CN105920591A - Preparation method for bright mung bean superoxide dismutase liposome - Google Patents
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Abstract
The invention provides a preparation method for a bright mung bean superoxide dismutase liposome, belonging to the technical field of superoxide dismutase. The preparation method comprises the following steps: crushing and drying bright mung bean; extraction of bright mung bean superoxide dismutase; hot bath treatment; centrifugal filtering so as to obtain supernatant I and filter residue I; fractional precipitation with ammonium sulfate; treatment with a G-200 sephadex column; extraction of the filter residue I with anhydrous petroleum ether; digestion of a mixture II in 95% ethanol; dissolving of paste I in ether; dissolving of paste II in acetone; crushing of a foamed solid; dissolving of powdery extract I in a mixed solvent; addition of cholesterol into a mixed solution I; injection of a superoxide dismutase phosphate buffer; pressure-reduced drying; dissolving in a phosphate buffered solution; etc. According to the invention, the filter residue extracted from the bright mung bean is used for preparation of the bright mung bean superoxide dismutase liposome; the method is simple; the prepared bright mung bean superoxide dismutase liposome has the characteristics of high encapsulation efficiency, stable properties, low embedding cost, wide sources of raw materials used for preparation, etc.; and the method is beneficial for comprehensive utilization of bright mung bean.
Description
Technical field
The present invention relates to a kind of Semen phaseoli radiati extract and preparation method thereof, particularly relate to a kind of bright Semen phaseoli radiati superoxide dismutase
The preparation method of enzyme liposome.
Background technology
Semen phaseoli radiati (Vigna radiate L.), for the ripe kind of leguminous herbaceous plant's Semen phaseoli radiati (Phaseolusradiatus)
Son.Modern pharmacological research shows that Semen phaseoli radiati has antibacterial bacteriostatic, antitumor, antioxidation, raising immunity, blood fat reducing and removing toxic substances etc.
Effect.
Mingkwang origin green bean (hereinafter referred to as " bright Semen phaseoli radiati "), main product in Mingguang City Ming Dong, masonry dam, Jian Xi, the small towns such as keep shop.With it
The features such as color and luster is sparkling and crystal-clear dark green, and grain great Pi is thin, soup is the most rotten, and delicate fragrance is good to eat, quality is the hat in the whole nation, is described as " bright green ", once
Through by as tribute.2008 " Mingkwang origin green bean " is listed in " national geography sign protection product ", and obtains registered trade mark.
After testing: in the seed of Mingkwang origin green bean, fat content is 0.90%, content of ashes is 3.70%, and amount of absolute moisture is
11.80%, protein content is 25.26%, and carbohydrate content is 58.34%.
Superoxide dismutase (Superoxide dismutase, SOD), is the eka-gold being widely present in organism
Proteases, has catalysis Superoxide anion free radical (O2 -) specific action of dismutation reaction, it is described as by medical circle that " human body is cleaned the street
Husband ".Superoxide dismutase can the fast and effeciently superoxide radical in purged body, preventing organism from superoxide radical
Damage, radioprotective, antitumor, raising body immunity, compressive resistance and fatigue, prevent ischemia reperfusion injury, treatment is dynamic
Arteries and veins blood vessel scleratheroma, treats seborrheic skin inflammation and delays the aspects such as body aging to play an important role, in food, agricultural
And the application of cosmetic industry is more and more extensive.But the past extracts mostly from blood, not only complex process, preparation cost
High, be difficult to room temperature and preserve, and there is the potential danger of the blood disease cross infections such as acquired immune deficiency syndrome (AIDS), WHO stops moving
The use of physical property superoxide dismutase.Plant Superoxide Dismutase then has higher safety in utilization, so, from plant source
Middle acquisition superoxide dismutase is just becoming focus.
Through retrieval, not yet retrieve the preparation method of bright Semen phaseoli radiati superoxide dismutase liposome.
Summary of the invention
It is an object of the invention to provide a kind of self component comprehensively utilizing bright Semen phaseoli radiati and prepare bright Semen phaseoli radiati superoxides discrimination
The method changing enzyme liposome.The invention provides the preparation method of a kind of bright Semen phaseoli radiati superoxide dismutase liposome, specifically flow
Journey step is:
A) pulverize and be dried bright Semen phaseoli radiati；
B) ultrasonic wave added pH7 extract with phosphate buffer bright Semen phaseoli radiati superoxide dismutase, obtains mixture I
C) by mixture I with the hot water water-bath 1 ~ 3h of 42 ~ 45 DEG C；
D) mixture I after water bath processing is centrifuged under conditions of temperature 2 ~ 6 DEG C, after being centrifuged, is filtrated to get supernatant I and filter
Slag I；
E) ammonium sulfate is joined in supernatant I, be configured to the solution that ammonium sulphate content is 30 ~ 38%, centrifugal after standing, filter
Obtaining supernatant II and filtering residue II, take supernatant II, continue to add ammonium sulfate, the ammonium sulphate content to supernatant II is 60 ~ 70%,
Being completely dissolved rear secondary centrifuging, secondary filter obtains supernatant III and precipitate I；
F) precipitate I is redissolved to water, dialyse with the phosphate buffer of pH6.8 ± 0.1, after having dialysed, cross G-
200 sephadex columns, obtain bright Semen phaseoli radiati superoxide dismutase phosphate buffer；
G) filtering residue I dry oil ether is extracted, after having extracted, take solid portion, referred to as mixture II；
H) mixture II is extracted with 95% ethanol solution, filter and remove insoluble matter and filter vacuum is concentrated, obtain paste
Ⅰ；
I) paste I that obtain is concentrated in vacuo with ether dissolution, filters after dissolving, and filtrate is placed in the environment of 20 ~ 30 DEG C
It is evaporated under reduced pressure, obtains paste II；
J) again with acetone solution paste II, filter after dissolving, and by filter vacuum lyophilization, obtain foaming solid；
K) foaming solid is pulverized, obtain Powder Extract I；
L) Powder Extract I is dissolved completely in the mixed solvent of methanol, ethanol, normal hexane, obtains mixed solution I；
M) in mixed solution I, add cholesterol, obtain mixed solution II；
N) stated clearly Semen phaseoli radiati superoxide dismutase phosphate buffer joins in mixed solution II, and solid-liquid ratio is 1:4 ~ 1:5,
Obtain mixed solution III；
O) solvent during drying under reduced pressure removes mixed solution III, obtains superoxide dismutase lipid film；
P) in superoxide dismutase lipid film, add the phosphate buffered solution of pH7.2 ± 0.1, make superoxide dismutase
Lipid film is completely dissolved, and obtains stable bright Semen phaseoli radiati superoxide dismutase liposome.
The preparation method of bright Semen phaseoli radiati superoxide dismutase liposome the most according to claim 1, it is characterised in that:
In step j, the proportioning of each composition of described mixed solvent is: methanol: ethanol: normal hexane=70:50:1.
The preparation method of bright Semen phaseoli radiati superoxide dismutase liposome the most according to claim 1, it is characterised in that:
In step k, the addition of described cholesterol is 0.3 ~ 2.5%(W/V).
The preparation method of bright Semen phaseoli radiati superoxide dismutase liposome the most according to claim 1, it is characterised in that:
In step m, the condition of described drying under reduced pressure is at 45 ~ 55 DEG C, drying under reduced pressure under conditions of 0.01 ~ 0.2MP.
Beneficial effect
Applicant, during the extraction of bright Semen phaseoli radiati superoxide dismutase, finds to use the filtering residue I during extracting as former
Material, further by 95% ethanol solution, after ether and acetone purify, then mixes with the superoxide dismutase extracted, cholesterol
After, through means such as magnetic agitation, drying under reduced pressure, the superoxide dismutase liposome that stability is higher can be prepared.?
To the active embedding rate of active sod liposome be: 88.73%, it is seen then that the superoxide dismutase of the present invention
Do not have influence on the activity of superoxide dismutase.
The present invention self searches out the method protecting bright Semen phaseoli radiati superoxide dismutase from bright Semen phaseoli radiati, can not only protect from
The superoxide dismutase extracted in bright Semen phaseoli radiati, and during fully utilizing the extraction of bright Semen phaseoli radiati superoxide dismutase
Waste material, the material of embedding is the natural component of bright Semen phaseoli radiati self, extracts while bright Semen phaseoli radiati superoxide dismutase extracting
Natural embedding composition in bright Semen phaseoli radiati, and continuously the superoxide dismutase extracted is carried out embedding treatment, prepare
It is high that superoxide dismutase liposome has envelop rate, stable in properties, embeds low cost, prepares the raw material feature such as generally.This system
The method of standby liposome is simple, merges completely in the extraction process of bright Semen phaseoli radiati superoxide dismutase, only with super oxygen
Garbage after the extraction of compound dismutase, the method that bright Semen phaseoli radiati superoxide dismutase is carried out embedding treatment.The present invention provides
A kind of preparation method of brand-new bright Semen phaseoli radiati superoxide dismutase liposome.
Accompanying drawing explanation
Bright Semen phaseoli radiati superoxide dismutase liposome electron microscope image prepared by Fig. 1: the method through embodiment 1；
The bright Semen phaseoli radiati superoxide dismutase electron microscope image of Fig. 2: ultrasound assisted extraction.
Detailed description of the invention
It is further elucidated with the present invention, it should be understood that these embodiments are merely to illustrate this below in conjunction with being embodied as example
Bright, rather than limit the scope of the present invention, after having read the present invention, those skilled in the art is various etc. to the present invention
The amendment of valency form all falls within the scope of the application claims defined.
Embodiment 1
A kind of preparation method of bright Semen phaseoli radiati superoxide dismutase liposome, using bright Semen phaseoli radiati, cholesterol is that raw material is prepared from,
Bright Semen phaseoli radiati is provided by Anhui Yanzhifang Food Co., Ltd, and cholesterol is food stage.
Concrete operation method comprises the steps:
1. pulverizing and be dried bright Semen phaseoli radiati: being pulverized by bright Semen phaseoli radiati with disintegrating apparatus, using vacuum dryer is 40 DEG C in temperature, very
Drying under reduced pressure under conditions of reciprocal of duty cycle 20-80kPa, and cross the sieve of 80 mesh, obtain bright Green Gram Seed.
2. extract bright Semen phaseoli radiati superoxide dismutase: bright Green Gram Seed is added pH7 phosphate buffer, use liquid-solid ratio
22:1(mL/g), ultrasonic power 120w, temperature be 44 DEG C under conditions of ultrasound assisted extraction bright Green Gram Seed 90min, extracted
Mixture I is obtained after one-tenth.
3. processed by hot bath: by said mixture I with the hot water water-bath 2h of 44 DEG C.
4. centrifugal: by the mixture I after water bath processing temperature 4 DEG C, centrifugal under conditions of centrifuge RPMs 10000r/min
20 min, are filtrated to get supernatant I and filtering residue I.
5. ammonium sulfate precipitation: solid ammonium sulfate powder is added in supernatant I, prepares ammonium sulfate with supernatant I and contain
Amount is the solution of 35%, magnetic agitation 30 min, stands 4 h, be centrifuged 20 with the revolution of 12000 r/min in the environment of 4 DEG C
min；Obtain supernatant II after Li Xin, take supernatant II, continue reinforcing body ammonium sulfate powder, to the ammonium sulphate content of supernatant II
It is 65%, then in the environment of 4 DEG C, continues to be centrifuged 20 min with the revolution of 12000 r/min, obtain supernatant III and precipitation
Thing I.
6. cross G-200 sephadex column: redissolved to water by precipitate I, in the phosphate buffer of pH 6.7 thoroughly
Analysis, crosses G-200 sephadex column, obtains bright Semen phaseoli radiati superoxide dismutase phosphate buffer.
7. dry oil ether extracts filtering residue: by filtering residue I under conditions of 40 DEG C, by dry oil ether reflux, extract, 6 ~ 12
H, filters after having extracted and makes its solid-liquid separation, and the solid portion obtained is referred to as mixture II.
8.95% alcohol steep mixture II: mixture II is joined in 95% ethanol solution, wherein, solid-liquid ratio is 1:
1.5(m/V), under the conditions of 40 DEG C, extract 4h, after having extracted, filter and remove insoluble matter and filter vacuum is concentrated, obtain paste
Thing I.
9. ether dissolution paste I: the paste I that obtain be concentrated in vacuo with ether dissolution, concussion stirring, when paste not
After minimizing, filter, and filtrate be placed in the environment of 25 DEG C reduction vaporization, obtain paste II,
10. acetone solution paste II: in like manner, then with acetone solution paste II, be sufficiently stirred for, when paste II does not reduces
After, filter, and by filter vacuum lyophilization, obtain foaming solid.
11. pulverize foaming solid: pulverized by foaming solid, Powder Extract I.
12. mixed solvent dissolved powders shape extracts I: Powder Extract I is dissolved completely in methanol, ethanol, just oneself
In the mixed solvent of alkane, solid-liquid ratio is: 20.82 mg/mL, obtains mixed solution I, wherein, joining of each composition of described mixed solvent
Ratio is: methanol: ethanol: normal hexane=70:50:1.
13. add cholesterol in mixed solution I: add 0.45%(m/V in mixed solution I) cholesterol, mixed
Solution II.
14. injection superoxide dismutase phosphate buffers: bright Semen phaseoli radiati step 6 obtained with syringe or injection device
Superoxide dismutase phosphate buffer is expelled in mixed solution II, and solid-liquid ratio is 1:4.1, magnetic agitation 30 min,
To mixed solution III.
15. rotary evaporations: at 55 DEG C, solvent during rotary evaporation removes mixed solution III under conditions of 0.1MP, obtain super oxygen
Compound dismutase lipid film.
16. phosphate buffered solution are dissolved: add the phosphate-buffered of pH 7.2 in superoxide dismutase lipid film
Solution, makes superoxide dismutase lipid film be completely dissolved, and obtains stable bright Semen phaseoli radiati superoxide dismutase liposome.
Applicant uses envelop rate to evaluate the effect of bright Semen phaseoli radiati superoxide dismutase liposome, and method is as follows:
Using pyrogallol method to measure superoxide dismutase enzymatic activity before encapsulating, method is shown in GB/T5009.171-2003.
After preparing liposome, take bright Semen phaseoli radiati superoxide dismutase liposome 1.0mL, add normal propyl alcohol to 3.0mL breakdown of emulsion,
Make class lipid bilayer dissolve thus discharge superoxide dismutase, use pyrogallol method to measure superoxides discrimination after fragmentation
Change enzymatic activity, and calculate the activity encapsulating percentage rate of bright Semen phaseoli radiati superoxide dismutase liposome.
The superoxide dismutase liposome that the present embodiment is prepared by applicant is placed in ultramicroscope, the structure obtained
Figure as it is shown in figure 1, and the structure chart of extracting directly superoxide dismutase out as shown in Figure 2.It is evident that super oxygen in figure
The smooth surface of compound dismutase liposome, tight tight, it can be seen that liposome is in the surface shape of superoxide dismutase
Become layer protecting film, thus the activity of protective enzyme is the most stable.Through detection, the superoxide dismutase that the present embodiment obtains
The active envelop rate of liposome is: 88.73%.Superoxide dismutase liposome prepared by visible whole preparation technology is to super oxygen
The active protection effect of compound dismutase is notable.
The superoxide dismutase liposome temperature tolerance of superoxide dismutase and the present embodiment is done by applicant
Contrast experiment, result shows, superoxide dismutase liposome is after 70 DEG C of constant temperature water bath 60 min, and remaining vigor is 90%,
And the remaining vigor that free superoxide dismutase in contrast is under similarity condition is 75%；80 DEG C of constant temperature water baths 150
Under min treatment conditions, the relative activity of the present embodiment superoxide dismutase liposome is 90%, free superoxide dismutase
Enzyme is only 2%, illustrates that superoxide dismutase is had by superoxide dismutase liposome prepared by the present invention and well protects work
With.
Applicant simulates human consumption's environment, respectively with the superoxides that pepsin is free under conditions of 37 DEG C
Dismutase and superoxide dismutase liposome, result shows, the activity of superoxide dismutase free after process is to process
Front 5.2%, and after processing, the activity of superoxide dismutase liposome is before treatment 72.6%, it can be seen that, system of the present invention
Standby bright Semen phaseoli radiati superoxide dismutase liposome has significant protective effect to bright Semen phaseoli radiati superoxide dismutase.
Embodiment 2
A kind of preparation method of bright Semen phaseoli radiati superoxide dismutase liposome, using bright Semen phaseoli radiati, cholesterol is that raw material is prepared from,
Bright Semen phaseoli radiati is provided by Anhui Yanzhifang Food Co., Ltd, and cholesterol is food stage.
Concrete operation method comprises the steps:
1. pulverizing and be dried bright Semen phaseoli radiati: being pulverized by bright Semen phaseoli radiati with disintegrating apparatus, using vacuum dryer is 50 DEG C in temperature, very
Drying under reduced pressure under conditions of reciprocal of duty cycle 20-80kPa, and cross the sieve of 100 mesh, obtain bright Green Gram Seed.
2. extract bright Semen phaseoli radiati superoxide dismutase: bright Green Gram Seed is added pH7 phosphate buffer, uses ultrasonic auxiliary
Help method to extract bright Green Gram Seed, after having extracted, obtain the mixture of bright Green Gram Seed and phosphate buffer, named: mixing
Thing I.
3. processed by hot bath: by mixture I with the hot water water-bath 3h of 45 DEG C.
4. centrifugal: the mixture I after water bath processing is centrifuged under conditions of temperature 6 DEG C, it is filtrated to get supernatant after being centrifuged
Liquid I and filtering residue I.
5. ammonium sulfate precipitation: solid ammonium sulfate powder is added in supernatant I, prepares ammonium sulfate with supernatant I and contain
Amount is the solution of 38%, magnetic agitation, stands in the environment of 6 DEG C, centrifugal after standing, obtains supernatant II and filtering residue II, takes
Clear liquid II, continues reinforcing body ammonium sulfate powder, and the ammonium sulphate content to supernatant II is 70%, is completely dissolved rear secondary centrifuging,
To supernatant III and precipitate I.
6. cross G-200 sephadex column: redissolved to water by precipitate I, in the phosphate buffer of pH 6.8 thoroughly
Analysis, crosses G-200 sephadex column, obtains bright Semen phaseoli radiati superoxide dismutase phosphate buffer.
7. dry oil ether extract filtering residue: by filtering residue I under conditions of 45 DEG C, with dry oil ether reverse backflow extract 3 ~
8 h, filter after having extracted and make its solid-liquid separation, and the solid portion obtained is referred to as mixture II.
8.95% alcohol steep mixture II: joined by mixture II in 95% ethanol solution, after extraction 6h, crosses and filters off
Concentrate except insoluble matter and by filter vacuum, obtain paste I.
9. ether dissolution paste I: the paste I that obtain is concentrated in vacuo with ether dissolution, filters after being completely dissolved, and will
Filtrate is placed in the environment of 20 ~ 30 DEG C reduction vaporization, obtains paste II,
10. acetone solution paste II: in like manner, then with acetone solution paste II, filter after being completely dissolved, and by filter vacuum
Lyophilization, obtains foaming solid.
11. pulverize foaming solid: pulverized by foaming solid, Powder Extract I.
12. mixed solvent dissolved powders shape extracts I: Powder Extract I is dissolved completely in methanol, ethanol, just oneself
In the mixed solvent of alkane, obtaining mixed solution I, wherein, the proportioning of each composition of described mixed solvent is: methanol: ethanol: normal hexane
=70:50:1。
13. add cholesterol in mixed solution I: add 0.3 ~ 2.5%(W/V in mixed solution I) cholesterol, obtain
Mixed solution II.
14. injection superoxide dismutase phosphate buffers: the bright Semen phaseoli radiati superoxide dismutase phosphoric acid that step 6 is obtained
Salt buffer joins in mixed solution II, and solid-liquid ratio is 1:4.1, stirs 30 min, obtains mixed solution III.
15. drying under reduced pressure: at 45 DEG C, solvent during drying under reduced pressure removes mixed solution III under conditions of 0.01MP, surpassed
Superoxide dismutase lipid film.
16. phosphate buffered solution are dissolved: add the phosphate-buffered of pH 7.2 in superoxide dismutase lipid film
Solution, makes superoxide dismutase lipid film be completely dissolved, and obtains stable bright Semen phaseoli radiati superoxide dismutase liposome.
Embodiment 3
A kind of preparation method of bright Semen phaseoli radiati superoxide dismutase liposome, using bright Semen phaseoli radiati, cholesterol is that raw material is prepared from,
Bright Semen phaseoli radiati is provided by Anhui Yanzhifang Food Co., Ltd, and cholesterol is food stage.
Concrete operation method comprises the steps:
1. pulverize and be dried bright Semen phaseoli radiati: with disintegrating apparatus, bright Semen phaseoli radiati being pulverized, using vacuum dryer to be dried bright Semen phaseoli radiati, and mistake
The sieve of 100 mesh, obtains bright Green Gram Seed.
2. extract bright Semen phaseoli radiati superoxide dismutase: bright Green Gram Seed is added pH7 phosphate buffer, uses ultrasonic auxiliary
Help method to extract bright Green Gram Seed, after having extracted, obtain the mixture of bright Green Gram Seed and phosphate buffer, named: mixing
Thing I.
3. processed by hot bath: by mixture I with the hot water water-bath 1h of 42 DEG C.
4. centrifugal: the mixture I after water bath processing is centrifuged under conditions of temperature 2 DEG C, it is filtrated to get supernatant after being centrifuged
Liquid I and filtering residue I.
5. ammonium sulfate precipitation: solid ammonium sulfate powder is added in supernatant I, prepares ammonium sulfate with supernatant I and contain
Amount is the solution of 30%, stirring, stands in the environment of 2 DEG C, centrifugal after standing, obtains supernatant II and filtering residue II, takes supernatant
II, continue reinforcing body ammonium sulfate powder, the ammonium sulphate content to supernatant II is 60%, is completely dissolved rear secondary centrifuging, obtain on
Clear liquid III and precipitate I.
6. cross G-200 sephadex column: redissolved to water by precipitate I, in the phosphate buffer of pH 6.8 ± 0.1
Middle dialysis, crosses G-200 sephadex column, obtains bright Semen phaseoli radiati superoxide dismutase phosphate buffer.
7. dry oil ether extracts filtering residue: by filtering residue I under conditions of 50 DEG C, extracts 10 ~ 18 h with dry oil ether, carries
Filtering after taking into and make its solid-liquid separation, the solid portion obtained is referred to as mixture II.
8.95% alcohol steep mixture II: joined by mixture II in 95% ethanol solution, after extraction 6h, crosses and filters off
Concentrate except insoluble matter and by filter vacuum, obtain paste I.
9. ether dissolution paste I: the paste I that obtain is concentrated in vacuo with ether dissolution, filters after being completely dissolved, and will
Filtrate is placed in the environment of 20 ~ 30 DEG C reduction vaporization, obtains paste II,
10. acetone solution paste II: in like manner, then with acetone solution paste II, filter after being completely dissolved, and by filter vacuum
Lyophilization, obtains foaming solid.
11. pulverize foaming solid: pulverized by foaming solid, Powder Extract I.
12. mixed solvent dissolved powders shape extracts I: Powder Extract I is dissolved completely in methanol, ethanol, just oneself
In the mixed solvent of alkane, obtaining mixed solution I, wherein, the proportioning of each composition of described mixed solvent is: methanol: ethanol: normal hexane
=70:50:1。
13. add cholesterol in mixed solution I: add 0.3 ~ 2.5%(W/V in mixed solution I) cholesterol gallbladder is admittedly
Alcohol, obtains mixed solution II.
14. injection superoxide dismutase phosphate buffers: the bright Semen phaseoli radiati superoxide dismutase phosphoric acid that step 6 is obtained
Salt buffer joins in mixed solution II, and solid-liquid ratio is 1:5, stirs 30 min, obtains mixed solution III.
15. drying under reduced pressure: at 50 DEG C, solvent during drying under reduced pressure removes mixed solution III under conditions of 0.2MP, obtain super oxygen
Compound dismutase lipid film.
16. phosphate buffered solution are dissolved: add the phosphate of pH7.2 ± 0.1 in superoxide dismutase lipid film
Buffer solution, makes superoxide dismutase lipid film be completely dissolved, and obtains stable bright Semen phaseoli radiati superoxide dismutase liposome.
Claims (4)
1. a preparation method for bright Semen phaseoli radiati superoxide dismutase liposome, described preparation method includes step: pulverizes and does
Dry bright Semen phaseoli radiati, ultrasonic wave added pH7 extract with phosphate buffer bright Semen phaseoli radiati superoxide dismutase, obtain mixture I；Its feature exists
In, described preparation method also comprises the steps:
By mixture I with the hot water water-bath 1 ~ 3h of 42 ~ 45 DEG C；
Mixture I after water bath processing is centrifuged under conditions of temperature 2 ~ 6 DEG C, after being centrifuged, is filtrated to get supernatant I and filtering residue
Ⅰ；
Ammonium sulfate is joined in supernatant I, be configured to the solution that ammonium sulphate content is 30 ~ 38%, centrifugal after standing, filter
To supernatant II and filtering residue II, taking supernatant II, continue to add ammonium sulfate, the ammonium sulphate content to supernatant II is 60 ~ 70%, complete
Secondary centrifuging after CL, secondary filter obtains supernatant III and precipitate I；
Precipitate I is redissolved to water, dialyses with the phosphate buffer of pH 6.8 ± 0.1, after having dialysed, cross G-200
Sephadex column, obtains bright Semen phaseoli radiati superoxide dismutase phosphate buffer；
Filtering residue I dry oil ether is extracted, after having extracted, takes solid portion, referred to as mixture II；
Mixture II is extracted with 95% ethanol solution, filters and remove insoluble matter and filter vacuum is concentrated, obtain paste I；
The paste I that obtain is concentrated in vacuo with ether dissolution, filters after dissolving, and filtrate is placed in the environment of 20 ~ 30 DEG C and subtracts
Pressure evaporation, obtains paste II；
Again with acetone solution paste II, filter after dissolving, by filter vacuum lyophilization, obtain foaming solid；
Foaming solid is pulverized, obtains Powder Extract I；
Powder Extract I is dissolved completely in the mixed solvent of methanol, ethanol, normal hexane, obtains mixed solution I；
In mixed solution I, add cholesterol, obtain mixed solution II；
Stated clearly Semen phaseoli radiati superoxide dismutase phosphate buffer joins in mixed solution II, and solid-liquid ratio is 1:4 ~ 1:5,
To mixed solution III；
Drying under reduced pressure removes solvent in mixed solution III, obtains superoxide dismutase lipid film；
In superoxide dismutase lipid film, add the phosphate buffered solution of pH 7.2 ± 0.1, make superoxide dismutase
Lipid film is completely dissolved, and obtains stable bright Semen phaseoli radiati superoxide dismutase liposome.
The preparation method of bright Semen phaseoli radiati superoxide dismutase liposome the most according to claim 1, it is characterised in that: in step
In rapid j, the proportioning of each composition of described mixed solvent is: methanol: ethanol: normal hexane=70:50:1.
The preparation method of bright Semen phaseoli radiati superoxide dismutase liposome the most according to claim 1, it is characterised in that: in step
In rapid k, the addition of described cholesterol is 0.3 ~ 2.5%(W/V).
The preparation method of bright Semen phaseoli radiati superoxide dismutase liposome the most according to claim 1, it is characterised in that: in step
In rapid m, the condition of described drying under reduced pressure is at 45 ~ 55 DEG C, drying under reduced pressure under conditions of 0.01 ~ 0.2MP.
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