CN108727511A - A kind of carboxy methylation mulberries polysaccharide and its preparation method and application - Google Patents
A kind of carboxy methylation mulberries polysaccharide and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of carboxy methylation mulberries polysaccharide, preparation method and its in the application of pharmacy.The carboxy methylation mulberries polysaccharide be by weight average molecular weight be 20.93kDa mulberries polysaccharide after carboxy methylation is modified gained, and be determined by experiment analyze its weight average molecular weight be 26.18kDa, degree of substitution 0.6-0.8.Meanwhile the present invention proves that the carboxy methylation mulberries polysaccharide has the function of activating alcohol dehydrogenase, scavenging capacity oxygen radical, prevention free radical to cyto-architectural destruction by pharmacological experiment, has a good application prospect.
Description
Technical field
The present invention relates to the field of Chinese medicines, and in particular to a kind of carboxy methylation mulberry that mulberries polysaccharide is obtained through carboxymethylation reaction
Shen polysaccharide and preparation method thereof and its purposes in preparing hepatic.
Background technology
Liver is intraperitoneal maximum parenchymatous organ, undertakes the important physiological function of human body, is the important removing toxic substances of human body
Organ.There are some in the Nature and human industry's production process to the virose chemical substance of liver, including alcohol, environment
In chemical toxic substances and some drugs, liver can be entered by gastrointestinal tract portal vein or body circulation and converted, can be caused
The different degrees of necrosis of liver cells of liver, fat deformation, hepatic sclerosis and liver cancer, seriously threaten human health.
Mulberries (Mori Fructus) are the mature fruit clusters of perennial woody plant mulberry (Morus alba L), are China
First is embodied in the kind of health ministry " integration of drinking and medicinal herbs " register, while being also the excellent health functional food resource in China
One of, have the effects that enhancing is immune, adjusts blood glucose and blood fat, protect liver.Mulberries polysaccharide is one of its main active, is had
Preferable hepatoprotective effect.The work (number of patent application 201510431557.5) of inventor early period also provides point of mulberries polysaccharide
Application from purification process and in preparing drug and health products.
The bioactivity of polysaccharide and its structure are closely related, and molecular modification is carried out to the structure of polysaccharide with method appropriate,
Its physiological activity can be improved to a certain extent, reduced certain toxic side effects or assigned its new physiological activity.Carboxy methylation
Polysaccharide (carboxymethyl polysaccharides, CP) refers to one or several hydroxyls in polysaccharide chain on monosaccharide molecule
A kind of polysaccharide derivates complicated, activity is various for being replaced and formed by carboxymethyl group.
Invention content
The object of the present invention is to provide a kind of carboxy methylation mulberries polysaccharide (C- that mulberries polysaccharide is obtained through carboxymethylation reaction
MFP) and preparation method thereof, and purposes of the carboxy methylation mulberries polysaccharide in preparing hepatic is studied, it is a kind of new to develop
Hepatic or health-care preparation.
The present invention adopts the following technical scheme that：
A kind of carboxy methylation mulberries polysaccharide, it is modified by the mulberries polysaccharide that weight average molecular weight is 20.93kDa through carboxy methylation
Gained afterwards.
The weight average molecular weight of carboxy methylation mulberries polysaccharide above-mentioned is 26.18kDa, degree of substitution 0.6-0.8.
The preparation method of carboxy methylation mulberries polysaccharide above-mentioned, includes the following steps：
(1) carboxymethyl derivative reaction：Mulberries polysaccharide after purification is taken, the isopropanol that 7-12 times of volume is added stirs and evenly mixs
The NaOH solution of 10-30 times of volume is added afterwards, stirring is slow added into the monoxone of 7-10 times of mulberries polysaccharide weight, reacts 3-
5h is waited for being cooled to room temperature after reaction, solution is neutralized to neutrality, obtains carboxyl methylation derivant；
(2) it dialyses：Carboxyl methylation derivant is transferred in bag filter, first flowing water dialysis, then is dialysed with distilled water, concentrated
Dialyzate；
(3) alcohol precipitation and drying：Ethyl alcohol is added into the dialyzate after concentration, carries out alcohol precipitation, centrifugation, taking precipitate first
Alcohol/acetone rinsing, vacuum freeze drying is to get carboxy methylation mulberries polysaccharide.
More specifically, the preparation method of carboxy methylation mulberries polysaccharide above-mentioned includes the following steps：
(1) carboxymethyl derivative reaction：The purifying mulberries polysaccharide of 300 parts by weight is taken, the isopropanol of 7-12 times of volume is added,
In the room temperature 20%-30%NaOH solution that 10-30 times of volume is added after magnetic stirrer 30-60min, stir at room temperature
1-3h is slowly added to the monoxone of 2100-3000 parts by weight, so that it is reacted 3-5h under the conditions of 55-65 DEG C, waits for after reaction
It is cooled to room temperature, is neutralized to neutrality with 20%-30%HCl solution, obtains carboxyl methylation derivant；
(2) it dialyses：Carboxyl methylation derivant is transferred in bag filter, first flowing water dialysis 12-36h, then saturating with distilled water
36-60h is analysed, dialyzate is concentrated under reduced pressure；
(3) alcohol precipitation and drying：Ethyl alcohol is added into the dialyzate after concentration, ethyl alcohol final concentration is made to reach 80% or more, from
The heart rinses sediment 1-3 times with methanol/acetone, and vacuum freeze drying is to get carboxy methylation mulberries polysaccharide.
In the preparation method of carboxy methylation mulberries polysaccharide above-mentioned, the mulberries polysaccharide is carried as follows
It takes：
1. extracting：Appropriate mulberries are taken, are homogenized, the water of 3-5 times of weight is added, 1-2 is extracted under the conditions of 80-90 DEG C of temperature
Secondary, each 1.5-2.5h is filtered, and merging filtrate is concentrated under reduced pressure into the 2/7-2/9 of original volume；
2. decolourizing：Suitable macroreticular resin progress adsorption bleaching is added in filtrate 1. step concentrates after, permanent at 45-50 DEG C
Temperature oscillation decoloration 20-40min, filtration repeat to elute the mulberries polysaccharide on macroreticular resin, and merging filtrate is concentrated under reduced pressure；
3. removing protein：It is added protease in filtrate after 2. being concentrated to step, at 40-70 DEG C, 1-2 is added in enzymolysis protein
Times chloroform of volume or the n-butanol of dichloromethane and 1/5-1/2 times of volume shake 20-120min, stand, and remove impurity
Albumen repeats 3-7 times up to no Precipitation, and centrifugation takes supernatant；
4. alcohol precipitation：Ethyl alcohol is added in supernatant to the final concentration of 60%-90% of ethyl alcohol, 4 DEG C stand 12-36h, and centrifugation takes
Precipitation is redissolved, again alcohol precipitation, is repeated 2-3 times, sediment absolute ethyl alcohol and acetone respectively wash 1-3 times；
5. dialysing：Sediment after washing is fitted into pretreated bag filter, is dialysed；Or the side using membrane filtration
Method filters, molecular cut off >=1500 of dialysis/membrane filtration, dry to get mulberries Thick many candies.
In the preparation method of carboxy methylation mulberries polysaccharide above-mentioned, the purifying of mulberries Thick many candies includes the following steps：
1. cellulose ion chromatographs
Prepare 8-12gL-1Mulberries Thick many candies solution, be transferred in cellulose ion exchange resin column, with distillation wash
It is de-, flow velocity 2-3mLmin-1, eluent is collected, reduced pressure is dialysed, vacuum freeze drying, and the mulberries for obtaining grading purification are more
Sugar；
2. gel filtration chromatography
The mulberries polysaccharide of grading purification is dissolved in ultra-pure water, solution is transferred in gel column, distills water elution, flow velocity 0.1-
0.4mL·min-1, eluent is collected, after reduced pressure, vacuum freeze drying is to get mulberries polysaccharide.
In the preparation method of carboxy methylation mulberries polysaccharide above-mentioned, the cellulose ion exchanger resin column packing is
DEAE-Cellulose 52 or DEAE-Cellulose 32；Preferably DEAE-Cellulose 52.
In the preparation method of carboxy methylation mulberries polysaccharide above-mentioned, the gel column packing be Sephadex G-50,
Sephadex G-100, Sephadex G-150 or Sephadex G-200；Preferably Sephadex G-100.
Application of the carboxy methylation mulberries polysaccharide above-mentioned in preparing hepatic or health products.
Application of the carboxy methylation mulberries polysaccharide above-mentioned in preparing alcoholic liver injury drug or health products.
Following experiment has been carried out to verify obtained carboxy methylation mulberries polysaccharide and its hepatoprotective effect effect, inventor：
1, the measurement of the infrared analysis of carboxy methylation mulberries polysaccharide and degree of substitution
The carboxy methylation mulberries polysaccharide that 2mg is fully dry is taken, be sufficiently mixed in the agate mortar with dry KBr (m: m=1:
100) after, grind into powder after tabletting, is scanned with infrared spectrometer, 4000~400cm of scanning range-1.It is shown by Fig. 2, with figure
The infared spectrum of sample is compared before 1 carboxy methylation, and C-MFP is in 1617cm-1And 1423cm-1There are two new strong absorptions in place, says
Bright MFP is succeeded by part carboxy methylation, and C-MFP is in 848.21cm-1There are new bands of a spectrum in place, shows the α types C-H vibrations of pyranose
Characteristic absorption peak.It is worth noting that, still having the characteristic absorption peak of polysaccharide in modification derivant.Therefore, in the present invention
Natural polysaccharide is successfully modified the molecular chain structure without changing polysaccharide itself.
The measurement of the degree of substitution of carboxy methylation mulberries polysaccharide
(1) sample treatment：Precision weighs 10mg carboxymethylated polysaccharides, and after 100 DEG C of dry 1h, 70% ethyl alcohol 3mL is added,
5min is placed in stirring, sequentially adds 10mL distilled water, and 0.1%NaOH solution is stirred after mixing until sample is completely dissolved.So
It uses 0.1%HCl solution to titrate afterwards, with phenolphthalein indicator, calculates mM number (A) of the HCl needed for every gram of carboxymethyl polysaccharide.
(2) degree of substitution (degree of substitution, DS) calculates：Degree of substitution refers to each saccharide ring in polysaccharide molecule
The upper substituted degree of hydroxyl.Calculation formula is as follows：
In formula：V0The NaOH solution volume being added, mL；V1The consumed HCl solution volume of blank determination, mL；V2Sample
Measure consumed HCl solution volume, mL；c0The NaOH solution concentration being added, M；C- measures HCl solution concentration used, M；M- is surveyed
Determine used sample quality, g.
It is analyzed by measuring, carboxy methylation mulberries polysaccharide molecular weight is about 26.18kDa, degree of substitution 0.6-0.8.
2, the activation of mulberries polysaccharide and carboxy methylation mulberries polysaccharide to alcohol dehydrogenase
Instrument：Microplate reader, climatic chamber, ten a ten thousandth balances.
Material and reagent：Oxidized coenzyme I (NicotinamideAdenine Dinucleotide, NAD+), ethyl alcohol is de-
Hydrogen enzyme (ADH), ethyl alcohol, sodium pyrophosphate buffer salt.
Using the Valle&Hoch methods detection ADH activity after improvement, the 100 μ L that pH is 8.8 are sequentially added in assay plate
Sodium pyrophosphate buffer solution, 75 μ LNAD+, 35 μ L ethyl alcohol, 10 μ L mulberries polysaccharide solutions, carboxy methylation mulberries polysaccharide solution, mixing,
Capping is incubated 5min at 37 DEG C.10 μ L ADH are added immediately, measure the absorbance value (A at 340nm after shaking up immediately340), every
10s readings are primary, and METHOD FOR CONTINUOUS DETERMINATION 5min records data.Ethanol solution zeroising is replaced with distilled water, is sky to be not added with medicine group
White group, using bifendate as positive control.ADH activity and activity ratio are calculated using following formula.
As shown in figure 3, the influence experimental result table of mulberries polysaccharide and carboxy methylation mulberries polysaccharide to alcohol dehydrogenase activity
Bright, mulberries polysaccharide significantly improves the activation of alcohol dehydrogenase after carboxy methylation is modified, and shows that it has good solution
The application prospect of wine protect liver.
3, the anti-acute alcohol-induced hepatic injury Effect study of mulberries polysaccharide and carboxy methylation mulberries polysaccharide
In order to further confirm that mulberries polysaccharide and carboxy methylation mulberries polysaccharide have hepatoprotective effect, chmice acute liver is established
Damage model.Steps are as follows for specific experiment：
(1) instrument：Microplate reader, climatic chamber.
(2) material and reagent：Glutamic-oxalacetic transaminease (AST), glutamic-pyruvic transaminase (ALT), lactic dehydrogenase (LDH), glycerine three
Ester (TG), total cholesterol (T-CHO), total bilirubin (TBIL) and low density lipoprotein cholesterol (LDL-C), malonaldehyde (MDA),
Superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) kit.
(3) experimental procedure：
1. by the healthy male KM mouse that 90 weight are 18-22g, temperature is 20 ± 2 DEG C indoors, relative humidity
50%, under conditions of natural lighting, raises one week, 9 groups, respectively blank control group (physiology salt are only randomly divided into every group 10
Water), pathological model group (EtOH), positive controls (EtOH+0.21gkg-1BW bifendate), purified mulberries polysaccharide
High, medium and low dosage group (EtOH+123.6mgkg-1MFP、EtOH+61.8mg·kg-1MFP、EtOH+30.9mg·kg- 1MFP), the high, medium and low dosage group (EtOH+123.6mgkg of carboxy methylation mulberries polysaccharide-1C-MFP、EtOH+61.8mg·
kg-1C-MFP、EtOH+30.9mg·kg-1C-MFP).Physiological saline/biphenyl pair is given according to different groups are oral first daily
Ester/MFP/C-MFP takes orally after 4h and gives physiological saline/alcohol, and all groups are continuously pressed above-mentioned sample and dosage gavage 7 days, experiment
It before terminating after mouse fasting 12h, plucks eyeball and blood, dislocation is taken to put to death, liver is taken out in rapid dissection, with ice normal saline flushing, filter
Paper weighs after blotting surface moisture, for calculating liver organ index.Then take left lobe of liver prepare liver homogenate in case measure malonaldehyde,
The liver biochemical indexes such as superoxide dismutase and glutathione peroxidase.In addition it takes out away from hepatomegaly leaf marginalisation 5cm
About 1.0 × 1.0 × 0.2cm3Liver organization is put into 10% neutral formalin solution and fixes, for making pathological section.
2. the preparation of liver homogenate：It takes murine liver tissue, is added the physiological saline of 1: 9 (W: V), under the conditions of ice-water bath, use group
Refiner homogenate is knitted, low-temperature centrifugation 10min takes supernatant spare.
3. liver coefficient determination：Liver organ index (%)=liver mass (g)/mouse weight (g) × 100%
4. the measurement of serum AST, ALT, LDH, TG, T-CHO, TBIL and LDL-C：After blood sample ice water is stood, centrifugation,
Upper serum is taken after 15min, the stringent method provided by kit specification measures every Biochemical Indices In Serum.
5. the measurement of liver tissue homogenate's MDA, SOD and GSH-PX level：10% liver tissue homogenate is diluted to appropriate dense
After degree, the stringent method by kit specification measures every liver biochemical indexes.
5. the making of histopathology slide：After dissecting mouse, liver is taken out rapidly, is rinsed with the physiological saline of precooling,
Blood is removed, filter paper is wiped dry, is fixed for 24 hours with 10% formalin behind every group of same position for cutting mouse liver, with flowing water mistake
Night rinses tissue block, and dehydration is transparent, embeds, slice, Hematoxylin-eosin dyeing, and mounting after natural air drying places glass slide
The morphological changes of various tissue components of hepatic tissue section is observed under an optical microscope.
(4) the anti-acute alcohol-induced hepatic injury Effect study result of mulberries polysaccharide and carboxy methylation mulberries polysaccharide
1. MFP and C-MFP is to the changes of weight of acute alcohol-induced hepatic injury mouse
Before and after experiment, naive mice weight average increases by 5.1%, and model group mouse weight significantly reduces (P < 0.01).
Each dosage groups of MFP and the reduction of each dosage groups of C-MFP and the weight of positive controls mouse but unobvious, the middle agent of wherein C-MFP
Amount, high dose group are better than positive controls.
2. MFP and C-MFP influences the liver index of acute alcohol-induced hepatic injury mouse
9 groups of mouse liver indexes do not make significant difference as can be seen from Table 1, can tentatively judge mulberries polysaccharide and carboxy methylation
Mulberries Polysaccharides on Mice is without dominant toxicity.Wherein the liver index of C-MFP high dose groups declines most strong.
Influences of the table 1MFP and C-MFP to acute alcohol-induced hepatic injury mouse liver coefficient
| Group | Liver/body (gg-1) |
| Blank group | 0.041±0.0067 |
| Pathological model group | 0.044±0.0027 |
| Positive controls | 0.044±0.0033 |
| MFP high dose groups | 0.045±0.0043 |
| MFP middle dose groups | 0.043±0.0054 |
| MFP low dose groups | 0.049±0.0052 |
| C-MFP high dose groups | 0.037±0.048 |
| C-MFP middle dose groups | 0.044±0.0035 |
| C-MFP low dose groups | 0.044±0.0053 |
(3) liver function influences of the MFP and C-MFP to acute alcohol-induced hepatic injury mice serum
As can be seen from Table 2, AST, ALT, LDH, TG, LDL-C, T-CH and TBIL level are significantly higher than blank in model group serum
Control group illustrates modeling success.Mulberries polysaccharide, each dosage group of carboxy methylation mulberries polysaccharide compared with model group, AST, ALT,
LDH, TG, LDL-C and TBIL level continuously decrease, and basic, normal, high dosage shows each index level of gradient reduction, illustrates more
Sugared concentration imitates relationship with vigor in a certain amount.The TG of the middle and high dosage groups of MFP and C-MFP high doses is horizontal, is less than positive control
Group, it is horizontal that prompt can significantly reduce TG in acute alcohol-induced hepatic injury mice serum.Compared with model group, C-MFP is high, medium and low
It is horizontal (P < 0.01) that dosage group can significantly reduce serum LDL-C.In conclusion MFP and C-MF are to acute alcohol-induced hepatic injury
The liver function influence of mice serum can effectively improve the damage situations of liver cell, and can effectively reduce the fat content in serum.
As can be seen from Table 2, AST, ALT, LDH, TG, LDL-C, T-CH and TBIL level are significantly higher than blank control group in model group serum,
Illustrate modeling success.Mulberries polysaccharide, each dosage group of carboxy methylation mulberries polysaccharide compared with model group, AST, ALT, LDH, TG,
LDL-C and TBIL levels continuously decrease, and basic, normal, high dosage shows gradient reduces each index level, illustrate polysaccharide concentration with
Vigor imitates relationship in a certain amount.The TG of the middle and high dosage groups of MFP and C-MFP high doses is horizontal, is less than positive controls, prompts
It is horizontal that TG in acute alcohol-induced hepatic injury mice serum can be significantly reduced.Compared with model group, the high, medium and low dosage groups of C-MFP are equal
It is horizontal (P < 0.01) to can significantly reduce serum LDL-C.In conclusion MFP and C-MFP is to acute alcohol-induced hepatic injury mouse blood
Clear liver function influence can effectively improve the damage situations of liver cell, and can effectively reduce the fat content in serum.
4. MFP and C-MFP is to the liver function influence such as table 3 of acute alcohol-induced hepatic injury murine liver tissue, anxious pathological model
The MDA levels of group mouse liver significantly increase (P < 0.01) in pole compared with blank control group, illustrate that hepatocellular injury is serious,
When the height of pre-administration MFP and C-MFP low dosage, middle dosage, the MDA levels of mouse liver cell significantly have dropped (P <
0.05).Wherein, wherein the MDA down ratios of C-MFP high dose groups mouse liver cell are better than positive control bifendate.In addition,
Bifendate and C-MFP high, middle dosage can significantly increase the content of SOD activity and GSH, wherein the effect of C-MFP high dose groups
Fruit is most significantly (P < 0.01).Illustrate that MFP and C-MFP to alcoholic liver injury, have scavenging capacity oxygen radical, prevent freely
Effect of the base to cyto-architectural destruction.
Comprehensive MFP and C-MFP is to this 9 kinds of AST, ALT, LDH, TG, LDL-C, T-CHO, TBIL, MDA, SOD and GSH-PX
The detection of liver function index in mice serum and liver, the results showed that, C-MFP is with more the protection to acute alcohol-induced hepatic injury
Effect.
Influences of table 2 MFP and C-MFP to acute alcohol-induced hepatic injury mice serum Biochemical Indexes
Note：* compared with blank group, P < 0.05, * * are compared with blank group, P < 0.01；Δ is compared with model group, P <
0.05, Δ Δ is compared with model group, P < 0.01.
Influences of table 3 MFP and C-MFP to acute alcohol-induced hepatic injury mouse liver Biochemical Indexes
Note：* compared with blank group, P < 0.05, * * are compared with blank group, P < 0.01；
Δ is compared with model group, P < 0.05, and Δ Δ is compared with model group, P < 0.01.
5. the pathological section of acute alcohol-induced hepatic injury murine liver tissue is analyzed
The murine liver tissue pathological section of each experimental group is shown in Fig. 4, presses each in sequence and table 3 from left to right, from top to bottom
Experimental group is corresponding, and blank control group liver cell structural integrity, nucleus is high-visible, and the uniform inorganization of cytoplasm is abnormal.
On the contrary, pathological model group has the apparent morphology table：Cell gross distortion, and cell is smudgy, swelling of liver cell.Sun
For property control group compared with Models of Acute Alcoholic Liver Injury group, bifendate can effectively mitigate alcohol to liver cell lesion
Symptom has apparent protecting effect to liver cell.The mouse liver cell of MFP high dose groups is and thin without apparent cellular swelling
Born of the same parents' edge clear is visible.The mouse liver cell cell integrity of C-MFP high dose groups is good, the basic crack-free of cell, and cell week
The lipid matter enclosed significantly reduces.Each dosage groups of MFP and C-MFP, have acute alcohol-induced hepatic injury certain therapeutic effect,
There are certain metering dependences for it.The high dose group of C-MFP has preferable effect for acute alcohol-induced hepatic injury, is solution
The potential drug of wine protect liver.
In conclusion pharmacological evaluation proves, the activity of carboxymethylated polysaccharides C-MFP energy large increases ADH shows that it has
The application prospect of good relieving alcoholism and protecting liver；Pharmacological evaluation is also shown that mulberries polysaccharide, each dosage group of carboxy methylation mulberries polysaccharide and mould
Type group is compared, and AST, ALT, LDH, TG, LDL-C and TBIL level continuously decrease, and basic, normal, high dosage shows gradient reduction
Each index level illustrates that polysaccharide concentration imitates relationship, also, the mouse of pre-administration MFP and C-MFP with vigor in a certain amount,
The MDA levels of liver cell significantly have dropped (P < 0.05), wherein the MDA of C-MFP high dose group mouse liver cells declines
Ratio is better than positive control bifendate.In addition, C-MFP high, middle dosage can significantly increase the content of SOD activity and GSH, say
Bright MFP and C-MFP has scavenging capacity oxygen radical, prevents free radical to cyto-architectural destruction to alcoholic liver injury
Effect.Meanwhile C-MFP has the function of scavenging capacity oxygen radical but also C-MFP is with before being prepared into anti-oxidation medicine
Scape.
In addition, the mouse liver cell of MFP high dose groups is without apparent cellular swelling, and cell edges are high-visible.C-
The mouse liver cell cell integrity of MFP high dose groups is good, the basic crack-free of cell, and the lipid matter of cell peripheral obviously subtracts
It is few.Each dosage groups of MFP and C-MFP, have acute alcohol-induced hepatic injury certain therapeutic effect, there are certain metering according to
The relationship of relying, it may be possible to the potential drug of relieving alcoholism and protecting liver.
The mulberries polysaccharide that the present invention is obtained for purifying has carried out carboxy methylation structural modification, obtains the mulberry of carboxy methylation
Shen polysaccharide.Research finds that there is this carboxy methylation mulberries polysaccharide very strong hepatoprotective effect, these results to open the depth of mulberries polysaccharide
Hair also provides scientific basic using foundation is provided to the research and development of new hepatic.
Description of the drawings：
Fig. 1 is the infared spectrum of mulberries polysaccharide sample before carboxy methylation.
Fig. 2 is the infared spectrum of carboxy methylation mulberries polysaccharide sample.
The influence of Fig. 3 mulberries polysaccharide and carboxy methylation mulberries polysaccharide to alcohol dehydrogenase activity.
Fig. 4 mulberries polysaccharide and carboxy methylation mulberries polysaccharide act on the disease of Models of Acute Alcoholic Liver Injury murine liver tissue
Manage slice map.
Specific implementation mode
The preparation method of 1 carboxy methylation mulberries polysaccharide of embodiment
(1) carboxymethyl derivative reaction：The mulberries polysaccharide for taking the weight average molecular weight of 300 parts by weight to be 20.93kDa, is added 7
The isopropanol of times volume, in the room temperature 20%NaOH solution that 10 times of volumes are added after magnetic stirrer 30min, at room temperature
1h is stirred, the monoxone of 2100 parts by weight is slowly added to, so that it is reacted 3h under the conditions of 55 DEG C, wait for being cooled to room after reaction
Temperature is neutralized to neutrality with 20%HCl solution, obtains carboxyl methylation derivant；
(2) it dialyses：Above-mentioned carboxyl methylation derivant is transferred in bag filter, flowing water dialysis 12h, then saturating with distilled water
36h is analysed, dialyzate is concentrated under reduced pressure；
(3) alcohol precipitation and drying：Ethyl alcohol is added into the dialyzate after above-mentioned concentration, make ethyl alcohol final concentration reach 80% with
On, centrifugation rinses sediment 1 time with methanol/acetone, and vacuum freeze drying is to get carboxy methylation mulberries polysaccharide.
The preparation method of 2 carboxy methylation mulberries polysaccharide of embodiment
(1) carboxymethyl derivative reaction：The mulberries polysaccharide for taking the weight average molecular weight of 300 parts by weight to be 20.93kDa, is added
The isopropanol of 10 times of volumes, in the room temperature 25%NaOH solution that 20 times of volumes are added after magnetic stirrer 45min, room temperature
Lower stirring 2h, is slowly added to the monoxone of 2500 parts by weight, it is made to react 4h under the conditions of 60 DEG C, waits for being cooled to after reaction
Room temperature is neutralized to neutrality with 25%HCl solution, obtains carboxyl methylation derivant；
(2) it dialyses：Above-mentioned carboxyl methylation derivant is transferred in bag filter, flowing water is dialysed for 24 hours, then saturating with distilled water
48h is analysed, dialyzate is concentrated under reduced pressure；
(3) alcohol precipitation and drying：Ethyl alcohol is added into the dialyzate after above-mentioned concentration, make ethyl alcohol final concentration reach 80% with
On, centrifugation rinses sediment 2 times with methanol/acetone, and vacuum freeze drying is to get carboxy methylation mulberries polysaccharide.
Wherein mulberries polysaccharide can be prepared in accordance with the following steps：
(1) mulberries Thick many candies extract
1. extracting：Appropriate mulberries are taken, are homogenized, the water of 3 times of weight is added, extract 1 time under the conditions of 80 DEG C of temperature, every time
1.5h, filtration, merging filtrate are concentrated under reduced pressure into the 2/7 of original volume；
2. decolourizing：Suitable macroreticular resin progress adsorption bleaching, the constant temperature at 45 DEG C is added in filtrate 1. step concentrates after
Oscillation decoloration 20min, filtration repeat to elute the mulberries polysaccharide on macroreticular resin, and merging filtrate is concentrated under reduced pressure；
3. removing protein：It is added protease in filtrate after 2. being concentrated to step, at 40 DEG C, 1 times of volume is added in enzymolysis protein
Chloroform and 1/5 times of volume n-butanol, shake 20min, stand, remove impurity protein, be repeated 3 times until without precipitation analyse
Go out, centrifuges, take supernatant；
4. alcohol precipitation：Ethyl alcohol is added in supernatant to ethyl alcohol final concentration of 60%, 4 DEG C of standing 12h centrifuge, precipitation are taken to redissolve,
Alcohol precipitation again is repeated 2 times, sediment absolute ethyl alcohol and acetone respectively washing 1 time；
5. dialysing：Sediment after will be washed is fitted into pretreated bag filter, dialysis；Or using membrane filtration
Method filters, molecular cut off >=1500 of dialysis/membrane filtration, dry to get mulberries Thick many candies；
(2) purifying of mulberries Thick many candies
1. cellulose ion chromatographs
Prepare 8gL-1Mulberries Thick many candies solution, be transferred to equipped with cellulose ion exchange resin column DEAE-Cellulose
In 52, with distillation water elution, flow velocity 2mLmin-1, eluent is collected, is concentrated under reduced pressure, dialysis, vacuum freeze drying, score
The mulberries polysaccharide of grade purifying；
2. gel filtration chromatography
Mulberries polysaccharide after 52 grading purifications of DEAE-Cellulose is dissolved in ultra-pure water, solution is transferred to gel column
In Sephadex G-50, water elution, flow velocity 0.1mLmin are distilled-1, eluent is collected, after reduced pressure, vacuum refrigeration is dry
It is dry to get mulberries polysaccharide.
Wherein Sephadex G-100, Sephadex G-150 or Sephadex G-200 also can be selected in gel column.
The preparation method of 3 carboxy methylation mulberries polysaccharide of embodiment
(1) carboxymethyl derivative reaction：The mulberries polysaccharide for taking the weight average molecular weight of 300 parts by weight to be 20.93kDa, is added
The isopropanol of 12 times of volumes, in the room temperature 30%NaOH solution that 30 times of volumes are added after magnetic stirrer 60min, room temperature
Lower stirring 3h, is slowly added to the monoxone of 3000 parts by weight, it is made to react 5h under the conditions of 65 DEG C, waits for being cooled to after reaction
Room temperature is neutralized to neutrality with 30%HCl solution, obtains carboxyl methylation derivant；
(2) it dialyses：Above-mentioned carboxyl methylation derivant is transferred in bag filter, flowing water dialysis 36h, then saturating with distilled water
60h is analysed, dialyzate is concentrated under reduced pressure；
(3) alcohol precipitation and drying：Ethyl alcohol is added into the dialyzate after above-mentioned concentration, make ethyl alcohol final concentration reach 80% with
On, centrifugation rinses sediment 3 times with methanol/acetone, and vacuum freeze drying is to get carboxy methylation mulberries polysaccharide.
Wherein mulberries polysaccharide can be prepared in accordance with the following steps：
(1) mulberries Polyose extraction
1. extracting：Appropriate mulberries are taken, are homogenized, the water of 5 times of weight is added, extract 2 times under the conditions of 90 DEG C of temperature, every time
2.5h, filtration, merging filtrate are concentrated under reduced pressure into the 2/9 of original volume；
2. decolourizing：Suitable macroreticular resin progress adsorption bleaching, the constant temperature at 50 DEG C is added in filtrate 1. step concentrates after
Oscillation decoloration 40min, filtration repeat to elute the mulberries polysaccharide on macroreticular resin, and merging filtrate is concentrated under reduced pressure；
3. removing protein：It is added protease in filtrate after 2. being concentrated to step, at 70 DEG C, 2 times of volumes are added in enzymolysis protein
Dichloromethane and 1/2 times of volume n-butanol, shake 120min, stand, remove impurity protein, be repeated 7 times up to no precipitation
It is precipitated, centrifugation takes supernatant；
4. alcohol precipitation：Ethyl alcohol is added in supernatant to ethyl alcohol final concentration of 90%, 4 DEG C of standing 36h centrifuge, precipitation are taken to redissolve,
Alcohol precipitation again is repeated 3 times, with absolute ethyl alcohol and acetone respectively washing 3 times；
5. dialysing：Sediment after will be washed is fitted into pretreated bag filter, dialysis；Or using membrane filtration
Method filters, molecular cut off >=1500 of dialysis/membrane filtration, dry to get mulberries Thick many candies；
(2) purifying of mulberries Thick many candies
1. cellulose ion chromatographs
Prepare 12gL-1Mulberries Thick many candies solution, be transferred to equipped with cellulose ion exchange resin column DEAE-
In Cellulose 32, with distillation water elution, flow velocity 3mLmin-1, eluent is collected, is concentrated under reduced pressure, dialysis, vacuum refrigeration
It is dry, obtain the mulberries polysaccharide of grading purification；
2. gel filtration chromatography
Mulberries Thick many candies after 32 grading purifications of DEAE-Cellulose are dissolved in ultra-pure water, solution is transferred to gel column
In Sephadex G-200, water elution, flow velocity 0.4mLmin are distilled-1, collect eluent, after reduced pressure, vacuum refrigeration
Drying is to get mulberries polysaccharide.
Wherein Sephadex G-50, Sephadex G-100 or Sephadex G-150 also can be selected in gel column.
The preparation method of 4 carboxy methylation mulberries polysaccharide of embodiment
(1) carboxymethyl derivative reaction：The mulberries polysaccharide for taking the weight average molecular weight of 300 parts by weight to be 20.93kDa, is added
The isopropanol of 10 times of volumes, in the room temperature 25%NaOH solution that 20 times of volumes are added after magnetic stirrer 45min, room temperature
Lower stirring 2h, is slowly added to the monoxone of 2600 parts by weight, it is made to react 4h under the conditions of 60 DEG C, waits for being cooled to after reaction
Room temperature is neutralized to neutrality with 25%HCl solution, obtains carboxyl methylation derivant；
(2) it dialyses：Above-mentioned carboxyl methylation derivant is transferred in bag filter, flowing water is dialysed for 24 hours, then saturating with distilled water
48h is analysed, dialyzate is concentrated under reduced pressure；
(3) alcohol precipitation and drying：Ethyl alcohol is added into the dialyzate after above-mentioned concentration, make ethyl alcohol final concentration reach 80% with
On, centrifugation rinses sediment 2 times with methanol/acetone, and vacuum freeze drying is to get carboxy methylation mulberries polysaccharide.
Wherein mulberries polysaccharide may be used the prior art and extract, and obtain mulberries Thick many candies, then in accordance with the following steps
Mulberries Thick many candies are purified：
1. cellulose ion chromatographs
Prepare 10gL-1Mulberries Thick many candies solution, be transferred to equipped with cellulose ion exchange resin column DEAE-
In Cellulose 52, with distillation water elution, flow velocity 3mLmin-1, eluent is collected, is concentrated under reduced pressure, dialysis, vacuum refrigeration
It is dry, obtain the mulberries polysaccharide of grading purification；
2. gel filtration chromatography
Mulberries Thick many candies after 52 grading purifications of DEAE-Cellulose are dissolved in ultra-pure water, solution is transferred to gel column
In Sephadex G-100, water elution, flow velocity 0.25mLmin are distilled-1, collect eluent, after reduced pressure, vacuum refrigeration
Drying is to get mulberries polysaccharide.
Wherein Sephadex G-50, Sephadex G-150 or Sephadex G-200 also can be selected in gel column.
The preparation method of 5 carboxy methylation mulberries polysaccharide of embodiment
(1) carboxymethyl derivative reaction：The mulberries polysaccharide for taking the weight average molecular weight of 300 parts by weight to be 20.93kDa, is added
The isopropanol of 10 times of volumes, in the room temperature 30%NaOH solution that 25 times of volumes are added after magnetic stirrer 60min, room temperature
Lower stirring 2h, is slowly added to the monoxone of 2200 parts by weight, it is made to react 4h under the conditions of 55 DEG C, waits for being cooled to after reaction
Room temperature is neutralized to neutrality with 20%HCl solution, obtains carboxyl methylation derivant；
(2) it dialyses：Above-mentioned carboxyl methylation derivant is transferred in bag filter, flowing water dialysis 36h, then saturating with distilled water
48h is analysed, dialyzate is concentrated under reduced pressure；
(3) alcohol precipitation and drying：Ethyl alcohol is added into the dialyzate after above-mentioned concentration, make ethyl alcohol final concentration reach 80% with
On, centrifugation rinses sediment 3 times with methanol/acetone, and vacuum freeze drying is to get carboxy methylation mulberries polysaccharide.
Wherein mulberries polysaccharide can extract in accordance with the following steps：
1. extracting：Appropriate mulberries are taken, are homogenized, the water of 4 times of weight is added, extract 2 times under the conditions of 85 DEG C of temperature, every time
2h, filtration, merging filtrate are concentrated under reduced pressure into the 16/63 of original volume；
2. decolourizing：Suitable macroreticular resin progress adsorption bleaching, the constant temperature at 48 DEG C is added in filtrate 1. step concentrates after
Oscillation decoloration 30min, filtration repeat to elute the mulberries polysaccharide on macroreticular resin, and merging filtrate is concentrated under reduced pressure；
3. removing protein：It is added protease in filtrate after 2. being concentrated to step, at 55 DEG C, 2 times of volumes are added in enzymolysis protein
Sevag reagents (VN-butanol∶VChloroform=1: 4), shaking 70min, stand, remove impurity protein, be repeated 5 times until being analysed without precipitation
Go out, centrifuges, take supernatant；
4. alcohol precipitation：Ethyl alcohol is added in supernatant to ethyl alcohol final concentration of 75%, 4 DEG C stand for 24 hours, and centrifugation takes precipitation to redissolve,
Alcohol precipitation again is repeated 2 times, with absolute ethyl alcohol and acetone respectively washing 2 times；
5. dialysing：Sediment after will be washed is fitted into pretreated bag filter, dialysis；Or using membrane filtration
Method filters, molecular cut off >=1500 of dialysis/membrane filtration, dry to get mulberries Thick many candies；
Then the mulberries Thick many candies that extraction obtains are purified using the prior art, further obtains mulberries polysaccharide.
The application of 6 carboxy methylation mulberries polysaccharide of embodiment
In mass ratio 8: 0.4: 8: 0.3 weighs carboxy methylation mulberries polysaccharide (embodiment 1 is made), dextrin, sucrose and carboxylic first
Granule is made in base sodium cellulosate, mixing.For preventing or treating acute alcohol-induced hepatic injury, daily measure：1~2g.
The application of 7 carboxy methylation mulberries polysaccharide of embodiment
In mass ratio 8: 0.4: 0.2: 0.3 weighs carboxy methylation mulberries polysaccharide (embodiment 2 is made), starch, sucrose and carboxylic
Tablet, 0.3~0.5g/ pieces is made in sodium carboxymethylcellulose pyce, mixing.For preventing or treating Asia property alcoholic liver injury, often take daily
Dosage：3~5.
The application of 8 carboxy methylation mulberries polysaccharide of embodiment
In mass ratio 8: 0.4: 6: 0.3 weighs carboxy methylation mulberries polysaccharide (embodiment 4 is made), dextrin, sucrose and carboxylic first
Granule is made in base sodium cellulosate, mixing.For prevention or medicine physical property hepatic injury, daily measure：1~2g.
The application of 9 carboxy methylation mulberries polysaccharide of embodiment
In mass ratio 8: 0.4: 0.1: 0.3 weighs carboxy methylation mulberries polysaccharide (embodiment 3 is made), starch, sucrose and carboxylic
Tablet, 0.3~0.5g/ pieces is made in sodium carboxymethylcellulose pyce, mixing.For relieving alcoholism and protecting liver, daily measure：3~5.
Claims (10)
1. a kind of carboxy methylation mulberries polysaccharide, it is characterised in that：The carboxy methylation mulberries polysaccharide is to be by weight average molecular weight
The mulberries polysaccharide of 20.93kDa gained after carboxy methylation is modified.
2. carboxy methylation mulberries polysaccharide according to claim 1, which is characterized in that the weight of the carboxy methylation mulberries polysaccharide
Average molecular weight is 26.18kDa, degree of substitution 0.6-0.8.
3. the preparation method of carboxy methylation mulberries polysaccharide as claimed in claim 1 or 2, which is characterized in that include the following steps：
(1) carboxymethyl derivative reaction：Take mulberries polysaccharide after purification, be added 7-12 times of volume isopropanol stir and evenly mix after plus
Enter the NaOH solution of 10-30 times of volume, stir, be slow added into the monoxone of 7-10 times of mulberries polysaccharide weight, reacts 3-5h, wait for
It is cooled to room temperature after reaction, solution is neutralized to neutrality, obtains carboxyl methylation derivant；
(2) it dialyses：Carboxyl methylation derivant is transferred in bag filter, first flowing water dialysis, then is dialysed with distilled water, concentration dialysis
Liquid；
(3) alcohol precipitation and drying：It is added ethyl alcohol into the dialyzate after concentration, carries out alcohol precipitation, centrifugation, taking precipitate is with methanol/the third
Ketone rinses, and vacuum freeze drying is to get carboxy methylation mulberries polysaccharide.
4. the preparation method of carboxy methylation mulberries polysaccharide according to claim 3, which is characterized in that include the following steps：
(1) carboxymethyl derivative reaction：The purifying mulberries polysaccharide of 300 parts by weight is taken, the isopropanol of 7-12 times of volume is added, in room
The temperature 20%-30%NaOH solution that 10-30 times of volume is added after magnetic stirrer 30-60min, stirs 1- at room temperature
3h is slowly added to the monoxone of 2100-3000 parts by weight, it is made to react 3-5h under the conditions of 55-65 DEG C, waits for cold after reaction
But to room temperature, it is neutralized to neutrality with 20%-30%HCl solution, obtains carboxyl methylation derivant；
(2) it dialyses：Carboxyl methylation derivant is transferred in bag filter, first flowing water is dialysed 12-36h, then is dialysed 36- with distilled water
Dialyzate is concentrated under reduced pressure in 60h；
(3) alcohol precipitation and drying：Ethyl alcohol is added into the dialyzate after concentration, ethyl alcohol final concentration is made to reach 80% or more, centrifuges, uses
Methanol/acetone rinses sediment 1-3 times, and vacuum freeze drying is to get carboxy methylation mulberries polysaccharide.
5. the preparation method of carboxy methylation mulberries polysaccharide according to claim 3 or 4, which is characterized in that the mulberries
Polysaccharide extracts as follows：
1. extracting：Appropriate mulberries are taken, are homogenized, the water of 3-5 times of weight is added, extract 1-2 times under the conditions of 80-90 DEG C of temperature, often
Secondary 1.5-2.5h, filtration, merging filtrate are concentrated under reduced pressure into the 2/7-2/9 of original volume；
2. decolourizing：Suitable macroreticular resin progress adsorption bleaching is added in filtrate 1. step concentrates after, and constant temperature shakes at 45-50 DEG C
Decoloration 20-40min is swung, filtration repeats to elute the mulberries polysaccharide on macroreticular resin, and merging filtrate is concentrated under reduced pressure；
3. removing protein：It is added protease in filtrate after 2. being concentrated to step, at 40-70 DEG C, 1-2 times of body is added in enzymolysis protein
The n-butanol of long-pending chloroform or dichloromethane and 1/5-1/2 times of volume shakes 20-120min, stands, and removes impurity egg
In vain, 3-7 times is repeated up to no Precipitation, and centrifugation takes supernatant；
4. alcohol precipitation：Ethyl alcohol is added in supernatant to the final concentration of 60%-90% of ethyl alcohol, 4 DEG C stand 12-36h, and centrifugation takes precipitation
It redissolves, again alcohol precipitation, repeats 2-3 times, sediment absolute ethyl alcohol and acetone respectively wash 1-3 times；
5. dialysing：Sediment after washing is fitted into pretreated bag filter, is dialysed；Or the method mistake using membrane filtration
Filter, molecular cut off >=1500 of dialysis/membrane filtration are dry to get mulberries Thick many candies.
6. the preparation method of carboxy methylation mulberries polysaccharide according to claim 5, which is characterized in that mulberries Thick many candies it is pure
Change includes the following steps：
1. cellulose ion chromatographs
Prepare 8-12gL-1Mulberries Thick many candies solution, be transferred in cellulose ion exchange resin column, with distillation water elution, stream
Speed is 2-3mLmin-1, eluent is collected, is concentrated under reduced pressure, dialysis, vacuum freeze drying obtains the mulberries polysaccharide of grading purification；
2. gel filtration chromatography
The mulberries polysaccharide of grading purification is dissolved in ultra-pure water, solution is transferred in gel column, distills water elution, flow velocity 0.1-
0.4mL·min-1, eluent is collected, after reduced pressure, vacuum freeze drying is to get mulberries polysaccharide.
7. the preparation method of carboxy methylation mulberries polysaccharide according to claim 6, which is characterized in that the cellulose ion
Exchanger resin column packing is DEAE-Cellulose 52 or DEAE-Cellulose 32；Preferably DEAE-Cellulose 52.
8. the preparation method of carboxy methylation mulberries polysaccharide according to claim 6, which is characterized in that the gel column packing
For Sephadex G-50, Sephadex G-100, Sephadex G-150 or Sephadex G-200；Preferably Sephadex
G-100。
9. application of the carboxy methylation mulberries polysaccharide as claimed in claim 1 or 2 in preparing hepatic or health products.
10. carboxy methylation mulberries polysaccharide answering in preparing alcoholic liver injury drug or health products as claimed in claim 1 or 2
With.
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| CN102321186A (en) * | 2011-08-02 | 2012-01-18 | 江苏大学 | Carboxymethylated auricularia auricula polysaccharide and crude polysaccharide, and preparation method and application thereof |
| CN104987431A (en) * | 2015-07-28 | 2015-10-21 | 华南理工大学 | Mulberry active polysaccharide and extracting method thereof |
| CN105147718A (en) * | 2015-07-22 | 2015-12-16 | 贵州师范大学 | Application of mulberry polysaccharide extract in preparing medicine or health product |
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| CN102321186A (en) * | 2011-08-02 | 2012-01-18 | 江苏大学 | Carboxymethylated auricularia auricula polysaccharide and crude polysaccharide, and preparation method and application thereof |
| CN105147718A (en) * | 2015-07-22 | 2015-12-16 | 贵州师范大学 | Application of mulberry polysaccharide extract in preparing medicine or health product |
| CN104987431A (en) * | 2015-07-28 | 2015-10-21 | 华南理工大学 | Mulberry active polysaccharide and extracting method thereof |
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