CN110256596B - Acidic notoginseng polysaccharide and extraction and purification method thereof - Google Patents
Acidic notoginseng polysaccharide and extraction and purification method thereof Download PDFInfo
- Publication number
- CN110256596B CN110256596B CN201910584252.6A CN201910584252A CN110256596B CN 110256596 B CN110256596 B CN 110256596B CN 201910584252 A CN201910584252 A CN 201910584252A CN 110256596 B CN110256596 B CN 110256596B
- Authority
- CN
- China
- Prior art keywords
- polysaccharide
- notoginseng
- acidic
- volume
- collecting
- 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.)
- Active
Links
Images
Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/30—Drugs for disorders of the nervous system for treating abuse or dependence
- A61P25/32—Alcohol-abuse
-
- 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
-
- 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
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Addiction (AREA)
- Pharmacology & Pharmacy (AREA)
- General Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Polymers & Plastics (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Materials Engineering (AREA)
- Sustainable Development (AREA)
- Gastroenterology & Hepatology (AREA)
- Psychiatry (AREA)
- Biomedical Technology (AREA)
- Neurology (AREA)
- Neurosurgery (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses an acidic notoginseng polysaccharide and a preparation method thereof, wherein the molecular weight of the acidic notoginseng polysaccharide is 2500-3000kDa, the monosaccharide composition and the molar ratio are Rha: Ara: Man: Glc: Gal: GalA =0.038:0.045:0.002:0.016:0.031:0.868, GalA is galacturonic acid, and the glycosidic bond type and the molar ratio thereof are as follows: ara-1 → Rha-1 → 5-Ara-1 → 4-GalA-1 → 3,6-Gal-1 → 0.06:0.04:0.05:0.80: 0.05; the acidic panax notoginseng polysaccharide is obtained by re-extracting the medicine residues after the panax notoginseng saponins are extracted, the medicinal value of panax notoginseng is fully utilized, the panax notoginseng is greatly utilized, the secondary development of panax notoginseng resources is significant, and the acidic panax notoginseng polysaccharide extracted by the invention has a significant effect on protecting alcoholic liver injury.
Description
Technical Field
The invention belongs to the field of medicines, and relates to acidic panax notoginseng polysaccharide and an extraction and purification method thereof.
Background
Polysaccharides are widely found in animals, plants and microorganisms. As one of the components of living materials, polysaccharides are widely involved in the regulation of various vital phenomena and physiological processes of cells. The physiological activities of the composition comprise antioxidation, anti-tumor, blood fat reduction, antibiosis and the like. The biological activity of the polysaccharide is closely related to the structural composition and spatial conformation of the polysaccharide. The research on the structural composition of the polysaccharide has important significance for the development and the utilization of the polysaccharide.
Notoginseng is a perennial herb of Panax of Araliaceae, is a precious medicinal material in China, and mainly contains Panax notoginsenosides, dencichine and the like as effective components. The Panax notoginsenosides are effective components for preventing and treating cardiovascular and cerebrovascular diseases. The total saponins of Panax notoginseng are obtained by extracting with 75% ethanol and separating as main raw material of Xueshuantong series products. The residue after extraction contains polysaccharides, which account for about 3-5% of the residue.
The annual planting area of the panax notoginseng is about 10 ten thousand mu, and the yield is 9000 tons. The amount of panax notoginseng used for extracting panax notoginseng saponins is about 1000 tons per year. The notoginseng residue is discarded after extraction, wherein about 30-50 ten thousand tons of notoginseng polysaccharide can not be effectively utilized. Therefore, the panax notoginseng polysaccharide extracted from the panax notoginseng residues and the structure identification have significant meaning for the secondary development of panax notoginseng resources.
The patent with the application number of 201710459344.2 discloses an extraction and purification method of neutral notoginseng polysaccharide and research and application of pharmacological activity for promoting cell proliferation. The patent prepares the pseudo-ginseng crude polysaccharide by a water extraction and alcohol precipitation method, and obtains the neutral pseudo-ginseng polysaccharide after purifying by adopting a DEAE anion exchange column. The polysaccharide is found to promote the proliferation of periodontal ligament stem cells, mouse osteoblasts and human skin epidermal cells. However, the acidic polysaccharide is not obtained, nor is its structural composition further purified and characterized.
The patent with the application number of 201611189464.7 discloses a preparation method and application of a small molecular pseudo-ginseng polysaccharide extract. The patent adopts trifluoroacetic acid or lysozyme for hydrolysis to obtain micromolecule polysaccharide crude liquid, and the micromolecule pseudo-ginseng polysaccharide extract is prepared by anion and cation exchange column chromatography, activated carbon decoloration and freeze drying. However, the patent does not further isolate and purify the polysaccharide extract nor identify the polysaccharide components and structural composition.
The patent with the application number of 201210481377.4 discloses a panax notoginseng polysaccharide extract, a preparation method, a preparation and an application thereof. The panax notoginseng polysaccharide extract is obtained by extracting, concentrating, refining, drying, crushing and inspecting raw materials from waste residues after extracting panax notoginseng saponins, and the polysaccharide extract is not further separated and purified and the structure of the polysaccharide extract is not identified.
Disclosure of Invention
The invention provides an acidic notoginseng polysaccharide, which is derived from notoginseng dregs after panax notoginseng saponins are extracted, has the molecular weight of 2500-3000kDa and contains carboxyl groups in the structure, so the notoginseng polysaccharide is the acidic notoginseng polysaccharide, the monosaccharide composition and the molar ratio of Rha to Ara to Man to Glc to Gal to GalA =0.038:0.045 to 0.002:0.016:0.031:0.868, and the GalA structure is galacturonic acid.
The acidic notoginseng polysaccharide is characterized in that the types of glycosidic bonds and the molar ratio thereof are analyzed through methylation: ara-1 → Rha-1 → 5-Ara-1 → 4-GalA-1 → 3,6-Gal-1 → =0.06:0.04:0.05:0.80:0.05, wherein → 4-GalA-1 → is a main structure in the acidic panax notoginseng polysaccharide, the main chain is GalA, Glc and Gal, the branched chain is Ara and Rha, and the structural connection is mainly → 4-GalA-1 →.
The invention also provides an extraction and purification method of the acidic panax notoginseng polysaccharide, which comprises the steps of extracting panax notoginseng crude polysaccharide from panax notoginseng dregs after extracting panax notoginseng saponins by a water extraction and alcohol precipitation method, removing pigments by an activated carbon method, removing proteins by a sevag method, purifying by anion exchange cellulose column chromatography, performing gradient elution by NaCl solutions with different concentrations, and further purifying by selecting a NaCl solution eluent with the highest concentration of the acidic panax notoginseng polysaccharide to obtain the acidic panax notoginseng polysaccharide.
Further purifying by using a G50 gel preparation system, collecting 30-36 tubes of samples in the first half part of a polysaccharide gel column separation spectrogram symmetric peak detected by the gel preparation system, dialyzing, performing rotary evaporation concentration at 45-50 ℃, and performing vacuum freeze drying to obtain a light yellow powdery solid, namely the acidic pseudo-ginseng polysaccharide.
The concentration of the NaCl solution during gradient elution is 0.2mol/L, 0.5mol/L, 1mol/L and 2mol/L in sequence, and the NaCl solution eluent with the highest concentration of the acidic notoginseng polysaccharide is the NaCl solution eluent with the concentration of 0.5 mol/L.
The invention has the beneficial effects that:
the invention obtains the notoginseng polysaccharide by re-extracting the dregs after extracting the panax notoginseng saponins, fully utilizes the medicinal value of the notoginseng, leads the notoginseng to be utilized more greatly and has significant meaning for the secondary development of the notoginseng resource. The acidic notoginseng polysaccharide extracted by the invention has obvious effect on protecting alcoholic liver injury.
Drawings
FIG. 1 the polysaccharide gel column separation spectrum of example 1;
FIG. 2 purity and relative molecular mass spectra of the polysaccharide of example 1;
FIG. 3 GC-MAS chromatogram of example 1;
fig. 4 pathological section (x 400) analysis chart ((a) blank control group, (b) model control group, (c) drug (positive) group, (d) notoginseng polysaccharide low dose group, (e) notoginseng polysaccharide medium dose group (f) notoginseng polysaccharide high dose group).
Detailed Description
The essential features of the invention are further elucidated below by means of examples and figures, without the scope of protection of the invention being limited in any way to the examples.
Example 1
Extracting and purifying panax notoginseng acidic polysaccharide:
(1) drying Notoginseng radix residue after extracting Notoginseng radix total saponin at 50 deg.C, pulverizing, sieving with 80 mesh sieve, adding 20 times volume of distilled water, extracting at 70 deg.C for 4 hr, filtering, collecting residue, adding 15 times volume of distilled water, repeatedly extracting for 4 hr, filtering, and mixing the two extractive solutions; deproteinizing by referring to a sevag method (sevag liquid is chloroform: n-butanol (V: V) =5: 1), namely adding sevag liquid with 0.3 times volume of an extracting solution, shaking vigorously for 15min, standing for layering, separating out a protein layer, repeating for four times until the protein is completely removed, placing the extracting solution at 50 ℃, performing rotary evaporation and concentration to 1/6 of the original volume, adding 3 times volume of absolute ethyl alcohol, precipitating, removing ethyl alcohol in a water bath at 55 ℃, re-dissolving, adding active carbon for decoloring (0.06 g/mL) (the re-dissolving volume and the volume of medicine residue are 1:1), performing water bath at 40 ℃ for 3h, centrifuging at 4000rpm for 10min, taking a supernatant, centrifuging again at 12000rpm for 15min, dialyzing at 3500Da for one night, and freeze-drying to obtain pseudo-ginseng crude polysaccharide NGP;
(2) weighing 300mg of the crude notoginseng polysaccharide prepared in the step (1) and dissolving the crude notoginseng polysaccharide in 20mL of deionized water, performing column chromatography through an anion exchange cellulose column DEAE-52(4cm multiplied by 60cm), eluting with 1 column volume of distilled water, then performing gradient elution by sequentially using NaCl solutions with the column volumes of 0.2mol/L, 0.5mol/L, 1.0mol/L and 2.0mol/L, wherein the flow rate is 24mL/h, collecting the eluted components in 1 test tube per 8mL, performing tracking detection by using a phenol-sulfuric acid method, collecting the NaCl solution with the highest acidic notoginseng polysaccharide concentration of 0.5mol/L, collecting the components, performing freeze drying to obtain freeze-dried powder, and naming the powder as NGP0.5M;
(3) weighing NGP0.5M mg obtained in the step (2), dissolving in 3mL of distilled water, centrifuging for 10min by using a high-speed centrifuge (the rotation speed of the centrifuge is 12000 rpm), collecting supernatant for subsequent sample loading, purifying by using a G-50 polysaccharide gel purification system, detecting and collecting by using a phenol-sulfuric acid method, collecting symmetrical peaks, collecting a gel column separation spectrogram as shown in figure 1, collecting only the left half peak part, namely 30-36 tubes, in the symmetrical peaks because the right half peak shows different peak overlaps and possibly contains other polysaccharides, rotating at 45 ℃, evaporating and concentrating, dialyzing by using a 3500Da dialysis bag, and freeze-drying to obtain the acidic notoginseng polysaccharide separated and purified by using the gel column, wherein the name of the acidic notoginseng polysaccharide is NGP0.5M-P.
Structural analysis of acidic notoginseng polysaccharide NGP0.5M-P:
(1) determination of molecular weight and purity:
detecting by using high performance liquid chromatograph (WATERS-515) combined with differential detector (WATERS-2414), using dextran (Mw: 5200, 11600, 23800, 48600, 148000, 273000, 410000, 668000) with different relative molecular masses as standard, and using Waters ultra-hydrogel250, 1000 and 2000 gel columns (7.8 × 300mm) in series, with sample concentration of 2.0mg/mL, sample amount of 20 μ L, and mobile phase of 20mMCH3COONH4Drawing a standard curve at the flow rate of 0.5 mL/min; NGP0.5M-P was then formulated at a concentration of 2mg/mL and tested according to the parameters described above. The detection result is shown in figure 2, NGP0.5M-P presents a uniform and symmetrical peak shape, the purity is determined to be 97.289% according to integration, and the three times of repetition are carried out, so that the molecular weights of the polysaccharides are 2617kDa, 2724kDa and 2686kDa respectively.
(2) Monosaccharide composition and molar ratio:
preparation of acetylated derivatives: accurately weighing 2mgNGP0.5M-P, adding 1mL of 2mol/L trifluoroacetic acid, charging nitrogen for protection, sealing the tube, hydrolyzing at 120 ℃ for 90min, evaporating to dryness by a rotary evaporator, adding 2mL of methanol, evaporating to dryness, repeating for 2 times to obtain NGP0.5M-P1 after acid hydrolysis; adding 10mg of hydroxylamine hydrochloride into NGP0.5M-P1 subjected to acid hydrolysis, dissolving the hydroxylamine hydrochloride by using 1.0mL of pyridine, sealing the tube, reacting for 90min at 90 ℃, cooling to room temperature, adding 0.5mL of anhydrous acetic anhydride, sealing the tube, and continuing to react for 60min at 100 ℃ for acetylation; after the reaction is finished, performing vacuum pumping, adding 3mL of methylbenzene, performing vacuum concentration and evaporation, repeating for 5 times to remove redundant acetic anhydride, fully dissolving the derivatized product by using chloroform, then transferring to a separating funnel, adding a small amount of deionized water for extraction, removing an aqueous solution, repeating for 4 times, collecting a chloroform layer, drying by using a proper amount of anhydrous sodium sulfate, and fixing the volume to 5mL for analysis; analyzing the components by gas chromatography-mass spectrometer, comparing with monosaccharide standard, and showing monosaccharide standard spectrogram and spectrogram NGP0.5M-P1 in figure 3; by comparison, the acetylated monosaccharide composition is: ara: Xyl: Man: Gal =0.123:0.338:0.258:0.056:0.225, NGP0.5M-P is an acidic polysaccharide, so the structure may contain an uronic acid structure, then the acidic notoginseng polysaccharide is reduced by adopting a carbodiimide method, the reduced NGP0.5M-P monosaccharide composition is analyzed by a gas chromatography-mass spectrometer, and the comparison calculation of the monosaccharide composition with the unreduced monosaccharide composition shows that the monosaccharide composition of NGP0.5M-P1 is the monosaccharide composition with the molar ratio of Rha: Ara: Man: Glc: Gal: GalA =0.038:0.045:0.002:0.016:0.031:0.868, which is the actual composition.
Placing an acidic panax notoginseng polysaccharide sample reduced by a carbodiimide method into a reaction flask, adding DMSO, quickly adding NaOH powder, sealing, dissolving under the action of ultrasound, adding methyl iodide for reaction, finally adding water into the mixture to terminate methylation reaction, taking methylated polysaccharide, hydrolyzing, acetylating, measuring an acetylated product sample by using a gas chromatography-mass spectrometer (GC-MS), wherein the methylation result is shown in the following table 1, and according to the result, NGP0.5M-P1 mainly comprises the following five glycosidic bond connections, namely Ara-1 →, Rha-1 →, → 5-Ara-1 →, → 4-GalA-1 →, and → 3,6-Gal-1 →, wherein the molar ratio of → 4-GalA-1 → 0.80, and the acetylated product can be inferred to be a main linked structure.
TABLE 1
(3) Partial acid hydrolysis
Accurately weighing 200mg of NGP0.5M-P sample, adding 0.1mg/mL of trifluoroacetic acid, hydrolyzing the sample at 80 ℃ for 5h, transferring the hydrolyzed sample into a 3500Da dialysis bag, dialyzing the dialyzed sample with deionized water for 24h, respectively collecting an inner phase sample and an outer phase sample, and performing rotary evaporation and concentration, wherein the samples are respectively named as 0.1N and 0.1W;
accurately weighing 200mg of NGP0.5M-P sample, adding 0.5mg/mL of trifluoroacetic acid, hydrolyzing the sample at 80 ℃ for 5h, transferring the hydrolyzed sample into a 3500Da dialysis bag, dialyzing the dialyzed sample for 24h against deionized water, respectively collecting an inner phase sample and an outer phase sample, and carrying out rotary evaporation concentration, wherein the samples are respectively named as 0.5N and 0.5W;
the four samples were analyzed by gas chromatography-mass spectrometer according to the method for measuring monosaccharide composition, and the results are shown in table 2 below.
TABLE 2
The result shows that NGP0.5M-P is hydrolyzed by trifluoroacetic acid of 0.1mg/mL, and the proportion of Rha and Ara is increased by comparing monosaccharide compositions of the inner and outer components of the dialysis bag; meanwhile, the proportion of Glc and Gal is reduced, and the samples hydrolyzed by 0.5mg/mL trifluoroacetic acid show the same trend, so that the main chains of the polysaccharide are Glc and Gal, and the branched chains are Ara and Rha.
The acidic notoginseng polysaccharide main chain is GalA, Glc and Gal, the branched chain is Ara and Rha, and the structural connection is mainly → 4-GalA-1 → according to the partial acid hydrolysis combined with GC-MS analysis.
(4) Study of pharmacological experiments
1. Laboratory animal
Kunming mice (SPF grade), male, 18-22g, purchased from Schlekschada laboratory animals Co., Ltd, Hunan, license number SCXK2016-002, were kept in a dry, ventilated and quiet environment with free access to water and ordinary feed for 1 week.
2. Drugs and agents
Acidic notoginseng polysaccharide (prepared in example 1); bifendate dripping pill (Xinchang pharmaceutical factory, Zhejiang pharmaceutical Co., Ltd.); diluting 56-degree white spirit with pure water to twice volume for later use by Beijing Hongxing Erguotou liquor industry Limited; the kit comprises a glutamic-pyruvic transaminase (ALT) determination kit, an glutamic-oxalacetic transaminase (AST) determination kit, a Triglyceride (TG) determination kit, a total protein quantification (BCA) determination kit, a Malondialdehyde (MDA) determination kit, a superoxide dismutase (SOD) determination kit, a Glutathione (GSH) determination kit, a glutathione peroxidase (GSH-Px) determination kit and an Alcohol Dehydrogenase (ADH) determination kit, which are purchased from Nanjing to build a bioengineering institute; normal saline, absolute ethyl alcohol, hematoxylin-eosin dye solution, sodium citrate and xylene, all of which are analytically pure.
3. Laboratory apparatus
An electronic balance AX124ZH, a DW-86L386 vertical ultra-low temperature storage box, a BMG LABTECH multifunctional microplate reader, a UV-2600 ultraviolet visible spectrophotometer, a microtome, a micro-pipette, a glass homogenizer and an ELISA plate.
4. Test drug and treatment method
Dissolving the notoginseng polysaccharide obtained in the example 1 with purified water to prepare three solutions with different concentrations of 10mg/mL, 15mg/mL and 20mg/mL, wherein the positive medicament is bifendate, the administration mode of the stomach irrigation is that the volume of the medicament is 1mL/10g, and the specific grouping is as follows:
blank control group (10 mice): normal mice were fed normally with distilled water by gavage every day;
model control group (10 mice): after 0.025mL/10g of diluted white spirit solution is adaptively infused into the stomach for 2 days, the volume of the diluted white spirit solution is changed into 0.05mL/10g of the diluted white spirit solution for 3 days, the volume of the diluted white spirit solution is changed into 0.08mL/10g of the diluted white spirit solution for 5 days, the volume of the diluted white spirit solution is changed into 0.10mL/10g of the diluted white spirit solution for 1 week, finally the volume of the diluted white spirit solution is increased to 0.12mL/10g of the diluted white spirit solution, the diluted white spirit solution is infused into the stomach for 29 days, and the diluted white spirit solution is infused into the stomach by 0.14mL/10g of the diluted white spirit solution in a shocking manner on 30 days; performing intragastric administration with distilled water before treating with Chinese liquor solution for 30min every day, and feeding normally;
drug (yang) group (10 mice): after 0.025mL/10g of diluted white spirit solution is adaptively infused into the stomach for 2 days, the volume of the diluted white spirit solution is changed into 0.05mL/10g of the diluted white spirit solution for 3 days, the volume of the diluted white spirit solution is changed into 0.08mL/10g of the diluted white spirit solution for 5 days, the volume of the diluted white spirit solution is changed into 0.10mL/10g of the diluted white spirit solution for 1 week, finally the volume of the diluted white spirit solution is increased to 0.12mL/10g of the diluted white spirit solution, the diluted white spirit solution is infused into the stomach for 29 days, and the diluted white spirit solution is infused into the stomach by 0.14mL/10g of the diluted white spirit solution in a shocking manner on 30 days; performing intragastric administration (150 mg/kg, each time every day) with bifendate before treating with Chinese liquor solution every day for 30min, and feeding normally;
pseudo-ginseng polysaccharide low group (10 mice): after 0.025mL/10g of diluted white spirit solution is adaptively infused into the stomach for 2 days, the volume of the diluted white spirit solution is changed into 0.05mL/10g of the diluted white spirit solution for 3 days, the volume of the diluted white spirit solution is changed into 0.08mL/10g of the diluted white spirit solution for 5 days, the volume of the diluted white spirit solution is changed into 0.10mL/10g of the diluted white spirit solution for 1 week, finally the volume of the diluted white spirit solution is increased to 0.12mL/10g of the diluted white spirit solution, the diluted white spirit solution is infused into the stomach for 29 days, and the diluted white spirit solution is infused into the stomach by 0.14mL/10g of the diluted white spirit solution in a shocking manner on 30 days; performing intragastric administration (100 mg/kg, every day) with Notoginseng radix polysaccharide before treating with Chinese liquor solution for 30min every day, and feeding normally;
pseudo-ginseng polysaccharide middle group (10 mice): after 0.025mL/10g of diluted white spirit solution is adaptively infused into the stomach for 2 days, the volume of the diluted white spirit solution is changed into 0.05mL/10g of the diluted white spirit solution for 3 days, the volume of the diluted white spirit solution is changed into 0.08mL/10g of the diluted white spirit solution for 5 days, the volume of the diluted white spirit solution is changed into 0.10mL/10g of the diluted white spirit solution for 1 week, finally the volume of the diluted white spirit solution is increased to 0.12mL/10g of the diluted white spirit solution, the diluted white spirit solution is infused into the stomach for 29 days, and the diluted white spirit solution is infused into the stomach by 0.14mL/10g of the diluted white spirit solution in a shocking manner on 30 days; performing intragastric administration (150 mg/kg, each time every day) with Notoginseng radix polysaccharide before treating with Chinese liquor solution every day for 30min, and feeding normally;
pseudo-ginseng polysaccharide middle group (10 mice): after 0.025mL/10g of diluted white spirit solution is adaptively infused into the stomach for 2 days, the volume of the diluted white spirit solution is changed into 0.05mL/10g of the diluted white spirit solution for 3 days, the volume of the diluted white spirit solution is changed into 0.08mL/10g of the diluted white spirit solution for 5 days, the volume of the diluted white spirit solution is changed into 0.10mL/10g of the diluted white spirit solution for 1 week, finally the volume of the diluted white spirit solution is increased to 0.12mL/10g of the diluted white spirit solution, the diluted white spirit solution is infused into the stomach for 29 days, and the diluted white spirit solution is infused into the stomach by 0.14mL/10g of the diluted white spirit solution in a shocking manner on 30 days; performing intragastric administration (200 mg/kg, every day) with Notoginseng radix polysaccharide before treating with Chinese liquor solution for 30min every day, and feeding normally; the experiment lasted for 30 days.
5. Experimental animal sample Collection
(1) Serum preparation: placing eyeball blood in a clean sterilized centrifugal tube, adding anticoagulant (sodium citrate) 0.09mol/L (volume ratio anticoagulant: blood =1: 9), standing for 2h, centrifuging at 3500r/min for 10min, and collecting supernatant as serum.
(2) Preparation of liver homogenate: after the mouse is dissected, the liver tissue is quickly taken out, the liver homogenate is collected, the weight is accurately weighed, 9 times of volume of physiological saline is added according to the proportion that the weight (g) is equal to the volume (mL) =1:9, the homogenate is carried out under the ice-water bath condition, the centrifugation is carried out for 10min at 2500r/min under the condition of 4 ℃, the supernatant is taken to be 10 percent tissue homogenate, and the liver homogenate is collected for testing liver indexes.
(3) Preparation of liver histopathological section
Dissecting a mouse, quickly taking out liver tissue, washing and wiping the liver tissue in ice-cold normal saline, directly fixing the liver tissue in 10% formaldehyde fixing solution for 24 hours, taking out the liver tissue from 10% formaldehyde, putting the liver tissue in a large beaker, washing for 24 hours, washing the fixing solution, dehydrating the liver tissue by using 50%, 70%, 80%, 90%, 95% and 100% gradient (from low-concentration alcohol to high-concentration alcohol) alcohol, putting a mixed solution of xylene and alcohol, namely 1:1 (V: V) for 15 minutes, putting the dehydrated tissue block in pure xylene for transparence, replacing the alcohol in the tissue block by the xylene, putting the transparent tissue in a paraffin-dissolved dipping cup for wax penetration twice, embedding the tissue block for 30 minutes each time at the temperature of 65-70 ℃, completely dipping the paraffin into the tissue block, and putting the material on an embedding machine, the section of the wax block is trimmed to be square, the wax block is cut into thin wax sheets with the diameter of 6 mu m on a slicer, then the slices are placed on the water surface at the temperature of 40 ℃ to be fully flattened, the thin wax sheets are laid on a glass slide for dyeing, the cell nucleus is dyed by hematoxylin according to the operation of the instruction of a kit, the cytoplasm is dyed by eosin, the cells are sealed after natural air drying, the glass slide is placed under an optical microscope to observe the pathological morphological change of the liver tissue section, and the pathological change of the liver is observed under a light microscope.
And (4) analyzing results:
1. influence of notoginseng polysaccharide on ALT, AST and TG levels of mice with chronic alcoholic liver injury:
ALT and AST are indexes for measuring hepatocyte damage, when hepatocyte degeneration, increased cell membrane permeability and hepatocyte necrosis occur, ALT is mainly escaped from cells, AST will obviously rise, and the damage condition of hepatocytes can be reflected. The results in table 3 show that ALT and AST in serum and liver tissues of the mice in the model control group are increased compared with those in the blank control group, wherein AST in liver tissues is obviously increased (P < 0.05), which indicates that the liver of the mice is damaged by long-term wine feeding, but the increase is not obvious, which indicates that the experimental alcohol concentration is insufficient and the damage is not serious; compared with a model control group, ALT and AST of each three doses of panax notoginseng polysaccharide are lower than those of the model control group, particularly, the serum ALT level reduction of each dose group has significance (P <0.05 or P < 0.01), but the influence on ALT of liver tissues is smaller, the serum AST activity in the dose group of panax notoginseng polysaccharide is obviously reduced (P < 0.05), and the result shows that the panax notoginseng polysaccharide component reduces ALT and AST levels of mice with chronic alcoholic liver injury, and the panax notoginseng polysaccharide component has a protective effect on the chronic alcoholic liver injury of the mice.
TABLE 3 Effect of Notoginseng radix polysaccharide on ALT and AST in chronic alcoholic liver injury mice: (
）
Note: n =10, compared to the blank control group: p <0.05, P < 0.01; # P <0.05, # P <0.01 compared to model control.
The synthesis and storage of triglyceride can be abnormal after a large amount of ethanol enters an organism, the TG level in serum is increased, the content of TG indirectly reflects the degree of liver damage, and Table 4 shows that compared with a blank control group, the content of triglyceride in the serum and liver tissues of a model control group mouse is obviously increased (P is less than 0.05 or P is less than 0.01), which indicates that the liver of the mouse is damaged by long-term drinking, so that the change of the content of triglyceride in the mouse is abnormal. The middle and high dose groups of notoginseng polysaccharide are lower than the model control group on the level of mouse serum TG; compared with a model control group, the TG content of each dose group in liver tissues is reduced, wherein the TG content (0.0079 +/-0.0023 mmol/gprot) of a pseudo-ginseng polysaccharide high dose group is lower than that of a drug (positive) group (P < 0.05), and is close to a blank control group, so that the pseudo-ginseng polysaccharide can reduce the TG content of a mouse with chronic alcoholic liver injury, and the result shows that the pseudo-ginseng polysaccharide has a certain protection effect on the mouse with chronic alcoholic liver injury.
TABLE 4 Effect of Notoginseng radix polysaccharide on TG in mice with chronic alcoholic liver injury: (
）
Note: n =10 compared to the blank control group: p <0.05, P < 0.01; # P <0.05, # P <0.01 compared to model control.
2. The influence of notoginseng polysaccharide on the oxidation and oxidation resistance level of liver tissues of a mouse with chronic alcoholic liver injury:
the determination of the amount of MDA can reflect the degree of lipid peroxidation in an organism and indirectly reflect the degree of cell damage, GSH and SOD play an important role in the oxidation and anti-oxidation balance of the organism, and GSH-Px can play a role in protecting the structural and functional integrity of cell membranes.
The results in table 5 show that the MDA content of the mice in the model control group is significantly increased (P < 0.05) compared with the blank control group; the GSH content in the blood serum of the mouse is obviously reduced (P < 0.05), which shows that the liver and blood serum of the chronic alcoholism mouse are in imbalance of oxidation and oxidation resistance, and the liver cells are seriously damaged, while compared with a model control group, the MDA content of each dose group of the notoginseng polysaccharide is reduced, wherein the MDA content in the dose group of the notoginseng polysaccharide is even lower than that in a drug (positive) group (0.33 +/-0.08 nmol/mgprot) (P < 0.05), and is closer to that in a blank control group (0.30 +/-0.05 nmol/mgprot); compared with a model control group, the serum GSH content of the medium and high dose groups is obviously increased, the significance is high (P is less than 0.01), the antioxidant capacity is enhanced, and the panax notoginseng polysaccharide can reduce the lipid peroxidation degree in a mouse, enhance the antioxidant capacity and protect the mouse from chronic liver injury.
The results in table 6 show that, compared with the blank control group, the antioxidant enzyme activity of GSH-Px in the liver tissue of the mouse in the model control group is obviously reduced (P < 0.01), the SOD in the serum of the mouse is also obviously reduced (P < 0.05), which indicates that the oxidation and the antioxidant balance in the liver and the serum of the chronic alcoholism mouse are imbalanced, the liver cell is seriously damaged, the modeling is successful, and the antioxidant enzyme activity of the liver tissue GSH-Px in each dose group of panax notoginseng polysaccharide is obviously improved (P < 0.01) compared with the model control group; the activity of serum SOD in the low and high dose groups of the notoginseng polysaccharide is obviously enhanced (P is less than 0.05), and the result shows that the notoginseng polysaccharide can enhance the oxidation resistance of liver tissues of mice, and the protection effect of the notoginseng polysaccharide on the chronic alcoholic liver injury of the mice is related to the improvement of the oxidation resistance of organisms of the mice.
TABLE 5 comparison of liver tissue MDA and serum GSH content in mice of each experimental group: (
）
TABLE 6 comparison of GSH-Px, SOD and serum SOD activity in liver tissue of mice in each experimental group: (
）
Pathological section is shown in fig. 4, blank control (fig. 4 a): the liver cells are polygonal and are arranged around the central blood vessel in a radial shape, the cell division is obvious, the cell nucleus is circular and is positioned in the center of the cell, and the cell apoptosis is not seen; model control group (fig. 4 b): the liver cord is disorderly arranged, liver cells are obviously swelled and even become necrotic, and the phenomenon of nucleus contraction can be seen; drug (yang) group (fig. 4 c): the hepatocyte structure and liver cable arrangement are basically close to normal and occasional liver cell swelling around central blood vessels; pseudo-ginseng polysaccharide low dose group (fig. 4 d): the liver cells have severe swelling, most cell nucleuses are fixed and shrunk and have irregular shapes, and obvious large-scale cell necrosis is not seen; medium dose group of notoginseng polysaccharides (fig. 4 e): compared with the lower dose group, the number and the number of the cells shrunk are reduced, and the degree is reduced; pseudo-ginseng high dose group (fig. 4 f): most of the hepatocytes are radially arranged around the central blood vessel, most of the cell nuclei are circular, and the hepatocyte swelling phenomenon is rarely seen; the results show that: the notoginseng polysaccharide has a certain protection effect on the liver injury of mice caused by alcohol.
And (4) conclusion: the invention adopts a gastric perfusion mode to establish a chronic alcoholic liver injury mouse model, and analyzes and discusses the protective effect of the notoginseng polysaccharide on alcoholic liver injury mice through comprehensive results by detecting various biochemical and enzymatic activity reaction indexes in serum and liver, such as glutamic-pyruvic transaminase (ALT), glutamic-oxalacetic transaminase (AST), Triglyceride (TG), Malondialdehyde (MDA), superoxide dismutase (SOD), Glutathione (GSH) and the like. The results show that: the ALT, AST and TG levels of the mice in the model control group are obviously increased compared with those in the blank control group, the SOD activity is reduced, the MDA content is increased, the GSH content is obviously reduced, the GSH-Px activity is obviously reduced, and the gavage alcohol of the mice has certain damage to the livers of the mice; the pseudo-ginseng polysaccharide dose groups have inhibiting effects of different degrees on the level increase of ALT, AST and TG, simultaneously inhibit the reduction of mouse SOD activity and the increase of MDA content to different degrees, and obviously enhance the activity of GSH-Px and improve the content of GSH. The panax notoginseng polysaccharide has a certain protection effect on the liver injury of the mice caused by alcohol, and the conclusion can be obtained by observing the pathological tissue morphology of the liver of the mice.
Claims (1)
1. An acidic notoginseng polysaccharide is characterized in that the molecular weight is 2500-ion 3000kDa, the monosaccharide composition and the molar ratio are Rha, Ara, Man, Glc, Gal, GalA =0.038, 0.045, 0.002, 0.016, 0.031, 0.868, and the GalA structure is galacturonic acid; the types and the molar ratio of the glycosidic bond are as follows: ara-1 → Rha-1 → 5-Ara-1 → 4-GalA-1 → 3,6-Gal-1 → 0.06:0.04:0.05:0.80: 0.05;
the preparation method of the acidic notoginseng polysaccharide comprises the following steps:
(1) drying Notoginseng radix residue after extracting Notoginseng radix total saponin at 50 deg.C, pulverizing, sieving with 80 mesh sieve, adding 20 times volume of distilled water, extracting at 70 deg.C for 4 hr, filtering, collecting residue, adding 15 times volume of distilled water, repeatedly extracting for 4 hr, filtering, and mixing the two extractive solutions; performing deproteinization by referring to a sevag method, wherein the sevag solution is chloroform and n-butanol =5:1 in volume ratio, namely adding sevag solution with 0.3 time volume of an extracting solution, shaking vigorously for 15min, standing for layering, removing a protein layer, repeating for four times until the protein is completely removed, placing the extracting solution at 50 ℃, performing rotary evaporation and concentration to 1/6 of the original volume, adding 3 times volume of absolute ethanol, precipitating and precipitating, removing ethanol by using 55 ℃ water bath, redissolving, adding activated carbon for decoloration, performing water bath at 40 ℃ for 3h, performing centrifugation at 4000rpm for 10min, taking supernatant, performing centrifugation again at 12000rpm for 15min, dialyzing at 3500Da for one night, and performing freeze drying to obtain crude panax notoginseng polysaccharide NGP;
(2) weighing 300mg of the crude notoginseng polysaccharide prepared in the step (1) and dissolving the crude notoginseng polysaccharide in 20mL of deionized water, performing column chromatography through an anion exchange cellulose column DEAE-52, wherein the size of the anion exchange cellulose column DEAE-52 is 4cm multiplied by 60cm, eluting with 1 column volume of distilled water, then sequentially performing gradient elution by using one column volume of NaCl solution with the concentration of 0.2mol/L, 0.5mol/L, 1.0mol/L and 2.0mol/L, wherein the flow rate is 24mL/h, collecting the eluate fraction in 1 test tube per 8mL by adopting a phenol-sulfuric acid method, collecting the eluate fraction of 0.5mol/L with the highest acidic notoginseng polysaccharide concentration, collecting the eluate fraction, and freeze-drying to obtain freeze-dried powder which is named as NGP0.5M;
(3) weighing NGP0.5M mg obtained in the step (2), dissolving in 3mL of distilled water, centrifuging for 10min by using a high-speed centrifuge at the rotating speed of 12000rpm, collecting the supernatant for subsequent sample loading, purifying by using a G-50 polysaccharide gel purification system, detecting and collecting by using a phenol-sulfuric acid method, collecting the left half peak part in the symmetrical peaks, namely 30-36 tubes, then performing rotary evaporation and concentration at 45 ℃, dialyzing by using a 3500Da dialysis bag, and freeze-drying to obtain the acidic notoginseng polysaccharide separated and purified by using a gel column.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910584252.6A CN110256596B (en) | 2019-07-01 | 2019-07-01 | Acidic notoginseng polysaccharide and extraction and purification method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910584252.6A CN110256596B (en) | 2019-07-01 | 2019-07-01 | Acidic notoginseng polysaccharide and extraction and purification method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110256596A CN110256596A (en) | 2019-09-20 |
| CN110256596B true CN110256596B (en) | 2021-08-31 |
Family
ID=67923473
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910584252.6A Active CN110256596B (en) | 2019-07-01 | 2019-07-01 | Acidic notoginseng polysaccharide and extraction and purification method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110256596B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111533820A (en) * | 2020-05-11 | 2020-08-14 | 昆明理工大学 | Notoginseng polysaccharide and its preparation method and use |
| CN114031694B (en) * | 2021-12-23 | 2023-02-03 | 云南三七科技有限公司 | Extraction method of pseudo-ginseng polysaccharide |
| CN115028752B (en) * | 2022-06-24 | 2023-04-07 | 黑龙江葵花药业股份有限公司 | Uniform water-soluble polysaccharide and preparation method and application thereof |
| CN115141289B (en) * | 2022-08-02 | 2023-06-16 | 云南西草资源开发有限公司 | Acidic pseudo-ginseng polysaccharide and preparation method and application thereof |
| CN115414379B (en) * | 2022-08-17 | 2023-11-17 | 广东药科大学 | Application of notoginseng polysaccharide SQP20 in preparation of medicine for treating intestinal injury and inflammatory infiltration |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102961424A (en) * | 2012-11-23 | 2013-03-13 | 云南云科药业有限公司 | Panax notoginseng polysaccharide extract and preparation method, preparations and applications thereof |
| CN103145872A (en) * | 2013-04-03 | 2013-06-12 | 昆明制药集团金泰得药业股份有限公司 | Panax notoginseng polysaccharide with low ash content and preparation method of polysaccharide |
| CN107286265A (en) * | 2017-06-16 | 2017-10-24 | 云南多糖生物科技有限公司 | The method for extraction and purification and promotion cell propagation pharmacology activity research of a kind of neutral notoginseng polysaccharide and application |
| CN107892721A (en) * | 2017-03-16 | 2018-04-10 | 楚雄云植药业有限公司 | A kind of low ash content notoginseng polysaccharide extraction preparation method |
-
2019
- 2019-07-01 CN CN201910584252.6A patent/CN110256596B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102961424A (en) * | 2012-11-23 | 2013-03-13 | 云南云科药业有限公司 | Panax notoginseng polysaccharide extract and preparation method, preparations and applications thereof |
| CN103145872A (en) * | 2013-04-03 | 2013-06-12 | 昆明制药集团金泰得药业股份有限公司 | Panax notoginseng polysaccharide with low ash content and preparation method of polysaccharide |
| CN107892721A (en) * | 2017-03-16 | 2018-04-10 | 楚雄云植药业有限公司 | A kind of low ash content notoginseng polysaccharide extraction preparation method |
| CN107286265A (en) * | 2017-06-16 | 2017-10-24 | 云南多糖生物科技有限公司 | The method for extraction and purification and promotion cell propagation pharmacology activity research of a kind of neutral notoginseng polysaccharide and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110256596A (en) | 2019-09-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110256596B (en) | Acidic notoginseng polysaccharide and extraction and purification method thereof | |
| Huang et al. | Studies on polysaccharides from three edible species of Nostoc (Cyanobacteria) with different colony morphologies: comparison of monosaccharide compositions and viscosities of polysaccharides from field colonies and suspension cultures | |
| Wang et al. | A novel acidic polysaccharide from the residue of Panax notoginseng and its hepatoprotective effect on alcoholic liver damage in mice | |
| WO2022016644A1 (en) | Acanthopanax senticosus harms homogeneous polysaccharide, preparation method therefor and use thereof | |
| CN110128562B (en) | An antitumor fructus Psoraleae polysaccharide, its extraction and separation method, and its application in preparing antitumor drugs | |
| Jin et al. | Physicochemical properties of polysaccharides separated from Camellia oleifera Abel seed cake and its hypoglycemic activity on streptozotocin-induced diabetic mice | |
| Ke et al. | Research progress on the extraction technology and activity study of Epimedium polysaccharides | |
| CN115721671A (en) | Method for extracting and separating effective components of liquorice root in full-industrial-chain cooperation manner | |
| CN105175566B (en) | Polyamide column and macroporous resin column connection post method remove the method for protein and pigment in Radix Panacis Quinquefolii polysaccharide extract | |
| Liu et al. | Advances in Polygonatum sibiricum polysaccharides: Extraction, purification, structure, biosynthesis, and bioactivity | |
| CN112175100A (en) | Neutral panax notoginseng polysaccharide NPPN purification method, structure characterization and application | |
| Zhang et al. | Preparation and structural characterization of acid-extracted polysaccharide from Grifola frondosa and antitumor activity on S180 tumor-bearing mice | |
| Yue et al. | Extraction, purification, structural features and biological activities of longan fruit pulp (Longyan) polysaccharides: A review | |
| Chen et al. | Extraction, structural characterization and biological activities of polysaccharides from mulberry leaves: A review | |
| Yang et al. | Polysaccharides from waste Zingiber mioga leaves: Ultrasonic-microwave-assisted extraction, characterization, antioxidant and anticoagulant potentials | |
| Chen et al. | Extraction, purification, structural characteristics and biological properties of the polysaccharides from Armillaria mellea (Vahl) P. Kumm.: A review | |
| WO2023036203A1 (en) | Cs-4 fermented mycelium heteropolysaccharide, preparation method therefor and use thereof | |
| CN113694104B (en) | Traditional Chinese medicine composition with protection effect on chemical liver injury and liver regeneration promotion function, preparation method and application thereof | |
| Chen et al. | Study on the extraction, purification, partial chemical characterization and anti-alcohol liver injury activity of Mori Fructus polysaccharides | |
| CN105753920B (en) | A kind of method that alpha-glucosidase restrainer is extracted from litchi pulp | |
| CN108948223B (en) | Myrtle polysaccharide P1, its separation method and application in preparing hypolipidemic medicine | |
| CN112794925B (en) | Amomum villosum polysaccharide and preparation method and application thereof | |
| CN113730466A (en) | Preparation method and application of blackberry extract | |
| CN110218263B (en) | Hericium erinaceus fungus chaff polysaccharide, preparation method and application | |
| Sun et al. | Isolation, structural properties and bioactivities of polysaccharides from Crataegus pinnatifida |
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 | ||
| CB03 | Change of inventor or designer information | ||
| CB03 | Change of inventor or designer information |
Inventor after: Wang Chengxiao Inventor after: Cui Xiuming Inventor after: Liu Shengnan Inventor after: Yang Ye Inventor after: Yang Xiaoyan Inventor after: Qu Yuan Inventor after: Liu Diqiu Inventor before: Cui Xiuming Inventor before: Liu Shengnan Inventor before: Wang Chengxiao Inventor before: Yang Ye Inventor before: Yang Xiaoyan Inventor before: Qu Yuan Inventor before: Liu Diqiu |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |