A preparation of Ganoderma lucidum and Pleurotus ostreatus with multiple effects of protecting neurons and lowering lipids, its preparation method and application

Abstract

This invention discloses a Ganoderma lucidum and Pleurotus ostreatus preparation with multiple effects of protecting neurons and lowering lipids, its preparation method and application. The Ganoderma lucidum and Pleurotus ostreatus preparation is composed of Ganoderma lucidum alcohol extract and Pleurotus ostreatus water extract. The Ganoderma lucidum alcohol extract and Pleurotus ostreatus water extract have a synergistic effect of protecting neurons in a mass ratio of 1:4 to 4:1. The Ganoderma lucidum alcohol extract and Pleurotus ostreatus water extract have a synergistic effect of lowering lipids in a mass ratio of 1:4 to 1:1.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a ganoderma lucidum and pholota adiposa preparation with multiple effects of protecting neurons and reducing fat, and a preparation method and application thereof. BACKGROUND

[0002] With the continuous improvement of medical technology and living standards, human life is also getting longer and longer, and the elderly diseases represented by Alzheimer's disease are becoming more and more a hot topic of world life science research; epidemiological surveys show that the prevalence rate of senile dementia among people over 65 years old worldwide is 13%, and 45% of people over 85 years old have senile dementia, and the prevalence rate is rising with the acceleration of population aging, which brings great threat to the life and health of the elderly; more surprisingly, the World Health Organization predicts that the incidence of AD may rise by more than 3 times in 50 years, and neurodegenerative diseases will surpass cancer to become the second leading cause of death in the world population by 2040. At the same time, the report of China's residents' diet and chronic disease status (2020) shows that the overall prevalence rate of hyperlipidemia among residents aged 18 and above in China is as high as 35.6%, causing serious disease burden; hyperlipidemia refers to the excessive content of lipids in the blood, including cholesterol, triglycerides, etc.; hyperlipidemia is an important risk factor for hypertension, diabetes, coronary heart disease, and stroke, and long-term hyperlipidemia can lead to atherosclerosis, increasing the incidence and mortality of cardiovascular diseases; although there are currently prescription drugs for reducing blood lipids and protecting neurons, long-term use of prescription drugs can easily induce side effects, and functional foods and health products with multiple effects of protecting neurons and reducing fat have not been seen on the market, so products with such multiple effects have great market prospects. SUMMARY

[0003] This section aims to summarize some aspects of the embodiments of the present application and briefly introduce some preferred embodiments.

[0004] As one aspect of the present application, the present application provides a ganoderma lucidum and pholota adiposa preparation with multiple effects of protecting neurons and reducing fat, wherein: the ganoderma lucidum and pholota adiposa preparation is composed of ganoderma lucidum ethanol extract and pholota adiposa water extract, and the mass ratio of the ganoderma lucidum ethanol extract to the pholota adiposa water extract is 1:4-4:1; the ganoderma lucidum ethanol extract and the pholota adiposa water extract synergistically act on protecting neurons and reducing fat.

[0005] As a preferred scheme of the ganoderma lucidum and pholota adiposa preparation with multiple effects of protecting neurons and reducing fat, the mass ratio of the ganoderma lucidum ethanol extract to the pholota adiposa water extract is 1:4-1:1.

[0006] As one of the aspects of the present application, the present application provides the preparation method of the Ganoderma lucidum and Pholiota nameko preparation with the multiple effects of protecting neurons and reducing fat, wherein: the preparation method of the Ganoderma lucidum ethanol extract is that Ganoderma lucidum fruiting bodies are extracted with 100% ethanol; and the preparation method of the Pholiota nameko water extract is that Pholiota nameko fruiting bodies are extracted with 100 ℃ boiling water bath.

[0007] As a preferred scheme of the preparation method of the Ganoderma lucidum and Pholiota nameko preparation with the multiple effects of protecting neurons and reducing fat, the preparation method of the Pholiota nameko water extract is that the Pholiota nameko fruiting bodies are crushed and then added into water, the solid-liquid ratio is 1:10, the extraction is carried out in a 100 ℃ boiling water bath for 2 h, the extraction is carried out twice, the filtrates of the two times of extraction are combined, the filter residue is discarded, the filtrate is concentrated and dried to obtain the Pholiota nameko water extract.

[0008] As a preferred scheme of the preparation method of the Ganoderma lucidum and Pholiota nameko preparation with the multiple effects of protecting neurons and reducing fat, the solid-liquid ratio is 1:10, the ultrasonic treatment is carried out for 30 min, the extraction is carried out twice, the filtrates of the two times of extraction are combined, the filter residue is discarded, the filtrate is concentrated and dried to obtain the Ganoderma lucidum ethanol extract.

[0009] As a preferred scheme of the preparation method of the Ganoderma lucidum and Pholiota nameko preparation with the multiple effects of protecting neurons and reducing fat, the ultrasonic power of the ultrasonic treatment is 500-700 W.

[0010] As a preferred scheme of the preparation method of the Ganoderma lucidum and Pholiota nameko preparation with the multiple effects of protecting neurons and reducing fat, the filtrate is obtained by filtering with a filter membrane with a pore size of 0.22 μm.

[0011] The present application also provides the application of the Ganoderma lucidum and Pholiota nameko preparation in the preparation of food with the multiple effects of protecting neurons and reducing fat.

[0012] The Ganoderma lucidum ethanol extract and the Pholiota nameko water extract have the synergistic effect of protecting neurons in the range of 1:4-4:1 in mass ratio; and the Ganoderma lucidum ethanol extract and the Pholiota nameko water extract have the synergistic effect of reducing fat in the range of 1:4-1:1 in mass ratio.

[0013] The fat reduction includes reducing the content of low-density lipoprotein.

[0014] Beneficial effects of this invention: This study found that Ganoderma lucidum alcohol extract and Oyster mushroom water extract have a synergistic protective effect on neuronal cells. When Oyster mushroom water extract and Ganoderma lucidum alcohol extract are combined at a mass ratio of 4:1 and 1:1, the CI values ​​are 0.573 and 0.321, respectively, indicating a certain synergistic effect. However, when combined at a mass ratio of 4:1, the CI value is 0.220, showing a strong synergistic effect. Ganoderma lucidum alcohol extract and Oyster mushroom water extract also have a synergistic effect in lowering low-density lipoprotein (LDL). When combined at a mass ratio of 1:1, the CI is 0.598, showing a certain synergistic effect. When combined at a mass ratio of 4:1, the CI is 0.275, showing a strong synergistic effect. Attached Figure Description

[0015] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. 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:

[0016] Figure 1 The inhibitory effect of each group of samples on Ox-LDL-induced lipid accumulation in RAW264.7 was investigated. Detailed Implementation

[0017] 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.

[0018] Example 1:

[0019] Preparation of Ganoderma lucidum alcohol extract and water extract:

[0020] Weigh 100g of dried Pleurotus ostreatus fruiting bodies (Shenxiu No. 1), pulverize them into 1000-mesh ultrafine powder, and then perform water extraction in a 100℃ water bath for 2 hours each time, twice. The material-to-liquid ratio is 1:10 each time. Filter the solution through a 0.22μm millpore membrane, combine the two filtrates, discard the filter residue, and concentrate the filtrate in a 100℃ water bath to a specific gravity of 1.5 (observe the concentration until no more precipitation occurs). Pack the solution into a self-sealing bag and freeze dry it in a freeze dryer to obtain the Pleurotus ostreatus water extract.

[0021] Weigh 100g of dried Ganoderma lucidum (Hu Nong No. 1) fruiting bodies to constant weight, pulverize them into 1000 mesh ultrafine powder, and extract them twice with 100% ethanol and ultrasonic treatment at 600W power for 30 minutes each time, with a material-to-liquid ratio of 1:10 each time. Filter the mixture through a 0.22μm pore size membrane (millpore), combine the two filtrates, discard the filter residue, and concentrate the filtrate using a rotary evaporator until there is no ethanol odor. Spread the concentrate evenly in a sterilized bag and place it in a 60℃ oven until the moisture is dried to obtain the Ganoderma lucidum ethanol extract.

[0022] Experimental sample preparation:

[0023] Sample group 1 was the water extract group of Pleurotus ostreatus, with sample concentrations of Pleurotus ostreatus water extract of 12.5, 25, 50 and 100 μg / mL, respectively;

[0024] Sample group 2 was the Ganoderma lucidum alcohol extract group, with Ganoderma lucidum alcohol extract sample concentrations of 12.5, 25, 50 and 100 μg / mL, respectively;

[0025] Sample group 3 was a 4:1 mixture, in which water extract of Pleurotus ostreatus and alcohol extract of Ganoderma lucidum were mixed at a mass ratio of 4:1, with sample concentrations of 12.5, 25, 50 and 100 μg / mL, respectively.

[0026] Sample group 4 was a 1:1 mixture group, in which water extract of Pleurotus ostreatus and alcohol extract of Ganoderma lucidum were mixed at a mass ratio of 1:1, with sample concentrations of 12.5, 25, 50 and 100 μg / mL, respectively.

[0027] Sample group 5 was a 1:4 mixture group, in which water extract of Pleurotus ostreatus and alcohol extract of Ganoderma lucidum were mixed at a mass ratio of 1:4, and the sample concentrations were 12.5, 25, 50 and 100 μg / mL.

[0028] Table 1 Composition of different experimental samples

[0029] Sample No. Component 1 Water extract of P. cubensis 2 Ethanol extract of G. lucidum 3 Water extract of P. cubensis: ethanol extract of G. lucidum = 4:1 4 Water extract of P. cubensis: ethanol extract of G. lucidum = 1:1 5 Water extract of P. cubensis: ethanol extract of G. lucidum = 1:4

[0030] Activity experiment:

[0031] Neuron protection experiment:

[0032] The experiment included a negative control group, a model group (containing only 6 mM scopolamine), and five sample concentration gradient groups (12.5, 25, 50, and 100 μg / mL), with three replicates for each group. Highly differentiated neural cell line PC12, in good logarithmic growth phase, was digested and suspended to obtain a concentration of 1 × 10⁻⁶ μg / mL. 5 Single-cell suspensions of 10 cells / mL were prepared at a ratio of 1 × 10⁶ cells / well. 4A certain number of cells (100 μL cell suspension) were seeded into 96-well plates. Cells were cultured in a cell culture incubator for 24 hours until complete adhesion. The culture medium was then removed. Except for the negative control group, the model group and each sample group were treated with 6 mM scopolamine solution for 6 hours. After complete cell adhesion, the cells were treated with sample medium for 24 hours. The negative control group received colorless RPMI-1640 medium containing 0.5% DMSO. After 24 hours, the cells were cultured using Alamar Blue. TM The experiment measured cell proliferation data for each group.

[0033]

[0034] Table 2. Effects of different samples on neuronal proliferation caused by scopolamine-induced neuronal damage.

[0035]

[0036]

[0037] To facilitate data analysis, the above alamarBlue... TM Cell proliferation data from each sample group in the experiment were converted into protective effects against scopolamine-induced neuronal damage. CompuSyn software was used to calculate the CI values. CI values ​​of 0.1–0.3 indicated a strong synergistic effect, while 0.3–0.7 showed a synergistic effect. Statistical analysis results are shown in Table 3. The experimental results show that, under conditions of simultaneous addition of scopolamine and the sample, higher values ​​indicate better repair effects on scopolamine-induced cell damage. Specifically, when the water extract of *Pleurotus ostreatus* and the alcohol extract of *Ganoderma lucidum* were combined at mass ratios of 4:1 and 1:1, the CI values ​​were 0.573 and 0.321, respectively, indicating a certain synergistic effect. However, when the water extract of *Pleurotus ostreatus* and the alcohol extract of *Ganoderma lucidum* were combined at a mass ratio of 4:1, the CI value was 0.220, demonstrating a strong synergistic effect.

[0038]

[0039] Table 3 Synergistic effects of different ratios of Ganoderma lucidum water extract and alcohol extract

[0040]

[0041] Lipid-lowering experiment:

[0042] The experiment consisted of a negative control group, a model group (containing only oxidized low-density lipoprotein Ox-LDL), and five sample concentration gradient groups (12.5, 25, 50, and 100 μg / mL), with three replicates per group. RAW264.7 macrophages in good logarithmic growth phase were digested and suspended to obtain a concentration of 2 × 10⁻⁶ μg / mL. 5Single-cell suspensions of 300 μL / mL were added to each well of a 24-well plate. After cell attachment, the supernatant was discarded for the experiment. Ox-LDL was added to the model group and sample group to a final concentration of 40 μg / mL. Different prepared samples were added to the sample group. An equal volume of PBS was added as a control to the negative control group. The final volume was 300 μL per well. The cells were incubated at 37°C with 5% CO2 for 24 hours. The cell supernatant was discarded, and the upper residual culture medium was gently washed away with PBS. 300 μL of Oil Red O reagent was added to each well for staining for 10-15 min, followed by washing with distilled water at room temperature for 5-20 s. Counterstaining with reagent II solution was performed for 3-5 min, followed by washing with distilled water for approximately 30-60 s. Finally, an aqueous mounting medium was added to the cell surface for mounting, and the cells were observed and counted under a microscope.

[0043]

[0044] CI values ​​were calculated using CompuSyn software. CI 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 4. The experimental results show that, under conditions of simultaneous addition of Ox-LDL and the sample, higher values ​​indicate better lipid-lowering effects. Specifically, when the water extract of *Pleurotus ostreatus* and the alcohol extract of *Ganoderma lucidum* were combined at a mass ratio of 1:1, the CI was 0.598, demonstrating a certain synergistic effect. However, when the water extract of *Pleurotus ostreatus* and the alcohol extract of *Ganoderma lucidum* were combined at a mass ratio of 4:1, the CI was 0.275, demonstrating a strong synergistic effect.

[0045] Table 4. Synergistic effect of different samples on inhibiting Ox-LDL-induced lipid accumulation in RAW264.7 macrophages

[0046]

[0047]

[0048] 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. Ganoderma and Hypsizygus marmoreus preparation having multiple effects of protecting neurons and reducing lipids, characterized by: The Ganoderma lucidum and Pholiota adiposa preparation is composed of Ganoderma lucidum alcohol extract and Pholiota adiposa water extract, and the mass ratio of the Ganoderma lucidum alcohol extract to the Pholiota adiposa water extract is 1:4 to 4:1; the Ganoderma lucidum alcohol extract and the Pholiota adiposa water extract synergistically act on neuron protection and lipid reduction; the preparation method of the Pholiota adiposa water extract is that Pholiota adiposa fruiting bodies are crushed and then added into water, the solid-liquid ratio is 1:10, extraction is carried out in a boiling water bath at 100 DEG C for 2 h, extraction is carried out twice, the filtrates of the two times of extraction are combined, the filter residue is discarded, the filtrate is concentrated and dried to obtain the Pholiota adiposa water extract; the preparation method of the Ganoderma lucidum alcohol extract is that Ganoderma lucidum fruiting bodies are crushed and then added into ethanol, the solid-liquid ratio is 1:10, ultrasonic treatment is carried out for 30 min, extraction is carried out twice, the filtrates of the two times of extraction are combined, the filter residue is discarded, the filtrate is concentrated and dried to obtain the Ganoderma lucidum alcohol extract. ​ 2. Ganoderma and Hypsizygus marmoreus preparation having multiple effects of protecting neurons and reducing lipids according to claim 1, characterized by: The mass ratio of the Ganoderma lucidum alcohol extract to the Pholiota adiposa water extract is 1:4 to 1:

1.

3. The method of preparing Ganoderma and Hypsizygus marmoreus preparation having multiple effects of protecting neurons and reducing lipids according to claim 1 or 2, characterized in that: The ultrasonic treatment is carried out at an ultrasonic power of 500 to 700 W.

4. The method of preparing Ganoderma and Hypsizygus marmoreus preparation having multiple effects of protecting neurons and reducing lipids according to claim 1 or 2, characterized in that: The filtrate is obtained by filtration with a filter membrane with a pore size of 0.22 µm. The filtrate is obtained by filtration with a filter membrane with a pore size of 0.22 µm.

Images