CN109549951A - Cage mesh kelp fucoidan sulfuric ester compound immunoenhancer - Google Patents
Cage mesh kelp fucoidan sulfuric ester compound immunoenhancer Download PDFInfo
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- CN109549951A CN109549951A CN201910071974.1A CN201910071974A CN109549951A CN 109549951 A CN109549951 A CN 109549951A CN 201910071974 A CN201910071974 A CN 201910071974A CN 109549951 A CN109549951 A CN 109549951A
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- ethyl alcohol
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/737—Sulfated polysaccharides, e.g. chondroitin sulfate, dermatan sulfate
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
Abstract
The present invention discloses a kind of cage mesh kelp fucoidan sulfuric ester compound immunoenhancer, it is characterised in that is mixed in mass ratio for 1:2:1 by cage mesh kelp fucoidan sulfuric ester, astragalus polyose and Codonopsis pilosula polysaccharide.The experimental results showed that cage mesh kelp fucoidan sulfuric ester compound immunoenhancer can increase GM-CSF in BALB/c mouse body, TNF-α, IL-10 secretion level, immunity of organism activity is improved by activation nuclear Factor-Kappa (NF- κ B) and Mitogen-activated Protein Kinase Cascades (MAPKs) approach；It can promote body nospecific immunity, while, serum hemolysin HC50, ConA induction Splenic vein hemodynamics abnormal to mouse delayed show significant difference, can adjust the specific cellular immunity and humoral immunity of body.
Description
Technical field
The present invention relates to a kind of immunopotentiator, especially a kind of cage mesh kelp fucoidan for being remarkably improved immunity
Sulfuric ester compound immunoenhancer.
Background technique
Fucoidan sulfuric ester (fucoidan) is the active heteroglycan that a kind of water solubility is combined with sulfate group, mainly at
It is divided into fucose and sulfate.Fucoidan sulfuric ester has extensive physiology and bioactivity, studies have shown that different extractions
The fucoidan sulfuric ester extracted in method, different algae, there are larger differences for constituent and structure etc..Fucoidan
The bioactivity of sulfuric ester depends not only on its constituent, structure, the content of sulfate group and its position, additionally depends on possibility
The polyphenol content linked with polysaccharide.Cage mesh kelp (Kjellmaniella crassifolia Miyabe) it is brown alga advantage germplasm
Resource, it belong in genealogical classification Laminariales (Laminariales), Laminariaceae (Laminariaceae),KjellmaniellaBelong to, is the high-quality brown alga that research team cultivates cultivation in recent years.On the one hand cage mesh kelp is rich in fucoidan
Sulfuric ester, on the other hand because the growing environment of low temperature makes it be rich in the high-quality fucoidan sulfuric ester based on U-fucoidan,
With multiple biological activities such as antitumor, reducing blood lipid, liver protectings.
Astragalus polyose and Codonopsis pilosula polysaccharide be respectively using Radix Astragali and Radix Codonopsis as raw material, it is extracted, concentration, purifying made of it is water-soluble
Property heteroglycan, can be used as immunopotentiating agent or regulator.
Although cage mesh kelp fucoidan sulfuric ester, astragalus polyose and Codonopsis pilosula polysaccharide all have the effect for improving immunity,
But there is no compound three rationally to significantly improve the relevant report of immunity so far.
Summary of the invention
The present invention is to provide one kind in order to solve above-mentioned technical problem present in the prior art and be remarkably improved immunity
Cage mesh kelp fucoidan sulfuric ester compound immunoenhancer.
The technical solution of the invention is as follows: a kind of cage mesh kelp fucoidan sulfuric ester compound immunoenhancer, special
Sign is to be mixed in mass ratio for 1:2:1 by cage mesh kelp fucoidan sulfuric ester, astragalus polyose and Codonopsis pilosula polysaccharide；
The cage mesh kelp fucoidan sulfuric ester is successively prepared in accordance with the following steps: using the cage mesh kelp after cleaning as raw material,
Complex enzyme is added into raw material, is digested 40 ~ 70 minutes under the conditions of pH4.0~pH5.5 and 40~60 DEG C of temperature, it is described compound
Enzyme additive amount be material quality 0.70~0.90%, then 96~100 DEG C heating water bath 3~4 hours, be down to room temperature, be centrifuged
Supernatant A, the complex enzyme by cellulase, pectase and protease be in mass ratio 5:5~6:0.3~0.5 mixing and
At；It is 20% generation precipitating that ethyl alcohol to ethyl alcohol mass concentration is added in supernatant A, is centrifuged to obtain supernatant B；Add in supernatant B
Entering ethyl alcohol to ethyl alcohol mass concentration is 60% generation precipitating, is centrifuged to obtain precipitating；Soluble in water, addition ethyl alcohol to ethyl alcohol matter will be precipitated
Measuring concentration is 30% generation precipitating, and centrifugation goes to precipitate to obtain supernatant C；Continue to add ethyl alcohol in supernatant C to ethyl alcohol mass concentration
Precipitating is generated for 70%, is centrifuged to obtain precipitating；Freeze-drying precipitates to obtain white powder；
The astragalus polyose and Codonopsis pilosula polysaccharide extract as follows: weighing 60 mesh Milkvetch Roots or Radix Codonopsis powder 5.00g, add 8
Times volumes of deionized water, 100 DEG C of 90 min of heating water bath；4500 r/min centrifugation, retains filtrate, in triplicate；Merge filter
Liquid, vacuum concentrated by rotary evaporation to 20 mL；Be added 95% ethyl alcohol of three times volume mass concentration, 4 DEG C of overnight alcohol precipitations, 4500 r/min from
The heart retains precipitating, and vacuum freeze drying respectively obtains astragalus polyose or Codonopsis pilosula polysaccharide.
The present invention is rationally to be compounded cage mesh kelp fucoidan sulfuric ester, astragalus polyose and Codonopsis pilosula polysaccharide, can be shown
It writes and improves immunity of organisms.
Detailed description of the invention
Fig. 1 is the embodiment of the present invention and control group carbonic clearance index schematic diagram.
Fig. 2 is the influence schematic diagram of the embodiment of the present invention Yu control group serum hemolysin HC50.
Specific embodiment
Embodiment 1:
Cage mesh kelp fucoidan sulfuric ester compound immunoenhancer of the invention, be by cage mesh kelp fucoidan sulfuric ester,
Astragalus polyose and Codonopsis pilosula polysaccharide mix in mass ratio for 1:2:1；
The cage mesh kelp fucoidan sulfuric ester is successively prepared in accordance with the following steps: using the cage mesh kelp after cleaning as raw material,
Complex enzyme is added into raw material, is digested 40 minutes under the conditions of pH5.5 and temperature 60 C, the complex enzyme additive amount is raw material
The 0.90% of quality, then 100 DEG C heating water bath 3 hours, be down to room temperature, be centrifuged to obtain supernatant A, the complex enzyme is to take
5.0g cellulase, 5.0g pectase and 0.3g protease mixed preparing form, the enzyme activity of cellulase, pectase and protease
Respectively 80,000u/g, 80,000 u/g and 1,000,000 u/g；Ethyl alcohol to ethyl alcohol mass concentration is added in supernatant A is
20% generates precipitating, is centrifuged to obtain supernatant B；It is 60% generation precipitating, centrifugation that ethyl alcohol to ethyl alcohol mass concentration is added in supernatant B
It must precipitate；To precipitate it is soluble in water, be added ethyl alcohol to ethyl alcohol mass concentration be 30% generate precipitating, centrifugation go to precipitate to obtain supernatant
C；Continue to add ethyl alcohol to ethyl alcohol mass concentration in supernatant C to be 70% generation precipitating, is centrifuged to obtain precipitating；Freeze-drying precipitating
Obtain white powder；
The astragalus polyose and Codonopsis pilosula polysaccharide extract as follows: weighing 60 mesh Milkvetch Roots or Radix Codonopsis powder 5.00g, add 8
Times volumes of deionized water, 100 DEG C of 90 min of heating water bath；4500 r/min centrifugation, retains filtrate, in triplicate；Merge filter
Liquid, vacuum concentrated by rotary evaporation to 20 mL；Be added 95% ethyl alcohol of three times volume mass concentration, 4 DEG C of overnight alcohol precipitations, 4500 r/min from
The heart retains precipitating, and vacuum freeze drying respectively obtains astragalus polyose or Codonopsis pilosula polysaccharide.
Embodiment 2:
Cage mesh kelp fucoidan sulfuric ester compound immunoenhancer of the invention, be by cage mesh kelp fucoidan sulfuric ester,
Astragalus polyose and Codonopsis pilosula polysaccharide mix in mass ratio for 1:2:1；
The cage mesh kelp fucoidan sulfuric ester is successively prepared in accordance with the following steps: using the cage mesh kelp after cleaning as raw material,
Complex enzyme is added into raw material, is digested 70 minutes under the conditions of pH4.0 and 40 DEG C of temperature, the complex enzyme additive amount is raw material
The 0.70% of quality, then 96 DEG C heating water bath 4 hours, be down to room temperature, be centrifuged to obtain supernatant A, the complex enzyme is to take 5.0g
Cellulase, 6.0g pectase and 0.5g protease mixed preparing form, the enzyme activity difference of cellulase, pectase and protease
For 80,000u/g, 80,000 u/g and 1,000,000 u/g；It is 20% that ethyl alcohol to ethyl alcohol mass concentration is added in supernatant A
Precipitating is generated, supernatant B is centrifuged to obtain；It is 60% generation precipitating that ethyl alcohol to ethyl alcohol mass concentration is added in supernatant B, is centrifuged
Precipitating；To precipitate it is soluble in water, be added ethyl alcohol to ethyl alcohol mass concentration be 30% generate precipitating, centrifugation go to precipitate to obtain supernatant C；
Continue to add ethyl alcohol to ethyl alcohol mass concentration in supernatant C to be 70% generation precipitating, is centrifuged to obtain precipitating；Freeze-drying precipitates
White powder；
The astragalus polyose and Codonopsis pilosula polysaccharide extract as follows: weighing 60 mesh Milkvetch Roots or Radix Codonopsis powder 5.00g, add 8
Times volumes of deionized water, 100 DEG C of 90 min of heating water bath；4500 r/min centrifugation, retains filtrate, in triplicate；Merge filter
Liquid, vacuum concentrated by rotary evaporation to 20 mL；Be added 95% ethyl alcohol of three times volume mass concentration, 4 DEG C of overnight alcohol precipitations, 4500 r/min from
The heart retains precipitating, and vacuum freeze drying respectively obtains astragalus polyose or Codonopsis pilosula polysaccharide.
Embodiment 3:
Cage mesh kelp fucoidan sulfuric ester compound immunoenhancer of the invention, be by cage mesh kelp fucoidan sulfuric ester,
Astragalus polyose and Codonopsis pilosula polysaccharide mix in mass ratio for 1:2:1；
The cage mesh kelp fucoidan sulfuric ester is successively prepared in accordance with the following steps: using the cage mesh kelp after cleaning as raw material,
Complex enzyme is added into raw material, is digested 50 minutes under the conditions of pH5.0 and temperature 50 C, the complex enzyme additive amount is raw material
The 0.80% of quality, then 98 DEG C heating water bath 3.5 hours, be down to room temperature, be centrifuged to obtain supernatant A, the complex enzyme is to take
5.0g cellulase, 5.5g pectase and 0.4g protease mixed preparing form, the enzyme activity of cellulase, pectase and protease
Respectively 80,000u/g, 80,000 u/g and 1,000,000 u/g；Ethyl alcohol to ethyl alcohol mass concentration is added in supernatant A is
20% generates precipitating, is centrifuged to obtain supernatant B；It is 60% generation precipitating, centrifugation that ethyl alcohol to ethyl alcohol mass concentration is added in supernatant B
It must precipitate；To precipitate it is soluble in water, be added ethyl alcohol to ethyl alcohol mass concentration be 30% generate precipitating, centrifugation go to precipitate to obtain supernatant
C；Continue to add ethyl alcohol to ethyl alcohol mass concentration in supernatant C to be 70% generation precipitating, is centrifuged to obtain precipitating；Freeze-drying precipitating
Obtain white powder；
The astragalus polyose and Codonopsis pilosula polysaccharide extract as follows: weighing 60 mesh Milkvetch Roots or Radix Codonopsis powder 5.00g, add 8
Times volumes of deionized water, 100 DEG C of 90 min of heating water bath；4500 r/min centrifugation, retains filtrate, in triplicate；Merge filter
Liquid, vacuum concentrated by rotary evaporation to 20 mL；Be added 95% ethyl alcohol of three times volume mass concentration, 4 DEG C of overnight alcohol precipitations, 4500 r/min from
The heart retains precipitating, and vacuum freeze drying respectively obtains astragalus polyose or Codonopsis pilosula polysaccharide.
It is more to embodiment 1, embodiment 2,3 gained cage mesh kelp fucoidan sulfuric ester of embodiment, astragalus polyose and Radix Codonopsis
Sugar is analyzed, and the purity of cage mesh kelp fucoidan sulfuric ester is 80.0% or more, and total reducing sugar quality is 50.0~65.0%, sulfuric acid
Root quality 21.0~35.0%, astragalus polyose purity and Codonopsis pilosula polysaccharide purity are respectively 78.00 % and 80.00 % or more.
Experiment:
Experimental animal grouping and administration are as shown in table 1.
The grouping of 1 experimental animal of table and administration
Positive group (Positive): radix astragali particle (AG)；Chinese medicine group (ACM): the Huang prepared according to the method for the embodiment of the present invention 3
Astragalus polysaccharides and Codonopsis pilosula polysaccharide are mixed according to mass ratio for 2:1；Fucoidan sulfuric ester (KF): according to the embodiment of the present invention 3
Method preparation cage mesh kelp fucoidan sulfuric ester；Compound low dose group (KCA-L), compound middle dose group (KCA-M) and
Compound high dose group (KCA-H) is 3 gained cage mesh kelp fucoidan sulfuric ester compound immunoenhancer of the embodiment of the present invention.
1. 3 cage mesh kelp fucoidan sulfuric ester compound immunoenhancer of the embodiment of the present invention is to BALB/c mouse serum
Immune factor secretion influences
According to the method for the prior art, immune factors in serum secretion experiment is carried out to experimental group BALB/c mouse,
Experimental result such as table 2.
Table 2, which secretes BALB/c mouse immune factors in serum, influences (n=8)
All data are with mean+SDIt indicates, is analyzed using single factor test variance (ANOVA), Duncan
Multiple range test between method group, *P< 0.05, * *P < 0.01。
Table 2 the result shows that, Positive group, ACM group, KF group do not make significant difference to IL-10 secretion level in Mice Body, but
3 groups of the embodiment of the present invention (KCA) can dramatically increase IL-10 content in serum.Meanwhile experiment also carries out GM-CSF, TNF-α
Research, the results showed that except Positive group in addition to, remaining each group can dramatically increase GM-CSF secretion level in Mice Body, remove empty
Outside, all pharmaceutical intervention groups can increase TNF-α secretion in Mice Body, but Positive group, ACM to white control group (Control)
Group, KF group and 3 groups of the embodiment of the present invention (KCA) are variant on secretion level.
Conclusion: 3 groups of the embodiment of the present invention (KCA) can increase GM-CSF in BALB/c mouse body, TNF-α, IL-10 secretion water
It is flat, immunity of organism activity is improved by activation nuclear Factor-Kappa (NF- κ B) and Mitogen-activated Protein Kinase Cascades (MAPKs) approach；
3 groups of the embodiment of the present invention (KCA) can promote body nospecific immunity, while KCA is molten to phagocytic index α, the serum of mouse
Sanguinin HC50 shows significant difference, can adjust the specific cellular immunity and humoral immunity of body.
2. influence of the 3 cage mesh kelp fucoidan sulfuric ester compound immunoenhancer of the embodiment of the present invention to carbonic clearance index
The phagocytic activity of mononuclear macrophage is the important indicator for reflecting animal non-specific immune function, and early stage research discovery exists
Carbon particle clearance rate and concentration of carbon exponentially functional relation in mouse blood in a certain range.According to the method for the prior art,
Carbonic clearance exponential experiment is carried out to 7 groups of BALB/c mouses listed in experiment 1, as a result as shown in Figure 1.
All data are with mean+SD in Fig. 1It indicates, using single factor test variance (ANOVA) point
Analysis, Duncan method carry out Multiple range test between group, *P< 0.05, * *P < 0.01。
By Fig. 1 result it is found that the phagocytosis of Positive group, KCA dosage group (KCA-L, KCA-M, KCA-H) BALB/c mouse
Index α is dramatically increased compared to blank control group (Control), respectively 4.67,4.73,5.77,5.23；And ACM group, KF
There was no significant difference compared to blank control group (Control) group by group mouse phagocytic index α.
Conclusion: the carbonic clearance index of normal BALB/c mouse can be by 3 cage mesh kelp fucoidan sulfuric acid of the embodiment of the present invention
Ester compound immunoenhancer significantly improves, it was demonstrated that the cage mesh kelp fucoidan sulfuric ester compound immune of the embodiment of the present invention enhances
Agent can promote body nospecific immunity.
3. 3 cage mesh kelp fucoidan sulfuric ester compound immunoenhancer of the embodiment of the present invention is to serum hemolysin HC50
Influence
Serum hemolysin HC50 directly reflects mouse specific b cells humoral immunity level, right according to the method for the prior art
Listed 7 groups of BALB/c mouses carry out the influence experiment of serum hemolysin HC50 in experiment 1, as a result as shown in Figure 2.
All data are with mean+SD in Fig. 2It indicates, using single factor test variance (ANOVA) point
Analysis, Duncan method carry out Multiple range test between group, *P< 0.05, * *P < 0.01。
Each experimental group HC50 value is dramatically increased compared to blank control group (Control) in Fig. 2.Wherein Positive
Group, ACM group, KF group increase separately 8.37 %, 5.66 %, 7.51 %, and each dosage group HC50 value of KCA significantly increases.
Conclusion: 3 cage mesh kelp fucoidan sulfuric ester compound immunoenhancer of the embodiment of the present invention is to mice serum haemolysis
Plain HC50 shows significant difference, shows cage mesh kelp fucoidan sulfuric ester compound immunoenhancer energy of the embodiment of the present invention
Enough adjust the specific cellular immunity and humoral immunity of body.
Claims (1)
1. a kind of cage mesh kelp fucoidan sulfuric ester compound immunoenhancer, it is characterised in that by cage mesh kelp fucoidan sulphur
Acid esters, astragalus polyose and Codonopsis pilosula polysaccharide mix in mass ratio for 1:2:1；
The cage mesh kelp fucoidan sulfuric ester is successively prepared in accordance with the following steps: using the cage mesh kelp after cleaning as raw material,
Complex enzyme is added into raw material, is digested 40 ~ 70 minutes under the conditions of pH4.0~pH5.5 and 40~60 DEG C of temperature, it is described compound
Enzyme additive amount be material quality 0.70~0.90%, then 96~100 DEG C heating water bath 3~4 hours, be down to room temperature, be centrifuged
Supernatant A, the complex enzyme by cellulase, pectase and protease be in mass ratio 5:5~6:0.3~0.5 mixing and
At；It is 20% generation precipitating that ethyl alcohol to ethyl alcohol mass concentration is added in supernatant A, is centrifuged to obtain supernatant B；Add in supernatant B
Entering ethyl alcohol to ethyl alcohol mass concentration is 60% generation precipitating, is centrifuged to obtain precipitating；Soluble in water, addition ethyl alcohol to ethyl alcohol matter will be precipitated
Measuring concentration is 30% generation precipitating, and centrifugation goes to precipitate to obtain supernatant C；Continue to add ethyl alcohol in supernatant C to ethyl alcohol mass concentration
Precipitating is generated for 70%, is centrifuged to obtain precipitating；Freeze-drying precipitates to obtain white powder；
The astragalus polyose and Codonopsis pilosula polysaccharide extract as follows: weighing 60 mesh Milkvetch Roots or Radix Codonopsis powder 5.00g, add 8
Times volumes of deionized water, 100 DEG C of 90 min of heating water bath；4500 r/min centrifugation, retains filtrate, in triplicate；Merge filter
Liquid, vacuum concentrated by rotary evaporation to 20 mL；Be added 95% ethyl alcohol of three times volume mass concentration, 4 DEG C of overnight alcohol precipitations, 4500 r/min from
The heart retains precipitating, and vacuum freeze drying respectively obtains astragalus polyose or Codonopsis pilosula polysaccharide.
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