CN113717293B - Soft coral polysaccharide, and preparation method and application thereof - Google Patents
Soft coral polysaccharide, and preparation method and application thereof Download PDFInfo
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- CN113717293B CN113717293B CN202110909696.XA CN202110909696A CN113717293B CN 113717293 B CN113717293 B CN 113717293B CN 202110909696 A CN202110909696 A CN 202110909696A CN 113717293 B CN113717293 B CN 113717293B
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- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
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- A—HUMAN NECESSITIES
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- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
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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
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- A—HUMAN NECESSITIES
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a soft coral polysaccharide and a preparation method and application thereof, wherein the monosaccharide components in the soft coral polysaccharide are as follows: mannose, rhamnose, glucuronic acid, glucose, galactose and xylose. In the invention, the inventor extracts and separates the soft coral polysaccharide from the pod soft coral (Lobophytum sp.) for the first time, finds that the soft coral polysaccharide can promote the proliferation of RAW264.7 macrophages, promote cells to secrete NO and cytokines such as IL-1 beta, TNF-alpha, IL-6 and the like, can improve the phagocytosis capacity of the cells, can be used as a better immunomodulator, provides effective technical reference for the development and improvement of the subsequent extraction process of the coral polysaccharide, and has important research significance and development value for the utilization of the coral polysaccharide.
Description
Technical Field
The invention belongs to the field of natural product extraction, and particularly relates to a soft coral polysaccharide, and a preparation method and application thereof.
Background
The soft pod coral (Lobophytum sp.) belongs to coelenterate (Coelenterata), Coralliacea (Anthiozoa), Octaginea (Octocollia), Corallina (Alcyonacea), Alcyriaceae (Alcyonidae) in taxonomy, is a marine low-grade invertebrate, and is commonly found in tropical and subtropical sea. The pod soft coral has various colors and high ornamental value, mainly takes plankton as food in the sea, and is a seabed attached organism. The pod soft coral has almost no offensive effect, and the single plant coral can survive for hundreds of years under the condition of proper survival conditions.
The pharmaceutical use of coral is described in compendium of materia Medica. The coral reef resources in the ocean are abundant, accounting for about 22.4% of the total amount of marine organisms, and in the related technology, the research on corals is only limited to secondary metabolites, while few researches on polysaccharides are available. Polysaccharide as a biological macromolecule with wide application prospect widely exists in plants, animals and microorganisms, and is one of four basic substances forming life. Since coral, a marine organism having a medicinal value, inevitably contains polysaccharides having a strong function, it is of great importance to accelerate the development of polysaccharides in coral in the fields of medicine and food.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides a soft coral polysaccharide, a preparation method and application thereof, the soft coral polysaccharide is successfully separated for the first time, has better immunoregulatory activity, has NO obvious cytotoxicity on RAW264.7 macrophage, can obviously promote the release of NO, and improves the phagocytic capacity of cells on neutral red.
In a first aspect of the present invention, there is provided a coral polysaccharide, wherein the coral polysaccharide comprises the following monosaccharide components: mannose, rhamnose, glucuronic acid, glucose, galactose and xylose.
According to a first aspect of the invention, in some embodiments of the invention, the coral polysaccharide is a pod soft coral polysaccharide.
Because of the particularity of marine environment, the polysaccharide structure, activity and activity of the polysaccharide produced by coral are different from those of polysaccharides in terrestrial organisms, and thus, the research on the polysaccharide of marine organisms is very important.
According to a first aspect of the present invention, in some embodiments of the present invention, the monosaccharide components of the coral polysaccharides are, by mass: rhamnose: glucuronic acid: glucose: galactose: xylose (0.15-0.20): (0.20-0.25): (0.01-0.05): (98.5-99.0): (0.45-0.50): (0.25-0.30).
In some preferred embodiments of the present invention, the monosaccharide components of the coral polysaccharide are, by mass: rhamnose: glucuronic acid: glucose: galactose: xylose-0.19: 0.21: 0.05: 98.8: 0.46: 0.26.
in some preferred embodiments of the invention, the coral polysaccharide has a hydrogen nuclear magnetic resonance spectrum of:
1 H NMR(600MHz,D 2 O)δ6.77–4.68(m,64H),4.78(s,3H),4.72(s,7H),4.70(s,45H),4.89–3.67(m,80H),3.46(d,J＝136.8Hz,15H),3.07(s,4H),3.05–2.68(m,2H)。
in some preferred embodiments of the invention, the coral polysaccharide has a nuclear magnetic resonance carbon spectrum of:
13 C NMR(151MHz,D 2 O)δ99.29(d,J＝212.4Hz),77.72(s),74.11(s),74.11(s),74.03–72.65(m),71.72(s),70.66(dd,J＝243.3,116.1Hz),61.51(d,J＝288.7Hz),38.71(s)。
in some preferred embodiments of the invention, the molecular weight of the coral polysaccharide is (4.90. + -. 0.2). times.10 6 Da。
In some preferred embodiments of the invention, the coral is a polyporeThe molecular weight of the saccharide is preferably 4.90X 10 6 Da。
The second aspect of the invention provides a preparation method of coral polysaccharide, which comprises the following steps:
(1) removing lipid components and pigments in the coral sample;
(2) heating, extracting with water, filtering, collecting filtrate, precipitating with ethanol, and centrifuging to obtain precipitate;
(3) re-dissolving the alcohol precipitation precipitate obtained in the step (2), adding protease for enzymolysis, centrifuging to remove precipitated protein, adding a deproteinization reagent to remove residual protein to obtain deproteinized coral crude polysaccharide;
(4) taking deproteinized coral crude polysaccharide, separating with DEAE-52 cellulose ion exchange column, and eluting with water or sodium chloride to obtain coral polysaccharide.
According to a second aspect of the invention, in some embodiments of the invention, the method is in particular:
(1) removing lipids from coral samples:
taking a coral sample, adding petroleum ether with 4 times volume for Soxhlet extraction, and removing lipid components and part of pigment molecules in the soft coral. Siphoning for 7-9 times at 85 ℃ to finish the reaction, and placing the treated coral sample in an oven for overnight drying.
(2) Water extraction and alcohol precipitation:
and (2) drying the coral sample in the step (1) according to the material-liquid ratio of 1 g: (7-30) mL of the extract is mixed with water, extracted at 60-95 ℃ for 4 hours, extracted repeatedly for 2-5 times, filtered, and the filtrates are combined. Concentrating under reduced pressure at 60 deg.C to obtain concentrated solution, adding 4 times volume of 95% ethanol to obtain 75% ethanol precipitation solution, and standing in refrigerator at 4 deg.C overnight.
In some preferred embodiments of the present invention, the feed-to-liquid ratio in step (2) is 1 g: 20 mL.
In some preferred embodiments of the present invention, the water extraction temperature in step (2) is 90 ℃.
In some preferred embodiments of the present invention, the number of water extractions in step (2) is 2.
(3) Protein removal:
and (3) taking the alcohol precipitation solution obtained in the step (2), centrifuging at 5000rpm for 20min, removing supernatant, obtaining precipitate, namely coral crude polysaccharide, redissolving the coral crude polysaccharide by using deionized water, adding protease, and continuously stirring and reacting at 60 ℃ for 4 h. Heating at 100 ℃ for 15min after the reaction is finished, cooling to room temperature (25-30 ℃), centrifuging at 5000rpm for 20min to obtain a precipitate as coral protein, discarding the precipitate, and taking the supernatant. Adding a deproteinizing reagent (chloroform: n-butanol: 4: 1, v/v) into the supernatant according to the volume ratio of (5-15): 1 (supernatant: deproteinizing reagent: 5-15): 1, vortexing for 5min, and centrifuging at 5000rpm for 15 min. The centrifuged solution will be divided into three layers, the lower layer being an organic reagent layer, the middle layer being a protein layer, and the upper layer being a coral polysaccharide layer. And (3) taking the upper coral polysaccharide layer, adding a deproteinizing reagent according to the volume ratio of (5-15): 1 (coral polysaccharide layer: deproteinizing reagent): 5-15): 1 again, and repeatedly extracting for 4-5 times to fully remove the coral proteins in the solution. And (3) spin-drying the coral polysaccharide layer solution with the coral proteins removed completely by using a rotary evaporator, removing an organic reagent in the coral polysaccharide layer solution, re-dissolving the coral polysaccharide layer solution by using a small amount of deionized water, and dialyzing the solution for 24 hours, wherein water is replaced every three hours. Vacuum freeze drying to obtain deproteinized coral crude polysaccharide.
In some preferred embodiments of the present invention, the supernatant in step (3): deproteinizing agent and coral polysaccharide layer: the volume ratio of the deproteinizing reagent is 10: 1.
(4) DEAE-52 cellulose ion exchange column separation:
taking the deproteinized coral crude polysaccharide (400mg) obtained in the step (3), and adding water for redissolving. 5mL of the sample was applied, and the sample was separated by using a DEAE-52 cellulose ion exchange column (2.6X 30cm (diameter. times. column height) packed with NaCl solutions having concentrations of 0, 0.05, 0.1, 0.2, 0.3, 0.5, 0.8 and 1.0M in this order, and eluted by using an automatic sampler at a rate of 0.8mL/min for 10min for each 30 tubes of the concentrated solution. The product was collected, concentrated and dialyzed for 24 hours, with water changed every three hours. Vacuum freeze-drying to obtain the final product.
According to a second aspect of the invention, in some embodiments of the invention, the method further comprises:
(5) the coral polysaccharide obtained by eluting with water was separated by Sephadex column chromatography and eluted with sodium chloride.
In some preferred embodiments of the present invention, step (5) is specifically:
and (3) taking the pod soft coral polysaccharide component (60mg) eluted by the NaCl solution (namely pure water) of 0M in the step (4), and adding water for redissolving. 5mL was used as a sample, and the column was separated by Sephacryl 300HR Sephadex (1.6X 90cm, filler purchased from Shanghai-source leaf Biotech), eluted with 0.1M NaCl solution, and collected by an autosampler at 60 tubes at a sampling rate of 0.5mL/min for 10min per tube. The product was collected, concentrated and dialyzed for 24 hours, with water changed every three hours. Vacuum freeze-drying to obtain purified polysaccharides LCPs-1-A of the pod soft coral polysaccharide.
According to the second aspect of the present invention, in some embodiments of the present invention, the alcohol solution used in the alcohol extraction in step (2) comprises: ethanol solution.
In some preferred embodiments of the present invention, the alcohol solution used in the alcohol extraction in step (2) is an ethanol solution.
According to a second aspect of the invention, in some embodiments of the invention, the protease in step (2) comprises: any one of papain, trypsin, pepsin and alkaline protease.
In some preferred embodiments of the invention, the protease in step (2) is papain at a final concentration of 1 mg/mL.
According to a second aspect of the invention, in some embodiments of the invention, the deproteinizing reagent in step (2) comprises: any one of Sevag reagent, trifluorotrichloroethane and trichloroacetic acid.
In some preferred embodiments of the invention, the deproteinizing reagent in step (2) is Sevag reagent.
In a third aspect of the present invention, there is provided a use of the coral polysaccharide of the first aspect of the present invention or the coral polysaccharide produced by the production process of the second aspect of the present invention in the production of a medicament.
According to a third aspect of the invention, in some embodiments of the invention, the medicament comprises an immunomodulator.
In some preferred embodiments of the invention, the immunomodulator is an immune activator.
According to a third aspect of the invention, in some embodiments of the invention, the medicament is for improving immunity, promoting immune cell proliferation, promoting expression and secretion of immune cytokines.
In some preferred embodiments of the invention, the immune cell is a macrophage, in particular a RAW264.7 macrophage.
In some preferred embodiments of the invention, the immunocytokines include NO and IL-1 β, TNF- α, IL-6.
In the invention, the inventor discovers the Lobophytum sp polysaccharide for the first time, and separates out a purified polysaccharide LCPs-1-A with good immune activity regulation activity from the Lobophytum sp polysaccharide, wherein the purified polysaccharide is a natural and safe extraction product, can promote RAW264.7 macrophage proliferation, promote cell to secrete NO and cytokines such as IL-1 beta, TNF-alpha, IL-6 and the like, can improve the phagocytosis capacity of cells, can be used as a better immune regulator, and has important research significance and development value.
In a fourth aspect of the present invention, there is provided use of the coral polysaccharide of the first aspect of the present invention or the coral polysaccharide produced by the production process of the second aspect of the present invention in the production of a food.
The invention has the beneficial effects that:
1. the invention extracts and separates the polysaccharides LCPs-1-A from the soft-shelled coral pods (Lobophytum sp.) for the first time, and provides an effective technical reference for the extraction process of the coral polysaccharides.
2. The pod soft coral polysaccharide LCPs-1-A can promote the proliferation of RAW264.7 macrophages, promote cells to secrete cell factors such as NO and IL-1 beta, TNF-alpha, IL-6 and the like, can improve the phagocytic capacity of the cells, can be used as a better immunomodulator, and has important research significance and development value.
Drawings
FIG. 1 shows the separation results of DEAE-52 cellulose column, a crude polysaccharide of Corallium japonicum Kishinouye in the example of the present invention;
FIG. 2 shows the separation of polysaccharide fractions eluted with 0M NaCl solution on Sephacryl 300HR Sephadex in the examples of the present invention;
FIG. 3 shows the proliferation and differentiation of RAW264.7 macrophages in different administration groups, wherein A is blank control, B is LCPs-1-A administration group, C is LPS group, and D is LCPs-1-B administration group;
FIG. 4 shows MTT assay results of RAW264.7 macrophages in different administration groups;
FIG. 5 shows the measurement results of NO content of RAW264.7 macrophage cells in different administration groups;
FIG. 6 shows the results of neutral erythrophagocytosis of RAW264.7 macrophages in different administration groups;
FIG. 7 shows the expression of IL-1 β, TNF- α and IL-6 in RAW264.7 macrophages of different administration groups, wherein A is IL-1 β, B is TNF- α and C is IL-6;
FIG. 8 is a molecular weight chromatogram of purified polysaccharides LCPs-1-A from Sarcophyton glaucum;
FIG. 9 is a chromatogram of monosaccharide composition, wherein A is a chromatogram of a mixed monosaccharide standard, B is a chromatogram of LCPs-1-A, and 1 is mannose; 2 is rhamnose; 3 is glucuronic acid; 4 is galacturonic acid; 5 is glucose; 6 is galactose; 7 is xylose; 8 is arabinose; 9 is fucose;
FIG. 10 is a nuclear magnetic spectrum of LCPs-1-A, wherein A is a hydrogen spectrum of LCPs-1-A, and B is a partial enlarged view of a part of the peak value of the hydrogen spectrum of LCPs-1-A;
FIG. 11 is a carbon spectrum of purified polysaccharides LCPs-1-A from Sarcophyton glaucum;
FIG. 12 is an infrared spectrum of purified soft coral polysaccharide LCPs-1-A;
FIG. 13 is a UV scanning spectrum of LCPs-1-A of purified polysaccharides of Sarcophyton glaucum.
Detailed Description
In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to specific embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.
The experimental materials and reagents used are, unless otherwise specified, all consumables and reagents which are conventionally available from commercial sources.
Preparation of pod soft coral polysaccharide
The method comprises the following specific steps:
(1) pretreatment (removal of lipid components and impurities of pigments):
taking fresh pod-soft coral, naturally drying and crushing, and sieving with a 40-mesh sieve to obtain pod-soft coral powder. Weighing 100g of powder of the soft coral pods into a filter paper cylinder, adding petroleum ether with 4 times of volume for Soxhlet extraction, and removing lipid components and part of pigment molecules in the soft coral. Siphoning for 7-9 times at 85 ℃, finishing the reaction, and placing the treated pod soft coral powder in an oven for overnight drying.
(2) Extracting pod soft coral polysaccharide:
and (2) drying the pod soft coral powder in the step (1) according to a material-liquid ratio of 1 g: mixing 20mL of the above water, extracting at 90 deg.C for 4 hr, repeating the extraction for 2 times, filtering, and mixing the filtrates obtained after 2 times. Concentrating under reduced pressure at 60 deg.C to obtain concentrated solution, adding 4 times volume of 95% ethanol to obtain 75% ethanol precipitation solution, and standing in refrigerator at 4 deg.C overnight.
(3) Protein removal:
and (3) taking the alcohol precipitation solution obtained in the step (2), centrifuging at 5000rpm for 20min, and removing the supernatant to obtain the precipitate, namely the coral crude polysaccharide. Redissolving the crude coral polysaccharide with deionized water, adding papain (final concentration of 1mg/mL), and reacting at 60 deg.C for 4h with stirring. Heating at 100 ℃ for 15min after the reaction is finished, cooling to room temperature (25-30 ℃), centrifuging at 5000rpm for 20min to obtain a precipitate as coral protein, discarding the precipitate, and taking the supernatant. Sevag reagent (chloroform: n-butanol: 4: 1, v/v) was added to the supernatant at a volume ratio of 10:1 (supernatant: Sevag reagent: 10:1), vortexed for 5min, and centrifuged at 5000rpm for 15 min. The centrifuged solution will be divided into three layers, the lower layer being an organic reagent layer, the middle layer being a protein layer, and the upper layer being a coral polysaccharide layer. And taking the upper coral polysaccharide layer, adding Sevag reagent again according to the volume ratio of 10:1 (the coral polysaccharide layer: Sevag reagent is 10:1), and repeatedly extracting for 4-5 times to fully remove the coral proteins in the solution. And (3) spin-drying the coral polysaccharide layer solution with the coral proteins removed completely by using a rotary evaporator, removing an organic reagent in the coral polysaccharide layer solution, re-dissolving the coral polysaccharide layer solution by using a small amount of deionized water, and dialyzing the solution for 24 hours, wherein water is replaced every three hours. Vacuum freeze drying to obtain protein-removed coral polysaccharide (named LCPs).
(4) Separating and purifying coral crude polysaccharide:
taking 0.4g of the LCPs obtained in the step (3), and adding water for redissolution (the final concentration is 80 mg/mL). 5mL of the sample was applied, and the sample was separated by using a DEAE-52 cellulose ion exchange column (2.6X 30cm, a packing material purchased from Beijing Solebao technologies, Ltd.), and eluted sequentially with NaCl solutions having concentrations of 0, 0.05, 0.1, 0.2, 0.3, 0.5, 0.8, and 1.0M, and each concentration solution was collected by an autosampler at a rate of 0.8mL/min for 10 min. Detecting whether the solution contains polysaccharide by a phenol-sulfuric acid method, collecting products, respectively concentrating and dialyzing for 24 hours, and changing water every three hours. Vacuum freeze-drying to obtain the final product.
The purification result of ion exchange column chromatography is shown in FIG. 1.
It can be found that the elution with NaCl solution of 0, 0.05, 0.1, 0.2M concentration has stronger ultraviolet absorption peak, i.e. LCPs can be separated by DEAE-52 cellulose ion exchange column to obtain 4 different fractions of pod soft coral polysaccharide.
(5) Separating and purifying the pod soft coral polysaccharide with different components:
and (4) respectively taking 60mg of the pod soft coral polysaccharide obtained in the step (4) and adding water to prepare a solution with the final concentration of 12 mg/mL. 5mL of the fraction was separated by Sephacryl 300HR Sephadex column (1.6X 90cm, filler purchased from Shanghai-source leaf Biotech), eluted with 0.1M NaCl solution, and each fraction of the pod-soft coral polysaccharide was collected in 60 tubes by an autosampler at a sampling rate of 0.5mL/min for 10min per tube. Detecting whether the solution contains polysaccharide by a phenol-sulfuric acid method, collecting products, respectively concentrating, dialyzing for 24 hours, changing water every three hours, and carrying out vacuum freeze-drying to obtain the purified polysaccharide of the pod soft coral polysaccharide with different components.
The gel column chromatography purification results are shown in fig. 2.
In the step, two purified pod coral polysaccharide polysaccharides can be obtained actually, wherein the two purified pod coral polysaccharide polysaccharides are derived from the component of the pod coral polysaccharide eluted by 0M NaCl solution (i.e. pure water) in the step (4), and the two purified pod coral polysaccharide polysaccharides obtained by the separation of the gel column in the step (5) are named as LCPs-1-A (17 th to 23 th tubes) and LCPs-1-B (27 th to 33 th tubes), wherein only the LCPs-1-A has immunoregulatory activity.
Immunoregulation activity detection of pod soft coral polysaccharide purified polysaccharide
In this example, the immunomodulatory activity of purified polysaccharides from Sophora pods Coralloides was examined on RAW264.7 macrophages.
The specific detection steps comprise:
(1) resuscitation and culture of RAW264.7 macrophages:
the RAW264.7 cell strain is taken, heated in a water bath at 37 ℃, and quickly shaken to be melted. The thawed cell sap was transferred to a 15mL sterile, enzyme-sterilized centrifuge tube, 2mL complete medium containing 10% FBS was added, and centrifugation was carried out at 1000rpm for 3 min. The supernatant was removed and 1mL of medium (complete medium containing 10% FBS, purchased from Gibco, USA) was added and blown up uniformly. The mixed cell fluid was transferred to 5mL of a medium (complete medium containing 10% FBS) to resuspend the cells, and 5% CO was added at 37 ℃ 2 And incubating under the condition of saturated humidity, and carrying out passage when the cell density is 80-90%.
(2) Effect of pod coral polysaccharide purified polysaccharide on growth of RAW264.7 macrophages:
RAW264.7 macrophages in logarithmic growth phase were seeded in 96-well plates at a concentration of 2 ten thousand per well, and 100. mu.L of cell culture medium (complete medium with 10% FBS) was added per well for adherent growth for 12 h. The old medium was discarded, and 100. mu.L of a medium containing purified polysaccharides from pod soft coral polysaccharide (complete medium containing 10% FBS) at final concentrations of 0, 2, 4, 6, 8, and 10. mu.g/mL was added thereto, and the culture was continued for 24 hours. And after the incubation is finished, observing the cell morphology under a microscope, discarding the culture medium after the observation is finished, adding 20 mu L of 5mg/mL MTT solution into each hole, incubating for 4h, sucking out the supernatant, adding 200 mu L of dimethyl sulfoxide solution into each hole, shaking on a horizontal shaking table for 10min, and measuring the absorbance at 490nm by using an enzyme labeling instrument.
Blank control and positive group were set (100. mu.L of 1. mu.g/mL Lipopolysaccharide (LPS) was used).
The results are shown in FIGS. 3 and 4.
As shown in FIG. 3, the cells of the control group and LCPs-1-B did not differentiate, while the cells of both LCPs-1-A and LPS group developed antennae, and it was thus found that LCPs-1A had a certain immunomodulatory activity, whereas LCPs-1-B did not.
(3) Effect of pod coral polysaccharide purified polysaccharide on the amount of NO secreted by RAW264.7 macrophages:
RAW264.7 macrophages in logarithmic growth phase were seeded in 96-well plates at a concentration of 20 ten thousand per well, and 100. mu.L of complete medium containing 10% FBS was added to each well for adherent growth for 12 h. Discarding the old culture medium, adding 100 μ L of complete culture medium containing 10% FBS and having final concentration of 0, 2, 4, 6, 8, 10, 12 μ g/mL podophyllum japonicum polysaccharide purified polysaccharide, respectively, adding 100 μ L of culture medium containing 1 μ g/mL LPS to the positive control LPS group, culturing for 24h, sucking 50 μ L of supernatant, and detecting NO content in each group according to the instruction of NO kit (Biyuntian NO detection kit).
A blank control was set.
The results are shown in FIG. 5.
As shown in FIG. 5, the amount of NO secreted from the cells of the administered group increased with the increase of the concentration of the drug, indicating that LCPs-1A has a certain immunomodulatory activity.
(4) Neutral Red (CAS: 553-24-2) assay for the Effect of the purified polysaccharides of Sophora pods Coralloides on RAW264.7 macrophage phagocytosis:
RAW264.7 macrophages in logarithmic growth phase were seeded in 96-well plates at a concentration of 10 ten thousand per well, and 100. mu.L of complete medium containing 10% FBS was added to each well for adherent growth for 12 h. The old medium was discarded, 100. mu.L of 10% FBS-containing complete medium containing purified polysaccharides from pod soft coral polysaccharide at final concentrations of 0, 2, 4, 6, 8, 10, and 12. mu.g/mL was added to each of the positive control LPS groups, and 100. mu.L of 1. mu.g/mL LPS-containing complete medium containing 10% FBS was added to each of the positive control LPS groups, and the culture was continued for 24 hours. The old medium was discarded, rinsed 3 times with PBS, and 100. mu.L of a neutral red-PBS solution at a final concentration of 0.75mg/mL was added to each well and incubated for 45 min. The supernatant was discarded, rinsed 3 times with PBS, and 100 μ L of cell lysate (ethanol: glacial acetic acid ═ 1:1, v/v) was added and reacted at room temperature for 2 hours. The absorbance was measured at 540 nm.
A blank control was set.
The results are shown in FIG. 6.
As shown in FIG. 6, the phagocytosis of neutral red by the cells of the administered group increased with the increase of the drug concentration, indicating that LCPs-1A has a certain immunomodulatory activity.
(5) Effect of pod Soft coral polysaccharide purified polysaccharide on the expression levels of IL-1 β, TNF- α and IL-6 of RAW264.7 macrophages:
RAW264.7 macrophages in logarithmic growth phase were seeded in 6-well plates at a concentration of 150 ten thousand per well, and 2mL of complete medium containing 10% FBS was added per well for adherent growth for 12 h. The old medium was discarded, 2mL of complete medium containing 10% FBS and containing purified polysaccharides from pod soft coral polysaccharide at final concentrations of 0, 4, 8, 10, and 12. mu.g/mL, respectively, was added to the positive control LPS group, and 2mL of medium containing 1. mu.g/mL LPS was added thereto, and the culture was continued for 24 hours. The old medium was discarded, rinsed 3 times with pre-cooled PBS, total cellular RNA was extracted by Trizol method, then RNA was converted into cDNA using reverse transcription kit (FSQ-301 reverse transcription kit, purchased from Toyobo scientific and biological Co., Ltd.), and fluorescent quantitative PCR analysis was performed to determine the expression of IL-1 β, TNF- α and IL-6.
The IL-1 beta, TNF-alpha and IL-6PCR amplification primers can be those conventional in the art or those in related kits. The internal reference is GADPH.
In this example, the reference primers are: upstream: 5'-GTGTTCCTACCCCCAATGTGT-3' (SEQ ID NO. 1); downstream: 5'-ATTGTCATACCAGGAAATGAGCTT-3' (SEQ ID NO. 2).
The IL-1 beta related primers are: upstream: 5'-CTTCAGGCAGGCAGTATCACTC-3' (SEQ ID NO. 3); downstream: 5'-TGCAGTTGTCTAATGGGAACGT-3' (SEQ ID NO. 4).
TNF-alpha related primers were: upstream: 5'-GATCGGTCCCCAAAGGGATG-3' (SEQ ID NO. 5); downstream: 5'-GTGGTTTGTGAGTGTGAGGGT-3' (SEQ ID NO. 6).
The IL-6 related primers were: upstream: 5'-GATCGGTCCCCAAAGGGATG-3' (SEQ ID NO. 7); downstream: 5'-GTGGTTTGTGAGTGTGAGGGT-3' (SEQ ID NO. 8).
A blank control was set.
The results are shown in FIG. 7.
As shown in FIG. 7, the mRNA expression of IL-1. beta., TNF-. alpha., and IL-6 in the cells of the administered group increased with the increase of the drug concentration, indicating that LCPs-1A has a certain immunomodulatory activity.
In conclusion, it can be found that only LCPs-1-A in the purified polysaccharides LCPs-1-A and LCPs-1-B obtained in the above examples have immunomodulatory activity, and LCPs-1-A has good immunomodulatory activity, shows concentration dependence, and has a certain application prospect.
Characterization of purified polysaccharides LCPs-1-A from Sophora pods Coralloides
Based on the conclusions in the above examples, in this example, only LCPs-1-A with immunomodulatory activity were characterized.
(1) Molecular weight determination of LCPs-1-A:
the purified polysaccharides LCPs-1-A of the pod Sarcophyton was prepared into a 1mg/mL solution with ultrapure water, filtered through a 0.45 μm pore size membrane, and the molecular weight of the sample was measured by HPLC, using the following chromatographic conditions: the HPLC is Shimadzu LC-20AT, the chromatographic column is PolySep-GFC-P4000 chromatographic column (Phenomenex, 300 x 7.8mm), the Detector is an Evaporative Light Scattering Detector (ELSD), the set temperature of the Detector is 60 ℃, the gain value is 10, the column temperature is 35 ℃, ultrapure water is used as a mobile phase, the flow rate is 1.0mL/min, and the sample injection amount is 20 muL for detection.
The same concentration of dextran standard was used as a control to obtain a calibration curve of molecular weight and retention time.
The results are shown in FIG. 8.
It can be found that in the molecular weight chromatogram of LCPs-1-A of purified polysaccharides of Sophora pods soft coral polysaccharide, the retention time of LCPs-1-A is about 5.11min, and the retention time is substituted into a standard curve obtained from a dextran standard with the same concentration.
Wherein, the standard curve is:
lgMw＝-0.8867T+11.316；
wherein Mw represents a molecular weight; t represents a retention time.
The molecular weight of the purified polysaccharides LCPs-1-A of the pod soft coral polysaccharide is 4.90 × 10 6 Da。
(2) Analysis of the monosaccharide composition of LCPs-1-A:
the specific analysis steps are as follows:
firstly, measuring a mixed monosaccharide standard product:
respectively adding 50 μ L of 2mM monosaccharide standard (mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose, fucose) into 400 μ L of 0.3mol/L NaOH solution and 400 μ L of 0.5 mol/L1-phenyl-3-methyl-5-pyrazolone (PMP) -methanol solution, mixing, and reacting at 70 deg.C for 30 min. After the reaction was completed, 410. mu.L of 0.3mol/L HCl was added for neutralization. 1mL of chloroform was added, centrifuged at 14000rpm for 8min, the supernatant was removed, and centrifuged again at 14000rpm for 8min, 1mL of chloroform, three times, to completely remove PMP. The supernatant was collected, filtered through a 0.45 μm filter and detected by HPLC, under the following chromatographic conditions: the HPLC was Shimadzu LC-20AT, the column chromatography was Symmetry C18(Waters, 4.6X 250mm), the detector was a diode array detector (detection wavelength: 250nm), 0.05M phosphate buffer (pH 6.7) -acetonitrile (V/V: 83:17), the flow rate was 1.0mL/min, and the sample injection amount was 20. mu.L.
Derivatization of PMP of LCPs-1-A:
5mg of the purified LCPs-1-A polysaccharide from the pod soft coral polysaccharide prepared in the above example were added with 2mL of 3mol/L trifluoroacetic acid (TFA) and reacted at 120 ℃ for 6 hours. And after the reaction is finished, adding 1-2 mL of methanol, spin-drying the reaction solution, adding 1-2 mL of methanol again, spin-drying, and repeating for 3 times to fully remove TFA. Add 800 μ L deionized water for redissolution. 100 mu L of the redissolved polysaccharide solution is taken, 100 mu L of 0.3mol/L NaOH and 100 mu L of 0.5mol/L PMP methanol solution are added, and the mixture is uniformly mixed and reacts for 30min at the temperature of 70 ℃. After the reaction was completed, 105. mu.L of 0.3mol/L HCl was added for neutralization. The solution was diluted by the addition of 200. mu.L of deionized water. Add 700. mu.L chloroform, centrifuge at 14000rpm for 8min, take the supernatant, add 700. mu.L chloroform again, centrifuge at 14000rpm for 8min, repeat three times to remove the PMP sufficiently. The supernatant was collected, filtered through a 0.45 μm filter and subjected to HPLC detection under the above-mentioned chromatographic conditions.
The results are shown in FIG. 9.
Comparing the chromatogram of the mixed monosaccharide standard product and the LCPs-1-A of the purified polysaccharides of the pod coral polysaccharide to obtain the monosaccharide composition of the LCPs-1-A of the purified polysaccharides of the pod coral polysaccharide as mannose: rhamnose: glucuronic acid: glucose: galactose: xylose-0.19: 0.21: 0.05: 98.8: 0.46: 0.26 (wt%).
(3) LCPs-1-A NMR analysis:
30mg of the purified polysaccharide LCPs-1-A of the pod soft coral polysaccharide prepared in the above example was dissolved in 0.55mL of heavy water and lyophilized repeatedly three times. The measurement was carried out using a 600MHz NMR spectrometer.
The results are shown in FIGS. 10 and 11.
The hydrogen spectrum of the purified polysaccharide LCPs-1-A of the pod soft coral polysaccharide is as follows:
1 H NMR(600MHz,D 2 O)δ6.77–4.68(m,64H),4.78(s,3H),4.72(s,7H),4.70(s,45H),4.89–3.67(m,80H),3.46(d,J＝136.8Hz,15H),3.07(s,4H),3.05–2.68(m,2H)。
the carbon spectrum is:
13 C NMR(151MHz,D 2 O)δ99.29(d,J＝212.4Hz),77.72(s),74.11(s),74.11(s),74.03–72.65(m),71.72(s),70.66(dd,J＝243.3,116.1Hz),61.51(d,J＝288.7Hz),38.71(s)。
as can be seen from FIGS. 10 and 11, LCPs-1-A has two major signal peaks in the anomeric proton region, 1 the two main signal peaks in the H-NMR spectrum were 5.29ppm and 4.90ppm, respectively, from which it was concluded that LCPs-1-A may have two sugar residues. 4.70ppm of signal from solventHeavy water, while the signal peaks between 3.0ppm and 4.5ppm add up, making it difficult to distinguish from attribution and therefore are generally not analyzed. 13 In a C-NMR spectrum, the chemical range of anomeric carbon is 90-110 ppm. As can be seen from the spectrum, the anomeric carbon region mainly has two signal peaks, respectively 99.99ppm and 98.58ppm, and the result corresponds to the hydrogen spectrum. The signal peaks of the hydrogen spectrum and the carbon spectrum are combined to show that both sugar residues of the LCPs-1-A are alpha-configuration glycosidic bonds (the signal peak of the carbon spectrum belongs to the alpha-configuration glycosidic bonds between 97 ppm and 103ppm, and the result corresponds to the hydrogen spectrum).
(4) Infrared analysis of LCPs-1-A:
after the background is adjusted to zero, taking a proper amount of purified polysaccharides LCPs-1-A of the pod soft coral polysaccharide prepared in the embodiment, and using an infrared spectrometer at 4000-400 cm -1 Is scanned.
The results are shown in FIG. 12.
It can be found that LCPs-1-A is 3326cm -1 The strong and wide steamed bun peak is caused by-OH stretching vibration and belongs to a characteristic absorption peak of polysaccharide. At 2900cm -1 The nearby peak is caused by C-H stretching vibration and belongs to another characteristic absorption peak of the polysaccharide. And 1640cm -1 The peak at (a) is a water peak, indicating that the polysaccharide contains a certain amount of bound water. 1356cm -1 The peak is caused by C-O stretching vibration and C-H bending vibration. 1152cm -1 And 1078cm -1 The characteristic peak at (A) indicates that LCPs-1-A are assigned to arabinofuranose of alpha configuration, 1025cm -1 The presence of the alpha- (1 → 6) glycosidic bond and pyranose can also be confirmed by the absorption peak at (E). In addition, 578cm -1 The presence of glycosidic linkages is also indicated by nearby absorbance peaks. From this, LCPs-1-A are typical polysaccharide compounds.
(5) UV scanning of LCPs-1-A:
taking a proper amount of the purified polysaccharides LCPs-1-A of the pod soft coral polysaccharide prepared in the above embodiments and water to prepare a solution of 1mg/mL, and carrying out ultraviolet scanning at 190-400 nm.
The results are shown in FIG. 13.
It can be found that LCPs-1-A has no obvious ultraviolet absorption at 260nm and 280nm, which indicates that LCPs-1-A has almost no nucleic acid and protein, and is a polysaccharide with high purity.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
SEQUENCE LISTING
<110> Zhongshan university
<120> soft coral polysaccharide, preparation method and application thereof
<130>
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<170> PatentIn version 3.5
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Claims (9)
1. The coral polysaccharide is characterized in that the coral polysaccharide is pod soft coral polysaccharide; according to the mass ratio, the monosaccharide components in the coral polysaccharide are as follows: mannose: rhamnose: glucuronic acid: glucose: galactose: xylose (0.15-0.20): (0.20-0.25): (0.01-0.05): (98.5-99.0): (0.45-0.50): (0.25 to 0.30);
the molecular weight of the coral polysaccharide is (4.90 + -0.2) x 10 6 Da。
2. The method for preparing coral polysaccharides in claim 1, comprising the steps of:
(1) removing lipid components and pigments in the coral sample;
(2) heating, extracting with water, collecting filtrate, and precipitating with ethanol to obtain precipitate;
(3) re-dissolving the alcohol precipitation precipitate obtained in the step (2), adding protease for enzymolysis, centrifuging to remove precipitated protein, adding a deproteinization reagent to remove residual protein to obtain deproteinized coral crude polysaccharide;
(4) taking deproteinized coral crude polysaccharide, separating with DEAE-52 cellulose ion exchange column, eluting with water or sodium chloride to obtain coral polysaccharide;
the coral polysaccharide is pod soft coral polysaccharide.
3. The method of manufacturing according to claim 2, further comprising:
(5) the coral polysaccharide obtained by eluting with water was separated by Sephadex column chromatography and eluted with sodium chloride.
4. The method according to claim 2, wherein the alcohol solution used in the alcohol precipitation in the step (2) is an ethanol solution.
5. The method according to claim 2, wherein the protease in the step (2) comprises: any one of papain, trypsin, pepsin and alkaline protease;
the deproteinizing reagent in the step (2) comprises: any one of Sevag reagent, trifluorotrichloroethane and trichloroacetic acid.
6. Use of the coral polysaccharide of claim 1 or the coral polysaccharide produced by the process of any one of claims 2 to 5 in the production of a medicament.
7. The use of claim 6, wherein the medicament comprises an immunomodulator.
8. The use of claim 7, wherein the immunomodulator is used for improving immunity, promoting immune cell proliferation, and promoting expression and secretion of immune cytokines.
9. Use of the coral polysaccharide of claim 1 or the coral polysaccharide produced by the production method of any one of claims 2 to 5 in the production of a food.
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