阅读说明：本技术 一种具有预防和治疗炎症的生物多糖及其应用 (Biological polysaccharide for preventing and treating inflammation and application thereof ) 是由 郭宏亮 庄秀园 郭瑞 林檬 王轩 张雯 叶榛 朱勤健 娄海霞 于 2018-05-23 设计创作，主要内容包括：本发明提供了一种具有预防和治疗炎症的生物多糖及其应用。具体地,β-葡聚糖,尤其是裂褶菌β-葡聚糖,是一种预防和/或治疗炎症的功效成分,一方面是β-葡聚糖对巨噬细胞具有免疫激活作用,增强抵御能力,从而有效预防外界病原微生物的侵染；另一方面,对于炎症,β-葡聚糖可直接作用于免疫细胞,通过调节炎性细胞因子的分泌水平等方式,防止炎症过度而造成损伤。(The invention provides a biological polysaccharide for preventing and treating inflammation and application thereof. Specifically, the beta-glucan, especially the Schizophyllum commune beta-glucan, is an effective component for preventing and/or treating inflammation, on one hand, the beta-glucan has an immune activation effect on macrophages and enhances the resistance capability, so that the infection of external pathogenic microorganisms is effectively prevented; on the other hand, for inflammation, beta-glucan can directly act on immune cells, and prevent injury caused by excessive inflammation by means of regulating secretion level of inflammatory cytokines and the like.)

1. a kind of purposes of beta glucan, which is characterized in that be used to prepare a preparation or composition, the preparation or composition are used In (a) immune cell activated；And/or (b) promote immune cell propagation.

2. purposes as described in claim 1, which is characterized in that the immunocyte is macrophage.

3. purposes as described in claim 1, which is characterized in that the preparation or composition are in immune cell activated MAPK signal path and/or NF- κ B signal access.

4. purposes as described in claim 1, which is characterized in that the beta glucan includes schizophyllum commune beta glucan.

5. purposes as described in claim 1, which is characterized in that the preparation or composition are also used to (a) and promote described be immunized Cell secretes NO, and/or (b) promotes or raise the iNOS expression in the immunocyte.

6. a kind of purposes of beta glucan, which is characterized in that be used to prepare the promotor of cell factor, or be used to prepare a preparation Or composition, the preparation or composition for promoting or raising the cytokine-expressing in cell, wherein the cell because Son is the immunity-associated cell factor.

7. a kind of ion vitro immunization immune cell activated and/or the method for promoting immune cell propagation, which is characterized in that the method Comprising steps of immunocyte is cultivated in the presence of beta glucan, thus immune cell activated and/or promotion immune cell propagation.

8. a kind of purposes of beta glucan, which is characterized in that be used to prepare a preparation or composition, the preparation or composition are used In

(a) promote the inflammatory reaction of individual, wherein the individual is no inflammation or without excessive inflammation；And/or

(b) prevent and/or treat excessive inflammation or inflammatory disease.

9. purposes as claimed in claim 8, which is characterized in that the inflammation has one or more features selected from the group below:

(a) macrophage abnormal secretion NO；

(b) in macrophage the immunity-associated cell factor unconventionality expression；And/or

(c) abnormal activation of the MAPK signal path in macrophage and/or NF- κ B signal access.

10. a kind of purposes of beta glucan, which is characterized in that be used to prepare a preparation or composition, the preparation or composition For in excessive inflammation:

(a) inhibit macrophage abnormal secretion NO；

(b) inhibit the unconventionality expression of the immunity-associated cell factor in macrophage；And/or

(c) inhibit the abnormal activation of the MAPK signal path and/or NF- κ B signal access in macrophage.

Technical field

The present invention relates to field of biotechnology more particularly to it is a kind of with prevent and treat inflammation biological polyoses and its answer With.

Background technique

Inflammation, being exactly is a kind of defense reaction of the body for stimulation usually " inflammation " described in people, show as it is red, Swollen, heat, pain and dysfunction.Inflammation can be infective inflammation caused by infection, non-caused by may not be due to infection Infective inflammation.Under normal conditions, inflammation is beneficial, is the automatic defense reaction of human body, but sometimes, inflammation Harmful, for example, to the attack of human body autologous tissue, occur in inflammation of hyaline tissue etc..

Macrophage is a kind of white blood cell in tissue, is originated from monocyte, and monocyte derives from marrow In precursor.Macrophage and monocyte are all phagocyte, and non-specific defence is participated in vertebrate (first Nature are immune) and specificity defence (cellular immunity).Their major function is in the form of fixing cell or free cell pair Cell debris and pathogen carry out phagocytosis (swallow and digest), and activate lymph corpuscle or other immunocytes, enable it It reacts to pathogen.

There is an urgent need in the art to develop a kind of drug that can be effectively prevented and treated inflammation.

Summary of the invention

The object of the present invention is to provide it is a kind of can effectively prevention and or treatment inflammation drug.

It is a further object of the present invention to provide a kind of with the biological polyoses for preventing and treating inflammation and its application.

The first aspect of the present invention provides a kind of purposes of beta glucan, is used to prepare a preparation or composition, described Preparation or composition are used for (a) immune cell activated；And/or (b) promote immune cell propagation.

In another preferred example, the immune cell activated includes activating macrophage, and it is thin to be preferably comprised stimulation macrophage Born of the same parents' immune activation.

In another preferred example, the immunocyte is macrophage.

In another preferred example, the preparation or composition in immune cell activated MAPK signal path and/or NF- κ B signal access.

In another preferred example, the immunocyte is RAW264.7 macrophage.

In another preferred example, the immune cell activated MAPK signal path includes promoting in MAPK signal path The phosphorylation of ERK, p38 and/or JNK.

In another preferred example, the immune cell activated NF- κ B signal access includes promoting IKK β and/or p65 albumen Phosphorylation.

In another preferred example, the beta glucan is callose.

In another preferred example, the beta glucan is β -1,3- glucan, it is preferable that there is β -1, the β -1 of 6- branch, 3- glucan.

In another preferred example, the structure of the beta glucan is shown in formula I,

Wherein, l is the integer of 0-50, preferably 0-10, more preferably 0-3, more preferably 1-2, more preferably 1；M be >=0 it is whole Number, it is more preferably 0 that preferably 0-19, preferably 0-4, which are more preferably 0-1,；The integer that n is >=3, preferably 30- 60000, more preferably 100-10000.

In another preferred example, the degree of branching (DB) of the beta glucan is 0.02-0.8, preferably 0.1-0.5, preferably Ground 0.25-0.4.

In another preferred example, par≤20 of the monosaccharide unit of the side chain of the beta glucan, preferably≤5, It preferably≤3, more preferably≤1.5, is more preferably 1.

In another preferred example, the beta glucan includes the beta glucan with three screw stereo structures.

In another preferred example, the content of the beta glucan of the three screw stereos structure is 80%, 90%, 95%, is pressed The total moles meter of beta glucan.

In another preferred example, β -1 of the beta glucan, 3- main chain are the main body of three screw stereo structures.

In another preferred example, β -1 of the beta glucan, 6- branch are located at the outside of three screw stereo structures.

In another preferred example, molecular weight >=2kD of the beta glucan, preferably 2kD-40000kD, more preferably 20kD-20000kD。

In another preferred example, the molecular weight of the beta glucan can be 5kD-35000kD；10kD-30000kD； 50kD-25000kD；100kD-20000kD；200kD-18000kD；400kD-16000kD；500kD-14000kD；1000kD- 12000kD；2000kD-4000kD；3000kD-5000kD；4000kD-6000kD；5000kD-7000kD；6000kD- 8000kD；7000kD-9000kD；Or 8000kD-10000kD.

In another preferred example, the beta glucan is selected from the group: schizophyllum commune beta glucan, β-lentinan, small nut Bacterium beta glucan, grifola frondosa beta-glucose, polysaccharide of picking up the ears, mushroom beta glucan, yeast beta-dextran, avenabeta glucosan or A combination thereof.

In another preferred example, the beta glucan is schizophyllum commune beta glucan.

In another preferred example, the β-lentinan is every 5 β -1, and the main chain of 3- has 2 β -1, point of 6- Branch, and the beta glucan of 1 glucose residue of each branch.

In another preferred example, purity >=70% of the beta glucan, preferably >=90%, more preferably >=95%, more Goodly >=99%.

In another preferred example, the beta glucan is with good stability.

In another preferred example, the beta glucan is in solid-state form or liquid form, such as beta glucan solid particle or Powder or beta glucan aqueous solution.

In another preferred example, partial size≤20mm of the beta glucan particle or powder, preferably 0.001-10mm, more Good ground 0.01-5mm, more preferably 0.1-2mm.

In another preferred example, the beta glucan is completely water-soluble beta glucan.

In another preferred example, the beta glucan (particle or powder) has good water-soluble and/or natural soluble Property.

In another preferred example, solubility of the beta glucan (particle or powder) in 25 DEG C of water (100g) >= 0.0001g, preferably 0.01-50g, more preferably 0.1-10g.

In another preferred example, solubility of the beta glucan (particle or powder) in 25 DEG C of water (100g) can be 0.1-100g；0.2-90g；0.5-80g；1-50g；Alternatively, the solubility can be 0.1-0.3g；0.2-0.4g；0.3- 0.5g；0.4-0.6g；0.5-0.7g；0.6-0.8g；0.7-0.9g；0.8-1g；Or 1-3g；2-4g；3-5g；4-6g；5-7g； 6-8g；7-9g；8-10g.

In another preferred example, the beta glucan solution is the solution of beta glucan in water, i.e. beta glucan is water-soluble Liquid.

In another preferred example, beta glucan (water) solution viscosity is big；Preferably, the β-that mass concentration is 0.5% Glucan aqueous solution (at 25 DEG C) viscosity >=40mPas, more preferably 100-10000mPas, more preferably 500-2000mPa s。

In another preferred example, beta glucan aqueous solution (25 DEG C) viscosity that the mass concentration is 0.5% can be 50- 10000mPa·s；100-9000mPa·s；200-8000mPa·s；300-7000mPa·s；400-6000mPa·s；450- 5000mPa·s；500-5000mPa·s；550-4000mPa·s；600-3000mPa·s；650-2000mPa·s；700- 1500mPa·s。

In another preferred example, the aqueous solution for the beta glucan that the mass concentration is 1% has high clarity or high saturating Light rate, light transmittance >=50% for the beta glucan aqueous solution that the mass concentration is 1%, preferably >=80%, preferably >= 85%, more preferably >=95%；

In another preferred example, the beta glucan solution is with good stability.

In another preferred example, the preparation or composition contain (a) beta glucan；And optional (b) pharmaceutically, food In conduct and learning, on cosmeceutical or the acceptable carrier of weapon exercises or excipient.

In another preferred example, the preparation or composition contain (a) schizophyllum commune beta glucan；And optional (b) medicine On, in bromatology, on cosmeceutical or the acceptable carrier of weapon exercises or excipient.

In another preferred example, the preparation or composition contain 0.0001-99wt%, preferably 0.001-90wt%, More preferably 0.01-50wt%, the more preferably beta glucan of 0.05-10wt%, by the total weight of preparation or composition.

In another preferred example, mass concentration of the beta glucan in the preparation or composition is >=1 μ g/mL, It is specifically as follows 1 μ g/mL-200mg/mL perhaps 1 μ g/mL-5mg/mL or 1 μ g/mL-1mg/mL.

In another preferred example, the preparation or composition are also used to enhance cutaneous immunisation power or Initiative Defense function.

In another preferred example, the preparation or composition be also used to prevent and/or treat skin and mucosa inflammation or other Inflammatory disease of the skin.

In another preferred example, the preparation or composition are also used to prevent and/or treat skin problem selected from the group below: Drying, red capillary, allergy, inflammation, microgroove, color spot, it is fuel-displaced, or combinations thereof.

In another preferred example, the dosage form of the composition or preparation is solid dosage forms, semisolid dosage form or liquid agent Type, such as solution, gel, cream, lotion.

In another preferred example, the composition is pharmaceutical composition or cosmetic composition, preferably external drug Dosage form.

In another preferred example, the preparation is external preparation or preparation capable of permeating skin (such as externally used solution agent, ointment, patch Deng).

In another preferred example, the preparation or composition include cosmetics, food, medical instrument or drug.

In another preferred example, the preparation or composition are also used to (a) promotion immunocyte secretion NO, and/or (b) promote or raise the iNOS expression in the immunocyte.

In another preferred example, the immunocyte is macrophage.

In another preferred example, the promotion macrophages secrete NO has platform effect.

In another preferred example, described " platform effect " refers to that preferably β-Portugal is poly- when beta glucan is more than a certain concentration When sugared concentration >=30 μ g/mL, more preferably 40-500 μ g/mL, more preferably 50-300 μ g/mL promote macrophages secrete NO effect Specific level can be maintained.

In another preferred example, the preparation or composition are for promoting macrophages secrete NO level to reach appropriate dense Degree, the appropriate finger will not cause inflammation, immunologic derangement or generate murder by poisoning to normal cell, preferably≤5000 μ g/mL, more preferably Ground 1-1000 μ g/mL.

The second aspect of the present invention provides a kind of purposes of beta glucan, is used to prepare the promotor of cell factor, or It is used to prepare a preparation or composition, the preparation or composition are used to promoting or raising the cytokine-expressing in cell, Described in cell factor be the immunity-associated cell factor.

In another preferred example, the beta glucan is selected from the group: schizophyllum commune beta glucan, β-lentinan, small nut Bacterium beta glucan, grifola frondosa beta-glucose, polysaccharide of picking up the ears, mushroom beta glucan, yeast beta-dextran, avenabeta glucosan or A combination thereof.

In another preferred example, the beta glucan is schizophyllum commune beta glucan.

In another preferred example, the beta glucan is completely water-soluble beta glucan.

In another preferred example, the beta glucan, which has, is selected from the group one or more features:

(1) purity >=70% of the beta glucan, preferably >=90%, more preferably >=95%, more preferably >=99%；

(2) beta glucan has good water-soluble, solubility and/or natural soluble；

(3) solubility >=0.0001g/100g water of the beta glucan (solid particle or powder) in 25 DEG C of water, compared with Good ground 0.01-50g/100g water, more preferably 0.1g-10g/100g water；

(4) aqueous solution of the beta glucan has high clarity or high transparency；Preferably, the mass concentration is Light transmittance >=50% of 1% beta glucan aqueous solution, preferably >=80%, preferably >=85%, more preferably >=95%；

(5) the beta glucan solution viscosity is big；Preferably, mass concentration is (25 DEG C of beta glucan aqueous solution of 0.5% When) viscosity >=40mPas, more preferably 100-10000, more preferably 600-2000mPas；

(6) aqueous solution of the beta glucan is with good stability；And/or

(7) molecular weight >=2kD of the beta glucan, preferably 2kD-40000kD, more preferably 20kD-20000kD.

In another preferred example, the cell factor comes from mammal.

In another preferred example, the mammal includes people and non-human mammal, is preferably comprised rodent (such as Mouse, rat), Primate (such as people).

In another preferred example, the cell factor includes the cell factor of immunocyte secretion or expression.

In another preferred example, the cell factor includes the cell factor of macrophages secrete or expression.

In another preferred example, the cell factor is selected from the group: TNF-α, IL-1 β, IL-6, IL-10, chemotactic factor (CF) MCP-1, CXCL10, or combinations thereof.

In another preferred example, the promotor includes the expression for promoting the cell factor, or improve the cell because The expression quantity of son.

The third aspect of the present invention provides a kind of ion vitro immunization immune cell activated and/or promotes immune cell propagation Method, the method includes the steps: in the presence of beta glucan, cultivate immunocyte, thus immune cell activated and/or rush Into immune cell propagation.

In another preferred example, the immunocyte is macrophage.

In another preferred example, when the concentration of the beta glucan >=30 μ g/mL, more preferably 40-500 μ g/mL, more preferably 50-200μg/mL。

In another preferred example, the method is non-therapeutic and nondiagnostic.

The fourth aspect of the present invention provides a kind of purposes of beta glucan, is used to prepare a preparation or composition, described Preparation or composition are used for

(a) promote the inflammatory reaction of individual and cope with disease, wherein the individual is no inflammation or without excessive inflammation； And/or (b) prevent and/or treat excessive inflammation or inflammatory disease.

In another preferred example, the beta glucan is selected from the group: schizophyllum commune beta glucan, β-lentinan, small nut Bacterium beta glucan, grifola frondosa beta-glucose, polysaccharide of picking up the ears, mushroom beta glucan, yeast beta-dextran, avenabeta glucosan or A combination thereof.

In another preferred example, the beta glucan is schizophyllum commune beta glucan.

In another preferred example, the beta glucan is completely water-soluble beta glucan.

In another preferred example, the beta glucan, which has, is selected from the group one or more features:

(1) purity >=70% of the beta glucan, preferably >=90%, more preferably >=95%, more preferably >=99%；

(2) beta glucan has good water-soluble, solubility and/or natural soluble；

(3) solubility >=0.0001g/100g water of the beta glucan (solid particle or powder) in 25 DEG C of water, compared with Good ground 0.01-50g/100g water, more preferably 0.1g-10g/100g water；

(4) aqueous solution of the beta glucan has high clarity or high transparency；Preferably, the mass concentration is Light transmittance >=50% of 1% beta glucan aqueous solution, preferably >=80%, preferably >=85%, more preferably >=95%；

(5) the beta glucan solution viscosity is big；Preferably, mass concentration is (25 DEG C of beta glucan aqueous solution of 0.5% When) viscosity >=40mPas, more preferably 100-10000, more preferably 600-2000mPas；

(6) aqueous solution of the beta glucan is with good stability；And/or

(7) molecular weight >=2kD of the beta glucan, preferably 2kD-40000kD, more preferably 20kD-20000kD.

In another preferred example, the individual includes people and non-human mammal.

In another preferred example, the preparation or composition are used for prevention of inflammation or inflammatory disease.

In another preferred example, the inflammation or inflammatory disease include the inflammation as caused by lipopolysaccharides or inflammatory disease.

In another preferred example, the inflammation includes skin (mucous membrane) inflammation or other inflammatory disease of the skin.

In another preferred example, the preparation or composition are excessive for anti-inflammatory, mitigation inflammatory reaction or prevention or treatment Immune (excessive inflammation).

In another preferred example, the inflammation has one or more features selected from the group below:

(a) macrophage abnormal secretion NO；

(b) in macrophage the immunity-associated cell factor unconventionality expression；And/or

(c) abnormal activation of the MAPK signal path in macrophage and/or NF- κ B signal access.

In another preferred example, the abnormal secretion NO includes secreting the diacrisis of excessive NO or NO to increase (or sharply Increase), specifically the secretion of NO is higher than normal level.

In another preferred example, the unconventionality expression of the cell factor includes the cell factor height expression or is higher than normal Horizontal (or mRNA expression of cytokines horizontal abnormality improves or is higher than normal level).

In another preferred example, the cell factor include TNF-α, IL-1 β, or combinations thereof.

In another preferred example, the abnormal activation of the MAPK signal path includes (exception) phosphoric acid of ERK and/or p38 Change.

In another preferred example, the abnormal activation of the NF- κ B signal access includes the (different of IKK β and/or p65 albumen Often) phosphorylation.

The fifth aspect of the present invention provides a kind of purposes of beta glucan, is used to prepare a preparation or composition, described Preparation or composition are used in excessive inflammation:

(a) inhibit macrophage abnormal secretion NO；

(b) inhibit the unconventionality expression of the immunity-associated cell factor in macrophage；And/or

(c) inhibit the abnormal activation of the MAPK signal path and/or NF- κ B signal access in macrophage.

In another preferred example, the cell factor include TNF-α, IL-1 β, or combinations thereof.

In another preferred example, the abnormal secretion, unconventionality expression or abnormal activation are as caused by lipopolysaccharides.

In another preferred example, it is described inhibition be before excessive inflammation, among, and/or later.

In another preferred example, described to inhibit to include prevention and/or treatment.

In another preferred example, the preparation or composition are used to inhibit or reduce (sharply) of macrophage NO level Increase.

In another preferred example, the preparation or composition are for inhibiting cell factor (TNF-α described in macrophage And/or IL-1 β) high expression, or reduce the expression of the cell factor.

In another preferred example, the preparation or composition are used to inhibit the phosphorylation of ERK and/or p38 in macrophage, Or reduce ERK and/or p38 phosphorylation level.

In another preferred example, the preparation or composition are used to inhibit the phosphorus of IKK β and/or p65 albumen in macrophage Acidification, or reduce IKK β and/or p65 protein phosphorylation level.

It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, In This no longer tires out one by one states.

Detailed description of the invention

Fig. 1 shows the Fourier transform infrared spectroscopy figure of the schizophyllum commune beta glucan prepared in embodiment 1.

Fig. 2 shows beta glucan stability contrast, and one bottle of the left side is commercially available 1.0% avenabeta glucosan solution, the right One bottle is 1.0% schizophyllum commune beta glucan prepared by the present invention.

Fig. 3 is that influence of the schizophyllum commune beta glucan to RAW264.7 macrophage survival rate (is handled with without beta glucan Blank control group compare, * p < 0.05, n=6).

Fig. 4 is that the schizophyllum commune beta glucan of various concentration expresses (A, n=3) to RAW264.7 macrophage iNOS mRNA With the influence (compared with blank control group, p < 0.01 * p < 0.05, * *) of NO release (C, n=4), wherein NO burst size is according to mark Directrix curve (B) is calculated.

Fig. 5 is the schizophyllum commune beta glucan of various concentration to RAW264.7 cytokine TNF-α (A), IL-1 β (B), IL-6 (C), IL-10 (D) and chemotactic factor (CF) MCP-1 (E), CXCL10 (F) mRNA expression influence (compared with blank control group, * p < 0.05, * p < 0.001 * p < 0.01, * * *；N=3).

Fig. 6 be the commercially available water dispersible yeast beta-dextran of various concentration to RAW264.7 cytokine TNF-α (A) and IL-1 β (B) mRNA expression influence (compared with blank control group, p < 0.05 *；N=3).

Fig. 7 is influence of the schizophyllum commune beta glucan of various concentration to RAW264.7 macrophage MAPK signal path.

Fig. 8 is influence of the schizophyllum commune beta glucan of various concentration to RAW264.7 macrophage NF- κ B signal access.

Fig. 9 is the influence that lipopolysaccharides generates RAW264.7 cell NO after the schizophyllum commune beta glucan of various concentration prevents (B, compared with blank control group, p < 0.01 * *；Compared with lipopolysaccharides stimulation group, ##p < 0.01；N=4), the amount of NO is according to mark Directrix curve (A) is calculated.

Figure 10 is after the schizophyllum commune beta glucan of various concentration prevents, and lipopolysaccharides is to RAW264.7 cytokine TNF-α (A) After influence and the prevention of commercially available soluble yeast ss-glucan with IL-1 β (C) mRNA expression, lipopolysaccharides to TNF-α (B) and IL-1 β (D) mRNA expression influence (compared with blank control group, p < 0.01 * *；Compared with lipopolysaccharides stimulation group, #p < 0.05, ##p < 0.01, ###p < 0.001；N=3).

Figure 11 is after the schizophyllum commune beta glucan of various concentration prevents, and lipopolysaccharides is to RAW264.7 macrophage MAPK signal The influence of access.

Figure 12 is after the schizophyllum commune beta glucan of various concentration prevents, and lipopolysaccharides believes RAW264.7 macrophage NF- κ B The influence of number access.

Figure 13 be various concentration schizophyllum commune beta glucan to lipopolysaccharides stimulation after RAW264.7 cytokine TNF-α (A), After the therapeutic effect of IL-1 β (C) and IL-6 (E) mRNA expression and commercial dispersants yeast beta-dextran stimulate lipopolysaccharides The therapeutic effect of RAW264.7 cytokine TNF-α (B), IL-1 β (D) and IL-6 (F) mRNA expression is (with blank control group phase Than p < 0.01 * p < 0.05, * *；Compared with lipopolysaccharides stimulation group, #p < 0.05, ##p < 0.01；N=3).

Specific embodiment

The present inventor after extensive and in-depth study, for the first time it was unexpectedly observed that beta glucan (preferably schizophyllum commune β-Portugal Glycan), especially there is natural soluble, high molecular weight, highly viscous schizophyllum commune beta glucan, there is immunostimulatory activity, And it being capable of effectively prevention and or treatment inflammation.Experiment shows that beta glucan being capable of immune activation macrophage, promotion Macrophage proliferation.Schizophyllum commune beta glucan can also treat or prevent inflammation or other inflammatory diseases simultaneously.On this basis, complete At the present invention.

The effect of beta glucan (preferably schizophyllum commune beta glucan) of the present invention is a kind of prevention and or treatment inflammation at Point, on the one hand it is that beta glucan has immunostimulation to macrophage, enhances resilience, thus the effectively extraneous disease of prevention Pathogenic microorganism infects, and have platform effect, will not overstimulation cause inflammation, immunologic derangement or to normal cell generate It poisons；On the other hand, for inflammation, beta glucan can be done directly on immunocyte, by point for adjusting inflammatory cytokine Bleeding Peer Mode prevents inflammation from excessively causing to damage.(stimulation is huge for beta glucan activated cell fundamental immunity function of the present invention Phagocyte activation and proliferation), while preventing and/or treating excessive inflammatory response, there is two-way immunoloregulation function, will not injure Patient's normal cell (such as skin) makes it generate drug resistance.

Term explanation

Unless otherwise defined, otherwise whole technologies used herein and scientific term all have such as fields of the present invention The normally understood identical meanings of those of ordinary skill.

As used herein, in use, term " about " means that the value can be from enumerating in mentioning the numerical value specifically enumerated Value changes not more than 1%.For example, as used herein, statement " about 100 " include 99 and 101 and between whole values (for example, 99.1,99.2,99.3,99.4 etc.).

As used herein, term " containing " or " including (including) " can be open, semi-enclosed and enclosed.It changes Yan Zhi, the term also include " substantially by ... constitute " or " by ... constitute ".

As used herein, the beta glucan that term " completely water-soluble " refers to the solid-state form, can be completely dissolved in water As beta glucan aqueous solution, i.e., solubility >=0.0001g of beta glucan in 25 DEG C of 100g water, preferably 0.01-50g, More preferably 0.1g-10g.

As used herein, term " natural soluble " refers to that beta glucan of native state itself is possessed, complete in water The property of fully dissolved formation aqueous solution." beta glucan of native state " refers to and produces to obtain (such as life using natural method Object fermentation) beta glucan, the beta glucan by any chemical modification and without by any physics and/ Or its long-chain molecule is interrupted and reduces its molecular mass by chemical and/or biological method.In another preferred example, of the invention Beta glucan is the beta glucan of native state.

Beta glucan

Beta glucan is a kind of natural polysaccharide, can find the beta glucan of suitable multiple types in the natural environment, usually It is present in the cell wall of the bacterium of Special Category, saccharomycete, fungi (ganoderma lucidum), also is present in the coating of higher plant seed In.There are mainly two types of the production methods of beta glucan, one is directly mentioning from the fructifications fungi such as the cereal such as oat or mushroom It takes；The second is processing is extracted via to fermentation liquid, by the liquid fermentation of fungi or bacterium to obtain beta glucan.

As used herein, " beta glucan of the present invention ", " biological polyoses of the present invention " are used interchangeably, and are primarily referred to as the present invention Beta glucan described in first aspect, the beta glucan are selected from the group: schizophyllum commune beta glucan, β-lentinan, small nut Bacterium beta glucan, grifola frondosa beta-glucose, polysaccharide of picking up the ears, mushroom beta glucan, yeast beta-dextran, avenabeta glucosan or A combination thereof；Preferably schizophyllum commune beta glucan.

As used herein, " schizophyllum commune beta glucan " refers to the beta glucan from schizophyllum commune.

In another preferred example, the structure of the beta glucan is shown in formula I.

In another preferred example, molecular weight >=2kD of the beta glucan, preferably 2kD-40000kD, more preferably 20kD-20000kD。

In another preferred example, the molecular weight of the beta glucan can be 5kD-35000kD；10kD-30000kD； 50kD-25000kD；100kD-20000kD；200kD-18000kD；400kD-16000kD；500kD-14000kD；1000kD- 12000kD；2000kD-4000kD；3000kD-5000kD；4000kD-6000kD；5000kD-7000kD；6000kD- 8000kD；7000kD-9000kD；Or 8000kD-10000kD.

In another preferred example, purity >=70% of the beta glucan, preferably >=90%, more preferably >=95%, more Goodly >=99%.

In another preferred example, the beta glucan is with good stability.

In another preferred example, the beta glucan is in solid-state form or liquid form, such as beta glucan solid particle or Powder or beta glucan aqueous solution.

In another preferred example, partial size≤20mm of the beta glucan particle or powder, preferably 0.001-10mm, more Good ground 0.01-5mm, more preferably 0.1-2mm.

In another preferred example, the beta glucan (particle or powder) has good water-soluble and/or natural soluble Property.

In another preferred example, solubility of the beta glucan (particle or powder) in 25 DEG C of water (100g) >= 0.0001g, preferably 0.01-50g, more preferably 0.1-10g.

In another preferred example, solubility of the beta glucan (particle or powder) in 25 DEG C of water (100g) can be 0.1-100g；0.2-90g；0.5-80g；1-50g；Alternatively, the solubility can be 0.1-0.3g；0.2-0.4g；0.3- 0.5g；0.4-0.6g；0.5-0.7g；0.6-0.8g；0.7-0.9g；0.8-1g；Or 1-3g；2-4g；3-5g；4-6g；5-7g； 6-8g；7-9g；8-10g.

In another preferred example, the beta glucan solution is the solution of beta glucan in water, i.e. beta glucan is water-soluble Liquid.

In another preferred example, beta glucan (water) solution viscosity is big；Preferably, the β-that mass concentration is 0.5% Glucan aqueous solution (at 25 DEG C) viscosity >=40mPas, more preferably 100-10000mPas, more preferably 500-2000mPa s。

In another preferred example, beta glucan aqueous solution (25 DEG C) viscosity that the mass concentration is 0.5% can be 50- 10000mPa·s；100-9000mPa·s；200-8000mPa·s；300-7000mPa·s；400-6000mPa·s；450- 5000mPa·s；500-5000mPa·s；550-4000mPa·s；600-3000mPa·s；650-2000mPa·s；700- 1500mPa·s。

In another preferred example, the aqueous solution for the beta glucan that the mass concentration is 1% has high clarity or high saturating Light rate, light transmittance >=50% for the beta glucan aqueous solution that the mass concentration is 1%, preferably >=80%, preferably >= 85%, more preferably >=95%；

In another preferred example, the beta glucan aqueous solution is with good stability.

In another preferred example, the beta glucan derives from higher plant or various bacteriums, fungi.

The embodiment of the present invention specifically by taking the tunning of schizophyllum commune as an example, but not limited to this.

The effect of beta glucan (preferably schizophyllum commune beta glucan) of the present invention is a kind of prevention and or treatment inflammation at Point, on the one hand it is that beta glucan has immunostimulation to macrophage, enhances resilience, thus the effectively extraneous disease of prevention Pathogenic microorganism infects, and have platform effect, will not overstimulation cause inflammation, immunologic derangement or to normal cell generate It poisons；On the other hand, for inflammation, beta glucan can be done directly on immunocyte, by point for adjusting inflammatory cytokine Bleeding Peer Mode prevents inflammation from excessively causing to damage.(stimulation is huge for beta glucan activated cell fundamental immunity function of the present invention Phagocyte activation and proliferation), while preventing and/or treating excessive inflammatory response, there is two-way immunoloregulation function, will not injure Patient's normal cell (such as skin) makes it generate drug resistance.

NO

NO is a kind of simple diatomic free radical of structure in organism, while being also important messenger molecule, has weight The physiological function wanted.Under normal circumstances, iNOS is not expressed, from the low of endothelium in type NOS (eNOS) and nervous system type NOS (nNOS) Horizontal NO has positive effect for the maintenance of body basic physiology function.The activation of immune system, especially body by To after inflammatory factor or pathogen stimulation, the high expression of iNOS will occurs in the immunocytes such as macrophage and NO is horizontal Increase.Suitable NO can enhance the innate immune function of body, such as macrophage be promoted to play cytotoxicity, to inhibit cause of disease The breeding of body, while the also proliferation and apoptosis of controllable immunocyte and inflammatory cell.But excessive NO, usually along with inflammation Phenomena such as disease and immunologic derangement.Local NO too high levels may generate cytotoxicity to neighbouring normal cell, cause itself group The damage knitted.Therefore the variation of NO level is an important indicator of macrophage innate immune activity.

MAPK signal path

The inflammatory reaction process of MAPK signal path participation body.The MAPK signal path having determined at present mainly has three Item: extracellular signal-regulated kinase ERK signal path, c-Jun N-terminal kinases JNK signal path and p38 signal path.Every letter Number access all has the specificity and independent function of height.When cell is by inflammatory factor and/or environmental stimuli, related pathways The phosphorylation level of albumen increases, and by regulating and controlling downstream albumen, increases inflammatory related gene expression, and then promote inductivity thin The secretion and expression of intracellular cytokine and inflammatory mediator.The phosphorylation level of Western blot detection GAP-associated protein GAP can be used.

NF- κ B signal access

NF- κ B is important transcription factor during inflammatory reaction, participates in the multiple functions for adjusting body.Cell is in quiet When breath state, NF- κ B is present in cytoplasm in conjunction with its inhibitor I κ B, when cell is stimulated by inflammatory factor, pathogen etc. When, NF- κ B signal access is activated, and for I κ B by its kinases IKK phosphorylation, the I κ B being finally phosphorylated passes through uiquitin-protease Body is degraded.The free NF- κ B being released regulates and controls the transcription of target gene into nucleus.During inflammatory reaction, NF- κ B can be played a significant role in inflammatory reaction by raising the expression of inflammatory mediator.It is detectable using western blotting method The p65 subunit of NF- κ B and the phosphorylation level of IKK β.

Lipopolysaccharides

Lipopolysaccharides is the main component of gram-negative bacteria cell wall, and high concentration lipopolysaccharides is the induction that body generates inflammation One of factor can cause a series of inflammatory reaction.Lipopolysaccharides interacts with host cell, by believing in active cell Number transmitting cascade reaction, and causes the change of cytokine profiles and inflammatory mediator level.Laboratory is frequently with lipopolysaccharides conduct Inflammatory reaction stimulant establishes the inflammatory model of cell or animal, is made with the prevention and treatment to inflammation of goal in research substance With.

Preparation or composition

The present invention provides a kind of preparation or composition for prevention and or treatment inflammation, and the preparation or composition contain There is (a) beta glucan；And optional (b) pharmaceutically, on cosmeceutical, in bromatology or the acceptable carrier of weapon exercises or Excipient.

In another preferred example, the preparation or composition contain (a) schizophyllum commune beta glucan；And optional (b) medicine On, on cosmeceutical, in bromatology or the acceptable carrier of weapon exercises or excipient.

In another preferred example, the preparation or composition contain 0.0001-99wt%, preferably 0.001-90wt%, More preferably 0.01-50wt%, the more preferably beta glucan of 0.05-10wt% (preferably schizophyllum commune beta glucan), by preparation or The total weight of composition.

In another preferred example, mass concentration of the beta glucan in the preparation or composition is >=1 μ g/mL, It is specifically as follows 1 μ g/mL-200mg/mlL perhaps 1 μ g/mL-5mg/mL or 1 μ g/mL-1mg/mL.

" active constituent " in preparation or composition of the present invention refers to beta glucan (preferably ground cleave of the present invention Gill fungus beta glucan).

" active constituent " of the present invention, preparation and/or composition can be used for prevention and or treatment inflammation.

In another preferred example, described " active constituent ", preparation and/or composition are also used to prevent and/or treat skin The preparation or composition of mucosal inflammation or other inflammatory disease of the skin.

In another preferred example, described " active constituent ", preparation and/or composition are also used to prevent and/or treat:

(a) macrophage abnormal secretion NO；

(b) in macrophage the immunity-associated cell factor unconventionality expression；And/or

(c) abnormal activation of the MAPK signal path in macrophage and/or NF- κ B signal access.

" safe and effective amount " refers to: the amount of active constituent is enough to be obviously improved the state of an illness or symptom, and is unlikely to generate tight The side effect of weight.

In general, pharmaceutical composition contains 1-2000mg active constituent/agent, more preferably, containing 10-200mg active constituent/ Agent.Preferably, described is " one " for a tablet or an injection.

" pharmaceutically acceptable carrier " refers to: one or more biocompatible solids or liquid filler or gelatinous mass, They are suitable for people's use and it is necessary to have enough purity and sufficiently low toxicity.

In " compatibility " referred to herein as composition each component energy and active constituent of the invention and they between mutually Blending, and significantly reduce the drug effect of active constituent.

Pharmaceutically acceptable carrier part example has cellulose and its derivates (such as sodium carboxymethylcellulose, ethyl Sodium cellulosate, cellulose ethanoate etc.), gelatin, talcum, solid lubricant (such as stearic acid, magnesium stearate), calcium sulfate, plant Oily (such as soya-bean oil, sesame oil, peanut oil, olive oil), polyalcohol (such as propylene glycol, glycerol, mannitol, sorbierite), emulsification Agent is (such as), wetting agent (such as lauryl sodium sulfate), colorant, flavoring agent, stabilizer, antioxidant, preservative, nothing Pyrogen water etc..

In another preferred example, beta glucan of the present invention can act on shape by nonbonding with macromolecular compound or macromolecule At compound.

In another preferred example, beta glucan of the present invention can be connected by chemical bond with macromolecular compound or macromolecule It connects.The macromolecular compound can be large biological molecule such as high glycan, albumen, nucleic acid, polypeptide etc..

The method of application of active constituent or composition of the invention is not particularly limited, representative method of application include but Be not limited to: be applied with, be oral, in tumor, rectum, parenteral (intravenous, intramuscular or subcutaneous) etc..

Solid dosage forms includes capsule, tablet, pill, powder and granule.

In these solid dosage forms, active constituent is mixed at least one conventional inert excipients or carrier, such as citric acid Sodium or Dicalcium Phosphate, or with following compositions are one or more mixes:

(a) filler or expanding material, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid；

(b) adhesive, for example, hydroxymethyl cellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and Arab Glue；

(c) moisturizer, for example, glycerol；

(d) disintegrating agent, for example, agar, calcium carbonate, potato starch or tapioca, alginic acid, certain composition silicates, And sodium carbonate；

(e) retarding solvent, such as paraffin；

(f) absorbsion accelerator, for example, quaternary ammonium compound；

(g) wetting agent, such as cetanol and glycerin monostearate；

(h) adsorbent, for example, kaolin；And/or

(i) lubricant, for example, talcum, calcium stearate, magnesium stearate, solid polyethylene glycol, lauryl sodium sulfate, or Its mixture.

In capsule, tablet and pill, dosage form also may include buffer.

Coating and shell material preparation also can be used in the solid dosage forms, such as casing and other materials well known in the art.It May include opacifying agent, also, in this composition active constituent release can in a delayed fashion it is in the digestive tract certain It is discharged in a part.The example of adoptable embedding component is polymeric material and wax material.

Liquid dosage form includes pharmaceutically acceptable lotion, solution, suspension, syrup or tincture.Other than active constituent, Liquid dosage form may include the inert diluent routinely used in this field, such as water or other solvents, solubilizer and emulsifier, example Such as, ethyl alcohol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-BDO, dimethylformamide and oil, especially Cottonseed oil, peanut oil, maize germ, olive oil, castor oil and sesame oil or the mixture of these substances etc..In addition to these inertia Outside diluent, composition also may include auxiliary agent, such as wetting agent, emulsifier and suspending agent, sweetener, corrigent and fragrance.

Other than active constituent, suspension may include suspending agent, for example, ethoxylation isooctadecane alcohol, polyoxyethylene mountain The pure and mild Isosorbide Dinitrate of pears, microcrystalline cellulose, aluminium methoxide and agar or the mixture of these substances etc..

Composition may include physiologically acceptable sterile, aqueous or anhydrous solution, dispersion liquid, suspension or lotion, and use In the aseptic powdery for re-dissolving into sterile Injectable solution or dispersion liquid.It is suitable aqueous and nonaqueous carrier, diluent, molten Agent or excipient include water, ethyl alcohol, polyalcohol and its suitable mixture.

It is that the present composition of safe and effective amount is applied to mammal in need for the treatment of when using pharmaceutical composition (such as people), wherein dosage is the effective dosage pharmaceutically thought when application, for the people of 60kg weight, day is to medicament Amount is usually 1~1000mg, preferably 10~200mg, more preferably 20~100mg.Certainly, specific dosage is also contemplated that administration way The factors such as diameter, patient health situation, within the scope of these are all skilled practitioners technical ability.

The present composition can be administered alone, or with other treatment administered in combination (as prepared in same drug In composition).

Pharmaceutical composition of the present invention can also be combined to the known other medicines for treating or improving similar symptom.Combine to When medicine, originally the administration mode of drug and dosage are remained unchanged, and subsequently or simultaneously use pharmaceutical composition of the present invention.Drug connection Pharmaceutical composition of the present invention and other one or more of known drugs are used with the period for being also included within overlapping.When medicine of the present invention When compositions and other one or more of drugs carry out drug combination, the dosage of pharmaceutical composition of the present invention or known drug can Dosage when can be than their independent medications is lower.

Main advantages of the present invention include:

(a) inflammation can effectively be prevented and/or be treated to beta glucan (especially schizophyllum commune beta glucan) of the present invention Disease, such as skin and mucosa inflammation or other inflammatory disease of the skin；Specifically, macrophage abnormal secretion NO, macrophage can be inhibited thin The unconventionality expression of the immunity-associated cell factor, and/or the MAPK signal path in macrophage and/or NF- κ B signal are logical in born of the same parents The abnormal activation on road.

(b) side effect of beta glucan of the present invention is very low, does not destroy the skin surface flora ecological balance, will not injure trouble Person's normal cell (skin) makes it generate drug resistance.

(c) beta glucan of the present invention can be not only used for prevention and or treatment inflammation, can also enhance Initiative Defense function, Antibacterial, anti-inflammatory and reparation (such as skin), prevention and/or treatment skin and mucosa inflammation or other inflammatory disease of the skin.β-of the present invention Glucan activated cell fundamental immunity function (stimulating expression of macrophage activation and proliferation), while preventing and/or treating excessive inflammation Reaction, have two-way immunoloregulation function, will not patient harm normal cell (such as skin) or make its generate drug resistance.

(d) beta glucan of the present invention is the biological polyoses in pure natural source, has natural soluble.Due to needing not move through The modification or modification of any chemistry and/or physics, beta glucan of the present invention completely remains the three-dimensional conformation of three spirals, this for The performance of its effect is most important.Beta glucan of the present invention has splendid stability, can coexist and protect with most of substances Its activity is held, thus application field is extensive, can be used in combination with other treatment drug or skin care item.

Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part, such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.Unless otherwise stated, no Then percentage and number are weight percent and parts by weight.

Experimental method used in following embodiments is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified, partially such as Shown in the following table 1-3:

Antibody needed for table 1

Title	Buy source
		Phospho-Ser536NF- κ B p65 rabbit polyclonal antibody	CST company
Phospho-Tyr199IKK β rabbit polyclonal antibody	Abcam company
		IKK β mouse polyclonal antibody	Proteintech company
ERK1/2 rabbit polyclonal antibody	Proteintech company
		Phospho-Thr202/Tyr204ERK/MAPK rabbit polyclonal antibody	Abgent company
JNK, rabbit polyclonal antibody	Proteintech company
		Phospho-Thrl83/Tyrl85JNK1/2/3 rabbit polyclonal antibody	Abgent company
P38 rabbit polyclonal antibody	Proteintech company
		Phospho-Thr180/Tyr182p38MAPK rabbit polyclonal antibody	Abgent company

Drug needed for table 2 and reagent

Primer needed for table 3