CN113975289A - Application of sulfated fucogalactomannoglucuronan polysaccharide from brown algae in anti-aging - Google Patents
Application of sulfated fucogalactomannoglucuronan polysaccharide from brown algae in anti-aging Download PDFInfo
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- CN113975289A CN113975289A CN202111382619.XA CN202111382619A CN113975289A CN 113975289 A CN113975289 A CN 113975289A CN 202111382619 A CN202111382619 A CN 202111382619A CN 113975289 A CN113975289 A CN 113975289A
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- polysaccharide
- sulfated
- aging
- sargassum
- brown algae
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Abstract
The invention discloses an application of sulfated fucogalactomannosyluronic acid polysaccharide from brown algae in anti-aging, which is used for solving the problems of large side effect and common effect of the existing anti-aging drugs, wherein the application of the sulfated fucogalactomannosyluronic acid polysaccharide from brown algae in anti-aging achieves the aim of anti-aging by enhancing cell activity, delaying cell aging, improving the expression of aging-related proteins of islet beta cells and improving the functions of the islet beta cells.
Description
Technical Field
The invention relates to the field of pharmacy, in particular to application of sulfated fucalactoglucuronan polysaccharide from brown algae in anti-aging.
Background
After the 21 st century, people have been confronted with a serious challenge of population aging, and many countries (including China) have entered the population aging society, and the elderly people aged at 60 or more account for more than 10% of the total population. At present, the elderly (including a part of middle-aged people) in China are seriously troubled by aging, and the health condition and the life quality of the elderly are directly influenced. How to completely explain the pathogenesis of aging and how to improve the health condition and the life quality of the old people is a problem which needs to be solved by researchers at present. In recent years, with the development of science and technology and the continuous improvement of research means, the focus of modern people is the western anti-aging mechanism and the medicinal potential of the Chinese herbal medicines, and the Chinese herbal medicines show great attractive prospects.
Cellular senescence refers to the irreversible, permanent state of cell cycle arrest that cells cause after stress. Cell damage itself does not directly lead to obvious signs of aging, but when the damage accumulates and reaches a certain limit, the cells stop proliferating, resulting in a macroscopically weak tissue and a physiological aging phenotype. Several senescence-inducing factors (including DNA damage, oncogenic mutations, active metabolites, high mitogen and trophic signals increase mTOR activity, protein toxicity stress) may act alone or in concert on cells, causing cellular senescence through the p16INK4a/Rb, p53/p21 pathway, or other pathways. These may lead to widespread gene expression changes and chromatin remodeling (formation of heterochromatin), which underlie SASP, senescence-associated growth arrest, and morphological changes.
Studies have shown that aging and diabetes are related. The prevalence rate of diabetes and pre-diabetes remarkably increases with age, and the proliferation potential of islet beta cells remarkably decreases with age, so that the prevalence rate of diabetes in the elderly is increased, and the diabetes is an age-related disease. With the aging, the secretion function of islet beta cells is reduced, the regeneration capacity of the cells is reduced, and the islet beta cells are aged and accumulated, so that the glucose tolerance is reduced and the diabetes mellitus is caused. At the same time, the aging can also cause the apoptosis of the islet beta cells to increase, and further influence the function of the islet beta cells. Aging of islet beta cells is not only age-related, but is also affected by other factors, such as obesity and abnormal glycolipid metabolism, which increase the number of aging cells in the body and accelerate cellular aging. When cells undergo senescence, a universal marker with the senescence phenotype, senescence-associated acid beta-galactosidase activity, p21Cis1， p16Ink4aIncreased expression in the senescent cells and,meanwhile, the proportion of Ki67 positive cells is obviously reduced, the compensatory hyperplasia of the pancreatic island is inhibited, and the secretion factor of the secretion spectrum (SASP) related to aging is increased. Although the relationship between islet beta cell senescence and diabetes is very clear, the concerns of diabetes caused by senescent islet cells are relatively low, and the development process of the medicine is restricted.
Chinese medicine resources are rich in China, and at present, a lot of anti-aging medicines are available, wherein the Chinese medicine polysaccharide has good anti-aging activity. Compared with traditional medicines, polysaccharide medicines are not medicines, do not have any toxicity, but can bring about various pharmacological activities, and nowadays when people pay more attention to the toxicity of medicines, polysaccharide can effectively resist various chronic diseases of chemical medicines which treat symptoms but not root causes. The marine polysaccharide is derived from organisms in a unique marine ecological environment, the synthesized components have distinct marine characteristics and unique chemical characteristics, and present unique pharmacological activity, the safety is high, the toxic and side effects are small, and the like, so that the marine polysaccharide becomes a new trend of drug development.
The marine brown algae has various polysaccharides with complex structures, and only sulfated polysaccharides include three major groups, one is fucoidan sulfate (also called fucoidan sulfate), which is a sulfated polysaccharide component with fucose as a constituent monosaccharide, the other is sulfated polysaccharide with fucose and galactose, namely galactofucoidan sulfate, besides, brown algae also commonly has a low sulfated heteroglycan rich in uronic acid, which has complex monosaccharide composition and contains various monosaccharides such as mannose, glucuronic acid, fucose and galactose, and when the sulfated polysaccharide component in brown algae is subjected to fractional purification by anion gel chromatography, the low sulfated heteroglycan component containing glucuronic acid is generally eluted by low-concentration electrolyte firstly. Due to the large difference of chemical characteristics, the biological activities of the low sulfated heteroglycan and the fucoidan sulfate are greatly different. For example, Croci et al reported that two sulfated polysaccharides were isolated from brown algae Laminaria saccharophila, one belonging to the low sulfated heteroglycan species known as fucomannoglucuronan (fucoidan) and the other fucoidan, and their comparative studies on the activity of the two different polysaccharides concluded that fucoidan, but not fucomannoglucuronan, determined the biological activity of the sulfated polysaccharides in brown algae Laminaria saccharophila, including anticoagulant, antitumor, anti-inflammatory, etc. activities. (fungi, but not fungal polysaccharides, degraded polysaccharides from Laminaria saccharona brown seaweed, PLoS ONE2011,6(2): 1-10). We found that low sulfated heteroglycan had significant efficacy in treating parkinson's disease and also in treating and/or preventing type 2 diabetes in the early days, and further found that sulfated fucogalactomannoglucuronan polysaccharide (structurally similar to low sulfated heteroglycan) had significant effects in anti-aging.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, clarifies a new application and an action mechanism of sulfated fucalactoglucuronan polysaccharide from brown algae according to multiple cell experiments, and performs experimental verification.
The invention is realized by the following technical scheme:
in order to achieve the purpose, the invention provides an application of sulfated fucogalactomannoglucuronate polysaccharide derived from brown algae in anti-aging, which is characterized in that the sulfated fucogalactomannoglucuronate polysaccharide derived from brown algae achieves the aim of anti-aging by enhancing cell activity, delaying cell aging, improving the expression of aging-related protein of islet beta cells and improving the function of the islet beta cells.
Preferably, the enhancing cell viability is enhancing the viability of the beta cell line.
Preferably, the delaying of cellular senescence is repairing cell cycle changes.
Preferably, the above-mentioned improvement of the expression of senescence-associated proteins of islet beta cells is to reduce the expression of senescence-associated proteins p53, p21 and p16, while alleviating senescence-associated secretory phenotypes IL-1 beta, TNFa, CXCL4 and CXCL 10.
Preferably, the function of the islet beta cells is to repair insulin synthesis damage of the islet cells.
Preferably, the sulfated fucogalactomannoglucuronate polysaccharide mainly comprises monosaccharides including glucuronic acid, mannose, galactose and fucose; the content of hydrolyzed sulfate group is 2-12%, the content of fucose is 3-10%, and the content of uronic acid is 20-40%.
The sulfated fucalactoglucuronan polysaccharide has the following structural characteristics:
(1) the component sugar comprises: mannose, glucuronic acid, galactose, fucose;
(2) mannose and glucuronic acid are linked by an alpha (1 → 4) glycosidic bond; glucuronic acid and mannose are linked by a β (1 → 2) glycosidic bond;
(3) the glucuronic acid and the glucuronic acid are connected by a beta (1 → 3) glycosidic bond;
(4) galactose is mainly connected by beta (1 → 6) glycosidic bond;
(5) fucose is connected with alpha (1 → 3) glycosidic bond, C2 or C4 site is sulfated; or mannose linked in a branched form by an alpha (1 → 3) glycosidic linkage;
(6) comprises the following structural units (I), (II), (III) and (IV) which are alternately connected with each other;
wherein R is1Is H or a sulfate radical, R2Is a sulfated fucose residue.
Preferably, the sulfated fucus galactomannosyluronic acid polysaccharide is derived from marine brown algae, wherein the marine brown algae comprises one or more of sea tangle, Undaria pinnatifida, Sargassum hemiphyllum, Sargassum thunbergii, Sargassum integrifolia, Cyrtymenia Sparsa, Sargassum salmonicum, Sargassum muelleri, and Sargassum muticum.
Preferably, the molecular weight of the sulfated fucogalactomannoglucuronan polysaccharide is 1kD to 1000 kD.
Preferably, the above method of application is the administration of an effective amount of sulfated fucgalactomannoglucuronan polysaccharide as a medicament.
Preferably, the above-mentioned medicament is a pharmaceutically acceptable carrier or dosage form.
The invention also provides a preparation method of the brown algae-derived sulfated fucgalactomannosylglucuronic acid polysaccharide, which comprises the following steps: extracting water-soluble polysaccharide from marine brown algae by water extraction; and finally, degrading by using high-concentration acid and precipitating by using alcohol to obtain the water-soluble polysaccharide with stable strong acid.
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention has obvious anti-aging effect, small toxic and side effect, safety and effectiveness, and can be used for preparing anti-aging medicaments.
(2) According to the invention, after research, the sulfated fucus galactomannogalacturonic acid polysaccharide from brown algae has a very good treatment effect on anti-aging, no other colleges or institutions have researched in the direction at present, and the technical progress of the scheme of the invention is relatively great, thereby being beneficial to market popularization.
Drawings
FIG. 1 is SA-beta-gal staining of different SFGGs
FIG. 2 is a diagram of cell motility; wherein A is the influence of SFGG-SJ addition on the vitality of MIN6 cells; b is H2O2Effect of addition of SFGG-SJ on cell viability after treatment.
FIG. 3 is a graph of the cell cycle changes caused by SFGG-SJ versus H2O 2; wherein S is: a DNA synthesis period; g1 is: DNA synthesis earlier stage; g2 is: late stage of DNA synthesis;
FIG. 4 is the expression of cellular senescence-associated proteins after SFGG-SJ treatment;
FIG. 5 is a graph of the change in senescence-associated secretory phenotype after SFGG-SJ treatment;
FIG. 6 is a model of aging after SFGG-SJ treatment.
Detailed Description
The present invention will be described in more detail with reference to examples. It is to be understood that the practice of the invention is not limited to the following examples, and that various changes or modifications may be made therein without departing from the scope of the invention; and the methods in the following examples are conventional in the art unless otherwise specified.
Example 1: preparation of Laminaria japonica-derived fucogalactomannoglucuronan (SFGG-SJ)
(1) Extracting 1kg of kelp by 15 times of water in a pressure cooker for 3 hours, and controlling the temperature to be 100-; extracting for 1 time. Removing algae, mixing extractive solutions, vacuum filtering with diatomite, concentrating the filtrate, adding 2mol/L MgCl2Making MgCl2Adding anhydrous ethanol to final mass concentration of 0.05mol/L to obtain ethanol with final weight concentration of 20%, stirring to obtain precipitate, centrifuging to remove precipitate, dialyzing with 3500Da molecular weight cutoff dialysis bag for 2 days, concentrating the solution in dialysis bag, and lyophilizing to obtain laminarin A with yield of 1.5%.
(2) Dissolving laminarin A in water to prepare a solution with the concentration of 1 percent (mass concentration), adding 4mol/L hydrochloric acid to ensure that the final concentration is 0.1mol/L, stirring for 2 hours in a water bath with the temperature of 80 ℃, then neutralizing with sodium hydroxide, concentrating, and pouring into absolute ethyl alcohol with the same volume. Centrifuging, and oven-drying with infrared to obtain laminarin B with yield of 53.5%.
(3) Dissolving laminarin B in water to prepare a solution with the concentration of 1 percent (mass concentration), adding 4mol/L hydrochloric acid to ensure that the final concentration is 0.5mol/L, stirring and degrading for 2 hours in a water bath with the temperature of 80 ℃, then neutralizing with sodium hydroxide, concentrating, and pouring into absolute ethyl alcohol with the same volume. Centrifuging, and oven-drying with infrared to obtain laminarin C with yield of 40.4%.
(4) Dissolving laminarin C (0.5G) in 4 ml of 0.2M ammonium bicarbonate, loading to column chromatography with Bio-Gel P-10 as a carrier, taking 0.2M ammonium bicarbonate as a mobile phase, collecting an elution component, concentrating the elution component, loading to column chromatography desalting with Sephadex G10 as a carrier, concentrating an eluent, and freeze-drying to obtain the fucogalactomannan uronic acid polysaccharide (SFGG-SJ) from the kelp, wherein the yield is 80.5%. The SFGG-SJ components were analyzed for chemical components, monosaccharide ratios, sulfate content and molecular weight, and the results are shown in table 1 below.
Example 2: preparation of sea-tangle-derived low-molecular-weight fucogalactomannoglucuronan polysaccharide (DSFGG-SJ)
(1) Dissolving laminarin A in water to prepare a solution with the concentration of 1 percent (mass concentration), adding 30mM ascorbic acid and 30mM hydrogen peroxide, and reacting for 2 hours at normal temperature. The reaction solution was dialyzed with a dialysis bag having a molecular weight cutoff of 3500Da in tap water and distilled water, respectively, and then concentrated and lyophilized. Obtaining the low molecular weight laminarin D.
(2) Dissolving low molecular weight laminarin D in water to prepare a solution with the concentration of 1% (mass concentration), adding 4mol/L hydrochloric acid to ensure that the final concentration is 0.1mol/L, stirring for 2 hours in a water bath of 80 ℃, then neutralizing with sodium hydroxide, concentrating, and pouring into absolute ethyl alcohol with the same volume. Centrifuging, and oven-drying with infrared to obtain laminarin E with yield of 55.7%.
(3) Dissolving laminarin E in water to prepare a solution with the concentration of 1 percent (mass concentration), adding 4mol/L hydrochloric acid to ensure that the final concentration is 0.5mol/L, stirring and degrading for 2 hours in a water bath with the temperature of 80 ℃, then neutralizing with sodium hydroxide, concentrating, and pouring into absolute ethyl alcohol with the same volume. Centrifuging, and oven-drying with infrared to obtain laminarin F with yield of 48.5%.
(4) Dissolving laminarin F (0.5G) in 4 ml of 0.2M ammonium bicarbonate, loading to column chromatography with Bio-Gel P-10 as a carrier, taking 0.2M ammonium bicarbonate as a mobile phase, collecting an elution component, concentrating the elution component, loading to column chromatography with Sephadex G10 as a carrier for desalting, concentrating an eluent, and freeze-drying to obtain the fucogalactomannan uronic acid polysaccharide (DSFGG-SJ) from the kelp, wherein the yield is 85.3%. The DSFGG-SJ component was analyzed for chemical components, monosaccharide ratio, sulfate content and molecular weight, and the results are shown in Table 1 below.
Example 3: preparation of fucogalactomannoglucuronan (SFGG-SH) derived from Sargassum hemiphyllum
(1) Extracting 1kg of half-leaf gulfweed with 15 times of water in a pressure cooker for 3 hours, and controlling the temperature to be 100-105 ℃; extracting for 1 time. Removing algae, mixing extractive solutions, vacuum filtering with diatomite, concentrating the filtrate, adding 2mol/L MgCl2Making MgCl2Adding anhydrous ethanol to final weight concentration of 20% to obtain ethanol final weight concentration of 0.05mol/L, stirring to obtain precipitate, centrifuging to remove precipitate, dialyzing with 3500Da molecular weight cutoff dialysis bag for 2 days, concentrating, and dialyzingThe inner solution is freeze-dried to obtain the sargassum semilaevis polysaccharide A, and the yield is 4.6%.
(2) Dissolving sargassum semialatum polysaccharide A in water to prepare a solution with the concentration of 1% (mass concentration), adding 4mol/L hydrochloric acid to enable the final concentration to be 0.1mol/L, stirring for 2 hours in a water bath at 80 ℃, then neutralizing with sodium hydroxide, concentrating, and pouring into anhydrous ethanol with the same volume. Centrifuging, and oven drying with infrared to obtain Sargassum hemiphyllum polysaccharide B with yield of 60.5%.
(3) Dissolving sargassum semialatum polysaccharide B in water to prepare a solution with the concentration of 1% (mass concentration), adding 4mol/L hydrochloric acid to enable the final concentration to be 0.5mol/L, stirring and degrading in a water bath at 80 ℃ for 2 hours, then neutralizing with sodium hydroxide, concentrating, and pouring into anhydrous ethanol with the same volume. Centrifuging, and oven-drying with infrared to obtain Sargassum hemiphyllum polysaccharide SFGG-SH with yield of 43.3%. The SFGG-SJ components were analyzed for chemical components, monosaccharide ratios, sulfate content and molecular weight, and the results are shown in table 1 below.
Example 4: preparation of fucogalactomannosyluronic acid polysaccharide (SFGG-SF) derived from Cyrtymenia Sparsa
(1) Extracting 1kg of sargassum fusiforme in a pressure cooker for 3 hours by using water with the mass 15 times, and controlling the temperature to be 100-105 ℃; extracting for 1 time. Removing algae, mixing extractive solutions, vacuum filtering with diatomite, concentrating the filtrate, adding 2mol/L MgCl2Making MgCl2Adding anhydrous ethanol to final mass concentration of 0.05mol/L to make ethanol final weight concentration 20%, stirring to generate precipitate, centrifuging to remove precipitate, dialyzing with 3500Da molecular weight cutoff dialysis bag for 2 days, concentrating the solution in dialysis bag, and lyophilizing to obtain Sargassum fusiforme polysaccharide A with yield of 3.5%.
(2) Dissolving sargassum fusiforme polysaccharide A in water to prepare a solution with the concentration of 1% (mass concentration), adding 4mol/L hydrochloric acid to enable the final concentration to be 0.1mol/L, stirring for 2 hours in a water bath at 80 ℃, then neutralizing with sodium hydroxide, concentrating, and pouring into anhydrous ethanol with the same volume. Centrifuging, and oven drying with infrared to obtain Sargassum fusiforme polysaccharide B with yield of 40.5%.
(3) Dissolving Cyrtymenia Sparsa polysaccharide B in water to obtain 1% (mass concentration) solution, adding 4mol/L hydrochloric acid to make final concentration 0.5mol/L, stirring in 80 deg.C water bath for degradation for 2 hr, neutralizing with sodium hydroxide, concentrating, and adding into equal volume of anhydrous ethanol. Centrifuging, and oven drying with infrared to obtain Sargassum hemiphyllum polysaccharide SFGG-SF with yield of 33.3%. The SFGG-SF components were analyzed for chemical components, monosaccharide ratios, sulfate content and molecular weight, and the results are shown in table 1 below.
Example 5: preparation of fucogalactomannosyluronic acid polysaccharide (SFGG-ST) derived from Sargassum thunbergii
(1) Extracting 1kg of sargassum thunbergii with 15 times of water in a pressure cooker for 3 hours, and controlling the temperature to be 100-105 ℃; extracting for 1 time. Removing algae, mixing extractive solutions, vacuum filtering with diatomite, concentrating the filtrate, adding 2mol/L MgCl2Making MgCl2Adding anhydrous ethanol to final mass concentration of 0.05mol/L to make ethanol final weight concentration 20%, stirring to generate precipitate, centrifuging to remove precipitate, dialyzing with 3500Da molecular weight cutoff dialysis bag for 2 days, concentrating solution in dialysis bag, and lyophilizing to obtain Sargassum thunbergii polysaccharide A with yield of 5.8%.
(2) Dissolving sargassum thunbergii polysaccharide A in water to prepare a solution with the concentration of 1% (mass concentration), adding 4mol/L hydrochloric acid to enable the final concentration to be 0.1mol/L, stirring for 2 hours in a water bath at 80 ℃, then neutralizing with sodium hydroxide, concentrating, and pouring into anhydrous ethanol with the same volume. Centrifuging, and oven drying with infrared to obtain Sargassum thunbergii polysaccharide B with yield of 37.3%.
(3) Dissolving sargassum thunbergii polysaccharide B in water to prepare a solution with the concentration of 1% (mass concentration), adding 4mol/L hydrochloric acid to enable the final concentration to be 0.5mol/L, stirring and degrading in a water bath at 80 ℃ for 2 hours, then neutralizing with sodium hydroxide, concentrating, and pouring into anhydrous ethanol with the same volume. Centrifuging, and oven drying with infrared to obtain Sargassum thunbergii polysaccharide SFGG-ST with yield of 31.2%. The SFGG-ST fraction was analyzed for chemical composition, monosaccharide ratio, sulfate content and molecular weight, and the results are shown in table 1 below.
Example 6: preparation of fucogalactomannoglucuronan (SFGG-UP) derived from Undaria pinnatifida
(1) Extracting 1kg of undaria pinnatifida in a pressure cooker for 3 hours by using 15 times of water, and controlling the temperature to be 100-105 ℃; extracting for 1 time. Removing algae, mixing extractive solutions, and adding diatomiteFiltering, concentrating the filtrate, adding 2mol/L MgCl2Making MgCl2Adding anhydrous ethanol to final mass concentration of 0.05mol/L to obtain ethanol with final weight concentration of 20%, stirring to obtain precipitate, centrifuging to remove precipitate, dialyzing with 3500Da dialysis bag for 2 days, concentrating the solution in the dialysis bag, and lyophilizing to obtain Undaria pinnatifida polysaccharide A with yield of 2.5%.
(2) Dissolving Undaria pinnatifida polysaccharide A in water to obtain a solution with a concentration of 1% (mass concentration), adding 4mol/L hydrochloric acid to make the final concentration of 0.1mol/L, stirring for 2 hours in 80-degree water bath, neutralizing with sodium hydroxide, concentrating, and pouring into anhydrous ethanol with the same volume. Centrifuging, and oven drying with infrared to obtain Undaria pinnatifida polysaccharide B with yield of 38.2%.
(3) Dissolving Undaria pinnatifida polysaccharide B in water to obtain a solution with a concentration of 1% (mass concentration), adding 4mol/L hydrochloric acid to make the final concentration of 0.5mol/L, stirring and degrading in 80 ℃ water bath for 2 hours, then neutralizing with sodium hydroxide, concentrating, and pouring into anhydrous ethanol with the same volume. Centrifuging, and oven drying with infrared to obtain Undaria pinnatifida polysaccharide SFGG-UP with yield of 30.8%. The SFGG-UP component was analyzed for chemical components, monosaccharide ratio, sulfate content and molecular weight, and the results are shown in Table 1 below.
Example 7: preparation of fucogalactomannoglucuronan (SFGG-SHO) derived from Sargassum muticum
(1) Extracting 1kg of sargassum muticum with 15 times of water in a pressure cooker for 3 hours, and controlling the temperature at 100 ℃ and 105 ℃; extracting for 1 time. Removing algae, mixing extractive solutions, vacuum filtering with diatomite, concentrating the filtrate, adding 2mol/L MgCl2Making MgCl2Adding anhydrous ethanol to a final mass concentration of 0.05mol/L to make the final weight concentration of ethanol be 20%, stirring to generate precipitate, centrifuging to remove precipitate, dialyzing with a dialysis bag with molecular weight cutoff of 3500Da for 2 days, concentrating the solution in the dialysis bag, and lyophilizing to obtain Sargassum muticum polysaccharide A with yield of 1.8%.
(2) Dissolving sargassum muticum polysaccharide A in water to prepare a solution with the concentration of 1% (mass concentration), adding 4mol/L hydrochloric acid to enable the final concentration to be 0.1mol/L, stirring for 2 hours in a water bath at 80 ℃, then neutralizing with sodium hydroxide, concentrating, and pouring into anhydrous ethanol with the same volume. Centrifuging, and oven-drying with infrared to obtain Sargassum muticum polysaccharide B with yield of 43.2%.
(3) Dissolving sargassum muticum polysaccharide B in water to prepare a solution with the concentration of 1% (mass concentration), adding 4mol/L hydrochloric acid to enable the final concentration to be 0.5mol/L, stirring and degrading in a water bath at 80 ℃ for 2 hours, then neutralizing with sodium hydroxide, concentrating, and pouring into anhydrous ethanol with the same volume. Centrifuging, and baking by infrared to obtain sargassum muticum polysaccharide SFGG-SHO with the yield of 35.2%. The SFGG-SHO components were analyzed for chemical components, monosaccharide ratios, sulfate content and molecular weight, and the results are shown in table 1 below.
Example 8: preparation of fucogalactomannoglucuronan (SFGG-SN) derived from Sargassum Ammoniacus
(1) Extracting 1kg of sargassum sal ammoniac with 15 times of water in a pressure cooker for 3 hours, and controlling the temperature at 100-; extracting for 1 time. Removing algae, mixing extractive solutions, vacuum filtering with diatomite, concentrating the filtrate, adding 2mol/L MgCl2Making MgCl2Adding anhydrous ethanol to final mass concentration of 0.05mol/L to obtain ethanol with final weight concentration of 20%, stirring to obtain precipitate, centrifuging to remove precipitate, dialyzing with 3500Da molecular weight cutoff dialysis bag for 2 days, concentrating the solution in the dialysis bag, and lyophilizing to obtain Sargassum Ammoniacus polysaccharide A with yield of 5.5%.
(2) Dissolving sargassum sal ammoniac polysaccharide A in water to prepare a solution with the concentration of 1% (mass concentration), adding 4mol/L hydrochloric acid to enable the final concentration to be 0.1mol/L, stirring for 2 hours in a water bath at 80 ℃, then neutralizing with sodium hydroxide, concentrating, and pouring into anhydrous ethanol with the same volume. Centrifuging, and oven-drying with infrared to obtain Sargassum salmianum polysaccharide B with yield of 50.2%.
(3) Dissolving sargassum sal ammoniac polysaccharide B in water to prepare a solution with the concentration of 1% (mass concentration), adding 4mol/L hydrochloric acid to enable the final concentration to be 0.5mol/L, stirring and degrading for 2 hours in a water bath at 80 ℃, then neutralizing with sodium hydroxide, concentrating, and pouring into anhydrous ethanol with the same volume. Centrifuging, and oven-drying with infrared to obtain Sargassum salmianum polysaccharide SFGG-SN with yield of 41.3%. The SFGG-SN components were analyzed for chemical components, monosaccharide ratios, sulfate content and molecular weight, and the results are shown in Table 1 below.
Example 9: preparation of fucogalactomannoglucuronan (SFGG-SI) derived from Sargassum integerrimum
(1) Extracting 1kg of sargassum integerrimum in 15 times of water in a pressure cooker for 3 hours, and controlling the temperature to be 100-105 ℃; extracting for 1 time. Removing algae, mixing extractive solutions, vacuum filtering with diatomite, concentrating the filtrate, adding 2mol/L MgCl2Making MgCl2The final mass concentration is 0.05mol/L, and simultaneously absolute ethyl alcohol is added to make the final weight concentration of the ethyl alcohol be 20%, the mixture is stirred to generate precipitate, the precipitate is removed by centrifugation, dialyzed for 2 days by a dialysis bag with the molecular weight cutoff of 3500Da, the solution in the dialysis bag is concentrated, and freeze-dried to obtain the sargassum integrifolium polysaccharide A, and the yield is 4.2%.
(2) Dissolving sargassum integrifolium polysaccharide A in water to prepare a solution with the concentration of 1% (mass concentration), adding 4mol/L hydrochloric acid to ensure that the final concentration is 0.1mol/L, stirring for 2 hours in a water bath at 80 ℃, then neutralizing with sodium hydroxide, concentrating, and pouring into absolute ethyl alcohol with the same volume. Centrifuging, and oven drying with infrared to obtain Sargassum integerrimum polysaccharide B with yield of 46.1%.
(3) Dissolving sargassum integrifolium polysaccharide B in water to prepare a solution with the concentration of 1% (mass concentration), adding 4mol/L hydrochloric acid to ensure that the final concentration is 0.5mol/L, stirring and degrading in a water bath at 80 ℃ for 2 hours, then neutralizing with sodium hydroxide, concentrating, and pouring into anhydrous ethanol with the same volume. Centrifuging, and oven drying with infrared to obtain Sargassum integrifolia polysaccharide SFGG-SI with yield of 39.8%. The SFGG-SI components are analyzed for chemical components, monosaccharide ratio, sulfate radical content, molecular weight and the like, and the results are shown in the following table 1.
Example 10: preparation of fucogalactomannoglucuronan (SFGG-SM) derived from Sargassum muelleri
(1) Extracting 1kg of gulfweed with 15 times of water in a pressure cooker for 3 hours, and controlling the temperature at 100-105 ℃; extracting for 1 time. Removing algae, mixing extractive solutions, vacuum filtering with diatomite, concentrating the filtrate, adding 2mol/L MgCl2Making MgCl2Adding anhydrous ethanol to final weight concentration of 20% to obtain ethanol final weight concentration of 0.05mol/L, stirring to obtain precipitate, centrifuging to remove precipitate, dialyzing with 3500Da molecular weight cutoff dialysis bag for 2 days, concentrating, and dialyzingThe solution is freeze-dried to obtain the sargassum morganii polysaccharide A, and the yield is 5.2%.
(2) Dissolving sargassum morganii polysaccharide A in water to prepare a solution with the concentration of 1% (mass concentration), adding 4mol/L hydrochloric acid to ensure that the final concentration is 0.1mol/L, stirring for 2 hours in a water bath at 80 ℃, then neutralizing with sodium hydroxide, concentrating, and pouring into anhydrous ethanol with the same volume. Centrifuging, and oven-drying with infrared to obtain Sargassum muelleri polysaccharide B with yield of 37.3%.
(3) Dissolving sargassum morganii polysaccharide B in water to prepare a solution with the concentration of 1% (mass concentration), adding 4mol/L hydrochloric acid to ensure that the final concentration is 0.5mol/L, stirring and degrading in a water bath at 80 ℃ for 2 hours, then neutralizing with sodium hydroxide, concentrating, and pouring into anhydrous ethanol with the same volume. Centrifuging, and oven-drying with infrared to obtain Sargassum muelleri polysaccharide SFGG-SM with yield of 35.2%. The SFGG-SM fraction was analyzed for chemical components, monosaccharide ratios, sulfate content and molecular weight, and the results are shown in table 1 below.
Example 11: effect of fucogalactomannosyluronic acid polysaccharide (SFGG) derived from brown algae on MIN6 cell viability
MIN6 is mouse islet beta cell line, has good insulin secretion function, and is a common cell line for researching islet function. After islet beta cells MIN6 were pretreated with hydrogen peroxide, 100. mu.g/ml SFGG from examples 1-10 was added for drug treatment for 24h, and CCK8 was used to test cell viability. Table 2 summarizes the effect of SFGG on improving the reduction in cell viability of MIN6 caused by hydrogen peroxide in examples 1-10.
TABLE 2 MIN6 cell viability following different SFGG treatments
Example 12: brown algae-derived fucogalactomannosyluronic acid polysaccharide (SFGG) for delaying MIN6 cell aging
After MIN6 cells were pretreated with hydrogen peroxide, SFGG was added at a concentration of 100. mu.g/ml in examples 1-10 for drug treatment for 24h, as shown in FIG. 1, staining with senescence-associated galactosidase SA-. beta. -gal clearly shows that SFGG can significantly reduce senescent cells.
Example 13: laminaria japonica-derived fucogalactomannosyluronic acid polysaccharide (SFGG-SJ) capable of enhancing cell viability of MIN6
After MIN6 cells were pretreated with hydrogen peroxide, different concentrations of the polysaccharides of example 1 were added for drug treatment for 24h, and the results in FIG. 2 show that different concentrations of SFGG-SJ have no toxic effect on cells; following hydrogen peroxide treatment, MIN6 cell viability was improved as the concentration of SFGG-SJ increased.
Example 14: fucogalactomannosyluronic acid polysaccharide (SFGG-SJ) derived from herba Zosterae Marinae for delaying MIN6 cell aging
As shown in FIG. 3, further examination of the cell cycle revealed that SFGG-SJ could improve H2O2The resulting cell cycle changes, the interphase of the cell cycle is divided into three phases, namely, a DNA synthesis early phase (G1 phase), a DNA synthesis phase (S phase) and a DNA synthesis late phase (G2 phase):
(1) the G1 phase (first gap) is a period from mitosis to before DNA replication, also called the pre-synthetic phase, during which RNA and ribosomes are mainly synthesized. The stage is characterized by active metabolism, rapid synthesis of RNA and protein, and significant increase of cell volume. The main meaning of this phase is to prepare the next stage S phase for DNA replication with respect to material and energy.
(2) The S phase (synthesis) is the DNA synthesis phase, during which, in addition to DNA synthesis, also histone proteins are synthesized. The enzymes required for DNA replication are all synthesized at this stage.
(3) The G2 phase (second gap) is a late stage of DNA synthesis and is a preparatory phase to mitosis. During this period, DNA synthesis is terminated, and RNA and proteins including tubulin and maturation-promoting factors are synthesized in large quantities.
As is clear from the results shown in FIG. 3, it was found that the addition of SFGG-SJ improved H2O2The cell cycle is changed, thereby achieving the effect of repairing.
Example 15: laminaria japonica-derived fucogalactomannosyluronic acid polysaccharide (SFGG-SJ) improves the expression of senescence-associated proteins in MIN6 cells
In normal human cells, inactivation of the p53 or p21 gene can prolong the replicative life of the cells, suggesting that the pathway is closely related to cellular senescence. p53 mediates the response of cells to DNA damage, p53 mediates both replicative senescence caused by telomere shortening and premature senescence caused by stress, and its downstream target molecule is p 21. p21 is an inhibitor of cyclin-dependent protein kinases and inhibits the cell cycle, causing the cell to enter irreversible growth arrest. p21 is a broad-spectrum cell cycle inhibitor, and can cause G1 phase arrest and G2 phase arrest by specifically inhibiting the protein kinase activity of cyclinD1-CDK4/CDK6, cyclinE-CDK2 and cyclinA-CDK 2.
Recent studies have found that p16INK4a has another important role in promoting the development of aging by inducing a decline in the function of stem cells in older mammals, as demonstrated at least in the bone marrow, forebrain, islet cells and keratinocytes. Treatment H with SFGG-SJ in example 12O2Model of induced aging. The senescence-associated proteins p53, p21, p16 also decreased with increasing concentration. From the above, it can be shown that SFGG-SJ can delay the aging of MIN6 cells with the increase of concentration. Also the QPCR results show that the senescence-associated metabolic phenotype is also restored.
As shown in FIG. 4, Western blot examined the expression of senescence-associated proteins p53, p21, and p16in cells, and the results showed that SFGG-SJ treatment restored senescence caused by H2O2, and that the senescence-associated proteins p53, p21, and p16 also decreased with increasing concentration.
As shown in fig. 5, qPCR detected senescence-associated secretory phenotypes such as IL-1 β, TNFa, CXCL4 and CXCL10 in cells, and it was found that the senescence-associated metabolic phenotype was also restored.
Example 16: laminaria japonica-derived fucogalactomannosyluronic acid polysaccharide (SFGG-SJ) improves function of MIN6 cell
Treatment H with SFGG-SJ in example 12O2The aging model elicited that the intracellular insulin levels increased with increasing concentrations of SFGG-SJ. Therefore SFGG-SJ can reverse H2O2Resulting in damage to insulin synthesis in islet cells.
DAPI (4', 6-diamidino-2-phenylindole): is a fluorescent dye capable of strongly binding to DNA.
Insulin is a protein hormone secreted by the beta cells of the islets of langerhans in the pancreas stimulated by endogenous or exogenous substances such as glucose, lactose, ribose, arginine, glucagon, etc.
Merge: the two fluorescence images were synthesized.
As shown in FIG. 6, intracellular insulin levels were significantly enhanced with increasing concentration of SFGG-SJ (25 → 200), while H2O2Can cause the damage of insulin synthesis, and indicates that SFGG-SJ can reverse/repair H2O2Resulting in damage to insulin synthesis in islet cells.
Claims (10)
1. The application of the sulfated fucogalactomannoglucuronan polysaccharide derived from brown algae in anti-aging is characterized in that the sulfated fucogalactomannoglucuronan polysaccharide derived from brown algae achieves the aim of anti-aging by enhancing cell activity, delaying cell aging, improving the expression of aging-related proteins of islet beta cells and improving the functions of the islet beta cells.
2. The use of claim 1, wherein the enhancing cell viability is enhancing the viability of a beta cell line.
3. The use of claim 1, wherein the retarding of cellular senescence is repairing cell cycle changes.
4. The use of claim 1, wherein said improvement in the expression of senescence-associated proteins in islet beta cells is a reduction in the expression of senescence-associated proteins p53, p21, and p16, while alleviating the senescence-associated secretory phenotypes IL-1 beta, TNFa, CXCL4, and CXCL 10.
5. The use of claim 1, wherein the improvement in islet beta cell function is repair of islet cell impairment of insulin synthesis.
6. The use of claim 1, wherein the sulfated fucgalactomannoglucuronan polysaccharide has the following characteristics: the main components of the beverage comprise monosaccharide including mannose, glucuronic acid, fucose and galactose, wherein the content of hydrolyzed sulfate groups is 2% -12%, the content of fucose is 3% -10%, and the content of uronic acid is 20% -40%.
The sulfated fucalactoglucuronan polysaccharide has the following structural characteristics:
(1) the component sugar comprises: mannose, glucuronic acid, galactose, fucose;
(2) mannose and glucuronic acid are linked by an alpha (1 → 4) glycosidic bond; glucuronic acid and mannose are linked by a β (1 → 2) glycosidic bond;
(3) the glucuronic acid and the glucuronic acid are connected by a beta (1 → 3) glycosidic bond;
(4) galactose is mainly connected by beta (1 → 6) glycosidic bond;
(5) fucose is connected with alpha (1 → 3) glycosidic bond, C2 or C4 site is sulfated; or mannose linked in a branched form by an alpha (1 → 3) glycosidic linkage;
(6) comprises the following structural units (I), (II), (III) and (IV) which are alternately connected with each other;
wherein R is1Is H or a sulfate radical, R2Is a sulfated fucose residue.
7. The use of claim 1, wherein the sulfated fucalachitomanniuronic acid polysaccharide is derived from marine brown algae including one or more of kelp, undaria pinnatifida, gulfweed, sargassum thunbergii, gulfweed, sargassum fusiforme, sargassum sal, sargassum morsella, and sargassum muticum.
8. The use of claim 1, wherein the sulfated fucgalactomannoglucuronan polysaccharide has a molecular weight of 1kD to 1000 kD.
9. The use of claim 1, wherein the method of use is the administration of an effective amount of a sulfated fucgalactomannoglucuronan as a medicament in a pharmaceutically acceptable carrier or dosage form.
10. A preparation method of sulfated fucogalactomannoglucuronan polysaccharide derived from brown algae is characterized by comprising the following steps: extracting water-soluble polysaccharide from marine brown algae by water extraction; and finally, degrading by using high-concentration acid and precipitating by using alcohol to obtain the water-soluble polysaccharide with stable strong acid.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115417934A (en) * | 2022-09-02 | 2022-12-02 | 威海迪普森生物科技有限公司 | Preparation method of kelp extract with high fucooligosaccharide content |
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| CN115844928A (en) * | 2022-12-16 | 2023-03-28 | 中国科学院上海药物研究所 | Seaweed sulfated polysaccharide and preparation method and application thereof |
| CN115844928B (en) * | 2022-12-16 | 2024-03-01 | 中国科学院上海药物研究所 | Seaweed sulfated polysaccharide and preparation method and application thereof |
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