CN106749731A - A kind of preparation method and application of small molecule notoginseng polysaccharide extract - Google Patents

A kind of preparation method and application of small molecule notoginseng polysaccharide extract Download PDF

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CN106749731A
CN106749731A CN201611189464.7A CN201611189464A CN106749731A CN 106749731 A CN106749731 A CN 106749731A CN 201611189464 A CN201611189464 A CN 201611189464A CN 106749731 A CN106749731 A CN 106749731A
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extract
small molecule
polysaccharide
notoginseng
preparation
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张文生
曾立品
罗金明
蔡群虎
辛文锋
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Yunnan 37 Technology Co Ltd
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Yunnan 37 Technology Co Ltd
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    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08BPOLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0003General processes for their isolation or fractionation, e.g. purification or extraction from biomass
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/185Magnoliopsida (dicotyledons)
    • A61K36/25Araliaceae (Ginseng family), e.g. ivy, aralia, schefflera or tetrapanax
    • A61K36/258Panax (ginseng)

Abstract

The invention discloses a kind of preparation method of small molecule notoginseng polysaccharide extract, the method is with pseudo-ginseng as raw material, the water extraction of the gained dregs of a decoction obtains pseudo-ginseng Thick many candies extract solution after ethanol solution is extracted after raw material crushing, ethanol precipitation, precipitates water-soluble rear plus trifluoroacetic acid or lysozyme hydrolysis obtain the thick liquid of micromolecular polysaccharide;The thick liquid of micromolecular polysaccharide remove isolating protein, pigment etc. through cation and anion exchange post after active carbon powder decolourizes, titanium rod filter is filtrated to get colourless low sticky micromolecular polysaccharide solution, concentration, freeze-drying are obtained small molecule notoginseng polysaccharide extract;This technological operation is simple, and low cost, industrialization level is high;Extract of the present invention can be promoted into macrophage proliferation, and it is ripe to be further differentiated into macrophage, the macrophage as activation, and it been significantly enhanced its phagocytic activity;Cellular immunity can be accelerated quickly to improve in cell culture immunity test;The extract has the application prospect for the treatment of immunological diseases, and preparation method is suitable to industrialized production.

Description

A kind of preparation method and application of small molecule notoginseng polysaccharide extract
Technical field
The present invention relates to a kind of preparation method and application of small molecule notoginseng polysaccharide extract, belong to active substance of plant and carry Take separation field.
Background technology
In plant sugar chemical research many reports point out to have cure the disease, the high activity polysaccharide of diseases prevention effect is essentially small point Sub- polysaccharide, macromolecular polysaccharide effect is weaker.Immune system is an extremely complex and important physiological systems, wherein phagocyte It is one of the first line of defence of body resistance cause pathogeny imcrobe infection, including neutrophil leucocyte, monocyte, macrophage etc., They are all the important components of innate immune system.Wherein macrophage being capable of non-specific phagocytosis and the killing micro- life of cause of disease Thing, while some inflammatory mediators can also be discharged and cell factor is killed, can also be supplied to T lymphs by pathogen in addition Cell, activation adaptive immunity reaction discharges various cell factors to play immunoregulation effect.Research finds GL-B (PSG) the ripe of immunocyte can be promoted to break up by NF~κ B signals path, and cytokine TNF~α, IL~1 β, IL~ 12, the expression of costimulatory molecules CD40, CD80, CD86, so as to be played a significant role in the immune response of body.
Pseudo-ginseng also known as invaluable, is Araliaceae, and using its root as medicinal effects, pseudo-ginseng main product is in the Yunnan of China Mountain of papers, notoginseng polysaccharide be extracted from pseudo-ginseng with multiple biological activities and baroque heteroglycan or it is compound Thing.Numerous studies prove that notoginseng polysaccharide has the multiple efficacies such as antitumor, hypoglycemic, anti-oxidant, strengthen immunity.China's mesh The preceding exploitation to notoginseng polysaccharide are simultaneously few, and efficacy study, product development of notoginseng polysaccharide etc. also have very big research Value and space.
The extracting method of active pseudo-ginseng micromolecular polysaccharide has no report, and it is always many that relevant report is concentrated mainly on pseudo-ginseng Sugared extracting method and pharmacology activity research aspect;Pseudo-ginseng total starches are extracted mainly to be had using dry pulverizing medicinal materials, degreasing, water extraction, second The alcohol precipitation method, de- albumen, pseudo-ginseng total starches are dried to obtain, but soluble cellulose are contained due to the macromolecular polysaccharide for extracting, is formed sediment Powder etc., drug activity is very low, and most of macromolecular polysaccharide molecular weight be all it is several absolutely, it is up to a million, human body is difficult to absorb, human body Really to absorb that play a role will be by being metabolized to small molecule composition, so the utilization of macromolecular polysaccharide is low.
Therefore, carried out the research to pseudo-ginseng micromolecular polysaccharide, can for further further investigation China peculiar pseudo-ginseng resource, Improve its added value, increase Planting household income and make positive contribution, with preferable economic and social benefit, can also give people to carry It is that human health contributes for new health products.
The content of the invention
It is an object of the invention to provide the preparation method of small molecule notoginseng polysaccharide extract, the method is with pseudo-ginseng as former Material, concrete operations are as follows:
(1)Ethanol solution 2~the 3h of extraction process of mass percent concentration 95~100% is added in Notoginseng Root after being pulverized, Filtering filter residue repeats to extract 1~2 time, abandons filtrate;
(2)In step(1)Water is added in filter residue and in being extracted 1~2 time at 90~95 DEG C, filtered after 1.5~2 hours extraction times Merging filtrate, abandons filter residue;
(3)By step(2)Filtrate is concentrated in vacuo to Baume degrees 1.1 at being placed in 40~60 DEG C after, high concentration ethanol solution is added to make Concentration of alcohol is 93%~97% in obtaining mixed liquor, after standing 3~5h hours, precipitation is collected by filtration;
(4)In step(3)Precipitation in add the pure water of 10~15 times of its quality to mix, be subsequently adding mixture quality 3~ 5% trifluoroacetic acid, in after 90~100 DEG C of 20~40min of closed hydrolysis, vacuum reclaims trifluoroacetic acid and obtains small point at 40~60 DEG C Sub- liquid of extracting polysaccharide;
Or in step(3)Precipitation in add the pure water of 10~15 times of its quality to mix, be subsequently adding mixture quality 2% Lysozyme, after being hydrolyzed 3~5 hours in 35 DEG C~45 DEG C, is warming up to 100 DEG C of enzyme 10min that go out and obtains micromolecular polysaccharide crude extract;
(5)By step(4)Micromolecular polysaccharide crude extract persulfonic acid base strong acid cation exchange resin column, collects trickle;
(6)By step(5)The liquid of collection crosses acrylic acid weak-base anion-exchange resin post, collects trickle;
(7)In step(6)The activated carbon powder of liquid quality 1~2% is added in the liquid of collection, is stood at 60~65 DEG C after mixing After 0.5h, low sticky small molecule notoginseng polysaccharide solution is filtered to obtain with titanium rod filter;
(8)By step(7)Small molecule notoginseng polysaccharide extract is obtained final product after the concentration of small molecule notoginseng polysaccharide solution, freeze-drying.
The step(1)The ethanol solution amount of middle addition is 4~6 times of Notoginseng Root quality.
The step(2)The water of middle addition is 6~10 times of filter residue quality.
The filter element filtering precision of the titanium rod filter is 0.25~0.5 micron.
The present invention is another object is that small molecule notoginseng polysaccharide obtained in the above method is applied to prepare treatment immune deficiency Or in hypoimmunity disease medicament, that is, prepare treatment immunological disorder drug.
One or more pharmaceutically acceptable auxiliary material, the auxiliary material can also be added in application of the present invention includes medicine The conventional filler in field, diluent, adhesive, excipient, sorbefacient, filler, surfactant and stabilizer Deng can also add flavouring agent, pigment and sweetener etc. if necessary;Or used cooperatively with the medicine of other treatment immunological diseases.
Application of the present invention can also be made pill, pulvis, tablet, granula, oral liquid and injection in addition to capsule is made The diversified forms such as liquid.
The inventive method coarse extraction is using alcohol degreasing, water extraction, the most of protein of ethanol precipitation removal, pectin, mucus The impurity such as matter, starch;Then using trifluoroacetic acid or lysozyme hydrolysis control hydrolysis process so that macromolecular polysaccharide is to small Molecular polysaccharide direction hydrolysis, are not converted into monose as far as possible, more gentle more controllable than classical acid hydrolysis, enzymatic hydrolysis reaction, Micromolecular polysaccharide yield is higher;Amino acid, protein, pigment, ash content in liquid are sloughed using cation and anion exchange post, is substituted and is passed The de- protein Process of the organic solvent of system, solves gained exceeded this problem of polysaccharide ash content in conventional method, with more preferable industry Change operability;Liquid freezing dries products therefrom, it is to avoid heated-air drying causes micromolecular polysaccharide component chemistry to change, and extracts Protein, starch residual are extremely low after testing for thing, and this technological operation is simple, and low cost, industrialization level is high.
The present invention uses the pseudo-ginseng extraction preparation is carried out for raw material, and gained pseudo-ginseng micromolecular polysaccharide high income, purity are high, color In vain;It is monose that conventional polysaccharide saccharic acid hydrolysis uses the products therefroms such as hydrochloric acid, sulfuric acid, enzyme hydrolysis major part, thoroughly loses it many Sugared activity, polysaccharide molecule cutting or lysozyme hydrolysis are carried out using trifluoroacetic acid, and it is small molecule to react more gentle products therefrom Polysaccharide, its hydrolysing reagent solution is removed without residual, does not influence product quality.
The extract obtained middle protein of the present invention, starch, ash content, heavy metal removing are more thorough, and polysaccharide pharmacological activity is higher;It is real Test result display the present invention it is extract obtained compare with pseudo-ginseng total starches control group, in cell propagation effect immunity test and cell Immune-enhancing effect has significant difference in Phagocytosis immunity test, illustrates small molecule notoginseng polysaccharide extract than pseudo-ginseng total starches Extract can more promote immunity to improve, the application prospect with treatment immunological diseases, and preparation method is suitable to industrialized production.
Specific embodiment
The present invention is described in further detail below by embodiment, but protection scope of the present invention be not limited to it is described Content.
Embodiment 1:The preparation method of small molecule notoginseng polysaccharide extract is as follows:
(1)The ethanol solution of mass percent concentration 95% of 5 times of its quality is added to extract 3h in 1kg Notoginseng Roots after being pulverized, Filtering filter residue repeats to extract 2 times, abandons filtrate and obtains the dregs of a decoction;
(2)In step(1)The water of 8 times of dregs of a decoction quality is added in the dregs of a decoction and in extraction 2 times, 2 hours extraction times at 90~93 DEG C After filter to get filtrate, abandon the dregs of a decoction;
(3)By step(2)Filtrate is concentrated in vacuo to Baume degrees 1.1 at being placed in 40 DEG C after, absolute ethyl alcohol is added so that in mixed liquor Concentration of alcohol is 93%, stands 3 hours, and filtering to precipitate;
(4)By step(3)The middle precipitation dissolved in purified water of 12 times of its quality, is subsequently adding the trifluoroacetic acid of mixed liquor quality 4% In after 100 DEG C of closed hydrolysis 20min, trifluoroacetic acid obtains micromolecular polysaccharide crude extract in vacuum withdrawal liquid at 60 DEG C;
(5)By step(4)Micromolecular polysaccharide crude extract persulfonic acid base storng-acid cation exchange resin (001X10) post, collects stream Go out liquid;
(6)By step(5)The liquid of collection crosses acrylic acid weak-base anion-exchange resin (D202) post, collects colourless outflow Liquid;
(7)Step(6)The liquid of collection adds the active carbon powder of liquid weight 2%, after soaking half an hour at 60 DEG C after mixing, Titanium rod filter(0.5 micron of filter stamen filtering accuracy)Filter to obtain low sticky small molecule notoginseng polysaccharide solution;
(8)By step(7)After small point of notoginseng polysaccharide solution is concentrated to original volume 1/3, it is many that freeze-drying obtains active small molecular pseudo-ginseng Sugar extract 31g, polyoses content 87%, ash content 2ppm, nitrogen content is not detected.
Embodiment 2:The preparation method of small molecule notoginseng polysaccharide extract is as follows:
(1)The absolute ethyl alcohol of 4 times of its quality is added to extract 2h in 1kg Notoginseng Roots after being pulverized, filtering filter residue repeats to extract 1 It is secondary, abandon filtrate and obtain the dregs of a decoction;
(2)In step(1)The water of 7 times of dregs of a decoction quality is added in the dregs of a decoction and in extraction 2 times, 1.5 hours extraction times at 90-93 DEG C After filter to get filtrate, abandon the dregs of a decoction;
(3)By step(2)Filtrate is concentrated in vacuo to Baume degrees 1.1 at being placed in 50 DEG C after, absolute ethyl alcohol is added so that in mixed liquor Concentration of alcohol is 97%, stands 4 hours, and filtering to precipitate;
(4)In step(3)Precipitation add 10 times of its quality dissolved in purified water, be subsequently adding the lysozyme of mixture quality 2%, After being hydrolyzed 5 hours in 35 DEG C, it is warming up to 100 DEG C of enzymes that go out and obtains micromolecular polysaccharide crude extract in 10 minutes;
(5)By step(4)Micromolecular polysaccharide crude extract persulfonic acid base storng-acid cation exchange resin (001X4) post, collects stream Go out liquid;
(6)By step(5)The liquid of collection crosses acrylic acid weak-base anion-exchange resin (D941) post, collects colourless efflux;
(7)Step(6)The liquid of collection adds the active carbon powder of liquid weight 1%, is used after soaking half an hour at 60 DEG C after mixing Titanium rod filter(0.3 micron of stamen of filter)Filter to obtain low sticky small molecule notoginseng polysaccharide solution;
(8)By step(7)After small molecule notoginseng polysaccharide solution is concentrated to original volume 1/3, freeze-drying obtains small molecule notoginseng polysaccharide 24 grams of extract, polyoses content 88%, ash content 2ppm, nitrogen content is not detected.
Embodiment 3:The preparation method of small molecule notoginseng polysaccharide extract is as follows:
(1)The absolute ethyl alcohol of 4 times of its quality is added to extract 2h in 1kg Notoginseng Roots after being pulverized, filtering filter residue repeats to extract 1 It is secondary, abandon filtrate and obtain the dregs of a decoction;
(2)In step(1)The water of 6 times of dregs of a decoction quality is added in the dregs of a decoction and in being extracted 1 time at 90~95 DEG C, extraction time 1.5 is small When after filter to get filtrate, abandon the dregs of a decoction;
(3)By step(2)Filtrate is concentrated in vacuo to Baume degrees 1.1 at being placed in 50 DEG C after, absolute ethyl alcohol is added so that in mixed liquor Concentration of alcohol is 97%, stands 4 hours, and filtering to precipitate;
(4)By step(3)The middle precipitation dissolved in purified water of 10 times of its quality, is subsequently adding the trifluoroacetic acid of mixed liquor quality 3% In after 90 DEG C of closed hydrolysis 30min, trifluoroacetic acid obtains micromolecular polysaccharide crude extract in vacuum withdrawal liquid at 50 DEG C;
(5)By step(4)Micromolecular polysaccharide crude extract persulfonic acid base storng-acid cation exchange resin (001X4) post, collects stream Go out liquid;
(6)By step(5)The liquid of collection crosses acrylic acid weak-base anion-exchange resin (D941) post, collects colourless efflux;
(7)Step(6)The liquid of collection adds the active carbon powder of liquid weight 1%, after soaking half an hour at 60 DEG C after mixing, Titanium rod filter(0.25 micron of filter stamen filtering accuracy)Filter to obtain low sticky small molecule notoginseng polysaccharide solution;
(8)By step(7)Small point of notoginseng polysaccharide solution is concentrated to after filtering after volume product 1/3, and freeze-drying obtains active small molecular Notoginseng polysaccharide extract 25g, polyoses content 84%, ash content 2ppm, nitrogen content is not detected.
Multiplication effect of the extract of the present embodiment 3 to RAW264.7 cells is detected using mtt assay:
1st, experimental cell:Mouse macrophage strain RAW264.7 is purchased from Shanghai cell institute, is incubated at containing 10% hyclone In RPMI1640 nutrient solutions(Containing 100 mgL-1Penicillin, 100 mgL-1Streptomysin), it is placed in 37 DEG C, 5%CO2Cell culture In case, liquid is changed daily, passed on 1 time every 2 days;
2nd, the preparation of medicine:Small molecule notoginseng polysaccharide extract is dissolved in a certain amount of cell culture fluid so that pseudo-ginseng is more in solution Final concentration of 5,10,20,40,80, the 160 μ g/mL of sugar, filtering(0.22 μm of filter membrane)Degerming, -20 DEG C of packing are preserved;
3rd, experimental technique
MTT analytic approach is with living cells metabolin reducing agent 3-(4,5)-dimethylthiahiazo (- z-y1) - 3,5-di-phenytetrazoliumromide, based on MTT tetramethyl azo azoles salts.MTT is yellow compound, It is a kind of hydrionic dyestuff of receiving, the respiratory chain in living cells mitochondria is may act on, in succinate dehydrogenase and cell color Tetrazolium ring opening in the presence of plain C, generates blue formazan crystallization, and the growing amount of formazan crystallization is only directly proportional to number of viable cells (Succinate dehydrogenase disappears in dead cell, it is impossible to reduce MTT);Reduction generation formazan crystallizations can be molten in DMSO lysates Solution, determines the optical density OD values at 570 nm, to reflect number of viable cells using ELIASA.
4th, analysis method:Regulation RAW264.7 cell concentrations are 5 × 104Individual/mL, the μ L of cell suspending liquid 100 are added per hole, Culture washes away non-attached cell after 4 hours, is then respectively adding small molecule notoginseng polysaccharide, the pseudo-ginseng total starches of various concentrations(Eventually Concentration is 5,10,20,40,80,160 μ g/mL), while setting the LPS of 1 μ g/mL, blank control wells;Every group is all provided with 6 again Hole;5%CO2, 37 DEG C are incubated 24 hours, inverted microscope observation, add the μ L of nutrient solution 20 containing 5% MTT, and training is terminated after 4 hours Support, carefully suck nutrient solution in hole, add 150 μ L dimethyl sulfoxides to shake per hole 10 minutes, crystallization is fully dissolved, ELIASA 570 nm wavelength detecting light absorption values, calculate comparative survival rate of cells.
Cell with respect to proliferation rate=(Experimental group light absorption value-control group light absorption value)/ control group light absorption value × 100%;
5th, experimental result is shown in Table 1:
Table 1:Proliferative effect of the present embodiment small molecule notoginseng polysaccharide extract to RAW264.7 cells
6th, conclusion
MTT experiment result shows, no matter pseudo-ginseng total starches, small molecule notoginseng polysaccharide extract, various dose(5,10,20,40, 80,160 μ g/mL)The growth of macrophage can be promoted after effect RAW264.7 cells 24h, and with the increase of dosage, increased The rate of growing gradually rises, and peaks(40 mg·L-1)Proliferation rate is gradually reduced afterwards, but still shows as promoting cell growth, says Bright small molecule notoginseng polysaccharide extract acellular poison effect;Than the pseudo-ginseng total starches of low concentration, small molecule notoginseng polysaccharide is extracted Thing has the effect for promoting cell propagation, and especially low concentration group is substantially better than total starches.
Embodiment 4:The preparation method of small molecule notoginseng polysaccharide extract is as follows:
(1)The ethanol solution of mass percent concentration 95% of 5 times of its quality is added to extract 3h in 1kg Notoginseng Roots after being pulverized, Filtering filter residue repeats to extract 2 times, abandons filtrate and obtains the dregs of a decoction;
(2)In step(1)The water of 8 times of dregs of a decoction quality is added in the dregs of a decoction and in extraction 2 times, 2 hours extraction times at 90~93 DEG C After filter to get filtrate, abandon the dregs of a decoction;
(3)By step(2)Filtrate is concentrated in vacuo to Baume degrees 1.1 at being placed in 40 DEG C after, absolute ethyl alcohol is added so that in mixed liquor Concentration of alcohol is 93%, stands 3 hours, and filtering to precipitate;
(4)By step(3)The middle precipitation dissolved in purified water of 12 times of its quality, is subsequently adding the trifluoroacetic acid of mixed liquor quality 4% In after 100 DEG C of closed hydrolysis 20min, trifluoroacetic acid obtains micromolecular polysaccharide crude extract in vacuum withdrawal liquid at 60 DEG C;
(5)By step(4)Micromolecular polysaccharide crude extract persulfonic acid base storng-acid cation exchange resin (001X10) post, collects stream Go out liquid;
(6)By step(5)The liquid of collection crosses acrylic acid weak-base anion-exchange resin (D202) post, collects colourless outflow Liquid;
(7)Step(6)The liquid of collection adds the active carbon powder of liquid weight 2%, after soaking half an hour at 60 DEG C after mixing, Titanium rod filter(0.4 micron of filter stamen filtering accuracy)Filter to obtain low sticky small molecule notoginseng polysaccharide solution;
(8)By step(7)Small point of notoginseng polysaccharide solution is concentrated to volume 1/3 after filtering, and freeze-drying obtains active small molecular pseudo-ginseng Polyoses extract 30g, polyoses content 89%, ash content 1ppm, nitrogen content is not detected.
Phagocytosis of the small molecule notoginseng polysaccharide extract of embodiment 4 to RAW264.7 cells are detected using dimethyl diaminophenazine chloride method;
1st, experimental cell:Mouse macrophage strain RAW264.7 is purchased from Shanghai cell institute, is incubated at containing 10% hyclone In RPMI1640 nutrient solutions(Containing 100 mgL-1Penicillin, 100 mgL-1Streptomysin), it is placed in 37 DEG C, 5%CO2Cell culture In case, liquid is changed daily, passed on 1 time every 2 days;
2nd, the preparation of medicine:Small molecule notoginseng polysaccharide extract is dissolved in a certain amount of cell culture fluid so that notoginseng polysaccharide polysaccharide Final concentration of 5,10,20,40,80,160 μ g/mL, filtering(0.22 μm of filter membrane)Degerming, -20 DEG C of packing are preserved;
3rd, experimental technique:
Under normal circumstances, macrophage is in quiescent condition, and phagocytic activity increases after being activated, and can be lived by detecting that the phase swallows Property weighs its immunocompetent increase.After the macrophage of quiescent condition is upset, phagocytic function enhancing, phagocytic activity Size reflects the pharmacological activity of medicine.Dimethyl diaminophenazine chloride method detection macrophage phagocytic function is red by macrophage centering Phagocytosis amount reflects the phagocytic activity of macrophage, is the conventional cell membrane for quickly screening macrophage phagocytic function modifying ingredients Type.
4th, analysis method:Pseudo-ginseng total starches, small molecule notoginseng polysaccharide extract(Final concentration of 5,10,20,40,80,160 µg/mL)Act on final concentration of 106 The h of/mL RAW264.7 macrophages 24, while setting the LPS of 1 μ g/mL, blank Hole.Supernatant is abandoned, 0.1% dimethyl diaminophenazine chloride is added(100 mg dimethyl diaminophenazine chloride/100 mL physiological saline)The μ L of solution 100/hole, cultivate 3 hours Washed 3 times with warm PBS afterwards, add cell pyrolysis liquid(Acetic acid:Ethanol=1:1)200 μ L/ holes, stand 30 min, are determined with ELIASA The dimethyl diaminophenazine chloride of macrophage phagocytic, with A540Value expression, calculates phagocytic index.
Phagocytic index=(Experimental group light absorption value/control group light absorption value -1)×100%;
5th, experimental result is shown in Table 2:
Macrophage plays the important effect without that can lack in body specific immunity, nospecific immunity, to invading The foreign matter of body and the cell of aging death have phagocytic function, are also the antineoplastic the first line of defence of body, and phagocytosis energy Masterpiece is the important indicator for detecting macrophage function.In order to detect that whether the phagocytosis that can strengthen macrophage in notoginseng polysaccharide is lived Property, therefore influence of the notoginseng polysaccharide to macrophage phagocytic function is determined in vitro.
Table 2:Proliferative effect of the notoginseng polysaccharide to RAW264.7 cells
6th, conclusion:
Experimental result shows, no matter pseudo-ginseng total starches, small molecule notoginseng polysaccharide extract(5,10,20,40,80,160 μ g/mL) After treatment RAW264.7 macrophages 24h, Macrophage Cell can be promoted to activate, significantly improve the ability of phagocytosis dimethyl diaminophenazine chloride, Activity is promoted to be gradually reduced and with the increase of drug concentration, the phagocytic activity of macrophage is also dramatically increased, during high concentration. Longitudinal comparison finds:Total starches and small molecule notoginseng polysaccharide extract(The > pseudo-ginseng of 57.34% > small molecules holosaccharide 54.76% is total Polysaccharide 53.96%), this show small molecule notoginseng polysaccharide can stimulating expression of macrophage, be further differentiated into macrophage ripe, into It is the macrophage of activation, and been significantly enhanced its phagocytic activity.

Claims (5)

1. a kind of preparation method of small molecule notoginseng polysaccharide extract, it is characterised in that step is as follows:
(1)Ethanol solution 2~the 3h of extraction process of mass percent concentration 95~100% is added in Notoginseng Root after being pulverized, Filtering filter residue repeats to extract 1~2 time, abandons filtrate;
(2)In step(1)Water is added in filter residue and in being extracted 1~2 time at 90~95 DEG C, filtered after 1.5~2 hours extraction times Merging filtrate, abandons filter residue;
(3)By step(2)Filtrate is concentrated in vacuo to Baume degrees 1.1 at being placed in 40~60 DEG C after, ethanol solution is added to cause mixing Concentration of alcohol is 93%~97% in liquid, after standing 3~5h hours, precipitation is collected by filtration;
(4)In step(3)Precipitation in add the pure water of 10~15 times of its quality to mix, be subsequently adding mixture quality 3~ 5% trifluoroacetic acid, in after 90~100 DEG C of 20~40min of closed hydrolysis, vacuum reclaims trifluoroacetic acid and obtains small point at 40~60 DEG C Sub- liquid of extracting polysaccharide;
Or in step(3)Precipitation in add the pure water of 10~15 times of its quality to mix, be subsequently adding mixture quality 2% Lysozyme, after being hydrolyzed 3~5 hours in 35~45 DEG C, is warming up to 100 DEG C of enzyme 10min that go out and obtains micromolecular polysaccharide crude extract;
(5)By step(4)Micromolecular polysaccharide crude extract persulfonic acid base strong acid cation exchange resin column, collects trickle;
(6)By step(5)The liquid of collection crosses acrylic acid weak-base anion-exchange resin post, collects trickle;
(7)In step(6)The activated carbon powder of liquid quality 1~2% is added in the liquid of collection, is soaked at 60~65 DEG C after mixing After 0.5h, low sticky small molecule notoginseng polysaccharide solution is filtered to obtain with titanium rod filter;
(8)By step(7)Small molecule notoginseng polysaccharide extract is obtained final product after small point of concentration of notoginseng polysaccharide solution, freeze-drying.
2. the preparation method of small molecule notoginseng polysaccharide extract according to claim 1, it is characterised in that:Step(1)In The ethanol solution amount of addition is 4~6 times of Notoginseng Root quality.
3. the preparation method of small molecule notoginseng polysaccharide extract according to claim 1, it is characterised in that:Step(2)In The water of addition is 6~10 times of filter residue quality.
4. the preparation method of small molecule notoginseng polysaccharide extract according to claim 1, it is characterised in that:Titanium rod filter Filter element filtering precision be 0.25~0.5 micron.
5. small molecule notoginseng polysaccharide extract obtained in the preparation method of the small molecule notoginseng polysaccharide extract described in claim 1 Application in treatment immunological disorder drug is prepared.
CN201611189464.7A 2016-12-21 2016-12-21 A kind of preparation method and application of small molecule notoginseng polysaccharide extract Pending CN106749731A (en)

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CN108250320A (en) * 2018-03-09 2018-07-06 杨凌萃健生物工程技术有限公司 A kind of low ash content ganoderan extract and preparation method thereof
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CN110092845A (en) * 2019-05-07 2019-08-06 山东大学 A kind of low molecular weight Sepia polysaccharide and preparation method and application
CN110105459A (en) * 2019-05-27 2019-08-09 云南绿戎生物产业开发股份有限公司 A kind of extracting method of notoginseng polysaccharide
CN110150286A (en) * 2019-05-27 2019-08-23 云南绿戎生物产业开发股份有限公司 A kind of pesticide containing notoginseng polysaccharide
CN114031694A (en) * 2021-12-23 2022-02-11 云南三七科技有限公司 Extraction method of pseudo-ginseng polysaccharide
CN114031694B (en) * 2021-12-23 2023-02-03 云南三七科技有限公司 Extraction method of pseudo-ginseng polysaccharide

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