CN107050044A - Medical application of the grey meat Russula polysaccharide in systemic lupus erythematosus - Google Patents

Abstract

There is regulatory T-cell subgroup activity the invention discloses the grey meat Russula polysaccharide PRG1 2 of medical application of the grey meat Russula polysaccharide in systemic lupus erythematosus, IL 10, ds DNA antibodies and Urine proteins are reduced, it is evident in efficacy, it is safe and reliable.The present invention for ash meat Russula polysaccharide PRG1 2 in preparation system lupus erythematosus application, with wide potential applicability in clinical practice.

Description

Medical application of the grey meat Russula polysaccharide in systemic lupus erythematosus

Technical field

The invention discloses medical application of the grey meat Russula polysaccharide in systemic lupus erythematosus, there is provided ash is flesh-coloured The new medical application of mushroom polysaccharide, belongs to pharmaceutical technology field.

Background technology

Systemic loupus erythematosus（Systemic Lupus Erythematosus, SLE）It is a kind of common itself of clinic Immunity disease, is produced, complement activation and immune complex deposit is major pathologic features using autoantibody.The master of SLE morbidities It is exactly that autoimmune tolerance is abnormal to want feature, therefore immunology factor is paid attention to extensively.Research shows, patient's SLE peripheral blood CD3 + CD4+T cells decline, and CD3+CD8+ rises, the up-regulation of IL-10 levels.IL-10 has immunostimulation and the dual work of immunosupress With.There are some researches show IL-10 stimulating system patients with SLE PBMCs is produced in Anti-hCG action, SLE patient's body Level is all significantly higher than healthy population in active stage and paracmasis.The Chinese SLE incidence of disease is 0.31%-0.70%, the course of disease It is long, delay repeatedly, disability rate is high, harm to the human body is very big.Because the SLE causes of disease and pathogenesis are still not clear, thus it is right at present SLE treatment not fully effective method, can only clinical remission can not cure.At present, treatment SLE main method is sharp Plain therapy, but Long-term Hormone side effect is big, and hormone subtracts and stops most likely resulting in aggravation.Therefore, one is found Planting new natural drug treatment SLE has very important realistic meaning and application prospect.

The flesh-coloured mushroom of ash（Russula griseocarnosa）It was registered as newly in China first by Wang Xianghua in 2009 Kind, and grey flesh-coloured mushroom is named as first.Originally the flesh-coloured mushroom of ash is mistaken as Russula vinosa（Russula vinosa）, there is no at present People isolates strain.Russula Basidiomycotina, Hymenomycetes, russule mesh, Russulaceae, Russula is a class macro fungi." this Careless detailed outline " is contained, and russule taste is clear, warm-natured, appetizing, antidiarrheal, removing toxic substances, nourishing, informal dress lengthen one's life." legendary god of farming's book on Chinese herbal medicine " is recorded, red Mushroom can be used for treatment eyes to fail to understand, can purging the liver of pathogenic fire through fire, radiating has a respite.Modern science and technology research also indicates that grey flesh-coloured mushroom has There are multiple pharmacological effect such as antibacterial action, antioxidation, improve anaemia, clearing damp strengthens immunity of organisms, prevents heart disease, Hypoglycemic, blood fat and anti-cancer function.

At present, China receives significant attention on the research of edible and medical fungi polysaccharide, due to its efficient, natural, nontoxic secondary work Wide research and application prospect are shown with feature.Vast territory and abundant resources for China, and a large amount of edible and medical fungis are urgently developed.Had no through retrieval Medical application file report of the grey meat Russula polysaccharide in systemic lupus erythematosus disease.

The content of the invention

It is flesh-coloured with ash the invention discloses a kind of medical application of grey meat Russula polysaccharide in systemic lupus erythematosus Mushroom dry product raw material, a kind of grey meat Russula polysaccharide is obtained using special extracting method and effective way of purification（Referred to as： PRG1- 2）, the medicine for preparing systemic lupus erythematosus.

It is an object of the invention to provide a kind of preparation method of grey meat Russula polysaccharide, comprise the following steps：

1）Crush grey flesh-coloured massee fruiting bodies：

Wild grey flesh-coloured massee fruiting bodies are dried to quality and no longer changed, are crushed using crusher, and carry out 100 mesh sieve mistakes Filter, sealing preserve is used to extract polysaccharide；

2）Water extraction extracts Thick many candies：

Ultra-pure water is added to grey flesh-coloured massee fruiting bodies powder, is extracted in high temperature bath after a period of time, leaching liquor is filtered with double-deck Paper is filtered, and gained filtrate is leaching liquor, and leaching liquor is concentrated into the 1/20 of initial volume using Rotary Evaporators；It is preferred that, weigh 40 g steps 1）Middle obtained powder；It is preferable in 90 DEG C of water-baths and extracts；It is preferred that, extract 3 h；It is preferred that, leaching liquor is concentrated into 50 mL；

3）Alcohol precipitation：

The absolute ethyl alcohol of 4 times of volume of concentrate is added into concentrate and is fully mixed, stands overnight, retains under the conditions of 4 DEG C Precipitation, and it is abundant using the washing of 75 % ethanol, precipitation is lyophilized standby；It is preferred that, 4 DEG C of standing 12h;

4）Sevage method deproteinizeds：

By step 3）Gained sediment is dissolved using ultra-pure water, is separately added into isometric n-butanol：Chloroform（1:4）Mixed solution Afterwards, centrifuge, retain supernatant, altogether in triplicate；It is preferred that, 2000 g centrifugations 20min；

5）Dialysis：

By step 4）After gained polysaccharide ultra-pure water dissolves, after being dialysed in ultra-pure water, freeze；

6）Alcohol precipitation：

By step 5）Polysaccharide sample is dissolved with ultra-pure water again, centrifuging and taking supernatant, then with the ethanol alcohol precipitation of final concentration 80%, centrifugation is gone Supernatant, precipitation is lyophilized standby;It is preferred that, 5000rpm is used after ultra-pure water dissolving, 10 min is centrifuged, takes supernatant；It is preferred that, After 80% ethanol alcohol precipitation, using 5000rpm, 10 min are centrifuged；

7）Cation exchange chromatography：

By 6）Alcohol precipitation sample dissolves the Thick many candies for obtaining grey flesh-coloured massee fruiting bodies using ultra-pure water, using ultrapure water balance DEAE- 52 posts, the slow loading of Thick many candies solution is first eluted with water, neutral sugar is got rid of, then be respectively adopted containing 0.1,0.2,0.3 The M NaCl aqueous solution elutes pillar, collects the sample under the elution of the 0.2 M NaCl aqueous solution；

8）Sieve chromatography is purified：

Using 0.2M NaCl aqueous equilibrium molecular sieves, the polysaccharide sample under being eluted containing the 0.2 M NaCl aqueous solution is carried out Sephadex G100 molecular sieve column chromatographies, and eluted using the 0.2 M NaCl aqueous solution, first eluting peak is collected, i.e., Obtain PRG1-2.

The positive effect of the present invention is：

There is provided grey meat Russula polysaccharide in the new medical application of systemic lupus erythematosus disease, by animal model test, Grey meat Russula polysaccharide has regulatory T-cell subgroup activity, IL-10, ds-DNA antibody and Urine proteins effect is reduced, for systematicness The treatment of lupus erythematosus has evident in efficacy, safe and reliable, the features such as toxic side effect is small, be the treatment of systemic loupus erythematosus again There is provided a kind of new medicine.

Brief description of the drawings

Fig. 1 is the flesh-coloured mushroom Thick many candies of DEAE chromatographies of the present invention ash；

Fig. 2 is that sieve chromatography of the present invention separates grey meat Russula polysaccharide PRG1.

Embodiment

The embodiment of the present invention is used for that the composition of the present invention is further described, rather than to the present invention's Limitation.

Embodiment 1

The preparation method of the flesh-coloured massee fruiting bodies Thick many candies of ash, is followed the steps below successively：

1）The grey flesh-coloured massee fruiting bodies for plucking fresh are dried to quality and no longer changed, moisture removal is thoroughly removed；

2）Using the fructification of crusher crushing drying, cross 100 mesh sieves and remove larger particles；

3）The flesh-coloured massee fruiting bodies powder of 40 g ashes is weighed, 1L ultra-pure waters is added, 3 h is extracted in 90 DEG C of water-bath.By leaching liquor Filtered with double-layer filter paper, gained filtrate is leaching liquor, and leaching liquor is concentrated into 50 mL using Rotary Evaporators；

4）The absolute ethyl alcohol of 4 times of volume of concentrate is slowly added into concentration leaching liquor and is fully mixed,

40 DEG C of standing 12-16 h, retain precipitation, and use 75 % ethanol to be freezed after fully washing；

5）Sediment is resuspended with 10 mL ultra-pure waters and dissolved, with 10 mL n-butanols, chloroform mixed liquor（N-butanol：Chloroform=4:1） Under mixing, normal temperature, fully shaking, 3000 g centrifuge 20 min, take upper strata aqueous phase, this step is repeated 3 times；

6）By step（5）After aqueous sample ultra-pure water dissolves, after being dialysed in ultra-pure water, freeze；

7）By step（6）Use ultra-pure water dissolves polysaccharide sample again, and 5000rpm, 10 min of centrifugation take supernatant, then dense with end 80% ethanol alcohol precipitation is spent, 5000rpm, 10 min of centrifugation remove supernatant, and precipitation is lyophilized standby.

Embodiment 2

The flesh-coloured plant mushroom fruit body polysaccharide PRG1-2 of ash isolation and purification method, is followed the steps below successively：

1）Ion-exchange chromatography：Grey flesh-coloured mushroom Thick many candies freeze-dried powder, 0.22 μM of membrane filtration are resuspended using ultra-pure water.Adopt in advance With ultrapure water balance DEAE-52 posts, by Thick many candies solution with the slow upper prop of 1mL/min speed, the milli-Q water of 2 times of column volumes Pillar, then be respectively adopted containing 0.1M NaCl, 0.2 M NaCl and 0.3M the NaCl aqueous solution is washed with 1.0mL/min flow velocity De- pillar, and all polysaccharide samples eluted are collected, 0.2 M NaCl are eluted into lower sample P RG1 carries out next step purifying；

2）The PRG1 of previous step is carried out into molecular sieve to isolate and purify.Use first and contain 0.2 M NaCl aqueous equilibriums Sephadex G100, PRG1 loadings are eluted with 1 mL/min flow velocitys, and it is PRG1-2 to collect second eluting peak.

Further prove grey meat Russula polysaccharide in the new medical of systemic lupus erythematosus disease by following zoopery Purposes：

Influences of the flesh-coloured massee fruiting bodies purified polysaccharide PRG1-2 of ash to systemic loupus erythematosus MRL/lpr mouse：

1st, 40 8 week old SLE female MRL/lpr mouse are randomly divided into following 4 groups, every group 10, daily gastric infusion connects Continuous administration 3 weeks；

2nd, mice serum IL-10 and ds-DNA level is determined：

Using IL-10 mouse ELISA kit (Invitrogen) and Mouse dsDNA ELISA Kit（Creative Diagnostics）IL-10 and ds-DNA antibody levels in each group mice serum after measure is administered 3 weeks；

3rd, mouse boosting cell T cell subgroup (CD3 is determined⁺CD4⁺, CD3⁺CD8⁺)：

Splitting erythrocyte processing is carried out using Mechanical Method separating mouse splenocyte, and to it, will using 2% FBS PBS Cell concentration is adjusted to 2 × 10⁶Cells/mL, every mouse prepares two sample cells, and often the μ L of pipe 100, are added by following form Enter streaming antibody（BD Biosciences）.Room temperature lucifuge is incubated 30min, using the FBS of 1mL 2% PBS（500g, 5min）Washing, and be resuspended using the FBS of 500 μ L 2% PBS, for flow cytometer detection；

4）Mouse urine protein level is determined：

Using Urine proteins detection kit（Beijing Strong Biotechnologies, Inc.）Determine each group mouse retention after being administered 3 weeks Protein level；

5）Experimental result：

Compared with SLE control mice groups（Table 1）, PRG1-2 administration groups can significantly reduce IL-10, ds-DNA antibody and Urine proteins Level, and with the increase of PRG1-2 dosage, IL-10, ds-DNA antibody and urine protein level reduction are more notable, this explanation present invention It is notable for SLE mouse therapeutic effects；

Influences of the PRG1-2 of table 1 to SLE mice serums IL-10, ds-DNA and urine protein level：

In addition, T cell subgroup analysis result shows, compared with SLE control mice groups（Table 2）, after gastric infusion PRG1-2, give The CD3 of medicine group mouse⁺CD4⁺Subset proportions rise in dose dependent fashion, and the CD3 of administration group mouse⁺CD8⁺Subset proportions are in Dose dependent fashion declines, with the effect for correcting SLE mouse T cell subset proportions imbalance states.

Influences of the PRG1-2 of table 2 to SLE mice serums IL-10, ds-DNA and urine protein level

Conclusion, PRG1-2 has reduction SLE mouse IL-10, ds-DNA, a Urine proteins effect, and it is unbalance to correct T cell subgroup, Illustrate that the PRG1-2 prepared by the present invention treats SLE mouse effect significantly, the medicine available for systemic lupus erythematosus In application.

Claims (2)

1. a kind of application of grey meat Russula polysaccharide in the medicine of systemic lupus erythematosus is prepared.

2. a kind of preparation method of grey meat Russula polysaccharide, comprises the following steps：

1）Crush grey flesh-coloured massee fruiting bodies：

Wild grey flesh-coloured massee fruiting bodies are dried to quality and no longer changed, are crushed using crusher, and carry out 100 mesh sieve mistakes Filter, sealing preserve is used to extract polysaccharide；

2）Water extraction extracts Thick many candies：

Ultra-pure water is added to grey flesh-coloured massee fruiting bodies powder, is extracted in high temperature bath after a period of time, leaching liquor is filtered with double-deck Paper is filtered, and gained filtrate is leaching liquor, and leaching liquor is concentrated into the 1/20 of initial volume using Rotary Evaporators；It is preferred that, weigh 40 g steps 1）Middle obtained powder；It is preferable in 90 DEG C of water-baths and extracts；It is preferred that, extract 3 h；It is preferred that, leaching liquor is concentrated into 50 mL；

3）Alcohol precipitation：

The absolute ethyl alcohol of 4 times of volume of concentrate is added into concentrate and is fully mixed, stands overnight, retains under the conditions of 4 DEG C Precipitation, and it is abundant using the washing of 75 % ethanol, precipitation is lyophilized standby；It is preferred that, 4 DEG C of standing 12h;

4）Sevage method deproteinizeds：

By step 3）Gained sediment is dissolved using ultra-pure water, is separately added into isometric n-butanol：Chloroform（1:4）Mixed solution Afterwards, centrifuge, retain supernatant, altogether in triplicate；It is preferred that, 2000 g centrifugations 20min；

5）Dialysis：

By step 4）After gained polysaccharide ultra-pure water dissolves, after being dialysed in ultra-pure water, freeze；

6）Alcohol precipitation：

By step 5）Polysaccharide sample is dissolved with ultra-pure water again, centrifuging and taking supernatant, then with the ethanol alcohol precipitation of final concentration 80%, centrifugation is gone Supernatant, precipitation is lyophilized standby;It is preferred that, 5000rpm is used after ultra-pure water dissolving, 10 min is centrifuged, takes supernatant；It is preferred that, After 80% ethanol alcohol precipitation, using 5000rpm, 10 min are centrifuged；

7）Cation exchange chromatography：

By 6）Alcohol precipitation sample dissolves the Thick many candies for obtaining grey flesh-coloured massee fruiting bodies using ultra-pure water, using ultrapure water balance DEAE- 52 posts, the slow loading of Thick many candies solution is first eluted with water, neutral sugar is got rid of, then be respectively adopted containing 0.1,0.2,0.3 The M NaCl aqueous solution elutes pillar, collects the sample under the elution of the 0.2 M NaCl aqueous solution；

8）Sieve chromatography is purified：

Using 0.2M NaCl aqueous equilibrium molecular sieves, the polysaccharide sample under being eluted containing the 0.2 M NaCl aqueous solution is carried out Sephadex G100 molecular sieve column chromatographies, and eluted using the 0.2 M NaCl aqueous solution, first eluting peak is collected, i.e., Obtain PRG1-2.