CN108727511B - Carboxymethylated mulberry polysaccharide and preparation method and application thereof - Google Patents
Carboxymethylated mulberry polysaccharide and preparation method and application thereof Download PDFInfo
- Publication number
- CN108727511B CN108727511B CN201810668387.6A CN201810668387A CN108727511B CN 108727511 B CN108727511 B CN 108727511B CN 201810668387 A CN201810668387 A CN 201810668387A CN 108727511 B CN108727511 B CN 108727511B
- Authority
- CN
- China
- Prior art keywords
- carboxymethylated
- polysaccharide
- mulberry polysaccharide
- mulberry
- preparation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- Materials Engineering (AREA)
- Polymers & Plastics (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- General Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Gastroenterology & Hepatology (AREA)
- Sustainable Development (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Epidemiology (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses carboxymethylated mulberry polysaccharide, a preparation method thereof and application thereof in pharmacy. The carboxymethylation mulberry polysaccharide is obtained by carboxymethylation modification of mulberry polysaccharide with the weight-average molecular weight of 20.93kDa, and the weight-average molecular weight of the mulberry polysaccharide is 26.18kDa and the substitution degree of the mulberry polysaccharide is 0.6-0.8 through experimental determination and analysis. Meanwhile, pharmacological experiments prove that the carboxymethylated mulberry polysaccharide has the effects of activating alcohol dehydrogenase, removing active oxygen free radicals and preventing the free radicals from damaging cell structures, and has a good application prospect.
Description
Technical Field
The invention relates to the field of traditional Chinese medicines, and in particular relates to carboxymethylation mulberry polysaccharide obtained by carrying out carboxymethylation reaction on mulberry polysaccharide, a preparation method of the carboxymethylation mulberry polysaccharide and application of the carboxymethylation mulberry polysaccharide in preparation of liver-protecting medicines.
Background
The liver is the largest parenchymal organ in the abdominal cavity, is responsible for important physiological functions of the human body, and is an important detoxifying organ of the human body. In the production process of both nature and human industry, some chemical substances which are toxic to the liver, including alcohol, chemical toxic substances in the environment and some drugs, can enter the liver through the portal vein of the gastrointestinal tract or systemic circulation for transformation, can cause different degrees of hepatocyte necrosis, fatty deformation, liver cirrhosis and liver cancer of the liver, and seriously threatens the human health.
Mulberries (Mori fruits) are mature fruit clusters of perennial woody plant mulberries (Morus alba L), are the first varieties of Chinese recorded in the famous records of homology of medicine and food of the national ministry of health, are one of excellent health-care functional food resources in China, and have the effects of enhancing immunity, regulating blood sugar and blood fat, protecting liver and the like. The mulberry polysaccharide is one of the main active ingredients, and has a good liver protection effect. The previous work of the inventor (patent application No. 201510431557.5) also provides a separation and purification method of the mulberry polysaccharide and application of the mulberry polysaccharide in preparing medicines and health products.
The biological activity of the polysaccharide is closely related to the structure of the polysaccharide, and the molecular modification is carried out on the structure of the polysaccharide by a proper method, so that the physiological activity of the polysaccharide can be improved to a certain extent, certain toxic and side effects are reduced, or new physiological activity is endowed. Carboxymethylated Polysaccharides (CP) refer to a class of structurally complex and active polysaccharide derivatives formed by substituting one or several hydroxyl groups on a monosaccharide molecule in a polysaccharide chain with carboxymethyl groups.
Disclosure of Invention
The invention aims to provide carboxymethylation mulberry polysaccharide (C-MFP) obtained by carrying out carboxymethylation reaction on mulberry polysaccharide and a preparation method thereof, and researches the application of the carboxymethylation mulberry polysaccharide in the preparation of a liver-protecting medicine so as to develop a new liver-protecting medicine or health-care product preparation.
The invention adopts the following technical scheme:
carboxymethylation mulberry polysaccharide is obtained by carboxymethylation modification of mulberry polysaccharide with the weight-average molecular weight of 20.93 kDa.
The weight average molecular weight of the carboxymethylated mulberry polysaccharide is 26.18kDa, and the substitution degree is 0.6-0.8.
The preparation method of the carboxymethylated mulberry polysaccharide comprises the following steps:
(1) and (3) carboxymethyl derivatization reaction: adding 7-12 times of isopropanol into the purified mulberry polysaccharide, uniformly stirring, adding 10-30 times of NaOH solution, stirring, slowly adding chloroacetic acid 7-10 times of the mulberry polysaccharide, reacting for 3-5h, cooling to room temperature after the reaction is finished, and neutralizing the solution to be neutral to obtain carboxymethylated derivatives;
(2) and (3) dialysis: transferring the carboxymethylated derivative into a dialysis bag, dialyzing with running water, dialyzing with distilled water, and concentrating the dialysate;
(3) alcohol precipitation and drying: and adding ethanol into the concentrated dialysate, carrying out alcohol precipitation and centrifugation, taking out precipitate, washing with methanol/acetone, and carrying out vacuum freeze drying to obtain the carboxymethylated mulberry polysaccharide.
More specifically, the preparation method of the carboxymethylated mulberry polysaccharide comprises the following steps:
(1) and (3) carboxymethyl derivatization reaction: adding 7-12 times volume of isopropanol into 300 parts by weight of purified mulberry polysaccharide, stirring for 30-60min at room temperature by using a magnetic stirrer, adding 10-30 times volume of 20-30% NaOH solution, stirring for 1-3h at room temperature, slowly adding 2100-3000 parts by weight of chloroacetic acid, reacting for 3-5h at 55-65 ℃, cooling to room temperature after the reaction is finished, and neutralizing to neutrality by using 20-30% HCl solution to obtain carboxymethylated derivatives;
(2) and (3) dialysis: transferring the carboxymethylated derivatives into a dialysis bag, dialyzing with running water for 12-36h, dialyzing with distilled water for 36-60h, and concentrating the dialysate under reduced pressure;
(3) alcohol precipitation and drying: adding ethanol into the concentrated dialysate to make ethanol concentration reach above 80%, centrifuging, washing precipitate with methanol/acetone for 1-3 times, and vacuum freeze drying to obtain carboxymethylated Mori fructus polysaccharide.
In the above preparation method of carboxymethylated mulberry polysaccharide, the mulberry polysaccharide is extracted according to the following method:
firstly, extraction: taking a proper amount of mulberry, homogenizing, adding 3-5 times of water by weight, extracting for 1-2 times at the temperature of 80-90 ℃ for 1.5-2.5h each time, filtering, combining filtrates, and concentrating under reduced pressure to 2/7-2/9 of the original volume;
② decoloring: adding a proper amount of macroporous resin into the concentrated filtrate for adsorption decoloration, oscillating and decoloring at constant temperature of 45-50 ℃ for 20-40min, filtering, repeatedly eluting the mulberry polysaccharide on the macroporous resin, merging the filtrate, and concentrating under reduced pressure;
removing protein: adding protease into the concentrated filtrate, performing enzymolysis at 40-70 deg.C, adding 1-2 times of chloroform or dichloromethane and 1/5-1/2 times of n-butanol, shaking for 20-120min, standing, removing impurity protein, repeating for 3-7 times until no precipitate is separated out, centrifuging, and collecting supernatant;
fourthly, alcohol precipitation: adding ethanol into the supernatant until the final concentration of ethanol is 60% -90%, standing at 4 deg.C for 12-36h, centrifuging, re-dissolving the precipitate, precipitating with ethanol again, repeating for 2-3 times, and washing the precipitate with anhydrous ethanol and acetone for 1-3 times respectively;
fifth, dialysis: putting the washed precipitate into a pre-treated dialysis bag, and dialyzing; or filtering by membrane filtration, wherein the cut-off molecular weight of dialysis/membrane filtration is not less than 1500, and drying to obtain crude mulberry polysaccharide.
In the preparation method of the carboxymethylated mulberry polysaccharide, the purification of the crude mulberry polysaccharide comprises the following steps:
(ii) cellulose ion chromatography
Preparation of 8-12 g.L-1The crude polysaccharide solution of Mori fructus is transferred into cellulose ion exchange resin column, and eluted with distilled water at flow rate of 2-3 mL/min-1Collecting eluent, concentrating under reduced pressure, dialyzing, and freeze-drying under vacuum to obtain fractionated and purified mulberry polysaccharide;
② gel column chromatography
Dissolving the purified mulberry polysaccharide in ultrapure water, transferring the solution into gel column, eluting with distilled water at flow rate of 0.1-0.4 mL/min-1Collecting the eluent, concentrating under reduced pressure, and vacuum freeze-drying to obtain the mulberry polysaccharide.
In the preparation method of the carboxymethylated mulberry polysaccharide, the Cellulose ion exchange resin column filler is DEAE-Cellulose52 or DEAE-Cellulose 32; preferably DEAE-Cellulose 52.
In the preparation method of the carboxymethylated mulberry polysaccharide, the gel column filler is Sephadex G-50, Sephadex G-100, Sephadex G-150 or Sephadex G-200; preferably Sephadex G-100.
The carboxymethylated mulberry polysaccharide is applied to preparation of liver-protecting medicines or health-care products.
The carboxymethylated mulberry polysaccharide is applied to preparation of drugs or health care products for alcoholic liver injury.
In order to verify the prepared carboxymethylated mulberry polysaccharide and the liver protection effect thereof, the inventor performs the following experiments:
1. infrared analysis of carboxymethylated mulberry polysaccharide and determination of substitution degree
Taking 2mg of fully dried carboxymethylated mulberry polysaccharide, fully mixing with dried KBr in an agate mortar (m: m is 1: 100), grinding into powder, tabletting, and scanning by using an infrared spectrometer within the scanning range of 4000-400 cm-1. Shown in FIG. 2, withFIG. 1 comparison of the IR spectra of the samples before carboxymethylation, C-MFP at 1617cm-1And 1423cm-1Two new strong absorptions appeared, indicating that MFP was partially carboxymethylated and C-MFP was 848.21cm-1A new band appears, which shows the characteristic absorption peak of the alpha-type C-H vibration of pyranose. Notably, the characteristic absorption peaks of the polysaccharide still exist in the modified derivative. Therefore, the natural polysaccharide of the present invention is successfully modified without changing the molecular chain structure of the polysaccharide itself.
Measurement of degree of substitution of carboxymethylated Mulberry polysaccharide
(1) Sample treatment: accurately weighing 10mg carboxymethylated polysaccharide, drying at 100 deg.C for 1h, adding 3mL 70% ethanol, stirring, standing for 5min, sequentially adding 10mL distilled water and 0.1% NaOH solution, mixing, and stirring until the sample is completely dissolved. The millimoles of HCl (A) per gram of carboxymethyl polysaccharide are then calculated by titration with a 0.1% HCl solution, using phenolphthalein as indicator.
(2) Degree of Substitution (DS) calculation: the degree of substitution refers to the degree to which the hydroxyl groups on each sugar ring in the polysaccharide molecule are substituted. The calculation formula is as follows:
in the formula: v0-volume of NaOH solution added, mL; v1-volume of HCl solution consumed for blank determination, mL; v2-volume of HCl solution consumed for sample determination, mL; c. C0-the concentration of the added NaOH solution, M; c-determining the concentration of HCl solution used, M; m-mass of sample used, g.
Through determination and analysis, the molecular weight of the carboxymethylated mulberry polysaccharide is about 26.18kDa, and the degree of substitution is 0.6-0.8.
2. Activation effect of mulberry polysaccharide and carboxymethylated mulberry polysaccharide on alcohol dehydrogenase
The instrument comprises the following steps: a microplate reader, a constant temperature and humidity box and a one hundred thousand balance.
Materials and reagents: oxidized coenzyme I (Nicotinamide coenzyme, NAD)+) Alcohol Dehydrogenase (ADH), ethanol, sodium pyrophosphate buffer salts.
By using a modified Valle&The ADH activity was determined by the Hoch method by adding 100. mu.L of sodium pyrophosphate buffer, pH 8.8, and 75. mu.L of LNAD buffer to the assay plate+Mixing 35 μ L ethanol, 10 μ L Mori fructus polysaccharide solution, and carboxymethylated Mori fructus polysaccharide solution, and incubating at 37 deg.C for 5 min. 10 μ L of ADH were added immediately and, after shaking up, the absorbance at 340nm was measured immediately (A)340) The measurement was continued for 5min every 10s and the data was recorded. Distilled water is used for replacing ethanol solution for zero adjustment, a group without medicine is used as a blank group, and bifendate is used as a positive control. ADH activity and activation rate were calculated using the following formulas.
As shown in fig. 3, the experiment results of the influence of the mulberry polysaccharide and the carboxymethylated mulberry polysaccharide on the activity of the alcohol dehydrogenase show that the activation effect of the mulberry polysaccharide on the alcohol dehydrogenase is obviously improved after the mulberry polysaccharide is modified by carboxymethylation, and the mulberry polysaccharide has a good application prospect in relieving alcoholism and protecting liver.
3. Research on acute alcoholic liver injury resistance of mulberry polysaccharide and carboxymethylated mulberry polysaccharide
In order to further prove that the mulberry polysaccharide and the carboxymethylated mulberry polysaccharide have the liver protection effect, a mouse acute liver injury model is established. The specific experimental steps are as follows:
(1) the instrument comprises the following steps: a microplate reader and a constant temperature and humidity chamber.
(2) Materials and reagents: glutamic-oxaloacetic transaminase (AST), glutamic-pyruvic transaminase (ALT), Lactate Dehydrogenase (LDH), Triglyceride (TG), total cholesterol (T-CHO), Total Bilirubin (TBIL), low-density lipoprotein cholesterol (LDL-C), Malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) kit.
(3) The experimental steps are as follows:
feeding 90 healthy male KM mice with the weight of 18-22g for one week under the indoor temperature of 20 +/-2 ℃, the relative humidity of 50% and natural lighting conditions, randomly dividing 10 mice into 9 groups, namely a blank control group (physiological saline), a pathological model group (EtOH) and a positive control group (EtOH +0.21 g.kg. kg)-1BW bifendate), high, medium, and low dose groups of purified mulberry polysaccharide (EtOH +123.6mg kg)-1MFP、EtOH+61.8mg·kg-1MFP、EtOH+30.9mg·kg- 1MFP), high, medium, low dose group of carboxymethylated Mulberry polysaccharides (EtOH +123.6mg kg)-1C-MFP、EtOH+61.8mg·kg-1C-MFP、EtOH+30.9mg·kg-1C-MFP). According to different groups, firstly, the normal saline/bifendate/MFP/C-MFP is orally taken every day, the normal saline/alcohol is orally taken after 4 hours, all groups are continuously irrigated to the stomach for 7 days according to the samples and the dosages, after a mouse is fasted for 12 hours before the test is finished, the eyeball is picked to draw blood, the mouse is dislocated and killed, the liver is quickly dissected and taken out, the mouse is washed by the ice normal saline, and the filter paper is weighed after absorbing the water on the surface and is used for calculating the liver organ index. Then taking the left liver leaf to prepare liver homogenate for measuring biochemical indexes of livers such as malonaldehyde, superoxide dismutase, glutathione peroxidase and the like. About 1.0X 0.2cm away from the border of the liver lobule by 5cm was additionally taken3Fixing liver tissue in 10% neutral formaldehyde solution, and making pathological section.
② preparing liver homogenate: taking mouse liver tissue, adding 1: 9 (W: V) physiological saline, homogenizing with a tissue homogenizer under ice water bath condition, centrifuging at low temperature for 10min, and taking supernatant for use.
Measuring liver coefficient: liver organ index (%) -. liver mass (g)/mouse weight (g). times.100%
Measurement of serum AST, ALT, LDH, TG, T-CHO, TBIL and LDL-C: standing blood sample with ice water, centrifuging, taking upper layer serum after 15min, and determining biochemical indexes of each serum strictly according to the method provided by the kit instruction.
Fifthly, measuring the levels of MDA, SOD and GSH-PX of the liver tissue homogenate: after 10% liver tissue homogenate is diluted to a proper concentration, various biochemical indexes of the liver are determined strictly according to the method of a kit instruction.
Preparing histopathology sections: after the mouse is dissected, the liver is quickly taken out, the liver is rinsed by precooled normal saline, blood is removed, filter paper is wiped dry, the same part of the liver of the mouse in each group is cut and fixed by 10% formalin for 24 hours, a tissue block is washed by running water overnight, dehydrated, transparent, embedded, sliced, stained by hematoxylin-eosin, naturally dried and then sliced, and the slide glass is placed under an optical microscope to observe the pathological morphological change of the liver tissue slice.
(4) Research result of anti-acute alcoholic liver injury effect of mulberry polysaccharide and carboxymethylated mulberry polysaccharide
(ii) weight changes of MFP and C-MFP on mice with acute alcoholic liver injury
Before and after the experiment, the weight of the blank group mice averagely increases by 5.1 percent, and the weight of the model group mice obviously decreases (P is less than 0.01). The weight of mice in each dose group of MFP and each dose group of C-MFP and the positive control group is reduced but is not obvious, wherein the medium dose and high dose groups of C-MFP are superior to the positive control group.
(II) Effect of MFP and C-MFP on liver index of mice with acute alcoholic liver injury
As can be seen from Table 1, the liver indexes of 9 groups of mice have no significant influence, and the mulberry polysaccharide and carboxymethylated mulberry polysaccharide can be preliminarily judged to have no dominant toxicity to the mice. The liver index decreased most strongly in the C-MFP high dose group.
TABLE 1 Effect of MFP and C-MFP on liver coefficients in mice with acute alcoholic liver injury
| Group of | Liver/body (g.g)-1) |
| Blank group | 0.041±0.0067 |
| Pathological model group | 0.044±0.0027 |
| Positive control group | 0.044±0.0033 |
| MFP high dose group | 0.045±0.0043 |
| Dose groups in MFP | 0.043±0.0054 |
| MFP Low dose group | 0.049±0.0052 |
| C-MFP high dose group | 0.037±0.048 |
| C-MFP Medium dose group | 0.044±0.0035 |
| C-MFP Low dose group | 0.044±0.0053 |
(3) Liver function Effect of MFP and C-MFP on serum of mice with acute alcoholic liver injury
As can be seen from Table 2, the serum levels of AST, ALT, LDH, TG, LDL-C, T-CH and TBIL in the model group are significantly higher than those in the blank control group, indicating successful modeling. Compared with the model group, the levels of AST, ALT, LDH, TG, LDL-C and TBIL of each dosage group of the mulberry polysaccharide and the carboxymethylated mulberry polysaccharide are gradually reduced, and the low, medium and high dosages show gradient reduction of each index level, which shows that the concentration of the polysaccharide and the activity have a certain dose-effect relationship. TG levels in the MFP medium-high dose group and the C-MFP high dose group are lower than those in the positive control group, which indicates that the TG level in the serum of the acute alcoholic liver injury mouse can be obviously reduced. Compared with the model group, the high, medium and low dose groups of C-MFP all can obviously reduce the LDL-C level of serum (P < 0.01). In conclusion, MFP and C-MF have effects on the liver function of the serum of the acute alcoholic liver injury mouse, can effectively improve the damage condition of liver cells, and can effectively reduce the fat content in the serum. As can be seen from Table 2, the serum levels of AST, ALT, LDH, TG, LDL-C, T-CH and TBIL in the model group are significantly higher than those in the blank control group, indicating successful modeling. Compared with the model group, the levels of AST, ALT, LDH, TG, LDL-C and TBIL of each dosage group of the mulberry polysaccharide and the carboxymethylated mulberry polysaccharide are gradually reduced, and the low, medium and high dosages show gradient reduction of each index level, which shows that the concentration of the polysaccharide and the activity have a certain dose-effect relationship. TG levels in the MFP medium-high dose group and the C-MFP high dose group are lower than those in the positive control group, which indicates that the TG level in the serum of the acute alcoholic liver injury mouse can be obviously reduced. Compared with the model group, the high, medium and low dose groups of C-MFP all can obviously reduce the LDL-C level of serum (P < 0.01). In conclusion, the MFP and C-MFP can effectively improve the damage condition of liver cells and effectively reduce the fat content in serum due to the influence of the MFP and the C-MFP on the liver function of the serum of the acute alcoholic liver injury mouse.
The liver function influence of MFP and C-MFP on liver tissues of mice with acute alcoholic liver injury is shown in Table 3, the MDA level of the liver of mice in an acute pathology model group is remarkably increased (P is less than 0.01) compared with that of a blank control group, the liver cell injury is serious, and the MDA level of the liver cell of the mice is remarkably reduced (P is less than 0.05) when the MDA level is pre-dosed with high and medium doses of low doses of MFP and C-MFP. Wherein the MDA reduction ratio of the liver cells of the C-MFP high-dose group mice is better than that of the positive control biphenyldicarboxylate. In addition, the bifendate and the C-MFP can obviously enhance the SOD activity and the content of GSH at high and medium doses, wherein the effect of the C-MFP high dose group is most obvious (P is less than 0.01). The MFP and C-MFP have the functions of eliminating active oxygen free radicals and preventing the damage of the free radicals to cell structures on alcoholic liver injury.
The results of the comprehensive detection of the liver function indexes in the serum and the liver of 9 mice, namely AST, ALT, LDH, TG, LDL-C, T-CHO, TBIL, MDA, SOD and GSH-PX by using MFP and C-MFP show that the C-MFP has a better protection effect on acute alcoholic liver injury.
TABLE 2 Effect of MFP and C-MFP on Biochemical indicators in serum of mice with acute alcoholic liver injury
Note: p < 0.05 compared to blank, P < 0.01 compared to blank; compared with the model group, P is less than 0.05, and compared with the model group, P is less than 0.01.
TABLE 3 Effect of MFP and C-MFP on Biochemical indicators in liver of mice with acute alcoholic liver injury
Note: p < 0.05 compared to blank, P < 0.01 compared to blank;
compared with the model group, P is less than 0.05, and compared with the model group, P is less than 0.01.
Pathological section analysis of liver tissue of mouse with acute alcoholic liver injury
Pathological sections of the liver tissue of the mice in each experimental group are shown in fig. 4, and correspond to the experimental groups in table 3 from left to right and from top to bottom, the hepatocyte structure of the blank control group is complete, the cell nucleus is clear and visible, and the cytoplasm is uniform without histological abnormality. In contrast, the pathological model group had obvious morphology the table: the cells had been severely deformed and the cells were blurred and the hepatocytes swollen. Compared with the acute alcoholic liver injury model group, the bifendate can effectively relieve the symptom of the liver cell lesion caused by alcohol and has obvious protective effect on liver cells. The hepatocytes of the mice in the high dose group of MFP had no significant cell swelling and the cell edges were clearly visible. The hepatocyte cells of the mice in the C-MFP high-dose group have good integrity, the cells are basically not broken, and lipid substances around the cells are obviously reduced. The MFP and C-MFP dose groups have certain treatment effect on acute alcoholic liver injury, and have certain metering dependence. The high-dose group of the C-MFP has a good effect on acute alcoholic liver injury, and is a potential drug for relieving alcoholism and protecting liver.
In conclusion, pharmacological experiments prove that the carboxymethylated polysaccharide C-MFP can greatly improve the activity of ADH, and the carboxymethylated polysaccharide C-MFP has a good application prospect in relieving alcoholism and protecting liver; pharmacological experiments also show that compared with a model group, the levels of AST, ALT, LDH, TG, LDL-C and TBIL of each dose group of the mulberry polysaccharide and the carboxymethylated mulberry polysaccharide are gradually reduced, and low, medium and high doses show gradient reduction of each index level, which shows that the polysaccharide concentration and the activity have a certain dose-effect relationship, and the MDA level of hepatocytes of mice pre-dosed with MFP and C-MFP is remarkably reduced (P is less than 0.05), wherein the MDA reduction ratio of the hepatocytes of the mice in the high dose group of the C-MFP is superior to that of the positive control biphenyldicarboxylate. In addition, the content of SOD activity and GSH can be obviously enhanced by high and medium dosage of C-MFP, which shows that MFP and C-MFP have the functions of eliminating active oxygen free radicals and preventing the damage of free radicals to cell structures on alcoholic liver injury. Meanwhile, the C-MFP has the function of eliminating active oxygen free radicals, so that the C-MFP has the prospect of preparing antioxidant drugs.
In addition, the hepatocytes of the high dose group of MFP had no significant cell swelling and the cell edges were clearly visible. The hepatocyte cells of the mice in the C-MFP high-dose group have good integrity, the cells are basically not broken, and lipid substances around the cells are obviously reduced. The MFP and C-MFP dosage groups have certain treatment effect on acute alcoholic liver injury, have certain metering dependence relationship, and can be potential drugs for relieving alcoholism and protecting liver.
According to the invention, carboxymethylation structure modification is carried out on the purified mulberry polysaccharide to obtain the carboxymethylated mulberry polysaccharide. Researches find that the carboxymethylated mulberry polysaccharide has a strong liver protection effect, and the results provide a basis for deep development and utilization of the mulberry polysaccharide and also provide a scientific basis for research and development of new liver protection medicaments.
Description of the drawings:
fig. 1 is an infrared spectrum of a mulberry polysaccharide sample before carboxymethylation.
Fig. 2 is an infrared spectrum of a carboxymethylated mulberry polysaccharide sample.
Figure 3 influence of mulberry polysaccharide and carboxymethylated mulberry polysaccharide on alcohol dehydrogenase activity.
Fig. 4 is a pathological section view of the action of mulberry polysaccharide and carboxymethylated mulberry polysaccharide on liver tissues of mice of acute alcoholic liver injury models.
Detailed Description
Example 1 preparation method of carboxymethylated Mulberry polysaccharide
(1) And (3) carboxymethyl derivatization reaction: taking 300 parts by weight of mulberry polysaccharide with the weight-average molecular weight of 20.93kDa, adding isopropanol with the volume being 7 times that of the mulberry polysaccharide, stirring the mixture for 30min at room temperature by using a magnetic stirrer, adding 20% NaOH solution with the volume being 10 times that of the mulberry polysaccharide, stirring the mixture for 1h at room temperature, slowly adding 2100 parts by weight of chloroacetic acid, reacting the mixture for 3h at the temperature of 55 ℃, cooling the mixture to room temperature after the reaction is finished, and neutralizing the mixture to be neutral by using 20% HCl solution to obtain carboxymethylated derivatives;
(2) and (3) dialysis: transferring the carboxymethylated derivatives into a dialysis bag, dialyzing with running water for 12h, dialyzing with distilled water for 36h, and concentrating the dialysate under reduced pressure;
(3) alcohol precipitation and drying: adding ethanol into the concentrated dialysate to make ethanol concentration reach above 80%, centrifuging, washing precipitate with methanol/acetone for 1 time, and vacuum freeze drying to obtain carboxymethylated Mori fructus polysaccharide.
Example 2 preparation method of carboxymethylated Mulberry polysaccharide
(1) And (3) carboxymethyl derivatization reaction: taking 300 parts by weight of mulberry polysaccharide with the weight-average molecular weight of 20.93kDa, adding 10 times of isopropanol, stirring for 45min at room temperature by using a magnetic stirrer, adding 20 times of 25% NaOH solution, stirring for 2h at room temperature, slowly adding 2500 parts by weight of chloroacetic acid, reacting for 4h at 60 ℃, cooling to room temperature after the reaction is finished, and neutralizing to neutrality by using 25% HCl solution to obtain carboxymethylated derivatives;
(2) and (3) dialysis: transferring the carboxymethylated derivatives into a dialysis bag, dialyzing with running water for 24h, dialyzing with distilled water for 48h, and concentrating the dialysate under reduced pressure;
(3) alcohol precipitation and drying: adding ethanol into the concentrated dialysate to make ethanol concentration reach above 80%, centrifuging, washing precipitate with methanol/acetone for 2 times, and vacuum freeze drying to obtain carboxymethylated Mori fructus polysaccharide.
The mulberry polysaccharide can be prepared according to the following steps:
(1) mulberry crude polysaccharide extraction
Firstly, extraction: taking a proper amount of mulberry, homogenizing, adding 3 times of water by weight, extracting for 1 time at the temperature of 80 ℃ for 1.5h each time, filtering, combining filtrates, and concentrating under reduced pressure to 2/7 of the original volume;
② decoloring: adding a proper amount of macroporous resin into the concentrated filtrate for adsorption decoloration, oscillating and decoloring at a constant temperature of 45 ℃ for 20min, filtering, repeatedly eluting the mulberry polysaccharide on the macroporous resin, merging the filtrates, and concentrating under reduced pressure;
removing protein: adding protease into the concentrated filtrate, performing enzymolysis at 40 ℃, adding 1 time of chloroform and 1/5 times of n-butanol, shaking for 20min, standing, removing impurity protein, repeating for 3 times until no precipitate is separated out, centrifuging, and collecting supernatant;
fourthly, alcohol precipitation: adding ethanol into the supernatant until the final concentration of ethanol is 60%, standing at 4 deg.C for 12 hr, centrifuging, re-dissolving the precipitate, precipitating with ethanol again, repeating for 2 times, and washing the precipitate with anhydrous ethanol and acetone for 1 time respectively;
fifth, dialysis: putting the washed precipitate into a pre-treated dialysis bag, and dialyzing; or filtering by membrane filtration, wherein the cut-off molecular weight of dialysis/membrane filtration is more than or equal to 1500, and drying to obtain crude mulberry polysaccharide;
(2) purification of crude mulberry polysaccharide
(ii) cellulose ion chromatography
Preparation of8g·L-1The crude polysaccharide solution of Mori fructus is transferred into DEAE-Cellulose52 column filled with Cellulose ion exchange resin, and eluted with distilled water at flow rate of 2 mL/min-1Collecting eluent, concentrating under reduced pressure, dialyzing, and freeze-drying under vacuum to obtain fractionated and purified mulberry polysaccharide;
② gel column chromatography
Dissolving the mulberry polysaccharide after DEAE-Cellulose52 fractional purification in ultrapure water, transferring the solution into gel column Sephadex G-50, eluting with distilled water at flow rate of 0.1 mL/min-1Collecting the eluent, concentrating under reduced pressure, and vacuum freeze-drying to obtain the mulberry polysaccharide.
Wherein the gel column can also be selected from Sephadex G-100, Sephadex G-150 or Sephadex G-200.
Example 3 preparation method of carboxymethylated Mulberry polysaccharide
(1) And (3) carboxymethyl derivatization reaction: taking 300 parts by weight of mulberry polysaccharide with the weight-average molecular weight of 20.93kDa, adding isopropanol with the volume being 12 times that of the mulberry polysaccharide, stirring the mixture for 60min at room temperature by using a magnetic stirrer, adding 30% NaOH solution with the volume being 30 times that of the mulberry polysaccharide, stirring the mixture for 3h at room temperature, slowly adding 3000 parts by weight of chloroacetic acid, reacting the mixture for 5h at 65 ℃, cooling the mixture to room temperature after the reaction is finished, and neutralizing the mixture to be neutral by using 30% HCl solution to obtain carboxymethylated derivatives;
(2) and (3) dialysis: transferring the carboxymethylated derivatives into a dialysis bag, dialyzing with running water for 36h, dialyzing with distilled water for 60h, and concentrating the dialysate under reduced pressure;
(3) alcohol precipitation and drying: adding ethanol into the concentrated dialysate to make ethanol concentration reach above 80%, centrifuging, washing precipitate with methanol/acetone for 3 times, and vacuum freeze drying to obtain carboxymethylated Mori fructus polysaccharide.
The mulberry polysaccharide can be prepared according to the following steps:
(1) mulberry polysaccharide extraction
Firstly, extraction: taking a proper amount of mulberry, homogenizing, adding 5 times of water by weight, extracting for 2 times at 90 ℃ for 2.5 hours each time, filtering, combining filtrates, and concentrating under reduced pressure to 2/9 of the original volume;
② decoloring: adding a proper amount of macroporous resin into the concentrated filtrate for adsorption decoloration, oscillating and decoloring at a constant temperature of 50 ℃ for 40min, filtering, repeatedly eluting the mulberry polysaccharide on the macroporous resin, merging the filtrates, and concentrating under reduced pressure;
removing protein: adding protease into the concentrated filtrate, performing enzymolysis at 70 ℃, adding 2 times of dichloromethane and 1/2 times of n-butanol, shaking for 120min, standing, removing impurity protein, repeating for 7 times until no precipitate is separated out, centrifuging, and taking supernatant;
fourthly, alcohol precipitation: adding ethanol into the supernatant until the final concentration of ethanol is 90%, standing at 4 deg.C for 36h, centrifuging, re-dissolving the precipitate, precipitating with ethanol again, repeating for 3 times, and washing with anhydrous ethanol and acetone for 3 times respectively;
fifth, dialysis: putting the washed precipitate into a pre-treated dialysis bag, and dialyzing; or filtering by membrane filtration, wherein the cut-off molecular weight of dialysis/membrane filtration is more than or equal to 1500, and drying to obtain crude mulberry polysaccharide;
(2) purification of crude mulberry polysaccharide
(ii) cellulose ion chromatography
Preparation of 12 g.L-1The crude polysaccharide solution of Mori fructus is transferred into DEAE-Cellulose32 loaded with Cellulose ion exchange resin column, and eluted with distilled water at flow rate of 3 mL/min-1Collecting eluent, concentrating under reduced pressure, dialyzing, and freeze-drying under vacuum to obtain fractionated and purified mulberry polysaccharide;
② gel column chromatography
Dissolving the crude polysaccharide of Mori fructus after DEAE-Cellulose32 fractional purification in ultrapure water, transferring the solution into gel column Sephadex G-200, eluting with distilled water at flow rate of 0.4 mL/min-1Collecting the eluent, concentrating under reduced pressure, and vacuum freeze-drying to obtain the mulberry polysaccharide.
Wherein the gel column can also be selected from Sephadex G-50, Sephadex G-100 or Sephadex G-150.
Example 4 preparation method of carboxymethylated Mulberry polysaccharide
(1) And (3) carboxymethyl derivatization reaction: taking 300 parts by weight of mulberry polysaccharide with the weight-average molecular weight of 20.93kDa, adding 10 times of isopropanol, stirring for 45min at room temperature by using a magnetic stirrer, adding 20 times of 25% NaOH solution, stirring for 2h at room temperature, slowly adding 2600 parts by weight of chloroacetic acid, reacting for 4h at 60 ℃, cooling to room temperature after the reaction is finished, and neutralizing to neutrality by using 25% HCl solution to obtain carboxymethylated derivatives;
(2) and (3) dialysis: transferring the carboxymethylated derivatives into a dialysis bag, dialyzing with running water for 24h, dialyzing with distilled water for 48h, and concentrating the dialysate under reduced pressure;
(3) alcohol precipitation and drying: adding ethanol into the concentrated dialysate to make ethanol concentration reach above 80%, centrifuging, washing precipitate with methanol/acetone for 2 times, and vacuum freeze drying to obtain carboxymethylated Mori fructus polysaccharide.
The mulberry polysaccharide can be extracted by adopting the prior art to obtain the mulberry crude polysaccharide, and then the mulberry crude polysaccharide is purified according to the following steps:
(ii) cellulose ion chromatography
Preparation of 10 g/L-1The crude polysaccharide solution of Mori fructus is transferred into DEAE-Cellulose52 filled with Cellulose ion exchange resin column, and eluted with distilled water at flow rate of 3 mL/min-1Collecting eluent, concentrating under reduced pressure, dialyzing, and freeze-drying under vacuum to obtain fractionated and purified mulberry polysaccharide;
② gel column chromatography
Dissolving the crude polysaccharide of Mori fructus after DEAE-Cellulose52 fractional purification in ultrapure water, transferring the solution into gel column Sephadex G-100, eluting with distilled water at flow rate of 0.25 mL/min-1Collecting the eluent, concentrating under reduced pressure, and vacuum freeze-drying to obtain the mulberry polysaccharide.
Wherein the gel column can also be selected from Sephadex G-50, Sephadex G-150 or Sephadex G-200.
Example 5 preparation method of carboxymethylated Mulberry polysaccharide
(1) And (3) carboxymethyl derivatization reaction: taking 300 parts by weight of mulberry polysaccharide with the weight-average molecular weight of 20.93kDa, adding 10 times of isopropanol, stirring for 60min at room temperature by using a magnetic stirrer, adding 25 times of 30% NaOH solution, stirring for 2h at room temperature, slowly adding 2200 parts by weight of chloroacetic acid, reacting for 4h at 55 ℃, cooling to room temperature after the reaction is finished, and neutralizing to neutrality by using 20% HCl solution to obtain carboxymethylated derivatives;
(2) and (3) dialysis: transferring the carboxymethylated derivatives into a dialysis bag, dialyzing with running water for 36h, dialyzing with distilled water for 48h, and concentrating the dialysate under reduced pressure;
(3) alcohol precipitation and drying: adding ethanol into the concentrated dialysate to make ethanol concentration reach above 80%, centrifuging, washing precipitate with methanol/acetone for 3 times, and vacuum freeze drying to obtain carboxymethylated Mori fructus polysaccharide.
The mulberry polysaccharide can be extracted according to the following steps:
firstly, extraction: taking a proper amount of mulberry, homogenizing, adding 4 times of water by weight, extracting for 2 times at the temperature of 85 ℃ for 2 hours each time, filtering, combining filtrates, and concentrating under reduced pressure to 16/63 of the original volume;
② decoloring: adding a proper amount of macroporous resin into the concentrated filtrate for adsorption decoloration, oscillating and decoloring at a constant temperature of 48 ℃ for 30min, filtering, repeatedly eluting the mulberry polysaccharide on the macroporous resin, merging the filtrates, and concentrating under reduced pressure;
removing protein: adding protease into the concentrated filtrate, performing enzymolysis at 55 deg.C, and adding 2 times volume of Sevag reagent (V)N-butanol∶VTrichloromethaneShaking at a ratio of 1: 4) for 70min, standing, removing impurity protein, repeating for 5 times until no precipitate is separated out, centrifuging, and collecting supernatant;
fourthly, alcohol precipitation: adding ethanol into the supernatant until the final concentration of ethanol is 75%, standing at 4 deg.C for 24 hr, centrifuging, re-dissolving the precipitate, precipitating with ethanol again, repeating for 2 times, and washing with anhydrous ethanol and acetone for 2 times respectively;
fifth, dialysis: putting the washed precipitate into a pre-treated dialysis bag, and dialyzing; or filtering by membrane filtration, wherein the cut-off molecular weight of dialysis/membrane filtration is more than or equal to 1500, and drying to obtain crude mulberry polysaccharide;
and purifying the extracted crude mulberry polysaccharide by adopting the prior art to further obtain the mulberry polysaccharide.
Example 6 application of carboxymethylated Mulberry polysaccharide
Carboxymethylated mulberry polysaccharide (prepared in example 1), dextrin, sucrose and sodium carboxymethylcellulose are weighed according to the mass ratio of 8: 0.4: 8: 0.3, uniformly mixed and prepared into granules. For preventing or treating acute alcoholic liver injury, the daily dose: 1-2 g.
Example 7 application of carboxymethylated Mulberry polysaccharide
Weighing carboxymethylated mulberry polysaccharide (prepared in example 2), starch, sucrose and sodium carboxymethylcellulose according to the mass ratio of 8: 0.4: 0.2: 0.3, uniformly mixing, and preparing into tablets of 0.3-0.5 g/tablet. The medicine is used for preventing or treating the subalcoholic liver injury, and the daily dose is as follows: 3-5 pieces.
Example 8 application of carboxymethylated Mulberry polysaccharide
Carboxymethylated mulberry polysaccharide (prepared in example 4), dextrin, sucrose and sodium carboxymethylcellulose are weighed according to the mass ratio of 8: 0.4: 6: 0.3, uniformly mixed and prepared into granules. For preventing or treating drug-induced liver injury, the daily dose: 1-2 g.
Example 9 application of carboxymethylated Mulberry polysaccharide
Weighing carboxymethylated mulberry polysaccharide (prepared in example 3), starch, sucrose and sodium carboxymethylcellulose according to the mass ratio of 8: 0.4: 0.1: 0.3, uniformly mixing, and preparing into tablets of 0.3-0.5 g/tablet. The medicine is used for relieving alcoholism and protecting liver, and the daily dose is as follows: 3-5 pieces.
Claims (8)
1. Carboxymethylated mulberry polysaccharide is characterized in that: the carboxymethylation mulberry polysaccharide is obtained by carboxymethylation modification of mulberry polysaccharide with the weight-average molecular weight of 20.93 kDa; the weight average molecular weight of the carboxymethylated mulberry polysaccharide is 26.18kDa, and the substitution degree is 0.6-0.8.
2. The preparation method of carboxymethylated mulberry polysaccharide of claim 1, comprising the steps of:
(1) and (3) carboxymethyl derivatization reaction: adding 7-12 times of isopropanol into the purified mulberry polysaccharide, uniformly stirring, adding 10-30 times of NaOH solution, stirring, slowly adding chloroacetic acid 7-10 times of the mulberry polysaccharide, reacting for 3-5h, cooling to room temperature after the reaction is finished, and neutralizing the solution to be neutral to obtain carboxymethylated derivatives;
(2) and (3) dialysis: transferring the carboxymethylated derivative into a dialysis bag, dialyzing with running water, dialyzing with distilled water, and concentrating the dialysate;
(3) alcohol precipitation and drying: adding ethanol into the concentrated dialysate, precipitating with ethanol, centrifuging, collecting precipitate, washing with methanol or acetone, and vacuum freeze drying to obtain carboxymethylated Mori fructus polysaccharide.
3. The preparation method of carboxymethylated mulberry polysaccharide according to claim 2, comprising the steps of:
(1) and (3) carboxymethyl derivatization reaction: adding 7-12 times volume of isopropanol into 300 parts by weight of purified mulberry polysaccharide, stirring for 30-60min at room temperature by using a magnetic stirrer, adding 10-30 times volume of 20-30% NaOH solution, stirring for 1-3h at room temperature, slowly adding 2100-3000 parts by weight of chloroacetic acid, reacting for 3-5h at 55-65 ℃, cooling to room temperature after the reaction is finished, and neutralizing to neutrality by using 20-30% HCl solution to obtain carboxymethylated derivatives;
(2) and (3) dialysis: transferring the carboxymethylated derivatives into a dialysis bag, dialyzing with running water for 12-36h, dialyzing with distilled water for 36-60h, and concentrating the dialysate under reduced pressure;
(3) alcohol precipitation and drying: adding ethanol into the concentrated dialysate to make ethanol concentration reach above 80%, centrifuging, washing precipitate with methanol or acetone for 1-3 times, and vacuum freeze drying to obtain carboxymethylated Mori fructus polysaccharide.
4. The preparation method of carboxymethylated mulberry polysaccharide according to claim 2 or 3, wherein the mulberry polysaccharide is extracted by the following method:
firstly, extraction: taking a proper amount of mulberry, homogenizing, adding 3-5 times of water by weight, extracting for 1-2 times at the temperature of 80-90 ℃ for 1.5-2.5h each time, filtering, combining filtrates, and concentrating under reduced pressure to 2/7-2/9 of the original volume;
② decoloring: adding a proper amount of macroporous resin into the concentrated filtrate for adsorption decoloration, oscillating and decoloring at constant temperature of 45-50 ℃ for 20-40min, filtering, repeatedly eluting the mulberry polysaccharide on the macroporous resin, merging the filtrate, and concentrating under reduced pressure;
removing protein: adding protease into the concentrated filtrate, performing enzymolysis at 40-70 deg.C, adding 1-2 times of chloroform or dichloromethane, simultaneously adding 1/2 times of n-butanol, shaking for 20-120min, standing, removing impurity protein, repeating for 3-7 times until no precipitate is separated out, centrifuging, and collecting supernatant;
fourthly, alcohol precipitation: adding ethanol into the supernatant until the final concentration of ethanol is 60% -90%, standing at 4 deg.C for 12-36h, centrifuging, re-dissolving the precipitate, precipitating with ethanol again, repeating for 2-3 times, and washing the precipitate with anhydrous ethanol and acetone for 1-3 times respectively;
fifth, dialysis: putting the washed precipitate into a pre-treated dialysis bag, and dialyzing; or filtering by membrane filtration, wherein the cut-off molecular weight of dialysis/membrane filtration is not less than 1500, and drying to obtain crude mulberry polysaccharide.
5. The preparation method of carboxymethylated mulberry polysaccharide according to claim 4, wherein the purification of crude mulberry polysaccharide comprises the following steps:
(ii) cellulose ion chromatography
Preparation of 8-12 g.L-1The crude polysaccharide solution of Mori fructus is transferred into cellulose ion exchange resin column, and eluted with distilled water at flow rate of 2-3 mL/min-1Collecting eluent, concentrating under reduced pressure, dialyzing, and freeze-drying under vacuum to obtain fractionated and purified mulberry polysaccharide;
② gel column chromatography
Dissolving the purified mulberry polysaccharide in ultrapure water, transferring the solution into gel column, eluting with distilled water at flow rate of 0.1-0.4 mL/min-1Collecting the eluent, concentrating under reduced pressure, and vacuum freeze-drying to obtain the mulberry polysaccharide.
6. The preparation method of carboxymethylated mulberry polysaccharide according to claim 5, wherein the Cellulose ion exchange resin column packing is DEAE-Cellulose52 or DEAE-Cellulose 32.
7. The preparation method of carboxymethylated mulberry polysaccharide according to claim 5, wherein the gel column packing is SephadexG-50, SephadexG-100, SephadexG-150 or SephadexG-200.
8. The use of the carboxymethylated mulberry polysaccharide of claim 1 in the preparation of a liver-protecting medicament or health product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810668387.6A CN108727511B (en) | 2018-06-26 | 2018-06-26 | Carboxymethylated mulberry polysaccharide and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810668387.6A CN108727511B (en) | 2018-06-26 | 2018-06-26 | Carboxymethylated mulberry polysaccharide and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108727511A CN108727511A (en) | 2018-11-02 |
| CN108727511B true CN108727511B (en) | 2021-04-02 |
Family
ID=63930763
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810668387.6A Expired - Fee Related CN108727511B (en) | 2018-06-26 | 2018-06-26 | Carboxymethylated mulberry polysaccharide and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108727511B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113397206B (en) * | 2021-07-09 | 2023-03-28 | 广西中烟工业有限责任公司 | Preparation method of carboxymethylated momordica grosvenori polysaccharide and application of carboxymethylated momordica grosvenori polysaccharide in tobacco humectant |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
2018
- 2018-06-26 CN CN201810668387.6A patent/CN108727511B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (1)
| Title |
|---|
| 桑葚多糖的分离纯化及组成分析;田仁君;《华西药学杂志》;20140815;第29卷(第4期);第401-404页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108727511A (en) | 2018-11-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Nie et al. | Bioactive polysaccharides from Cordyceps sinensis: Isolation, structure features and bioactivities | |
| Niu et al. | In Vitro Antioxidant activities and anti-diabetic effect of a polysaccharide from Schisandra sphenanthera in rats with type 2 diabetes | |
| He et al. | Research progress on extraction, purification, structure and biological activity of Dendrobium officinale polysaccharides | |
| CN104710538B (en) | A kind of sanchi flower arabogalactan and its production and use | |
| CN108047343B (en) | Preparation method and application of fritillaria pallidiflora total polysaccharide | |
| CN108727509B (en) | Moso bamboo shoot shell arabinogalactan and preparation and application thereof | |
| CN114874344B (en) | Preparation method and application of alkali-extracted polysaccharides of highland barley tender leaves | |
| Yang et al. | Antioxidant activities of sulfated Codonopsis polysaccharides in acute oxidative stress | |
| CN108727511B (en) | Carboxymethylated mulberry polysaccharide and preparation method and application thereof | |
| Zhang et al. | Preparation and structural characterization of acid-extracted polysaccharide from Grifola frondosa and antitumor activity on S180 tumor-bearing mice | |
| CN102408494A (en) | Grifola frondosa polysaccharide ZZK component and preparation method thereof | |
| JP7285596B2 (en) | Kudzu root polysaccharide and its production method and use | |
| CN104107187A (en) | Application of apocynum venetum acidic polysaccharide to medicines for treating diabetes | |
| CN102988457A (en) | Total flavone extract of lonicera macranthoides leaves, and preparation method and application thereof | |
| CN112898446A (en) | Kudzu root polysaccharide extract and extraction method and application thereof | |
| CN109232756B (en) | Suaeda salsa polysaccharide extract and preparation method and application thereof | |
| CN116375902A (en) | Grape residue polysaccharide for improving immune function and preparation method and application thereof | |
| CN112807322A (en) | Application of oligomannuronate in preparing medicine and functional food for delaying skin senility and resisting immune inflammation | |
| CN105753920B (en) | A kind of method that alpha-glucosidase restrainer is extracted from litchi pulp | |
| CN117500842A (en) | Cs-4 fermentation mycelium heteropolysaccharide and preparation method and application thereof | |
| CN101643515A (en) | Method for separating and purifying lentinan for injection | |
| CN113694104A (en) | Traditional Chinese medicine composition with protection effect on chemical liver injury and liver regeneration promotion function, preparation method and application thereof | |
| CN113637091A (en) | Extraction method of high-purity low-molecular-weight astragalus polysaccharide, product and application thereof | |
| CN108948223B (en) | Myrtle polysaccharide P1, its separation method and application in preparing hypolipidemic medicine | |
| CN103550243B (en) | The application of Rhizoma Atractylodis polysaccharide in the medicine improving kuffer cellular immune function or health product |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20210402 Termination date: 20210626 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |