CN108359021B - Method for rapidly preparing flaxseed polysaccharide with antiviral and immunoregulatory activities - Google Patents
Method for rapidly preparing flaxseed polysaccharide with antiviral and immunoregulatory activities Download PDFInfo
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Abstract
The invention belongs to the field of biological medicines and functional foods, and particularly relates to a method for quickly preparing flaxseed polysaccharide with antiviral and immunoregulatory activities. The invention discloses a method for rapidly preparing flaxseed polysaccharide with antiviral and immunoregulatory activities, which comprises the following steps: 1) separating the flaxseed shell and kernel; 2) extracting linseed polysaccharide; 3) deproteinizing and precipitating with ethanol; 4) and (5) performing column chromatography purification. The preparation method of the flaxseed polysaccharide FHP-2 is simple, efficient and rapid, can be popularized as a general process for preparing flaxseed polysaccharide, and the novel flaxseed polysaccharide FHP-2 has anti-hepatitis B virus and immunoregulation activity and has potential to be developed into novel medicaments and functional food additives.
Description
Technical Field
The invention belongs to the field of biological medicines and functional foods, and particularly relates to a method for quickly preparing flaxseed polysaccharide with antiviral and immunoregulatory activities.
Background
Flax (Linustationssimum L.), also known as flax and kenaf, belongs to an annual herbaceous plant of flax of the family Linaceae, is widely distributed in more than 50 countries in the world, the main production places are countries such as Canada, China, India, America, Elaeagna, and the like, and the planting area is 260 million hectares in the world. Flax seeds are the seeds of flax, often used as an important oil seed, and the annual output worldwide is about 230 ten thousand tons. The flax seeds are rich in nutritional ingredients such as fat, protein, dietary cellulose, vitamins, mineral substances and the like, and functional factors such as omega-3/6 unsaturated fatty acid, lignan, plant polysaccharide, cyclopeptide and the like, and have various nutritional functions and health care effects. The linseed polysaccharide is a natural high molecular polysaccharide extracted from linseed and is used as soluble dietary fiber, and has the effects of reducing blood cholesterol level, reducing coronary arteriosclerosis, reducing diabetes, preventing colon cancer and the like. Researches show that the linseed polysaccharide consists of acidic polysaccharide and neutral polysaccharide, wherein the acidic polysaccharide mainly consists of rhamnose, fucose and galacturonic acid; the neutral polysaccharide mainly comprises arabinose, xylose and galactose.
Polysaccharides are natural high molecular compounds derived from the cell membranes of higher plants and animals and the cell walls of microorganisms, and are one of four major basic substances constituting life. In recent years, due to the growing interest in important physiological functions, research on plant polysaccharides has become one of the hot spots for pharmaceutical research. A large number of experimental studies show that polysaccharide components are important information substances in human bodies and participate in a plurality of physiological and pathological processes of the human bodies. The plant polysaccharide has wide sources, and the polysaccharide of hundreds of plants is separated and extracted at present, so the polysaccharide has the greatest advantage of no cytotoxicity and small toxic and side effects when used as a medicament, and therefore, a great deal of research focusing on the plant polysaccharide and the structure and activity thereof is carried out at home and abroad. However, the reports of the present research on the linseed polysaccharide are still relatively rare, and there are currently disclosed patents related to the extraction or preparation of the linseed polysaccharide: chinese patent application publication nos. CN1221771, CN1242952 and CN1263139, which relate to improvements of the conventional hot water extraction process of flaxseed gum, and do not relate to the structure and biological activity of flaxseed polysaccharide. Patent application No. CN201510391862.6 researches the preparation of novel flaxseed polysaccharide FHP-1 and the immunoregulation and antiviral activity of the flaxseed polysaccharide, but the technical process is very complicated, the preparation efficiency is low, the product yield is not high, and the research of quickly and efficiently preparing the high-purity flaxseed polysaccharide with the antiviral and immunoregulation activities is not reported yet.
Disclosure of Invention
Aiming at the problem of low efficiency of the traditional method for preparing the flaxseed polysaccharide, the invention aims at providing a simple and rapid preparation process of the novel flaxseed polysaccharide FHP-2, and the invention aims at obtaining the novel flaxseed polysaccharide FHP-2 by adopting the rapid preparation method.
The purpose of the invention is realized by the following technical scheme:
a method for rapidly preparing flaxseed polysaccharides with antiviral and immunomodulatory activity, comprising the steps of:
1) separating flaxseed shell and kernel: the separation of the flaxseed shell and kernel is realized by adopting an ethanol wet filtration technology. Fully crushing flax seeds, washing and degreasing the flax seeds by adopting ethanol, sieving the mixed feed liquid, and drying the screened and intercepted part of the flax seeds to obtain defatted flax seed hulls which are used for extracting flax seed polysaccharide;
2) extracting linseed polysaccharide: taking the dried flaxseed hulls obtained in the step 1) as a raw material, obtaining a flaxseed crude polysaccharide extracting solution by adopting an ultrasonic-assisted hot water extraction method, filtering to remove the flaxseed hulls, collecting filtrate, centrifuging to remove insoluble substances, taking supernatant, concentrating, dehydrating and freeze-drying to obtain flaxseed crude polysaccharide;
3) deproteinization and alcohol precipitation: dissolving the linseed crude polysaccharide obtained in the step 2) in water, removing free and bound proteins in the linseed crude polysaccharide by using papain, carrying out enzyme inactivation at high temperature after enzyme treatment, carrying out centrifugation to remove denatured enzyme precipitate, concentrating supernatant to an appropriate concentration, removing alcohol-soluble micromolecules by using an ethanol precipitation method, and carrying out centrifugation, redissolution and freeze drying to obtain primarily purified linseed polysaccharide;
4) and (3) column chromatography purification: loading the primarily purified linseed polysaccharide obtained in the step 3) on a DEAE-Sepharose flow column, sequentially carrying out step-type elution by using 0-2.0 mol/LNaCl solution, collecting by tubes, and detecting the polysaccharide content by using a phenol-sulfuric acid method to obtain an elution curve; collecting the second peak component sugar solution, rotary evaporating for concentration, dialyzing for 48h, and vacuum freeze drying; preparing the dried polysaccharide into a 2mg/mL solution, loading the solution on a Sephadex G-100 Sephadex column, eluting with a corresponding eluent, collecting polysaccharide components, dialyzing again, collecting trapped fluid, concentrating, and freeze-drying to obtain the purified flaxseed polysaccharide FHP-2.
As mentioned above, the ethanol wet filtration technology of step 1) is to rapidly prepare linseed polysaccharide with antiviral and immunoregulatory activities, and the ethanol wet filtration technology is to: adding 95% ethanol water solution or anhydrous ethanol with volume 3-5 times of the crushed flaxseed weight, stirring and washing at 40-70 deg.C for 0.5-2.0 h, stirring the feed liquid uniformly, sieving with 425 μm sieve, retaining flaxseed shell, and vacuum or normal pressure drying to obtain flaxseed shell.
In the method for rapidly preparing the flaxseed polysaccharide with antiviral and immunoregulatory activities, the process conditions of the ultrasonic-assisted hot water leaching in the step 2) are as follows: adopting an ultrasonic extractor to mix the linseed shells and water according to a mass ratio of 1: 20-1: mixing at 50 deg.C to 100 deg.C, ultrasonic power of 150-; filtering with 425 μm sieve; the centrifugation speed is 3000-; the concentration is carried out in vacuum.
In the method for rapidly preparing the linseed polysaccharide with antiviral and immunoregulatory activities as described above, the enzymatic deproteinization process conditions in the step 3) are as follows: dissolving the crude linseed polysaccharide in water until the mass percentage of the crude linseed polysaccharide is 1-5%, adding papain equivalent to 0.5-2.5% of the mass of the crude linseed polysaccharide, and performing oscillatory enzymolysis at 50-70 ℃ for 0.5-2.5 h, wherein the enzyme deactivation temperature is 100 ℃; the alcohol precipitation is to precipitate absolute ethanol with 4 times volume of the crude concentrated sugar solution at 4 ℃ for 8-12 h, regularly stirring the precipitate during the alcohol precipitation, and centrifuging to remove the ethanol under the condition of 2500-.
In the method for rapidly preparing the linseed polysaccharide with antiviral and immunoregulatory activities, the specific conditions of the DEAE-Sepharose fast flow ion exchange column chromatography in the step 4) are as follows: the sample loading concentration is 5mg/mL, the eluent is 0-2.0 mol/LNaCl solution, the elution speed is 1mL/min, 30 tubes are provided with one gradient, and 5mL is collected in each tube; the dialysis is carried out in deionized water for 48h by using a dialysis bag with Mw (half-weight) of 3500Da, wherein water is replaced every 8h, the concentration is carried out in vacuum, and the freeze-drying time is 12-24 h.
The linseed polysaccharide with antiviral and immunoregulatory activity obtained by the preparation method is a white fiber (flocculent) natural polymer macromolecular plant polysaccharide, the yield is 1.32% (relative to the whole seed), the components are uniform, the relative molecular weight is 1182kDa by gel permeation chromatography, FHP-2 is composed of 7 monosaccharides and 1 uronic acid by gas chromatography, and the relative molecular weight is respectively: l-rhamnose, L-arabinose, L-fucose, D-xylose, galacturonic acid, D-mannose, D-glucose, and D-galactose.
The flaxseed polysaccharide having antiviral and immunomodulatory activity as described above has a triple helix structure, and simultaneously has anti-hepatitis b virus and immunomodulatory activity. The novel flaxseed polysaccharide FHP-2 has certain anti-hepatitis B virus activity, and compared with a blank control group, a medicine group added with the FHP-2 can obviously reduce the expression of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) and inhibit the expression level of hepatitis B virus DNA, and has a dose-effect relationship. The M1 type macrophage polarization test result of the flaxseed polysaccharide FHP-2 shows that after macrophages are stimulated by LPS, M1/M2 balance begins to develop towards M1, RAW264.7 cells mainly express the function of M1 type macrophages, and when M1 is formed and activated, a medium-high dose FP-2 group promotes the secretion of various inflammatory factors IL-6, IL-10, TGF-beta, TNF-alpha and iNOS mRNA of RAW264.7 cells to a certain extent, and the immune function of the cells is greatly increased.
The preparation method obtains a novel flaxseed polysaccharide FHP-2 with anti-hepatitis B virus and immunoregulation activity. The preparation method has simple process, the ethanol wet-process dehulling and enzymatic deproteinization process greatly reduces the workload and shortens the process time, and the ultrasonic-assisted extraction technology greatly improves the extraction efficiency of the flaxseed polysaccharide compared with the traditional hot water extraction technology.
The preparation method of the flaxseed FHP-2 is simple, efficient and rapid, and can be popularized as a general process for preparing flaxseed polysaccharide, and the novel flaxseed polysaccharide FHP-2 has anti-hepatitis B virus and immunoregulation activity and has potential to be developed into novel medicaments and functional food additives.
Compared with the prior art, the invention has the following advantages:
(1) the preparation method has the following three advantages in the preparation process of the flaxseed polysaccharide: firstly, the separation of the flaxseed hull and kernel is realized by adopting an ethanol wet filtration technology for the first time, and compared with the solvent density method reported in the patent application No. CN201510391862.6, the method has the characteristics of simple process, rapidness, high efficiency, high hull yield and less solvent residue; compared with the traditional hot water extraction method, the ultrasonic-assisted extraction method can extract the linseed polysaccharide (gum), can utilize the physical action of ultrasonic waves and generate cavitation, mechanical action and thermal action through high-frequency oscillation, so that oscillation, growth and contraction are generated inside an object, the movement frequency and speed of molecules are increased, plant tissues and cells are deformed and broken, and inclusions are released, so that the dissolution of the molecules in the cells is promoted, the extraction efficiency of the linseed polysaccharide is greatly improved, and the time is saved by 6-8 times compared with the traditional hot water extraction method. Thirdly, the Sevage protein removal process reported in patent application No. CN201510391862.6 is very complicated and the removal of protein is not thorough, the invention firstly utilizes papain to remove the protein in the flaxseed polysaccharide, the conditions are mild, the method is simple, the protein removal efficiency is high (free protein and binding protein can be effectively removed), no solvent residue is generated, and the bioactivity of the flaxseed polysaccharide is ensured.
(2) Compared with the flaxseed polysaccharide FHP-1 reported in the patent application number CN201510391862.6, the flaxseed polysaccharide FHP-2 obtained by the invention has the characteristics of smaller molecular weight and easy digestion and absorption by a human body; FHP-2 is a novel polysaccharide which is not reported, is formed by connecting 7 monosaccharide molecules through specific glycosidic bonds, has a double-helix structure, and proves that FHP-2 has anti-hepatitis B virus activity and immunoregulation activity for the first time, can obviously reduce the expression of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) and inhibit the expression level of hepatitis B virus DNA, can obviously improve the secretion of tumor necrosis factor TNF-a, interleukin IL-6, IL-12 and inflammatory factor NO by macrophage Raw264.7 of a mouse, and can be used as a novel medicament for preventing HBV and food and health care products for improving the immune function of organisms.
(3) The invention adopts the common oil seed flaxseed as the raw material, has convenient and economic raw material foundation, simple and reliable preparation method, green and safe performance, has excellent physicochemical property and anti-hepatitis B virus and immunoregulation activity, can be considered as a novel medicament or functional food ingredient, widens the application range of the oil seed flaxseed, enriches the varieties of polysaccharide substances, and provides theoretical reference and practical basis for the deep development of flaxseed series products.
Drawings
FIG. 1 shows the DEAE-Sepharose fast flow elution curve of FHP-2.
FIG. 2 is a Sephadex G-100 elution curve of FHP-2.
FIG. 3 is a molecular weight distribution curve of FHP-2.
FIG. 4 is a gas chromatogram of the monosaccharide composition of FHP-2, and A, B is a gas chromatogram of the monosaccharide standard and the monosaccharide composition of FHP-2, respectively.
FIG. 5 shows the results of FHP-2's fresh fruit red test, i.e., the variation of the maximum absorption wavelength of the mixture of fresh fruit red and FP-2 at different alkali concentrations.
FIG. 6 is a graph of the effect of flax seed sulfate polysaccharide (FHP-2) on the antiviral activity of HepG2.2.15 cells at a positive control lamivudine concentration of 20 μ g/mL, where A is the effect of flax seed FHP-2 on HepG2.2.15 cell survival; B. c, D is a graph showing the effect of the flax seed sulfate polysaccharide FHP-2 on HbeAg, HBsAg and DNA, respectively.
FIG. 7 is a graph showing the effect of FHP-2 on the expression of M1 polarization factor (A) IL-6, (B) IL-10, (C) TGF-. beta., (D) TNF-. alpha.and (E) iNOS mRNA in RAW264.7 cells.
Detailed Description
The invention is further described below by means of specific examples, but said invention is not in any way restricted to the scope of the invention as claimed.
Example 1: preparation of flaxseed polysaccharide FHP-2
A method for rapidly preparing flaxseed polysaccharides with antiviral and immunomodulatory activity, comprising the steps of:
1) separating flaxseed shell and kernel: cleaning flaxseeds, removing impurities, fully crushing to obtain flaxseed powder, washing with ethanol twice, performing wet screening, taking the screened and intercepted part, and fully drying to obtain flaxseed shells. The ethanol washing conditions were: washing at 50 deg.C for 30min with a material-to-liquid ratio of 1:4, and stirring continuously during alcohol washing; the wet screening comprises the following specific operations: fully stirring the mixture of the alcohol washing system to enable the solid material to be evenly suspended in the ethanol system, then quickly sieving the mixed material through a 425-micron sieve to enable the alcohol extract and the fine linseed kernel particles to pass through sieve pores, and intercepting the linseed shells on the sieve due to the larger particles. The yield of the flax seed shell treated by the method is 23.12 percent.
2) Extracting linseed polysaccharide: taking dried linseed shells as a raw material, extracting by adopting a QQ-T650CT type ultrasonic extractor, and controlling the material-liquid ratio to be 1: 30, the temperature is 80 ℃, the ultrasonic power is 250W, and the extraction time is 60 minutes. Sieving the extractive solution with 425 μm sieve, centrifuging for 10min at 5000r/min for 10min, collecting supernatant, concentrating at 60 deg.C to appropriate volume, freezing at-80 deg.C, and freeze drying to obtain crude semen Lini polysaccharide. Under the condition, the yield of the linseed crude polysaccharide is 7.62 percent.
3) Deproteinization and alcohol precipitation: dissolving crude linseed polysaccharide in water until the mass percentage of the crude linseed polysaccharide is 0.5%, adding papain with the mass equivalent to 2% of that of linseed gum, performing oscillatory enzymolysis for 1h at 60 ℃, boiling at 100 ℃ after the enzymolysis is finished to inactivate enzyme, centrifuging to remove denatured enzyme precipitate, concentrating supernate to a proper volume, adding 4 times of absolute ethanol for precipitation for 12h at 4 ℃, centrifuging to remove ethanol, dissolving precipitate in water, freezing at-80 ℃, and performing freeze drying to obtain the primarily purified linseed polysaccharide. Under the condition, the removal rate of the linseed polysaccharide protein is 92.34%, and the linseed polysaccharide protein hardly absorbs at ultraviolet spectrums of 260nm and 280 nm.
4) And (3) column chromatography purification: dissolving primarily purified flaxseed polysaccharide in water to prepare a 5mg/mL solution, loading the solution on a DEAE-Sepharose fast flow column, sequentially carrying out step-type elution by using 0-2.0 mol/LNaCl solution, keeping the flow rate at 1mL/min, carrying out gradient treatment by using 30 tubes, collecting 5mL of the solution in each tube, carrying out tube-by-tube collection, detecting the polysaccharide content by using a phenol-sulfuric acid method, combining sugar reaction positive collected liquid, carrying out rotary evaporation concentration at 50 ℃, dialyzing for 48h (3500Da), and carrying out vacuum freeze drying; preparing the dried polysaccharide into a 2mg/mL solution, loading the solution on a Sephadex G-100 Sephadex column, eluting with a corresponding eluent, collecting a second polysaccharide component, dialyzing again, collecting trapped fluid, concentrating, and freeze-drying to obtain the purified linseed polysaccharide FHP-2. The column chromatography purification results are shown in figures 1 and 2, and the obtained FHP-2 is the second elution component (figure 1).
Example 2: characterization of flaxseed polysaccharide FHP-2
(1) Determination of relative molecular masses
FHP-2 was dissolved in ultrapure water to prepare a sample solution of 2.5mg/mL, and the purity and molecular weight were measured by GPC. The specific test conditions and methods are as follows: adopting TSK G-5000PWXLAnd TSK G-3000PWXLThe chromatographic columns are connected in series, the 2414 type refractive index detector is used, the column temperature is 35 ℃, and the mobile phase is KH with 0.02mol/L2PO4The flow rate was 0.6 mL/min. The result shows that the FHP-2 component is uniform, and the relative molecular weight is 1182 kDa.
(2) Monosaccharide composition analysis
Hydrolysis of polysaccharide samples: taking 10mg FHP-2 sample, adding 4.0mL of 2mol/L trifluoroacetic acid, hydrolyzing at 110 ℃ for 8h, spin-drying the hydrolyzed solution, washing with methanol for 3 times, and using N while hot2And (5) drying.
Derivatization of monosaccharides: adding 10mg of hydroxylamine hydrochloride, 1mg of internal standard inositol and 0.5mL of pyridine into the hydrolyzed sample, reacting at 90 ℃ for 0.5h, cooling, adding 0.5mL of acetic anhydride, reacting at 90 ℃ for 0.5h, cooling, adding water and chloroform, extracting for 3 times, taking the chloroform layer, evaporating to dryness, dissolving the residue with chloroform, filtering with a 0.22-micron organic phase filter membrane, taking the filtered clear solution, and analyzing by GC.
Gas Chromatography (GC) conditions were HP-5 quartz capillary column (30m × 0.32.32 mm × 0.25.25 μm) with N as carrier gas2(ii) a Sample introduction amount: 1 mu L of the solution; the flow rate is 1 mL/min; no split flow, injection port temperature: 250 ℃; the temperature of the FID detector was set at 250 ℃; temperature programming: the initial temperature is 100 ℃, the temperature is kept for 0.5min, the temperature is increased to 160 ℃ at the speed of 3 ℃/min, the temperature is increased continuously. The temperature was raised to 250 ℃ at a rate of 10 ℃/min and maintained for 5 min.
As shown in the attached figure 4, the result shows that FHP-2 consists of 7 monosaccharides and 1 uronic acid, and the molar percentage contents are respectively as follows: l-rhamnose (11.27%), L-arabinose (4.03%), L-fucose (2.82%), D-xylose (7.12%), galacturonic acid (55.75%), D-mannose (2.99%), D-glucose (4.41%), D-galactose (11.61%).
(3) Methylation analysis
Weighing 20mgFHP-2 in a serum bottle, adding 6mL dimethyl sulfoxide, ultrasonically dissolving, adding 200mg sodium hydroxide, continuously ultrasonically treating for 30min, and sealing overnight.
Methylation: 3.6mL of methyl iodide was added to the serum bottle, and after 12 hours of reaction in the dark, 2mL of distilled water was added to terminate the reaction. Adding chloroform for repeated extraction for 5 times, combining organic phases, repeatedly adding distilled water to wash the organic phases for 5 times, collecting the organic phases, and spin-drying. Washing with chromatographically pure methanol for 3 times to obtain methylated polysaccharide.
And (3) hydrolyzing methylated polysaccharide: reference is made to the method of hydrolysis of a sample during analysis of the monosaccharide composition in example 2.
Reduction: adding 4mL of distilled water, gradually adding a sodium hydroxide solution to adjust the pH value to 10-12, adding 100mg of sodium borohydride, and oscillating at constant temperature and low speed overnight. Adjusting the pH value of the sample solution to 5.5 by using glacial acetic acid, and spin-drying the reaction solution. Adding chromatographic pure methanol, evaporating to dryness, and repeating for 3 times.
Derivatization: derivatization was performed according to the acetylation procedure in the analysis of monosaccharide composition in example 2, and GC-MS analysis was performed after passing through a 0.22 μm organic phase filter.
Gas chromatography-mass spectrometry (GC-MS) conditions: TR-5MS elastic capillary column (30m × 0.25mm × 0.25 um); the carrier gas is helium; the sample injection amount is 1 uL; the flow rate is 1mL/min, and the sample injection split ratio is 10: 1; sample inlet temperature: 250 ℃; temperature programming: the initial temperature is 150 ℃, the temperature is kept for 2min, the temperature is increased to 180 ℃ at the speed of 10 ℃/min, the temperature is kept for 2min, the temperature rising speed is changed, and the temperature rising is continued. The temperature was raised to 260 ℃ at a rate of 15 ℃/min and maintained for 5 min.
Methylation analysis results (see table below) indicate that FP-2 is mainly composed of → 4) -Galp- (1 →, → 2,4) -Galp- (1 →, → 2) -Rhap- (1 → and Glup- (1 → wherein → 2) -Rhap- (1 → takes up 28.06%, → 4) -Galp- (1 →, → 2,4) -Galp- (1 → takes up 31.21% and 21.53%, respectively, indicating that FP-2 polysaccharide is a 1 → 4 linked galactose as the backbone.
TABLE 1 FP-2 analysis of polysaccharide methylation products
4) Analysis of triple helix Structure
Preparing FHP-2 solution of 1mg/mL, mixing Congo red reagent of which the concentration is 100 mu mol/L with 2mL polysaccharide solution, and gradually adding NaOH solution of which the concentration is 4mol/L to gradually increase the concentration of NaOH in the mixed solution from 0mol/L to 0.5 mol/L. And (3) taking Congo red solution without polysaccharide as a reference, scanning within the wavelength range of 400-800 nm, and comparing the change of the maximum absorption wavelength. As shown in figure 4, the result shows that FHP-2 can form a complex with Congo red, the maximum absorption wavelength of the complex can be red-shifted compared with that of Congo red, and the three-helix conformation exists in FHP-2, so that better biological activity is possible.
Example 3: anti-hepatitis B virus activity of FHP-2
(1) MTT assay
Taking activated HepG2.2.15 cells in logarithmic growth phase as 1 × 105Inoculating in 96-well plate at one/mL concentration, and placing in 5% CO2Culturing at 37 deg.C, after 24 hr, adding 150 μ L of FHP-2, and culturing with the group without drug as blank control. After 24h, 20. mu.L of MTT solution was added, the culture was terminated after 4h of further culture, and the culture medium in the wells was aspirated. Add 150. mu.L of dimethyl sulfoxide into each well, and shake for 10min at low speed on a shaking bed to dissolve the crystals sufficiently. Measuring the absorbance value of the sample at a wavelength of 450nm by using an enzyme-linked immunosorbent assay, and calculating the Survival Rate (SR) of the cells according to the following formula:
survival rate SR (%) ═ A1/A0× 100% wherein A is1Absorbance values for parallel tests of sample sets, A0The absorbance of the control group parallel test is shown.
The results show that the influence of FHP-2 on the survival rate of HepG2.2.15 cells is shown in FIG. 6A, the survival rate is more than 90%, and the results show that FHP-2 has no inhibition effect on HepG2.2.15 cells.
(2) Effect of FHP-2 on secretion of HBeAg and HBsAg from HepG2.2.15 cells and HBV-DNA replication
Activated HepG2.2.15 cells in the logarithmic growth phase were taken and cultured in 6cm plates, each of which had a cell count of 5X 105 ten thousand. After 24h, the flaxseed polysaccharide FHP-2 is added as a drug group, a drug-free group is used as a blank control, and 20ug/mL lamivudine is added as a positive control. Culturing for 24h, collecting culture supernatant, and adding new cell liquid for culturing. The supernatant was diluted 100 times to detect HBsAg, 10 times to detect HBeAg, and the HBV-DNA content in the supernatants of the different concentration groups was determined. The detection of HBeAg and HBsAg is carried out according to the corresponding kit instructions, and the detection of HBV-DNA is carried out according to the kit instructions of the ordinary QPCR. The absorbance of each well was measured at 450nm in a microplate reader.
The results show (fig. 6B, C, D), the drug group added with FHP-2 significantly inhibited HBeAg, HBsAg secretion and HBV-DNA replication compared to the blank control, had significant effect against HBV virus, and had dose-effect relationship.
Example 4: immunomodulatory Activity of FHP-2
(1) MTT assay
RAW264.7 cells 1 × 106Inoculate in 96-well plates at one/mL concentration in 5% CO2Culturing at 37 deg.C for 24 hr, removing supernatant, replacing with fresh culture solution, adding 20 μ L of oleum Lini sulfate polysaccharide drug, adding 20 μ g/mL lipopolysaccharide as positive control, and culturing. After 24h, 20. mu.L of MTT solution was added, the culture was terminated after 4h of further culture, and the culture medium in the wells was aspirated. Add 150. mu.L of dimethyl sulfoxide into each well, and shake for 10min at low speed on a shaking bed to dissolve the crystals sufficiently. The results are shown in FIG. 7A, which indicates that the survival rate of the RAW264.7 cells affected by FHP-2 is more than 95%, and indicates that FHP-1 has no inhibitory effect on the RAW264.7 cells.
(2) Regulation of polarized M1-type macrophages by FP-2 polysaccharide
Taking RAW264.7 cells with activated logarithmic growth phase as 1 × 106Inoculate in 6-well plates at a concentration of one/mL in 5% CO2Culturing at 37 deg.C, attaching to wall, stimulating with LPS at proper concentration for a certain time, discarding supernatant, washing cells with PBS, extracting RNA, detecting the expression of mRNA corresponding to the immune factors TGF- β, IL-6, IL-10, TNF- α and iNOS with RT-PCR, and determining the result as shown in FIG. 7B, C, D, E, F1/M2Equilibrium is started to M1The RAW264.7 cells mainly express the function of M1 type macrophages, and when M1 is activated, the medium-high dose FP-2 group promotes the secretion of various inflammatory factors IL-6, IL-10, TGF- β, TNF- α and iNOS mRNA of the RAW264.7 cells to a certain extent, thereby greatly increasing the immune function of the cells.
Claims (5)
1. A method for rapidly preparing flaxseed polysaccharides having antiviral and immunomodulatory activity, comprising the steps of:
1) separating flaxseed shell and kernel: separating the flaxseed shell and kernel by adopting an ethanol wet filtration technology: fully crushing flax seeds, washing and degreasing the flax seeds by adopting ethanol, sieving the mixed feed liquid, and drying the screened and intercepted part of the flax seeds to obtain defatted flax seed hulls which are used for extracting flax seed polysaccharide;
2) extracting linseed polysaccharide: taking the dried flaxseed hulls obtained in the step 1) as a raw material, obtaining a flaxseed crude polysaccharide extracting solution by adopting an ultrasonic-assisted hot water extraction method, filtering to remove the flaxseed hulls, collecting filtrate, centrifuging to remove insoluble substances, taking supernatant, concentrating, dehydrating and freeze-drying to obtain flaxseed crude polysaccharide;
3) deproteinization and alcohol precipitation: dissolving the linseed crude polysaccharide obtained in the step 2) in water, removing free and bound proteins in the linseed crude polysaccharide by using papain, carrying out enzyme inactivation at high temperature after enzyme treatment, carrying out centrifugation to remove denatured enzyme precipitate, concentrating supernatant to an appropriate concentration, removing alcohol-soluble micromolecules by using an ethanol precipitation method, and then carrying out centrifugation, redissolution and freeze drying to obtain primarily purified linseed polysaccharide;
4) and (3) column chromatography purification: loading the primarily purified linseed polysaccharide obtained in the step 3) on a DEAE-Sepharose fast flow column, sequentially carrying out step-type elution by using 0-2.0 mol/LNaCl solution, collecting by tubes, and detecting the polysaccharide content by using a phenol-sulfuric acid method to obtain an elution curve; collecting the second peak component sugar solution, rotary evaporating for concentration, dialyzing for 48h, and vacuum freeze drying; preparing the dried polysaccharide into a 2mg/mL solution, loading the solution on a Sephadex G-100 Sephadex column, eluting with a corresponding eluent, collecting polysaccharide components, dialyzing again, collecting trapped fluid, concentrating, and freeze-drying to obtain purified linseed polysaccharide FHP-2;
the linseed polysaccharide with the antiviral and immunoregulatory activities is uniform in component, the relative molecular weight is 1182kDa through gel permeation chromatography, and FHP-2 is composed of 7 monosaccharides and 1 uronic acid through gas chromatography determination, wherein the two monosaccharides are as follows: l-rhamnose, L-arabinose, L-fucose, D-xylose, D-mannose, D-glucose, D-galactose and galacturonic acid.
2. A method for the rapid preparation of flaxseed polysaccharides with antiviral and immunomodulatory activity according to claim 1, wherein: the ethanol wet filtration technology in the step 1) refers to: adding 95% ethanol water solution or anhydrous ethanol with volume 3-5 times of the crushed flaxseed weight, stirring and washing at 40-70 deg.C for 0.5-2.0 h, stirring the feed liquid uniformly, sieving with 425 μm sieve, retaining flaxseed shell, and vacuum or normal pressure drying to obtain flaxseed shell.
3. A method for the rapid preparation of flaxseed polysaccharides with antiviral and immunomodulatory activity according to claim 1, wherein: the process conditions of the ultrasonic-assisted hot water leaching in the step 2) are as follows: adopting an ultrasonic extractor to mix the linseed shells and water according to a mass ratio of 1: 20-1: mixing at 50 deg.C to 100 deg.C, ultrasonic power of 150-; filtering with 425 μm sieve; the centrifugation speed is 3000-; the concentration is carried out in vacuum.
4. A method for the rapid preparation of flaxseed polysaccharides with antiviral and immunomodulatory activity according to claim 1, wherein: the conditions of the enzymatic deproteinization process in the step 3) are as follows: dissolving the crude linseed polysaccharide in water until the mass percentage of the crude linseed polysaccharide is 1-5%, adding papain equivalent to 0.5-2.5% of the mass of the crude linseed polysaccharide, and performing oscillatory enzymolysis at 50-70 ℃ for 0.5-2.5 h, wherein the enzyme deactivation temperature is 100 ℃; the alcohol precipitation is to precipitate absolute ethanol with 4 times volume of the crude concentrated sugar solution at 4 ℃ for 8-12 h, regularly stirring the precipitate during the alcohol precipitation, and centrifuging to remove the ethanol under the condition of 2500-.
5. A method for the rapid preparation of flaxseed polysaccharides with antiviral and immunomodulatory activity according to claim 1, wherein: the DEAE-Sepharose fast flow ion exchange column chromatography of the step 4) has the following specific conditions: the sample loading concentration is 5mg/mL, the eluent is 0-2.0 mol/LNaCl solution, the elution speed is 1mL/min, 30 tubes are provided with one gradient, and 5mL is collected in each tube; the dialysis is carried out in deionized water for 48h by using a dialysis bag with Mw (half-weight) of 3500Da, wherein water is replaced every 8h, the concentration is carried out in vacuum, and the freeze-drying time is 12-24 h.
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| CN1263139A (en) * | 1999-02-12 | 2000-08-16 | 清华大学 | Wet extraction method of flax glue from flax seed |
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| CN101899174A (en) * | 2010-08-11 | 2010-12-01 | 珈侬生化科技(中国)有限公司 | Bletilla polysaccharide compound serving as cosmetic raw material and preparation method thereof |
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