TWI733005B - Use of a ganoderma extract in manufacturing a composition for increasing bioenergetic healthy index and promoting cellular differentiation - Google Patents

Abstract

A use of a ganoderma extract in manufacturing a composition for increasing the bioenergetics healthy index and promoting cellular differentiation. This composition, which includes the ganoderma extract, is able to improve the synaptic growth of nerve cells.

Description

Ganoderma lucidum extract is used to prepare and improve the bioenergy health index and promote cell progress Use of differentiated composition

The present invention relates to a composition for improving the bioenergy health index and promoting cell differentiation, in particular the use of a Ganoderma lucidum extract for preparing a composition for improving the bioenergy health index and promoting cell differentiation.

Mitochondria is the main place for oxidative phosphorylation and synthesis of adenosine triphosphate (ATP) in cells. Since adenosine triphosphate is the energy source for cell activities, mitochondria are also called "cell energy factories". In addition to providing energy for cells, mitochondria are also involved in processes such as cell differentiation, cell information transmission and cell apoptosis, and have the ability to regulate cell growth cycles. When the mitochondria are in a better state of health, the cells have a higher bioenergy health index, which means that the mitochondria can provide sufficient energy to the cells, and the mitochondria have better flexibility in regulating energy supply. In this way, when cells with a higher bioenergy health index are under stress, mitochondria can provide energy according to the needs of the cells, so that the cells have sufficient energy to cope with the stress.

However, some of the by-products produced by the mitochondria during the oxidative phosphorylation reaction are harmful to the inner membrane of the mitochondria. The pressure generated by the long-term accumulation of these harmful substances will adversely affect the energy supply of mitochondria and the elasticity of regulating energy supply, which will reduce the cell's bio-energy health index. What's more, the severely damaged mitochondrial inner membrane will trigger the disintegration of mitochondria, which in turn triggers cell apoptosis. Therefore, how to improve the bioenergy health of cells Indexes have become an important subject.

The present invention provides a use of Ganoderma lucidum extract for preparing a composition that improves the bioenergy health index and promotes cell differentiation. By administering this composition, the bioenergy health index can be increased, and sufficient cells can be obtained during differentiation. Energy supply.

An embodiment of the present invention discloses the use of a Ganoderma lucidum extract for preparing a composition for improving the bioenergy health index and promoting cell differentiation.

Another embodiment of the present invention discloses the use of a Ganoderma lucidum extract for preparing a composition for promoting cell differentiation.

In some embodiments of the present invention, the aforementioned cells are nerve cells, and the Ganoderma lucidum extract can promote the synaptic growth of nerve cells.

In some embodiments of the present invention, the concentration of Ganoderma lucidum extract is 100 to 300 micrograms per milliliter (μg/ml).

In some embodiments of the present invention, the effective dose of Ganoderma lucidum extract is 1.081 to 3.244 milligrams (mg).

The use of the Ganoderma lucidum extract disclosed in the present invention for the preparation of a composition that improves the bioenergy health index and promotes cell differentiation. When the composition containing the Ganoderma lucidum extract is provided to cells under stress, the Ganoderma lucidum extract Substances can improve the bioenergy health index and ensure that cells can get sufficient energy supply for differentiation. In this way, mitochondria have the ability to provide energy according to the needs of the cells, so that the cells have sufficient energy to deal with stress and carry out various life activities.

The above description of the disclosure and the following description of the embodiments are used to demonstrate and explain the spirit and principle of the present invention, and to provide a further explanation of the scope of the patent application of the present invention.

Figure 1 is a schematic diagram of the amount of free radicals produced by the rat adrenal medulla chromaffin cells of Examples A1 to A4, Comparative Example A and Control Example A under oxidative pressure.

2 is a schematic diagram of the measurement results of the rat adrenal medulla chromaffin cells of Examples A2 to A4, Comparative Example A and Control Example A by a hippocampal bioenergy meter.

Fig. 3 is a schematic diagram of the basic oxygen consumption of mitochondria in the rat adrenal medulla chromaffin cells of Examples A2 to A4, Comparative Example A and Control Example A.

Figure 4 is a schematic diagram of the oxygen consumption of synthetic nematode triphosphate in the mitochondria of rat adrenal medulla chromaffin cells in Examples A2 to A4, Comparative Example A and Control Example A.

Fig. 5 is a schematic diagram of the oxygen consumption of mitochondria in the rat adrenal medulla chromaffin cells of Examples A2 to A4, Comparative Example A and Control Example A to overcome hydrogen ion leakage.

Fig. 6 is a schematic diagram of the maximum oxygen consumption capacity of mitochondria in the rat adrenal medulla chromaffin cells of Examples A2 to A4, Comparative Example A and Control Example A.

Fig. 7 is a schematic diagram of pre-existing oxygen consumption capacity of mitochondria in rat adrenal medulla chromaffin cells of Examples A2 to A4, Comparative Example A and Control Example A.

Fig. 8 is a schematic diagram of the mediator efficiency of mitochondria in rat adrenal medulla chromaffin cells of Examples A2 to A4, Comparative Example A and Control Example A.

Fig. 9 is a schematic diagram showing the oxygen consumption of non-mitochondria in the rat adrenal medulla chromaffin cells of Examples A2 to A4, Comparative Example A and Control Example A.

Fig. 10 is a schematic diagram of the bioenergy health index of the rat adrenal medulla chromaffin cells of Examples A2 to A4, Comparative Example A and Control Example A.

Figure 11 is a schematic diagram of the differentiation index of rat adrenal medulla chromaffin cells treated with Ganoderma lucidum extract.

Figures 12 to 14 are electron micrographs of rat adrenal medulla chromaffin cells treated with different treatments.

The detailed features and advantages of the present invention will be described in detail in the following embodiments. The content is sufficient to enable anyone familiar with the relevant art to understand the technical content of the present invention and implement it accordingly, and according to the content disclosed in this specification, the scope of the patent application and the drawings, anyone familiar with the relevant art can easily understand the related purpose of the present invention And advantages. The following examples further illustrate the viewpoints of the present invention in detail, but do not limit the scope of the present invention by any viewpoint.

The Ganoderma lucidum extract used in the present invention is extracted from the fruit body or mycelium of Ganoderma lucidum. The Ganoderma lucidum extract contains triterpenoids and Ganoderma lucidum polysaccharide (triterpenoid: Ganoderma lucidum polysaccharide) in a weight ratio of 1:2.5 to 1:3.0. Triterpenoids include Ganoderic acid, Ganoderenic acid, etc. The method of obtaining the Ganoderma lucidum extract used in the present invention is, for example, carbon dioxide or pure water is used as a supercritical fluid to extract Ganoderma lucidum, or pure water, methanol, ethanol, acetone, ethyl acetate, chlorinated with a concentration of 0.1 to 5% by weight Sodium aqueous solution, potassium chloride aqueous solution with a weight percentage of 0.1 to 5%, calcium chloride aqueous solution with a weight percentage of 0.1 to 5%, magnesium chloride aqueous solution with a weight percentage of 0.1 to 5%, and a weight percentage of 0.1 to 5 % Sodium chloride ethanol solution, 0.1 to 5% by weight potassium chloride ethanol solution, 0.1 to 5% by weight calcium chloride ethanol solution or 0.1 to 5% by weight magnesium chloride ethanol The solution is used as a solvent to extract the Ganoderma lucidum to obtain the initial extract of Ganoderma lucidum. Then, the Ganoderma lucidum primary extract is filtered and purified to obtain the Ganoderma lucidum extract used in the present invention. Phyllanthus emblica extract can be dried by spray drying, vacuum drying or freeze-drying to obtain Ganoderma lucidum extract powder that is easy to store.

In the present invention, when a composition containing Ganoderma lucidum extract is given to cells under stress, the Ganoderma lucidum extract at a concentration of 100 to 300 micrograms per milliliter (μg/ml) can improve the bioenergy health index and ensure the cells Sufficient energy supply is available for differentiation. In this way, mitochondria have the ability to provide energy according to the needs of the cells, so that the cells have sufficient energy to deal with stress and carry out various life activities.

In detail, under pressure, the mitochondria in cells treated with Ganoderma lucidum extract have increased the amount of mitochondria synthesized by the oxidative phosphorylation reaction, and the mitochondria’s maximum oxygen consumption capacity (Maximal Respiration) , Oxygen consumption of synthetic nematode triphosphate (ATP Production), Spare Respiratory Capacity, and Coupling Efficiency (Coupling Efficiency) are all increased, while the Proton Leakage of mitochondria decreases.

Furthermore, the calculation method of the Bioenergetic Healthy Index (BHI) value of the cell is as follows: BHI=[oxygen consumption of synthetic triphosphate x pre-stored oxygen consumption]/[oxygen consumption to overcome hydrogen ion leakage x Non-mitochondrial oxygen consumption]. The bio-energy health index of the cells treated with Ganoderma lucidum extract is improved, so that the cell's ability to respond to stress is also improved.

The method of supplying the Ganoderma lucidum extract to the cells is, for example, oral ingestion of a composition or preparation containing the Ganoderma lucidum extract. When the Ganoderma lucidum extract is administered to the cells orally, the effective human dose of the Ganoderma lucidum extract ranges from 1.081 grams (g) to 3.244 grams. The effective dose here is calculated according to the conversion formula of the effective dose of the cell experiment and the kilogram of the human body. The conversion formula is as follows: human effective dose = effective dose of cell experiment × weight of mice × conversion coefficient × kilograms of human body. Among them, suppose that the weight of the mouse is 20 grams, the kilogram of the human body is 60 kilograms, and the conversion coefficient is 9.01. The conversion coefficient is obtained from the conversion coefficient table of the dose per kg body weight of the animal and the human body.

To facilitate the oral intake of Ganoderma lucidum extract, the composition containing the Ganoderma lucidum extract can be made into, for example, liquid, solid, granular, powder, paste or gel preparations.

In some embodiments of the present invention, the preparation may only contain Ganoderma lucidum extract. In another part of the embodiments of the present invention, without affecting the effects and objectives that the present invention can produce, the preparation may also contain other ingredients or additives, such as carriers, diluents, adjuvants, and excipients. Shape agent or taste agent. The excipient can make the preparation convenient and practical, and the flavoring agent can enhance the flavor of the preparation.

This preparation can be a hard capsule, so that it contains Ganoderma lucidum extract in the form of coated dry powder Take the composition. The preparation can also be a soft capsule to coat the composition containing the Ganoderma lucidum extract in the form of a solution, suspension, paste, powder or granules.

The fats and oils used in the soft capsule to dissolve or disperse the composition containing the Ganoderma lucidum extract are, for example, calyx pear oil, almond oil, linseed oil, cumin oil, perilla oil, olive oil, olive squalene, and orange oil. , Orange roughy oil, sesame oil, garlic oil, cocoa butter, pumpkin seed oil, chamomile oil, carrot oil, courgette oil, tallow fatty acid, macadamia stone oil, bilberry oil, brown rice germ oil, rice oil , Wheat germ oil, safflower oil, shea oil, liquid shea oil, perilla oil, soybean oil, evening primrose oil, camellia oil, corn oil, rapeseed oil, saw palmetto extract oil, Coix oil, peach kernel oil, parsley oil, castor oil, sunflower oil, grape seed oil, borage oil, Australian walnut oil, meadowfoam oil, cottonseed oil, peanut oil, tortoise oil, mink oil, Egg butter, fish oil, palm oil, palm kernel oil, wood wax, coconut oil, long-chain/medium-chain/short-chain fatty acid triglycerides, diglycerides, tallow, lard, squalene, squalane , Pristane, and hydrides of these oils and fats. Among them, borage oil and evening primrose oil contain a large amount of Gamma-Linolenic Acid (GLA). Gamma-Linolenic Acid is an essential fatty acid for the human body. It has moisturizing properties, promotes cell regeneration, and promotes brown fat activity. Degree to promote the function of fat burning.

Excipients are, for example, wheat starch, rice starch, corn starch, potato starch, dextrin, cyclodextrin and other starches; crystalline cellulose; lactose, glucose, granulated sugar, reduced maltose, maltose, fructo-oligosaccharide, emulsified oligosaccharide Other sugars; sugar alcohols such as sorbitol, erythritol, xylitol, lactitol, and mannitol.

Examples of flavoring agents include various fruit juice extracts such as longan extract, lychee extract and grapefruit extract; various fruit juices such as apple juice, orange juice, lemon juice, etc.; various flavors such as peach flavor, plum flavor, and yogurt flavor; acetosulfonamide Various sweeteners such as potassium acid, sucralose, erythritol, oligosaccharides, mannose, xylitol, and isomerized sugars; various sour agents such as citric acid, malic acid, tartaric acid, gluconic acid, etc.; green tea, oolong tea, Banaba tea, Various tea ingredients such as eucommia tea, Tieguanyin tea, coix tea, horse chestnut tea, water chestnut tea, kelp tea, etc.

In addition, coloring agents, preservatives, tackifiers, binders, disintegrating agents, dispersants, stabilizers, gelling agents, antioxidants, surfactants, preservatives, pH adjusters, etc. meet the requirements of government agencies. The substance can also be added to the preparations containing Ganoderma lucidum extract in accordance with the dosage standards prescribed by the government unit and the requirements of processing and production.

The following examples and comparative examples of the present invention are used to illustrate the use of the Ganoderma lucidum extract disclosed in the present invention to increase the bioenergy health index and promote cell differentiation under stress conditions, and to conduct experimental tests to illustrate the Ganoderma lucidum extract disclosed in the present invention It has the effect of improving the bioenergy health index and promoting the differentiation of cells in a stressful environment.

The method of obtaining the Ganoderma lucidum extract used in each embodiment of the present invention is as follows.

First, the fruit bodies of Ganoderma lucidum (Ganoderma lucidum, Antler) are ground into powdered Ganoderma lucidum powder. The grinding method can be a round of grinding with a grinder for 10 seconds and then a pause of 30 seconds, and a total of three rounds of grinding are performed. In this way, it is avoided that the high temperature generated by continuous grinding damages the components contained in the Ganoderma lucidum, thereby affecting the quality of the Ganoderma lucidum extract.

Next, the Ganoderma lucidum powder is water-extracted. The water extraction method is to put the Ganoderma lucidum powder into water and heat it at a temperature of 100°C for 60 minutes. The weight ratio of Ganoderma lucidum powder to water is, for example, 1:40. The Ganoderma lucidum extract used in each embodiment of the present invention is obtained by water quenching 5 g of Ganoderma lucidum powder and 200 g of pure water. In this way, the Ganoderma lucidum polysaccharide (0.5wt%) and triterpenoids (0.18wt%) in the Ganoderma lucidum are dissolved in water to obtain the Ganoderma lucidum primary extract.

Next, the Ganoderma lucidum primary extract is centrifuged with a centrifuge to obtain the Ganoderma lucidum supernatant. The centrifugal method is, for example, 205 grams of Ganoderma lucidum primary extract is set at 4 with a centrifugal force of 8000 rpm. Centrifuge at ℃ for 30 minutes.

Next, the Ganoderma lucidum supernatant is freeze-dried to obtain a powdered Ganoderma lucidum extract. Take the Ganoderma lucidum supernatant from the Ganoderma lucidum primary extract after centrifugation, and then use the Ganoderma supernatant Freeze-drying was performed for 3 days to obtain 0.12 g of powdered Ganoderma lucidum extract. This powdered Ganoderma lucidum extract is dissolved in water and is the aqueous solution of Ganoderma lucidum extract used in subsequent experiments. The aqueous solution of Ganoderma lucidum extract contains 0.18% by weight of triterpenoids and 0.5% by weight of Ganoderma lucidum polysaccharides.

The cells used in each experiment of the present invention are rat adrenal medulla chromaffin cells (PC-12), purchased from the Biological Resources Conservation and Research Center (BCRC) of the Food Industry Research Institute, and the BCRC number is 60048. The preparation method of the cells for the experiment is to implant 20,000 rat adrenal medulla chromaffin cells (PC-12) in each hole of the 24-well plate, and culture for 24 hours for subsequent analysis experiments.

The rat adrenal medulla chromaffin cells used in the free radical detection experiment and the hippocampal bioenergy measurement and analysis experiment were cultured with 10% horse serum and 5% fetal serum. Roswell Park Memorial Institute 1640 (RPMI 1640) medium of bovine serum. The rat adrenal medulla chromaffin cells used in the neural differentiation experiment were cultured with RPMI 1640 medium containing 1% horse serum and 0.5% fetal bovine serum.

In the free radical detection experiment and the hippocampal bioenergy measurement and analysis experiment, the PC-12 cell line of Example A1 was treated with an aqueous solution of Ganoderma lucidum extract at a concentration of 100 micrograms per milliliter (μg/ml). The treatment method is to add 1 microliter (μl) of Ganoderma lucidum extract aqueous solution at a concentration of 100μg/ml to the well plate where PC-12 cells that have been cultured for 24 hours are located, so that the concentration of PC-12 cells is 100μg/ml (μg/ml) Ganoderma lucidum extract aqueous solution soaked for 24 hours. The PC-12 cells of Example A2 to Example A4 are treated in a manner similar to the PC-12 cells of Example A1. The difference lies in that the concentrations of Example A2 to Example A4 are respectively 150μg/ml, 200μg/ml and 250μg/ PC-12 cells treated with ml of Ganoderma lucidum extract aqueous solution. The PC-12 cell lines of Comparative Example A and Control Example A were not treated with Ganoderma lucidum extract aqueous solution.

The following first describes the operation method and detection results of the free radical detection experiment.

First, remove the aqueous solution in the wells where the PC-12 cells of Example A1 to Example A4 and Comparative Example A are located. _{Next, an aqueous solution of hydrogen peroxide (H 2} O ₂ ) with a concentration of 20 μM was added to the wells where the PC-12 cells of Example A1 to Example A4 and Comparative Example A were located, and soaked for 60 minutes. Next, remove the aqueous solution in the well where the PC-12 cells of Example A1 to Example A4, Comparative Example A and Control Example A are located, and wash the PC in the well with Phosphate buffered saline (PBS) -12 cells. Next, a Dimethyl sulfoxide (DMSO) solution containing 20 mM DCFH-DA (Dichlorofluorescin-DA) was added to the wells and soaked for 45 minutes. Finally, the fluorescence intensity in the PC-12 cells of Example A1 to Example A4, Comparative Example A, and Control Example A were measured with a spectrophotometer to obtain the amount of free radicals generated.

Please refer to Figure 1 for the results of the free radical detection experiment. Figure 1 is a schematic diagram of the amount of free radicals produced by the rat adrenal medulla chromaffin cells of Examples A1 to A4, Comparative Example A and Control Example A under oxidative pressure. As shown in Figure 1, the amount of free radicals produced by the PC-12 cells of Example A1 to Example A4 under oxidative pressure is significantly lower than the amount of free radicals produced by the PC-12 cells of Comparative Example A under oxidative pressure. It can be seen that the Ganoderma lucidum extract aqueous solution with a concentration of 100 μg/ml to 300 μg/ml has the effect of reducing the amount of free radicals generated by cells under oxidative pressure.

Next, the operation method and test results of the mitochondrial activity detection experiment are explained.

First, remove the aqueous solution in the wells where the PC-12 cells of Example A1 to Example A4 and Comparative Example A are located. _{Next, an aqueous solution of hydrogen peroxide (H 2} O ₂ ) with a concentration of 50 μM was added to the wells where the PC-12 cells of Example A1 to Example A4 and Comparative Example A were located, and soaked for 60 minutes. Next, remove the aqueous solution in the well where the PC-12 cells of Example A1 to Example A4, Comparative Example A and Control Example A are located, and wash the PC in the well with Phosphate buffered saline (PBS) -12 cells. Finally, the hippocampal bioenergy meter was used to detect the oxygen consumption of the PC-12 cells of Example A1 to Example A4, Comparative Example A, and Control Example A, and calculate the mitochondrial activity in the PC-12 cells.

The measurement principle and process of the hippocampus bioenergy meter are as follows. First, replace the medium in the well with the medium for analysis. Next, detect the basal oxygen consumption of the cells in the well. Next, a nematosine triphosphate synthase inhibitor is added to inhibit the mitochondria from producing nematode triphosphate, and the reduced oxygen consumption at this time is the oxygen consumption for synthesizing nematode triphosphate. The nematic triphosphate synthase inhibitor is, for example, oligomycin (Oligomycin). Then, an appropriate concentration of anti-coupling agent is added to evaluate the maximum oxygen consumption capacity of the mitochondria by allowing the mitochondria to idle at the limit without damaging the electron transport chain of the mitochondrial inner membrane. The anti-coupling agent is, for example, Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP). Finally, the addition of an electron transport chain inhibitor has completely shut down the oxygen consumption of the mitochondria, thereby confirming the measured background value, that is, the non-mitochondrial oxygen consumption (Non-mitochondrial Respiration). The electron transport chain inhibitor is, for example, a combination of Rotenone and Antimycin A.

The basal oxygen consumption of the mitochondria is equal to the basic oxygen consumption of the cell minus the non-mitochondrial oxygen consumption. The basal oxygen consumption of mitochondria minus the amount of oxygen consumed by synthetic triphosphate is equal to the oxygen consumption to overcome the hydrogen ion leakage (Proton Leakage). The maximum oxygen consumption capacity of the mitochondria minus the basic oxygen consumption of the mitochondria is equal to the pre-stored oxygen consumption capacity of the mitochondria. The mediator efficiency of mitochondrial nematode triphosphate is equal to the oxygen consumption of synthetic nematode triphosphate divided by the basal oxygen consumption of mitochondria.

Please refer to Figure 2 to Figure 9 and Table 1 for the detection results of mitochondrial activity in PC-12 cells. 2 is a schematic diagram of the measurement results of the hippocampal bioenergy meter of the rat adrenal medulla chromaffin cells of the example, the comparative example A and the control example A.

As shown in the section from 0 minutes to 30 minutes in Figure 2, the mitochondria in cells treated with Ganoderma lucidum extract aqueous solution can still maintain a high oxygen consumption for synthesizing clementine triphosphate under oxidative pressure. . As shown in the section from 60 minutes to 80 minutes in Figure 2, the mitochondria in cells treated with the aqueous solution of Ganoderma lucidum extract have a significantly higher maximum oxygen consumption capacity under oxidative pressure.

Fig. 3 is a schematic diagram showing the pre-existing oxygen consumption capacity of mitochondria in the rat adrenal medulla chromaffin cells of Examples A2 to A4, Comparative Example A and Control Example A. 4 is a schematic diagram of the basic oxygen consumption of mitochondria in human fibroblasts of Examples A2 to A4, Comparative Example A and Control Example A. 5 is a schematic diagram of the maximum oxygen consumption capacity of mitochondria in human fibroblasts of Examples A2 to A4, Comparative Example A and Control Example A. Figure 6 is a schematic diagram of the oxygen consumption of synthetic nematode triphosphate in the mitochondria of human fibroblasts in Examples A2 to A4, Comparative Example A and Control Example A. Fig. 7 is a schematic diagram of the mediator efficiency of mitochondria of human fibroblasts in Examples A2 to A4, Comparative Example A and Control Example A. 8 is a schematic diagram of the oxygen consumption of mitochondria in normal human fibroblasts of Examples A2 to A4, Comparative Example A and Control Example A to overcome hydrogen ion leakage. Fig. 9 is a schematic diagram of non-mitochondrial oxygen consumption in normal human fibroblasts of Examples A2 to A4, Comparative Example A and Control Example A. Table 1 shows the concentration and experimental measurement results of the composition containing Ganoderma lucidum extract in Examples A2 to A4, Comparative Example A and Control Example A.

As shown in FIG. 2 and FIG. 3, the basic oxygen consumption of Example A2 to Example A4 and Comparative Example A have no significant difference. As shown in FIG. 2 and FIG. 4, the oxygen consumption of synthetic nematode triphosphate of Example A2 to Example A4 is higher than the oxygen consumption of synthetic nematode triphosphate of Comparative Example A. As shown in Figures 2 and 5, the oxygen consumption for overcoming hydrogen ion leakage in Example A2 is lower than the oxygen consumption overcoming hydrogen ion leakage in Comparative Example A, and the consumption of overcoming hydrogen ion leakage in Example A3 and Example A4 The amount of oxygen was significantly lower than that of Comparative Example A to overcome the leakage of hydrogen ions (p<0.05). As shown in Figure 2 and Figure 6, the maximum oxygen consumption capacity of Example A2 and Example A3 is higher than the maximum oxygen consumption capacity of Comparative Example A, and the maximum oxygen consumption capacity of Example A4 is significantly higher than that of Comparative Example A. Ability (p<0.05). As shown in Figure 2 and Figure 7, the pre-stored oxygen consumption capacity of Example A2 and Example A3 is higher than the pre-stored oxygen consumption capacity of Comparative Example A, and the pre-stored oxygen consumption capacity of Example A4 is significantly higher than that of Comparative Example A Oxygen capacity (p<0.05). As shown in Fig. 2 and Fig. 8, the mediating efficiency of the nematode triphosphate of Example A2 and Example A3 is higher than that of the comparative example A, and the mediating efficiency of the nematode triphosphate of Example A4 is obvious. The mediator efficiency of nematode triphosphate is higher than that of Comparative Example A (p<0.05). As shown in Figures 2 and 9, the non-mitochondrial oxygen consumption of Examples A2 to A4 is higher than that of Comparative Example A.

Then, the values in Table 1 are taken into the following formula to calculate the Bioenergetic Healthy Index (BHI) values of Example A2 to Example A4, Comparative Example A and Control Example A.

BHI=[oxygen consumption of synthetic triphosphoric acid xanthoside×pre-stored oxygen consumption capacity]/[oxygen consumption to overcome hydrogen ion leakage×non-mitochondrial oxygen consumption]

It can be calculated from the above formula that the BHI value of Example A2 is 1.05, the BHI value of Example A3 is 1.07, the BHI value of Example A4 is 1.15, and the BHI value of Comparative Example A is 0.92.

Fig. 10 is a schematic diagram of the bioenergy health index of the rat adrenal medulla chromaffin cells of Examples A2 to A4, Comparative Example A and Control Example A. It can be seen from FIG. 10 that the bioenergy health index of Example A2 and Example A3 is higher than the bioenergy health index of Comparative Example A, and the bioenergy health index of Example A4 is significantly higher than that of Comparative Example A (p <0.05).

According to the above experimental test results, the cells treated with the Ganoderma lucidum extract aqueous solution are compared with the cells without the Ganoderma lucidum extract aqueous solution treatment. Despite being under oxidative pressure, the mitochondria’s maximum oxygen consumption capacity and pre-stored oxygen consumption capacity are And the media and efficiency have been improved, so that the mitochondria can self-adjust the energy supply when they encounter changes or pressures, thereby increasing their flexibility to withstand changes or pressures.

Next, the operation method and experimental results of the cell differentiation experiment are explained.

First, the processing methods of the PC-12 cells of Example B, Comparative Example B, and Control Example B used in the neural differentiation experiment will be described. After the PC-12 cells of Example B were cultured in a 24-well plate for 24 hours, the RPMI 1640 medium containing 1% horse serum and 0.5% fetal bovine serum was replaced with no serum RPMI 1640 medium and continue to culture for 24 hours. Then, the serum-free RPMI 1640 medium was replaced with horse serum with a concentration of 1% by volume, fetal bovine serum with a concentration of 0.5% by volume, and nerve growth with a concentration of 100 nanograms per milliliter (ng/ml). RPMI factor (Nerve Growth factor, NGF) 1640 medium, and added to a concentration of 100mg / ml aqueous extract of Ganoderma lucidum 1.5 microliters (μ l) continued for 48 hours (Ganoderma lucidum extract concentration in the medium was 20μg / ml). Next, update the wells containing horse serum with a volume percentage of 1%, fetal bovine serum with a volume percentage of 0.5%, and nerve growth factor (Nerve Growth Factor, NGF) at a concentration of 100 nanograms per milliliter (ng/ml). ) RPMI 1640 medium, and re-add 1.5 microliters of Ganoderma lucidum extract aqueous solution with a concentration of 100mg/ml for 36 hours (the concentration of Ganoderma lucidum extract in the medium is 20μg/ml). Next, measure the content of Acetylcholinesterase (AChE) in the wells.

The PC-12 cells of Comparative Example B are treated in a similar manner to the PC-12 cells of Example B, and the difference is that the PC-12 cell line of Comparative Example B does not use the Ganoderma lucidum extract aqueous solution for treatment of PC-12 cells. The processing method of PC-12 cells of control example B is similar to that of PC-12 cells of example B. The difference is that the PC-12 cell line of control example B does not use nerve growth factor and Ganoderma lucidum extract aqueous solution to process PC-12 cell.

Please refer to Figure 11 for the test results of Acetylcholinesterase (AChE) content. Figure 11 is a schematic diagram of the differentiation index of rat adrenal medulla chromaffin cells treated with Ganoderma lucidum extract. In Figure 11, the concentration of pure acetylcholinesterase is used as the reference value for detection. Acetylcholinesterase is mainly present in the synapses of nerve cells, so the content of acetylcholinesterase can be used as an indicator of the degree of differentiation of nerve cells. As shown in Figure 11, the concentration of acetylcholinesterase produced by PC-12 cells of Example B was significantly higher than the concentration of acetylcholinesterase produced by PC-12 cells of Comparative Example B (p<0.05) The concentration of acetylcholinesterase produced by the PC-12 cells of Example B was also significantly higher than the concentration of acetylcholinesterase produced by the PC-12 cells of Control Example B (p<0.001).

Figures 12 to 14 are electron micrographs of rat adrenal medulla chromaffin cells treated with different treatments. As shown in Figure 12, no outwardly extending nerve synapses were seen on the PC-12 cells of Control Example B. As shown in Fig. 13, the PC-12 cells of Comparative Example B showed slightly outwardly extending nerve synapses. As shown in FIG. 14, the PC-12 cells of Example B can see obvious outwardly extending nerve synapses, and the length of the nerve synapses extending can reach several times the size of the PC-12 cells.

From the results of the above cell differentiation experiment, it can be seen that the Ganoderma lucidum extract can ensure that the cells Get sufficient energy supply for cells to differentiate. In addition, it can be seen from the foregoing experimental results that the Ganoderma lucidum extract has the effect of ensuring that the cells receive sufficient energy supply so that the cells can differentiate into nerve cells. Therefore, Ganoderma lucidum extract is also useful for treating degenerative brain or neurological diseases, or slowing down the deterioration of degenerative brain or neurological diseases.

The use of the Ganoderma lucidum extract disclosed in the present invention for the preparation of a composition that improves the bioenergy health index and promotes cell differentiation. When the composition containing the Ganoderma lucidum extract is provided to cells under stress, the Ganoderma lucidum extract Substances can improve the bioenergy health index and ensure that cells can get sufficient energy supply for differentiation. In this way, mitochondria have the ability to provide energy according to the needs of the cells, so that the cells have sufficient energy to deal with stress and carry out various life activities.

Furthermore, supplying the Ganoderma lucidum extract to the cells can reduce the amount of free radicals produced in the cells, thereby protecting the inner membrane of the mitochondria from damage by free radicals, thereby delaying the disintegration of the mitochondria. In this way, supplying the Ganoderma lucidum extract to the cells can slow down the disintegration of mitochondria in the cells and trigger cell apoptosis.

Although the present invention is disclosed in the foregoing embodiments, it is not intended to limit the present invention. All changes and modifications made without departing from the spirit and scope of the present invention fall within the scope of the patent protection of the present invention. For the scope of protection defined by the present invention, please refer to the attached scope of patent application.

Claims (12)

A kind of Ganoderma lucidum extract is used to prepare a composition for improving the bioenergy health index and promoting cell differentiation under pressure, wherein the Ganoderma lucidum extract contains triterpenoids and Ganoderma lucidum polysaccharides in a weight ratio of 1:2.5 to 1:3 . The use of a Ganoderma lucidum extract for preparing a composition for improving the bioenergy health index under a pressure environment, wherein the Ganoderma lucidum extract contains triterpenoids and Ganoderma lucidum polysaccharides in a weight ratio of 1:2.5 to 1:3. The use according to claim 1 or claim 2, wherein the Ganoderma lucidum extract contains 0.18% by weight of triterpenoids and 0.5% by weight of Ganoderma lucidum polysaccharides. The use according to claim 1, wherein the cells are nerve cells, and the Ganoderma lucidum extract promotes synaptic growth of nerve cells. The use according to claim 1 or claim 2, wherein the concentration of the Ganoderma lucidum extract is 100 to 300 micrograms per milliliter (μg/ml). The use according to claim 1 or claim 2, wherein the effective dose of the Ganoderma lucidum extract is 1.081 to 3.244 grams (g). The use according to claim 1, wherein, under the pressure environment, the Ganoderma lucidum extract further has the purpose of reducing the hydrogen ion leakage (Proton Leakage) of a plurality of mitochondria in the cells. The use according to claim 1, wherein, under the pressure environment, the Ganoderma lucidum extract has the purpose of enhancing the maximal respiration of a plurality of mitochondria in the cells. The use according to claim 1, wherein, under the pressure environment, the Ganoderma lucidum extract has the purpose of increasing the Spare Respiratory Capacity of a plurality of mitochondria in the cells. The use according to claim 1, wherein, under the pressure environment, the Ganoderma lucidum extract further has the purpose of improving the coupling efficiency of the multiple mitochondria in the cells. The use according to claim 1, wherein, under the pressure environment, the Ganoderma lucidum extract further has the purpose of increasing the oxygen consumption rate of ATP Production by a plurality of mitochondria in the cells. The use according to claim 1 or claim 2, wherein the composition further comprises a pharmaceutically or pharmaceutically acceptable carrier, excipient, diluent or adjuvant.

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