CN116925251A - Zijuan tea polysaccharide ZTPW with immunoregulatory activity and preparation method and application thereof - Google Patents
Zijuan tea polysaccharide ZTPW with immunoregulatory activity and preparation method and application thereof Download PDFInfo
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- CN116925251A CN116925251A CN202310886615.8A CN202310886615A CN116925251A CN 116925251 A CN116925251 A CN 116925251A CN 202310886615 A CN202310886615 A CN 202310886615A CN 116925251 A CN116925251 A CN 116925251A
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- polysaccharide
- zijuan tea
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Abstract
The invention belongs to the field of tea polysaccharide research, and discloses a Zijuan tea polysaccharide ZTPW with immunoregulatory activity, and a preparation method and application thereof. ZTPW is a homogeneous polysaccharide obtained by hot water extraction and alcohol precipitation of Zijuan tea, and separation and purification by ion exchange column and gel column, and has the main repeating unit structure shown below and weight average molecular weight of 7.73kDa. Through in vitro immunoregulatory activity research, ZTPW can be identified by pattern recognition receptors TLR2 and TLR4 to activate macrophages, the activity of mouse mononuclear macrophage RAW264.7 is obviously enhanced, and NO and cytokines are generated by mediating mouse mononuclear macrophage RAW264.7 through MAPKs and NF- κB signal pathway to play a role in immunoregulation. The polysaccharide of the invention can be used as a potential immunomodulator for developing functional foods or medicines.
Description
Technical Field
The invention belongs to the field of tea polysaccharide research, and in particular relates to Zijuan tea polysaccharide ZTPW with immunoregulatory activity, and a preparation method and application thereof.
Background
Tea polysaccharide (Tea polysaccharides, TPS) is a very important bioactive macromolecule in tea, and has various functional activities including immunoregulation, anti-tumor, antioxidation, anti-inflammatory, blood sugar and blood lipid reduction and the like. The immune regulation of polysaccharide can play an important role in disease prevention and control, and research on the chemical structure and action mechanism of immunocompetent polysaccharide by scientific researchers has been greatly progressed in recent years. The polysaccharide can regulate and improve the immune system of the organism by directly or indirectly activating macrophages and T/B lymphocytes, promoting and supplementing interferon, interleukin, tumor necrosis factor and the like, and is related to other anticancer, antiviral and other functions, so the polysaccharide has important application value in the development of functional foods and medicines.
In China, zijuan tea is a rare special tea tree resource, belongs to small arbor type and large leaf type medium bud species, and has the unique feature of purple stem, purple leaf and purple bud. The bud leaves are purple, red or red purple, have high anthocyanin content which can reach more than 1 percent of dry weight generally, the anthocyanin content in the common green bud leaves is only about 0.1 percent, the Zijuan tea is rich in anthocyanin, and the bud leaves have the functions of antioxidation, anti-aging and anticancer, the antioxidation performance is 50 times higher than vitamin E and 20 times higher than vitamin C, and the bud leaves have direct or indirect preventive treatment effects on more than 100 diseases. The current research on Zijuan tea mainly focuses on chemical component analysis and anthocyanin extraction technology of different tea products. In recent years, the breeding of variety of Zijuan tea and the research and development of products thereof are increasingly focused, and although the chemical composition analysis and anthocyanin extraction technologies of Zijuan tea are widely recorded, the structural analysis and immunoregulatory activity of Zijuan tea polysaccharide are not studied deeply.
Disclosure of Invention
In order to overcome the defects and the shortcomings of the prior art, the primary aim of the invention is to provide the Zijuan tea polysaccharide with immunoregulatory activity.
The invention also aims at providing a preparation method of the Zijuan tea polysaccharide with immunoregulatory activity; the invention takes the Zijuan tea as a research object, separates and purifies the Zijuan tea by hot water extraction and alcohol precipitation extraction through a DEAE ion exchange column and a molecular sieve, researches the biological activity of the Zijuan tea, and analyzes the monosaccharide composition, the glycosidic bond connection mode and the chain structure of the new component of the Zijuan tea polysaccharide ZTPW.
The invention also aims at providing an application of the Zijuan tea polysaccharide with immunoregulatory activity.
The aim of the invention is achieved by the following technical scheme:
a main repeating unit of Zijuan tea polysaccharide ZTPW with immunoregulatory activity comprises the following structure 1 and structure 2, and has weight average molecular weight (Mw) of 7.73kDa, number average molecular weight (Mn) of 7.53kDa, and dispersion index (Mw/Mn) of 1.03;
the sugar content of the Zijuan tea polysaccharide ZTPW is 85.59wt%.
The Zijuan tea polysaccharide ZTPW mainly comprises arabinose, galactose, glucose, xylose and mannose with the molar ratio of 18.48:15.92:32.99:3.32:29.29.
The preparation method of the Zijuan tea polysaccharide ZTPW comprises the following steps:
(1) Degreasing the crushed and sieved Zijuan tea powder, and drying to obtain Zijuan tea degreased powder;
(2) Adding deionized water into the defatted powder of Zijuan tea, extracting for multiple times, filtering, mixing the supernatants, and concentrating under reduced pressure to obtain concentrated solution;
(3) Precipitating the concentrated solution with ethanol, redissolving, removing protein, dialyzing, and lyophilizing to obtain crude polysaccharide lyophilized powder;
(4) Preparing crude polysaccharide freeze-dried powder into crude polysaccharide solution, separating and eluting by an ion exchange column, measuring by a phenol sulfuric acid method, collecting a target component peak elution product, concentrating, dialyzing, and freeze-drying to obtain a ZTPW component;
(5) Further separating ZTPW component by molecular sieve, detecting polysaccharide content by phenol-sulfuric acid method, collecting target component peak eluting product, concentrating the eluate, dialyzing, and freeze drying to obtain Zijuan tea polysaccharide ZTPW.
The step (1) is specifically carried out according to the following steps: grinding Zijuan tea, and sieving with a 40-mesh sieve to obtain Zijuan tea powder, wherein the feed liquid ratio is 1g:10mL, adding 85vt% ethanol into Zijuan tea powder, extracting at 70deg.C for 8h, repeating the extraction operation for three times, filtering, collecting precipitate, and oven drying at 60deg.C to obtain Zijuan tea defatted powder;
the step (2) is specifically carried out according to the following steps: according to the feed liquid ratio of 1g:15mL, adding deionized water into the defatted powder of Zijuan tea, extracting at 80deg.C for 2 hr to obtain water extractive solution, filtering with absorbent cotton yarn, repeating the extraction operation for 3 times to obtain residue, mixing filtrates, and concentrating under reduced pressure at 60deg.C to obtain concentrated solution;
the step (3) is specifically carried out according to the following steps: adding absolute ethanol into the concentrated solution to make the final concentration of ethanol 80vt%, standing at 4deg.C for 16h to precipitate insoluble polysaccharide, centrifuging at 6000r/min for 10min, and collecting precipitate; dissolving the precipitate in deionized water to obtain a crude polysaccharide solution; adding the same volume of Sevage reagent into the crude polysaccharide solution, wherein the Sevage reagent is chloroform-n-butanol mixed solution with the volume ratio of 4:1, stirring for 40min by using a magnetic stirrer, centrifuging at 6000rpm for 10min at room temperature, removing protein, collecting supernatant, repeatedly removing protein until white floccules are not formed at the boundary of two phases, and removing organic solvent by rotary evaporation; and then dialyzing the protein-removed polysaccharide solution for 72 hours by using a 3.5kDa dialysis bag, and freeze-drying to obtain crude polysaccharide freeze-dried powder.
The step (4) is specifically carried out according to the following steps: preparing crude polysaccharide freeze-dried powder into a solution with the concentration of 30mg/mL, separating by a DEAE-Sepharose fast flow ion exchange column, eluting by adopting ultrapure water, wherein the eluting flow rate is 2mL/min, the eluting time is 4 min/tube, receiving the front 45 tubes of eluting solution by adopting a full-automatic partial collector, detecting the polysaccharide content of each tube by adopting a phenol-sulfuric acid method, drawing an eluting curve, collecting 19-24 tubes of main eluting peak solution according to the eluting curve, dialyzing for 72h by adopting a 3.5kDa dialysis bag after concentrating, and freeze-drying to obtain a ZTPW component;
the step (5) is specifically carried out according to the following steps: preparing ZTPW component into solution with concentration of 10mg/mL, separating and purifying by Sephacryl S-200HR gel permeation column, eluting with phosphate buffer solution with pH of 7.4 containing 0.15MNaCl, eluting with flow rate of 1mL/min, eluting time of 8 min/tube, collecting the eluate of the first 60 tubes by using fully automatic part collector, measuring polysaccharide content by phenol-sulfuric acid method, drawing elution curve, collecting the main eluting peak solution of 35-50 tubes according to the elution curve, concentrating, dialyzing for 72h by 3.5kDa dialysis bag, and lyophilizing to obtain Zijuan tea polysaccharide ZTPW.
The application of the Zijuan tea polysaccharide ZTPW in preparing functional food and immunomodulator medicine is provided.
The functional food and the immunomodulator drug have the activity of enhancing the activity of macrophages, and/or increasing the NO production of the macrophages, and/or increasing the cytokine production of the macrophages.
The cytokines include TNF-alpha, IL-6.
The immunomodulator can be recognized by pattern recognition receptors TLR2 and TLR4, and can mediate macrophages to promote the production of NO and/or cytokines to play a role in regulating the immunity by activating Mitogen Activated Protein Kinase (MAPKs) and nuclear transcription factor kappa B (NF-kappa B) signaling pathways.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. the invention takes Zijuan tea as a research object, carries out hot water extraction and alcohol precipitation extraction, and separates and purifies the Zijuan tea polysaccharide ZTPW through a DEAE ion exchange column and a molecular sieve, and determines the specific activity application thereof;
2. according to the determination, the weight average molecular weight of the Zijuan tea polysaccharide ZTPW extracted and separated by the invention is 7.73kDa, and the Zijuan tea polysaccharide ZTPW mainly comprises arabinose, galactose, glucose, xylose and mannose, and the molar ratio of the Zijuan tea polysaccharide ZTPW to the mannose is 18.48:15.92:32.99:3.32:29.29; the method has the characteristic absorption peaks of saccharide compounds and alpha-and beta-configuration pyranose, and combines methylation and nuclear magnetic analysis to analyze the chemical structure of ZTPW, so as to determine the Zijuan tea polysaccharide ZTPW as a new substance;
3. the Zijuan tea polysaccharide ZTPW obtained by extraction and separation is in the concentration range of 25-200 mug/mL, and has no cytotoxicity compared with a control group; can obviously promote macrophage RAW264.7 to generate NO; can remarkably increase the production of macrophage RAW264.7 cytokines (TNF-alpha and IL-6); ZTPW is recognized by pattern recognition receptors TLR2 and TLR4, and exerts its immunomodulatory functions by activating MAPKs and NF- κb signaling pathways to mediate macrophages and promote production of NO, cytokines.
Drawings
FIG. 1 is an ultraviolet absorption spectrum of Zijuan tea polysaccharide ZTPW;
FIG. 2 is an HPGPC chart of Zijuan tea polysaccharide ZTPW;
FIG. 3 is a monosaccharide composition of Zijuan tea polysaccharide ZTPW;
fig. 4 is an infrared spectrum of the Zijuan tea polysaccharide ZTPW;
FIG. 5 is a ZTPW of Zijuan tea polysaccharide 1 H NMR spectrum;
FIG. 6 is a ZTPW of Zijuan tea polysaccharide 13 C NMR spectrum;
FIG. 7 shows the polysaccharide ZTPW of Zijuan tea 1 H- 1 HCOSY profile;
FIG. 8 is a HSQC spectrum of Zijuan tea polysaccharide ZTPW;
fig. 9 is an HMBC profile of the Zijuan tea polysaccharide ZTPW;
FIG. 10 is a graph showing the cytotoxicity effect of Zijuan tea polysaccharide ZTPW on macrophage RAW 264.7;
FIG. 11 is the effect of the Zijuan tea polysaccharide ZTPW on RAW264.7 cell culture supernatant (A) NO, (B) IL-6, (C) TNF- α expression;
FIG. 12 is the effect of the Zijuan tea polysaccharide ZTPW on mRNA expression of RAW264.7 cell culture supernatant (A) iNOS, (B) IL-6, (C) TNF- α;
FIG. 13 is a graph showing the effect of TLR2, TLR4 and Dectin-1 antibodies on the production of (A) NO, (B) IL-6 and (C) TNF- α by the ZtPW-induced macrophage RAW 264.7;
FIG. 14 is a graph showing the effects of four inhibitors of SP600125, U0126, SB203580, BAY11-7082 on the production of NO, IL-6 and TNF- α by the ZtPW-induced macrophage RAW 264.7.
Detailed Description
The invention is described in further detail below with reference to the drawings and the specific examples, but the embodiments of the invention are not limited thereto. Unless otherwise specified, the reagents, apparatus and methods employed in the present invention are those conventionally commercially available in the art and those conventionally used.
Example 1
Preparation of Zijuan tea polysaccharide ZTPW
Grinding Zijuan tea by using a mortar, sieving with a 40-mesh sieve to obtain Zijuan tea powder, wherein the feed liquid ratio is 1g: adding 85vt% ethanol into Zijuan tea powder 10mL, extracting at 70deg.C for 8 hr, shaking and mixing once every two hours, repeating the extraction operation for three times, filtering with filter paper, collecting precipitate, and oven drying at 60deg.C to obtain Zijuan tea defatted powder;
according to the feed liquid ratio of 1g:15mL, adding deionized water into the defatted powder of Zijuan tea, extracting at 80deg.C for 2 hr to obtain water extractive solution, filtering with absorbent cotton yarn, repeating the extraction operation for 3 times to obtain residue, mixing filtrates, and concentrating under reduced pressure at 60deg.C to obtain concentrated solution;
adding absolute ethanol into the concentrated solution to make the final concentration of ethanol 80vt%, standing at 4deg.C for 16h to precipitate insoluble polysaccharide, centrifuging at 6000r/min for 10min, and collecting precipitate; dissolving the precipitate in deionized water to obtain a crude polysaccharide solution;
adding the same volume of Sevage reagent into the crude polysaccharide solution, wherein the Sevage reagent is chloroform-n-butanol mixed solution with the volume ratio of 4:1, stirring for 40min by using a magnetic stirrer, centrifuging at 6000rpm for 10min at room temperature, removing protein, collecting supernatant, repeatedly removing protein until white floccules are not formed at the boundary of two phases, and removing organic solvent by rotary evaporation; and then dialyzing the protein-removed polysaccharide solution for 72 hours by using a 3.5kDa dialysis bag, and freeze-drying to obtain crude polysaccharide freeze-dried powder.
Preparing crude polysaccharide freeze-dried powder into a solution with the concentration of 30mg/mL, separating by a DEAE-Sepharose fast flow ion exchange column, eluting by adopting ultrapure water, wherein the eluting flow rate is 2mL/min, the eluting time is 4 min/tube, receiving the front 45 tubes of eluting solution by adopting a full-automatic partial collector, detecting the polysaccharide content of each tube by adopting a phenol-sulfuric acid method, drawing an eluting curve, collecting 19-24 tubes of main eluting peak solution according to the eluting curve, dialyzing for 72h by adopting a 3.5kDa dialysis bag after concentrating, and freeze-drying to obtain a ZTPW component;
preparing ZTPW component into solution with concentration of 10mg/mL, separating and purifying by Sephacryl S-200HR gel permeation column, eluting with phosphoric acid buffer solution with pH of 7.4 containing 0.15M NaCl, eluting with flow rate of 1mL/min, eluting time of 8 min/tube, collecting the eluate of the first 60 tubes by using fully automatic part collector, measuring polysaccharide content by phenol-sulfuric acid method, drawing elution curve, collecting the main elution peak solution of 35-50 tubes according to the elution curve, concentrating, dialyzing with 3.5kDa dialysis bag for 72h, and lyophilizing to obtain Zijuan tea polysaccharide ZTPW.
Two) content determination of Zijuan tea polysaccharide ZTPW
Preparing ZTPW into 0.1mg/mL solution, measuring by phenol sulfuric acid method, sucking 150 μL polysaccharide solution into 2mL centrifuge tube, adding 75 μL 6wt% phenol, mixing by vortex oscillator, adding 375 μL concentrated sulfuric acid, mixing, standing on ice for 3min, boiling water bath for 10min, cooling to room temperature, measuring its absorbance at 490nm, substituting standard curve y=4.1975x+0.0217 (R) 2 = 0.9979), the ZTPW sugar content was calculated to be 85.59wt%.
Three) ultraviolet spectrum analysis of Zijuan tea polysaccharide ZTPW
A certain amount of the obtained Zijuan tea polysaccharide ZTPW is dissolved in primary water to prepare a solution with the concentration of 1mg/mL, and the solution is scanned and analyzed on a U-2910 spectrophotometer, the wavelength range is 200-400nm, the result is shown as figure 1, and no characteristic absorption peak exists at 260nm and 280nm, so that the ZTPW does not contain nucleic acid and protein.
Fourth) determination of molecular weight of Zijuan tea polysaccharide ZTPW
The molecular weight of the obtained Zijuan tea polysaccharide ZTPW is measured by gel chromatography-differential-multi-angle laser light scattering system, and liquid phase systemThe differential detector is Optilab T-rEX, and the laser light scattering detector is DAWN HELEOS II. The column temperature was 45℃and the mobile phase was NaNO, using a gel exclusion chromatography column (Ohpak SB-805HQ (300X 8 mm), ohpak SB-804HQ (300X 8 mm) and Ohpak SB-803HQ (300X 8 mm) in series 3 (100 mM), flow rate was 0.4mL/min, and ZTPW molecular weight was calculated by software ASTRA 6.1 analysis, and the results are shown in FIG. 2.
Five) monosaccharide composition analysis of Zijuan tea polysaccharide ZTPW
Taking a proper amount of the obtained Zijuan tea polysaccharide ZTPW, hydrolyzing with 1mL of 2M trifluoroacetic acid at 121 ℃ for 2h, introducing nitrogen, and drying. Adding 99.99% methanol for cleaning, drying, and repeating methanol cleaning for 2-3 times. Adding sterile water for dissolving, and transferring into chromatographic bottle for testing.
A Siemens ion chromatography system (ICS 5000) was used, a Dionex CarboPac PA (3.0 mm. Times.150 mm) liquid chromatography column. Mobile phase a: water; mobile phase B:0.1M NaOH, mobile phase C:0.1M NaOH, 0.2M NaAc; sample injection volume 5. Mu.L; the flow rate is 0.5mL/min; the column temperature was 30℃and the detection was performed by an electrochemical detector.
Wherein the standard substance is: fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, guluronic acid, galacturonic acid, glucuronic acid, mannuronic acid. The results are shown in FIG. 3.
As shown by the ion chromatography result, the monosaccharide composition of the Zijuan tea polysaccharide ZTPW mainly comprises arabinose, galactose, glucose, xylose and mannose, and the molar ratio of the monosaccharides is 18.48:15.92:32.99:3.32:29.29.
Six) infrared spectrum analysis of Zijuan tea polysaccharide ZTPW
Mixing 1mg of the obtained Zijuan tea polysaccharide ZTPW with 100mg of dry potassium bromide powder, grinding in agate mortar, tabletting, and making into tablet with a thickness of 4000-400cm -1 Is scanned by infrared spectrum in the range of (2) and the result is shown in FIG. 4 at 3397.52cm -1 、2926.36cm -1 Characteristic absorption peak of saccharide appears at 1151.33cm -1 、1060.50cm -1 、1027.06cm -1 The absorption of (C) indicates that the characteristic moiety is in the pyranose configuration at 895.52cm -1 And 799.37cm -1 The absorption peak at this point indicates that both beta-and alpha-configured glycosidic linkages are present in ZTPW.
Seven) methylation analysis of Zijuan tea polysaccharide ZTPW
Placing the obtained Zijuan tea polysaccharide ZTPW sample in a reaction bottle, adding dimethyl sulfoxide and NaOH powder, immediately sealing and dissolving, adding methyl iodide in dark place, methylation for 1h, and finally adding deionized water to terminate the reaction. After the methylation reaction was completed, the mixture was extracted 3 times with chloroform and spin-evaporated to dryness.
The methylated polysaccharide was hydrolyzed with 2M trifluoroacetic acid at 121℃for 90min, and the excess acid was removed by rotary evaporation with methanol. Adding 2M ammonia water and 1M NaBD4 into the hydrolysate, mixing, reacting at room temperature for 2.5h, adding glacial acetic acid after the reaction is finished to neutralize redundant NaBD 4 Drying by nitrogen, washing by methanol twice, and drying by nitrogen. Acetic anhydride is added for acetylation at 100 ℃ for 2.5h, the mixture is cooled to room temperature, and water is added for standing for 10min. The acetylated product is extracted by 3mL of chloroform, a small amount of distilled water is added for full washing for 3 times, after the chloroform layer is removed for volume fixing, an Agilent 7890A-5977B gas chromatography-mass spectrometer is adopted for measuring an acetylated product sample.
GC-MS conditions: BPX70 column 30m x 0.25mm x 0.25 μm; the temperature programming conditions are as follows: the sample injection amount is 1 mu L, the split ratio is 10:1, and the carrier gas is high-purity helium; the initial temperature of the column oven was kept at 140℃for 2.0min, and the temperature was programmed to 230℃at 3℃per minute for 3min. The results are shown in Table 1 below.
TABLE 1 Zijuan tea polysaccharide ZTPW methylation analysis results
Eight) nuclear magnetic resonance analysis of Zijuan tea polysaccharide ZTPW
Dissolving 50mg of the obtained Zijuan tea polysaccharide ZTPW in 0.5mL of heavy water, sufficiently dissolving, freeze-drying, repeating for 3 times, transferring into a nuclear magnetic tube after the last dissolving, and detecting by using a Bruce nuclear magnetic resonance spectrometer. The results of the nuclear magnetic resonance analysis are shown in fig. 5 to 9, and chemical shift values of each carbon and hydrogen of each residue are assigned according to the nuclear magnetic patterns of fig. 5 to 9, and the assigned results are shown in table 2 below.
TABLE 2 assignment of Hydrogen and carbon signals to sugar residues in Zijuan tea polysaccharide ZTPW
The main repeating units of the Zijuan tea polysaccharide ZTPW obtained by combining monosaccharide composition, infrared spectrum, methylation and nuclear magnetic resonance analysis comprise the following structures 1 and 2:
example 2
First), toxic effects of Zijuan tea polysaccharide ZTPW on macrophage RAW264.7
RAW264.7 cell density was adjusted to 5X 10 5 mu.L of each well was inoculated into a 96-well plate at 37℃and 5% CO per mL 2 Culturing in incubator for 24 hr, discarding the culture medium, adding ZTPW (25, 50, 100, 200, 400 μg/mL) of the obtained Zijuan tea polysaccharide of example 1, adding culture medium and LPS (100 ng/mL) respectively into blank control group and positive control group, and culturing for 24 hr; taking out 96-well plate, discarding old medium, adding DMEM medium containing 1.5% CCK-8 (3 μl of CCK-8 stock solution per 200 μl of serum-free medium), adding at 37deg.C and 5% CO 2 Culturing in incubator for 90min, and measuring 450nm absorbance by enzyme-labeled instrument, wherein ZTPW has no toxicity to macrophage RAW264.7 at concentration range of 25-200 μg/mL as shown in figure 10.
Two) influence of Zijuan tea polysaccharide ZTPW on macrophage RAW264.7 to produce NO, cytokine and related gene expression
RAW264.7 cells were cultured at 5X 10 5 Density of individual/wells was seeded in 96-well plates at 37 ℃ with 5% CO 2 Culturing in an incubator for 24 hours; the old culture solution was discarded, and the ZTPW (25, 50, 100, 200. Mu.g/mL) of the Zijuan tea polysaccharide obtained in example 1 was added at various concentrations, and the control group and the positive control group were respectively added with medium and LPS (100 ng/mL), and after culturing for 24 hours, cell supernatants were collected for measuring the production amounts of NO and cytokines. NO was measured using Griess reagent, 100. Mu.L of cell supernatant was mixed with an equal volume of Griess reagent, allowed to stand in the dark for 10min, and absorbance at 542nm was measured using a microplate reader. The cytokine production and gene expression level are respectively measured by ELASA kit and qRT-PCR technique, and the result is shown in figure 11-12, and ZTPW can obviously up-regulate gene expression level of iNOS, TNF-alpha and IL-6 in the concentration range of 25-200 mug/mL, thereby promoting macrophage RAW264.7 to release NO and cytokines (TNF-alpha and IL-6).
Three) identification of macrophage RAW264.7 surface pattern recognition receptor by Zijuan tea polysaccharide ZTPW
RAW264.7 cells (5×10) 5 Seed/well) was inoculated in 96-well plate, pretreated with 10. Mu.g/mL TLR2, TLR4 and Dectin-1 antibody for 1h, 200. Mu.g/mL of the obtained Zijuan tea polysaccharide ZTPW of example 1 was added, and cultured for 24h; cell culture supernatants were collected for determination of NO, IL-6 and TNF- α. NO was measured using Griess reagent, 100. Mu.L of cell supernatant was mixed with an equal volume of Griess reagent, allowed to stand in the dark for 10min, and absorbance at 542nm was measured using a microplate reader. IL-6 and TNF- α levels were measured using ELISA kits, and the results are shown in FIG. 13, where TLR2 antibody pretreatment significantly reduced the production of NO, IL-6 and TNF- α compared to ZTPW alone, TLR4 antibody pretreatment significantly reduced the production of NO and IL-6, while Dectin-1 antibody pretreatment had NO significant effect on the production of NO, IL-6 and TNF- α; this result indicates that ZTPW can be recognized by pattern recognition receptors TLR2 and TLR4, activate macrophages and promote the production of NO, cytokines, exerting its immunomodulatory function.
Four) Effect of Zijuan tea polysaccharide ZTPW on secretion of related factors by macrophage RAW264.7 treated with Signal pathway inhibitor
To verify ZTPW activated macrophagiaCell-associated signaling pathways, the effect of 4 pathway inhibitors on ZTPW (200 μg/mL) induced RAW264.7 cells to secrete related factors (NO, TNF- α and IL-6) was further determined. RAW264.7 cells (5×10) 5 Individual/well) was inoculated in 96-well plates at 37℃with 5% CO 2 Culturing in an incubator for 24 hours, discarding old culture medium, and adding a signal pathway protein specific inhibitor: SP600125 (JNK), U0126 (ERK) and BAY11-7082 (NF- κB) are pretreated for 1h, the Zijuan tea polysaccharide ZTPW (the final concentration is 200 μg/mL) obtained in example 1 is added, and the mixture is co-cultured in a 96-well plate for 24h, wherein a culture medium and 100ng/mL LPS are respectively a control group and a positive control group; cell culture supernatants were collected and assayed for NO, TNF- α, IL-6 production, as shown in fig. 14, and the results showed a significant decrease in NO, TNF- α, and IL-6 in the 4 inhibitors and ZTPW co-treated groups compared to ZTPW alone, indicating that these four pathway inhibitors were effective in inhibiting ZTPW-induced RAW264.7 cell secretion of NO, TNF- α, and IL-6. The results show that ZTPW can mediate related immune factor secretion in RAW264.7 cells through MAPKs and NF- κB signaling pathway, and exert immune regulation activity.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (9)
1. The Zijuan tea polysaccharide ZTPW with immunoregulatory activity is characterized in that: the main repeating units of the Zijuan tea polysaccharide ZTPW comprise the following structure 1 and structure 2, the weight average molecular weight is 7.73kDa, the number average molecular weight is 7.53kDa, and the dispersion index is 1.03;
structure 1:
structure 2:
2. the Zijuan tea polysaccharide ZTPW with immunoregulatory activity of claim 1, which is characterized in that: the sugar content of the Zijuan tea polysaccharide ZTPW is 85.59wt%.
3. The Zijuan tea polysaccharide ZTPW with immunoregulatory activity of claim 1, which is characterized in that: the Zijuan tea polysaccharide ZTPW mainly comprises arabinose, galactose, glucose, xylose and mannose with the molar ratio of 18.48:15.92:32.99:3.32:29.29.
4. A preparation method of the Zijuan tea polysaccharide ZTPW according to any one of claims 1 to 3, which is characterized by comprising the following steps:
(1) Degreasing the crushed and sieved Zijuan tea powder, and drying to obtain Zijuan tea degreased powder;
(2) Adding deionized water into the defatted powder of Zijuan tea, extracting for multiple times, filtering, mixing the supernatants, and concentrating under reduced pressure to obtain concentrated solution;
(3) Precipitating the concentrated solution with ethanol, redissolving, removing protein, dialyzing, and lyophilizing to obtain crude polysaccharide lyophilized powder;
(4) Preparing crude polysaccharide freeze-dried powder into crude polysaccharide solution, separating and eluting by an ion exchange column, measuring by a phenol sulfuric acid method, collecting a target component peak elution product, concentrating, dialyzing, and freeze-drying to obtain a ZTPW component;
(5) Further separating ZTPW component by molecular sieve, detecting polysaccharide content by phenol-sulfuric acid method, collecting target component peak eluting product, concentrating the eluate, dialyzing, and freeze drying to obtain Zijuan tea polysaccharide ZTPW.
5. The preparation method of the Zijuan tea polysaccharide ZTPW according to claim 4, which is characterized in that: the step (1) is specifically carried out according to the following steps: grinding Zijuan tea, and sieving with a 40-mesh sieve to obtain Zijuan tea powder, wherein the feed liquid ratio is 1g:10mL, adding 85vt% ethanol into Zijuan tea powder, extracting at 70deg.C for 8h, repeating the extraction operation for three times, filtering, collecting precipitate, and oven drying at 60deg.C to obtain Zijuan tea defatted powder;
the step (2) is specifically carried out according to the following steps: according to the feed liquid ratio of 1g:15mL, adding deionized water into the defatted powder of Zijuan tea, extracting at 80deg.C for 2 hr to obtain water extractive solution, filtering with absorbent cotton yarn, repeating the extraction operation for 3 times to obtain residue, mixing filtrates, and concentrating under reduced pressure at 60deg.C to obtain concentrated solution;
the step (3) is specifically carried out according to the following steps: adding absolute ethanol into the concentrated solution to make the final concentration of ethanol 80vt%, standing at 4deg.C for 16h to precipitate insoluble polysaccharide, centrifuging at 6000r/min for 10min, and collecting precipitate; dissolving the precipitate in deionized water to obtain a crude polysaccharide solution; adding the same volume of Sevage reagent into the crude polysaccharide solution, wherein the Sevage reagent is chloroform-n-butanol mixed solution with the volume ratio of 4:1, stirring for 40min by using a magnetic stirrer, centrifuging at 6000rpm for 10min at room temperature, removing protein, collecting supernatant, repeatedly removing protein until white floccules are not formed at the boundary of two phases, and removing organic solvent by rotary evaporation; and then dialyzing the protein-removed polysaccharide solution for 72 hours by using a 3.5kDa dialysis bag, and freeze-drying to obtain crude polysaccharide freeze-dried powder.
6. The preparation method of the Zijuan tea polysaccharide ZTPW according to claim 4, which is characterized in that: the step (4) is specifically carried out according to the following steps: preparing crude polysaccharide freeze-dried powder into a solution with the concentration of 30mg/mL, separating by a DEAE-Sepharose fast flow ion exchange column, eluting by adopting ultrapure water, wherein the eluting flow rate is 2mL/min, the eluting time is 4 min/tube, receiving the front 45 tubes of eluting solution by adopting a full-automatic partial collector, detecting the polysaccharide content of each tube by adopting a phenol-sulfuric acid method, drawing an eluting curve, collecting 19-24 tubes of main eluting peak solution according to the eluting curve, dialyzing for 72h by adopting a 3.5kDa dialysis bag after concentrating, and freeze-drying to obtain a ZTPW component;
the step (5) is specifically carried out according to the following steps: preparing ZTPW component into solution with concentration of 10mg/mL, separating and purifying by Sephacryl S-200HR gel permeation column, eluting with phosphoric acid buffer solution with pH of 7.4 containing 0.15M NaCl, eluting with flow rate of 1mL/min, eluting time of 8 min/tube, collecting the eluate of the first 60 tubes by using fully automatic part collector, measuring polysaccharide content by phenol-sulfuric acid method, drawing elution curve, collecting the main elution peak solution of 35-50 tubes according to the elution curve, concentrating, dialyzing with 3.5kDa dialysis bag for 72h, and lyophilizing to obtain Zijuan tea polysaccharide ZTPW.
7. Use of the Zijuan tea polysaccharide ZTPW according to any one of claims 1-3 in the preparation of functional foods and immunomodulator medicaments.
8. The use according to claim 7, characterized in that: the functional food and the immunomodulator drug have the activity of enhancing the activity of macrophages, and/or increasing the NO production of the macrophages, and/or increasing the cytokine production of the macrophages.
9. The use according to claim 8, characterized in that: the cytokines include TNF-alpha, IL-6.
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