CN114984067A - Application of flavone extract of phellinus sophorae in antioxidation and immunity enhancement - Google Patents

Abstract

The invention provides application of a flavone extract of phellinus sophorae in oxidation resistance and immunity enhancement. The flavone extract of the phellinus sophorae is analyzed by hydroxyl radical scavenging capacity and total antioxidant capacity, and the influence of polysaccharide on the in vitro proliferation of 3 immune cells, namely Jurkat cells, Raji cells and macrophages and the influence of medicaments on the phagocytic capacity of the macrophages are detected; the effect of lymphokine secretion by T lymphocytes in vitro and the effect of cytokine secretion by Jurkat cells and RAW264.7 cells; the flavone extract of the phellinus sophorae has in vitro antioxidant activity by the methods of influencing the hemolysin content recovery of the mice with low immunity, influencing the immune function of the mice with low immunity and the like; has effects in promoting cell proliferation and enhancing immunity.

Description

Application of flavone extract of phellinus sophorae in antioxidation and immunity enhancement

Technical Field

The invention belongs to the field of biological medicine, and particularly relates to application of a phellinus igniarius flavone extract in oxidation resistance and immunity enhancement.

Background

Phellinus igniarius is used as a traditional medicine-food homologous fungus in China, and has a lot of precious biological activity and application value. Because the generation of the medicinal fungus active substances has a close relationship with the growth environment of the medicinal fungus active substances, the structures, components and contents of the active substances extracted from phellinus linteus from different habitat sources are different, the biological activities also have great differences, and particularly, the species difference of the phellinus linteus parasitic plants has great influence on the extracted active ingredients, thereby directly influencing the biological efficacy and the application potential. In the core area of loess plateau in Yangan province of northern Shaanxi, the vegetation is mainly wild pagoda tree, the distribution of phellinus igniarius is wide, the yield is large, and the development is worth to be carried out. At present, the research on Phellinus igniarius of Yanan Sophora japonica is rarely reported, and the nutritional value and the medicinal value of an active product are deeply researched and developed.

Disclosure of Invention

The invention aims to provide the application of the flavone extract of phellinus igniarius in oxidation resistance and immunity enhancement, and the flavone extract has obvious effects of oxidation resistance and immunity enhancement.

The technical scheme adopted by the invention is that the flavonoid extract of the phellinus igniarius is applied to oxidation resistance and immunity enhancement.

Yet another feature of the present invention is that,

the flavone extract of Phellinus sophorae is fruiting body flavone.

The flavone extract of Phellinus sophorae is mycelium flavone.

The application has the beneficial effects that the phellinus igniarius flavone extract of sophora japonica is used for clinically treating oxidation resistance, preventing aging and regulating the immunity of organisms to treat cancers, thereby providing a theoretical basis for the development and utilization of phellinus igniarius of northern Shaanxi; the compound is used as a raw material of foods, health-care foods, foods with special medical application and medicines, and provides a very high research value for clinical research on treatment of aging and low immunity.

Drawings

FIG. 1(a) is a graph of the hydroxyl radical scavenging ability of Phellinus sophorae total flavonoids ZHFA; FIG. 1(b) is a JHFA hydroxyl radical scavenging force analysis plot;

FIG. 2(a) is a diagram of analysis of the total antioxidant capacity of phellinus linteus fungus total flavonoids ZHFA from northern Shaanxi; FIG. 2(b) is a JHFA Total antioxidant capacity analysis graph;

FIG. 3(a) is a graph comparing the effect of Phellinus linteus fungus Total Flavonoids ZHFA on T lymphocyte (Jurkat cell line) proliferation; FIG. 3(b) is a graph comparing the effect of JHFA on T lymphocyte (Jurkat cell line) proliferation;

FIG. 4(a) is a graph comparing the effect of Phellinus linteus fungus Total Flavonoids ZHFA on B lymphocyte (Raji cell line) proliferation; FIG. 4(B) is a graph comparing the effect of JHFA on B lymphocyte (Raji cell line) proliferation;

FIG. 5(a) is a graph comparing the effect of Phellinus linteus fungus Total Flavonoids ZHFA on the proliferation of macrophages (Raw264.7 cell line); FIG. 5(b) is a graph comparing the effect of JHFA on macrophage (Raw264.7 cell line) proliferation;

FIG. 6(a) is a graph comparing the effect of Phellinus linteus fungus Total Flavonoids ZHFA on the phagocytic ability of RAW264.7 cell line; FIG. 6(b) is a graph comparing the effect of JHFA on the phagocytic capacity of RAW264.7 cell lines;

FIG. 7 is a comparison graph of the effect of Phellinus linteus total flavonoids in northern Shaanxi on in vitro secretion of lymphokines by T lymphocytes;

FIG. 8 is a graph comparing the effect of total flavonoids from Phellinus linteus in northern Shaanxi on cytokine secretion by Jurkat cells and RAW264.7 cells;

FIG. 9 is a comparison graph of the effect of the phellinus linteus general flavone ZHFA and JHFA in northern Shaanxi on the content recovery of hemolysin in immunocompromised mice;

FIG. 10 is a graph comparing the effect of phellinus linteus general flavone ZHFA and JHFA in northern Shaanxi on the immune function of immunocompromised mice;

FIG. 11 is a comparison graph of the effect of the phellinus linteus general flavone ZHFA and JHFA in northern Shaanxi on the serum cytokines of immunocompromised mice;

FIG. 12 is a comparison graph of the effect of the total flavonoids of Phellinus linteus from Shaanbei on the content change of short-chain fatty acids in intestinal tracts of mice.

Detailed Description

The invention is based on the analysis of the antioxidation and immunity-improving effect of the phellinus igniarius flavone extract, and the invention is explained in detail by combining the attached drawings and the specific embodiment.

The flavone extract of the phellinus sophorae comprises fruit body flavone ZHFA and mycelium flavone JHFA, hydroxyl radical scavenging capacity analysis and total oxidation resistance analysis are carried out, and the influence of polysaccharide on Jurkat cells, Raji cells and 3 kinds of immune cells of macrophages in vitro proliferation and the influence of medicaments on macrophage phagocytosis capacity and optimal action concentration are detected; the effect of lymphokine secretion by T lymphocytes in vitro and the effect of cytokine secretion by Jurkat cells and RAW264.7 cells; the flavone extract of the phellinus sophorae has in vitro antioxidant activity by the methods of influencing the hemolysin content recovery of the mice with low immunity, influencing the immune function of the mice with low immunity and the like; has effects of promoting cell proliferation and enhancing immunity; can be used as raw materials of foods, health-care foods, foods with special medical application and medicines, and is a new application of Yanan sophora phellinus igniarius.

In order to better understand the essence of the present invention, the pharmacological experiments of the phellinus igniarius flavone extract will be used to illustrate the application of the phellinus igniarius flavone extract in oxidation resistance and immunity enhancement.

The collection of the Phellinus linteus sporocarp around Yanan belongs to the semi-humid and drought-prone climate in the warm temperature zone due to the loess plateau at northern Shaanxi and the average altitude of 1200 m. The method comprises the steps of establishing a laboratory mycelium culture method by using the shape and molecular classification to perform species identification, separately culturing the phellinus linteus mycelium of northern Shaanxi into a PDA solid culture medium, taking a small amount of phellinus linteus mycelium and phellinus linteus sporophore in the culture medium, extracting total DNA, performing PCR amplification on the extracted total DNA of the wild phellinus linteus and the cultured mycelium DNA by using ITS primers, and analyzing by 1% agarose gel electrophoresis, wherein the wild phellinus linteus is more in L shape and hard in texture, and the obtained product has a dark brown color and a horseshoe shape. The phylogenetic tree (NJ tree) B constructed by Mega7.0 software shows that the similarity of the collected Phellinus linteus from northern Shaanxi and the nucleotide sequence of the wild Phellinus igniarius NW518 from northern Shaanxi loess plateau submitted in a database is as high as 100%. Therefore, the isolated strain of this experiment was named Phellinus igniarius YASH 1.

The method comprises the following specific steps of extracting the total flavonoids of phellinus igniarius: oven drying Phellinus Linteus fruiting body and mycelium at 56 deg.C to constant weight, and pulverizing to 100 mesh with pulverizer; weighing a certain amount of fruiting body and mycelium powder respectively, extracting with 70% ethanol at room temperature overnight, vacuum filtering, extracting the residue with 70% ethanol again overnight, and repeating for 3 times. Rotary evaporating the ethanol extractive solution at 40 deg.C to obtain extract, re-dissolving with ethyl acetate to obtain total flavone solution, placing in a fume hood to volatilize ethyl acetate to obtain total flavone dry powder, and storing at-20 deg.C; respectively naming fruit body flavone in the flavone dry powder as ZHFA, and naming mycelium flavone as JHFA;

NaOH reaction: dissolving a small amount of total flavone extract in 0.5ml of 70% ethanol in a test tube to obtain a total flavone solution, adding 0.25ml of 1% NaOH solution, carrying out color development reaction, observing the color change (yellow orange or red) of the solution, and carrying out reaction on different flavones and NaOH to obtain different color changes. By utilizing the characteristic of color reaction between flavone and NaOH and taking rutin as a reference, the extracted phellinus igniarius total flavone is respectively reacted with 1 percent of NaOH, and as a result, the original solution is orange yellow after the phellinus igniarius sporocarp total flavone ZHFA is dissolved by ethanol and is changed into brownish red after being reacted with the NaOH; the phellinus igniarius mycelium total flavone JHFA solution is orange yellow after being dissolved by ethanol, and becomes turbid and light after being reacted with NaOH to carry out color reaction.

Adopting a boric acid complexation method to determine the purity of the total flavonoids of the phellinus igniarius: weighing 2mg of rutin reference substance, dissolving with 70% ethanol, and diluting to 10ml as stock solution to prepare a standard solution with the concentration of 0.2 mg/ml; weighing 0.8g of boric acid and 1.0g of sodium acetate, dissolving with 70% ethanol, and fixing the volume to 100ml to serve as a complexing reagent; the measurement wavelength is selected by accurately sucking 200 μ L rutin standard solution into 25ml measuring flask, and adding 70% ethanol to desired volume. Sucking 2ml, adding 2ml boric acid complexing reagent, mixing, scanning from 200nm to 500nm with corresponding reagent 70% ethanol as blank, and recording the maximum absorption wavelength under 292 nm; standard curve, respectively sucking standard rutin solution of 0.5, 1.0, 1.5, 2.0 and 2.5ml, adding 70% ethanol to constant volume to 2.5 ml. Precisely absorbing 2.5ml of the solution respectively, adding 2.5ml of complexing agent, mixing uniformly, and measuring the light absorption value at 292 nm. And drawing a standard curve by taking the light absorption value A as a vertical coordinate and the rutin concentration C (mg/ml) as a horizontal coordinate to obtain a relationship curve of the rutin concentration (C) and the light absorption value (A).

And (3) analyzing the purity of the total flavonoids of the phellinus igniarius: taking rutin standard solution concentration (mg/ml) as an abscissa and absorbance value (OD292) as an ordinate to draw a standard curve, wherein the obtained regression equation is that y is 3.85x +0.6926, R2 is 0.9957, and the absorbance value of the flavone solution is substituted into the standard curve, so that the purity of the ZHFA is 42% and the purity of the JHFA is 22.7%.

Qualitative and quantitative analysis (LC-MS) of Phellinus linteus total flavone

Sample treatment: weighing 20mg flavone sample, placing in 15ml centrifuge tube, adding 70% methanol water solution 5.0ml, fully oscillating, ultrasonic dissolving for 30min, centrifuging at 10000rpm, collecting upper layer extractive solution, and filtering with 0.22 μm filter membrane for use.

LC-MS (liquid chromatography-mass spectrometer): LC-MS analysis; a chromatographic column: agilent Poroshell 120EC-C182.7 μm (3X 50mm), mobile phase A: 0.5% formic acid water mobile phase C: acetonitrile solution, flow rate: 0.6ml/min, sample size: 10 μ L, column temperature: 35 ℃ is carried out.

Mass spectrum detection: multiple Reaction Monitoring (MRM) detection conditions: the spraying voltage is 4.5-5.5 kv, the desolventizing temperature is 500 ℃, and the desolventizing gas (N2) is 1000L/h.

Mass spectrometry scan conditions included ESI + mode: the spraying voltage is 5.5 kv; the desolventizing temperature is 500 ℃; desolventizing gas (N2) 1000L/h; scanning range: 100-1000 m/Z; ESI-mode: the spraying voltage is 4.5 kv; the desolventizing temperature is 500 ℃; desolventizing gas (N2) 1000L/h; scanning range: 100-1000 m/Z;

qualitative and quantitative analysis of phellinus igniarius total flavonoids: according to the existing flavone component comparison of a database, the corresponding known flavone component is obtained, the total flavone component from phellinus igniarius in Shaanbei is complex, and the composition and the content of the wild phellinus igniarius sporocarp total flavone ZHFA and the laboratory-cultured phellinus igniarius mycelium total flavone JHFA are different, as shown in Table 1:

TABLE 1 qualitative and quantitative determination of Phellinus linteus Total Flavonoids

Compared with the mycelium phellinus igniarius total flavone, the phellinus igniarius sporocarp total flavone contains unique components such as procyanidin B1, anthocyanin B2, p-coumaric acid 1, dihydroquercetin-7-0-rhamnoside, glycyrrhizic acid 1, cyanidin 2 and the like, but lacks flavone raw material components such as quercetin 1, 2, 4, 6-tribromophenol 2, vanillin 2, trans-resveratrol 2 and the like.

The method for detecting hydroxyl radical scavenging force (TOC) of the phellinus sophorae extract comprises the following steps: weighing fruiting body flavone ZHFA and mycelium flavone JHFA 100mg respectively in 4 EP tubes, and dissolving in 0.5ml 70% ethanol to obtain stock solution with concentration of 200 mg/ml; diluting ZHFA with 70% ethanol to 30.0, 20.0, 10.0, 1.0 mg/ml; diluting JHFA mother liquor with 70% ethanol to 190.0, 150.0, 110.0, 70.0, 30.0 mg/ml; opening the microplate reader for preheating for 30min, and adjusting the wavelength to 536 nm; the reagents were added in 0.5ml EP tubes, operating according to Table 2:

TABLE 2 statistical table of operating doses

The reagents in each EP tube were mixed, incubated at 37 ℃ for 60min, and then centrifuged at 10000rmp for 10min at room temperature. After centrifugation, 200. mu.L of the supernatant was applied to a 96-well plate, and the absorbance value was measured at 536 nm. The absorbance values of blank tube, control tube and measuring tube are recorded as A blank, A pair and A measuring respectively.

Hydroxyl radical clearance D ═ 100% (pair a measured-a)/(pair a empty-a) ×

As shown in figure 1(a), the total flavone ZHFA in the concentration range of 14-30 mg/ml has hydroxyl radical clearance rate increasing with the increasing of the concentration and has concentration dependency, the clearance rate is 26.97%, 60.11%, 65.63%, 87.41%, 99.58% when the concentration is 14mg/ml, 18mg/ml, 22mg/ml, 26mg/ml, 30mg/ml, and (P <0.0001), and as shown in figure 1(b), the hydroxyl radical clearance rate is increasing with the increasing of the concentration when the concentration is 30-190 mg/ml, and has concentration dependency, the clearance rate is 2.18%, 10.25%, 22.69%, 35.85%, 54.51% when the concentration is 30mg/ml, 70mg/ml, 110mg/ml, 150mg/ml, 190mg/ml, and (P < 0.0001). Hydroxyl radical scavenging ability kit (BC1325-100T/96S) is used for analyzing the hydroxyl radical scavenging ability of phellinus igniarius total flavonoids from different sources respectively, and the phellinus igniarius fungal flavonoids ZHFA and JHFA have the hydroxyl radical scavenging ability.

Detecting the total antioxidant capacity (T-AOC) of the phellinus igniarius total flavone extract: preparing total flavone samples into solutions with different concentrations; preheating an enzyme-labeling instrument for more than 30min, and adjusting the wavelength to 593 nm; diluting the standard solution with distilled water to 0.15, 0.1, 0.05, 0.025, 0.0125 μmol/ml, sucking 100 μ L of standard solution (distilled water as blank), adding 100 μ L of reagent II, mixing well, reacting for 10min, measuring absorbance at 593nm, calculating A ═ A standard-A blank, at this time Fe ²⁺ Final concentration 0.075, 0.05, 0.025. mu. mol/ml in Fe ²⁺ The final concentration is an abscissa (x) and A is an ordinate (y), and a standard curve is drawn; the sample solution comprises the following components in percentage by weight: adding 180 μ L of sample solution into blank tube and measuring tube, respectively, adding 6 μ L of sample solution into measuring tube, mixing, adding 70% ethanol into blank tube, adding 18 μ L of 70% ethanol into measuring tube, and mixing. Mixing, reacting for 10min, sucking 200 μ L into 96-well plate, measuring absorbance at 593nm, calculating A-A blank, and obtaining flavone ZHFA and JHFA with antioxidant effect.

As shown in figure 2(a), when the concentration of the ZHFA is in the range of 0.125-1 mg/ml, the total antioxidant capacity is increased along with the increase of the concentration, and has certain concentration dependence, and when the concentration is 0.125mg/ml, 0.25mg/ml, 0.5mg/ml, 0.75mg/ml and 1mg/ml, the antioxidant capacity is respectively 0.088 mu mol/g, 0.21 mu mol/g, 0.265 mu mol/g, 0.266 mu mol/g and 0.403 mu mol/g, (P < 0.0001); as shown in figure 2(b), the JHFA has a concentration range of 0.5-2.5 mg/ml, the total antioxidant capacity is increased along with the increase of the concentration, and has a certain concentration dependency, and when the concentration is 0.5mg/ml, 1mg/ml, 1.5mg/ml, 2mg/ml and 2.5mg/ml, the antioxidant capacity is respectively 0.165 mu mol/g, 0.226 mu mol/g, 0.342 mu mol/g, 0.475 mu mol/g and 0.521 mu mol/g, and the P is less than 0.0001. The total antioxidant capacity (T-AOC) detection method of the flavonoid extract of Phellinus sophorae has proved that Phellinus sophorae fungal flavonoids ZHFA and JHFA have obvious antioxidant capacity.

Cell processing

Cell recovery: taking out the freezing storage tube of the 3 cell strains from a refrigerator at the temperature of-80 ℃, immediately putting the freezing storage tube into a water bath kettle at the temperature of 37 ℃, quickly shaking for 1-2min to quickly dissolve the freezing storage tube, and then transferring the freezing storage tube into a disinfected ultra-clean workbench; transferring the cells in the frozen tube into a centrifuge tube by using a pipette, centrifuging for 5min at 1000rmp, removing a supernatant after centrifugation, and adding 1ml of complete culture medium to resuspend cell precipitates; inoculating the resuspended cell suspension into a cell culture flask filled with cell culture fluid, and placing the cell culture flask in a medium containing 5% CO ₂ The cells were cultured at 37 ℃.

Cell passage: carrying out cell passage when RAW264.7 cell growth density covers 80-90% of the bottom area of the culture bottle; discarding the original culture medium, adding PBS, rinsing gently for 3 times, and discarding; adding 1000 mu L of trypsin, slightly shaking, placing in a cell culture box for 3-5 min, observing cell shape rounding under a microscope, and adding a fresh complete culture medium to terminate digestion; blowing and beating the cells on the wall of the culture bottle by using a pipette, transferring the cells into a centrifuge tube, centrifuging the centrifuge tube at 1000rpm for 5min, removing supernatant, adding 1ml of complete culture medium to suspend cell sediment, and subpackaging the cell sediment into two culture bottles; t cells and B cells were suspension cells, and trypsinization was omitted and the procedure was the same as for RAW 264.7.

Freezing and storing cells: preparing cells in a good growth state into cell suspension, centrifugally washing, adding freezing solution containing 10% DMSO for resuspension, and transferring into a cell freezing tube; and (3) putting the cryopreservation tube into a cryopreservation box containing isopropanol, and transferring the cryopreservation tube into a refrigerator at the temperature of 80 ℃ below zero to cryopreserve cells.

Cell counting: taking well-grown cells in log phaseGently rinsing RAW264.7 macrophage with PBS for three times, digesting with pancreatin, and counting cells; the number of four large square lattice cells at four corners of the cell counting plate is observed under a microscope, and the total number of the cells is expressed by the following formula: cell number (one/ml) 4 cells in large grid (N)/4X 10 ⁴ X dilution factor.

Detecting the influence of the total flavonoids on the proliferation rate of immune cells (CCK-8 method): through preliminary experiments, 4000 cells/hole inoculated by RAW264.7 macrophage and 10000 cells/hole inoculated by Raji cells and Jurkat cells are determined; respectively counting 3 cell strains, inoculating the cell strains to a 96-well plate, and inoculating the cell strains to the 96-well plate with 5% CO ₂ Culturing at 37 deg.C for 24 hr; weighing a small amount of total flavonoids, dissolving the total flavonoids in DMSO, and then completely culturing and diluting the total flavonoids to 1600 mu g/ml mother solution; LPS was diluted to 40, 20, 10.5. mu.g/ml solution with complete medium; diluting flavone mother liquor to 100, 50, 25, 12.5, 6.25 and 3.125 mu g/ml solution in complete culture medium; after the cells are cultured in the incubator for 24h, 100 mu L of medicines with different concentrations are added, RAW264.7 macrophage cells are continuously cultured in the incubator for 48h, and after the rest two cells are cultured in the incubator for 72h, OD of each hole is measured ₄₅₀ An absorbance value. (6 multiple wells per concentration setting)

Cell proliferation rate (%) ═ (OD) _Medicine -OD _{Blank space} )-(OD _{Negative control} -OD _{Blank space} )/(OD _{Negative control} -OD _{Blank space} )×100％

As shown in fig. 3(a), flavone ZHFA of phellinus linteus fruiting body has obvious effect on proliferation of Jurkat cells, the stimulation effect is strongest when the concentration is 12.5 μ g/ml, the proliferation rate is 88.85%, and the statistical significance (P <0.001) is achieved compared with a negative control group, so that a better stimulation effect can be achieved at a lower concentration; as shown in FIG. 3(b), the JHFA, a total flavonoid of mycelium at a concentration of 50. mu.g/ml, was statistically significant (P <0.05) in both DMSO group and negative control group, and had a good stimulatory effect on Jurkat cells.

As shown in fig. 4(a), ZHFA had a significant effect on proliferation of Raji cell lines, and at a concentration of 50 μ g/ml, the stimulation was strongest, and the cell proliferation rate was 56.14%, which was statistically significant compared to the negative control group (P < 0.0001); as shown in fig. 4(b), JHFA had a good stimulating effect on Raji cell lines at a concentration of 25 μ g/ml, had a cell proliferation rate of 54.29%, and had statistical significance (P <0.001) compared to the negative control group, and when the concentration continued to increase, inhibited cell proliferation, exhibited toxic and side effects on cells, and had statistical significance (P <0.0001) compared to the negative control group.

As shown in fig. 5(a), ZHFA has a significant effect on proliferation of raw264.7 cell line, and has the strongest stimulation effect when the concentration is 6.25 μ g/ml, the cell proliferation rate is 100.47%, and the ZHFA has statistical significance (P is less than 0.0001) compared with a negative control group and can play a better effect when the concentration is low; as shown in fig. 5(b), JHFA had a good stimulating effect on raw264.7 cell line at a concentration of 3.125 μ g/ml, and the proliferation rate of raw264.7 cell line was 48.36%, which was statistically significant (P <0.0001) compared to the negative control group, and the cell proliferation decreased as the concentration increased.

Trypan blue method for detecting phagocytic capacity of RAW264.7 macrophages: preparing 0.4%, 0.04%, 0.01%, 0.004% trypan blue solution for use; plating 4000/hole plates (100 mu L per hole) after counting macrophage cells, and culturing in an incubator for 48 hours; the culture solution was discarded, and 100. mu.L of each prepared trypan blue solution of each concentration and 5% CO were added ₂ After incubation at 37 ℃ for 15min in an incubator, trypan blue solution was removed, each well was rinsed gently three times with 200. mu.L LPBS, PBS was discarded, 100. mu.L of cell lysate was added to each well for lysis for 2h, and OD was measured at 450nm, 490nm, and 540 nm. Screening out obvious OD values for detection; after counting macrophage cells, laying plates at 4000 per hole (100 mu L per hole), culturing in an incubator for 24h, adding LPS (low-pressure lipoprotein) into a positive control, adding drugs (100 mu L per hole) with the optimal concentration of each screened drug into an experimental group, adding 100 mu L of cell culture solution into a negative control group, and continuously culturing for 48h after adding the drugs; discard the culture medium, add 100. mu.L trypan blue solution, incubate in incubator for 15min, remove the trypan blue solution, wash each well with 200. mu.L LPBS, wash 3 times, discard PBS, add 100. mu.L cell lysate to each well to lyse for 2h, measure OD at 540 nm.

Phagocytosis ratio P ═ 100% (a drug-a negative)/(a negative-a blank) ×

As shown in FIGS. 6(a) and (b), the phagocytic activity of macrophages was improved after the stimulation of macrophages by Phellinus linteus total flavonoids, and was statistically significant (P <0.05) compared to the negative control.

As shown in fig. 7, the effect on lymphokine secretion by T lymphocytes in vitro: 10000 cells/hole are plated (100 mu L per hole) after Jurkat cells are counted, LPS (2.5mg/ml) is added into a positive control group after the cells are cultured in an incubator for 24 hours, the optimal concentration of each drug is added into an experimental group, and the incubator is continued to culture for 72 hours after the drugs are added; collecting cell culture fluid, centrifuging in 1.5ml EP tube at 4 deg.C at 1000rmp for 10min, centrifuging, and packaging supernatant. The sample can be stored in a refrigerator at 4 ℃ within 24 h; taking the ELISA kit out of the refrigerator, and standing at room temperature for 0.5 h; calculating the volume of the required washing liquid, and diluting the concentrated washing liquid by 20 times with ultrapure water for use; taking out the standard substance and the standard substance diluent, dissolving the standard substance with 1ml of the standard substance diluent, wherein the concentration of the standard substance is 1000pg/ml, and then diluting according to 500, 250, 125, 62.5, 31.25, 15.625, 7.8 and 0 pg/ml; taking out the enzyme label plate, washing the plate for three times by using prepared washing liquid and drying the plate by spin; adding 100 μ L of standard substance and cell supernatant into the reaction well, sealing with rubber paper, and incubating at 37 deg.C for 90 min; after incubation is finished, taking out the enzyme label plate, washing the plate for 4 times, patting the plate dry, diluting the concentrated biotinylation antibody by 100 times by using an antibody diluent, adding the diluted biotinylation antibody into each reaction hole within 0.5h, sealing each reaction hole by 100 mu L, sealing the plate with a paper seal plate, and incubating for 60min in an incubator at 37 ℃; after incubation, taking out the enzyme label plate, washing the plate for 4 times and patting dry, diluting the concentrated enzyme conjugate by 100 times by using an enzyme conjugate diluent, adding the diluted enzyme conjugate into each reaction hole within 0.5h, sealing the reaction holes by 100 mu L each, and incubating for 30min in an incubator at 37 ℃ after sealing the plate with an adhesive paper plate; after incubation, taking out the enzyme label plate, washing the plate for 5 times and drying by patting, adding 100 mu L of color development liquid into each hole, sealing the plate with a rubber paper seal plate, and incubating for 15min in an incubator at 37 ℃ in a dark place; (11) after incubation, 50. mu.L of stop buffer was added to each well and OD was measured within 5min ₄₅₀ 。

Effect of each drug on lymphokine secretion by RAW264.7 macrophage cells in vitro: as shown in FIG. 8, A in FIG. 8 shows that the IL-2 content in Jurkat cells was 0.29pg/ml without drug stimulation, and the IL-2 content in LPS group, ZHFA group and JHFA group was increased to 3.5, 1.56 and 2.36pg/ml, respectively, after drug stimulation, which was statistically significant (P <0.01) compared to the negative control group; FIG. 8C shows that the IFN-. gamma.content of Jurkat cells was 1.68pg/ml when Jurkat cells were not stimulated with drugs, and the IFN-. gamma.content of LPS group, ZHFA group and JHFA group was increased to 2.28, 1.9 and 2.2pg/ml after drug stimulation, respectively, which was statistically significant (P <0.01) compared with the negative control group; b of FIG. 8 shows that the IL-6 content in RAW264.7 cells was 0.276pg/ml in the case of RAW264.7 cells without drug stimulation, and the IL-6 content in LPS group, ZHFA group and JHFA group was increased to 3.63, 1.56 and 0.64pg/ml, respectively, after drug stimulation, which was statistically significant (P <0.01) compared with the negative control group; FIG. 8D shows that TNF- α content in RAW264.7 cells was 0.154pg/ml without drug stimulation, and TNF- α content in LPS group, ZHFA group, and JHFA group was decreased to 0.1, 0.062, and 0.079pg/ml after drug stimulation, respectively, which was statistically significant (P <0.01) compared to the negative control group.

As shown in fig. 9, the effect of ZDPS, ZHFA immunocompromised mice on immune function: 25 Kunming mice weighing 18-22g, after being acclimatized to 1W in laboratory conditions, were culled 4 mice with higher or lower body weights, and the remaining 21 mice were randomly divided into 4 groups of 3 mice each, namely, normal group (KB), model group (CTX), low-dose group of ZHFA (ZHFA50), high-dose group of ZHFA (ZHFA 200);

animal treatment: the mice of all groups except KB group were intraperitoneally injected with CTX (100mg/kg) 1 time per day for 3 consecutive days, and 4 th group with 0.2ml of 6% chicken red blood cells, while the mice of all groups except KB group and CTX group were initially individually administered by gavage 1 time per day for 15 consecutive days. The KB group and the CTX group were perfused with saline for 15 days. After 15 days, each group of mice is sacrificed after the eyepit blood is taken, and the weight ratio of organs is measured, a serum hemolysin experiment is carried out, and the change of each cytokine in the serum is measured;

organ index measurement: after the mice in each group are fed for the last time, the mice are fasted and are not forbidden for 12 hours, after the mice are weighed, blood is taken from the eye sockets, then the mice are dislocated and killed, the heart, the liver, the spleen, the lung, the kidney and the thymus are taken out, after blood stains are wiped dry, the weight of each organ is weighed by an electronic balance, the organ index is calculated according to the following formula: each organ index is weight of organ (g)/weight (g)

The effects of the drug on the organs of the mice are shown in FIG. 9, in which A to F represent the thymus index, spleen index, heart index, liver index, lung index and kidney index of the test mice, respectively. Compared with the KB group, the CTX group has significantly reduced thymus index and spleen index and has statistical significance (P <0.01), and compared with the KB group, the CTX group has obviously atrophied thymus and spleen, which indicates that the mice are successfully modeled. Compared with the KB group, the differences of the heart index, the kidney index, the liver index and the lung index of the CTX group have no statistical significance, the organ index of each administration group has no obvious increase or decrease and no statistical significance compared with the CTX group, and the organs of each administration group have no obvious hyperplasia or atrophy compared with the CTX group, which indicates that the CTX can not cause obvious influence on the heart, the liver, the lung and the kidney of the mice.

Determination of mouse serum IL-2, IL-6, IFN-. gamma.and TNF-. alpha.: mice at 4d are intraperitoneally injected with 0.2ml of 6% chicken erythrocyte for sensitization, after the administration is finished, orbital blood is taken out from an anticoagulation tube, after the standing for 1h at room temperature, the centrifugation is carried out at 4 ℃ and 2000rmp for 20min, 50 muL of supernatant is taken out from an EP tube, 250 muL of each of 6% CRBC and 10% guinea pig serum is added, the incubation is carried out at 37 ℃ for 1h in an incubator, after the incubation is finished, the reaction is stopped for 15min on ice, then the centrifugation is carried out at 4 ℃ and 2000rmp for 20min, 200 muL of supernatant is taken out from a 96-well plate, and the OD value is measured at 540 nm. Determination of mouse serum IL-2, IL-6, IFN-gamma, TNF-alpha.

The detection principle is as follows: enzyme-linked immunosorbent assay technology of double antibody sandwich method is adopted. After the monoclonal antibody of the mouse is coated on the ELISA plate, each sample solution is added, so that each factor in the sample solution can be fully combined with the coated antibody on the ELISA plate. After the plate is fully washed, a biotinylated antibody is added, and the antibody can be specifically combined with each factor in a sample captured by the coated antibody in the ELISA plate; and (3) adding horseradish peroxidase (HRP) -labeled streptavidin after fully washing the plate again, wherein the streptavidin and the biotin can be subjected to high-strength non-covalent binding, adding a chromogenic reagent substrate TMB after fully washing the plate again, and changing the streptavidin into blue with different degrees if cytokines with different concentrations exist in reaction holesA color substance, which turns yellow in the well after the addition of the stop solution, and then the OD is measured ₄₅₀ And the concentration of the cell factor in the reaction hole is in direct proportion to the OD value, and the corresponding concentration of the cell factor is calculated according to the drawn standard curve.

Collecting a serum sample: collecting blood from mouse orbit, standing at room temperature for 1 hr, centrifuging at 4 deg.C for 10min at 1000rmp, packaging the supernatant, and storing in a refrigerator at 4 deg.C for 24 hr or-20 deg.C for more than 24 hr;

as shown in fig. 11, the anti-chicken erythrocyte antibody hemolysin produced by B lymphocytes in mice reflects the humoral immunity level of mice to a certain extent, and by detecting the hemolysin level of mice treated differently, the hemolysin level of CTX group mice was reduced compared to KB group, and there was a statistical difference (P <0.05), and CTX could reduce the humoral immunity of the body. Compared with the CTX group, the hemolysin level of mice in each administration group is improved, and the statistical significance is achieved (P is less than 0.05); the increase of the ZHFA low-dose group and the ZHFA high-dose group is obvious and has statistical significance (P is less than 0.001), and each drug can restore the humoral immunity of the mice to a certain degree. After modeling, the IL-2 level in the serum of a mouse model with low immunity treated by the phellinus igniarius total flavonoids is reduced obviously and has statistical significance (P is less than 0.01) in the group A of the CTX compared with the group KB by the IL-2 detection kit, and after administration, the IL-2 level of each drug group is recovered and has statistical significance (P is less than 0.05) compared with the CTX group; the levels of IL-6 in CTX group B in FIG. 11 were significantly lower than those in KB group and statistically significant (P <0.05), and after administration, IL-6 levels in each drug group were significantly higher than those in CTX group and statistically significant (P < 0.05); the CTX group mice in FIG. 11C had significantly lower IFN-. gamma.levels than the KB group, were statistically significant (P <0.0001), were not significantly elevated in IFN-. gamma.levels in the drug groups after administration, and were also significantly reduced in TNF-. alpha.levels in the CTX group mice in FIG. 11D compared to the KB group, were statistically significant (P <0.01), and were not elevated and restored in TNF-. alpha.levels in the ZHFA low dose group and the high dose group after administration compared to the CTX group.

Detecting the content of unsaturated fatty acid in the mouse excrement: 25 Kunming mice weighing 18-22g were culled after acclimation to laboratory conditions of 1W, and the remaining mice were randomly divided into 4 groups of 3 mice each, namely, normal group (KB), model group (CTX), ZHFA low dose group (ZHFA50), ZHFA high dose group (ZHFA 200). Except for the KB group injected with physiological saline (NS), each of the other mice was intraperitoneally injected with CTX (100mg/kg) 1 time a day for 3 days continuously to construct an immunocompromised mouse model. Beginning at 4d, KB and CTX groups were gavaged with saline, and the remaining groups of mice were gavaged 1 time daily for 15 consecutive days. After 15 days, the tail of the mouse is pulled to automatically defecate the mouse, and the feces are collected by a sterile centrifuge tube and are frozen and stored at the temperature of-80 ℃. A liquid chromatography tandem mass spectrometry (LC-MS/MS) detection technology is adopted, and the basic technical process comprises the following steps: sample pretreatment (grinding, purification, enrichment and purification), UPLC separation, MS/MS detection, MRM detection, data analysis and the like. As shown in fig. 12, as a result of detecting that the short-chain fatty acids are 7 content changes of acetic acid, propionic acid, butyric acid, valeric acid, hexanoic acid, isobutyric acid, and isovaleric acid in response to a-G, the metabolite concentrations of 7 SCFAs (intestinal short-chain fatty acids) of different groups of mice showed that CTX had a large effect on the content of SCFAs in the intestinal tract of mice, wherein among the 7 fatty acids, the acetic acid content was the greatest in each group of mice, the isovaleric acid was the second, and the hexanoic acid content was 0 in all the groups of mice administered. Comparing the short-chain fatty acid content of each group with CTX, after CTX modeling, the content of 7 short-chain fatty acids of mice in the CTX group is obviously reduced compared with that in the KB group, and the short-chain fatty acid has statistical significance (P is less than 0.001); after administration to each group of mice, the acetic acid content of the mice in the low-dose group of ZHFA in fig. 12 a was significantly recovered and statistically significant (P <0.05) compared to the CTX group, while the difference was not observed between the high-dose group of ZHFA and the CTX group, indicating that there was no effect of the high-dose of ZHFA in recovering the acetic acid level in the intestinal tract of the immunocompromised mice; the propionic acid content in B in figure 12 is changed, and each administration group can recover the propionic acid level of the immunocompromised mice, and has statistical significance (P < 0.05); as can be seen from the change in mouse butyrate content in fig. 12C, ZHFA promoted the recovery of intestinal butyrate levels, which was statistically significant compared to CTX (P < 0.0001). As shown by the change in the valeric acid content in the mice in fig. 12D, the levels of valeric acid in the ZHFA low dose group were lower than those in the CTX group, and statistically (P <0.05), no significant difference was observed between the ZHFA (200) group and the CTX group; in FIG. 12E, it is seen that the caproic acid content in the mice of each administration group was 0, and it is seen that the production of caproic acid was suppressed at both low and high doses; the isobutyric acid content changes in fig. 12F, with the low dose ZHFA group promoting intestinal isobutyric acid production in mice, statistically significant (P <0.001) compared to the CTX group, and the high dose ZHFA had no effect on the recovery of isobutyric acid content; the change in isovaleric acid content in G of fig. 12 was shown to be that the low ZHFA dose group promoted intestinal isovaleric acid production in mice with statistically significant differences (P <0.01) compared to the CTX group, whereas the high ZHFA dose had no effect on the recovery of isovaleric acid content.

And (3) detecting diversity of fecal intestinal flora of the mice: the mice are treated in groups and feces are collected, stored and transported as described above. Flora diversity analysis the nucleotide sequence difference of the V3V4 variable region of 16s rRNA in fecal samples was analyzed by high throughput procedures, and the diversity of the fecal intestinal flora of each group of mice was analyzed by corresponding bioinformatics software. Analysis shows that sequencing data of all test groups reach a plateau stage, namely the obtained 16s rRNA sequence information of the mouse intestinal flora can sufficiently cover the number of all the intestinal flora in a sample, and the diversity and the abundance of the intestinal flora of the sample can be reflected.

In the experiment, the mutual influence, activity and extraction rate in the extraction process of various components are fully considered, the extracted flavone has high extraction quantity and high purity, the utilization rate of the phellinus igniarius is greatly improved, the extraction time is obviously shortened, the cost is greatly saved, no special equipment is needed in the extraction process, the cost is low, a way is provided for combined extraction and separation of polysaccharide, a new way is provided for utilization of phellinus igniarius in northern Shaanxi, and the phellinus igniarius in Shaanxi has good social and economic benefits.

Claims (3)

1. The flavone extract of Phellinus sophorae can be used for resisting oxidation and enhancing immunity.

2. The use of the Sophora phellinus igniarius flavone extract as claimed in claim 1 for antioxidation and immunity enhancement, wherein the Sophora phellinus igniarius flavone extract is fruit body flavone.

3. The use of the Sophora phellinus igniarius flavone extract as claimed in claim 1 for antioxidation and immunity enhancement, wherein the Sophora phellinus igniarius flavone extract is mycelium flavone.

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