CN113861306A - Degradation method of porphyra haitanensis polysaccharide and anti-aging activity application thereof - Google Patents
Degradation method of porphyra haitanensis polysaccharide and anti-aging activity application thereof Download PDFInfo
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- CN113861306A CN113861306A CN202111363901.3A CN202111363901A CN113861306A CN 113861306 A CN113861306 A CN 113861306A CN 202111363901 A CN202111363901 A CN 202111363901A CN 113861306 A CN113861306 A CN 113861306A
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- polysaccharide
- porphyra haitanensis
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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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- 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
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
- A61K9/0056—Mouth soluble or dispersible forms; Suckable, eatable, chewable coherent forms; Forms rapidly disintegrating in the mouth; Lozenges; Lollipops; Bite capsules; Baked products; Baits or other oral forms for animals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/20—Pills, tablets, discs, rods
- A61K9/2004—Excipients; Inactive ingredients
- A61K9/2013—Organic compounds, e.g. phospholipids, fats
- A61K9/2018—Sugars, or sugar alcohols, e.g. lactose, mannitol; Derivatives thereof, e.g. polysorbates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
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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
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0036—Galactans; Derivatives thereof
Abstract
The invention belongs to the field of marine medical health care products, and relates to a porphyra haitanensis polysaccharide degradation method and application thereof in anti-aging. The method is characterized in that a degradation agent is directly added into porphyra haitanensis polysaccharide extracting solution, and uniform porphyra haitanensis polysaccharide with low molecular weight is obtained through degradation. The polysaccharide has obvious effect of delaying cell senescence, and is shown in improving the cell viability of a cell senescence model, namely human embryo lung diploid fibroblast WI-38, effectively reducing the staining rate of SA-beta-gal related to senescence, and obviously reducing the expression levels of senescence-related proteins p53 and p 21. The invention has the advantages of simple operation, stable preparation process, low manufacturing cost and the like. The invention is suitable for various health care product dosage forms such as buccal tablets and the like.
Description
The application is a divisional application with the application date of 2016, 11 and 22, the application number of 201611028733.1, and the name of 'a degradation method of porphyra haitanensis polysaccharide and anti-aging activity application thereof'.
Technical Field
The invention belongs to the field of marine medical health care products, and particularly relates to a porphyra haitanensis polysaccharide degradation method and application thereof in anti-aging.
Background
After the 21 st century, humans face a serious challenge of aging population. The proportion of the elderly over 65 years of age in the world has reached 7% of the total population. Aging is the source of 'all diseases' of the elderly. According to statistics, only 2015 is 1 to 9 months, 990 million senile dementia patients are newly added in the world. In the elderly population of 65 years and older in the United states, there are 1 Alzheimer's disease patient in every 9, and 1 in every 3 deaths of the elderly has been diagnosed with Alzheimer's disease or other forms of dementia. Therefore, finding effective ways to intervene in aging measures, explore aging mechanisms and improve the life and life quality of the elderly becomes a major topic in life science.
Porphyra haitanensis (Porphyra haitanensis), commonly known as Wucai, is a unique artificial cultivated seaweed in China. The vegetable is listed as tribute in three years as early as Song Dynasty Taiping Xingsheng, is a nutritional health food with extremely high grade, and is called longevity vegetable and Shenxiancai vegetable. Porphyra haitanensis Galactan Sulfate (GSP) mainly comprises galactose, 3, 6-lactonic galactose, sulfate and the like, accounts for 20-40% of dry weight of the Porphyra haitanensis, and is one of main components of the Porphyra haitanensis. Besides the traditional industrial value, the compound has multiple biological activities of anticoagulation, blood sugar reduction, blood fat regulation, cell immunity and humoral immunity enhancement and the like.
The porphyra haitanensis polysaccharide has large molecular weight, hundreds of thousands or even millions, and influences the biological activity to a certain extent. In order to obtain porphyra haitanensis polysaccharide with proper molecular weight, a certain method is adopted to degrade the porphyra haitanensis polysaccharide. In the current domestic published application patent, for example, the low molecular weight porphyra polysaccharide reported in CN101152207 is a high molecular weight polysaccharide sample extracted from porphyra haitanensis firstly, and then is degraded, so that the process is complex and is not suitable for production and application.
Disclosure of Invention
The invention aims to provide a preparation method for degrading stock solution to obtain low-molecular porphyra haitanensis polysaccharide aiming at the defects of the prior art. The invention uses porphyra haitanensis extract to directly add a degradation agent, and obtains low molecular weight polysaccharide through purification and separation, and the low molecular weight polysaccharide is applied to anti-aging health care products.
Another objective of the invention is to provide an anti-aging medicament or health product.
In order to achieve the purpose, the technical scheme is as follows:
the invention provides a porphyra haitanensis polysaccharide degradation method, which comprises the following degradation steps:
a method for degrading porphyra haitanensis polysaccharide is characterized by comprising the following degradation steps:
weighing dry porphyra haitanensis, crushing, putting into a microwave extraction tank, adding 30 times of distilled water for soaking for 1h, starting microwave radiation with the power of 250-; filtering the extractive solution with gauze to remove algae residue, and then filtering with diatomite to obtain clarified extractive solution; concentrating the clear extract, adding ascorbic acid to make the final concentration reach 20mM, stirring at room temperature for 1 hr, placing the extract in a dialysis bag with molecular weight cutoff of 3500Da, dialyzing with running water for two days, dialyzing with distilled water for one day, concentrating the dialysate under reduced pressure, adding anhydrous ethanol to make the final concentration of ethanol reach 75%, and collecting flocculent white precipitate; washing the precipitate with anhydrous ethanol, and oven drying to obtain low molecular weight porphyra haitanensis polysaccharide.
The invention also provides the porphyra haitanensis polysaccharide with low molecular weight prepared by the degradation method in the technical scheme.
The invention also provides application of the porphyra haitanensis polysaccharide with low molecular weight in preparation of anti-aging active drugs.
Preferably, the influence of the low molecular weight porphyra haitanensis polysaccharide on the aging process of WI-38 cells and related proteins; the concentration of the low molecular weight porphyra haitanensis polysaccharide is 10 mug/mL.
The invention also provides a health-care product prepared from the low-molecular-weight porphyra haitanensis polysaccharide by the technical scheme, wherein the health-care product consists of the low-molecular-weight porphyra haitanensis polysaccharide and a pharmaceutically acceptable carrier.
Preferably, the health-care product is in the type of polysaccharide buccal tablets.
The invention also provides a preparation method of the polysaccharide buccal tablet, which comprises the following steps: respectively pulverizing low molecular weight porphyra haitanensis polysaccharide and auxiliary materials xylitol, aspartame and menthol by a pulverizer, fully mixing, then adding distilled water as a wetting agent, stirring and granulating, drying, uniformly granulating, adding silica gel and magnesium stearate, mixing, tabletting by a tabletting machine, and packaging under a purification condition to obtain the polysaccharide buccal tablet.
The invention has the following advantages: the preparation process of the low molecular weight porphyra haitanensis polysaccharide is simple, stable and efficient, and is suitable for large-scale production. The product is used as a natural polysaccharide product derived from edible seaweed laver, has high safety and obvious effect in an anti-aging experiment.
Drawings
FIG. 1 molecular weight distribution of polysaccharide LSP in high performance gel chromatogram;
FIG. 2 polysaccharide LSP pair H2O2The effect of induced WI-38 cell viability and morphology;
FIG. 3 Effect of polysaccharide LSP on senescence-associated beta-galactosidase (SA-beta-gal);
FIG. 4 effect of polysaccharide LSPs on chromatin structure in senescent cells;
FIG. 5 Effect of polysaccharide LSP on senescent cell-associated proteins p53 and p 21.
Detailed Description
Example 1: preparation of low molecular weight porphyra haitanensis polysaccharide LSP
Weighing dry porphyra haitanensis, crushing, putting into a microwave extraction tank, and adding 30 times of distilled water for soaking for 1 h. The microwave radiation is started, the power is 250- & ltwbr/& gt 350W, and the radiation time is 10-12 min. Filtering the extractive solution with gauze to remove algae residue, and then filtering with diatomite to obtain clarified extractive solution. Concentrating the clear extractive solution, adding ascorbic acid to make final concentration reach 20mM, stirring at room temperature for 1 hr, placing the extractive solution in a dialysis bag with molecular weight cutoff of 3500Da, dialyzing with running water for two days, dialyzing with distilled water for one day, concentrating the dialysate under reduced pressure, and adding anhydrous ethanol to make ethanol final concentration reach 75% to obtain flocculent white precipitate. Washing the precipitate with absolute ethanol, and drying to obtain low molecular weight porphyra haitanensis polysaccharide LSP.
Example 2: LSP pair H2O2Effect of induced WI-38 cell viability
Young WI-38 cells (28PD) were taken at logarithmic growth phase at 3 xl 0 per well3Each cell (100. mu.L) was inoculated into a 96-well plate, cultured for 24 hours, and freshly prepared 200. mu. M H was added2O2After 2H of action, the culture medium is discarded, fresh culture medium is replaced, LSP obtained in example 1 with gradient concentration is added, each concentration is provided with 6 parallel holes, a blank control group without medicine is arranged, and H is added only2O2And a cytolytic control group without cells. After 3 days of incubation, 20. mu.L of MTT (5mg/mL) was added to each well at 37 ℃ with 5% CO2Culturing for 4h under the condition, carefully cleaning the liquid in the wells, adding 150 μ L DMSO in each well, gently shaking on a shaking table for 10min to fully dissolve the crystals, measuring the light absorbance at 570nm, and calculating the relative cell viability, wherein the cell viability of the blank group without the drug is 100%.
The result shows that the low molecular weight porphyra haitanensis polysaccharide LSP prepared by the invention can up-regulate the cell activity in a dose-dependent manner, has the best effect at 10 mu g/mL, is up-regulated by about 28 percent, and is changed to be youthful in morphology.
Example 3: effect of LSP on senescence-associated beta-galactosidase (SA-beta-gal)
Young WI-38 cells were taken at logarithmic growth phase for each well5×104Inoculating to 24-well cell culture plate, culturing for 24 hr, and adding 200 μ M H2O2Acting for 2H, replacing fresh culture solution, adding LSP obtained in example 1 with different concentrations, and adding blank control group without medicine and H only2O2The group of lesions of (1). Age-related SA- β -gal staining was performed after 5d of culture (3 d intervals with fresh culture).
The result shows that the SA-beta-gal staining positive rate of the young WI-38 cells is not more than 5 percent, H2O2The treated young WI-38 cells begin to stain after 3 days, the SA-beta-gal staining positive rate after 5 days is about 55%, the staining rate can be effectively reduced after LSP intervention, the staining rate at 10 mu g/mL is about 24%, and the effect is obvious. Simultaneous discovery, LSP intervention H2O2After the WI-38 cells are treated, the chromatin structure is subjected to characteristic changes and tends to be uniformly distributed, which indicates that the LSP has the activity of interfering the aging process.
Example 4: effect of LSP on senescence-associated proteins p53 and p21
Total WI-38 cell proteins were extracted, immunoblotted, developed according to the instructions of the ECLPLUS immunoblot kit, and images captured by the Chemi lmager 5500 gel imaging system. The results show that H2O2After the treatment, the protein levels of p53 and p21 in cells are increased, and LSP can obviously reduce the expression levels of the two proteins, and the reduction is most obvious at 10 mu g/mL.
Example 5: preparation of polysaccharide LSP buccal tablet
Pulverizing polysaccharide LSP and adjuvants (including xylitol, aspartame, and Mentholum) respectively, mixing, adding distilled water as wetting agent, stirring, granulating, drying, grading, adding silica gel and magnesium stearate, mixing, tabletting, and packaging under purifying condition to obtain LSP polysaccharide buccal tablet.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are included in the scope of the present invention.
Claims (7)
1. A method for degrading porphyra haitanensis polysaccharide is characterized by comprising the following degradation steps:
weighing dry porphyra haitanensis, crushing, putting into a microwave extraction tank, adding 30 times of distilled water for soaking for 1h, starting microwave radiation with the power of 250-; filtering the extractive solution with gauze to remove algae residue, and then filtering with diatomite to obtain clarified extractive solution; concentrating the clear extract, adding ascorbic acid to make the final concentration reach 20mM, stirring at room temperature for 1 hr, placing the extract in a dialysis bag with molecular weight cutoff of 3500Da, dialyzing with running water for two days, dialyzing with distilled water for one day, concentrating the dialysate under reduced pressure, adding anhydrous ethanol to make the final concentration of ethanol reach 75%, and collecting flocculent white precipitate; washing the precipitate with anhydrous ethanol, and oven drying to obtain low molecular weight porphyra haitanensis polysaccharide.
2. The low molecular weight porphyra haitanensis polysaccharide prepared by the degradation method of claim 1.
3. Use of low molecular weight porphyra haitanensis polysaccharides according to claim 2 for the preparation of anti-ageing active drugs.
4. The use according to claim 3, wherein the low molecular weight porphyra haitanensis polysaccharide has an effect on the WI-38 cell senescence process and associated proteins; the concentration of the low molecular weight porphyra haitanensis polysaccharide is 10 mug/mL.
5. The health product made of porphyra haitanensis polysaccharide with low molecular weight as claimed in claim 2, wherein the health product is composed of the porphyra haitanensis polysaccharide with low molecular weight and a pharmaceutically acceptable carrier.
6. The health product according to claim 5, wherein the type of the health product is polysaccharide lozenge.
7. The preparation method of the polysaccharide buccal tablet as claimed in claim 6, comprising the following steps: respectively pulverizing low molecular weight porphyra haitanensis polysaccharide and auxiliary materials xylitol, aspartame and menthol by a pulverizer, fully mixing, then adding distilled water as a wetting agent, stirring and granulating, drying, uniformly granulating, adding silica gel and magnesium stearate, mixing, tabletting by a tabletting machine, and packaging under a purification condition to obtain the polysaccharide buccal tablet.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202111363901.3A CN113861306A (en) | 2016-11-22 | 2016-11-22 | Degradation method of porphyra haitanensis polysaccharide and anti-aging activity application thereof |
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| CN201611028733.1A CN106565851A (en) | 2016-11-22 | 2016-11-22 | Degradation method of porphyra-haitanensis polysaccharide and anti-senescence activity application thereof |
| CN202111363901.3A CN113861306A (en) | 2016-11-22 | 2016-11-22 | Degradation method of porphyra haitanensis polysaccharide and anti-aging activity application thereof |
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| CN102417545B (en) * | 2011-11-03 | 2013-07-24 | 沈阳科思高科技有限公司 | Extracting method for active polysaccharide in higher plant or edible and medicinal fungi |
| CN102417548B (en) * | 2011-11-03 | 2013-07-24 | 沈阳科思高科技有限公司 | Method for extracting active polysaccharides from brown algae |
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Application publication date: 20211231 |