honeysuckle, Radix Isatidis, Herba Andrographis, the Radix Astragali, Radix Notoginseng etc., contain the multiple physiology biologically active substance of human body, strengthen the panimmunity function, making original immunologic hypofunction or immunodeficiency obtain reconstruction, is the ideal medicament that suppresses and treat early metaphase tumour, cancer; It is main raw material with the tea polyphenols complex that patent CN1114207 has applied for a kind of, to the immunostimulant of the equal tool significant curative effect of disease caused by toxic reaction of excessive free radicals in the body; Japanese Patent 03819036 discloses, the dihydro-pyrazolo pyridine compounds shows optionally and the inhibition activity of intensive to glycogen synthase kinase-3 β (GSK-3 β), and useful as drug, be used to prevent and/or treat diabetes, diabetic complication and nerve degeneration disease, or as immunostimulant.
Lanzhou lily is nutritious, is unique edible strain in the national four giant lily strains (Yixing, Jiangsu, Luoyang, Henan, Hunan fossilia dentis mastodi, Lanzhou, Gansu), also is the unique sweet lily of China.In nourishing YIN and clearing away lung-heat, clearing away the heart fire and tranquillizing, invigorating the spleen and replenishing QI, there is very high pharmaceutical use aspects such as treatment deficiency of Yin chronic cough, dysphoria, insomnia.China's famous plant classification scholar hole constitution force is thought: " the lanzhou lily flavor is extremely sweet, and fiber seldom has no bitter taste again, and is not only well-known throughout the country, also can claim the first in the world ".
But, and be that the method for the lily polysaccharide of a kind of strengthening immunity of feedstock production yet there are no document and mentions with the lanzhou lily.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of preparation method of lily polysaccharide of strengthening immunity.Method of the present invention is to be raw material with the lily bulb, the natural polysaccharide of a kind of strengthening immunity of step preparations such as process extraction, purifying, separation, deproteinated, can obviously improve the phagocytic activity of immunologic hypofunction mouse macrophage, promote its non-specific immune function, antagonism endoxan and the cell-mediated cell immune response of recovery T, improve the lymphocytic answering of T, promptly non-specific immunity and specific immunity (humoral immunization and cellular immunization) are all had remarkable influence.Compare with other enhancing immunity product, advantage such as it is simple to have production technique, easy to use, has no side effect, effective, and characteristic is with the obvious advantage, and resource reserve abundance, market outlook are very good, and economic benefit is wide.
For solving the problems of the technologies described above, the preparation method of the lily polysaccharide of a kind of strengthening immunity of the present invention comprises the steps:
1) take by weighing a certain amount of fresh Bulbus Lilii, pulverize, add water, the ratio of described fresh Bulbus Lilii and water is 1: 8~12; At power 55~65W, under the condition that temperature is 65~75 ℃, use ultrasonic extraction 80~120 minutes; Suction filtration under the pressure of 0.07~0.09MPa; Filter residue extracts 1 time under these conditions again; Merge 2 times filtrate, filtrate is at 60~70 ℃, 0.07 be evaporated to 1/2.5~3.5 of original volume under the condition of~0.09MPa, in concentrated solution, add ethanol, described ethanol content accounts for and adds 75~80% of overall solution volume behind the ethanol, at room temperature left standstill 10~12 hours, under 3500~4500 rev/mins rotating speed centrifugal 4~6 minutes, collecting precipitation; At 40~55 ℃, drying is 22~26 hours under the condition of 0.07~0.09MPa, gets substance A then;
2) under 60~70 ℃ of bath temperatures, the water of substance A with 80~120 times of weight is dissolved, under 3500~4500 rev/mins rotating speed centrifugal 4~6 minutes, discard insolubles; Centrifugal repeatedly in solution, can not detect starch with the IKI method till, add ethanol in the solution after centrifugal, described ethanol content accounts for and adds 75~80% of overall solution volume behind the ethanol, left standstill 10~12 hours, under 3500~4500 rev/mins rotating speed centrifugal 4~6 minutes, the collecting precipitation thing; At 40~55 ℃, drying is 20~24 hours under the condition of 0.07~0.09MPa, gets substance B then;
3) under 60~70 ℃ of bath temperatures, the water of substance B with 80~100 times of weight is dissolved, adding 3~6u/100ml papain enzymolysis 10~12 hours, is that 1: 3~5 chloroform and propyl carbinol mixed solution extract collection upper water solution 3~5 times with volume ratio again; At 40~50 ℃, be evaporated to 1/2.5~3.5 of original volume under the condition of 0.07~0.09MPa; Concentrated solution is put into molecular weight cut-off 8000~14000Da dialysis tubing dialyse, flowing water flushing dialysis 20~28 hours, the distilled water flushing dialysis is 20~24 hours then; Add ethanol in the dialyzate then in bag, described ethanol content accounts for and adds 75~80% of overall solution volume behind the ethanol, leaves standstill 10~12 hours; Under 3500~4500 rev/mins rotating speed centrifugal 4~6 minutes, collecting precipitation; Throw out is successively with dehydrated alcohol, acetone, ether washing, and then at 40~55 ℃, drying is 18~20 hours under the condition of 0.07~0.09MPa, gets lily polysaccharide.Utilize method of the present invention, the 100g fresh Bulbus Lilii can make 2.8~3.3g lily polysaccharide.
The lily polysaccharide physicochemical characteristic of method preparation of the present invention is as follows:
Color: white powder solid;
Reduced viscosity: be 81.698cm
3/ g;
PH value: 6.93.
Lily polysaccharide has strong absorption peak through UV scanning at the 200nm place, at equal free nucleic acid of 260nm, 280nm and proteinic charateristic avsorption band, impurity such as not containing nucleic acid and protein is described; Ninhydrin reaction, IKI reaction and biuret reaction are all negative, and no protein and starch are described; Infrared spectra is presented at 3432cm
-1One characteristic peak is arranged is the stretching vibration peak of O-H at the place, and it is that the angle vibration of c h bond in the molecule causes that 874.47cm-1 then is β-D-Glucopyranose absorption peak that a characteristic peak is arranged at 2925cm-1 place, and these absorption peaks are sugared charateristic avsorption bands; Measuring total sugar content with the phenol sulfuric acid process is 96.84%; Adopting its viscosity-average molecular weight of determination of ubbelohde viscometer is 20093.12; Fehling reagent reaction and sulfuric acid-phynol reaction are positive, illustrate that polysaccharide is the reductibility polysaccharide; Use the sedimentary polysaccharide of different concentration ethanol, in the time of 20 ℃, have different specific rotatioies, show the non-homogeneous component of this polysaccharide; Hot analysis revealed lily polysaccharide fusing point, second-order transition temperature are not obvious, and no crystal conversion temperature is the indefiniteness powder.
The lily polysaccharide strengthening immunity experiment that method of the present invention is prepared:
Kunming mice, body weight 18-22g, male, be 6 groups by weight average, 10 every group, grouping and filling stomach dosage such as table 1.Each group is pressed 20ml/kg and is irritated stomach, every day 1 time, 7-14 days continuously, body weight of 2 days titles.Administration the 2nd day, according to different experiments, abdominal cavity or subcutaneous injection endoxan 2-3 days, accumulative total 120-160mg/kg sets up the immunocompromised model.Measuring it cytophagously cleans up ear swelling degree, the PHA that index, immune organ weight, delayed type hypersensitivity cause and stimulates lymphocyte transformation rate and serum hemolysin.
Table 1 grouping and filling stomach dosage
The result is shown in accompanying drawing 4~7, and lily polysaccharide can obviously improve the phagocytic activity of immunologic hypofunction mouse macrophage, promotes its non-specific immune function; Can significantly recover DTH reaction of immunologic hypofunction mouse and the DTH that antagonism Cy reduces, the ear swelling degree of mouse is recovered, significantly improving PHA stimulates lymphocyte transformation, promptly improves the lymphocytic answering of T, the function of enhancing body specific cellular immunity; Lily polysaccharide can recover immunocompromised animal serum hemolysin O D value significantly, improves the specific humoral immunity function.Lily polysaccharide all has enhancement for immunosuppressed mice to its non-specific immunity and specific immunity (humoral immunization and cellular immunization), the obviously immunosuppression that causes of antagonism endoxan.
Beneficial effect of the present invention is as follows:
The present invention makes full use of the bulb of Lanzhou, Gansu domestic establishing in large scale plant lily, make the pure natural polysaccharide, compare with other enhancing immunity product, it is simple to have production technique, easy to use, have no side effect, advantage such as effective, and characteristic is with the obvious advantage, the resource reserve abundance, market outlook are very good, and economic benefit is wide.
Description of drawings
Below in conjunction with accompanying drawing the specific embodiment of the present invention is described in further detail
The infrared scan figure of the lily polysaccharide that Fig. 1 prepares for the present invention;
The thermogram of the lily polysaccharide that Fig. 2 prepares for the present invention;
The lily polysaccharide UV scanning figure that Fig. 3 prepares for the present invention;
The lily polysaccharide that Fig. 4 prepares for the present invention is to the influence of immunocompromised clearance in mice index (K);
The lily polysaccharide that Fig. 5 prepares for the present invention is to the influence of immunocompromised mice ear degree;
The lily polysaccharide that Fig. 6 prepares for the present invention is to the influence of immunocompromised mouse lymphocyte transformation efficiency;
The lily polysaccharide that Fig. 7 prepares for the present invention is to the influence of immunocompromised mice serum hemolysin.
Annotate: in Fig. 4~7, * represents P<0.05 (significantly); * represents P<0.01 (extremely remarkable).
Embodiment
Embodiment 1
The preparation method of the lily polysaccharide of a kind of strengthening immunity of the present invention comprises the steps:
1) take by weighing a certain amount of fresh Bulbus Lilii, pulverize, add water, the ratio of described fresh Bulbus Lilii and water is 1: 8; At power 55W, under the condition that temperature is 65 ℃, use ultrasonic extraction 80 minutes; Suction filtration under the pressure of 0.07MPa; Filter residue extracts 1 time under these conditions again; Merge 2 times filtrate, filtrate is at 60 ℃, 0.07MPa condition under be evaporated to 1/2.5 of original volume, in concentrated solution, add ethanol, described ethanol content accounts for and adds 75% of overall solution volume behind the ethanol, at room temperature left standstill 10 hours, under 3500 rev/mins rotating speed centrifugal 4 minutes, collecting precipitation; At 40 ℃, drying is 22 hours under the condition of 0.07MPa, gets substance A then;
2) under 60 ℃ of bath temperatures, the water of substance A with 80 times of weight is dissolved, under 3500 rev/mins rotating speed centrifugal 4 minutes, discard insolubles; Centrifugal repeatedly in solution, can not detect starch with the IKI method till, add ethanol in the solution after centrifugal, described ethanol content accounts for and adds 75% of overall solution volume behind the ethanol, leaves standstill 10 hours, under 3500 rev/mins rotating speed centrifugal 4 minutes, the collecting precipitation thing; At 40 ℃, drying is 20 hours under the condition of 0.07MPa, gets substance B then;
3) under 60 ℃ of bath temperatures, the water of substance B with 80 times of weight is dissolved, added the 3u/100ml papain enzymolysis 10 hours, be that 1: 3 chloroform and propyl carbinol mixed solution extracts collection upper water solution 3 times with volume ratio again; At 40 ℃, be evaporated to 1/2.5 of original volume under the condition of 0.07MPa; Concentrated solution is put into molecular weight cut-off 8000~14000Da dialysis tubing dialyse, flowing water flushing dialysis 20 hours, the distilled water flushing dialysis is 20 hours then; Add ethanol in the dialyzate then in bag, described ethanol content accounts for and adds 75% of overall solution volume behind the ethanol, leaves standstill 10 hours; Under 3500 rev/mins rotating speed centrifugal 4 minutes, collecting precipitation; Throw out is successively with dehydrated alcohol, acetone, ether washing, and then at 40 ℃, drying is 18 hours under the condition of 0.07MPa, gets lily polysaccharide.
Embodiment 2
The preparation method of the lily polysaccharide of a kind of strengthening immunity of the present invention comprises the steps:
1) take by weighing a certain amount of fresh Bulbus Lilii, pulverize, add water, the ratio of described fresh Bulbus Lilii and water is 1: 12; At power 65W, under the condition that temperature is 75 ℃, use ultrasonic extraction 120 minutes; Suction filtration under the pressure of 0.09MPa; Filter residue extracts 1 time under these conditions again; Merge 2 times filtrate, filtrate is at 70 ℃, 0.09MPa condition under be evaporated to 1/3.5 of original volume, in concentrated solution, add ethanol, described ethanol content accounts for and adds 80% of overall solution volume behind the ethanol, at room temperature left standstill 12 hours, under 4500 rev/mins rotating speed centrifugal 6 minutes, collecting precipitation; At 55 ℃, drying is 26 hours under the condition of 0.09MPa, gets substance A then;
2) under 70 ℃ of bath temperatures, the water of substance A with 120 times of weight is dissolved, under 4500 rev/mins rotating speed centrifugal 6 minutes, discard insolubles; Centrifugal repeatedly in solution, can not detect starch with the IKI method till, add ethanol in the solution after centrifugal, described ethanol content accounts for and adds 80% of overall solution volume behind the ethanol, leaves standstill 12 hours, under 4500 rev/mins rotating speed centrifugal 6 minutes, the collecting precipitation thing; At 55 ℃, drying is 24 hours under the condition of 0.09MPa, gets substance B then;
3) under 70 ℃ of bath temperatures, the water of substance B with 100 times of weight is dissolved, added the 6u/100ml papain enzymolysis then 12 hours, be that 1: 5 chloroform and propyl carbinol mixed solution extracts 5 times with volume ratio again, collect upper water solution; At 50 ℃, be evaporated to 1/3.5 of original volume under the condition of 0.09MPa; Concentrated solution is put into molecular weight cut-off 8000~14000Da dialysis tubing dialyse, flowing water flushing dialysis 28 hours, the distilled water flushing dialysis is 24 hours then; Add ethanol in the dialyzate then in bag, described ethanol content accounts for and adds 80% of overall solution volume behind the ethanol, leaves standstill 12 hours; Under 4500 rev/mins rotating speed centrifugal 6 minutes, collecting precipitation; Throw out is successively with dehydrated alcohol, acetone, ether washing, and then at 55 ℃, drying is 20 hours under the condition of 0.09MPa, gets lily polysaccharide.
Embodiment 3
The preparation method of the lily polysaccharide of a kind of strengthening immunity of the present invention comprises the steps:
1) take by weighing a certain amount of fresh Bulbus Lilii, pulverize, add water, the ratio of described fresh Bulbus Lilii and water is 1: 10; At power 60W, under the condition that temperature is 70 ℃, use ultrasonic extraction 100 minutes; Suction filtration under the pressure of 0.08MPa; Filter residue extracts 1 time under these conditions again; Merge 2 times filtrate, filtrate is at 65 ℃, 0.08MPa condition under be evaporated to 1/3 of original volume, in concentrated solution, add ethanol, described ethanol content accounts for and adds 78% of overall solution volume behind the ethanol, at room temperature left standstill 11 hours, under 4000 rev/mins rotating speed centrifugal 5 minutes, collecting precipitation; At 45 ℃, drying is 24 hours under the condition of 0.08MPa, gets substance A then;
2) under 65 ℃ of bath temperatures, the water of substance A with 100 times of weight is dissolved, under 4000 rev/mins rotating speed centrifugal 5 minutes then, discard insolubles; Centrifugal repeatedly in solution, can not detect starch with the IKI method till, add ethanol in the solution after centrifugal, described ethanol content accounts for and adds 78% of overall solution volume behind the ethanol, leaves standstill 11 hours, under 4000 rev/mins rotating speed centrifugal 5 minutes then, the collecting precipitation thing; At 45 ℃, drying is 22 hours under the condition of 0.08MPa, gets substance B then;
3) under 65 ℃ of bath temperatures, the water of substance B with 90 times of weight is dissolved, added the 5u/100ml papain enzymolysis 11 hours, be that 1: 4 chloroform and propyl carbinol mixed solution extracts collection upper water solution 4 times with volume ratio again; At 45 ℃, be evaporated to 1/3 of original volume under the condition of 0.08MPa; Concentrated solution is put into molecular weight cut-off 8000~14000Da dialysis tubing dialyse, flowing water flushing dialysis 24 hours, distilled water flushing dialysis 22 hours; Add ethanol in the dialyzate in bag, described ethanol content accounts for and adds 78% of overall solution volume behind the ethanol, leaves standstill 11 hours; Under 4000 rev/mins rotating speed centrifugal 5 minutes, collecting precipitation; Throw out is used dehydrated alcohol, acetone, ether washed twice successively, and then at 45 ℃, drying is 19 hours under the condition of 0.08MPa, gets lily polysaccharide.
Obviously, the above embodiment of the present invention only is for example of the present invention clearly is described, and is not to be qualification to embodiments of the present invention.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here can't give exhaustive to all embodiments.Everyly belong to the row that conspicuous variation that technical scheme of the present invention extends out or change still are in protection scope of the present invention.