Application of an edible fungus extract in the preparation of a drug for treating benign prostatic hyperplasia

Abstract

This invention discloses an edible fungus extract, its preparation method, and its application in the preparation of products that enhance immunity and lower lipids. The edible fungus extract is composed of Ganoderma lucidum water extract and enoki mushroom water extract. The research of this invention shows that the mixture of Ganoderma lucidum water extract and enoki mushroom water extract has the effect of synergistically enhancing immune regulation and lowering lipids, and can be added as a functional food ingredient, providing a basis for the research and development of high-value-added edible fungus products.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an edible fungus extract, its preparation method, and its application in the preparation of products that enhance immunity and lower lipids. Background Technology

[0002] The strength of the body's immune function directly determines the probability of developing related diseases. Specifically, a stable immune function serves as a powerful natural defense system, significantly resisting external threats and threats to health. Currently available medications either have immune-regulating effects or lipid-lowering effects; there are no medications that can regulate immunity and lower blood lipids simultaneously. Furthermore, such medications often have side effects and are not suitable for long-term use by individuals with weakened immunity or high blood lipid levels.

[0003] Edible fungi, characterized by their naturalness, safety, and nutritional value, are an excellent source of both food and medicine for the development of nutritional and functional foods. They are not only one of the most important sources of protein and nutrients in people's daily lives, but also contain a large number of active ingredients with immune-regulating, lipid-lowering, liver-protecting, gut microbiota-regulating, and anti-tumor effects, thus becoming a hot topic in the development of functional foods and health products. As the nutritional components, chemical composition, biological activity, and mechanisms of action of edible and medicinal fungi are continuously revealed, their edible, health-promoting, and medicinal value are receiving increasing attention. Due to their combined nutritional, functional, and safe characteristics, edible and medicinal fungi have a clear advantage as raw materials for functional foods. Among the approved functional foods, those using edible and medicinal fungi as raw materials account for more than 15%. Summary of the Invention

[0004] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0005] As one aspect of the present invention, the present invention provides an edible fungus extract, wherein the edible fungus extract is composed of Ganoderma lucidum water extract and Enoki mushroom water extract.

[0006] As a preferred embodiment of the edible fungus extract described in this invention, the mass ratio of Ganoderma lucidum water extract to enoki mushroom water extract is 1:0.1-10.

[0007] The present invention also provides a method for preparing the edible fungus extract: after crushing the fruiting bodies of Ganoderma lucidum and enoki mushrooms respectively, ethanol is added, ultrasonic treatment is performed, the ethanol is removed, water is added, and water bath extraction is performed to obtain Ganoderma lucidum water extract and enoki mushroom water extract, which are then freeze-dried. The Ganoderma lucidum water extract and enoki mushroom water extract are then mixed to obtain the edible fungus extract.

[0008] As a preferred embodiment of the preparation method of the edible fungus extract of the present invention: the water bath extraction has a material-to-liquid ratio of 1:5 to 15.

[0009] As a preferred embodiment of the preparation method of the edible fungus extract of the present invention, the ultrasonic treatment time is 20-40 min.

[0010] As a preferred embodiment of the preparation method of the edible fungus extract of the present invention: the water bath extraction is performed at a temperature of 100°C.

[0011] As a preferred embodiment of the preparation method of the edible fungus extract described in this invention, the mass ratio of Ganoderma lucidum water extract and enoki mushroom water extract is 1:0.1-10.

[0012] As a preferred embodiment of the preparation method of the edible fungus extract described in this invention, the mass ratio of Ganoderma lucidum water extract and enoki mushroom water extract is 1:1.

[0013] Beneficial effects of the present invention: The present invention shows that the mixture of Ganoderma lucidum water extract and Enoki mushroom water extract has the effect of synergistically improving immune regulation and lipid-lowering activity, and can be used as a functional food ingredient, providing a basis for the research and development of high value-added edible fungi products. Attached Figure Description

[0014] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0015] Figure 1 The effect of eleven edible fungi on the phagocytic rate of microspheres in Raw264.7 cells (compared to the negative control group).

[0016] Figure 2 The inhibitory effects of eleven edible fungi on lipid deposition in Raw264.7 cells were studied.

[0017] Figure 3 The effects of different formulations of Ganoderma lucidum and Enoki mushroom on the phagocytic rate of microspheres in Raw264.7 cells.

[0018] Figure 4To investigate the inhibitory effects of different formulations of Ganoderma lucidum and Enoki mushroom on lipid deposition in Raw264.7 cells. Detailed Implementation

[0019] To make the above-mentioned objectives, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to specific examples.

[0020] Example 1:

[0021] Weigh 100g each of dried Ganoderma lucidum, shiitake mushroom, maitake mushroom, enoki mushroom, king oyster mushroom, oyster mushroom, cordyceps militaris, deer antler mushroom, button mushroom, shiitake mushroom, and straw mushroom fruiting bodies (all materials are commercially available products or existing technology strains). After pulverizing, add ethanol and ultrasonically treat for 30 minutes at a material-to-liquid ratio of 1:10. After treatment, remove the ethanol and add distilled water for water extraction in a water bath at 100℃ for 2 hours each time, twice, with a material-to-liquid ratio of 1:10 each time. Combine the two filtrates and concentrate in a water bath at 100℃, observing the concentration until no more precipitation occurs. Freeze-dry in a freeze dryer to obtain the water extracts of each edible fungus.

[0022] Accurately weigh Ganoderma lucidum water extract and shiitake mushroom water extract and mix them according to different mass ratios. Place each extract sample in an Eppendorf centrifuge tube, add PBS solution under aseptic conditions to prepare a solution with a concentration of 5 mg / mL, centrifuge at 12000×g for 30 min, and use the supernatant as the mother liquor for later use. Before the experiment, add PBS solution to dilute to the corresponding concentration for the experiment.

[0023] Cytotoxicity assay:

[0024] Select well-grown logarithmic growth phase mouse RAW264.7 cells, digest them, and prepare them into 5×10⁶ cells using colorless 1640 medium containing 10% FBS. 4 Cell suspension was seeded at 100 μL / mL in 96-well plates, with 200 μL of cell suspension added to each well. The plates were incubated at 37°C and 5% CO2 for 24 h. After complete adhesion, the culture medium was removed, and different extracts prepared above were added. PBS was used as the negative control, and LPS (10 μg / mL) was used as the positive control. Each sample was tested in quadruplicates. After incubation at 37°C and 5% CO2 for 48 h, the culture medium was removed, and 25 mg / mL of Alamar Blue was weighed out. TM Then, it was prepared to a concentration of 0.0075 mg / mL for Alamar Blue. TM For the reagent, pipette 200 μL of the prepared reagent into a 96-well plate and incubate for 2-6 hours. After a significant color change in the reagent in the negative control group, measure the absorbance at 570 nm and 600 nm using a microplate reader. Calculate the cell viability using the following formula:

[0025] Survival rate (%) = [117216 × D] 570 (Sample)-80586× D600 (sample)]÷[117216×D 570 (Blank)-80586×D 600 (blank)].

[0026] Experimental results showed that neither Ganoderma lucidum water extract nor Enoki mushroom water extract was toxic and had no significant effect on cell survival rate.

[0027] RAW264.7 macrophage phagocytosis assay:

[0028] Select well-grown RAW264.7 macrophages in the logarithmic growth phase, digest them, and prepare them into 1×10⁶ cells / cells using colored DMEM medium containing 10% FBS. 6 Cells were seeded at a density of 500 μL / mL in 24-well plates. After incubation at 37°C and 5% CO2 for 4 hours, 500 μL of different prepared extracts, PBS (negative control), and LPS (10 μg / mL) (positive control) were added to each well. Three replicates were used for each sample. After incubation at 37°C and 5% CO2 for 4 hours, 1×10⁻⁶ cells / mL were added to each well. 8 200 μL of fluorescent microspheres were added and cultured in a 37°C, 5% CO2 incubator for 1 h. The culture medium was aspirated, and 1 mL of PBS was added to wash away excess fluorescent microspheres. Trypsin was added, and the mixture was digested in a 37°C, 5% CO2 incubator for 2 min. Digestion was terminated by adding DMEM medium, and the cells were transferred to centrifuge tubes and centrifuged at 1000 rpm for 3 min. The supernatant was discarded, and the cells were washed 1-2 times with 1 mL of PBS, centrifuged at 1000 rpm for 3 min, and the supernatant was discarded. Finally, the cells were resuspended in 500 μL of PBS and analyzed by flow cytometry. The phagocytic rate and phagocytic index of the above samples were calculated using the following formula.

[0029] Phagocytosis percentage (PP) = Macrophages phagocytosing fluorescent microspheres / Total number of macrophages × 100%.

[0030] Lipid-deposited cells: RAW264.7 macrophages in good growth condition were digested with trypsin and transferred to 15 mL centrifuge tubes. Digestion was stopped by adding culture medium at a ratio of 1 mL trypsin to 2 mL culture medium. The cells were centrifuged at 3000 rpm for 3 min, and the supernatant was carefully discarded. The cells were then resuspended in fresh DMEM medium and the cell concentration was adjusted to 2 × 10⁶ cells / mL using a cell counter. 5Cells were added at a concentration of 300 μL / mL to each well of a 24-well plate. After cell attachment, the supernatant was discarded for further experiments. The experiment consisted of a negative control group (Control), a model group (Ox-LDL), and a sample group, with three replicates for each group. The model and sample groups were each added with Ox-LDL to a final concentration of 40 μg / mL. The sample group was added with 500 μL of different prepared extracts. The negative control group was controlled with an equal volume of PBS. The final volume was 300 μL per well. Cells were incubated at 37°C with 5% CO2 for 24 hours, followed by Oil Red O staining. The cell supernatant was discarded, and the upper residual culture medium was gently washed away with PBS. 300 μL of Oil Red O reagent-application solution was added to each well for staining for 10-15 min, followed by washing with distilled water at room temperature for 5-20 s. After staining with reagent two counterstain for 3-5 minutes, wash with distilled water for about 30-60 seconds, and finally add water-based mounting agent to the cell surface to mount the slide. Observe and photograph using a fluorescence microscope.

[0031] Experimental results are as follows Figure 1 As shown, Figure 1 The effects of the following extracts at experimental concentrations on the phagocytic rate of microspheres in Raw264.7 cells were investigated: a negative control group (PBS), Ganoderma lucidum, Lentinus edodes, Grifola frondosa, Enoki mushroom, King oyster mushroom, Pleurotus ostreatus, Cordyceps militaris, Pleurotus eryngii, Agaricus bisporus, Pleurotus ostreatus, Strawberry mushroom, and a positive control group (10 μg / mL LPS). The concentrations of the extracts in each experimental group were 50 μg / mL. Figure 1 It can be seen that seven edible fungi, namely Ganoderma lucidum, shiitake mushroom, maitake mushroom, enoki mushroom, cordyceps militaris, king oyster mushroom, and deer antler mushroom, have a good activating effect on RAW264.7, among which Ganoderma lucidum has the best effect.

[0032] Experimental results are as follows Figure 2 As shown, Figure 2 The inhibitory effects of extracts from Ganoderma lucidum, Lentinus edodes, Grifola frondosa, Enoki mushroom, King oyster mushroom, Pleurotus ostreatus, Cordyceps militaris, Pleurotus eryngii, Agaricus bisporus, Pleurotus ostreatus, and Strawberry mushroom on lipid deposition in Raw264.7 cells were investigated at various experimental concentrations, serving as the negative control group (PBS), model group (40 μg / mL Ox-LDL), and positive control group (1 mmol / L simvastatin). The concentrations of the extracts in each experimental group were 50 μg / mL. Figure 2 It can be seen that five kinds of edible fungi, namely Ganoderma lucidum, shiitake mushroom, maitake mushroom, enoki mushroom, and cordyceps militaris, have good lipid-lowering effects, among which enoki mushroom has the best effect.

[0033] Experimental results are as follows Figure 3 As shown, Figure 3The effects of the negative control group (PBS), 100 ug / mL Ganoderma lucidum water extract, different Ganoderma lucidum and enoki mushroom formulations, 100 ug / mL enoki mushroom water extract, and the positive control group (10 ug / mL LPS) on the microsphere phagocytosis rate of Raw264.7 cells at the experimental concentrations were investigated. The concentrations of the Ganoderma lucidum and enoki mushroom formulations were 12.5 ug / mL (Ganoderma lucidum) + 87.5 ug / mL (enoki mushroom), 25 ug / mL (Ganoderma lucidum) + 75 ug / mL (enoki mushroom), 50 ug / mL (Ganoderma lucidum) + 50 ug / mL (enoki mushroom), 75 ug / mL (Ganoderma lucidum) + 25 ug / mL (enoki mushroom), and 87.5 ug / mL (Ganoderma lucidum) + 12.5 ug / mL (enoki mushroom). The three groups of 50ug / mL (Ganoderma lucidum) + 50ug / mL (Flammulina velutipes), 75ug / mL (Ganoderma lucidum) + 25ug / mL (Flammulina velutipes), and 87.5ug / mL (Ganoderma lucidum) + 12.5ug / mL (Flammulina velutipes) all showed better phagocytic stimulation of RAW264.7 microspheres than the 100ug / mL Ganoderma lucidum water extract.

[0034] Experimental results are as follows Figure 4 As shown, Figure 4 The study investigated the inhibitory effects of the following formulations on lipid deposition in Raw264.7 cells at experimental concentrations: negative control group (PBS), model group (40 μg / mL Ox-LDL), 100 μg / mL Ganoderma lucidum water extract, different formulations of Ganoderma lucidum and Enoki mushroom, 100 μg / mL Enoki mushroom water extract, and positive control group (10 μg / mL PBS). The concentrations of the Ganoderma lucidum and Enoki mushroom formulations were 12.5 μg / mL (Ganoderma lucidum) + 87.5 μg / mL (Enoki mushroom), 25 μg / mL (Ganoderma lucidum) + 75 μg / mL (Enoki mushroom), 50 μg / mL (Ganoderma lucidum) + 50 μg / mL (Enoki mushroom), 75 μg / mL (Ganoderma lucidum) + 25 μg / mL (Enoki mushroom), and 87.5 μg / mL (Ganoderma lucidum) + 12.5 μg / mL (Enoki mushroom). Figure 4 It was observed that the three groups of 12.5ug / mL (Ganoderma lucidum) + 87.5ug / mL (Flammulina velutipes), 25ug / mL (Ganoderma lucidum) + 75ug / mL (Flammulina velutipes), and 50ug / mL (Ganoderma lucidum) + 50ug / mL (Flammulina velutipes) had better lipid-lowering effects, all of which were better than the 100ug / mL Flammulina velutipes water extract.

[0035] This study used edible fungi fruiting bodies as the main raw material to compare the immunomodulatory and lipid-lowering effects of water extracts from eleven kinds of edible fungi fruiting bodies and different proportions of Ganoderma lucidum and enoki mushroom water extracts. The results showed that the formula with a mass ratio of Ganoderma lucidum water extract to enoki mushroom water extract of 1:1 has the effect of synergistically improving immunomodulatory and lipid-lowering activities. It can be used as a functional food ingredient and provides a basis for the research and development of high-value-added edible fungi products.

[0036] Benign prostatic hyperplasia experiment:

[0037] This experiment consisted of four sample groups: Sample 1, Sample 2, Sample 3, and Sample 4. Sample 1 was a Ganoderma lucidum water extract group with concentrations of 12.5, 25, 50, and 100 μg / mL; Sample 2 was a Flammulina velutipes water extract group with concentrations of 12.5, 25, 50, and 100 μg / mL; Sample 3 was a 3:1 mixture group, where Ganoderma lucidum and Flammulina velutipes water extracts were mixed at a mass ratio of 3:1, with total concentrations of 12.5, 25, 50, and 100 μg / mL; Sample 4 was a 1:1 mixture group, where Ganoderma lucidum and Flammulina velutipes water extracts were mixed at a mass ratio of 1:1, with total concentrations of 12.5, 25, 50, and 100 μg / mL; and Sample 5 was a 1:3 mixture group, where Ganoderma lucidum and Flammulina velutipes water extracts were mixed at a mass ratio of 1:3, with total concentrations of 12.5, 25, 50, and 100 μg / mL.

[0038] Prepare a 100mM phosphate buffer solution by dissolving 0.26g KH₂PO₄ and 1.41g K₂HPO₄ in ultrapure water and bringing the volume to 100mL. Prepare a MgCl₂ solution by dissolving 47.5mg MgCl₂ in 20mL of ultrapure water and bringing the volume to 50mL. Prepare a finasteride solution by dissolving 7.45mg finasteride in 75% methanol and bringing the volume to 2mL. Prepare a reduced coenzyme II solution by dissolving 1mg NADPH in 200μL of MgCl₂ solution. Prepare a testosterone solution by dissolving 1.44mg testosterone in 1mL of 60% methanol. Detect the inhibition rate of 5α-reductase in different samples using high-performance liquid chromatography (HPLC). The reaction system was carried out in 1.5 mL centrifuge tubes with a total volume of 200 μL, consisting of 160 μL of liver microsomal solution, 10 μL of the test sample, 10 μL of testosterone solution, 20 μL of reduced coenzyme II solution, and 10 μL of the test sample. After vortexing and mixing, the mixture was placed in a 37°C water bath for 80 min, and then methanol solution was added to terminate the reaction. After centrifugation at 12000 r / min for 30 min, the supernatant was collected, filtered through a 0.22 μm membrane, and analyzed by high performance liquid chromatography. The inhibitory activity of 5α-reductase was calculated by the change in the concentration of the substrate testosterone.

[0039]

[0040] The Cl values ​​were calculated using CompuSyn software. Cl values ​​of 0.1–0.3 indicated a strong synergistic effect, while those of 0.3–0.7 showed a synergistic effect. Statistical analysis results are shown in Table 1. The experimental results show that, under conditions of simultaneous addition of 5α-reductase and the sample, higher values ​​indicate a better inhibitory effect of the sample on 5α-reductase. When Ganoderma lucidum water extract and Fenotaphragmus violaceus water extract were combined at a mass ratio of 3:1, the Cl value was 0.156, exhibiting a strong synergistic effect; while when the mass ratios of Ganoderma lucidum water extract and Fenotaphragmus violaceus water extract were combined at 1:1 and 1:3, the Cl values ​​were 0.420 and 0.683, respectively, showing a certain degree of synergistic effect.

[0041] Table 1 Synergistic effect of different water extracts of Ganoderma lucidum and enoki mushroom on the inhibition of 5α-reductase

[0042]

[0043]

[0044] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. The application of an edible fungus extract in the preparation of a drug for treating benign prostatic hyperplasia, characterized in that: The edible fungus extract is composed of Ganoderma lucidum water extract and enoki mushroom water extract; the mass ratio of Ganoderma lucidum water extract to enoki mushroom water extract is 3:1; the edible fungus extract treats benign prostatic hyperplasia by inhibiting 5α-reductase activity. The preparation methods for the Ganoderma lucidum water extract and the enoki mushroom water extract are as follows: 100g each of dried Ganoderma lucidum fruiting bodies and enoki mushroom fruiting bodies are pulverized and then subjected to ultrasonic treatment with ethanol for 30min, with a material-to-liquid ratio of 1:

10. After treatment, the ethanol is removed, and distilled water is added for water extraction in a water bath at 100℃ for 2h each time. The water extraction is performed twice, and the two filtrates are combined. The filtrates are concentrated in a water bath at 100℃ and then freeze-dried in a freeze dryer to obtain the Ganoderma lucidum water extract and the enoki mushroom water extract, respectively.

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