CN110742900B - Chlorella extracellular polysaccharide compound with immunoregulation activity and preparation method and application thereof - Google Patents
Chlorella extracellular polysaccharide compound with immunoregulation activity and preparation method and application thereof Download PDFInfo
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- CN110742900B CN110742900B CN201911155767.0A CN201911155767A CN110742900B CN 110742900 B CN110742900 B CN 110742900B CN 201911155767 A CN201911155767 A CN 201911155767A CN 110742900 B CN110742900 B CN 110742900B
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- chlorella
- culture
- culture medium
- polysaccharide
- exopolysaccharide
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- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/02—Algae
- A61K36/05—Chlorophycota or chlorophyta (green algae), e.g. Chlorella
Abstract
The invention discloses a chlorella extracellular polysaccharide compound with immunoregulation activity and a preparation method and application thereof. The extracellular polysaccharide complex is extracted from Chlorella protothecoides CS-41 fermentation liquor, is obtained by separation and purification procedures such as anion exchange chromatography, gel exclusion chromatography and the like after protein is removed by filtering, centrifuging, concentrating, alcohol precipitating and Sevage method, and is white powder, the average relative molecular weight of the polysaccharide complex is 170kDa, and the polysaccharide complex consists of lipid and polysaccharide, wherein the lipid contains palmitic acid and stearic acid, and the polysaccharide consists of rhamnose, xylose, mannose and glucose. The extracellular polysaccharide complex disclosed by the invention is clear in composition, has an immunoregulation effect, can obviously reduce the levels of inflammatory factors (ROS, iNOS, IL-6 and TNF-alpha), and has a wide application prospect in clinical and related medical care fields.
Description
Technical Field
The invention relates to the field of functional health-care food or biological medicine, in particular to a chlorella extracellular polysaccharide compound with immunoregulation activity and a preparation method and application thereof.
Background
Microalgae biodiesel has become a third generation sustainable biofuel recognized worldwide, and the development and application of microalgae biodiesel have important significance for solving the problems of shortage of fossil fuels and air pollution at present. However, the microalgae energy industry still faces the dilemma of high cost, is still in the starting stage and is difficult to be commercially applied. Improving the accumulation rate of oil in algal cells and increasing the oil yield is one of effective solutions for reducing the cost of microalgae biodiesel.
Around the growth and oil accumulation characteristics of algae cells at home and abroad, by changing environmental conditions such as temperature, illumination, nutritional conditions, salinity, metal ions and the like, great achievements are obtained in the research field of improving the oil yield of the microalgae cells, for example, the oil accumulation of the algae cells can be greatly promoted by adopting an exponential terminal-short time high temperature stress mode to culture Scenedesmus quadricauda (Scenedesmus quadratus), so that the lipid content and the yield of the algae cells respectively reach 33.5 percent and 23.2mg L-1d-1(environ. technol.,2016,37: 2649-; the oil yield of Chlorella (Chlorella sp.) can be increased to 15.67mg L by inducing the stress of low-phosphorus environment-1d-1(J.appl.Phycol.,2013,25: 311-318.); faizol Bux et al, using ferric ammonium citrate stress, increased the lipid content and lipid productivity of KJ671624 of Fulvox Fusarium (Ankistrodesmus falcatus) to 59.6% and 74.07mg L, respectively-1d-1(biochem, eng.j, 2015,94, 22-29). Although the oil production efficiency of algae cells is greatly improved through the regulation and control of culture modes and culture conditions, so that the yield of the algae cells is improved, the price of microalgae biodiesel is still too expensive compared with the price of petroleum (Biofuels,2010,1: 763-784). In fact, microalgae not only accumulate oil in the cell during their growth, but also secrete a variety of metabolites, such as polysaccharides, proteins, lipids, pigments, vitamins, inhibitory and stimulatory factors, etc., into the extracellular environment. Most of the secondary metabolites have biological activity and have great development and application prospects, whereinThe microalgae polysaccharide and the microalgae polysaccharide complex have various types, also have important biological activities of oxidation resistance, tumor resistance, virus resistance, blood fat reduction, blood sugar reduction and the like, and show great application potential in the fields of medicine and clinic. The development and utilization of secondary microalgae metabolites and the acquisition of high-value active substances are important ways for reducing the price of microalgae diesel and accelerating the industrialization process of microalgae.
Chlorella contains abundant polysaccharides with anti-tumor activity, pathogenic bacteria resistance, virus infection resistance and immunity enhancement and compounds thereof, but the research on polysaccharide substances of Chlorella protothecoides is less at present, and only Mario and the like analyze the cell wall polysaccharide composition (Arch. Mikrobiol.,1973,92,227 one 233) and George and the like to optimize the extraction process of intracellular polysaccharides (Chin.J.Appl.Environ.biol.,2014,20(4):615 one 620), so far, no related reports on the application values of the original extracellular polysaccharides and the compounds thereof exist. The polysaccharide compound with the immunoregulatory activity is separated and prepared from Chlorella protothecoides CS-41 fermentation liquor, so that the extraction and preparation of Chlorella oil cannot be influenced, a product with a medical and health care prospect can be obtained by high-valued utilization of extracellular metabolites, and the polysaccharide compound has important significance for comprehensive development of microalgae resources and reduction of microalgae oil cost.
Disclosure of Invention
The invention aims to obtain high-value byproducts of chlorella, provides a chlorella extracellular polysaccharide compound with immunoregulatory activity and a preparation method thereof, and also provides the biological activity application of the immunoregulatory polysaccharide.
The technical scheme of the invention is as follows:
the chlorella extracellular polysaccharide complex with the immunoregulatory activity is white powder, has the average relative molecular weight of 170kDa, and consists of 16.52% of lipid and 82.58% of polysaccharide in percentage by mass, wherein the lipid consists of 39.44% of palmitic acid (C16:0) and 60.56% of stearic acid (C18:0) in percentage by mass, and the polysaccharide component consists of 4 monosaccharides, namely rhamnose, xylose, mannose and glucose in percentage by mole, and is 10.22%, 46.19%, 17.98% and 25.62% respectively.
The preparation method of the chlorella exopolysaccharide compound with the immunoregulation activity comprises the following steps:
(1) inoculating Chlorella protothecoides CS-41 to a solid culture medium for rejuvenation, then transferring to a liquid culture medium to obtain a primary seed solution, and then continuing transferring to a fresh liquid culture medium for amplification culture to obtain a secondary seed solution;
(2) continuously carrying out amplification culture on the chlorella secondary seed liquid obtained in the step (1) under the conditions of fresh liquid culture medium and salt stress and mixotrophic culture to obtain chlorella fermentation liquid;
(3) collecting the supernatant of the fermentation liquor obtained in the step (2), precipitating with absolute ethanol, collecting the precipitate for redissolution, adding Sevage reagent into the obtained sample to remove protein, dialyzing, and freeze-drying to obtain crude extracellular polysaccharide of chlorella;
(4) separating the crude extracellular polysaccharide of the chlorella obtained in the step (3) by anion exchange chromatography, performing gradient elution by using a Tris-HCl buffer solution containing 0-1 mol/L NaCl, collecting a target elution peak, dialyzing and freeze-drying;
(5) separating the elution peak obtained in the step (4) by using a molecular exclusion chromatography, collecting elution components, dialyzing, and freeze-drying to obtain single-component polysaccharide, namely the chlorella extracellular polysaccharide with the immunoregulatory activity, which is named as CPEPS-2.
Further, in the step (1), the solid culture medium is a Basal culture medium, the solid culture and the first-class seed liquid culture are mixotrophic culture, the illumination intensity is 2000Lux, the illumination period is 12h illumination: performing dark alternate culture for 12h at 28 deg.C for 5 days; the secondary seed liquid culture is heterotrophic culture, the inoculation amount is 5%, the culture temperature is 28 ℃, and the culture time is 5 days.
Further, in the step (2), the liquid culture medium is a Basal culture medium, the salt stress concentration is 10 per mill salinity, the inoculation amount is 1%, the illumination intensity is 2000Lux, and the illumination period is 8h illumination: the cultivation is carried out alternately in darkness for 16h at 28 ℃ and 160rpm for 7 days.
Further, in the step (3), the Sevage reagent is prepared by mixing the following components in a volume ratio of 4:1, trichloromethane-isopropanol mixed solution; the dosage of the Sevage reagent is equal to the volume of the sample, and the cut-off molecular weight of a dialysis bag adopted for dialysis is 3500 Da.
Further, in the step (4), an anion exchange chromatography column is DEAE Sepharose Fast Flow, the size is 2.0cm × 30cm, the gradient eluent is 300mL Tris-HCl buffer solutions containing NaCl with different concentrations, the pH value is 7.4, the NaCl concentration gradient is 0, 0.3mol/L and 0.5mol/L, and the collected elution peak is a component eluted by 0.5mol/L NaCl.
Further, in the step (5), the size exclusion chromatography is performed by using Sephadex G-75 with a well-balanced 0.01mol/L NaCl solution, the size is 1.0cm × 50cm, and the eluent is 0.01mol/L NaCl solution.
The chlorella extracellular polysaccharide compound is applied to inhibiting inflammation.
Experimental results show that the chlorella extracellular polysaccharide has an obvious effect of inhibiting inflammation, and can obviously reduce the generation of inflammatory factors ROS, iNOS, IL-6 and TNF-alpha.
The invention has the beneficial effects that:
(1) the chlorella extracellular polysaccharide compound has immunoregulation activity, is extracted from chlorella supernatant and is a natural extract, and has good safety;
(2) the chlorella extracellular polysaccharide compound CPEPS-2 obtained by the invention is a pure product;
(3) the chlorella extracellular polysaccharide compound obtained by the invention has good inflammation inhibiting effect;
(4) the chlorella extracellular polysaccharide compound obtained by the invention is simple to prepare, and can be combined with intracellular grease extraction, so that the cost of the microalgae grease is further reduced.
Drawings
FIG. 1 is an ion exchange chromatogram of DEAE Sepharose Fast Flow of the Chlorella vulgaris exopolysaccharide complex of the present invention.
FIG. 2 is a Sephadex G-75 gel column analysis diagram of the chlorella exopolysaccharide complex of the present invention.
FIG. 3 is a high performance liquid chromatography of the chlorella exopolysaccharide complex of the present invention.
FIG. 4 is a Fourier infrared spectrum of the Chlorella exopolysaccharide complex of the present invention.
FIG. 5 is the nuclear magnetic resonance hydrogen spectrum of the chlorella exopolysaccharide complex of the present invention.
FIG. 6 is a graph showing the effect of the complex of extracellular polysaccharides of Chlorella vulgaris of the present invention on the production of ROS by macrophage RAW264.7 under stimulation of LPS, wherein the graph indicates a significant difference (P < 0.05) compared to the blank control group and indicates a very significant difference (P < 0.01); a represents a significant difference (P < 0.05) compared with the LPS group, and B represents a very significant difference (P < 0.01) (the same below).
FIG. 7 shows the effect of the complex of extracellular polysaccharides of Chlorella vulgaris of the present invention on iNOS in macrophage RAW264.7 under stimulation of LPS.
FIG. 8 shows the effect of the complex of extracellular polysaccharides of Chlorella vulgaris of the present invention on the production of IL-6 by the macrophage RAW264.7 under LPS stimulation.
FIG. 9 shows the effect of the Chlorella exopolysaccharide complex of the present invention on TNF- α production by macrophage RAW264.7 upon stimulation with LPS.
Detailed Description
The present invention will be further described with reference to specific examples, but the present invention is not limited thereto.
Example 1
Preparation of chlorella exopolysaccharide complex with immunoregulatory activity
(1) Inoculating Chlorella protothecoides CS-41 to Basal solid culture medium, and mixotrophic culturing at 28 deg.C under 2000Lux illumination for 5 days to rejuvenate the strain; selecting rejuvenated chlorella, transferring into common Basal liquid culture medium, culturing with 2000Lux (12h light: 12h dark alternately) at 28 deg.C and 160rpm for 5 days to obtain first-stage seed solution; inoculating 5% of the first-stage seed solution into a fresh liquid culture medium, and performing heterotrophic culture for 5 days to obtain a second-stage seed solution;
(2) taking 1% secondary seed liquid to perform amplification culture in a basic liquid culture medium containing 10 per mill NaCl salinity (about 2.784g/L), wherein the illumination intensity is 2000Lux, and the illumination period is 8 h: culturing at 28 deg.C and 160rpm for 7 days in 16h dark (alternately light and dark) to obtain chlorella fermentation broth;
(3) centrifuging the chlorella fermentation liquid obtained in the step (2) at 8000rpm for 15min, and collecting supernatant; concentrating under reduced pressure at 60 deg.C, adding anhydrous ethanol 4 times the volume of the concentrated solution, and precipitating at 4 deg.C overnight; collecting the precipitate, redissolving, adding an equal volume of Sevage reagent (chloroform: isopropanol-4: 1(v/v)) and collecting an upper solution by vigorous shaking; repeating for 5 times to remove protein; dialyzing with dialysis bag with molecular weight cutoff of 3500Da in distilled water at 4 deg.C for 48h, and changing water for 5 times; freeze drying to obtain crude extracellular polysaccharide of Chlorella;
(4) and (2) taking 20mg of crude extracellular polysaccharide of the chlorella obtained in the step (3), dissolving in 1mL of Tris-HCl buffer solution (pH 7.4), loading the solution into a DEAE Sepharose Fast Flow anion exchange column (2.0cm multiplied by 30cm), performing gradient elution by using 300mL of Tris-HCl buffer solution containing 0, 0.3mol/L and 0.5mol/L NaCl respectively, performing Flow rate of 2mL/min, collecting the solution in 5 min/tube, measuring the absorbance of each tube by adopting an anthrone-sulfuric acid method, drawing an elution curve (shown in a figure 1), collecting the elution peak of 0.5mol/L NaCl solution, dialyzing and freeze-drying.
(5) Dissolving the eluted peak components obtained in the step (4) in 0.01mol/L NaCl solution, eluting by Sephadex G-75 size exclusion chromatography (1.0cm × 50cm) with 0.01mol/L NaCl solution as mobile phase at a flow rate of 1mL/min for 3 min/tube, and tracing and monitoring the sugar concentration of each tube by anthrone-sulfuric acid method to draw an elution curve (as shown in FIG. 2). After the elution components are collected, distilled water is dialyzed (the molecular weight cutoff is 3500Da) and then freeze-dried to obtain single-component polysaccharide, namely the chlorella extracellular polysaccharide with the immunoregulation activity, which is named as CPEPS-2.
Example 2
Determination of component composition, purity and molecular weight of chlorella exopolysaccharide complex with immunoregulatory activity
(1) Determination of sugar content
Respectively taking 0mL, 0.05mL, 0.1mL, 0.15mL, 0.2mL and 0.25mL of glucose standard solution (100 mu g/mL), adding distilled water to 0.25mL, adding 1mL of anthrone (with the mass percent of 0.2%) -sulfuric acid solution into each tube under the ice bath condition, and uniformly mixing; after boiling water bath for 10min, the reaction was terminated by rapid ice-bath and then equilibrated to room temperature. Detecting its absorbance at 620nm with spectrophotometer, and measuring the absorbance with dextranGlucose concentration as abscissa, in OD620For the ordinate, a standard curve was prepared. Meanwhile, 2 mu L of chlorella extracellular polysaccharide complex aqueous solution (10mg/mL) is taken, distilled water is supplemented to 0.25mL, other steps are the same as the above, and the sugar content is calculated according to a standard curve.
(2) Fat content determination
Preparing serial cholesterol solutions with concentration of 10mg/mL, 20mg/mL, 30mg/mL, 40mg/mL and 50mg/mL with 0.2mL concentrated sulfuric acid, boiling in water bath for 10min, cooling at room temperature, adding 3mL phosphor vanillin solution (1.2mg/L), mixing, standing at room temperature for 10min, and determining OD520Values and standard curves were plotted. And meanwhile, dissolving 10mg of chlorella extracellular polysaccharide complex CPEPS-2 by 0.2mL of concentrated sulfuric acid, measuring the light absorption value of the sample solution at 520nm according to the method, and calculating the fat content in the sample according to a standard curve.
(3) Purity and molecular weight
Dissolving chlorella extracellular polysaccharide compound CPEPS-2 with double distilled water to prepare a solution with the concentration of 10mg/mL, and filtering the solution by using a 0.22 mu m filter membrane; loading 20 μ L polysaccharide solution on HPLC-SUGAR KS-804 SUGAR column-evaporation photodetector (ELSD) system, and eluting with double distilled water at flow rate of 1mL/min at room temperature; collecting elution pattern (as shown in FIG. 3), determining sample purity and molecular weight according to peak appearance, and FIG. 3 shows single narrow symmetrical peak, which can determine that the polysaccharide is pure product, retention time is 6.155min, and molecular weight of Chlorella vulgaris extracellular polysaccharide is 170kDa according to polysaccharide molecular weight standard curve y ═ 54x + 502.77.
Example 3
Monosaccharide composition and fatty acid composition analysis of chlorella exopolysaccharide complex with immunoregulatory activity
(1) Monosaccharide composition
Weighing 20mg of dried chlorella exopolysaccharide CPEPS-2, adding 10mL of trifluoroacetic acid (2mol/L), and hydrolyzing at 110 ℃ for 6 h; drying the hydrolysate, adding 2mL of methanol, evaporating to dryness under reduced pressure, and repeating for 3-4 times to remove trifluoroacetic acid; then mixing the sample with 10mg of hydroxylamine hydrochloride and 0.6mL of pyridine, and carrying out water bath reaction at 90 ℃ for 30 min; after cooling, adding 0.5mL of acetic anhydride to continue the reaction for 30min at 90 ℃; the reaction product was dried and dissolved in 1mL of chloroform for GC-MS analysis. Chromatographic conditions are as follows: HP-5 chromatographic column, sample amount: 2 μ L, injection port temperature: the flow rate is 1mL/min at 250 ℃; temperature rising procedure: keeping the temperature at 110 ℃ for 2min, heating the temperature to 5-220 ℃ per minute, and keeping the temperature for 6 min. The GC-MS analysis results show that: the chlorella exopolysaccharide CPEPS-2 consists of rhamnose, xylose, mannose and glucose, and the mole percentage of the four is 10.22: 46.19: 17.98: 25.62.
(2) fatty acid composition
Adding 100 mu L of nonadecanoic acid (1g/L) -dichloromethane solution and 2mL of potassium hydroxide (40g/L) -methanol solution into 10mg of chlorella exopolysaccharide complex CPEPS-2, uniformly mixing, carrying out water bath at 75 ℃ for 15min, adding 2mL of boron trifluoride (40g/L) -methanol solution (volume ratio is 1:1) after the reaction solution is cooled to room temperature, carrying out water bath at 75 ℃ for 15min, respectively adding 1mL of saturated sodium chloride solution and 2mL of n-hexane after cooling, carrying out vortex mixing, centrifuging at 4000rpm for 8min, taking 1mL of supernatant, filtering through a 0.22 mu m filter membrane, and carrying out GC-MS analysis. Chromatographic conditions are as follows: HP-5 chromatographic column, sample amount: 2 μ L, injection port temperature: the flow rate is 1mL/min at 250 ℃; temperature rising procedure: keeping the temperature at 150 ℃ for 2min, heating the temperature to 8-250 ℃ per minute, and keeping the temperature for 6 min. The NIST08 spectrum library automatically retrieves fatty acid information, and calculates the relative content of fatty acid according to peak area.
Table 1 shows the results of fatty acid component analysis of chlorella exopolysaccharide complex with immunoregulatory activity, chlorella exopolysaccharide complex CPEPS-2 contains 16.52% lipid component containing 39.44% palmitic acid (C16:0) and 60.56% stearic acid (C18: 0).
TABLE 1
Example 4
Infrared spectroscopic analysis of Chlorella extracellular polysaccharide complexes having immunomodulatory activity
Mixing 2mg of dried Chlorella extracellular polysaccharide complex CPEPS-2 with KBr powder by tabletting method, grinding, and tabletting into 1mm sheet with tabletting machine for infrared spectrum analysis, wherein the detected wave number range isIs 4000cm-1～500cm-1。
The fourier infrared spectrum of CPEPS-2 (as shown in fig. 4) shows the characteristic absorption peaks of the polysaccharide: O-H stretching vibration (3428.59 cm)-1) C-H stretching vibration (2934.81 cm)-1) And C ═ O in COO-and (1619.57 cm)-1Is the peak of absorption of the hydrated sample of sugar); wave number of 1732.33cm-1(C ═ O vibration) and 1414.87cm-1(symmetrical stretching vibration of COO-) indicates that CPEPS-2 is an acidic polysaccharide; 1384.39cm-1Is of CH3-symmetric variable angle vibration; 1254.84cm-1C-O stretching vibration is adopted, and the C-O-H and the glycosidic bond C-O-C are positioned on a sugar ring; 1062.42cm-1Absorption peaks of C-O-C and C-O-H single bonds of pyranose rings indicate that the CPEPS-2 has pyranoside-form monosaccharide; wave number of 911.37cm-1The position is an absorption peak of a beta-glycosidic bond, which indicates that the bond type of the polysaccharide is the beta-glycosidic bond; 804.21cm-1Is the characteristic absorption peak of the mannopyranose at 775.21cm-1The peak is the absorption peak of the D-glucose ring, indicating that CPEPS-2 contains mannose and glucose, consistent with the results for the monosaccharide composition.
Example 5
Nuclear magnetic resonance hydrogen spectrum analysis of chlorella extracellular polysaccharide compound with immunoregulation activity
10mg of dried chlorella extracellular polysaccharide compound CPEPS-2 is dissolved in 0.5mL of heavy water and is transferred into a nuclear magnetic tube, and nuclear magnetic resonance hydrogen spectrum analysis is carried out by taking Tetramethylsilane (TMS) as an internal standard and the working frequency of 400 MHz.
Process for preparing CPEPS-2 polysaccharide1The H-NMR results are shown in FIG. 5: 5 proton signals exist in a delta 4.5-5.5 ppm area, wherein a heavy hydrogen signal exists at the delta 4.60ppm position, and the rest 4 signal peaks represent that the polysaccharide contains 4 monosaccharides, and the result is consistent with that of GC-MS; a signal at δ 5.06ppm of greater than 5.0ppm indicates the presence of the α -form sugar ring configuration, and signal peaks at δ 4.53ppm, δ 4.85ppm, and δ 4.98ppm are each less than 5.0ppm, indicating the presence of the β -sugar ring configuration in CPEPS-2.
Example 6
Immunomodulatory activity of chlorella exopolysaccharide complex having immunomodulatory activity
(1) Construction of a model of cellular inflammation
Macrophage RAW264.7 in log phase was resuspended in 1640 minimal medium containing 10% (volume fraction) fetal bovine serum and 1X 10 was added to 6-well plates4(ii) individual cells; after overnight culture for pre-adherence, removing the old culture medium, and adding fresh culture media containing medicines with different concentrations; after 1 hour, LPS (lipopolysaccharide) of 1. mu.g/mL was added to the wells to construct an inflammatory cell model, and the culture was continued for 24 hours.
(2) Effect of Chlorella exopolysaccharide complexes on ROS production by macrophage RAW264.7 under inflammatory conditions
Diluting DCFH-DA (2',7' -dichlorofluorescein diacetate, 1:1000) with serum-free medium to a final concentration of 10. mu. mol/L; after collecting cells by trypsinization, the cells were resuspended in 1mL of diluted DCFH-DA solution and incubated at 37 ℃ for 20 min; fully washing the cells for 3 times by using a serum-free 1640 culture medium to remove DCFH-DA; resuspend cells with 100 μ Ι _ of PBS solution (pH 7.4) and add to 96-well plate; and (3) detecting the fluorescence signal intensity of each hole by using a fluorescence microplate reader, wherein the excitation wavelength is 488nm and the emission wavelength is 525nm during detection. The fluorescence intensity in fig. 6 represents ROS content, and the results show that intracellular ROS content is significantly increased after LPS stimulates macrophage RAW 264.7; however, the ROS content in the cells after being treated by the polysaccharide compound CPEPS-2 shows a descending trend, and the polysaccharide CPEPS-2 has the function of inhibiting the ROS.
(3) Effect of Chlorella exopolysaccharide Complex on iNOS in macrophage RAW264.7 under inflammatory conditions
Digesting the cells in each well by pancreatin, washing the cells by PBS solution after collection and suspending to 100 mu L; repeatedly freezing and thawing the cells to lyse the cells to form cell homogenate, and detecting the content of iNOS in macrophage RAW264.7 according to the instruction of a kit (Nanjing Jiang A014-1), wherein the specific operation is as follows: sucking 100 mu L of cell homogenate, simultaneously adding a reagent six (100 mu L), a substrate buffer solution (200 mu L), an accelerant (10 mu L) and a color developing agent (100 mu L), uniformly mixing, carrying out water bath at 37 ℃ for 15min, and using double distilled water instead of a sample as a control tube; adding a clearing agent (100 mu L) and a terminating agent (2000 mu L) into the reaction solution, and uniformly mixing; the absorbance value of each tube measured at 530nm was the intracellular iNOS content (see FIG. 7). The result shows that LPS stimulates macrophage RAW264.7 to synthesize a large amount of inflammatory factor iNOS; the polysaccharide compound CPEPS-2 can reduce the content of iNOS in cells along with the increase of the dosage of CPEPS-2, thereby achieving the effect of inhibiting inflammation.
(4) Effect of Chlorella exopolysaccharide complex on IL-6 and TNF-alpha production by macrophage RAW264.7 under inflammatory conditions
Collecting the supernatant of each well cell, centrifuging at 3000rpm for 5min, collecting the supernatant, and detecting the content of IL-6 and TNF-alpha secreted by macrophage RAW264.7 according to the kit instructions (Shanghai Meixuan organism MEXN-M0047, MEXN-M0042), specifically operating as follows: absorbing 50 mu L of supernatant liquid, adding the supernatant liquid into an enzyme label plate, and simultaneously adding 50 mu L of standard substances (IL-6 or TNF-alpha) with different concentrations into the enzyme label plate; then adding 100 mu L of enzyme-labeled reagent into each hole, and reacting for 30min at 37 ℃; discarding liquid in each hole, washing the ELISA plate for 5 times by using a washing solution, and patting dry; adding 50 μ L of color development liquid A/B respectively, and incubating for 15min in dark; adding 50 μ L of stop solution, and detecting OD value at 450nm with microplate reader within 30 min. The ordinate of fig. 8 and fig. 9 represents the content of inflammatory factors IL-6 and TNF- α secreted out from macrophage RAW264.7, respectively, and the results show that as the dose of polysaccharide complex CPEPS-2 is increased, the amount of IL-6 and TNF- α secreted by cells into the supernatant is significantly reduced, and inflammation is inhibited.
Claims (6)
1. A chlorella exopolysaccharide complex having immunomodulatory activity, comprising: the chlorella extracellular polysaccharide compound is white powder, has the molecular weight of 170kDa, and consists of 16.52% of lipid and 82.58% of polysaccharide in percentage by mass, wherein the lipid comprises palmitic acid (C16: 039.44%) and stearic acid (C18: 060.56%), and the polysaccharide component comprises 4 monosaccharides including rhamnose, xylose, mannose and glucose in percentage by mass, and the molar percentages are 10.22%, 46.19%, 17.98% and 25.62%, respectively;
the extracellular polysaccharide compound is extracted from chlorella fermentation liquor and is obtained by separation and purification, and the acquisition of the chlorella fermentation liquor comprises the following steps:
(1) inoculating Chlorella protothecoides CS-41 to a solid culture medium for rejuvenation, then transferring to a liquid culture medium to obtain a primary seed solution, and then continuing transferring to a fresh liquid culture medium for amplification culture to obtain a secondary seed solution;
(2) transferring the secondary chlorella seed solution obtained in the step 1) to a fresh liquid culture medium, and continuously carrying out amplification culture under the conditions of salt stress and mixotrophic culture to obtain chlorella fermentation liquor.
2. The method for preparing chlorella exopolysaccharide complexes with immunomodulatory activity of claim 1, wherein the method comprises the steps of: the extracellular polysaccharide compound is extracted from chlorella fermentation liquor and is obtained by separation and purification, and the acquisition of the chlorella fermentation liquor comprises the following steps:
(1) inoculating Chlorella protothecoides CS-41 to a solid culture medium for rejuvenation, then transferring the Chlorella protothecoides to a liquid culture medium to obtain a primary seed solution, and then continuing transferring the Chlorella protothecoides to a fresh liquid culture medium for amplification culture to obtain a secondary seed solution;
(2) transferring the secondary chlorella seed solution obtained in the step 1) to a fresh liquid culture medium, and continuously carrying out amplification culture under the conditions of salt stress and mixotrophic culture to obtain chlorella fermentation liquor.
3. The method for preparing chlorella exopolysaccharide complexes with immunomodulatory activity of claim 2, wherein the method comprises the steps of: in the step (1), the culture medium is a Basal culture medium, the solid culture and the first-stage seed liquid culture are mixotrophic culture, the illumination intensity is 2000Lux, the illumination period is 12h illumination: performing dark alternate culture for 12h at 28 deg.C for 5 days; the secondary seed liquid culture is heterotrophic culture, the inoculation amount is 5%, the culture temperature is 28 ℃, and the culture period is 5 days.
4. The method for preparing chlorella exopolysaccharide complexes with immunomodulatory activity as claimed in claim 2, wherein the method comprises the steps of: in the step (2), the inoculation amount of the chlorella secondary seed solution is 1%, the used liquid culture medium is a Basal culture medium, the salt stress concentration is 10 per mill salinity, the illumination intensity is 2000Lux, and the light dark period is 8h illumination: the cultivation is carried out alternately in 16h of darkness and at the temperature of 28 ℃ and the rotation speed of 160rpm for 7 days.
5. Use of the chlorella exopolysaccharide complex of claim 1 in the preparation of a medicament for inhibiting inflammation.
6. The use according to claim 5, wherein the Chlorella exopolysaccharide complex significantly reduces the production of inflammatory factors ROS, iNOS, IL-6 and TNF- α.
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