CN111548429B

CN111548429B - Rana japonica oil polysaccharide component and application thereof

Info

Publication number: CN111548429B
Application number: CN202010452299.XA
Authority: CN
Inventors: 张扬; 虞晓瑾; 刘蕾; 张姝姝; 王佳怡; 唐月瑶; 任冰倩
Original assignee: Changshu Institute of Technology
Current assignee: Changshu Institute of Technology
Priority date: 2020-05-26
Filing date: 2020-05-26
Publication date: 2021-10-19
Anticipated expiration: 2040-05-26
Also published as: CN111548429A

Abstract

The invention provides a wood frog oil polysaccharide component, which belongs to a sugar-protein compound, and has total sugar content of 80.87 +/-1.17%, uronic acid content of 10.19 +/-0.43% and protein content of 6.42 +/-0.16%; the wood frog oil polysaccharide component belongs to heteropolysaccharide and consists of mannose, glucosamine, galacturonic acid, galactose and fucose; the wood frog oil polysaccharide component belongs to polydisperse polysaccharide, the weight average molecular weight of the wood frog oil polysaccharide component is 999.586kDa, the number average molecular weight of the wood frog oil polysaccharide component is 244.366kDa, and the polydispersity index of the wood frog oil polysaccharide component is 4.09; the rana japonica oil polysaccharide component belongs to pyranose, and the connection mode comprises alpha-and/or beta-configuration glycosidic bonds. The oviductus ranae polysaccharide component provided by the invention has obvious effects of resisting oxidation and enhancing the immunity of the organism, and can be used for preparing functional products such as tablets, capsules, oral liquid and the like.

Description

Rana japonica oil polysaccharide component and application thereof

Technical Field

The invention belongs to the field of preparation and application of natural products, and particularly relates to a rana japonica oil polysaccharide component and application thereof.

Background

The effect of Reactive Oxygen Species (ROS) on the immune system is twofold: controlling the attack of autoreactive T cells on self tissues, organs and cells by regulating immune response and cell proliferation at low concentration; at high concentrations, the immune system directly attacks activated T cells and destroys the structure of immune cell membranes, thereby causing immune dysfunction. Although endogenous antioxidant enzymes and related substances can reduce the damage of excess ROS to the body to a certain extent, the endogenous antioxidant enzymes and substances often do not reach the lowest effective concentration in the face of oxidative stress. Therefore, exogenous antioxidant supplementation under oxidative stress is essential to the repair of the autoimmune system and its function.

Antioxidant is one of the activities that most natural polysaccharides have. Recent studies have demonstrated that: antioxidant polysaccharides tend to show stronger immune enhancement, such as pleurotus sajor-caju mycelium polysaccharide (Carbohydr. polym.92(2) (2013)2187-92), quinoa polysaccharide (int.J.biol.Macromol.99(2017) 622-. The method not only further proves the correlation between the antioxidant effect and the immune enhancement effect, but also provides a basis for the preparation and the separation of the immunoregulation polysaccharide based on the antioxidant activity.

Wood frog oil (Oviductusranae), also known as oviduct fat, is a dried product of oviduct of female adult wood frog, and is a precious traditional tonifying traditional Chinese medicinal material. The Chinese pharmacopoeia records: oviductus ranae is used for tonifying kidney, replenishing vital essence, nourishing yin and moistening lung. Can be used for treating weakness after illness, listlessness, palpitation, insomnia, night sweat, tuberculosis, cough and hemoptysis. Based on traditional application, more than 20 rana japonica oil health foods approved by the national drug administration on the market have the health-care functions of enhancing the immunity of the organism, resisting fatigue and delaying senility. The oviductus Ranae is rich in various active ingredients beneficial to human body, such as protein, unsaturated fatty acid, phospholipid, polysaccharide, vitamin, nucleic acid, hormone and trace elements necessary for human body such as ferrum, manganese, selenium, etc. The content of protein in the rana japonica oil is as high as more than 50 percent, the protein is a main active ingredient, and the physicochemical properties and the pharmacological actions of the rana japonica oil are widely researched and proved (oxid. med. cell. langev.2019 (2012)4739450), while the polysaccharide active ingredient in the rana japonica oil is rarely researched.

Zhengsatellite, Yangjing, Chengliang and the like respectively report the process conditions for extracting the crude polysaccharide of the wood frog oil by enzyme method assistance and the development of the crude polysaccharide beverage, but do not research the activity and efficacy components thereof (the university of Jilin, 2011,33(4): 403-; yangjing reports the extraction and anti-tumor and anti-fatigue effects of crude rana japonica oil polysaccharide, but does not separate and characterize the effective components (Jilin agriculture university, 2011, Master academic paper); patent 200910152923.8 discloses a method for isolating rana japonica oil polysaccharide, but still lacks effective structural characterization and activity evaluation.

In conclusion, the current rana japonica oil polysaccharide is not deeply researched, and the tracking separation of the efficacy components and the evaluation of the structural characteristics and the activity are yet to be further explored. The invention provides a forest frog oil immunoregulation polysaccharide component obtained based on antioxidant activity guiding separation and product application thereof by combining the correlation of antioxidant and immunoregulation functions.

Disclosure of Invention

The invention aims to provide a wood frog oil polysaccharide component and product application thereof.

According to a first aspect of the present invention there is provided a oviductus ranae polysaccharide fraction belonging to the class of saccharide-protein complexes comprising 80.87 ± 1.17% total saccharide, 10.19 ± 0.43% uronic acid, 6.42 ± 0.16% protein; the wood frog oil polysaccharide component belongs to heteropolysaccharide and is composed of mannose, glucosamine, galacturonic acid, galactose and fucose in a molar ratio of 1:2.28:2.40:3.00: 2.23; the oviductus Ranae polysaccharide component belongs to polydisperse polysaccharide, and has weight average molecular weight (M)_W) 999.586kDa, number average molecular weight (M)_N) Is 244.366kDa, polydispersity index (M)_W/M_N) Is 4.09; the rana japonica oil polysaccharide component belongs to pyranose, and the connection mode comprises alpha-and/or beta-configuration glycosidic bonds.

The preparation method of the oviductus ranae polysaccharide component comprises the following steps:

1) extraction of crude polysaccharide

Referring to the method mentioned in the Yangjing Master thesis (forest frog oil crude polysaccharide extraction condition optimization and pharmacological activity research, Jilin agriculture university, 2011), 10g of forest frog oil defatted powder is weighed, soaked in distilled water for 12h according to the liquid-material ratio of 100:1, homogenized, the pH is adjusted to 6.8, and 0.2% (w/w) of papain is added to be incubated at 37 ℃ for 2 h; inactivating enzyme in boiling water bath for 10min, cooling, centrifuging to obtain supernatant, adjusting pH to 7.0, concentrating to 1/4, adding 3 times volume of 95V% ethanol water solution, and standing at 4 deg.C for 12 hr; centrifuging, collecting precipitate, and freeze drying to obtain crude oviductus Ranae polysaccharide with yield of 5.09 + -0.21% and total sugar content of 23.08 + -1.56%.

2) Purification of crude polysaccharide

Deproteinizing 3 times with Sevag reagent (n-butanol: chloroform: 4: 1); dialyzing for 3 times after removing residual Sevag reagent; freeze drying the dialyzate to obtain purified oviductus Ranae polysaccharide with total sugar content of 55.42 + -1.21%.

3) Separation of polysaccharide fractions

Weighing 100mg of oviductus Ranae purified polysaccharide, dissolving in 20mL of deionized water, and loading onto Diethylaminoethyl (DEAE) cellulose-52 chromatographic column (2.6cm × 60 cm); sequentially carrying out gradient elution by using deionized water and 0.1-0.5 mol/L NaCl solution at the flow rate of 1mL/min and 5mL per tube; detecting the absorbance of 490nm wavelength by phenol-sulfuric acid method, and drawing elution curve; selectively collecting the eluent corresponding to the main peak with the largest peak area according to the indication of the elution curve, namely combining the eluents corresponding to 0.3mol/L NaCl solution; dialyzing the eluate with deionized water for 3 times; freeze drying to obtain the wood frog oil polysaccharide component; the polysaccharide fraction contains 80.87 + -1.17% total sugar, 10.19 + -0.43% uronic acid, and 6.42 + -0.16% protein.

According to another aspect of the invention, the invention provides the application of the oviductus ranae polysaccharide component in preparing a product with functions of resisting oxidation and enhancing the immunity of the organism; can be used for preparing tablet, capsule, oral liquid, etc.

The invention has the following beneficial effects:

based on the correlation between the antioxidation and the immunoregulation, the invention adopts the antioxidation activity to guide and separate the forest frog oil immunoregulation polysaccharide, and the provided polysaccharide component has the obvious functions of antioxidation and organism immunity enhancement.

Secondly, the oviductus ranae polysaccharide component belongs to a sugar-protein compound, and the function of enhancing the immunity of the organism is usually higher than that of the simple polysaccharide (int.J.biol.Macromol.65(2014) 441-.

The rana japonica oil polysaccharide component can obviously enhance the phagocytic capacity of macrophages and increase the generation amount of Nitric Oxide (NO) under low dosage; but has no proliferation and inhibition effect on macrophage growth, and shows good safety characteristics.

Drawings

FIG. 1 DEAE-52 elution curve of purified polysaccharide from oviductus Ranae;

FIG. 2 is a high performance liquid chromatogram of POR-3 monosaccharide composition of oviductus ranae polysaccharide component (Man, mannose; GlcN, glucosamine; GalA, galacturonic acid; Gal, galactose; Fuc, fucose);

FIG. 3 shows the molecular weight distribution of polysaccharide POR-3 of oviductus Ranae;

FIG. 4 is a UV scanning diagram of a oviductus ranae polysaccharide component POR-3;

FIG. 5 is an infrared spectrum of a wood frog oil polysaccharide component POR-3;

FIG. 6. preparation of polysaccharide component POR-3 of oviductus Ranae¹H-nuclear magnetic resonance spectrogram;

FIG. 7. preparation of polysaccharide component POR-3 of oviductus Ranae¹³C-nuclear magnetic resonance spectrogram;

FIG. 8. Effect of the oviductus Ranae polysaccharide fraction POR-3 on macrophage proliferation (NC, blank control; LPS, lipopolysaccharide);

FIG. 9. Effect of oviductus Ranae polysaccharide fraction POR-3 on phagocytic capacity of macrophages (NC, blank control; LPS, lipopolysaccharide);

FIG. 10 is a graph showing the effect of the polysaccharide fraction POR-3 of oviductus Ranae on the amount of NO produced (NC, blank control; LPS, lipopolysaccharide).

Detailed Description

The oviductus ranae degreasing powder is purchased from Jilin gardenia pharmaceutical industry Co., Ltd; 1, 1-diphenyl picrylphenylhydrazine (DPPH), Vitamin C (VC), monosaccharide standard, T-series glucan standard and the like are purchased from national medicine group chemical reagent limited; calf serum (FBS), Lipopolysaccharide (LPS), 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazole bromide salt (MTT), dimethyl sulfoxide (DMSO), NO kit and the like are purchased from Nanjing to build a bioengineering research institute; RAW264.7 macrophage (ATCC TIB-71) supplied by Abcam corporation; all experiments were performed in triplicate, data were expressed as mean ± SD, statistical analysis of the data was performed using t-test or ANOVA, P <0.05 considered statistically different.

Example 1 extraction and purification of crude polysaccharides from oviductus Ranae

Referring to the method mentioned in the Yangjing Master thesis (forest frog oil crude polysaccharide extraction condition optimization and pharmacological activity research, Jilin agriculture university, 2011), 10g of forest frog oil defatted powder is weighed, soaked in distilled water for 12h according to the liquid-material ratio of 100:1, homogenized, the pH value is adjusted to 6.8, and papain with the content of 0.2% w/w of the forest frog oil defatted powder is added to incubate for 2h at 37 ℃; inactivating enzyme in boiling water bath for 10min, cooling, centrifuging to obtain supernatant, adjusting pH to 7.0, concentrating to 1/4, adding 3 times volume of 95V% ethanol water solution, and standing at 4 deg.C for 12 hr; centrifuging, collecting precipitate, and freeze drying to obtain crude oviductus Ranae polysaccharide with yield of 5.09 + -0.21% and total sugar content of 23.08 + -1.56%. Crude polysaccharide was purified by deproteinizing 3 times with Sevag reagent (n-butanol: chloroform: 4: 1); dialyzing for 3 times after removing residual Sevag reagent; the dialyzate is frozen and dried to obtain the purified polysaccharide of the rana japonica oil, the total sugar content is 55.42 +/-1.21 percent, and the determination of the total sugar content adopts a phenol-sulfuric acid method (anal. chem.28(3) (1956) 350-.

Example 2 separation of the polysaccharide component of oviductus Ranae

Weighing 100mg of oviductus Ranae purified polysaccharide, dissolving in 20mL of deionized water, and loading onto DEAE cellulose-52 chromatographic column (2.6cm × 60 cm); sequentially carrying out gradient elution by using deionized water and 0.1-0.5 mol/L NaCl solution at the flow rate of 1mL/min and 5mL per tube; and detecting the absorbance at 490nm by a phenol-sulfuric acid method, and drawing an elution curve. As shown in FIG. 1, when NaCl solutions with different concentrations are adopted for gradient elution, 3 elution peaks are respectively obtained, namely elution peaks POR-1-3 corresponding to elution of 0.1-0.3 mol/L NaCl solution; respectively collecting and combining the eluents corresponding to POR-1-3 to obtain 3 polysaccharide components of POR-1(0.1mol/L NaCl solution), POR-2(0.2mol/L NaCl solution) and POR-3(0.3mol/L NaCl solution); dialyzing 3 polysaccharide fractions with deionized water for 3 times; freeze drying to obtain polysaccharide components POR-1-3; then, the antioxidant activities of the 3 polysaccharide fractions were compared to determine the fraction with the highest activity.

Example 3 compositional analysis and comparison of antioxidant Activity of the polysaccharide component of Rana temporaria chensinensis oil

The total sugar content of each polysaccharide fraction was determined using the phenol-sulfuric acid method (anal. chem.28(3) (1956) 350-); the protein content is determined by Coomassie Brilliant blue method (anal. biochem.72(1976) 248-254); the uronic acid content was determined by the m-hydroxybiphenyl method (anal. biochem.54(2) (1973) 484-489).

Determination of hydroxyl radical clearance: taking 2mL of polysaccharide component solutions with different concentrations (0.2 mg/mL-1.0 mg/mL), 1mL of phosphate buffer solution (pH 7.4) containing 0.75mmol/L phenanthroline and 1mL of 0.75mmol/L FeSO₄Mixing the solution, adding 1ml of 0.12% H₂O₂The reaction was started in solution, incubated at 37 ℃ for 60min and the absorbance at 536nm was measured. The sample control tube replaces the sample with distilled water, and the bottom tube of the sample replaces H with distilled water₂O₂The hydroxyl radical clearance was calculated by substituting the following equation:

hydroxyl radical scavenging rate (%) - (A)_s-A₀)×100/(A_c-A₀)

Wherein A is_sIs the absorbance value of the test sample; a. the₀The absorbance value of the blank tube; a. the_cAbsorbance values for the background of the sample.

Determination of DPPH radical scavenging Rate: taking 2mL of polysaccharide component solutions with different concentrations (0.2 mg/mL-1.0 mg/mL) and 2mL of ethanol solution containing 0.1mmol/L of DPPH, mixing uniformly, reacting for 30min in a dark place at room temperature, and measuring the absorbance value at 517 nm. The blank control is obtained by replacing the sample with absolute ethyl alcohol, the sample control is obtained by replacing DPPH with absolute ethyl alcohol, and the DPPH free radical clearance rate is calculated by substituting the following formula:

DPPH radical scavenging ratio (%) - (A)_s-A_c)×100/A₀

As shown in Table 1, the polysaccharide fraction POR-3 of wood frog has the highest total sugar and uronic acid content; POR-2 has the highest total protein content; POR-1 has the same ability as POR-2 to scavenge hydroxyl radicals and DPPH radicals (P >0.05), but significantly lower than POR-3(P <0.05, P < 0.01). The stronger antioxidant effect of POR-3 is probably contributed by its higher content of uronic acid (Carbohydr. Polym.152(2016) 222-230).

TABLE 1 comparison of chemical composition of polysaccharide components POR-1-3 of wood frog with antioxidant activity

Data are expressed as mean ± SD (n ═ 3); in the same column, the different symbols of the superscript represent the presence of a statistical difference (P)<0.05 or P<0.01)；IC₅₀ ^HRepresents the half inhibitory concentration for scavenging hydroxyl radicals; IC (integrated circuit)₅₀ ^DRepresents the half inhibitory concentration for scavenging DPPH radicals.

EXAMPLE 4 separation of oviductus Ranae polysaccharide fraction POR-3

Weighing 100mg of oviductus Ranae purified polysaccharide, dissolving in 20mL of deionized water, and loading onto Diethylaminoethyl (DEAE) cellulose-52 chromatographic column (2.6cm × 60 cm); sequentially carrying out gradient elution by using deionized water and 0.1-0.5 mol/L NaCl solution at the flow rate of 1mL/min and 5mL per tube; detecting the absorbance of 490nm wavelength by phenol-sulfuric acid method, and drawing elution curve; selectively collecting eluent corresponding to a main peak (POR-3) with the largest peak area according to the indication of an elution curve, namely combining the eluent corresponding to 0.3mol/L NaCl solution; dialyzing the eluate with deionized water for 3 times; freeze drying to obtain oviductus Ranae polysaccharide component POR-3; the polysaccharide fraction contains 80.87 + -1.17% total sugar, 10.19 + -0.43% uronic acid, and 6.42 + -0.16% protein.

Example 5 structural characterization of the Wood frog polysaccharide component POR-3

And (3) monosaccharide composition determination: precisely weighing 2mg POR-3, putting the POR-3 into a hydrothermal reactor, adding 2mL of 2mol/L trifluoroacetic acid (TFA) aqueous solution, sealing the hydrothermal reactor, and hydrolyzing for 6h at 120 ℃; removing residual TFA, adding 200 mu L of 0.5mol/L methanol solution of 1-phenyl-3-methyl-5-pyrazolone (PMP) and 200 mu L of 0.3mol/L NaOH aqueous solution in sequence, mixing, and reacting at 70 ℃ for 60 min; cooling to room temperature, and stopping the reaction by using 0.3mol/L HCl aqueous solution; extracting with chloroform for three times, collecting water layer, and obtaining PMP marked monosaccharide; filtered through a 0.45 μm microporous membrane and analyzed by an instrument. Chromatographic conditions are as follows: a Thermo Ultimate3000 High Performance Liquid Chromatograph (HPLC) equipped with Thermo U3000 Diode Array Detector (DAD) with Supersil ODS2 column (5 μm, 4.6 mm. times.250 mm); sample introduction amount: 20 mu L of the solution; mobile phase: PBS (pH 6.8): acetonitrile 82:18 (v/v); a flow rate; 0.8 mL/min; column temperature: 30 ℃; detector wavelength 245 nm; operating time: and 85 min.

And (3) measuring the molecular weight: 20 μ L of 2mg/mL POR-3 solution was passed through a 0.45 μm microporous filter and then injected into an Elite P230IIHPLC equipped with Shodex SUGAR KS-804 chromatography column (8.0 mm. times.300 mm) and a differential Refractive Index Detector (RID); recording and processing data using an N2000GPC chromatography workstation; dextran with different molecular weights is used as standard sugar to prepare a series of standard sugar solutions with different molecular weights, and a three-order calibration curve for polysaccharide molecular weight determination is established according to retention time and molecular weight values. Chromatographic conditions are as follows: mobile phase: ultrapure water; flow rate: 1.0 ml/min; column temperature: 50 ℃; detector temperature: 35 ℃; operating time: and (3) 30 min.

Ultraviolet (UV) spectroscopy: the POR-3UV spectrum is measured by using a 2700 UV-visible spectrophotometer from Shimadzu, and the scanning range is 200 nm-400 nm.

Infrared (FT-IR) spectroscopy: mixing 2mgPOR-3 with 100mg dry potassium bromide, tabletting, measuring with FTIR-650 infrared spectrometer, and scanningDrawing range is 4000cm^-1～400cm^-1。

Nuclear Magnetic Resonance (NMR) spectroscopy: weighing dry sample POR-380mg, and adding D₂Dissolving the sample, freeze-drying, and repeating for 3 times; with 500. mu. L D₂The sample was dissolved by O and transferred to a nuclear magnetic tube for measurement. For POR-3, respectively, using Bruker's Advance 400 NMR spectrometer¹H-NMR (400MHz) and¹³C-NMR (100MHz) spectra were measured.

As shown in FIG. 2, POR-3 is mainly composed of mannose, glucosamine, galacturonic acid, galactose and fucose, and the molar ratio obtained by dividing the respective peak areas by the molecular weight of the above monosaccharides is 1:2.28:2.40:3.00: 2.23; as shown in FIG. 3, POR-3 belongs to the polydisperse polysaccharide fraction, and the weight average molecular weight (M) of POR-3 is determined by processing the data through a N2000GPC workstation_W) 999.586kDa, number average molecular weight (M)_N) 244.366kDa, polydispersity index (M)_W/M_N) It was 4.09.

As shown in FIG. 4, from the UV spectrum of POR-3, an absorption peak around 280nm was observed, indicating that free and/or bound proteins (Carbohydr. Polym.197(2018)9-16) were present in POR-3, which is consistent with the results of Table 1. Considering that all the polysaccharide fractions of rana japonica oil listed in table 1 contain protein, and 3 deproteinization operations in the purification step can substantially remove the free protein (carbohydrate. polym.229(2020) 115355). Therefore, it was confirmed that the protein in POR-3 is a polysaccharide-bound protein and not a free protein.

As shown in FIG. 5, 3417cm was observed from the infrared spectrum of POR-3^-1A broad peak exists nearby, which is caused by the stretching vibration of-OH; 2925cm^-1And 2854cm^-1Nearby peaks are respectively assigned to-CH₂-asymmetric and symmetric stretching vibrations; 1646cm^-1And 1548cm^-1The nearby strong peaks are absorption peaks characteristic of typical carbonyl, amide I and amide II, further confirming the presence of uronic acid and binding protein in POR-3; 1121cm^-1、1078cm^-1And 1036cm^-1The nearby absorption peak belongs to the stretching vibration of the pyran ring; 765cm^-1Nearby absorption peaks ascribe to the ring-to-D- (1-4) and α -D- (1-6) connected ring-to-ring-to-ring-to-ring-to-ring-and-to-ring-to-ring-to-ring-to-ring-to-ring-to-ring-to-ring-to-ring-to-ring-to-ring-to-. The infrared analysis result shows that POR-3 conforms to the structural characteristics of the pyran polysaccharide.

FIGS. 6 and 7 are those of POR-3¹H-NMR and¹³C-NMR spectrum chart. As shown in FIG. 6, the peaks at δ 4.63ppm and δ 4.54 are assigned to the anomeric hydrogen on the δ 0-glycosidic bond; a signal peak between δ 14.17ppm and δ 23.38ppm ascribes a hydrogen on the pyranose ring; the signal peak between δ 42.91ppm and δ 50.85ppm is assigned to the hydrogen on the methyl and/or methylene group on the monosaccharide residue and/or on the amino acid residue in the binding protein. As shown in fig. 7, the peak at δ 6173.86ppm was assigned to the carbon on the binding protein amide; the signal peak between δ 7105.01ppm and δ 101.21ppm was assigned to the β -anomeric carbon; and the signal peak between delta 99.95ppm and delta 96.15ppm is assigned to delta 3-anomeric carbon; strong signal peak attribution between delta 71.70ppm and delta 60.53ppm is C2-C6; in addition, signal peaks between δ 22.24ppm and δ 15.34ppm are assigned to methyl carbons on monosaccharide residues and/or amino acid residues in the binding protein.

The results of NMR analysis are basically consistent with the results of monosaccharide composition and infrared analysis, but there is a certain difference in the judgment of glycosidic bond configuration. The infrared spectrum is identified as alpha-glycosidic bond;¹the H-NMR spectrum was identified as a beta-glycosidic bond; to get from¹³The characteristic carbon atoms of the alpha-and beta-glycosidic bonds are observed simultaneously in the C-NMR spectrum. This is probably due to the complex structure of the polysaccharide itself, and the mutual influence of some characteristic peaks, which results in the loss of corresponding characteristic peaks. The types of POR-3 glycosidic linkages are, with caution, assigned to alpha-and/or beta-glycosidic linkages.

EXAMPLE 6 immunomodulating action of polysaccharide fraction POR-3 of Wood frog

Effect of POR-3 on macrophage proliferation: RAW264.7 cell suspension at 1X 10⁵cells/mL were seeded in 96-well plates at 37 ℃ with 5% CO₂Culturing for 24h under the condition of (1); discarding the supernatant, adding 100 mu L of POR-3 solution to make the final concentration 25-800 mu g/mL; the positive control was Lipopolysaccharide (LPS), 100. mu.L of the treated cells at a final concentration of 25. mu.g/mL were also added, and the blank control was performed using a medium without sample; after 24h incubation, 10. mu.L of 5mg/mL M was added to each wellContinuously culturing the TT solution for 4 hours; the supernatant was discarded, 100 μ L of DMSO was added per well, and formazan deposited in the cells was dissolved in DMSO; gently shake the 96-well plate and place in the dark for 10min to fully dissolve the purple crystals; the OD of each well at 490nm was measured and the macrophage proliferation index was calculated.

Effect of POR-3 on macrophage phagocytosis: RAW264.7 cells were treated with 25. mu.g/mL to 800. mu.g/mL POR-3 solution and 25. mu.g/mL LPS, respectively, and after 24 hours of incubation, 100. mu.L of the supernatant was transferred to a new 96-well plate for determination of NO content (see NO assay). Phagocytic capacity of RAW264.7 cells was determined by neutral erythrophagocytosis: removing the redundant supernatant, and washing twice with PBS; adding 100 mu L of neutral red solution into each hole, and culturing for 1 h; removing the supernatant; PBS was washed twice again; add 100. mu.L of cell lysate per well (ethanol: 1.0mol/L acetic acid 1:1) overnight at room temperature; the OD of each well at 490nm was measured and the macrophage phagocytosis index was calculated.

Effect of POR-3 on the release of NO by macrophages: taking cell supernatant respectively treated by POR-3 and LPS, and mixing with Griess Reagent I (1% sulfanilamide) and Griess Reagent II (0.1% N-1-naphthyl ethylenediamine dihydrochloride dissolved in 5% phosphoric acid) with equal volume for 10 min; determining the OD value of each hole at 540 nm; at different concentrations of NaNO₂A standard curve is drawn, and the amount of NO produced is calculated.

As shown in FIG. 8, POR-3 treated macrophages from 25. mu.g/mL to 800. mu.g/mL neither proliferated nor inhibited (P >0.05) compared to the blank control (NC). This indicates that POR-3 has no proliferative effect on macrophages, but is highly safe and no cytotoxicity is observed.

As shown in FIG. 9, compared with NC, POR-3 of 25 μ g/mL to 800 μ g/mL has the effect of remarkably promoting phagocytosis of macrophages (P < 0.01); when the concentration of POR-3 reaches 50 mug/mL, the phagocytosis index is 1.32 +/-0.074, which is almost equivalent to that of positive control LPS (1.31 +/-0.017); and when the concentration is more than 50 mu g/mL, the effect of POR-3 in promoting macrophage phagocytosis is not enhanced (P > 0.05). These results indicate that POR-3 has a potent macrophage phagocytosis promoting effect, and can achieve an effect equivalent to that of LPS at low concentrations.

As shown in FIG. 10, in the concentration range of 25. mu.g/mL-800. mu.g/mL, the effect of POR-3 in promoting NO release is concentration-dependent (P < 0.01); when the concentration of POR-3 reaches 800 mug/mL, the NO release amount reaches 16.72 +/-0.38 mu mol/L, which is obviously higher than that of LPS (10.77 +/-0.72 mu mol/L, P is less than 0.01). This indicates that POR-3 also exhibits a potent ability to promote NO release.

The result shows that POR-3 plays a role in enhancing the immunity of the organism by strongly promoting phagocytosis of macrophages and increasing NO release amount; meanwhile, the growth of macrophages is not inhibited, and good safety is shown.

EXAMPLE 7 preparation of Wood frog polysaccharide component POR-3 tablets

Mixing 3g of wood frog polysaccharide component POR-3 with 200mg of polyvinylpyrrolidone and 1.8mg of calcium hydrogen phosphate, grinding, and sieving with 100 mesh sieve; adding 5% polyvidone K30 in 95% ethanol solution while stirring to obtain soft material, and wet granulating (sieving with 16 mesh sieve); drying at 60 deg.C, sieving, grading, adding magnesium stearate 1 wt% and silica gel micropowder 2 wt%, mixing, and tabletting to obtain POR-3 tablet.

EXAMPLE 8 preparation of Wood frog polysaccharide component POR-3 capsules

Taking 3g of wood frog polysaccharide component POR-3 which is sieved by a 40-mesh sieve, spraying 90% ethanol according to the proportion of 1:1, mixing uniformly, adding 10% wt of starch of the wood frog oil polysaccharide component which is sieved by the 40-mesh sieve to prepare a soft material, sieving by the 20-mesh sieve to granulate, drying in an oven at 60 ℃ for 50min, sieving by the 20-mesh sieve to granulate, and filling in a No. 3 empty capsule under the environment that the relative humidity is lower than 63% to obtain the wood frog polysaccharide component POR-3 capsule.

EXAMPLE 9 preparation of Wood frog polysaccharide component POR-3 oral liquid

Adding 3g of wood frog polysaccharide component POR-3 into 100mL of purified water, stirring at room temperature until the mixture is dissolved, adding 0.5g of xylitol and 0.2g of potassium sorbate, stirring uniformly, canning, instantly sterilizing, bottling, and sealing to obtain the wood frog polysaccharide component POR-3 oral liquid.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A wood frog oil polysaccharide component with immunoregulation function is characterized in that: the oviductus ranae polysaccharide component belongs to a sugar-protein compound and comprises 80.87 +/-1.17% of total sugar, 10.19 +/-0.43% of uronic acid and 6.42 +/-0.16% of protein;

the rana japonica oil polysaccharide component belongs to heteropolysaccharide, and comprises mannose: glucosamine: galacturonic acid: galactose: fucose =1: 2.28:2.40:3.00: 2.23; the wood frog oil polysaccharide component belongs to polydisperse polysaccharide, the weight average molecular weight of the wood frog oil polysaccharide component is 999.586kDa, the number average molecular weight of the wood frog oil polysaccharide component is 244.366kDa, and the polydispersity index of the wood frog oil polysaccharide component is 4.09; the wood frog oil polysaccharide component belongs to pyranose, and the connection mode comprisesα-and/orβ-a glycosidic bond of configuration;

the preparation method of the wood frog oil polysaccharide component with the immunoregulation function comprises the following steps:

1) extraction of crude polysaccharide

Weighing 10g of rana japonica oil defatted powder, soaking in distilled water at a liquid-to-material ratio of 100:1 for 12h, homogenizing, adjusting pH to 6.8, adding papain of which the content is 0.2% w/w of the rana japonica oil defatted powder, and incubating at 37 ℃ for 2 h; inactivating enzyme in boiling water bath for 10min, cooling, centrifuging to obtain supernatant, adjusting pH to 7.0, concentrating to 1/4, adding 3 times volume of 95V% ethanol water solution, and standing at 4 deg.C for 12 hr; centrifuging, collecting precipitate, and freeze drying to obtain crude oviductus Ranae polysaccharide with yield of 5.09 + -0.21% and total sugar content of 23.08 + -1.56%;

2) purification of crude polysaccharide

Deproteinizing with Sevag reagent for 3 times, removing residual Sevag reagent, and dialyzing for 3 times; freeze drying the dialyzate to obtain purified oviductus Ranae polysaccharide with total sugar content of 55.42 + -1.21%;

3) separation of polysaccharide fractions

Weighing 100mg of oviductus ranae purified polysaccharide, dissolving in 20mL of deionized water, and loading to a diethylaminoethyl cellulose-52 chromatographic column; sequentially carrying out gradient elution by using deionized water and 0.1-0.5 mol/L NaCl solution at the flow rate of 1mL/min and 5mL per tube; detecting the absorbance of 490nm wavelength by phenol-sulfuric acid method, and drawing elution curve; selectively collecting the eluent corresponding to the main peak with the largest peak area according to the indication of the elution curve, namely combining the eluents corresponding to 0.3mol/L NaCl solution; dialyzing the eluate with deionized water for 3 times; freeze drying to obtain the wood frog oil polysaccharide component.

2. Use of the polysaccharide fraction of rana japonica oil with immunoregulatory function according to claim 1, wherein: is used for preparing functional products for resisting oxidation and enhancing the immunity of organisms.

3. Use according to claim 2, characterized in that: the functional product is a preparation of tablets, capsules and oral liquid.

4. Use according to claim 3, characterized in that: the preparation method of the tablet comprises the following steps: mixing 3g of oviductus Ranae polysaccharide component with immunoregulation function of claim 1 with 200mg of polyvinylpyrrolidone and 1.8mg of calcium hydrogen phosphate, grinding, and sieving with 100 mesh sieve; adding 5% wt polyvidone K30 95% ethanol solution while stirring to obtain soft material, wet granulating, and sieving with 16 mesh sieve; drying at 60 deg.C, sieving, grading, adding 1 wt% magnesium stearate and 2 wt% silica gel micropowder, mixing, and tabletting.

5. Use according to claim 3, characterized in that: the preparation method of the capsule comprises the following steps: taking 3g of the oviductus ranae polysaccharide component which is sieved by a 40-mesh sieve and has the immunoregulation function, spraying 90% V ethanol water solution according to the weight ratio of 1:1, uniformly mixing, adding 10% wt of starch of the oviductus ranae polysaccharide component sieved by the 40-mesh sieve to prepare a soft material, sieving by a 20-mesh sieve for granulation, drying in an oven at 60 ℃ for 50min, sieving by the 20-mesh sieve for granulation, and filling in a No. 3 empty capsule under the environment that the relative humidity is lower than 63% to obtain the oviductus ranae polysaccharide component.

6. Use according to claim 3, characterized in that: the preparation method of the oral liquid comprises the following steps: adding 3g of the oviductus Ranae polysaccharide component with immunoregulation function of claim 1 into 100mL of purified water, stirring at room temperature until the polysaccharide component is dissolved, adding 0.5g of xylitol and 0.2g of potassium sorbate, stirring uniformly, canning, instantly sterilizing, bottling, and sealing to obtain the product.