CN109620823B - Application of Hypholomine B in preparation of immune activator - Google Patents

Application of Hypholomine B in preparation of immune activator Download PDF

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CN109620823B
CN109620823B CN201811572386.8A CN201811572386A CN109620823B CN 109620823 B CN109620823 B CN 109620823B CN 201811572386 A CN201811572386 A CN 201811572386A CN 109620823 B CN109620823 B CN 109620823B
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hypholomine
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phellinus igniarius
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hypalone
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蒋福升
李美芽
丁滨
丁志山
李松涛
窦晓兵
吕圭源
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Zhejiang Chinese Medicine University ZCMU
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Abstract

The invention discloses an application of Hypholomine B in preparation of an immune activator, the Hypholomine B can effectively promote macrophages to secrete cytokines such as INF-gamma and the like, and the cytokines such as INF-gamma and the like have strong antiviral, immunoregulation and antitumor activities; we further prove that the Hypholomine B can effectively enhance the phagocytic activity of macrophages on Escherichia coli and the killing effect on tumor cells; hypalone B has the effects of promoting the proliferation of RAW264.7 cells and promoting the phagocytic activity of the RAW264.7 cells, and Hypalone B can promote the killing activity of macrophages on tumor cells in a dose-dependent manner. Therefore, the novel application of Hypholomine B in the aspect of immunoregulation can be used as a novel index of the medicinal components of the phellinus igniarius, and can also provide a thought for further scientific utilization of the phellinus igniarius, so that the economic benefit of the phellinus igniarius is effectively improved.

Description

Application of Hypholomine B in preparation of immune activator
(I) technical field
The invention relates to a new application of a medicinal monomer component Hypholomine B of phellinus igniarius in immunoregulation.
(II) background of the invention
Phellinus linteus is a valuable medicinal fungus with more than 2000 years of history, and the earliest herbal literature of China, Shennong's herbal classic, has records. It is slightly bitter in taste and cold in nature, and is mainly used for treating metrorrhagia, bloody stranguria, rectocele and bloody diarrhea, leukorrhagia, amenorrhea and the like in the traditional Chinese medicine. Modern pharmacological studies show that phellinus igniarius has the effects of resisting tumors, angiogenesis and fibrosis, protecting the liver, reducing blood sugar and blood fat, resisting oxidation, resisting bacteria, regulating immunity and the like, and is a medicinal fungus with the highest effective rate in the currently internationally recognized biological cancer treatment medicaments. The chemical components of the composition comprise polysaccharide, fatty acid, polyphenol, triterpenes, various amino acids, various enzymes and the like. The phellinus igniarius polysaccharide is the most studied component, is a relatively well-known medicinal component of phellinus igniarius at present, is also a main component for the phellinus igniarius to exert immunoregulation, and has not been reported in other research reports about immunoregulation components in phellinus igniarius. In recent years, research shows that the phellinus igniarius contains more polyphenols, such as flavone, styrene pyrone and other compounds, besides polysaccharide, and has strong effects of resisting oxidation, reducing blood fat, reducing blood sugar and the like, and the phellinus igniarius is very likely to be a potential medicinal component in the phellinus igniarius.
Phellinus linteus is a rare medicinal fungus, and hypha polysaccharide of Phellinus linteus has strong immunoregulation and anti-tumor activity, so that in production practice, if only the hypha polysaccharide is extracted, great resource waste is caused. The research finds that the phellinus igniarius hypha contains high-content small molecular components such as styryl pyrone besides polysaccharide components, wherein the compound hypalone B is particularly remarkable; therefore, the reasonable utilization of the compounds has important significance for effectively improving the actual production benefit. The research shows that Hypholomine B has stronger free radical scavenging activity and neuraminidase inhibition activity, so that the Hypholomine B has potential antioxidant and antiviral effects, and the research reports in other aspects, especially the immunoregulation effect are not related.
Disclosure of the invention
The invention aims to provide the application of a monomer component Hypalonamine B in phellinus igniarius in immune activation; it can effectively promote macrophage to release cell factors such as INF-gamma, and effectively promote macrophage phagocytosis activity to Escherichia coli and killing effect to tumor cells.
The technical scheme adopted by the invention is as follows:
the invention provides application of Hypholomine B in preparation of an immune activator.
Further, the immune activator is a drug promoting secretion of inflammatory factors including IL-6, IL-10, INF-gamma, IL-12, MCP-1 or TNF-alpha.
Further, the immune activator is a drug for improving macrophage activity, wherein the macrophage activity comprises phagocytosis escherichia coli activity or tumor cell killing activity.
The structural formula of Hypholomine B is as follows:
Figure BDA0001915881660000021
compared with the prior art, the invention has the following beneficial effects:
the invention discloses a new application of a compound Hypalone B in the aspect of immunoregulation, wherein the Hypalone B can effectively promote macrophages to secrete cytokines such as INF-gamma and the like, and the cytokines such as INF-gamma and the like have strong antiviral, immunoregulation and antitumor activities; we further prove that the Hypholomine B can effectively enhance the phagocytic activity of macrophages on Escherichia coli and the killing effect on tumor cells; hypalone B has the effects of promoting the proliferation of RAW264.7 cells and promoting the phagocytic activity of the RAW264.7 cells, and Hypalone B can promote the killing activity of macrophages on tumor cells in a dose-dependent manner. Therefore, the novel application of Hypholomine B in the aspect of immunoregulation can be used as a novel index of the medicinal components of the phellinus igniarius, and can also provide a thought for further scientific utilization of the phellinus igniarius, so that the economic benefit of the phellinus igniarius is effectively improved.
(IV) description of the drawings
FIG. 1 effect of Hypholomine B on RAW264.7 cell proliferation.
FIG. 2 shows the flow results of the phagocytosis activity of macrophage on E.coli by Hypholomine B, and a-f are control group, LPS 1. mu.g/mL treatment group and Hypholomine B10, 20, 40 and 60. mu.g/mL treatment groups, respectively. A is a flow cytometry detection result graph of each group, and B is an average fluorescence intensity histogram of each group.
FIG. 3 shows the results of confocal high content imaging analysis of phagocytic activity of Escherichia coli by using Hypholomine B, wherein a-f are respectively a control group, an LPS 1. mu.g/mL treatment group and Hypholomine B10, 20, 40 and 60. mu.g/mL treatment groups.
FIG. 4 Hypholomine B promotes macrophage toxication, with different letters indicating significant differences compared between groups, P < 0.01.
(V) detailed description of the preferred embodiments
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
example 1: hypholomine B on RAW264.7 cell proliferation
RAW264.7 cells (purchased from Shanghai department of sciences cell Bank) were cultured in DMEM medium (Gibco) containing 10% fetal bovine serum and diabodies (streptomycin 100. mu.g/mL and penicillin 100U/mL) at 37 ℃ in 5% CO2Culturing in an incubator. Taking RAW264.7 cells in exponential growth phase, washing twice with 4 ℃ precooled PBS, air-whipping to collect the cells, diluting to 1 × 10 with DMEM medium containing 10% fetal calf serum and double antibody (streptomycin 100 μ g/mL and penicillin 100U/mL)5cells/mL, seeded in 96-well plates at 200. mu.L/well, 37 ℃ 5% CO2The incubator is kept still for overnight culture. The cell viability was calculated by adding Hypophomine B at various final concentrations (0, 10, 20, 40, 60, 80, 100, 120. mu.g/mL) for an additional 44h, adding 10. mu.L of CCK8 reagent per well, incubating for an additional 4h, measuring absorbance at 450 nm. Under the same conditions, the blank was prepared by adding an equal amount of physiological saline instead of hypalone B.
Figure BDA0001915881660000031
After the treatment of the hypalone B with different concentrations for 48 hours, the cell survival rate of the treatment group with the concentration of 20-100 mug/mL is obviously higher than that of the control group, and the cell survival rate is increased in a dose-dependent manner under the treatment concentration of 10-60 mug/mL, which indicates that the hypalone B can promote the proliferation of RAW264.7 cells; however, the survival rate began to decrease at concentrations above 80. mu.g/mL, and the cell survival rate at 120. mu.g/mL was significantly lower than that of the control-treated group (see FIG. 1).
Example 2: hypholomine B promotes mouse abdominal cavity macrophage and RAW264.7 cell to secrete INF-gamma and other cytokines
ICR mice, cervical dislocation and sacrifice, 75% ethanol soaking and aseptic operation collecting abdominal cavity macrophage, using 10% fetal calf serum and two antibodies (streptomycin 100U g/mL and penicillin 100U/mL) DMEM medium dilution to 1 x 105cells/mL, inoculating a 24-well plate, 0.5mL per well; another RAW264.7 cells in exponential growth phase were diluted to 1X 10 with DMEM medium containing 10% fetal bovine serum and diabodies (streptomycin 100. mu.g/mL and penicillin 100U/mL)5cells/mL, inoculated in 12 well plates, 1mL per well, placed at 37 ℃ in 5% CO2Standing and culturing in an incubator overnight; the culture was continued by adding Hypholomine B at each of the final concentrations (10, 20, 40, 60. mu.g/mL), LPS at 1. mu.g/mL was used as a positive control, an equal amount of the culture medium was added as a negative control, and after 12 hours, the supernatant was taken and the level of each inflammatory factor was measured by the flow cytometry CBA method, and the results are shown in tables 1 and 2.
As can be seen from the data in Table 1, the positive control LPS can significantly increase the levels of other factors compared with the negative control group except for IL-12 INF-gamma; each dose group of Hypholomine B significantly increased the levels of each factor and showed a dose-dependent increase except for IL-12. From the results in Table 2, LPS can significantly increase the levels of various factors compared with the control group except for IL-12; while Hypholomine B shows certain inhibition effect on MCP-1 factor and dose-dependent promotion effect on other factors. Compared with LPS treatment group, Hypholomine B can remarkably promote IL-10, INF-gamma and IL-12, and shows immune activation effect.
TABLE 1 mouse peritoneal macrophage inflammatory factor levels
Figure BDA0001915881660000041
Note: HB is hypalone B abbreviation;*the expression is extremely significant compared with the control groupDifference in sex, P<0.01；#Indicates that the LPS group has extremely significant difference, P<0.01。
TABLE 2 RAW264.7 cell cytokine levels
Figure BDA0001915881660000042
Note: HB is hypalone B abbreviation;*indicates that there is a very significant difference, P, from the control group<0.01；#Indicates that the LPS group has extremely significant difference, P<0.01。
Example 3: hypholomine B promotes phagocytic activity of macrophages on escherichia coli
The supernatant of example 2 was subjected to a flow cytometry measurement for determining the phagocytic activity of the cells based on the intensity of fluorescence emitted by the cells after phagocytosing the bacteria, and the results are shown in FIG. 2. The average fluorescence intensity of the normal group is (4075.17 +/-253.36), the peak value of the normal group is shifted to the right by 3.8 percent after being stimulated by 1 mu g/mL LPS for 12 hours, the average fluorescence intensity is (4990.95 +/-207.35), and although the average fluorescence intensity is different from the control, the average fluorescence intensity does not show very remarkable phagocytosis activity; while Hypholomine B shows a remarkable dosage effect, when the concentration of 60 mu g/mL is treated, the peak value is integrally shifted to the right by 17.2 percent, the average fluorescence intensity is (11289.71 +/-477.71), and the Hypholomine B shows a remarkable phagocytosis promoting effect.
The fluorescence photography results of the confocal high content imaging analysis system are shown in fig. 3, the LPS should be increased in intensity compared to the control, while the fluorescence intensity of the hypalone B-treated group showed a significant dose-dependent increase, which is generally consistent with the flow results, indicating that hypalone B indeed has the phagocytosis activity of RAW264.7 cells.
Example 4: hypholomine B macrophage cytotoxic effect
ICR mice, cervical dislocation and sacrifice, 75% ethanol surface sterilization, aseptic operation to collect abdominal cavity macrophages, use 10% fetal bovine serum and two antibodies (streptomycin 100U g/mL and penicillin 100U/mL) DMEM medium dilution to 1 x 106cells/mL, seeded in 96-well plates at 0.1mL per well, added at different final concentrations (10, 20, 40, 6, respectively)0. mu.g/mL) of Hypholomine B at 37 ℃ with 5% CO2Standing and culturing for 24h in an incubator, adding mouse melanoma B16 target cells (purchased from Shanghai academy of sciences cell bank, with an effective-to-target ratio of 10:1) into each well, continuing culturing for 48h, adding CCK8 reagent 4h before terminating culturing, continuously incubating for 4h, measuring absorbance value at 450nm, and calculating inhibition rate. Under the same conditions, control was performed without hypalone B.
Figure BDA0001915881660000051
From the results in FIG. 4, it can be seen that Hypholomine B dose-dependently promotes the killing activity of macrophages on tumor cells.

Claims (3)

1. A method of increasing macrophage activity for non-diagnostic or therapeutic purposes comprising contacting macrophages with hypalonamine B.
2. The method of claim 1, wherein said macrophage activity comprises the activity of secreting: IL-6, IL-10, INF-gamma, IL-12, MCP-1 or TNF-alpha.
3. The method of claim 1, wherein said macrophage activity comprises phagocytic e.
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KR101446179B1 (en) * 2010-08-24 2014-10-01 전북대학교산학협력단 A composition for Inflammatory Disorder comprising the polyphenol extracts from Phellinus baumii

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KR101446179B1 (en) * 2010-08-24 2014-10-01 전북대학교산학협력단 A composition for Inflammatory Disorder comprising the polyphenol extracts from Phellinus baumii

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Anti-influenza activities of polyphenols from the medicinal mushroom Phellinus baumii;Hwang, Byung Soon等;《BIOORGANIC & MEDICINAL CHEMISTRY LETTERS》;20150815;第25卷(第16期);第3256-3260页 *
Styrylpyrones from the medicinal fungus Phellinus baumii and their antioxidant properties;In-Kyoung Lee等;《Bioorganic & Medicinal Chemistry Letters》;20100725;第20卷(第18期);第5459-5461页 *

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