CN108546728A - A kind of method that porcine haemoglobin enzymolysis decolourizes and its application in preparation relieves fatigue drug and antitumor drug - Google Patents
A kind of method that porcine haemoglobin enzymolysis decolourizes and its application in preparation relieves fatigue drug and antitumor drug Download PDFInfo
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Abstract
The present invention relates to pharmaceutical technology fields, and in particular to porcine haemoglobin digests discoloration method and its preparing the application in relieving fatigue drug and antitumor drug.It is decolourized to porcine hemoglobin using the method for papain enzymolysis, and the low dosage hydrogen peroxide of low concentration is combined to carry out secondary decolourization, obtain porcine haemoglobin enzymolysis decoloration powder.Porcine haemoglobin of the present invention enzymolysis discoloration method is easy to operate, and production cost is low, is made that Swine blood protein molecular weight is small, and no bitter taste is conducive to absorb, the porcine haemoglobin enzymolysis decoloration powder of preparation have the function of being relieved mouse movement it is tired, to liver cancer H22Solid tumor mouse tumor inhibiting rate is respectively 33.62%, 45.14%, compared with negative control group significant difference, and there was no significant difference compared with negative control group for spleen, thymus index.
Description
Technical field
The present invention relates to pharmaceutical technology fields, and in particular to porcine haemoglobin digests discoloration method and its relieved fatigue in preparation
Application in drug and antitumor drug.
Background technology
Pig blood is the by-product of food industry of high nutritive value that is a kind of cheap and can largely obtaining, is great exploitation
The animal protein resource of application.Contain a large amount of protein, abundant amino acid and suitable minerals, dimension life in pig blood
Element, hormone, enzyme and other biological activity substance.In terms of medicine, existing research applies modern extraction and separation technology from pig blood
It is middle to extract a variety of biochemical drugs and biochemical reagents, it is used successfully to clinic.Separately some researches show that, edible pork blood can treat headache,
The diseases such as nausea, dizziness, not hungry deficiency of food, vomiting.Porcine hemoglobin is mainly processed into blood meal as animal protein feed at present, but
Blood meal palatability is poor, it is difficult to digest and assimilate the waste for causing resource.Porcine hemoglobin as edible high-quality protein, due to
It is difficult to the fishy smell weight that decolourizes, easy oxidation discoloration limits its application in field of food.Therefore, the decoloration of porcine hemoglobin is it
The key problem in technology of application, the research for porcine hemoglobin decoloration domestic at present are relatively more, and main method can be divided into physics
Decoloration, oxidative decoloration, adsorption bleaching, organic solvent extraction, hydrolysis decoloration etc..
Hydrolysis decoloration is the most methods used at present, not only separates ferroheme and globin after hydrolysis, and
And protein itself is degraded to peptide, peptone etc., digestibility is greatly improved, and Hydrolyze method is divided into Acid hydrolysis and enzyme process water
Solution.Enzymatic hydrolysis must compare fully, and more commonly used enzyme has neutral proteinase, pancreatin, papain, microbial protease.
But the product individually obtained with enzyme hydrolysis may not decolourize completely, some also have bitter taste, peculiar smell etc..
And the enzyme solution of this research papain decolourizes to hemoglobin, and combine the low dosage of low concentration double
The secondary decolourization method of oxygen water can not only decolourize completely, not destroying the nutritive value of protein, and the product obtained is without hardship
Taste, peculiar smell, and easy to operate, are produced into low, this just provides feasibility to industrialized production.
Invention content
For the deficiencies in the prior art, the purpose of the present invention is to provide a kind of porcine haemoglobins to digest discoloration method
And its application in relieving fatigue drug and antitumor drug is being prepared, it is realized especially by following technical scheme：
A kind of porcine haemoglobin enzymolysis discoloration method, includes the following steps successively：
(1) pig blood is weighed, adds water that solution is made, centrifuges to obtain pig blood ball liquid, 3-7 times of pig blood ball liquid product is then added
Water (preferably 6-7 times), stirring makes blood cell rupture；
(2) papain of pig blood ball liquid weight (0.02-0.08) % is added into mixed liquor obtained by step (1),
1-8h is digested at 40-55 DEG C, hydrochloric acid is added dropwise in enzymolysis process makes the pH of reaction solution be 3.0-7.0；
(3) after digesting, enzymolysis reaction centrifuges enzymolysis liquid after cooling, and waste obtains supernatant；
(4) H is added into supernatant2O2, make H2O2A concentration of (0.1-0.3) % of aqueous solution, carries out at 45-65 DEG C
Decolourize 30-120min, and decoloration to enzymolysis liquid becomes faint yellow, its pH value is then adjusted to neutrality with NaOH, through ultrafiltration membrane ultrafiltration,
After concentration, it is spray-dried to obtain porcine haemoglobin enzymolysis decoloration powder.
Further, stirring described in step (1) is that rotating speed 200rpm stirs 30min, and centrifugation described in step (3) is：
Enzymolysis liquid is centrifuged into 4min with 8000r/min.
Further, the operation of enzymolysis reaction is that enzymolysis liquid is placed in boiling water bath 10min to be passivated in step (3)
Papain terminates reaction.
Further, hydrochloric acid is added dropwise in the step (2) in enzymolysis process makes the pH of reaction solution be 5.0.
Further, the wood of pig blood ball liquid weight 0.05% is added in the step (2) into mixed liquor obtained by step (1)
Melon protease digests 3-5h (preferably 4h) at 45 DEG C.
Further, H is added into supernatant in the step (4)2O2, make H2O2Concentration of aqueous solution is (0.2-
0.3) % decolourizes at 60-65 DEG C, and twice, H is preferably added in each 60min into supernatant for decoloration altogether2O2, make
H2O2Aqueous solution final concentration of 0.3%, decolourizes at 60 DEG C, decolourizes altogether twice, each 60min.
There is provided porcine haemoglobin enzymolysis decoloration powder prepared by the above method to prepare alleviation for second object of the present invention
Application in tired product.
The porcine haemoglobin enzymolysis decoloration powder prepared third object of the present invention is to provide the above method is anti-swollen in preparation
Application in tumor medicine, the especially application in the drug for the treatment of liver cancer.
Compared with prior art, the present invention has the following advantages and effects：
The present invention decolourizes to porcine hemoglobin using the enzyme solution of papain, and combines the low use of low concentration
The secondary decolourization method for measuring hydrogen peroxide can not only decolourize completely, the product for not destroying the nutritive value of protein, and obtaining
Without bitter taste, free from extraneous odour, and it is easy to operate, production cost is low, this just provides feasibility to industrialized production.
The porcine haemoglobin enzymolysis discoloration method of the present invention is easy to operate, and production cost is low, and Swine blood protein molecular weight is made
Become smaller, is conducive to absorb, no bitter taste, good mouthfeel；Alleviate mouse movement fatigue, to rat liver cancer H by exploring it22Solid tumor
Inhibition and extend liver cancer H22The effect of ascites tumor mouse survival time.As a result, it has been found that：High dose group, which has, is relieved mouse
The effect of sports fatigue, the significant difference compared with blank group；Low dose group, high dose group are to liver cancer H22Solid tumor mouse is swollen
Tumor inhibiting rate is respectively 33.62%, 45.14%, compared with negative control group significant difference, spleen, thymus index and the moon
Property control group compared to there was no significant difference.
Description of the drawings
Difference pH hemoglobins digest the effect that liquid centrifugation is decolourized and compare during Fig. 1 is the 2.2 of embodiment 1, from left to right pH
It is followed successively by 3.0,4.0,5.0,6.0,7.0.
Fig. 2 be embodiment 1 2.5 in 0.3%H2O2Under aqueous solution, differential responses temperature digests liquid oxygen to hemoglobin
Change the effect of decoloration, temperature time is from left to right：45℃、50℃、55℃、60℃、65℃.
Fig. 3 be embodiment 1 2.6 in 0.3% H2O2Aqueous solution, at 60 DEG C, the differential responses time is to hemoglobinase
Solve the influence of liquid decoloration.
The pig blood enzymolysis decoloration albumen powder outside drawing that Fig. 4 is obtained in being the 2.7 of embodiment 1.
The molecular weight distribution testing result for the pig blood enzymolysis decoloration albumen amyloid proteins that Fig. 5 is obtained in being the 2.7 of embodiment 1
Figure.
Specific implementation mode
Applicant will in conjunction with specific embodiments be described in further detail technical scheme of the present invention below.Ying Li
Solution, the following contents should not be construed as in any way the limitation for claims of the present invention being claimed range.
The reagent of part used and the source of material etc. are as follows in the embodiment of the present invention：
Experiment mouse used is Kunming mouse, is provided by Wuhan Biological Products Inst.'s Experimental Animal Center, licensing
Number：SCXK (Hubei Province)：2012-0033.
Liver cancer H22Ascit form：It is purchased from Wuhan University's China typical culture collection center (CCTCC), by this laboratory
Intraperitoneal inoculation is in Kunming mouse and passes on conservation.
Pig blood：It is provided by Wuhan that Bioisystech Co., Ltd that try to win favour, fresh pig blood is added to sodium citrate anti-freezing
It is placed on freezen protective in -20 DEG C of refrigerator-freezers.
Papain (200,000 units/grams：Guangxi Pang Bo Bioisystech Co., Ltd)；Quinine (Shanghai source leaf biology section
Skill Co., Ltd)；5 FU 5 fluorouracil injection (Shanghai Xudong Hipu Medicine Co., Ltd).
H used2O2The initial concentration of aqueous solution is 30%, the H described in present specification2O2The percentage of aqueous solution
Concentration refers both to mass percent.
A kind of 1 porcine haemoglobin of embodiment digests discoloration method, includes the following steps：
1 hemoglobin powder, preparation method thereof
(1) quantitative pig blood is taken, adds water that solution is made, pig blood ball liquid (substrate) is centrifuged to obtain, according to certain volume ratio
Add water, then so that blood cell is ruptured with rotating speed 200rpm stirrings 30min；
(2) quantitative papain constant temperature is added and reacts certain time, be added dropwise during the reaction the hydrochloric acid of 2mol/L with
Keep the pH constant (± 0.1pH units) of reaction；
(3) after enzymolysis to the predetermined time, enzymolysis liquid is placed in boiling water bath 10min to be passivated papain and terminates reaction,
Enzymolysis liquid is centrifuged into 4min with 8000r/min after cooling, waste obtains supernatant；
(4) H is added into supernatant2O2, decolourize at a certain temperature, become yellowish to enzymolysis liquid through decolourizing twice
Then its pH value is adjusted to neutrality by color with NaOH solution, after ultrafiltration membrane ultrafiltration, concentration, be spray-dried to obtain porcine haemoglobin enzyme
Free toner.
Index determining
Protein content determination：Coomassie Brilliant Blue.
Protein recovery measures：Enzymolysis measures the protein content of the front and back sample of enzymolysis, and pig blood is calculated according to following equation
The rate of recovery (P) of Lactoferrin：P=C1*m1/C0*m0(C1For the albumen concentration of the decoloration powder sample after enzymolysis, m1After enzymolysis
Decolourize powder sample quality, C0For the albumen concentration of the pig blood ball liquid sample before enzymolysis, m0For the pig blood ball liquid sample matter before enzymolysis
Amount).
Hemoglobin bleaching level measures：Solution of the hemoglobin after decolorization is suitably diluted with water, with blood plasma egg
Whiteness instrument measures the chromatic value of solution, and it is better to be worth lower expression decoloration.
The distribution for haemoglobin molecule amount of being decolourized using PAGE electrophoretic determinations.
2 experimentations
According to described in 1 preparation method and assay method carry out following experiment.
The influence that 2.1 concentration of substrate decolourize to hemoglobinase solution
Take fresh pig blood ball liquid respectively by pig blood ball liquid and water volume ratio 1:3、1:4、1：5、1:6、1：7 add water, then
30min splitting erythrocytes are stirred with 200rpm, the papain of pig blood ball liquid weight 0.05% is added, 4h is digested in 45 DEG C,
The pH that the hydrochloric acid holding reaction solution of 2mol/L is added dropwise in enzymolysis process is 5.0.After reaction, enzymolysis liquid is placed in boiling water bath
10min terminates reaction to be passivated papain, enzymolysis liquid is centrifuged 4min with 8000rpm after cooling, centrifugate dilutes 1 times of use
Chromascope measures chromatic value.
Experimental result is as shown in table 1, with volume ratio 1：6、1：7 add the centrifugate chromatic value after water small, illustrate pig blood
Erythrocyte splitting is abundant, and centrifugate decolorizing effect is good after enzymolysis, and from the considerations of reducing water angle, other factors experiment uses
Pig blood ball liquid and water volume ratio 1：6 water addition ratio example splitting erythrocyte.
The enzymolysis decolorizing effect of 1 hemoglobin difference amount of water of table processing
Note：Chromatic value unit degree of being in the present invention.
The influence that 2.2pH values decolourize to hemoglobinase solution
Take pig blood ball liquid by the volume ratio 1 of pig blood ball liquid and water:6 add water, 200rpm to stir 30min splitting erythrocytes, add
Enter the papain of pig blood ball liquid weight 0.05%, 45 DEG C digest 4h, use 2M hydrochloric acid to adjust reaction solution respectively in enzymolysis process
PH be fixed as 3.0,4.0,5.0,6.0,7.0, after reaction, enzymolysis liquid is placed in boiling water bath 10min to be passivated pawpaw egg
White enzyme terminates reaction, enzymolysis liquid is centrifuged 4min with 8000rpm after cooling, centrifugate dilutes 1 times and measures chromatic value with chromascope.
Experimental result is as shown in table 2, Fig. 1, the results showed that pH centrifuges hemoglobin enzymolysis liquid the influence ten of decolorizing effect
Divide significantly, adjust the centrifugation decolorizing effect of pH=5.0 best, other factors are tested in enzymolysis process and adjust reaction solution with 2M hydrochloric acid
PH=5.0.
The enzymolysis decolorizing effect of 2 hemoglobin difference pH processing of table
The influence that 2.3 enzyme concentrations decolourize to hemoglobinase solution
Take pig blood ball liquid by pig blood ball liquid and water volume ratio 1:6 add water, 200rpm to stir 30min splitting erythrocytes, respectively
The papain of pig blood ball liquid weight 0.02%, 0.04%, 0.05%, 0.06%, 0.08%, 45 DEG C of enzymolysis 4h, enzyme is added
It is 5.0 that the pH of 2M hydrochloric acid tune reaction solutions is used in solution preocess, after reaction, enzymolysis liquid sample is respectively placed in boiling water bath 10min
Reaction is terminated to be passivated papain, enzymolysis liquid is centrifuged into 4min with 8000rpm after cooling, centrifugate dilutes 1 times and uses coloration
Instrument measures chromatic value.
Experimental result is as shown in table 3, the results showed that and enzyme concentration has certain influence to hemoglobinase solution decolorizing effect,
The centrifugation decolorizing effect of the hemoglobin enzymolysis liquid of 0.05% Papain enzymatic treatment is best, can be compared with ferroheme at this concentration
It is fully detached from from hemoglobin related.Hemoglobin uses the pawpaw of pig blood ball liquid weight 0.05% in other factors experiment
Protease hydrolyzed.
The enzymolysis decolorizing effect of 3 hemoglobin difference enzyme concentration of table processing
The influence that 2.4 hydrolysis temperatures decolourize to hemoglobinase solution with the time
Take pig blood ball liquid by pig blood ball liquid and water volume ratio 1:6 add water, 200rpm to stir 30min splitting erythrocytes, are added
The papain of pig blood ball liquid weight 0.05% digests 1- at 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C respectively according to the condition of table 4
8h with the pH of 2M hydrochloric acid tune reaction solutions is 5.0 in enzymolysis process, takes 1 hemoglobin to digest liquid sample every 1h, by enzymolysis liquid
Sample is placed in boiling water bath 10min and terminates reaction to be passivated papain, and enzymolysis liquid is centrifuged 4min with 8000rpm after cooling,
Centrifugate dilutes 1 times and measures chromatic value with chromascope.
Experimental result is as shown in table 4, the results showed that and there are larger impact in hydrolysis temperature, time to the decoloration of hemoglobinase solution,
There are one suitable enzymolysis bleaching time ranges for hemoglobin under different hydrolysis temperatures, and the effect of 45 DEG C of enzymolysis decolorations is compared with 40
DEG C, 50 DEG C, 55 DEG C it is more preferable, and 45 DEG C of 3~5h of enzymolysis can obtain good decolorizing effect, while the centrifugation of hemoglobin enzymolysis liquid
Liquid is light reddish brown color.
The decolorizing effect of 4 hemoglobin difference hydrolysis temperature of table, time-triggered protocol
The influence of 2.5 hydrogen peroxide concentrations, temperature to enzymolysis hemoglobin oxidation decoloration
Take pig blood ball liquid by pig blood ball liquid and water volume ratio 1:6 add water, 200rpm to stir 30min splitting erythrocytes, are added
The papain of pig blood ball liquid weight 0.05%, 45 DEG C digest 4h, adjust the pH=of enzymolysis liquid in enzymolysis process with 2M hydrochloric acid
5.0, after reaction, enzymolysis liquid is placed in boiling water bath 10min to be passivated papain and terminates reaction, by enzymolysis liquid after cooling
4min is centrifuged with 8000rpm, hydrogen peroxide is separately added into centrifugate makes hydrogen peroxide final concentration of 0.1%, 0.2%, 0.3%,
Then respectively decolourize in 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C of water-baths 60min, is digested after measuring decoloration with chromascope
The chromatic value of liquid.
Experimental result is as shown in table 5, the results showed that oxidation of the concentration, reaction temperature of hydrogen peroxide to enzymolysis haemoglobin liquid
Decoloration has larger impact, and the raising decolorizing effect with the concentration of hydrogen peroxide, reaction temperature is better, and final concentration of 0.2%, 0.3%
Hydrogen peroxide can obtain good decolorizing effect in 60 DEG C, 65 DEG C of reaction 60min, and the decolorations effect of 60 DEG C, 65 DEG C reaction 60min
Fruit difference is little.
The decolorizing effect that 5 hemoglobin of table digests the different hydrogen peroxide concentrations of liquid, reaction temperature is handled
Influence of the 2.6 dioxygen treatment time of water to oxidative decoloration
Take pig blood ball liquid by pig blood ball liquid and water volume ratio 1:6 add water, 200rpm to stir 30min splitting erythrocytes, are added
The papain of pig blood ball liquid weight 0.05% digests 4h at 45 DEG C, adjusts the pH of enzymolysis liquid in enzymolysis process with 2M hydrochloric acid
It is 5.0, after reaction, enzymolysis liquid is placed in boiling water bath 10min to be passivated papain and terminates reaction, it will enzymolysis after cooling
Liquid centrifuges 4min with 8000rpm, and hydrogen peroxide is added into centrifugate makes hydrogen peroxide final concentration of 0.3%, reacts 30- in 60 DEG C
120min samples every 30min, its chromatic value is directly measured with chromascope.
Experimental result is as shown in Figure 3, the results showed that hemoglobinase solution liquid is with final concentration of 0.3% hydrogen peroxide in 60 DEG C
Reaction 60min can be obtained good decolorizing effect, reacts and tends towards stability after 60min, and hemoglobin digests liquid oxidation decoloration
Chromatic value maintains 2.0.
The detection for the porcine hemoglobin enzymolysis decoloration albumen powder index that 2.7 Optimizing Technicals are prepared
Take pig blood ball liquid by pig blood ball liquid and water volume ratio 1:6 add water, 200rpm to stir 30min splitting erythrocytes, are added
The papain of pig blood ball liquid weight 0.05%, 45 DEG C digest 4h, and it is 5.0 that 2M salt acid for adjusting pH is used in enzymolysis process, reaction
After, enzymolysis liquid is placed in boiling water bath 10min to be passivated papain and terminates reaction, it is cooling after by enzymolysis liquid with
8000rpm centrifuges 4min, be added in centrifugate hydrogen peroxide make its final concentration of 0.3%, (primary decoloration twice of decolourizing at 60 DEG C
Add after the completion hydrogen peroxide make its final concentration of 0.3%, carry out secondary decolourization), each 60min, secondary decolourization liquid 2M
NaOH adjusts pH to 7.0, is concentrated by ultrafiltration to the 30% of stoste volume with the ultrafiltration membrane of molecular cut off 1000Da, concentrate is through spray
Mist is dry that hemoglobinase frees chromoprotein powder.
Obtained pig blood enzymolysis decoloration albumen powder appearance is shown in that Fig. 4, the molecular weight distribution testing result of albumen are shown in Fig. 5,
The testing result of correlated quality technical indicator is as shown in table 6.
The prevailing quality technical indicator of the enzymolysis decoloration albumen powder of table 6
2 porcine haemoglobin enzymolysis polypeptide of embodiment relieves fatigue Effect study
Drug in the present embodiment and following embodiment is the 2.7 of the embodiment 1 hemoglobinase solution decoloration prepared
Albumen powder is formulated with purified water.
Experiment mice carries out adaptability raising after a week, and mouse is randomly divided into 3 groups：Blank group, low dose group, high dose
Group, every group of 6 mouse.Blank group gavage gives distilled water, and daily gavage gives dosage and is respectively for low dose group, high dose group
The drug of 300mg/kg, 900mg/kg.10 days successive administration time.After last gavage 30min, it is placed in water temperature (25.0 ± 1.0)
DEG C, in the water tank of depth 30cm, record mouse for its swimming time and calculates fortune from swimming to time when sinking to bottom 8s
Dynamic endurance rate elongation, the results are shown in Table 7.
Exercise tolerance rate elongation (%)=(m- blank group average swim time when experimental group average swim)/blank group is flat
Equal swimming time × 100%.
7 porcine haemoglobin enzymolysis polypeptide of table to mouse movement endurance influence (N=6)
Note：Compared with blank group：*P<0.01。
As can be seen from Table 7, various dose group has the tendency that being obviously prolonged exercise tolerance, compared with blank group, high agent
There is amount group significant difference, exercise tolerance rate elongation to reach 95.9%, illustrates that porcine haemoglobin polypeptide has and extends mouse movement
The effect of endurance.
The antitumous effect of 3 porcine haemoglobin enzymolysis polypeptide of embodiment is studied
Mouse is fed into 7d under conditions of water and feed abundance, after weighing, Mice Inoculated liver cancer H22Ascites tumor strain, after 7d
Tumor liquid is aseptically extracted, with 10 times of normal saline dilution, liver cancer cells suspension is formed after mixing, by liver cancer cells suspension
It is diluted with 0.02% Yihong of Fresh, is counted on blood count disk after mixing.Red colouration person is dead cell, is not dyed
Person is living cells, calculates viable count.When every milliliter of suspension has no less than 2.0 × 107When a oncocyte living, suspension is used
0.25mL syringes are inoculated in subject animal with every 0.2mL.It is divided into two projects of solid tumor and ascites tumor, solid tumor project is
It is inoculated in subject animal oxter, is fabricated to solid tumor animal model.Ascites tumor project is to be inoculated in the abdominal cavity of animal, is fabricated to abdomen
Hydatoncus animal model.After inoculation for 24 hours, mouse is divided into 4 groups by each project at random：Negative control group (model group), positive control
Group, low dose group, high dose group, every group of 6 mouse.Distilled water 0.2mL/g gavages, positive control is given once daily in negative control group
5 FU 5 fluorouracil (20mg/kg) 0.2mL/g intraperitoneal injections are given once daily in group, and low dose group, high dose group give agent daily respectively
Amount is 300mg/kg, 900mg/kg drug, 0.2mL/g.The mouse successive administration of solid tumor project 9 days, at the 10th day de- neck
Extremely, it takes tumour, spleen, thymus gland to weigh and calculates tumor control rate and Immune Organs Index；The mouse of ascites tumor project is continuously given
Medicine 12 days observes mouse survival situation, records the mouse survival time and calculates increase in life span.
Tumor control rate (%)=[1- (experimental group average knurl weight/negative control group average knurl weight)] × 100%；
Increase in life span (%)=(experimental group the average survival time number of days-negative control group the average survival time number of days)/negative control
Group the average survival time number of days × 100%；
Index and spleen index=spleen weight (mg)/weight (g)；
Thymus index=thymic factor D injection (mg)/weight (g)；
Concrete outcome is shown in Table 8-10, and as can be seen from Table 8, various dose group has tumor suppression trend, with negative control group ratio
Compared with, have significant difference (*P<0.05), wherein high dose group inhibiting rate reaches 45.14%, illustrates that porcine haemoglobin polypeptide has
Antitumor activity.9 result of table is shown：There was no significant difference with negative control group for the index and spleen index of experimental group, with positive controls
Significant difference；There was no significant difference with negative control group for the spleen index of experimental group, with the significant difference of positive controls；
Positive controls and the significant difference of negative control group.10 result of table is shown：High dose group, positive controls are small to ascites tumor
The life of mouse have extension effect (*P<0.05), high dose group is 30.61% to the increase in life span of ascites tumor mouse, with the positive
Control group compared to there was no significant difference (*P>0.05)。
8 porcine haemoglobin enzymolysis polypeptide of table is to liver cancer H22Solid tumor mouse antitumor activity influence (N=6)
Note：Compared with negative control group：*P<0.05。
9 porcine haemoglobin polypeptide of table is to liver cancer H22Solid tumor mice spleen, thymus index influence (N=6)
Note：Compared with negative control group：*P<0.05.
10 porcine haemoglobin polypeptide of table is to liver cancer H22Ascites tumor Bearing Mice Life Prolongation rate influence (N=6)
Note：Compared with negative control group：*P<0.05；In table it is forward and backward show medicine before, the number of mice survived after 12 days of administration
Amount.
Claims (10)
1. a kind of porcine haemoglobin digests discoloration method, which is characterized in that the method includes the following steps successively：
（1）Pig blood ball liquid is weighed, the water of 3-7 times of its volume is added, then stirring makes blood cell rupture；
（2）To step（1）The papain of pig blood ball liquid weight 0.02%-0.08% is added in gained mixed liquor, at 40-55 DEG C
Lower enzymolysis 1-8h, hydrochloric acid is added dropwise in enzymolysis process makes the pH of reaction solution be 3.0-7.0；
（3）After enzymolysis, enzymolysis reaction centrifuges enzymolysis liquid after cooling, and waste obtains supernatant；
（4）Hydrogen peroxide is added into supernatant, makes a concentration of 0.1%-0.3% of hydrogen peroxide, decoloration 30- is carried out at 45-65 DEG C
120min, decoloration become faint yellow to enzymolysis liquid, its pH value are then adjusted to neutrality with NaOH, through ultrafiltration membrane ultrafiltration, concentrates it
Afterwards, it is spray-dried to obtain porcine haemoglobin enzymolysis decoloration powder.
2. according to the method described in claim 1, it is characterized in that, step（1）Described in stirring for rotating speed 200rpm stir
30min, step（3）Described in centrifugation be：Enzymolysis liquid is centrifuged into 4min with 8000r/min.
3. according to the method described in claim 2, it is characterized in that, step（3）The operation of middle enzymolysis reaction is that will digest
Liquid is placed in boiling water bath 10min and terminates reaction to be passivated papain.
4. according to the method described in claim 3, it is characterized in that, the step（1）The middle volume that water is added is pig blood ball liquid
6-7 times of volume.
5. according to the method described in claim 4, it is characterized in that, the step（2）Hydrochloric acid is added dropwise in middle enzymolysis process to be made instead
It is 5.0 to answer the pH of liquid.
6. according to the method described in claim 5, it is characterized in that, the step（2）It is middle to step（1）Add in gained mixed liquor
The papain for entering pig blood ball liquid weight 0.05%, 3-5h is digested at 45 DEG C.
7. according to the method described in claim 6, it is characterized in that, the step（4）It is middle that hydrogen peroxide is added into supernatant, make
Hydrogen peroxide concentration is（0.2-0.3）% decolourizes at 60-65 DEG C, decolourizes altogether twice, each 60min.
8. porcine haemoglobin enzymolysis decoloration powder prepared by any methods of claim 1-7 is in preparation relieves fatigue drug
Using.
9. porcine haemoglobin enzymolysis decoloration powder prepared by any methods of claim 1-7 answering in the preparation of antitumor drugs
With.
10. application according to claim 9, which is characterized in that the drug is the drug for treating liver cancer.
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