CN113278091A - Porphyridium polysaccharide and preparation method and application thereof - Google Patents
Porphyridium polysaccharide and preparation method and application thereof Download PDFInfo
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- CN113278091A CN113278091A CN202110725721.9A CN202110725721A CN113278091A CN 113278091 A CN113278091 A CN 113278091A CN 202110725721 A CN202110725721 A CN 202110725721A CN 113278091 A CN113278091 A CN 113278091A
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- porphyridium
- polysaccharide
- culture
- porphyridium polysaccharide
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Classifications
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- 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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- 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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- C—CHEMISTRY; METALLURGY
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Abstract
The invention relates to the technical field of plant polysaccharide, and particularly relates to porphyridium polysaccharide and a preparation method and application thereof. The polysaccharide in the porphyridium comprises guluronic acid, mannose, ribose, glucuronic acid, glucosamine, glucose, galactose, xylose, arabinose and L-fucose, has no influence on the survival rate of cells within the range of 12.5-400 mu g/mL, can promote the phagocytic capacity of macrophages, can promote the release of nitric oxide when acting on the macrophages alone, and can increase the secretion of IL-6 and TNF-alpha. After the inflammation is induced by LPS, the porphyridium polysaccharide groups with different concentrations can reduce the release of NO in inflammatory cells, and the porphyridium polysaccharide groups with different concentrations can obviously reduce the release of IL-6 and TNF-alpha in the inflammatory cells. Therefore, the porphyridium polysaccharide has good anti-inflammatory effect and can be widely applied to the fields of medicines and health care products.
Description
Technical Field
The invention relates to the technical field of plant polysaccharide, and particularly relates to porphyridium polysaccharide and a preparation method and application thereof.
Background
Porphyridium (Porphyridium) is a marine unicellular alga belonging to Rhodophyta, Rhodophyceae, Porphyridiales, which has strong environmental adaptability and rapid reproductive ability, and can produce many bioactive substances such as phycobiliprotein, polyunsaturated fatty acid and extracellular polysaccharide substance as a relatively original unicellular alga of Rhodophyceae. The porphyridium cell can accumulate 20% -50% biomass polysaccharide, the polysaccharide is a polymer formed by monosaccharides such as xylose, glucose and galactose, and the polysaccharide has unique colloid performance and high viscosity, and has a structure similar to algin and brown algae starch. However, in recent years, studies on algal exopolysaccharides mainly include their use as adsorbents, because of their advantages of good adsorption ability of algal cells, fast adsorption speed and low possibility of causing secondary pollution; for example, CN105664862A discloses an adsorbent for extracellular polysaccharide of porphyridium and a preparation method thereof, and CN104591334A discloses a method for adsorbing metal ions by extracellular polysaccharide of porphyridium. Based on this, the development of the porphyridium polysaccharide is necessary to research the performance of other fields.
Disclosure of Invention
The invention aims to provide porphyridium polysaccharide, a preparation method thereof and application thereof in anti-inflammatory and/or immunoregulation medicaments. The research on the anti-inflammatory activity of the polysaccharide component in the porphyridium is carried out after the polysaccharide component is subjected to targeted extraction, and the result shows that the porphyridium polysaccharide can play the anti-inflammatory and/or immunoregulation role, and the porphyridium polysaccharide can be widely applied to the fields of medicines and health care products.
In one technical scheme of the invention, the porphyridium polysaccharide comprises guluronic acid, mannose, ribose, glucuronic acid, glucosamine, glucose, galactose, xylose, arabinose and L-fucose.
Further, the average molecular weight of the porphyridium polysaccharide is 4467770Da, and the proportion of guluronic acid, mannose, ribose, glucuronic acid, glucosamine, glucose, galactose, xylose, arabinose and L-fucose is 0.26%, 2.48%, 3.28%, 1.82%, 1.7%, 29.35%, 18.21%, 40%, 1.57% and 0.97% by mass fraction.
According to the second technical scheme, the preparation method of the porphyridium polysaccharide adopts porphyridium as a raw material, and the porphyridium polysaccharide is obtained by dialysis and concentration of obtained algae liquid after amplification culture.
Further, the method specifically comprises the following steps:
and (3) amplification culture of porphyridium: after activated culture, the porphyridium is inoculated in an f/2 liquid culture medium for shake culture for 7-10d, transferred into an f/2 seawater culture medium for continuous culture for 7 days, then expanded culture is carried out for 10-15d, algae liquid is collected, and the supernatant A and algae bodies are obtained after standing and centrifugation;
preparation of porphyridium polysaccharide: and (3) dialyzing the supernatant A after primary concentration and then carrying out secondary concentration, collecting the concentrated solution, adding trichloroacetic acid solution with the same volume to obtain mixed solution, standing at low temperature and centrifuging, taking the supernatant B, regulating the pH value to 7, carrying out tertiary concentration, carrying out alcohol precipitation, and centrifuging to obtain precipitate, namely crude polysaccharide.
Further, in the course of the amplification culture of porphyridium:
the activation culture specifically comprises the following steps: culturing porphyridium at 25 deg.C under 3000Lux for 24 hr;
the shake culture conditions are as follows: the temperature is 25 plus or minus 2 ℃, the illumination is 3000 plus or minus 100Lux, the rotating speed is 120 plus or minus 10r/min, and the light-dark ratio is 12h:12 h;
the amplification culture conditions are as follows: the temperature is 25 ℃ plus or minus 2 ℃, the illumination is 3000Lux plus or minus 100Lux, the rotating speed is 120r/min plus or minus 10, and the light-dark ratio is 12h:12 h.
Further, in the preparation process of the porphyridium polysaccharide:
the dialysis specifically comprises the following steps: dialyzing with running water for 24 +/-2 h, and dialyzing in single distilled water for 24 +/-2 h;
the mass fraction of trichloroacetic acid in the mixed solution is 10 percent;
the low-temperature placement specifically comprises the following steps: standing at 4 deg.C for 12-24 h;
the centrifugation is as follows: centrifuging at 8000rmp for 20 min;
the alcohol precipitation is as follows: three volumes of 95% ethanol were added for overnight precipitation.
In the third technical scheme of the invention, the porphyridium polysaccharide is applied to anti-inflammatory drugs and/or immune regulation drugs.
According to the fourth technical scheme, the anti-inflammatory drug comprises the porphyridium polysaccharide and auxiliary materials.
Compared with the prior art, the invention has the beneficial effects that:
the porphyridium polysaccharide prepared by the invention has no influence on the cell survival rate within the range of 12.5-400 mu g/mL, and can also promote the phagocytic capacity of macrophages. When the porphyridium polysaccharide acts on macrophages independently, the release of nitric oxide can be promoted, and the secretion of IL-6 and TNF-alpha is increased. After the inflammation is induced by LPS, the porphyridium polysaccharide groups with different concentrations can reduce the release of NO in inflammatory cells, and the porphyridium polysaccharide groups with different concentrations can obviously reduce the release of IL-6 and TNF-alpha in the inflammatory cells. The DTH result of the mouse shows that the porphyridium polysaccharide with different concentrations can effectively inhibit ear swelling caused by DNFB; western blot results show that porphyridium polysaccharide can reduce the expression of phosphorylated P38, phosphorylated P65 and phosphorylated I kappa B alpha protein; therefore, the porphyridium polysaccharide has good anti-inflammatory and immunoregulatory effects and can be widely applied to the fields of medicines and health care products.
The porphyridium polysaccharide itself may not have some activity or some biological activity is poor, but the structure of the porphyridium polysaccharide is modified to change the biological activity. Selenium is a trace element necessary for human body, is an active center of a plurality of antioxidant enzymes in a living body, and has the functions of removing free radicals, resisting aging and the like. Selenium-containing biomacromolecules, such as protein and polysaccharide, have anticancer, immunity enhancing, and antioxidant effects. The microalgae is used as a carrier, and inorganic selenium is converted into organic selenium through microorganisms, so that the method is simple and effective. The porphyridium can synthesize macromolecular substances such as protein, polysaccharide, lipid and the like, selenium can be combined with the protein and the lipid in the selenizing process of the porphyridium, the porphyridium is used as a carrier to prepare the selenized polysaccharide to form a polysaccharide derivative, so that the toxicity caused by supplementing inorganic selenium is avoided, the original activity of the polysaccharide is also kept, the porphyridium and the polysaccharide play a synergistic effect, and the anti-inflammatory effect is better. The method provides a foundation for converting inorganic nutrient elements into polysaccharide derivatives with higher economic value by using porphyridium as an intermediate through biotransformation.
Drawings
FIG. 1 is a schematic diagram of a process for preparing polysaccharides from supernatant of Porphyridium in step (2) in example 1 of the present invention;
FIG. 2 is a GPC chart of porphyridium polysaccharide in step (3) of example 1 of the present invention;
FIG. 3 is an infrared analysis spectrum of porphyridium polysaccharide in step (4) of example 1 of the present invention;
FIG. 4 is a graph showing the effect of different concentrations of Porphyridium polysaccharide on cell viability in step (1) of inventive example 1;
FIG. 5 is a graph showing the effect of different concentrations of porphyridium polysaccharide on the stimulation of NO release from macrophages in step (4) of example 1 of the present invention;
FIG. 6 is a graph showing the effect of porphyridium polysaccharide of different concentrations on phagocytic power of macrophages in step (5) of example 1 of the present invention;
FIG. 7 is a graph showing the effect of porphyridium polysaccharide of different concentrations on TNF-. alpha.secretion from macrophages in step (6) of example 1 of the present invention;
FIG. 8 is a graph showing the effect of porphyridium polysaccharide of different concentrations on IL-6 secretion from macrophages in step (6) of example 1 of the present invention;
FIG. 9 is a graph showing the effect of porphyridium polysaccharide on the NO secretion amount of inflammatory cells after LPS induces inflammation in step (3) of example 2 according to the present invention;
FIG. 10 is a graph showing the effect of porphyridium polysaccharide on the amount of TNF-. alpha.secreted from inflammatory cells after LPS-induced inflammation in step (4) of example 2 according to the present invention;
FIG. 11 is a graph showing the effect of porphyridium polysaccharide on the IL-6 secretion amount of inflammatory cells after LPS-induced inflammation in step (4) of example 2 according to the present invention;
FIG. 12 is a graph showing the effect of porphyridium polysaccharide on the degree of swelling of mouse ears in step (5) of example 2 according to the present invention;
FIG. 13 shows the effect of porphyridium polysaccharide on mouse body quality in step (5) of example 2 of the present invention;
FIG. 14 is a graph showing the effect of porphyridium polysaccharide on mouse spleen in step (5) of example 2 of the present invention;
FIG. 15 is a graph showing the effect of porphyridium polysaccharide on thymus in mice in step (5) of example 2 of the present invention.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
The invention relates to the configuration of the f/2 seawater culture medium used in the following examples:
the following components are prepared into mother liquor with initial concentration by using single distilled water, the corresponding amount of the mother liquor is added with the single distilled water to 1L to prepare f/2 nutrient solution, and the f/2 nutrient solution is prepared into an f/2 seawater culture medium by mixing with seawater according to a ratio of 1:1000 when in use. After the preparation, the mixture was filtered through a sterilizing filter, and sterilized seawater was added thereto, and the formulation of f/2 seawater medium is shown in tables 1 to 3.
TABLE 1 f/2 seawater culture Medium
TABLE 2 microelement mother liquor
TABLE 3 vitamin mother liquors
Example 1 porphyridium polysaccharide preparation and characterization
(1) And (3) performing expanded culture of porphyridium: firstly, the alga seeds (Porphyridium which is a gift from southern science and technology Co., Ltd. of Hengxing, Guangdong) stored in a refrigerator at 4 ℃ are placed in a constant-temperature illumination incubator (25 ℃, illumination 3000Lux) for 24h, then inoculated in 60mL f/2 liquid culture medium, and placed in an illumination shaking table for culture. The culture conditions were: the temperature is 25 ℃ plus or minus 2 ℃, the illumination is 3000Lux plus or minus 100Lux, the rotating speed is 120r/min plus or minus 10, and the light-dark ratio is 12h:12 h. After one week of culture, it was transferred to 200mL of f/2 seawater medium and the culture was continued. After they had grown well, they were transferred to 3000mL f/2 seawater medium. Finally, the culture medium is transferred to a 20L photoreactor for amplification culture (the temperature is 25 ℃ plus or minus 2 ℃, the illumination is 3000Lux plus or minus 100Lux, the rotating speed is 120r/min plus or minus 10, and the light-dark ratio is 12h:12 h). After culturing for about 10 days, collecting algae liquid, standing for precipitation, centrifuging and filtering, and respectively collecting supernatant and algae.
(2) Concentrating the supernatant in a rotary evaporator, adding the concentrated liquid into a dialysis bag, dialyzing in running water for 1 day, and dialyzing in single distilled water for 1 day. The dialyzed liquid was concentrated in a rotary evaporator. The concentrate was collected, added with an equal volume of 10% trichloroacetic acid to a final concentration and left overnight in a refrigerator at 4 ℃. 8000rmp centrifugation, 20min, supernatant was taken, the supernatant was adjusted to pH 7.0, concentrated on a rotary evaporator and then precipitated overnight by adding three volumes of 95% ethanol. Centrifuging the solution after precipitation, and taking the precipitate. Washing the precipitate with anhydrous ethanol and acetone for 2 times, and freeze drying to obtain crude polysaccharide. The preparation process route of polysaccharide in porphyridium supernatant is shown in figure 1.
(3) The average molecular weight of porphyridium polysaccharide was determined by Gel Permeation Chromatography (GPC): the chromatographic column is Ultrahydro gel TMLinear300 mm multiplied by 7.8mm, the mobile phase is 0.1% moL/L sodium nitrate water solution, the flow rate is 0.8mL/min, the column temperature is 30 ℃, and the sample injection amount is 20 mu L. Detector RID 2410. Dissolving dextran standards (2000000, 133800, 36800, 9700, 2700, 180) with different molecular weights and sample with a concentration of 0.5% with mobile phase, and detecting by sample injection. And (4) making a standard curve according to the retention time and the logarithm of the molecular weight, and substituting the retention time of the polysaccharide into the standard curve to obtain the average molecular weight of the sample.
GPC measurement results show that a relatively small peak appears within 14-16 minutes, two connected peaks appear within 21-24 minutes, and a peak appears within 24-26 minutes; the peak inversion occurred after 26 minutes as the solvent peak. The total number of the samples is 4, which indicates that the polysaccharide extracted in the step (2) is a polymer, and the molecular weight of the polymer is the average molecular weight; a larger peak appears between 24 and 26 minutes, which indicates that the content of the component is the maximum; this provides certain information for further exploring the component composition and content of each component of the porphyridium polysaccharide. The GPC chart is specifically shown in FIG. 2. Making a standard curve according to the retention time and the logarithm of the molecular weight of the standard substance to obtain a linear regression equation as follows: LogM 13.5-0.441T, and the sample retention time was substituted to obtain an average molecular weight of 4467770Da for the sample.
(4) Infrared spectrum analysis: weighing 1.0mg of dried porphyridium polysaccharide, tabletting by using KBr, carrying out infrared scanning, and determining the characteristic absorption peak of polysaccharide. At 3397.22cm-1A strong broad peak is positioned and is a stretching vibration peak of hydroxyl (-OH); at 2927.74cm-1A weaker peak appears, which is a C-H stretching vibration peak; 1431.99cm-1Is the variable angle shock peak of C-H; these two groups of C-H peaks are characteristic absorption peaks of polysaccharides. 1091.91cm-1Is the C-O stretching vibration peak; 1147.23cm-1Is the asymmetric stretching vibration peak of the sulfate radical. The infrared absorption peaks of porphyridium polysaccharide are shown in figure 3.
(5) And (3) measuring monosaccharide components and component ratios by an HPLC method: a10 mg sample of polysaccharide was weighed into a 20mL jar, 5mL of 2mol/L TFA was added, and N-charged2Sealing the tube, and hydrolyzing in an oven at 110 ℃ for 6 hours; after cooling, the lid was opened, 1mL of methanol was added to 1mL of the solution, and the mixture was heated in a 70 ℃ water bath with N2Blow drying, repeating the steps of adding methanol and using N2Blow-drying for 2 times to remove TFA; adding 1mL of 0.3mol/L NaOH solution to fully dissolve residues to obtain polysaccharide hydrolysate.
Respectively putting 400 mu L of mixed monosaccharide standard solution and polysaccharide hydrolysate into a 5mL test tube with a plug, adding 400 mu L of PMP methanol solution, and mixing uniformly by vortex; reacting for 2 hours in a water bath at 70 ℃; taking out, standing and cooling to room temperature; adding 400 mu L of 0.3mol/L HCl for neutralization (pH is 6-7); adding water 1200 μ L, adding equal volume of chloroform, vortex mixing, shaking, standing, discarding chloroform phase, and extracting for 2 times. The aqueous phase was filtered through a 0.45 μm microporous membrane (aq) and analyzed by HPLC injection. The results show that: the porphyridium polysaccharide is composed of guluronic acid, mannose, ribose, glucuronic acid, glucosamine, glucose, galactose, xylose, arabinose and L-fucose; the proportion of each component is 0.26%, 2.48%, 3.28%, 1.82%, 1.7%, 29.35%, 18.21%, 40%, 1.57% and 0.97%, wherein the content of xylose is the highest.
Effect test example 1 verification of immune-modulating Activity of Porphyridium polysaccharide
(1) Determination of RAW264.7 cell viability by porphyridium polysaccharide
Porphyridium polysaccharide is prepared into concentration gradients of 12.5 mu g/mL, 25 mu g/mL, 50 mu g/mL, 100 mu g/mL, 200 mu g/mL and 400 mu g/mL, and acts on macrophage RAW264.7 (purchased from Shanghai cell research institute) for 24h and 48h respectively, specifically: collecting cells, adjusting cell concentration to 5 × 103one/mL of the cells were inoculated into a 96-well plate at 100. mu.L/well and placed at 37 ℃ in 5% CO2Culturing in an incubator. Adding 100 μ L of diluted porphyridium polysaccharide solution with different concentrations into each hole, and continuously culturing for 24h and 48 h; the plate was removed and 100. mu.L MTT was added to each well to a final concentration of 0.5mg/mL, incubation was continued for 4h, the supernatant was discarded, 150. mu.L DMMSO was added to each well, the crystals were completely dissolved by shaking at low speed for 10min, and the absorbance at 490nm was measured.
The results are shown in figure 4, and figure 4 shows that the porphyridium polysaccharide mass concentration is within the range of 12.5-400 mu g/mL after the drug-adding culture for 24h and 48h, and has no statistical significance and no obvious toxicity to cells compared with a blank control group. Indicating that the porphyridium polysaccharide in this concentration range is safe and non-toxic to cells.
(2) Screening LPS action concentration by Griess method: collecting cells, adjusting cell concentration to 5 × 104one/mL of the cells were inoculated into a 96-well plate at 100. mu.L/well and placed at 37 ℃ in 5% CO2Culturing in an incubator. Designing a blank group, adding 100 mu L of LPS group (0.5 mu g/ml, 1 mu g/ml, 2 mu g/ml, 4 mu g/ml and 8 mu g/ml) into each hole, and continuing to culture for 24 h; pipette 50. mu.L of supernatant from each well into a new 96-well plate using Griess methodThe content of NO was measured. Thus determining the concentration of LPS action;
the result shows that LPS can effectively induce the release of cell NO, so the release amount of NO is selected to screen the action concentration of LPS.
(3) MTT assay to determine LPS effect on RAW264.7 cell viability: collecting cells, adjusting cell concentration to 5 × 103one/mL of the cells were inoculated into a 96-well plate at 100. mu.L/well and placed at 37 ℃ in 5% CO2Culturing in an incubator. A blank group was designed, and LPS group (0.5. mu.g/mL, 1. mu.g/mL, 2. mu.g/mL, 4. mu.g/mL, 8. mu.g/mL) was added with 100. mu.L per well, and continuously incubated for 24 hours, after the incubation was completed, the plate was taken out, the culture solution was removed, MTT 100. mu.L was added to a final concentration of 0.5mg/mL, incubation was continued for 4 hours, the supernatant was added with 150. mu.L of DMSO per well, and the plate was shaken at a low speed for 10min to completely dissolve the crystals, and the absorbance at 490nm was discarded.
The results show that LPS has no obvious influence on the cell viability in the set concentration range, has no statistical significance and no toxic effect on the cells compared with a blank control group, but the cell viability is slightly reduced after the concentration of 4 mug/mL.
(4) Determination of the NO content in the cell supernatants: preparation of cell concentration of 1X 104Macrophage suspension/mL, inoculated in a 96-well plate at 100. mu.L/well, placed at 37 ℃ in 5% CO2Culturing in an incubator. A blank group, an LPS group and a porphyridium polysaccharide group are designed, 100 mu L of the solution is added into each hole, after the solution is cultured for 24 hours, 50 mu L of the solution is absorbed from each hole of supernatant fluid into a new 96-hole plate, and the content of NO is measured by a Griess method.
The results are shown in fig. 5, and the LPS and the porphyridium polysaccharide with different concentrations can promote the cells to release NO, and the promotion degree is statistically significant compared with the blank group.
(5) Detection of the ability of macrophages to phagocytose neutral red: preparation of cell concentration of 1X 104Macrophage suspension/mL, inoculated in a 96-well plate at 100. mu.L/well, placed at 37 ℃ in 5% CO2Culturing in an incubator. Adding medicine according to the step (4), culturing for 24h, adding 0.05% neutral red 100 μ L/well, culturing for 5min, decanting the neutral red, washing with warm PBS for three times, adding cell lysate (absolute ethanol: glacial acetic acid ═ 1:1)200 μ L. Standing in a refrigerator at 4 ℃ for 2-3 h, measuring the light absorption value at 540nm after cell lysis, and calculating the phagocytosis index.
The results show (fig. 6): LPS and porphyridium polysaccharide with different concentrations can promote macrophages to phagocytose neutral red, and compared with a normal control group, the porphyridium polysaccharide shows significant difference in phagocytosis index within the mass concentration range of 12.5-400 mug/mL.
(6) The ELISA kit detects the release condition of the cytokine: preparation of cell concentration of 1X 104Macrophage suspension/mL, inoculated in 96-well plate at 200. mu.L/well, placed at 37 ℃ and 5% CO2Culturing in an incubator. Adding medicine according to the step (4), culturing for 24h, collecting cell supernatant, and centrifuging at 3000rmp for 10 min. Supernatants were removed and run according to mouse TNF-. alpha.and IL-6 kit instructions. And (3) carrying out dual-wavelength detection by using a microplate reader, measuring the maximum absorption wavelength at 450nm, taking 570nm as the reference wavelength, and subtracting the OD value of the reference wavelength from the OD value of the maximum wavelength at the last OD.
The results show that: the porphyridium polysaccharide and the LPS with different concentrations can promote the secretion of the TNF-alpha of the cells compared with a blank group, and have significant difference (p is less than 0.05). The secretion of TNF-alpha shows a trend of increasing firstly and then decreasing, and when the mass concentration is in the range of 12.5-50 mug/mL, the secretion quantity is increased in a dose-dependent manner; in contrast, the secretion amount is suppressed to some extent in the range of 100. mu.g/mL to 400. mu.g/mL. The secretion amount trend is shown in FIG. 7.
The effect of porphyridium polysaccharides on IL-6 secretion is different. The porphyridium polysaccharide and the LPS with different concentrations can promote the secretion of IL-6 of cells, but the secretion amount of the IL-6 tends to decrease along with the increase of the concentration of the porphyridium polysaccharide, but the difference still has statistical significance, as shown in figure 8.
The conclusion is drawn from the above: based on mouse abdominal cavity macrophage RAW264.7, the influence of porphyridium polysaccharide on the survival rate, the phagocytic capacity, the NO release capacity, the secretion capacity of cell factors TNF-alpha and IL-6 is preliminarily researched. LPS can well activate macrophages and is a common positive control drug for researching the immunoregulation effect of the macrophages, so LPS is selected as a positive control, and the regulation of the immunoregulation effect of LPS and porphyridium polysaccharide on the macrophages is jointly researched through relevant indexes of immunoregulation. The experimental results show that: when the porphyridium polysaccharide is acted for 24 hours, compared with a blank control group, the effect on the cell viability is not statistically significant, but after 48 hours of action, the cell viability is slightly reduced compared with 24 hours although the cell viability is not statistically significant compared with the blank group. Therefore, in order to eliminate the interference of the drug itself to the experiment, the drug action time taken in the subsequent content is 24 h. The results show that the porphyridium polysaccharide and the LPS with different concentrations can stimulate cells to release NO, the NO release amount of the porphyridium polysaccharide group is lower than that of the LPS group, but the release amount of the porphyridium polysaccharide group and the LPS group is higher than that of the blank group, and the statistical significance is achieved compared with the blank group (p is less than 0.05); indicating that the porphyridium polysaccharide can activate macrophages. The experimental result of the invention shows that the porphyridium polysaccharide with different concentrations can enhance the phagocytic capacity of macrophages, and the phagocytic capacity is enhanced along with the increase of the concentration of the porphyridium polysaccharide, and has significant difference (p is less than 0.05) compared with a blank group; the porphyridium polysaccharide has a concentration range of 12.5-50 mug/mL, the phagocytosis capacity is obviously enhanced, but the phagocytosis capacity is less than the enhancement effect of LPS; at concentrations of 100. mu.g/mL to 400. mu.g/mL, although the phagocytic activity increased with the increase in the concentration of porphyridium polysaccharide, the increased activity tended to be gentle and almost similar to the phagocytic activity of LPS group. Therefore, the porphyridium polysaccharide in the low concentration range can better enhance the phagocytic capacity of macrophages. Research shows that polysaccharide receptor is type III complement receptor, belongs to integrin family, and is mainly present on macrophage, natural killer cell and polymorphonuclear neutrophilic granulocyte surface. The polysaccharide binds to receptors with several characteristics: reversibility, saturation, specificity and presence of active centers. Based on this, there may be a saturation value of the interaction of porphyridium polysaccharide with macrophages, and the saturation value may be related to cell membrane surface receptors, so that the concentration is increased, and the effect of enhancing the phagocytic capacity of macrophages is not obvious. The porphyridium polysaccharide with different concentrations can stimulate macrophage to release TNF-alpha, and has significant difference compared with a blank control group (p < 0.05). However, the TNF-alpha release amount tends to increase first and then decrease, and the concentration of the porphyridium polysaccharide corresponding to the optimal release amount is 50 mug/mL. The porphyridium polysaccharide of each concentration can stimulate the release of IL-6, but the release amount of IL-6 is reduced along with the increase of the concentration of the porphyridium, but the release amount still has a significant difference (p <0.05) compared with a blank control group. The concentration of the porphyridium polysaccharide is in the interval of 12.5-100 mug/mL, the promotion effect on the release amount of IL-6 is obvious, the release amount between groups is not greatly different, and the promotion effect of the concentration in the interval of 200-400 mug/mL is obviously lower than that in the interval of 12.5-100 mug/mL. In conclusion, the concentration of the porphyridium polysaccharide is 50 mug/mL, which is the optimal concentration for promoting the release of TNF-alpha and IL-6.
Study subjects: SPF-grade male BALB/c mice (18-22g) were purchased from the Guangdong provincial animal center for medical laboratory (permit: SCXK 2013000, Production batch: NO 44007200044305), and RAW264.7 cells were purchased from Shanghai cell research institute.
(1) MTT assay for effects of dexamethasone on RAW264.7 cell viability: collecting cells, adjusting cell concentration to 5 × 103one/mL, inoculated in a 96-well plate at 100. mu.L/well, placed at 37 ℃ in 5% CO2Culturing in an incubator. Designing a blank group, continuously culturing a dexamethasone group (12.5ng/mL, 25ng/mL, 50ng/mL, 100ng/mL and 200ng/mL) for 24 hours, taking out the plate after the culture is finished, removing the culture solution, adding 100 mu L MTT with the final concentration of 0.5mg/mL, continuing culturing for 4 hours, abandoning the supernatant, adding 150 mu L DMSO into each hole, shaking at a low speed for 10min to completely dissolve crystals, and detecting the light absorption value at 490 nm.
The results show that the dexamethasone concentration is in the range of 12.5 ng/mL-200 ng/mL, and the dexamethasone concentration is not toxic to the cells and does not influence the cell survival rate compared with a blank control group.
(2) Screening dexamethasone anti-inflammatory concentration by Griess method: collecting cells, adjusting cell concentration to 5 × 104one/mL of the cells were inoculated into a 96-well plate at 100. mu.L/well and placed at 37 ℃ in 5% CO2Culturing in an incubator. A blank, LPS model, dexamethasone (12.5ng/ml, 25ng/ml, 50ng/ml, 100ng/ml, 200ng/ml) was designed to add 100. mu.L per well. Ground plug for first useProtecting cells for 2 hours by the Misongsong, and continuously culturing for 24 hours by using LPS; 50 μ L of the supernatant from each well was pipetted into a new 96-well plate and the NO content was determined by the Griess method. Thereby determining the anti-inflammatory concentration of dexamethasone.
The results show that the LPS model group has obviously increased release amount of cell NO relative to the blank group; compared with LPS groups, dexamethasone with different concentrations can reduce the release amount of NO to different degrees, and the difference has statistical significance.
(3) Determination of the NO content in the cell supernatants: preparation of cell concentration of 1X 104Macrophage suspension/mL, inoculated into 96-well plate at 100. mu.L/well, placed at 37 ℃ and 5% CO2Culturing in an incubator. A blank control group, an LPS + dexamethasone group, an LPS model group and an LPS + porphyridium polysaccharide group are designed. During the test, the LPS + dexamethasone group and the LPS + porphyridium polysaccharide group are firstly added with porphyridium polysaccharide (100 mu g/mL, 200 mu g/mL and 400 mu g/mL) with different concentrations and dexamethasone to protect cells for 2 hours, then, LPS with the final concentration of 2 mu g/mL is added for co-culture for 24 hours, 50 mu L of supernatant of each well is sucked into a new 96-well plate, and the content of NO is measured by a Griess method.
The results show that: compared with a blank control group, the amount of nitric oxide released by cells in the LPS group is obviously increased; compared with the LPS group, the porphyridium polysaccharide group and the dexamethasone group both can significantly inhibit the release of NO, and the difference has statistical significance (fig. 9).
(4) The ELISA kit detects the release condition of the cytokine: preparation of cell concentration of 1X 104Macrophage suspension/mL, inoculated in a 96-well plate at 100. mu.L/well, placed at 37 ℃ in 5% CO2Culturing in an incubator. A blank control group, an LPS + dexamethasone group, an LPS model group and an LPS + porphyridium polysaccharide group are designed. In the test, dexamethasone and porphyridium polysaccharide (100 mu g/mL, 200 mu g/mL and 400 mu g/mL) are added into the LPS + dexamethasone group and the LPS + porphyridium polysaccharide group to protect cells for 2 hours, and then LPS with the final concentration of 2 mu g/mL is added to culture for 24 hours. The cell supernatant was collected. 3000rmp centrifugation for 10min, supernatant and according to mouse TNF-alpha and IL-6 kit instructions. Performing dual-wavelength detection with microplate reader, measuring maximum absorption wavelength at 450nm, and 570nm as referenceWavelength, final OD the OD of the reference wavelength is subtracted from the OD of the maximum wavelength.
The result shows that compared with a blank group, the secretion amount of TNF-alpha and IL-6 of the cells of the LPS model group is obviously increased; compared with the model group, the porphyridium polysaccharide group can obviously inhibit the secretion of TNF-alpha and IL-6 of cells, and the differences have statistical significance (figures 10-11).
(5) Mouse delayed allergy (DTH) assay: 60 balb/c mice were randomly assigned to 6 groups (10 per group): the test results were divided into a normal control group (normal saline group), a positive control group (dexamethasone acetate group, 30mg/kg), a porphyridium polysaccharide high dose group (320mg/kg), a medium dose group (80mg/kg), a low dose group (20mg/kg) and a model group. The experimental process comprises the following steps: the administration is 0.2mL/10g by gavage and is continued for 7 days. On the first day of administration, the abdomen was depilated, and 1 hour after the second day of administration, 5% Dinitrotoluene (DNFB) was applied to the depilated site, 50. mu.L/mouse, and the same site was again applied with an equivalent amount of DNFB-enriched every other day. After 1h on day 6, 1% DNFB, 20. mu.L/mouse, was applied to both sides of the right ear and to both sides of the left ear using the corresponding solvent as a control. On day 8, the mice were sacrificed, both ears were cut off, the ears were punched with a punch having a diameter of 8mm, and weighed. Calculating the degree of ear swelling, namely the difference between the weights of the ears on two sides.
The results show that: compared with the normal saline group, the ear swelling degree of the model group mice is obviously greater than that of the normal saline group. Compared with the model group, the high, medium and low dose groups of porphyridium polysaccharide have the advantages that the ear swelling degree of mice in each group is obviously reduced, and the difference has statistical significance (p is less than 0.05). Comparative trends for each group are shown in fig. 12. The analysis of the body constitution of the mice shows that the body constitution of the mice of each group has no obvious difference and no statistical significance. Comparative trends for each group are shown in fig. 12. Comparing the spleen index and the thymus index of each test group of mice respectively, the spleen index of each test group of mice is not greatly different; the thymus indices of the experimental groups were also not significantly different and were not statistically different. Specific comparative trends are shown in fig. 14-15.
From the above, it is known that porphyridium polysaccharide has an influence on NO release from macrophages after inflammation is induced by LPS. After 24 hours of drug action, compared with a blank group, the model group is stimulated by LPS with 2 mu g/mL, and the NO release amount is remarkably increased. Compared with a model group, the NO release amount of the porphyridium polysaccharide pretreatment group can obviously inhibit the NO release; porphyridium polysaccharide can inhibit excessive TNF-alpha and IL-6 released by macrophage stimulated by LPS; compared with LPS group, the porphyridium polysaccharide group with different dosages has obviously reduced ear swelling degree; indicating that different doses of porphyridium polysaccharide are able to inhibit DNFB-induced ear swelling; meanwhile, the invention also researches the influence of the porphyridium polysaccharide on the body weight and immune organs of the mouse. As a result, it was found that the body weight of the model group mice was not significantly different from that of the normal saline group 7 days after the administration; the body weights of mice in each porphyridium polysaccharide group are not obviously different from those in the model group; the research result of the immunoregulation and anti-inflammatory effect of the porphyridium polysaccharide shows that the porphyridium polysaccharide can promote the phagocytic capacity of macrophages when acting alone, can promote the release of NO and the release of tumor necrosis factor, and shows the immune enhancement effect. However, when an LPS-induced inflammation model is established in vitro, porphyridium polysaccharide can reduce excessive NO, TNF-alpha and IL-6 generated by LPS induction, and an anti-inflammatory effect is shown. Therefore, the porphyridium polysaccharide has the bidirectional regulation function.
In addition to the above research contents, the present invention also researches the anti-inflammatory mechanism of the porphyridium polysaccharide, and the conclusion is as follows:
activation of the P38 MAPK signaling pathway promotes the production of inflammatory factors, exacerbating the inflammatory response. In the presence of inflammation, P38 is phosphorylated, thereby activating its downstream pathway and causing a series of cascade reactions. The amount of phosphorylated P38 expressed may be responsive to the degree of inflammation. Inhibition of P38 phosphorylation is effective in reducing inflammatory responses. The experimental result of P38 phosphorylated western blot shows that the expression level of phosphorylated P38 protein is obviously increased in an inflammation model LPS group compared with a blank control group; after the porphyridium polysaccharide pretreatment, compared with the LPS group, the expression level of the phosphorylated P38 protein of the porphyridium polysaccharide groups of 100 mu g/mL, 200 mu g/mL and 400 mu g/mL is obviously reduced.
The expression of phosphorylated P65 and phosphorylated I κ B α proteins was determined by western blot. The experimental result shows that compared with a blank control group, the expression level of phosphorylated P65 and phosphorylated I kappa B alpha protein in an inflammation model LPS group is obviously increased; after the porphyridium polysaccharide pretreatment, the expression quantity of phosphorylated P65 and phosphorylated I kappa B alpha protein is obviously reduced compared with that of LPS group in porphyridium polysaccharide groups of 100 mug/mL, 200 mug/mL and 400 mug/mL.
Therefore, the protein expression of phosphorylated P38, phosphorylated P65 and phosphorylated I kappa B alpha can be reduced by the porphyridium polysaccharide, and the porphyridium polysaccharide is proved to play an anti-inflammatory role by reducing the NF-kappa B and P38 MAPK pathway.
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 intended to be included therein.
Claims (7)
1. A porphyridium polysaccharide comprising guluronic acid, mannose, ribose, glucuronic acid, glucosamine, glucose, galactose, xylose, arabinose, and L-fucose.
2. The porphyridium polysaccharide of claim 1 wherein the average molecular weight of the porphyridium polysaccharide is 4467770Da, and the ratio of guluronic acid, mannose, ribose, glucuronic acid, glucosamine, glucose, galactose, xylose, arabinose, and L-fucose is 0.26%, 2.48%, 3.28%, 1.82%, 1.7%, 29.35%, 18.21%, 40%, 1.57%, and 0.97% by mass, respectively.
3. A process for preparing the porphyridium polysaccharide as claimed in any one of claims 1-2, wherein the porphyridium polysaccharide is prepared from the raw material of porphyridium through enlarging culture and dialyzing and concentrating the obtained alga liquid.
4. The method for preparing porphyridium polysaccharide according to claim 3, which comprises the following steps:
and (3) amplification culture of porphyridium: after activated culture, the porphyridium is inoculated in an f/2 liquid culture medium for shake culture for 7-10d, transferred into an f/2 seawater culture medium for continuous culture for 7 days, then expanded culture is carried out for 10-15d, algae liquid is collected, and the supernatant A and algae bodies are obtained after standing and centrifugation;
preparation of porphyridium polysaccharide: and (3) dialyzing the supernatant A after primary concentration, then carrying out secondary concentration, collecting the concentrated solution, adding trichloroacetic acid solution with the same volume to obtain mixed solution, standing at low temperature, centrifuging, taking the supernatant B, adjusting the pH value to 7, carrying out tertiary concentration, carrying out alcohol precipitation, and centrifuging to obtain precipitate, namely crude polysaccharide.
5. The method for preparing porphyridium polysaccharide according to claim 4, wherein in the course of amplification culture of porphyridium:
the activation culture specifically comprises the following steps: culturing porphyridium at 25 deg.C under 3000Lux for 24 hr;
the shake culture conditions are as follows: the temperature is 25 ℃ plus or minus 2 ℃, the illumination is 3000Lux plus or minus 100Lux, the rotating speed is 120r/min plus or minus 10, and the light-dark ratio is 12h:12 h;
the amplification culture conditions are as follows: the temperature is 25 ℃ plus or minus 2 ℃, the illumination is 3000Lux plus or minus 100Lux, the rotating speed is 120r/min plus or minus 10, and the light-dark ratio is 12h:12 h.
6. The method of claim 4, wherein the preparation process of porphyridium polysaccharide comprises:
the dialysis specifically comprises the following steps: dialyzing for 24 hours +/-2 hours in running water, and dialyzing for 24 hours +/-2 hours in single distilled water;
the mass fraction of trichloroacetic acid in the mixed solution is 10 percent;
the low-temperature placement specifically comprises the following steps: standing at 4 deg.C for 12-24 h;
the centrifugation is as follows: centrifuging at 8000rmp for 20 min;
the alcohol precipitation is as follows: three volumes of 95% ethanol were added for overnight precipitation.
7. Use of porphyridium polysaccharides according to any one of claims 1-2 in anti-inflammatory and/or immunomodulatory drugs.
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