CN113244258A

CN113244258A - Preparation method and application of spirulina polysaccharide proinflammatory enzyme inducer

Info

Publication number: CN113244258A
Application number: CN202110523467.4A
Authority: CN
Inventors: 黄日明; 陆颖芳; 张晓勇; 雷红涛; 罗辉; 李向梅; 罗连响
Original assignee: Guangdong Zhanjiang Institute Of Marine Medicine; South China Agricultural University
Current assignee: Guangdong Zhanjiang Institute Of Marine Medicine; South China Agricultural University
Priority date: 2021-05-13
Filing date: 2021-05-13
Publication date: 2021-08-13

Abstract

The invention relates to the technical field of biochemistry, and discloses a preparation method of a spirulina polysaccharide proinflammatory enzyme inducer. The preparation method of the spirulina polysaccharide proinflammatory enzyme inducer has the advantages of reasonable design, simple process, feasible operation, good repeatability, no need of using a large amount of toxic solvents in the preparation process, good safety, mild preparation conditions, stable structure of the obtained product, high purity, uniform molecular weight and the like, can promote the protein expression of COX-2 and iNOS of macrophages when being applied to cellular immunoregulation, is suitable for deep research and popularization, and provides a scientific research reference basis for further research, development and application of spirulina polysaccharide.

Description

Preparation method and application of spirulina polysaccharide proinflammatory enzyme inducer

Technical Field

The invention relates to the technical field of biochemistry, and particularly relates to a preparation method and application of a spirulina polysaccharide proinflammatory enzyme inducer.

Background

In inflammatory reaction, proinflammatory enzymes (iNOS and COX-2) play a key role, catalytic products of the proinflammatory enzymes can remarkably promote the progress of inflammatory reaction, and the generation of a large amount of secreted NO is an important index for detecting the inflammatory reaction. At present, regulation of the expression of NO synthase and its inducible synthase, iNOS, is considered to be an important means for treating inflammatory diseases. Generally, tissue cells have low COX-2 activity and need to be activated after induction by relevant factors, so as to mediate inflammatory and pain reactions by regulating synthesis of Prostaglandin (PG), but at present, proinflammatory enzyme inducers for activating the immune system are rarely reported.

Microalgae are plants in the ocean with simple structures, contain rich nutrition and various bioactive substances, extract effective active ingredients from the microalgae, and are amplified, prepared and processed into healthy products with economic benefits, thereby having great research and development values and application prospects. Research shows that the polysaccharide extracted from the microalgae has multiple varieties and rich resources, and has the characteristics of bacteriostasis, antioxidation, antivirus, immunity enhancement, low toxic and side effects and the like.

Spirulina (Spirulina) as a kind of microalgae belongs to the phylum Cyanophyta, class Cyanophyceae, genus Spirulina, and is an ancient low-grade prokaryotic unicellular or multicellular aquatic plant. Spirulina is rich in various nutrient elements and is a nutritional food, and the potential health benefits of spirulina have attracted much attention in recent years.

A large number of researches show that the spirulina is a pure natural food rich in various nutrient substances, the protein content in the nutrient protein dry powder is up to more than 60 percent, 8 kinds of amino acids necessary for human bodies are contained, and in addition, the contents of vitamins, mineral substances, trace elements and other nutrients are also very rich. The spirulina polysaccharide is a water-soluble macromolecular polysaccharide extracted and separated from spirulina, is a main form of the spirulina in which carbohydrates exist, and accounts for about 14-16% of dry weight. In the current research on spirulina, the microalgae polysaccharide is a polysaccharide with various activities, such as various biological activities of radiation resistance, virus resistance, tumor resistance, oxidation resistance and the like.

Therefore, it is necessary to develop a spirulina polysaccharide proinflammatory enzyme inducer which can be used in humans or animals.

Disclosure of Invention

The preparation method and the application of the spirulina polysaccharide proinflammatory enzyme inducer provided by the invention are mainly used for solving the problems that although some researches refer to that spirulina has an immunoregulation effect, the preparation method is not described in detail, and the problem of providing the spirulina polysaccharide proinflammatory enzyme inducer applied to human or animals is not reported.

In order to achieve the purpose, the technical scheme provided by the invention is as follows:

a preparation method of spirulina polysaccharide proinflammatory enzyme inducer comprises the following steps:

s1, hot water leaching: mixing spirulina powder with distilled water according to a material-liquid ratio of 1g: 15-25 mL, uniformly stirring, and leaching for 1-3 hours at 80-95 ℃;

s2, concentration and centrifugation: filtering the leaching solution obtained in the step S1, rotatably evaporating the obtained filtrate at 50-55 ℃ to 20-25% of the original volume, then centrifuging for 15-25 min, and collecting supernatant;

s3, alcohol precipitation treatment: adding anhydrous ethanol with three times volume into the supernatant obtained in the step S2, precipitating overnight at 4 ℃, centrifuging and collecting precipitate;

s4, removing free protein: redissolving the collected precipitate with distilled water, mixing the obtained solution and an elution reagent according to a ratio, stirring at room temperature for 50-65 min, then centrifuging for 10-20 min, collecting supernatant, repeating the steps until no protein layer appears in the middle, and combining the collected supernatant;

s5, dialysis freeze-drying: and transferring the supernatant into a dialysis bag, dialyzing with distilled water for 1-3 days, taking out the dialysis retentate, standing overnight at-80 ℃, and then putting into a vacuum freeze-drying machine for vacuum freeze-drying to obtain the spirulina polysaccharide proinflammatory enzyme inducer.

Further, in step S1, the ratio of spirulina powder to distilled water was 1g:20 mL.

Further, in step S1, after the spirulina powder is mixed with distilled water and stirred uniformly, it is leached at 90 ℃ for 2 hours, and the leaching is repeated twice.

Further, in step S2, after the leaching solution was filtered, the resulting filtrate was rotary evaporated at 52 ℃ to 25% of the original volume, and then centrifuged to collect the supernatant.

Further, in step S4, the redissolved solution and the elution reagent are mixed at a volume ratio of 4:1, stirred at room temperature for 60min, centrifuged for 15min, the supernatant is collected, and after repeated elution, the collected supernatants are combined.

Further, in step S4, the elution reagent is prepared by mixing chloroform and n-butanol at a volume ratio of 4: 1.

Further, in step S5, the dialysis bag used had a molecular weight cut-off of 3000Da and was dialyzed with distilled water for 3 days, during which the dialysis water was changed every 6 hours.

Further, in steps S2 and S4, the rotation speed of the centrifugal process is 12000 rpm.

The invention also aims to disclose the application of the spirulina polysaccharide proinflammatory enzyme inducer prepared by the preparation method in cellular immune regulation.

Further, the spirulina polysaccharide proinflammatory enzyme inducer is used for promoting the protein expression of COX-2 and iNOS of macrophages in cellular immune regulation.

Compared with the prior art, the technical scheme of the invention has the beneficial effects that:

1. the preparation method of the spirulina polysaccharide proinflammatory enzyme inducer has the advantages of reasonable design, simple process, feasible operation, good repeated reproducibility, low protein content in the obtained product and higher purity;

2. the spirulina polysaccharide proinflammatory enzyme inducer does not need to use a large amount of toxic solvents in the preparation process, has good safety and mild preparation conditions, the obtained product has a stable structure, the bioactivity of the spirulina polysaccharide proinflammatory enzyme inducer can be ensured, and raw materials, instruments and reagents used in the preparation method are easy to obtain, so that the preparation cost is low, and the preparation method has good repeatability and is suitable for popularization;

3. the spirulina polysaccharide proinflammatory enzyme inducer can be applied to cellular immunoregulation and can promote the expression of COX-2 and iNOS proteins of macrophages, so that the spirulina polysaccharide proinflammatory enzyme inducer can be used as a scientific research reference basis for further research and development and application of spirulina polysaccharide.

Drawings

FIG. 1 is a high performance gel permeation chromatogram of a Spirulina polysaccharide proinflammatory enzyme inducer;

FIG. 2 is an ion chromatogram of a spirulina polysaccharide proinflammatory enzyme inducer;

FIG. 3 is an infrared spectrum of a spirulina polysaccharide proinflammatory enzyme inducer;

FIG. 4 is a Western blotting test result chart of the pro-inflammatory enzyme inducer of spirulina polysaccharide.

Detailed Description

The present invention is further illustrated by the following detailed description, which is a preferred embodiment of the invention. It should be understood that the embodiments described in the specification are only for the purpose of illustrating the present invention and not for the purpose of limiting the same, parameters, proportions, etc. of the embodiments may be selected as appropriate without substantially affecting the result, and any modification made within the scope of the claims of the present invention is within the scope of the claims of the present invention, and thus the scope of the claims of the present invention is not limited thereto.

Unless otherwise specified, the technical means used in the examples of the present invention are conventional means well known to those skilled in the art, and materials, reagents and the like used therein are commercially available.

Example 1:

s1, hot water leaching: mixing spirulina powder with distilled water according to a material-liquid ratio of 1g:20mL, uniformly stirring, leaching for 2 hours at 90 ℃, and repeatedly leaching twice;

s2, concentration and centrifugation: filtering the leaching solution obtained in the step S1, rotatably evaporating the obtained filtrate at 52 ℃ to 25% of the original volume, centrifuging for 20min, and collecting the supernatant;

s4, removing free protein: redissolving the collected precipitate with distilled water, mixing the obtained solution and an elution reagent according to the volume ratio of 4:1, stirring at room temperature for 60min, then centrifuging for 15min, collecting supernatant, repeating the steps until no protein layer appears in the middle, and combining the collected supernatant;

wherein the elution reagent is prepared by mixing trichloromethane and n-butanol according to the volume ratio of 4: 1;

s5, dialysis freeze-drying: transferring the supernatant into a dialysis bag with molecular weight cutoff of 3000Da, dialyzing with distilled water for 3 days, changing dialysis water every 6 hours, taking out the dialysis retentate, standing overnight at-80 ℃, and vacuum freeze-drying in a vacuum freeze-drying machine to obtain the spirulina polysaccharide proinflammatory enzyme inducer, PSP for short.

Example 2:

s1, hot water leaching: mixing spirulina powder with distilled water according to a material-liquid ratio of 1g:15mL, uniformly stirring, leaching for 3 hours at 80 ℃, and repeatedly leaching twice;

s2, concentration and centrifugation: filtering the leaching solution obtained in the step S1, rotatably evaporating the obtained filtrate at 50 ℃ to 25% of the original volume, centrifuging for 25min, and collecting the supernatant;

s4, removing free protein: redissolving the collected precipitate with distilled water, mixing the obtained solution and an elution reagent according to the volume ratio of 4:1, stirring for 50min at room temperature, then centrifuging for 10min, collecting supernatant, repeating the steps until no protein layer appears in the middle, and combining the collected supernatant;

s5, dialysis freeze-drying: transferring the supernatant into a dialysis bag with molecular weight cutoff of 3000Da, dialyzing with distilled water for 2 days, changing dialysis water every 6 hours, taking out the dialysis retentate, standing overnight at-80 ℃, and vacuum freeze-drying in a vacuum freeze-drying machine to obtain the spirulina polysaccharide proinflammatory enzyme inducer, PSP for short.

Example 3:

s1, hot water leaching: mixing spirulina powder with distilled water according to a material-liquid ratio of 1g:25mL, uniformly stirring, leaching for 1 hour at 95 ℃, and repeatedly leaching twice;

s4, removing free protein: redissolving the collected precipitate with distilled water, mixing the obtained solution and an elution reagent according to the volume ratio of 4:1, stirring at room temperature for 65min, then centrifuging for 20min, collecting supernatant, repeating the steps until no protein layer appears in the middle, and combining the collected supernatant;

To identify the components of the spirulina polysaccharide proinflammatory enzyme inducer (PSP) of the present invention, the spirulina polysaccharide proinflammatory enzyme inducer (PSP) prepared in example 1 of the present invention is exemplified, and the relative molecular mass, the basic component determination, the monosaccharide composition determination, and the functional group analysis are performed, and the respective corresponding determination or analysis methods are as follows:

1. determination of relative molecular masses: the relative molecular weight of the PSP was determined by High Performance Gel Permeation Chromatography (HPGPC). The sample was weighed and prepared as a 5mg/mL solution. Centrifuge at 12000rpm for 10 min. The supernatant was filtered through a 0.22m microporous membrane. The samples were then transferred to 1.8ml injection vials. Establishing a regression equation by using the logarithm of the relative molecular mass of the dextran standard product to the elution volume to obtain a standard curve, and calculating the relative molecular weight of the PSP according to the standard curve. The high performance gel permeation chromatography of Spirulina polysaccharide proinflammatory enzyme inducer (PSP) is shown in FIG. 1.

As can be seen from the analysis of FIG. 1, the Spirulina polysaccharide proinflammatory enzyme inducer (PSP) contains 3 polysaccharides with different molecular weights, which are 30062.75kDa, 4493.66kDa and 97.35kDa respectively.

2. Basic component determination: determining the total sugar content of PSP by using mannose as a standard substance and adopting a phenol-sulfuric acid method; bovine serum albumin is used as a standard substance, and the protein content of PSP is determined by adopting a Coomassie brilliant blue method.

The total sugar content in the spirulina polysaccharide proinflammatory enzyme inducer (PSP) is 43.08 percent by measuring through a phenol-sulfuric acid method;

the protein content in the spirulina polysaccharide proinflammatory enzyme inducer (PSP) was 3.4% as determined by the coomassie brilliant blue method.

3. The monosaccharide composition of PSP was determined by ion chromatography: 16 monosaccharide standards (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, galactosamine hydrochloride, glucosamine hydrochloride, N-acetyl-D glucosamine, guluronic acid, mannuronic acid) were formulated into 10mg/ml standard solutions. 5mg of PSP was weighed out accurately and placed in an ampoule, 10mL of a 2mol/L trifluoroacetic acid (TFA) solution was added, and the mixture was hydrolyzed at 120 ℃ for 3 hours. Accurately absorbing the acid hydrolysis solution, transferring to a tube, blowing to dry by nitrogen, adding 5ml of water, mixing uniformly by vortex, absorbing 100uL of deionized water, adding 900uL of deionized water, centrifuging for 5min at the rotating speed of 12000rpm, and taking the supernatant to perform ion chromatographic analysis, wherein an ion chromatogram is shown in figure 2.

The measurement conditions of the ion chromatograph were as follows:

a chromatographic column: dionex carbopaac tmp 20(3 × 150);

mobile phase: a is H₂O；B:250mM NaOH；C:50mM NaOH&500mM NaOAC；

Flow velocity: 0.3 mL/min;

sample feeding amount: 5 mu L of the solution;

column temperature: at 30 ℃.

As shown in fig. 2, it was determined and analyzed that spirulina polysaccharide proinflammatory enzyme inducer (PSP) was composed of rhamnose (69.84%), glucose (15.41%), galactose (3.26%), glucuronic acid (3.19%), galacturonic acid (2.87%), glucosamine hydrochloride (2.25%), xylose (1.48%), fucose (0.90%), mannose (0.79%), wherein rhamnose was the main component, followed by glucose.

4. Functional group analysis: fourier transform infrared (FT-IR) is adopted to reach 4000-400 cm^-1The PSP functional group and sugar ring configuration are analyzed in the spectrophotometer range, firstly, 1-2 mg of fully dried PSP sample is weighed, mixed with spectrum-grade potassium bromide powder (100mg), then extruded into particles of 1mm, and measured by a VERTEX 70FT-IR infrared spectrometer, and the obtained infrared spectrogram is shown in figure 3.

As can be seen by analysis in conjunction with FIG. 3, the infrared spectrum is 3355cm^-1The strong absorption peak is due to the stretching vibration of O-H, 2935cm^-1The nearby absorption peaks are generated by C-H stretching vibration and bending vibration respectively, which indicates that the spirulina polysaccharide proinflammatory enzyme inducer (PSP) has a typical polysaccharide characteristic absorption peak; 1655cm^-1The absorption peak is ascribed to C ═ O stretching vibration, which indicates that spirulina polysaccharide proinflammatory enzyme inducer (PSP) contains uronic acid; 1042cm^-1The absorption peak is ascribed to the stretching vibration of C-O, which indicates that the spirulina polysaccharide proinflammatory enzyme inducer (PSP) has a pyran ring structure.

Experimental example:

in order to further research and verify the action mechanism of the spirulina polysaccharide proinflammatory enzyme inducer (PSP) on the cell immunoregulation, the spirulina polysaccharide proinflammatory enzyme inducer (PSP) prepared in the embodiment 1 of the invention is taken as an example, the spirulina polysaccharide proinflammatory enzyme inducer (PSP) intervenes in macrophages, and the cell immunoregulation effect of the PSP is verified by detecting the protein expression conditions of iNOS and COX-2.

1. Subject: mouse-derived mononuclear macrophages (RAW264.7 cells)

2. The main reagents used in the experiment are shown in table 1:

TABLE 1 test reagents

Culture and passage of RAW264.7 cells:

adding 10% (v/v) fetal calf serum and 1% into DMEM medium(v/v) double antibody as a complete culture medium for RAW264.7 cells. Resuspending and mixing with complete culture medium, placing in sterile culture flask, and placing at 37 deg.C and 5% CO₂Culturing in the incubator, and carrying out passage when the cells are attached to the wall and almost fully grow at the bottom of the culture bottle.

When in passage, the old culture medium is firstly sucked and discarded, washed for 2 times by PBS buffer solution, 5mL of fresh complete culture medium is added, cells are blown down from the bottle wall, the cell suspension is passed to a new cell culture bottle according to a certain proportion, and the new cell culture bottle is put into a cell culture box for continuous culture.

Verification of the immunomodulatory effects of PSP cells:

protein expression of nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) in RAW264.7 cells was detected by Western blotting (Western blot) and was performed as follows:

setting up an experimental group, a blank group and a control group respectively, wherein the experimental conditions of each group are as follows:

experimental groups: RAW264.7 cells were plated at 4X 10⁵Inoculating each cell/mL into a 6-well plate, intervening with PSP solution with the concentration of 0, 50, 100 and 200 mu g/mL after the cells grow to 70-80%, and washing the RAW264.7 cells twice with ice-cold PBS buffer after 24 h.

Blank group: RAW264.7 cells did not intervene;

control group: the RAW264.7 cells are respectively interfered by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with the concentrations of 50 mug/mL, 100 mug/mL and 200 mug/mL;

② RAW264.7 cells of the experimental group, the blank group and the control group are collected and lysed with 100 mul of cell lysis buffer at 4 ℃ for 10 minutes to extract cell protein. The mixture was centrifuged at 12000rpm for 10 minutes, then the protein concentration was determined and the protein sample was boiled with the loading buffer for 10 minutes. The samples were separated by SDS-PAGE (8%) and then transferred to a 0.45 μm nitrocellulose membrane, which was blocked with 5% skim milk for 1 hour at room temperature, the primary antibody (COX-2, iNOS), diluted 1:1000 in TBST buffer and incubated overnight at 4 ℃, the membrane was washed 3 times with it, and incubated with a secondary antibody conjugated with horseradish peroxidase (1:5000) for 1 hour at room temperature, and developed. The results of the western blot detection of each group are shown in fig. 4.

As can be seen from the analysis of FIG. 4, the relative expression amounts of iNOS and COX-2 proteins in the RAW264.7 cells in the PSP solutions with different concentrations are different, and the expression amounts of iNOS and COX-2 proteins in the RAW264.7 cells are obviously increased with the increase of the PSP concentration, and the expression amounts of the two proteins are the highest when the PSP concentration is 100 μ g/ml. iNOS and COX-2 are important downstream effectors in the NF-. kappa.B signaling pathway. Nuclear transcription factor NF-. kappa.B family members usually form complexes with their inhibitory proteins Iκ Bs in homodimeric or heterodimeric form, exist in the cytoplasm in inactive form, and NF-. kappa.B can only be activated by various activating factors. NF-. kappa.B proteins typically form homo/heterodimers with p65 and p50 and are inactivated in the cytoplasm by the formation of a trimeric complex upon binding to the inhibitor Iκ B. Upon binding of the upstream signaling factor TNF to a cell membrane surface receptor, the receptor conformationally changes and transmits a signal to the IKK kinase, which in turn phosphorylates and dissociates the ikb protein from the trimer. Then NF-kB dimer exposes nuclear localization sequence, rapidly enters into nucleus from cytoplasm, and combines with specific sequence on nuclear DNA to promote transcription of related gene. According to Western Blot results, the protein expression levels of iNOS and COX-2 are obviously increased, so that the spirulina polysaccharide proinflammatory enzyme inducer (PSP) is deduced to play a role in cell immunoregulation by activating an NF-kB pathway, increasing the expression of iNOS and COX-2 and promoting the secretion of cytokines.

The above-mentioned embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. It will be apparent to those skilled in the art that other variations and modifications may be made in the foregoing description, and it is not necessary or necessary to exhaustively enumerate all embodiments herein. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A preparation method of a spirulina polysaccharide proinflammatory enzyme inducer is characterized by comprising the following steps:

s4, removing free protein: redissolving the collected precipitate with distilled water, mixing the obtained solution and an elution reagent in proportion, stirring at room temperature for 50-65 min, then centrifuging for 10-20 min, and collecting supernatant;

s5, dialysis freeze-drying: and transferring the supernatant into a dialysis bag, dialyzing with distilled water for 1-3 days, taking out the dialysis retentate, standing overnight at-80 ℃, and then freeze-drying in vacuum to obtain the spirulina polysaccharide proinflammatory enzyme inducer.

2. The method for preparing a spirulina polysaccharide proinflammatory enzyme inducer according to claim 1, wherein in step S1, the feed-to-liquid ratio of spirulina powder to distilled water is 1g:20 mL.

3. The method of claim 2, wherein the spirulina powder is mixed with distilled water and stirred uniformly in step S1, and then extracted at 90 ℃ for 2 hours, and the extraction is repeated twice.

4. The method of claim 1, wherein the extract solution is filtered in step S2, the filtrate is rotary evaporated at 52 ℃ to 25% of the original volume, and then centrifuged to collect the supernatant.

5. The method of claim 1, wherein in step S4, the re-dissolved solution and the elution reagent are mixed at a volume ratio of 4:1, stirred at room temperature for 60min, centrifuged for 15min, the supernatant is collected, and the collected supernatants are combined after repeated elution.

6. The method of claim 4, wherein in step S4, the elution reagent is a mixture of chloroform and n-butanol at a volume ratio of 4: 1.

7. The method of claim 1, wherein the dialysis bag used in step S5 has a molecular weight cut-off of 3000Da and is dialyzed against distilled water for 3 days.

8. The method for producing a pro-inflammatory enzyme inhibitor of spirulina platensis according to claim 1, wherein the rotation speed of the centrifugal treatment is 12000rpm in steps S2 and S4.

9. Use of the spirulina polysaccharide proinflammatory enzyme inducer prepared by the preparation method according to any one of claims 1-8 in cellular immune regulation.

10. The use of a spirulina polysaccharide proinflammatory enzyme inducer according to claim 9 for cellular immunomodulation, wherein the spirulina polysaccharide proinflammatory enzyme inducer is used to promote protein expression of COX-2 and iNOS in macrophages.