CN113621088A - Crude polysaccharide of sargassum pallidum, preparation method, separation and purification method and application thereof - Google Patents
Crude polysaccharide of sargassum pallidum, preparation method, separation and purification method and application thereof Download PDFInfo
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- CN113621088A CN113621088A CN202111009537.0A CN202111009537A CN113621088A CN 113621088 A CN113621088 A CN 113621088A CN 202111009537 A CN202111009537 A CN 202111009537A CN 113621088 A CN113621088 A CN 113621088A
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- Prior art keywords
- polysaccharide
- sargassum pallidum
- crude polysaccharide
- sargassum
- pallidum
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- C—CHEMISTRY; METALLURGY
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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
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- 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
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Abstract
The invention discloses a crude polysaccharide of sargassum pallidum and a preparation method thereof, wherein the crude polysaccharide is extracted by taking sargassum pallidum as a raw material, and monosaccharide comprises the following components: galactose, fucose and mannose, structurally having typical S ═ O symmetric stretching vibration peaks and glycosidic linkages in the α configuration. The preparation method of the crude polysaccharide of the sargassum pallidum comprises the following steps: the crude polysaccharide of the sargassum pallidum is obtained by carrying out hot water extraction, concentration, dialysis, alcohol precipitation and freeze drying on sargassum pallidum. The invention also discloses a separation and purification method of the crude polysaccharide, which comprises the following steps: removing protein from the crude polysaccharide by a Sevag reagent, eluting and separating by a DEAE-cellulose anion exchange column, and then concentrating, dialyzing, precipitating with ethanol, and drying to obtain the powder of each component of the sargassum pallidum polysaccharide. The sargassum pallidum polysaccharide obtained by the invention has the effects of improving the immunity of the organism and inhibiting the growth of tumor cells, and can be applied to health food and/or pharmaceutical preparations for the auxiliary treatment of tumors or the improvement of the immunoregulation capability of the organism.
Description
Technical Field
The invention relates to the technical field of extraction and purification of natural products, and in particular relates to crude polysaccharide of sargassum pallidum, a preparation method, a separation and purification method and application thereof.
Background
Sargassum pallidum is a kind of algae of sargassum of brown algae, widely distributed in coastal areas, and mostly grows on low tide rocks. The sargassum pallidum is one of the traditional Chinese medicine seaweeds recorded in the herbal medicine of all generations, and has high medicinal value and development potential. Pharmacological studies have shown that polysaccharides are one of the main active substances of sargassum pallidum. However, the extraction rate of the crude polysaccharide of the sargassum pallidum is low, and the crude polysaccharide contains more components, so that the wide application of the sargassum pallidum polysaccharide in the aspect of medicines is seriously influenced. Therefore, the preparation method of the crude polysaccharide of the sargassum pallidum with high extraction rate and the further separation and purification method thereof are necessary for researching the activity and action mechanism of the polysaccharide of the sargassum pallidum, improving the utilization efficiency of the polysaccharide of the sargassum pallidum and expanding the application range of the polysaccharide of the sargassum pallidum.
Disclosure of Invention
The invention aims to provide crude polysaccharide of sargassum pallidum, a preparation method and a separation and purification method thereof, and sargassum pallidum polysaccharide obtained by separating and purifying the crude polysaccharide of sargassum pallidum, so that the defects of the prior art for extracting and purifying the polysaccharide of sargassum pallidum are overcome, and the polysaccharide of sargassum pallidum with anti-tumor and immunity enhancement activities is particularly obtained by the invention and is expected to be applied to tumor adjuvant therapy and organism immunoregulation therapy.
According to one technical scheme, the crude polysaccharide of the sargassum pallidum is obtained by extracting sargassum pallidum serving as a raw material, wherein the total sugar content of the crude polysaccharide of the sargassum pallidum is 24.32%; the monosaccharide composition comprises: galactose, fucose and mannose; the structure has typical S ═ O symmetric stretching vibration peak and alpha configuration glycosidic bond.
Further, the molar ratio of mannose, galactose and fucose in the crude sargassum pallidum polysaccharide is 46:1000: 140.
Further, the sulfate radical content of the crude sargassum pallidum polysaccharide is 5.39%.
The second technical scheme of the invention is that the preparation method of the crude polysaccharide of sargassum pallidum comprises the following steps:
(1) extracting sargassum pallidum with hot water to obtain crude polysaccharide stock solution;
(2) concentrating and dialyzing the crude polysaccharide stock solution obtained in the step (1) to obtain a crude polysaccharide solution;
(3) and (3) precipitating the crude polysaccharide solution obtained in the step (2) with ethanol, and freeze-drying to obtain the crude polysaccharide of the sargassum pallidum.
Further, the sargassum pallidum is soaked in hot water, and is dried and crushed in advance to prepare the algae powder with the granularity less than or equal to 100 meshes.
Further, the feed-liquor ratio of the hot water leaching method in the step (1) is 1g:30mL, the leaching temperature is 93 ℃, and the leaching time is 3 h.
Further, the dialysis time in step (2) is 3d, wherein the 1d is running water dialysis, the 2 nd to 3d are distilled water dialysis, and the molecular weight cut-off of dialysis is 3500 kD.
Further, the alcohol adopted in the alcohol precipitation in the step (3) is absolute ethyl alcohol, and the volume ratio of the crude polysaccharide solution to the absolute ethyl alcohol is 1: 4.
Further, the alcohol precipitation is specifically carried out by placing the mixed solution at 4 ℃ overnight.
The extraction rate of the crude polysaccharide obtained by the preparation method of the invention is 6.74%.
In the third technical scheme of the invention, the separation and purification method of the crude polysaccharide of sargassum pallidum comprises the following steps: dissolving crude polysaccharide of sargassum pallidum, removing protein by a Sevag method, and then eluting and separating by a DEAE-cellulose anion exchange column to obtain sargassum pallidum polysaccharide eluent; concentrating, dialyzing, precipitating with ethanol, and drying to obtain polysaccharide powder of Artemisia annua L.
Further, the solid-to-liquid ratio of the crude sargassum pallidum polysaccharide in the dissolving process is 1g to 10ml, and the volume ratio of the Sevag reagent to the crude polysaccharide solution in the protein removal by the Sevag method is 4 to 3; and NaCl solutions with different concentrations are used as eluent in elution separation to elute from small to large according to the concentration in sequence, and the concentration of the NaCl solution is as follows from small to large: 0. 0.1, 0.3, 0.5, 0.7, 1.0, 1.3, 1.5, 1.7, 2.0 mol/L.
Further, the Sevag reagent is a mixed solution of n-butanol and chloroform, and the volume ratio of n-butanol: chloroform-1: 4.
Further, the rotation speed of centrifugation in the process of removing protein by the Sevag method is 7000r/min, and the centrifugation time is 10 min.
According to the fourth technical scheme, the polysaccharide of the sargassum pallidum is obtained by separation and purification according to the separation and purification method of the crude polysaccharide of the sargassum pallidum, and monosaccharide composition of the polysaccharide of the sargassum pallidum comprises: one or more of rhamnose, glucose, fucose, mannose and galactose; the sargassum pallidum polysaccharide contains carboxyl groups and alpha-configuration glycosidic bonds.
Further, the total sugar content of the sargassum pallidum polysaccharide is 43% -68%; the sulfate radical content is 5-14%.
5 sargassum pallidum polysaccharides with different components can be obtained under the elution of NaCl solutions with different concentrations, wherein the polysaccharide components are respectively SPPS-0.3, SPPS-0.5, SPPS-0.7, SPPS-1 and SPPS-2, and the total sugar content is 43-68 percent; the sulfate radical content is between 5 and 14 percent; mainly comprises rhamnose, glucose, fucose, mannose and galactose; the 5 polysaccharides have carboxyl group and alpha-configuration glycosidic bond, and the rest polysaccharides except SPPS-0.5 polysaccharide are 1250cm-1And obvious S-O symmetrical stretching vibration peaks are formed nearby.
In the fifth technical scheme, the sargassum pallidum polysaccharide is applied to food and/or medicine preparation, the food is used for assisting tumor treatment or improving the immune regulation capability of an organism, and the medicine is used for assisting tumor treatment or improving the immune regulation capability of the organism.
The sargassum pallidum polysaccharide prepared by the invention has stronger anti-tumor activity, has the functions of inhibiting the proliferation of lung cancer cells A549, melanoma cells B16 and liver cancer cells HepG2, can promote the apoptosis process of tumor cells, can improve the expression of anti-tumor related factors, and is shown by transcriptome sequencing, and the sargassum pallidum polysaccharide component prepared by the invention can obviously improve the expression of anti-tumor related genes and pathways.
The sargassum pallidum polysaccharide prepared by the invention has stronger immune enhancement effect and can promote the proliferation of macrophage (RAW264.7) and mouse spleen lymphocytes; the expression of immune activating factors (IL-6, IL-1 beta, iNOS and TNF-alpha) can be enhanced, the expression activity of immune related factors can be improved, and transcriptome sequencing shows that the sargassum pallidum polysaccharide prepared by the invention can obviously improve the expression of immune related genes and pathways.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention provides a preparation method of crude polysaccharide of sargassum pallidum, which is characterized in that sargassum pallidum is used as a raw material, and the crude polysaccharide of sargassum pallidum is obtained after drying, grinding, hot water extraction, rotary evaporation concentration, dialysis concentration, alcohol precipitation and freeze drying. The crude polysaccharide of sargassum pallidum provided by the invention has high oxidation activity, good immune enhancement activity and good anti-tumor effect.
(2) The invention provides a separation and purification method of crude polysaccharide of sargassum pallidum, which comprises the steps of removing protein in the crude polysaccharide of the prepared crude polysaccharide of the sargassum pallidum through a Sevag reagent, eluting and separating the crude polysaccharide in NaCl solutions with different concentrations through a DEAE-cellulose anion exchange column, and obtaining sargassum pallidum polysaccharide powder through concentration, dialysis, alcohol precipitation and drying. The sargassum pallidum polysaccharide obtained by separation and purification by the method has high purity and yield, and sargassum pallidum polysaccharides with different molecular weights can be obtained by classification. The extraction and purification method provided by the invention is beneficial to further research and development of sargassum pallidum polysaccharide.
The sargassum pallidum polysaccharide extraction and purification component is applied to health food and medicine preparations for regulating body immunity. It is expressed as follows: the sargassum pallidum polysaccharide obtained by the invention has anti-tumor and immunity-improving activities, can obviously inhibit the proliferation of lung cancer cells (A549), mouse melanoma cells (B16) and liver cancer cells (HepG2), and proves that the sargassum pallidum polysaccharide is harmless to normal cells; meanwhile, the proliferation of macrophages and splenic lymphocytes of the mice can be obviously improved, the generation of cell factors is promoted, the expression of immune related genes is up-regulated, and the cellular immune response is activated; the result of transcriptome shows that the polysaccharide component of sargassum pallidum can activate the expression of anti-tumor and immunity-related genes and pathways.
(3) The algal polysaccharide provided by the invention is derived from large-scale economic algal sargassum pallidum, and has the advantages of abundant raw material sources and low production cost. Sargassum pallidum polysaccharide belongs to green and environment-friendly products, and has proved to be harmless to normal cells; the sargassum pallidum polysaccharide obtained by separation and purification by the separation and purification method can obviously inhibit the proliferation of lung cancer cells (A549), mouse melanoma cells (B16) and liver cancer cells (HepG2), and is proved to be harmless to normal cells; meanwhile, the proliferation of macrophages and splenic lymphocytes of the mice can be obviously improved, the generation of cell factors is promoted, the expression of immune related genes is up-regulated, and the cellular immune response is activated; the result of the transcriptome shows that the sargassum pallidum polysaccharide can activate the expression of anti-tumor and immunity-related genes and pathways; the fluorescent quantitative PCR verification shows that immune related factors such as: interleukin-6 (IL-6), interleukin-1 (IL-1 beta), nitric oxide synthase (iNOS) and tumor necrosis factor (TNF-alpha) are released to up-regulate immune related gene expression and activate cellular immune response. The sargassum pallidum polysaccharide obtained by the invention has excellent anti-tumor activity and immunity enhancement activity, can be applied to health food and pharmaceutical preparations with the tumor adjuvant therapy effect and the organism immunity adjustment effect, and has wide application prospect.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is an elution profile of polysaccharide component from Artemisia annua;
FIG. 2 is a standard curve of total sugar content;
FIG. 3 is a standard curve for sulfate content;
FIG. 4 is an infrared spectrum of crude polysaccharide from Artemisia annua;
FIG. 5 is an infrared spectrum of SPPS-0.3 component polysaccharide;
FIG. 6 is an infrared spectrum of SPPS-0.5 component polysaccharide;
FIG. 7 is an infrared spectrum of SPPS-0.7 component polysaccharide;
FIG. 8 is an infrared spectrum of SPPS-1 component polysaccharide;
FIG. 9 is an infrared spectrum of SPPS-2 component polysaccharide;
FIG. 10 is a graph comparing the inhibitory activity of various components of sargassum pallidum polysaccharide (SPPS-0.3, SPPS-0.5, SPPS-0.7, SPPS-1, SPPS-2) on A549 cells;
FIG. 11 is a graph comparing the inhibitory activity of various fractions of sargassum pallidum polysaccharide (SPPS-0.3, SPPS-0.5, SPPS-0.7, SPPS-1, SPPS-2) on B16 cells;
FIG. 12 is a graph comparing the inhibitory activity of various components of sargassum pallidum polysaccharide (SPPS-0.3, SPPS-0.5, SPPS-0.7, SPPS-1, SPPS-2) against HepG2 cells;
FIG. 13 is a graph comparing the effect of SPPS-0.7 at different concentrations on the induction of apoptosis in A549 tumor cells;
FIG. 14 is a graph showing the proliferation activity of the polysaccharide of Artemisia annua (SPPS-0.3, SPPS-0.5, SPPS-0.7, SPPS-1, SPPS-2) on mouse macrophage RAW 264.7;
FIG. 15 is a graph showing the comparison of the proliferation activity of sargassum pallidum polysaccharides of five components (SPPS-0.3, SPPS-0.5, SPPS-0.7, SPPS-1, and SPPS-2) on mouse spleen lymphocytes;
FIG. 16 is a graph showing the effect of SPPS-0.7 on the relative expression levels of mRNA of mouse macrophages RAW264.7 cytokines IL-6(A), IL-1 β (B), TNF- α (C) and iNOS (D);
FIG. 17 is a KEGG enrichment analysis histogram of SP25 vs CK;
FIG. 18 is a KEGG enrichment analysis histogram of SP100 vs CK;
FIG. 19 is a KEGG enrichment analysis histogram of SP400 vs CK.
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 description and examples are intended to be illustrative only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
Example 1
Preparation of crude polysaccharide of sargassum pallidum
Pulverizing sargassum pallidum, and sieving with 100 mesh sieve to obtain algae powder. Extracting algae powder by a hot water extraction method, extracting at 91 ℃ for 5h according to a material-liquid ratio of 1:30(g/mL), centrifuging the extracted mixture (2000r/min,15min), removing precipitates, taking supernate, performing rotary evaporation and concentration to 1/4 of the original volume, dialyzing for 3d by a dialysis bag with the molecular weight cutoff of 3500kD (24h running water dialysis, 48h distilled water dialysis), and removing inorganic salt micromolecule substances to obtain dialysate. And then performing rotary evaporation concentration again, concentrating to 1/4 volume of the dialysate, adding 4 times volume of anhydrous ethanol for alcohol precipitation, standing overnight at 4 ℃, then centrifuging (7000r/min,15min), and freeze-drying the precipitate to obtain crude polysaccharide of sargassum pallidum, wherein the calculated yield is 6.74%.
Example 2
Separation and purification of crude polysaccharide of sargassum pallidum
(1) Elution separation of crude polysaccharide from sargassum pallidum
Weighing 1g of crude sargassum pallidum polysaccharide prepared in example 1, dissolving the crude sargassum pallidum polysaccharide in 10mL of distilled water, adding 7.5mL of Sevag reagent (mixed solution of n-butanol and chloroform in a volume ratio of 1: 4), fully shaking and uniformly mixing, and centrifuging (7000r/min,15min) to separate a supernatant, namely a polysaccharide solution. Passing the polysaccharide solution through DEAE-cellulose anion exchange column, sequentially eluting with filtered 0M, 0.1M, 0.3M, 0.5M, 0.7M, 1M, 1.3M, 1.5M, 1.7M, and 2.0M sodium chloride solution, wherein the elution volume of each concentration of sodium chloride solution is 2L, collecting with automatic collector, collecting 10mL per tube, detecting with phenol-sulfuric acid method, measuring absorbance at 485nm, and drawing elution curve according to absorbance (as shown in FIG. 1). The polysaccharide with 5 components can be obtained by an elution curve, and are sequentially named as SPPS-0.3, SPPS-0.5, SPPS-0.7, SPPS-1 and SPPS-2. After the components are determined, the components are eluted and separated by using sodium chloride solutions with the concentrations of 0.3, 0.5, 0.7, 1 and 2mol/L to obtain polysaccharides of each component.
(2) Concentrating and drying the eluate
And (2) performing rotary evaporation concentration on the eluent of the 5 fractions obtained by elution in the step (1) respectively, dialyzing for 3d (24h running water dialysis and 48h distilled water dialysis) by a dialysis bag with the molecular weight cutoff of 3500kD, performing rotary evaporation concentration again, performing alcohol precipitation by using absolute ethyl alcohol with the volume of 4 times, standing overnight at the temperature of 4 ℃, centrifuging (7000r/min,15min), and performing freeze drying on the precipitate to obtain the component powder of the polysaccharide of the sargassum pallidum.
Effect verification:
1. measurement of crude polysaccharide of sargassum pallidum and physicochemical properties of components
(1) Phenol-sulfuric acid method for measuring total sugar content
Taking a glucose standard substance to prepare a glucose solution of 0.5 mg/mL. Three groups of 6 test tubes are taken, and the test tubes are respectively marked with numbers of 0, 1, 2, 3, 4 and 5. To 6 tubes were added 0, 0.2, 0.4, 0.6, 0.8, 1mL of the glucose standard solution in that order, followed by addition of distilled water to 1 mL. Then, 1mL of 5% phenol solution and 5mL of concentrated sulfuric acid were added in this order, shaken up, and allowed to stand for 20 min. The absorbance at 485nm was measured using a 0 cuvette as a control, and a standard curve was plotted (as shown in FIG. 2).
Preparing crude polysaccharide of sargassum pallidum and polysaccharide of each component into 0.1% solution, putting 100 μ L into a colorimetric tube, adding distilled water, and the final volume is 1 mL. 1mL of 5% phenol solution and 5mL of concentrated sulfuric acid were sequentially added, and after standing for 30min, the absorbance at 485nm was measured. The steps are repeated for three times, the average value of the absorbance is taken and is substituted into a glucose standard curve, and the total sugar content of the crude polysaccharide of the sargassum pallidum is measured to be 24.32 percent (see table 1). The total sugar content of the polysaccharides of each fraction was measured to be between 43% and 68% (see table 2).
TABLE 1 yield, chemical composition and monosaccharide molar ratio of crude polysaccharide from Artemisia annua
Man is mannose; rha is rhamnose; gal is galactose; GlcA is glucuronic acid; glc is glucose; xyl is xylose; fuc fucose
TABLE 2 yield, chemical composition and monosaccharide molar ratio of each component polysaccharide
Man is mannose; rha is rhamnose; gal is galactose; GlcA is glucuronic acid; glc is glucose; xyl is xylose; fuc fucose
(2) Determination of sulfate ion content by gelatin turbidimetry
Adding 1g gelatin into 100mL distilled water, dissolving at 60-70 deg.C, standing overnight at 4 deg.C, adding 0.5g BaCl the next day2Dissolving in the solution, standing for 40min, and using.
Will K2SO4Drying at 105-110 deg.C for 3h until the weight is not reduced, completely removing water, cooling to room temperature in a drier, accurately weighing 0.04-0.05g to prepare 100mL solution, and converting into 0.1g SO4 2-(mg/mL). Respectively take K2SO40, 50, 100, 150 and 200 mu L of standard solution, diluting to 200 mu L, taking 100 mu L, adding distilled water to 200 mu L, adding 3.8mL of 0.2M HCl solution and gelatin BaCl2The solution was 1mL, and the absorbance was measured at 500nm after 30 min. A standard curve is plotted (as shown in figure 3).
Hydrolyzing sargassum pallidum polysaccharide with acid, and neutralizing with ammonia water to pH 6-7. Decolorized and filtered, and the volume is adjusted to 25 mL. Then taking 100 mu L of the sample to be detected, adding distilled water to 200 mu L, and then sequentially adding 3.8mL of 0.2M HCl solution and gelatin BaCl2The solution was 1mL, and the absorbance was measured at 500nm after 30 min.
From the standard curve of fig. 3, a regression equation was calculated: y is 0.0004X, R2SO was calculated from the absorbance of the sample (X: sulfate concentration, Y: absorbance at 500 nm) by 0.9991 ═ g (X: sulfate concentration, Y: absorbance at 500 nm)4 2-The concentration of (c).
The results show a crude polysaccharide sulfate content of 5.39% (see table 1); the sulfate content of each component was between 5% and 14% (see table 2).
(3) Monosaccharide composition
Weighing 10mg sargassum pallidum polysaccharide in an ampoule, adding 1mL of water and 1mL of 4M trifluoroacetic acid (TFA), and hydrolyzing at 105-110 ℃ for 2-6 h. Neutralizing with NaOH to pH 5-6, diluting to 10mL, taking 0.2mL sample, and adding 0.2mL ribose standard as internal standard. 0.12mL of 0.5M 1-phenyl-3-methyl-5-pyrazolone (PMP) solution and 0.1mL of 0.3M NaOH solution were added to 0.1mL of the mixture, and the mixture was mixed and reacted at 70 ℃ for 60 min. Cooled to room temperature, 0.1mL of 0.3M HCl solution was added, mixed well, 0.5mL of chloroform was added, centrifuged (7000r/min, 5min), the organic layer was discarded, and the process was repeated three times. 0.1mL of the supernatant was aspirated for detection by High Performance Liquid Chromatography (HPLC). Column YMC-Pack ODS-AQ (250 mm. times.4.6 mm, 5 μm), column temperature 25 ℃, flow rate 1.0mL/min, detection wavelength 254nm, mobile phase A: 0.4% aqueous triethylamine solution: acetonitrile 9: 1. Mobile phase B: 0.4% aqueous triethylamine solution: acetonitrile 4: 6.
The analysis result of monosaccharide composition shows that the crude polysaccharide of sargassum pallidum mainly comprises galactose, fucose and mannose (see table 1). The determination of the monosaccharide composition of the polysaccharide component of the sargassum pallidum shows that SPPS-0.3 mainly comprises rhamnose, glucose, xylose, fucose and mannose; SPPS-0.5 mainly comprises fucose, mannose, galactose, glucose and xylose; SPPS-0.7 mainly comprises fucose, mannose and galactose; SPPS-1 mainly comprises rhamnose, glucose, xylose and galactose; SPPS-2 is mainly composed of rhamnose, galactose and fucose. As the eluent concentration increased, there was a tendency for the monosaccharide species to decrease (see Table 2).
(4) Infrared Spectroscopy (FT-IR)
Weighing crude polysaccharide of Artemisia annua L about 2mg, tabletting with potassium bromide (KBr), and making into 4000cm thick gel in infrared spectrum wavelength range of 400--1The infrared spectrum of crude polysaccharide of a lower scanning sample shows that the analysis result shows 400-4000cm-1The absorption peak at (A) is a characteristic peak typical of polysaccharides. At 3498.24cm-1Peak at 2932.27cm-1The weak absorption peaks at (A) are stretching characteristic peaks of O-H and C-H, respectively. At 1614.57cm-1And 1418.32cm-1The two absorption peaks at (a) represent the asymmetric stretching vibration and the symmetric stretching vibration peaks of C ═ O, indicating the presence of carboxyl groups in the sargassum polysaccharide. 1255.73cm-1The peak is S ═ O symmetric stretching vibration peak. 1039.92cm-1The absorption peak of (a) is attributed to the C-O-H deformation vibration. 831.17cm-1The peaks indicate that the polysaccharide of sargassum pallidum has alpha glycosidic bonds (the infrared spectrum of crude polysaccharide of sargassum pallidum is shown in FIG. 4).
SPPS-0.3, SPPS-0.5, SPPS-0.7, SPPS-1 and SPPS-2 polysaccharide samples in the infrared spectrum wavelength range of 400cm-1-4000cm-1The lower infrared spectra are shown in FIGS. 5 to 9, respectively. 3600-3200cm in infrared spectrum-1The peaks between the two peaks all belong to O-H stretching vibration peaks and are positioned at 2923cm-1The nearby peak belongs to the stretching vibration peak of-CH and is located at 1630cm-1The nearby peaks belong to the vibration peaks of the bound water. 1416cm-1The nearby peaks indicate that carboxyl groups exist in the polysaccharides SPPS-0.3, SPPS-0.5, SPPS-0.7, SPPS-1 and SPPS-2, and except the polysaccharide SPPS-0.5, the rest of SPPS-0.3, SPPS-0.5, SPPS-0.7, SPPS-1 and SPPS-2 are 1250cm in length-1S ═ O is obviously symmetrical and telescopic vibration nearby, and the vibration frequency is 1000cm at 1200--1Peaks appearing in the range of C-O-H stretching vibration peak and C-O-C stretching vibration peak of ether bond in pyran ring, wherein the peak is at 832cm-1The peaks appeared in the vicinity of the surface of the substrate indicate that SPPS-0.3, SPPS-0.5, SPPS-0.7, SPPS-1 and SPPS-2 have a glycosidic bond in the alpha configuration.
2. Anti-tumor experiment of sargassum pallidum polysaccharide component
(1) Experiment of sargassum pallidum polysaccharide component for inhibiting lung cancer A549 cells
Lung cancer A549 cells at 2 × 104The cells were seeded at a density of 100. mu.L/well in 96-well cell culture plates. Incubate at 37 ℃ for 24h, treat with 25, 100, 400 μ g/mL of sargassum pallidum polysaccharide in different fractions for 24h, add equal volume of medium to the blank, set in triplicate for each concentration. After the treatment was completed, the medium was removed from each well and washed twice with PBS, and a mixture of CCK-8 reagent and medium was added. After 1h, the absorbance at 450nm was measured under a microplate reader. The antitumor inhibition ratio of the polysaccharide fraction was calculated by the following formula: inhibition (%) (-) [1- (A1/A2)]X 100%, where a1 and a2 are the absorbance of the treated and control groups, respectively.
The results show that the five components of the sargassum pallidum polysaccharide show stronger inhibition effect on A549 cells, wherein the SPPS-0.7 inhibition effect is the best, and the inhibition rates are respectively 58.55%, 64.28% and 56.68% at the concentrations of 25 mu g/mL, 100 mu g/mL and 400 mu g/mL, and show the trend of increasing firstly and then decreasing. The inhibition effect of SPPS-1 and SPPS-2 on A549 is less than that of SPPS-0.5 and SPPS-0.3 on A549 (the inhibition activity of different components of sargassum pallidum polysaccharide on A549 cells is shown in figure 10).
(2) Experiment of sargassum pallidum polysaccharide component for inhibiting melanoma B16 cells
The inhibitory activity of different fractions of sargassum pallidum polysaccharides on B16 cells when B16 cells were treated according to the method of (1) is shown in FIG. 11. As can be seen from FIG. 11, the inhibitory activity of different fractions of sargassum pallidum polysaccharides on melanoma cells was different, among which, the inhibitory activity of SPPS-0.7 was the best, the inhibitory rate was 30.06% at 25. mu.g/mL, and the inhibitory rates at 100. mu.g/mL and 400. mu.g/mL were 26.95% and 17.14%, respectively, showing a decreasing trend. The inhibitory activity of SPPS-0.3 is the second, and the inhibitory activity of three components, namely SPPS-0.5, SPPS-1 and SPPS-2 is the weakest.
(3) Experiment for sargassum pallidum polysaccharide component for inhibiting liver cancer HepG2 cells
The HepG2 cells were treated according to the method of (1), the inhibitory activities of different fractions of sargassum polysaccharide on HepG2 cells are shown in FIG. 12, and it can be seen from FIG. 12 that the inhibition rates of different fractions of sargassum polysaccharide on liver cancer cells are different, among which, the inhibitory activity of SPPS-1 is the best, and the inhibition rates are 32.78%, 39.09% and 39.26% in sequence at three concentrations of low dose, medium dose and high dose. The inhibitory activity of three components, SPPS-0.7 and SPPS-0.5, was the weakest. In all component experiments, the inhibition rate increased with increasing polysaccharide concentration.
3. Experiment for promoting tumor cell apoptosis by sargassum pallidum polysaccharide component
(1) Experiment for promoting A549 cell apoptosis by sargassum pallidum polysaccharide component
The influence of sargassum pallidum polysaccharide on tumor cell apoptosis is researched by selecting the sargassum pallidum polysaccharide component SPPS-0.7 with better activity. Treating lung cancer cell A549, and treating A549 cell at 1 × 105SPPS-0.7(25, 100, 400. mu.g/mL) at various concentrations and densities of one/mL were mixed and plated in cell culture plates, 4mL per well, with the wells containing medium alone being blank pairsAnd (4) performing group control. After 48h, the cells were washed with PBS, digested and collected into a centrifuge tube, centrifuged (1700r/min, 3min) and the supernatant discarded. Washing with PBS, centrifuging (1700r/min, 3min), collecting cells, counting, and measuring cell concentration at 2 × 105～106The amount is one/mL; after treatment of the cells according to the kit instructions, apoptosis was analyzed by flow cytometry within 1 h.
Apoptosis was monitored by flow cytometry facscalibur (bd biosciences) and results were expressed as percent mean ± SD (n ═ 3), and the effects of different concentrations of SPPS-0.7 on inducing apoptosis in a549 tumor cells are shown in fig. 13(a) - (D) (a-D are controls, 25 μ g/mL, 100 μ g/mL and 400 μ g/mL treatment groups, respectively). In FIG. 13, the apoptosis rates of lung cancer cell A549 induced by blank group and treated group (25, 100, 400. mu.g/mL) were 1.12%, 7.99%, 8.01%, and 3.62%, respectively. It is shown that the sargassum pallidum polysaccharide SPPS-0.7 component can induce apoptosis. The cell survival rate of the polysaccharide concentration in the control group, the low dose and the medium dose is respectively 98.26%, 88.82% and 81.33%. The results show that SPPS-0.7 can inhibit the proliferation of the lung cancer cell A549 by inducing the apoptosis of the lung cancer cell A549.
4. Artemisia annua polysaccharide component immune cell experiment
(1) Mouse macrophage (RAW264.7) relative survival assay
RAW264.7 cells in logarithmic growth phase at a density of 5X 105Each/mL was inoculated into a 96-well plate, and the fractions were treated with different concentrations of polysaccharide (25, 100, 400. mu.g/mL) for 24 h. Next, 20. mu.L of CCK-8 was added to each well and the cells were incubated at 37 ℃ and 5% CO2Incubate for 4 hours. Finally, the absorbance at 450nm was measured to evaluate the cell viability.
The proliferation activity of the five components (SPPS-0.3, SPPS-0.5, SPPS-0.7, SPPS-1 and SPPS-2) sargassum pallidum polysaccharide on mouse macrophage RAW264.7 is shown in figure 14, and as can be seen from figure 14, the sargassum pallidum polysaccharide of each component has proliferation effect on RAW264.7 cells, wherein the SPPS-0.7 has the most obvious effect, the relative survival rate of the cells shows an increasing trend along with the increase of the polysaccharide concentration, and the relative survival rate of the cells is 139% at 400 mu g/mL. SPPS-0.3 and SPPS-2 have the second time on the proliferation of RAW264.7, and the relative survival rate of cells is 131% and 125% respectively at 400 mu g/mL. SPPS-1 and SPPS-0.5 have weak proliferation effect on RAW264.7, and the relative survival rate of cells is 113% and 105% respectively at 400 mu g/mL. The results show that the SPPS-0.7 has the most obvious effect of promoting the proliferation of RAW264.7 cells at 400 mu g/mL.
(2) Proliferation assay for splenic lymphocytes in mice
To evaluate the potential immunomodulatory effects of the polysaccharide component of sargassum pallidum, proliferation assays were performed using mouse splenic lymphocytes. Mice were sacrificed under sterile conditions, spleens were quickly removed, red blood cells were removed, and homogeneous splenocytes were prepared. Placing spleen lymphocytes in 96-well culture plates at a density of 2 × 106one/mL, volume 100. mu.L/well. Add complete medium containing sargassum pallidum polysaccharide SPPS-0.7 at 100. mu.L per well to final concentrations of 0, 25, 100, 400. mu.g/mL. 37 ℃ and 5% CO2After 48 hours of incubation, 20. mu.L of CCK-8 solution was added to each well and the plate was incubated for an additional 4 hours. The absorbance was measured at 450nm with a microplate reader.
The proliferation activity of five kinds of component (SPPS-0.3, SPPS-0.5, SPPS-0.7, SPPS-1, SPPS-2) sargassum pallidum polysaccharide on mouse spleen lymphocytes is shown in figure 15, and it can be known from figure 15 that five kinds of sargassum pallidum polysaccharide can stimulate mouse spleen lymphocytes to proliferate, wherein the proliferation effect of SPPS-0.7 on spleen lymphocytes is the best, and the proliferation rates are gradually increased at 25 mug/mL, 100 mug/mL and 400 mug/mL, and are respectively 32.68%, 46.28% and 59.43%. SPPS-0.3, SPPS-1 and SPPS-2 promote the proliferation rate of splenic lymphocytes of mice to be second, and the proliferation rate is increased along with the increase of sugar concentration. The results show that SPPS-0.7 has the best effect on the proliferation of splenic lymphocytes.
5. Research on immune enhancement mechanism of polysaccharide component of sargassum pallidum
(1) Immune-related factor assay
IL-6 is an immune proinflammatory factor that triggers an inflammatory response, and its main functions are to activate B cell proliferation and secretion of antibodies, stimulate T cell proliferation, and activate cytotoxic T lymphocytes, while promoting the differentiation of th17 cells. IL-6 can stimulate NF-kB to release and transport to nucleus, induce gene transcription and expression of inflammatory cell factor, including IL-1B, TNF-a and other downstream factors, and further enhance immune response. The immune related factors can be used as marker factors for measuring the immune level.
The active component SPPS-0.7 is selected to study the influence of sargassum pallidum polysaccharide on immune related factors. Logarithmic phase RAW264.7 cells were seeded in six well plates at a density of 3X 105Each well, the test is respectively provided with a test group and a control group, and the drug concentration is 25 mug/mL, 100 mug/mL and 400 mug/mL in sequence. Control groups were added with equal volume of complete medium. Incubate for 48h, discard the supernatant and rinse with PBS. Extracting total RNA by using a cell extraction kit, carrying out reverse transcription to generate cDNA, detecting the effect of SPPS-0.7 on the relative expression quantity of mRNA of IL-6, IL-1 beta, iNOS and TNF-alpha by using qPCR (quantitative polymerase chain reaction), carrying out Q-PCR analysis, and carrying out Q-PCR analysis according to a formula 2-ΔΔCTCalculating the relative expression amount of the immune related factor. The primer sequences of the target gene primer and the endogenous reference beta source reference gene during the Q-PCR reaction are shown as follows:
beta-actin: 5'-GCAGAAGGAGATCACTGCCCT-3' and
5‘-GCTGATCCACATCTGCTGGAA-3’；
IL-1. beta.: 5'-GGGATGATGATGATAACCTG-3' and
5‘-TTGTCGTTGCTTGGTTCTCCT-3’；
IL-6: 5'-CATGTTCTCTGGGAAATCGTGG-3' and
5‘-AACGCAACTAGGTTTGCCGAGTA-3’；
iNOS: 5'-GGTCTTCCTGGGCTCGATCTG-3' and
5‘-GCCGTGGCCAACATGCTACT-3’；
TNF- α: 5'-GATCTCAAAGCAAACCAACTAGTG-3' and
5‘-CTCCAGCTGGAAGACTCCCAG-3’。
the effect of SPPS-0.7 on the relative expression levels of mRNA of mouse macrophages RAW264.7 cytokines IL-6(A), IL-1 beta (B), TNF-alpha (C) and iNOS (D) is shown in FIGS. 16(A) - (D), and the results in FIGS. 16(A) and (B) show that the mRNA expression levels of IL-1 beta and IL-6 are both significantly increased compared with the control group and show dose correlation. IL-1. beta. and IL-6 mRNA expression after treatment with SPPS-0.7 at 400. mu.g/mL were 20.68-fold and 2.69-fold, respectively, that of the control group. Similarly, the mRNA expression levels of iNOS and TNF-. alpha.were also significantly increased in the graphs (C) and (D) as compared with the control group. When the concentration of SPPS-0.7 polysaccharide is 100 mug/mL, the expression quantity of iNOS and TNF-alpha is 3.16 times and 2.36 times of that of a control group respectively.
(2) Transcriptome analysis
Transcriptome sequencing is a newly developed technology for analyzing transcriptome by using a next-generation sequencing technology, and can comprehensively and quickly obtain sequence information and expression information of almost all transcripts of a specific cell or tissue in a certain state. The invention explores the anti-tumor and immune mechanisms of polysaccharide components of the sargassum pallidum by transcriptome analysis, uses SPPS-0.7 (named as CK, SP25, SP100 and SP400 respectively) with the concentration of 0, 25, 100 and 400 mu g/mL and macrophages to co-culture for 48h, adds 2mL of transZol to fully crack cells, and carries out transcriptome sequencing.
Figure 17, figure 18 and figure 19 list the top 20 pathways most significantly up-regulated and down-regulated in enrichment of the three groups (SP25, SP100, SP400 representing 25, 100, 400 μ g/mL treatment, respectively). The results showed that the SP25 group significantly upregulated pathway-related genes such as NF- κ B signaling pathway and significantly reduced PPAR signaling pathway compared to the CK group (as shown in fig. 17, wherein (a) is a pathway significantly upregulated in expression compared to CK, and (B) is a pathway significantly downregulated in expression compared to CK); in the SP100 group, pathway-related genes such as lysosome were significantly up-regulated and pathway-related genes such as spliceosome were significantly down-regulated, as compared with the CK group (as shown in fig. 18, wherein (a) represents a pathway whose expression is significantly up-regulated as compared with CK, and (B) represents a pathway whose expression is significantly down-regulated as compared with CK);
in the SP400 group, compared with the CK group, the immune-related pathway NF- κ B and iNOS and the phagosome-related pathway were highly expressed (as shown in fig. 19, wherein (a) is a pathway whose expression is significantly upregulated as compared with CK, and (B) is a pathway whose expression is significantly downregulated as compared with CK). Research shows that NF-kB and iNOS channels activate immune cells to participate in immune reaction through cascade reaction, the NF-kB belongs to an important transcription factor, especially plays a key role in cells, mediates the specific reaction of various inflammatory conduction pathways, can regulate the immune reactions of neutrophils, lymphocytes and the like of an organism, plays strong bridging and starting functions, and Inducible Nitric Oxide Synthase (iNOS) is used as a key signal binding factor at the downstream of the NF-kB, participates in the pathogenic process of various inflammatory factors, can mediate various immune cell reactions, and the two are highly expressed to activate the immune reaction of macrophages; in addition, the high expression of the related channels of the phagocyte can strengthen the phagocytosis of the macrophage so as to enhance the immunity of the body.
SP400 compared to the CK group, anti-tumor associated pathways: tumor Necrosis Factor (TNF), C-type lectin receptor (CLRs) signaling pathway, related genes exhibited high expression status (as shown in fig. 19). The immune cells secrete tumor necrosis factors by activating a TNF signal channel, and the tumor necrosis factors can directly kill tumor cells or inhibit the proliferation of the tumor cells; hypoxia necrosis inside tumor tissue can also be caused by inhibiting angiogenesis. Research shows that Dectin-1 receptors in CLRs can trigger strong antitumor immune response of immunocytes through beta-glucan activation, and CLRs can also promote the body to generate immune response to tumor cells through presenting tumor cell-related antigens.
In conclusion, the sargassum pallidum polysaccharide component can obviously improve the immune enhancement activity and the anti-tumor activity of RAW264.7, and enables genes in immune-related pathways such as iNOS and NF-kB signal pathways and anti-tumor-related pathways such as CLRs and TNF signal pathways to be highly expressed.
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 (10)
1. The crude polysaccharide of the sargassum pallidum is characterized in that the total sugar content of the crude polysaccharide of the sargassum pallidum is 24.32 percent, and the crude polysaccharide of the sargassum pallidum is extracted by taking sargassum pallidum as a raw material; the monosaccharide composition comprises: galactose, fucose and mannose; the structure has S ═ O symmetric stretching vibration peak and alpha configuration glycosidic bond.
2. The crude polysaccharide of sargassum pallidum as claimed in claim 1, wherein the molar ratio of mannose, galactose and fucose in the crude polysaccharide of sargassum pallidum is 46:1000: 140.
3. A method for preparing crude polysaccharide of sargassum pallidum according to any one of claims 1 to 2, comprising the steps of:
(1) extracting sargassum pallidum with hot water to obtain crude polysaccharide stock solution;
(2) concentrating and dialyzing the crude polysaccharide stock solution obtained in the step (1) to obtain a crude polysaccharide solution;
(3) and (3) precipitating the crude polysaccharide solution obtained in the step (2) with ethanol, and freeze-drying to obtain the crude polysaccharide of the sargassum pallidum.
4. The method for preparing crude polysaccharide of sargassum pallidum as claimed in claim 3, wherein the ratio of the raw materials in the hot water extraction method in step (1) is 1g:30mL, the extraction temperature is 93 ℃, and the extraction time is 3 h.
5. The method for preparing crude polysaccharide of sargassum pallidum as claimed in claim 3, wherein the dialysis time in step (2) is 3 days, wherein the 1 st day is running water dialysis, the 2 nd to 3 rd days are distilled water dialysis, and the cut-off molecular weight of dialysis is 3500 kD.
6. The method for preparing crude polysaccharide of sargassum pallidum as claimed in claim 3, wherein the alcohol used in the alcohol precipitation in step (3) is absolute ethanol, and the volume ratio of the crude polysaccharide solution to the absolute ethanol is 1: 4.
7. The method for separating and purifying crude polysaccharide of sargassum pallidum as claimed in any one of claims 1 to 2, comprising the steps of: dissolving crude polysaccharide of sargassum pallidum, removing protein by a Sevag method, and then eluting and separating by a DEAE-cellulose anion exchange column to obtain sargassum pallidum polysaccharide eluent; concentrating, dialyzing, precipitating with ethanol, and drying to obtain polysaccharide powder of Artemisia annua L.
8. The method for separating and purifying crude polysaccharide of sargassum pallidum as claimed in claim 7, wherein the solid-to-liquid ratio of the crude polysaccharide of sargassum pallidum is 1g:10ml, and the volume ratio of the Sevag reagent to the crude polysaccharide solution in the Sevag method deproteinization is 4: 3; NaCl solutions with different concentrations are used as eluent in the elution separation, elution is carried out from small to large according to the concentration, and the concentration of the NaCl solution is as follows from small to large: 0. 0.1, 0.3, 0.5, 0.7, 1.0, 1.3, 1.5, 1.7, 2.0 mol/L.
9. The polysaccharide of sargassum pallidum obtained by separation and purification according to any one of claims 7 to 8, wherein the monosaccharide composition of the polysaccharide of sargassum pallidum comprises one or more of rhamnose, glucose, fucose, mannose, and galactose; the sargassum pallidum polysaccharide contains carboxyl groups and alpha-configuration glycosidic bonds.
10. The use of the polysaccharide of sargassum pallidum as claimed in claim 9 in food and/or pharmaceutical preparations, wherein the food is for the adjuvant treatment of tumor or for enhancing the immunoregulatory ability of the body.
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Inventor after: Ju Hao Inventor after: Chen Ran Inventor after: Gao Yi Inventor after: Niu Yunze Inventor after: Li Yizhen Inventor after: Song Lin Inventor before: Gao Yi Inventor before: Niu Yunze Inventor before: Li Yizhen Inventor before: Song Lin Inventor before: Ju Hao Inventor before: Chen Ran |
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