An extract of Poria cocos fruit body and its application

Abstract

The present invention discloses a kind of Poria cocos fruiting body extract and its application.Described Poria cocos fruiting body extract preparation method is that Poria cocos strain is inoculated in the tissue culture bottle containing PDA culture medium, under the condition of relative humidity being 70 80%, temperature being 26 30 DEG C, first carry out 7 d dark culture, carry out 12 h light and dark alternation culture afterwards until produce fruiting body;Water or 75% ethanol is added to the fruiting body and soaked and ultrasonically extracted 50 80min, filtered, and obtained Poria cocos fruiting body extract after the filtrate is concentrated.The Poria cocos fruiting body extract provided by the present invention is higher than Poria cocos sclerotium in reducing NO content, improving the phagocytic activity of macrophages, and promoting the proliferation ability of macrophages, etc., illustrate that the Poria cocos fruiting body extract provided by the present invention is better than sclerotium in immunomodulation effect, for the development of immunomodulatory drugs or functional foods provides new direction, is conducive to the development and utilization of Poria cocos fruiting body products, can prevent the destruction of ecological resources at the same time, with ecological resource protection value.

Description

Technical Field

[0001] The invention belongs to the technical field of natural product extraction, and particularly relates to a Poria cocos fruiting body extract and application thereof. Background Art

[0002] my country is a major producer and consumer of edible and medicinal fungi, and was one of the first countries to recognize and utilize them. Poria cocos, a traditional Chinese medicinal and edible fungus, has high research and development value and broad application prospects in the pharmaceutical and health care product industries, generating enormous economic value. Currently, research on Poria cocos primarily focuses on its sclerotia. As China intensifies its efforts to build an ecological civilization, the area of Poria cocos cultivation has declined sharply, and production has plummeted, putting the Poria cocos industry in a difficult position. Therefore, finding a substitute for Poria cocos sclerotia is particularly important. The Poria cocos fruiting body, the reproductive organ of the plant, is difficult to collect under natural conditions, and artificial cultivation is the primary method, which, under certain conditions, can reduce the waste of natural resources.

[0003] The present invention aims to provide a Poria cocos fruiting body extract with strong immunoregulatory activity. Summary of the Invention

[0004] The first purpose of the present invention is to provide a Poria cocos fruiting body extract, and a further purpose is to provide a preparation method and application thereof.

[0005] The first object of the present invention is achieved by providing a Poria cocos fruiting body extract, which is prepared by the following method: 1) inoculating a Poria cocos strain into a tissue culture bottle containing PDA medium, and culturing in the dark for 7 days under the conditions of a relative humidity of 70-80% and a temperature of 26-30°C, followed by culturing in a light-dark cycle for 12 hours until fruiting bodies are produced;

[0006] 2) Add water or 75% ethanol to the fruiting body, soak and ultrasonically extract for 50-80 minutes, filter, and concentrate the filtrate to obtain the Poria cocos fruiting body extract.

[0007] A further object of the present invention is achieved by using the Poria cocos fruiting body extract in the preparation of immunomodulatory drugs.

[0008] The beneficial effects of the present invention are as follows: the Poria cocos fruiting body extract provided by the present invention is more effective than Poria cocos sclerotia in reducing NO content, improving the phagocytic activity of macrophages, and promoting the proliferation ability of macrophages, indicating that the Poria cocos fruiting body extract provided by the present invention is more effective than sclerotia in immunoregulation, providing a new direction for the development of immunoregulatory drugs or functional foods, which is beneficial to the development and utilization of Poria cocos fruiting body products, and at the same time can prevent the damage to ecological resources, and has ecological resource protection value. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 This is a graph showing the effects of different concentrations of Poria cocos fruiting body extracts prepared in Example 1 on the survival rate of RAW264.7 cells;

[0010] Figure 2 This is a graph showing the effects of different concentrations of Poria cocos fruiting body and sclerotium extracts prepared in Example 1 on the NO secretion level of RAW264.7 cells;

[0011] Figure 3 This is a graph showing the results of measuring the phagocytic activity of RAW264.7 cells by extracts of Poria cocos fruiting bodies and sclerotia at different concentrations prepared in Example 1;

[0012] Figure 4 This is a graph showing the results of measuring the proliferation ability of RAW264.7 cells with extracts of different concentrations of Poria cocos fruiting bodies and sclerotia prepared in Example 1;

[0013] Figure 5 These are morphological diagrams of the fruiting bodies cultured in Example 1 at the early, middle and late stages. DETAILED DESCRIPTION

[0014] The present invention is further described below with reference to the embodiments, but the present invention is not limited in any way. Any changes or substitutions made based on the teachings of the present invention fall within the scope of protection of the present invention.

[0015] The present invention provides a Poria cocos fruiting body extract, and its preparation method is as follows:

[0016] 1) Inoculate the Poria cocos strain at a volume of 2 cm 2 The cells were inoculated into a tissue culture bottle containing 60 mL of PDA medium and cultured in the dark for 7 days at a relative humidity of 70-80% and a temperature of 26-30°C, followed by 12 h of alternating light and dark culture until fruiting bodies were produced.

[0017] 2) Add water or 75% ethanol to the fruiting body, soak and ultrasonically extract for 50-80 minutes, filter, and concentrate the filtrate to obtain the Poria cocos fruiting body extract.

[0018] In step 1), the PDA culture medium contains the following ingredients: 200 g potato, 20 g glucose, 1 g KH2PO4, 0.5 g MgSO4, 10 mg vitamin B1, 18 g agar, and 1000 mL water.

[0019] In step 2), the ultrasonic frequency is 50-60 kHz.

[0020] The present invention also provides the use of the Poria cocos fruiting body extract in preparing immunomodulatory drugs.

[0021] The present invention further provides an immunomodulatory drug, which uses the Poria cocos fruiting body extract as a main active ingredient or one of the active ingredients.

[0022] Example 1

[0023] The mycelia of the 5.78 Poria cocos strain purchased from the Culture Collection Management Center were picked up with an inoculation needle and inoculated into a 9 cm diameter culture dish filled with PDA medium for activation for 7-10 days. A hole was punched with a punch and a 2 cm 2 The bacterial block was inoculated into a tissue culture bottle containing 60 mL of PDA medium. Under the conditions of relative humidity of 70-80% and temperature of 28℃, dark culture was carried out for 7 days, followed by 12 hours of light and dark alternating culture for 8 weeks to produce fruiting bodies. 7 days after the fruiting body is formed, the period when no pore tubes are formed is the early fruiting body stage. 15 days after the fruiting body is formed, the period when some pore tubes are formed is the mid-fruiting body stage. 25 days after the fruiting body is formed, the period when all pore tubes are formed is the late fruiting body stage. Figure 5 shown.

[0024] Example 2

[0025] The Poria cocos strain was inoculated at a size of 2 cm 2 The cells were inoculated into a tissue culture bottle containing 60 mL of PDA medium and cultured in the dark for 7 days at a relative humidity of 70% and a temperature of 26°C. Then, a 12-h light-dark alternation culture was performed for 54 days to produce fruiting bodies.

[0026] Example 3

[0027] The Poria cocos strain was inoculated at a size of 2 cm 2 The cells were inoculated into a tissue culture bottle containing 60 mL of PDA medium and cultured in the dark for 7 days at a relative humidity of 80% and a temperature of 30°C. The cells were then cultured in a 12-h light-dark alternating cycle for 60 days to produce fruiting bodies.

[0028] Example 4 Preparation of Poria cocos fruiting body extract

[0029] 1. Preparation of aqueous extract: The Poria cocos fruiting bodies at different stages in Example 1 were freeze-dried and ultrasonically extracted with water as the solvent at a liquid-to-solid ratio of 40:1 ml / g for 50 min. The extracts were then filtered through a 0.22 μm filter, concentrated and freeze-dried, and the freeze-dried powder was stored at -20°C for later use to obtain aqueous extracts of Poria cocos fruiting bodies at different stages.

[0030] 2. Preparation of alcohol extract: The freeze-dried Poria fruiting bodies and sclerotia at different stages were subjected to ultrasonic-assisted extraction with 75% ethanol as solvent at a liquid-to-solid ratio of 20:1 ml / g for 50 min. The extracts were then filtered through a 0.22 μm filter and concentrated and freeze-dried. The freeze-dried powder was stored at -20°C for later use to obtain alcohol extracts of Poria fruiting bodies at different stages.

[0031] Example 5

[0032] 1. Preparation of aqueous extract: The Poria cocos fruiting bodies at different stages in Example 1 were freeze-dried and ultrasonically extracted with water as the solvent at a liquid-to-solid ratio of 40:1 ml / g for 60 min. The extracts were then filtered through a 0.22 μm filter, concentrated and freeze-dried, and the freeze-dried powder was stored at -20°C for later use to obtain aqueous extracts of Poria cocos fruiting bodies at different stages.

[0033] 2. Preparation of alcohol extract: The freeze-dried Poria fruiting bodies and sclerotia at different stages were subjected to ultrasonic-assisted extraction with 75% ethanol as solvent at a liquid-to-solid ratio of 20:1 ml / g for 60 min. The extracts were then filtered through a 0.22 μm filter and concentrated and freeze-dried. The freeze-dried powder was stored at -20°C for later use to obtain alcohol extracts of Poria fruit bodies at different stages.

[0034] Example 6

[0035] 1. Preparation of aqueous extract: The Poria cocos fruiting bodies at different stages in Example 1 were freeze-dried and ultrasonically extracted with water as the solvent at a liquid-to-solid ratio of 40:1 ml / g for 80 min. The extracts were then filtered through a 0.22 μm filter, concentrated and freeze-dried, and the freeze-dried powder was stored at -20°C for later use to obtain aqueous extracts of Poria cocos fruiting bodies at different stages.

[0036] 2. Preparation of alcohol extract: The freeze-dried Poria fruiting bodies and sclerotia at different stages were subjected to ultrasonic-assisted extraction with 75% ethanol as solvent at a liquid-to-solid ratio of 20:1 ml / g for 80 min. The extracts were then filtered through a 0.22 μm filter and concentrated and freeze-dried. The freeze-dried powder was stored at -20°C for later use to obtain alcohol extracts of Poria fruit bodies at different stages.

[0037] Comparative Example 1 Preparation of Poria cocos Sclerotium Extract

[0038] 1. Preparation of water extract: 5.78 sclerotia of Poria cocos from Shuangbai area were freeze-dried and water was used as solvent. Ultrasonic-assisted extraction was performed for 50 min at a liquid-to-solid ratio of 40:1 g / ml. The extract was then filtered through a 0.22 μm filter and concentrated and freeze-dried. The freeze-dried powder was stored at -20°C for later use to obtain the water extract of Poria cocos sclerotia.

[0039] 2. Preparation of alcohol extract: The Poria sclerotia were extracted with 75% ethanol as solvent at a liquid-to-solid ratio of 20:1 g / ml for 50 min by ultrasonic-assisted extraction. The extract was then filtered through a 0.22 μm filter, concentrated and freeze-dried, and the freeze-dried powder was stored at -20°C for later use to obtain the Poria sclerotia alcohol extract.

[0040] Experimental Example 1 Detection of immunomodulatory activity of the aqueous and ethanol extracts of the Poria cocos fruiting bodies prepared in Example 4

[0041] 1. Effects on the survival rate of RAW264.7 cells

[0042] Experimental methods:

[0043] 1. Cell recovery: Take out the RAW264.7 cell cryovial from the liquid nitrogen tank, quickly place it in a 37 ℃ water bath and shake it repeatedly to thaw within 1 min; immediately transfer it to the clean bench, quickly transfer the RAW264.7 cryovial to a 15 mL sterile centrifuge tube, add 3 mL complete high-glucose DMEM medium, centrifuge (800 r / min, 5 min), and remove the supernatant; add 9 mL complete high-glucose DMEM medium to the centrifuge tube, mix thoroughly, and transfer to a 50 mL cell culture flask; place it in a 5% CO2 incubator at 37 ℃; observe the cell state and density; when the cells are in good growth condition (attached growth as a monolayer and about 80% of the bottom of the culture flask is covered by cells), wash twice with PBS (2 mL PBS each time, the purpose is to wash away dead cells and metabolic products), add 2 mL complete high-glucose DMEM medium and pipette the flask wall with a pipette, mix thoroughly, and transfer to a 15 mL sterile centrifuge tube, 800 Centrifuge at 400 rpm for 5 minutes. Discard the supernatant and add 3 mL of fresh complete high-glucose DMEM medium. Mix thoroughly by pipetting, then divide the cells into 3 flasks (one for each cell). Add 8 mL of fresh medium to each flask. Incubate the cells in a 5% CO2 incubator at 37°C for subculture.

[0044] 2. The water extracts of the Poria cocos fruiting bodies at different stages prepared in Example 1 and the water extracts of the sclerotia prepared in Comparative Example 1 were added to the culture medium for dissolution, and the alcohol extracts of the fruiting bodies and the sclerotia at different stages were added with 0.1% DMSO and fully dissolved before being prepared. The preparation concentration was 1 mg / mL and filtered and sterilized using a 0.22 μm filter head for subsequent operations.

[0045] 3. When the cells are in the logarithmic growth phase, collect the cells and make a single cell suspension. Adjust the cell concentration to 1X10 5 The cells were then aliquoted into a 96-well plate at 100 μL / well. The edge wells were filled with PBS. The cells were cultured in a 5% CO2, 37°C incubator for 24 h.

[0046] 4. The experiment was divided into a control group (sclerotium water extract group and sclerotium alcohol extract group) and different extract concentration groups (early fruiting body water extract group, early fruiting body alcohol extract group, middle fruiting body water extract group, middle fruiting body alcohol extract group, late fruiting body water extract group, late fruiting body alcohol extract group). The extract concentration of each group was set at 50, 100, 200, 300, 400, 600, 800, and 1000 μg / ml, with 6 replicates in each group.

[0047] 5. After 24 hours, when the cell density reaches about 50%, add culture medium containing the above-mentioned drug concentrations and continue culturing in a 5% CO2, 37°C incubator for 24 hours.

[0048] 6. Weigh a certain amount of MTT powder and dissolve it in PBS to a working concentration of 5 mg / ml.

[0049] 7. After 24 hours of incubation, add 20 μL of MTT solution to each well. After 120 minutes, read the OD value of each well at 490 nm using a microplate reader.

[0050] Calculation of cell viability: Cell viability (%) = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) * 100%.

[0051] MTT assay showed that when the drug concentration was greater than 100 μg / ml, the cell survival rate in the alcohol extraction group was less than 80%, indicating cytotoxicity. Therefore, 100 μg / ml was selected as the highest drug concentration, and 25 μg / ml was selected as the lowest drug concentration according to the multiple method.

[0052] Results: As Figure 1 As shown, the water and alcohol extracts of the fruiting body had a certain impact on cell viability with increasing dose concentration, with the alcohol extract having a greater effect. Cell viability can indirectly reflect the toxicity of the test drug, so the toxicity of the alcohol extract of the sclerotium was more significant than that of the fruiting body. Therefore, the toxicity of Poria fruiting bodies is lower than that of sclerotia.

[0053] 2. Effects on NO Secretion in RAW264.7 Cells

[0054] 1. When the cells are in the logarithmic growth phase, collect the cells and prepare a cell suspension. Adjust the cell concentration to 2X10 5 The cells were then aliquoted into a 24-well plate at 500 μL / well. Normal wells, model wells, and dosing wells were set up and cultured in a 5% CO2, 37°C incubator for 24 h.

[0055] After 24 hours, when the cell density reached approximately 50%, water extracts, alcohol extracts, and water and alcohol extracts of Poria cocos fruiting bodies at different stages, as well as Poria cocos sclerotia, were diluted from their initial concentrations to low (25 μg / ml) and high (100 μg / ml) concentrations, respectively. The supernatants were discarded, and 500 μL of culture medium was added to the normal wells, 480 μL of culture medium and 20 μL of LPS (25 μg / ml) were added to the model wells. The positive drug group was treated with 480 μL of diluted 0.1 μm mol / L dexamethasone and 20 μL of LPS, and the drug wells were treated with 480 μL of diluted extract and 20 μL of LPS. Six replicates were added to each group. The cells were incubated in a 5% CO2, 37°C incubator for 24 hours.

[0056] After 24 hours, label the supernatant from each group and centrifuge at 12,000 rpm for 5 minutes at 4°C. Follow the instructions for the total nitric oxide assay kit. Read the OD value of each well at 540 nm on a microplate reader and calculate the NO content in each well according to the following formula. Calculate the NO concentration in each group using the standard curve.

[0057] Result analysis: Macrophages release cytotoxic molecules NO during immune regulation, but excessive release of NO not only kills pathogenic microorganisms but also damages normal cells in the body. Figure 2 It can be seen that both fruiting bodies and sclerotia have the effect of reducing NO concentration, but the effect of fruiting bodies is more significant than that of sclerotia, indicating that fruiting body extracts can inhibit the overexpression of NO and have an immunomodulatory effect on LPS-stimulated RAW264.7 cells.

[0058] The positive drug dexamethasone also has significant effects, but dexamethasone is a glucocorticoid drug and long-term use will cause serious side effects. The Poria fruiting body is the reproductive organ of the medicinal and edible fungus Poria cocos, which has certain safety and fewer side effects than dexamethasone.

[0059] 3. Effects on the phagocytic activity of RAW264.7 cells

[0060] 1. When the cells are in the logarithmic growth phase, collect the cells and make a single cell suspension. Adjust the cell concentration to

[0061] 1x10 5 The cells were then aliquoted into a 96-well plate at 100 μL / well. The edge wells were filled with PBS. The cells were cultured in a 5% CO2, 37°C incubator for 24 h.

[0062] After 24 hours, when the cell density reached approximately 50%, water extracts, alcohol extracts, and water and alcohol extracts of Poria cocos fruiting bodies at different stages, as well as Poria cocos sclerotia, were diluted from their initial concentrations to low (25 μg / ml) and high (100 μg / ml) concentrations, respectively. The supernatants were discarded, 100 μL of culture medium was added to the control wells, 96 μL of culture medium and 4 μL of LPS (25 μg / ml) was added to the model wells, and 100 μL of each diluted extract was added to the dosing wells. Six replicates were added to each group. The cells were incubated in a 5% CO2, 37°C incubator for 24 hours.

[0063] 3. Discard the supernatant, add 100 μL of 0.075% neutral red solution, incubate for 30 minutes, and then use pre-cooled

[0064] The cells were washed three times with PBS, 100 μL of cell lysis buffer was added, and the cells were lysed for 4 h. The OD value of each well was read at 570 nm using a microplate reader.

[0065] Cell phagocytosis rate % = OD value of the drug group / OD value of the blank group

[0066] The results are as follows Figure 3 As shown, the water extract and alcohol extract of the fruiting body are more effective than those of the sclerotium in promoting cell phagocytosis activity. Phagocytosis is one of the important physiological functions of macrophages. The stronger the phagocytic ability, the better the immune regulation effect of macrophages. Therefore, the immune regulation effect of the fruiting body is higher than that of the sclerotium.

[0067] IV. Effects on the proliferation ability of RAW264.7 cells

[0068] 1. When the cells are in the logarithmic growth phase, collect the cells and make a single cell suspension. Adjust the cell concentration to

[0069] 1x10 5 The cells were then aliquoted into a 96-well plate at 100 μL / well. The edge wells were filled with PBS. The cells were cultured in a 5% CO2, 37°C incubator for 24 h.

[0070] 2. After 24 hours, when the cell density reaches approximately 50%, dilute the initial extract to a low concentration (25 μg / ml) and a high concentration (100 μg / ml). Discard the original supernatant, add 100 μL of culture medium to the control wells, and 100 μL of the diluted extract to the dosing wells. Repeat six times per group. Incubate in a 5% CO2, 37°C incubator for 24 hours.

[0071] 3. After 24 hours of incubation, add 20 μL of CCK-8 solution to each well. After 90 minutes, read the OD value of each well at 450 nm using a microplate reader.

[0072] Calculation of cell viability: Cell viability (%) = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) * 100%.

[0073] The results are as follows Figure 4 As shown in the results, the water extract and alcohol extract of the fruiting body have a higher effect on macrophage proliferation than the sclerotium. By evaluating the activation effect of drugs on macrophages based on the proliferation ability of cells, it can be concluded that the activation effect of Poria fruiting body on macrophages is better than that of sclerotium.

[0074] This experiment explored the immunomodulatory ability of Poria cocos fruiting bodies and sclerotia by detecting cell phagocytosis activity, cell proliferation ability and NO content in cell supernatant. Figure 1-4 As shown, both aqueous and alcoholic extracts of Poria cocos fruiting bodies and sclerotia can reduce NO levels, enhance macrophage phagocytosis, and promote macrophage proliferation. Poria cocos fruiting bodies are more effective than sclerotia in reducing NO levels, enhancing macrophage phagocytosis, and promoting macrophage proliferation. Therefore, Poria cocos fruiting bodies are more effective than sclerotia in immunomodulation, providing a theoretical basis for future research on the fruiting bodies in pharmaceutical or functional food applications.

Claims

1. An application of a Poria cocos fruiting body extract in the preparation of a drug for improving immunomodulatory activity, characterized in that: The Poria cocos fruiting body extract is an ethanol extract of the early Poria cocos fruiting body, which can reduce the NO secretion of macrophages or increase the phagocytic activity of macrophages; the preparation method of the early Poria cocos fruiting body ethanol extract is as follows: 1) Use an inoculation needle to pick up the mycelium of the Poria cocos strain and inoculate it into a culture dish containing PDA medium for activation for 7 to 10 days. Use a hole puncher to make a 2 cm hole. 2 The culture block was inoculated into a tissue culture bottle containing 60 mL of PDA medium, and dark culture was performed for 7 days at a relative humidity of 70-80% and a temperature of 28°C, followed by 12-hour light-dark alternating culture for 8 weeks to produce fruiting bodies. The early stage of Poria fruiting body was 7 days after the fruiting body was formed and the period of no pore tube formation was considered; the PDA medium contained 200 g of potato, 20 g of glucose, 1 g of KH2PO4, 0.5 g of MgSO4, 10 mg of vitamin B1, 18 g of agar, and 1000 mL of water; 2) Take the early stage Poria cocos fruiting body, add 75% ethanol at a liquid-to-solid ratio of 20:1 mL / g, soak and ultrasonically extract for 50 min, filter with a 0.22 μm filter, and concentrate and lyophilize the filtrate to obtain the early stage Poria cocos fruiting body ethanol extract.

2. The use according to claim 1, characterized in that The frequency of ultrasonic extraction in step 2) is 50~70kHz.

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