USING GINSENG EXTRACT TO REDUCE FATIGUE OR IMPROVE COGNITIVE FUNCTION

Abstract

A use of a ginseng extract, which is a use thereof in the preparation of a composition for resisting fatigue or improving cognitive function performance. The ginseng extract is extracted from the root of ginseng (Panax ginseng C.A.Meyer) by using water as a solvent.

Description

Use of ginseng extract in preparing anti-fatigue or cognitive function performance enhancing composition Technical Field

[0001] The present invention relates to a use of a ginseng extract, and in particular to using the ginseng extract to prepare a composition for resisting fatigue or improving cognitive function performance. Background Art

[0002] Fresh ginseng, freshly dug from the ground, is high in water and difficult to preserve. It's rarely found on the market and is only occasionally used in cooking near its source. Ginseng is typically processed and prepared in different ways, with fresh ginseng being classified as red ginseng, white ginseng, or Taiji ginseng.

[0003] While red ginseng, white ginseng, and Taiji ginseng are generally considered to have greater efficacy and higher saponin content than fresh ginseng, they each have different properties and applicability, and their quality varies due to differences in processing techniques.

[0004] Summary of the Invention

[0005] In view of this, in order to avoid over-processing or unknown effects caused by the processing process, the present invention extracts a ginseng extract from ginseng that has not been dried and fully retains the activity of the original ginseng components. The ginseng extract can be used to prepare an anti-fatigue or cognitive function enhancement composition.

[0006] In some embodiments, a ginseng extract is used to prepare an anti-fatigue composition, wherein the ginseng extract is extracted from Panax ginseng CA Meyer using water as a solvent.

[0007] In some embodiments, ginseng extract promotes mitochondrial activity.

[0008] In some embodiments, ginseng extract promotes skeletal muscle cell proliferation.

[0009] In some embodiments, a ginseng extract is used to prepare a composition for improving cognitive performance, wherein the ginseng extract is extracted from Panax ginseng CA Meyer using water as a solvent.

[0010] In some embodiments, ginseng extract is used to enhance hypoxia tolerance.

[0011] In some embodiments, ginseng extract is used to increase blood oxygen saturation.

[0012] In some embodiments, ginseng extract increases MDA, a marker of lipid peroxidation.

[0013] In some embodiments, ginseng extract enhances total antioxidant capacity (TAC).

[0014] In some embodiments, the composition is a food composition, and the composition comprises at least 5 mL of ginseng extract, that is, the effective amount of ginseng extract is 5 mL / day.

[0015] In some embodiments, the ginseng extract contains at least 13.3 mg of ginsenoside Rg3 per 100 ml.

[0016] In summary, the ginseng extract of any embodiment can be used to prepare a composition for fatigue relief or cognitive performance enhancement. The ginseng extract of any embodiment can be used to prepare a composition for promoting mitochondrial activity, promoting skeletal muscle cell proliferation, enhancing hypoxia tolerance, increasing blood oxygen saturation, increasing the lipid peroxidation marker MDA, increasing total antioxidant capacity (TAC), improving hypoxia tolerance, and enhancing judgment and short-term memory. The ginseng extract of any embodiment contains at least 13.3 mg of ginsenoside Rg3 per 100 ml. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is a diagram of the fingerprint analysis results.

[0018] FIG2 is a graph showing the relative ginsenoside Rg3 content test results.

[0019] FIG3 is a graph showing the relative total alkaloid content test results.

[0020] FIG4 is a graph showing the relative antioxidant efficiency test results.

[0021] FIG5 is a graph showing the relative mitochondrial membrane potential test results.

[0022] FIG6 is a graph showing the relative bone cell proliferation test results.

[0023] FIG7 is a graph showing the test results of blood oxygen saturation concentration in a human trial of ginseng extract.

[0024] FIG8 is a graph showing the test results of lipid peroxidation index in human trials of ginseng extract.

[0025] FIG9 is a graph showing the test results of the total antioxidant capacity of ginseng extract in human trials.

[0026] FIG10 is a graph showing the results of the time spent answering questions in the human trial of ginseng extract.

[0027] FIG11 is a graph showing the results of the number of correct answers in the human test of ginseng extract.

[0028] FIG12 is a graph showing the accuracy of answering questions in a human trial of ginseng extract.

[0029] Wherein, the reference numerals:

[0030] Rg3: Ginsenoside Rg3 DETAILED DESCRIPTION

[0031] As used herein, the term "extract" refers to a product prepared by extraction. The extract may be in the form of a solution dissolved in a solvent, or the extract may be in the form of a concentrate or essence that is free or substantially free of solvent.

[0032] As used herein, "ginseng" refers to the fleshy root of fresh Panax ginseng (scientific name: Panax ginseng), also known as Korean ginseng. In some embodiments, ginseng refers to ginseng roots that have not been dried or steamed. In some embodiments, ginseng is a root that has been frozen or refrigerated within seven days of harvest. This may further include roots that have been whole, chopped, diced, ground, milled, or otherwise processed to affect the size or physical integrity of the raw material.

[0033] In some embodiments, the ginseng is ginseng grown in Northeast China. In some embodiments, the ginseng is ginseng that has been artificially cultivated for less than five years.

[0034] In some embodiments, the ginseng extract is obtained by extracting ginseng using water as a solvent. In some embodiments, the ginseng extract is obtained by heating ginseng using water as a solvent. In some embodiments, heating extraction refers to heating the mixed solution obtained by mixing water and ginseng raw materials and maintaining it at 85±5°C for 50 to 80 minutes. In other words, the extraction temperature is 85±5°C and the extraction time is 50 to 80 minutes. In some embodiments, the extraction temperature can be 85°C and the extraction time can be 60 minutes. Here, the extraction is to soak the ginseng in heated water.

[0035] In some embodiments, the weight ratio of water to ginseng is 1:5 to 15. In some embodiments, the weight ratio of water to ginseng is 1:5.

[0036] In some embodiments, the ginseng extract is prepared by extracting ginseng with water as a solvent to obtain a primary extract, and then filtering the primary extract through a sieve to remove solids. For example, the sieve can be a 400 mesh sieve.

[0037] In some embodiments, the ginseng extract contains at least 13.3 mg of ginsenoside Rg3 per 100 ml.

[0038] In some embodiments, ginseng extract can be used to prepare anti-fatigue compositions. In some embodiments, ginseng extract achieves anti-fatigue effects by promoting mitochondrial activity. In some embodiments, ginseng extract achieves anti-fatigue effects by promoting skeletal muscle cell proliferation. In other words, ginseng extract can enhance muscle cell force conversion.

[0039] In some embodiments, ginseng extract can be used to prepare a composition for enhancing cognitive function. In some embodiments, ginseng extract enhances cognitive function by increasing hypoxia tolerance. In some embodiments, ginseng extract enhances cognitive function by increasing blood oxygen saturation. In some embodiments, ginseng extract enhances cognitive function by increasing MDA, a marker of lipid peroxidation. In some embodiments, ginseng extract enhances cognitive function by increasing total antioxidant capacity (TAC).

[0040] In some embodiments, the composition is a food composition, and the composition comprises at least 5 mL of ginseng extract.

[0041] In some embodiments, the aforementioned composition may be a pharmaceutical product, that is, the pharmaceutical product comprises an effective amount of ginseng extract.

[0042] In some embodiments, the aforementioned pharmaceutical products can be manufactured into a dosage form suitable for enteral or oral administration using techniques well known to those skilled in the art. Such dosage forms include, but are not limited to, tablets, troches, lozenges, pills, capsules, dispersible powders or granules, solutions, suspensions, emulsions, syrups, elixirs, slurries, and the like.

[0043] In some embodiments, the aforementioned pharmaceutical product can be manufactured into a dosage form suitable for parenteral or topical administration using techniques well known to those skilled in the art, including, but not limited to, injections, sterile powders, external preparations, and the like. In some embodiments, the pharmaceutical product can be administered parenterally via a route selected from the group consisting of subcutaneous injection, intraepidermal injection, intradermal injection, and intralesional injection.

[0044] In some embodiments, the pharmaceutical product may further include a pharmaceutically acceptable carrier widely used in pharmaceutical manufacturing techniques. For example, the pharmaceutically acceptable carrier may include one or more of the following agents: a solvent, a buffer, an emulsifier, a suspending agent, a decomposer, a disintegrating agent, a dispersing agent, a binding agent, an excipient, a stabilizing agent, a chelating agent, a diluent, a gelling agent, a preservative, a wetting agent, a lubricant, an absorption delaying agent, a liposome, and the like. The selection and amount of these agents are within the professional knowledge and routine skills of those skilled in the art.

[0045] In some embodiments, the pharmaceutically acceptable carrier comprises a solvent selected from the group consisting of water, normal saline, phosphate buffered saline (PBS), and an aqueous solution containing alcohol.

[0046] In some embodiments, the aforementioned composition may be an edible composition for non-medical purposes. In some embodiments, the edible composition may be prepared as a food product or a food additive, meaning that the composition is added to food ingredients during food preparation using conventional methods or during the production of the food product. In this context, the food product may be formulated with edible materials for human or animal consumption.

[0047] In some embodiments, the food product may be, but is not limited to, beverages, fermented foods, bakery products, health foods for non-medical purposes, and dietary supplements for non-medical purposes.

[0048] Example 1: Preparation of test samples

[0049] 1-1. Preparation of Ginseng Extract: Grind the entire fleshy root of Panax ginseng (cultivated for less than five years, fresh ginseng harvested within seven days and frozen) into a powder. Sieve the powder through a 12mm mesh sieve to produce ginseng powder with a size of less than 12mm. Next, use water as the solvent, mix the ginseng powder with water in a weight ratio of 1:5, and extract at 85°C for 1 hour to produce a primary extract. This primary extract is then passed through a 400-mesh sieve to produce the ginseng extract.

[0050] 1-2. Preparation of White Ginseng Extract: After white ginseng is pulverized into a powder, it is sieved through a 12mm mesh sieve to produce white ginseng powder with a size of less than 12mm. Next, the white ginseng powder is mixed with water in a weight ratio of 1:5, and extracted at 85°C for 1 hour to produce a primary extract. This primary extract is then passed through a 400-mesh sieve to produce the white ginseng extract. The white ginseng is obtained by peeling fresh ginseng and drying it with hot air at 45°C to 50°C.

[0051] 1-3. Preparation of red ginseng extract: After the red ginseng is crushed into powder, it is sieved through a 12mm mesh screen to obtain red ginseng powder with a size of less than 12mm. Next, using water as a solvent, the red ginseng powder and water are mixed in a weight ratio of 1:5 and extracted at 85°C for 1 hour to form a primary extract. Subsequently, the extracted primary extract is passed through a 400 mesh screen to obtain red ginseng extract. Here, red ginseng is obtained by steaming unpeeled fresh ginseng at 90°C to 100°C for at least 1 hour, and then drying it with hot air at 45°C to 50°C.

[0052] Example 2: Component Analysis of Ginseng Extract

[0053] To understand the differences in the composition of ginseng compared to white ginseng and red ginseng obtained by different processing methods, the content of ginsenoside Rg3 and its fingerprints of ginsenoside Rg3 in ginseng, white ginseng and red ginseng were analyzed below.

[0054] 2-1. The equipment, setting method and equipment source are described as follows:

[0055] 1D and 2D spectra were obtained using an Ascend 400 MHz NMR spectrometer (Bruker Co., Germany). Chemical shifts are expressed in ppm using δ.

[0056] Mass spectrometer (MS) tandem mass spectrometry-two-dimensional ion trap tandem Fourier transform mass spectrometry and ESI-MS / MS: measured using a Bruker amaZon SL system, the unit is m / z.

[0057] Medium pressure liquid chromatography (MPLC): Rf + ,Teledyne ISCO, Lincoln, NE, High Performance Liquid Chromatography (HPLC): High Performance Liquid Chromatography (HPLC): Agilent 1200 series; degasser: Agilent vacuum degasser 1322A; elution solvent delivery: Agilent quaternary pump G1311A; multiple wavelength detector (MWD): Agilent G1314B; diode array detector (DAD): Agilent 1260 Infinity DAD VL G1315D, detection wavelengths: 210 nm, 280 nm, 320 nm, 365 nm (Agilent Germany).

[0058] Analytical column: C18(2) (250 x 10 mm, Phenomenex, USA).

[0059] Column chromatography packing materials: Sephadex LH-20 (Pharmacia, Piscataway, NJ, USA), Diaion HP-20 (Mitsubishi Chemical Co., Japan), Merck Kieselgel 60 (40-63 μm, Art. 9385), Merck RP-18(40-63um,Art.0250).

[0060] Thin-layer chromatography (TLC) was performed using TLC aluminum sheets (Silica gel 60F254, 0.25 mm, Merck, Germany) and TLC aluminum sheets (RP-18F254-S, 0.25 mm, Merck, Germany).

[0061] Solvents used and their sources: n-hexane, ethyl acetate, acetone, methanol, ethanol, acetonitrile (purchased from Merck, Taiwan), chloroform-d1 (deuteration degree 99.5%), methanol-d4 (deuteration degree 99.5%), deuterium oxide (deuteration degree>99.8%), and dimethyl sulfoxide-d6 (deuteration degree>99.9%) (Merck, Taiwan).

[0062] 2-2. Analysis process:

[0063] First, the ginseng extract, white ginseng extract, and red ginseng extract prepared in Example 1 were analyzed using high performance liquid chromatography (HPLC) to obtain their respective fingerprints, as shown in FIG1 .

[0064] The relevant settings of the above-mentioned liquid chromatography are as follows: the solvents used are methanol (A) and water (B), with the additional addition of 0.1% formic acid, the flow rate is 1 mL / min, the column temperature is 40°C, the injection volume is 10 μL, and the ginseng extract is filtered through a 0.22 μm polyvinylidene fluoride membrane before injection.

[0065] Evaporation conditions: A:B = 2:98 at 0 minutes; A:B = 2:98 at 10 minutes; A:B = 70:30 at 40 minutes; A:B = 100:0 at 50 minutes; A:B = 100:0 at 60 minutes.

[0066] Ginsenoside Rg3 quantitative analysis was also performed on ginseng extracts, red ginseng extracts, and white ginseng extracts. First, a calibration curve was generated using a ginsenoside Rg3 standard compound. The ginsenoside Rg3 standard compound was purchased from Merck, Taiwan (CAS No. 14197-60-5, purity >98%).

[0067] Preparation of the standard curve: 10 mg of ginsenoside Rg3 was accurately weighed and diluted to a 10 mL volumetric flask with methanol / water (1 / 1 volume ratio). This yielded a 100 mg / 100 mL ginsenoside Rg3 standard solution. Calibration curve for ginsenoside Rg3: Ginsenoside Rg3 solutions were prepared at concentrations of 40, 20, 10, 5, and 1 mg / 100 mL, respectively. HPLC analysis was performed using a Mightysil RP-18GP 250 column (250 x 4.6 mm, 5 μm, Kanto, Tokyo, Japan) with a detection wavelength of 210 nm. The peak areas at 40, 20, 10, 5, and 1 mg / 100 ml were 2206.5, 1108.1, 477.7, 230.4, and 48.1, respectively. Linear regression yielded a standard curve of ginsenoside Rg3 concentration (x-axis) - peak area (y-axis): y = 43.84x + 16.275 (r^2 = 0.9998).

[0068] According to the above fingerprint, the peak areas of ginseng extract, red ginseng extract, and white ginseng extract were 599.4, 208.7, and 92.1, respectively. Through the standard curve and calculation, it was found that the ginsenoside Rg3 content of each extract was 13.3 mg / 100 ml, the ginsenoside Rg3 content of red ginseng extract was 4.389 mg / 100 ml, and the ginsenoside Rg3 content of white ginseng extract was 1.792 mg / 100 ml. The relative percentages are shown in Figure 2.

[0069] 2-3. Test Results

[0070] Referring to Figure 1, the bioactive substance represented by the peak at 27 minutes is ginsenoside Rg3. Figure 1 clearly shows that the ginseng extract exhibits the highest peak at 27 minutes, indicating that this ginseng extract has the highest ginsenoside Rg3 content. The fingerprint of the red ginseng extract also exhibits a small peak around 27 minutes, indicating that while the red ginseng extract also contains ginsenoside Rg3, its content is lower than that of the ginseng extract. The white ginseng extract also exhibits a small peak around 27 minutes, indicating that its ginsenoside Rg3 content is also low.

[0071] Refer to Figure 2. The value of the ginseng extract (experimental group) is considered 100%. Compared to the ginseng extract, the relative content of ginsenoside Rg3 in the red ginseng extract (control group B) was 13.0%, and the relative content of ginsenoside Rg3 in the white ginseng extract (control group A) was 33.0%.

[0072] Example 3: Total alkaloid content test

[0073] 3-1. Material Description:

[0074] DR solution: Prepared by mixing the first solution (0.8 g of bismuth nitrate pentahydrate in 40 mL of distilled water and 10 mL of glacial acetic acid) and the second solution (8.0 g of potassium iodide in 20 mL of distilled water).

[0075] Standard bismuth nitrate (Bi(NO3)3.5H2O) solution: Dissolve 10 mg of bismuth nitrate in 5 mL of concentrated nitric acid, then dilute to 100 mL with distilled water.

[0076] 3% thiourea solution: prepared by dissolving 3 g of thiourea in 100 ml of distilled water.

[0077] 1% disodium sulfide solution: Dissolve 1 g of disodium sulfide in 100 mL of distilled water.

[0078] 3-2. Prepare standard curve:

[0079] First, the standard bismuth nitrate solution was diluted to nine different concentrations as standard samples by transferring 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, and 9 mL of standard bismuth nitrate solution into separate 10 mL standard flasks and diluting to the 10 mL mark with distilled water. 1 mL of each of the nine different concentrations of bismuth nitrate solution (standard samples) was taken, and 5 mL of thiourea solution was added to each standard sample to form nine yellow standard samples. The absorbance of these nine yellow standard samples was then measured at 435 nm relative to a colorless reagent blank.

[0080] 3-3. Test process:

[0081] 5 mL of sample was taken from each group, and HCl was added to maintain the pH value at 2-2.5. Here, the sample of the experimental group was the ginseng extract prepared in Example 1, the sample of the control group A was the white ginseng extract prepared in Example 1, and the sample of the control group B was the red ginseng extract prepared in Example 1.

[0082] 2 mL of DR solution was added to each group and centrifuged to form a centrifuge containing a precipitate. At the same time, DR solution was added to the centrifuge to check whether the centrifuge was completely precipitated.

[0083] After centrifugation, the liquid of the centrifuge was decanted, and the precipitate was taken out and further washed with alcohol.

[0084] After removing the filtrate, the precipitate was treated with 2 mL of disodium sulfide solution, and a brown-black precipitate was formed. Meanwhile, the completion of precipitation was checked by adding another 2 drops of disodium sulfide.

[0085] The residue was dissolved in 2 mL of concentrated nitric acid, with heating if necessary to achieve complete dissolution.

[0086] This solution was diluted to 10 mL with distilled water and placed in a standard flask. 1 mL was then taken out and 5 mL of thiourea solution was added to it, and the absorbance was measured at 435 nm.

[0087] The absorbance of the sample was compared with the standard curve to obtain the total alkaloid content of the sample. The results are shown in FIG3 .

[0088] 3-4. Test Results

[0089] Continued reference to Figure 3. The values ​​of the experimental group were considered 100%. Compared to the experimental group, the relative content of total alkaloids in the control group A was 67.3%, and the relative content of total alkaloids in the control group B was 21.3%.

[0090] Example 4: Antioxidant efficiency test

[0091] 4-1. Materials and equipment description:

[0092] Cell line: Skeletal muscle cells C2C12 (hereinafter referred to as C2C12 cells) were used. C2C12 cells were purchased from the American Type Culture Collection. ) C2C12 cell line (ATCC CRL-1772 TM ).

[0093] Cell culture medium: Minimum essential medium (MEM, hereinafter referred to as MEM medium) (Gibco, Catalog No. 11095080) supplemented with 10% fetal bovine serum (FBS; Gibco, Catalog No. 10437-028), 1% penicillin-streptomycin (Antibiotic-Antimycotic) (Gibco, Catalog No. 15140122), and 1 mM sodium pyruvate (Gibco, Catalog No. 11360-070).

[0094] Phosphate buffered saline (PBS solution): purchased from Gibco, product number 10437-028.

[0095] DCFH-DA solution: Dissolve 2,7-dichloro-dihydro-fluorescein diacetate (DCFH-DA; product number SI-D6883, purchased from Sigma) in dimethyl sulfoxide (DMSO, purchased from Sigma, product number SI-D6883-50MG) to prepare a 5 μg / mL DCFH-DA solution.

[0096] Flow cytometry: BD Accuri C6Plus.

[0097] Hydrogen peroxide (H2O2): purchased from Sigma-Aldrich, product model 95299-1L.

[0098] Trypsin-EDTA: 10X Trypsin-EDTA (purchased from Gibco) was diluted 10-fold in 1X PBS solution.

[0099] 4-2. Test process:

[0100] First, take 2×10 5 C2C12 cells were seeded into 6-well cell culture plates at a density of 100 cells / well, 2 mL of cell culture medium was added to each well, and the cells were cultured in a carbon dioxide incubator at 37°C for 24 hours.

[0101] After the C2C12 cells adhered to the bottom of the cell culture dish, they were divided into experimental group, control group A, and control group B. The cell culture medium of each group was removed and replaced with 500 μl / well test medium, and then incubated at 37°C for 1 hour.

[0102] The test medium for the experimental group consisted of a cell culture medium containing 0.25% of the ginseng extract obtained in Example 1 and 1 mM hydrogen peroxide. The test medium for the control group A consisted of a cell culture medium containing 0.25% of the white ginseng extract obtained in Example 1 and 1 mM hydrogen peroxide. The test medium for the control group B consisted of a cell culture medium containing 0.25% of the red ginseng extract obtained in Example 1 and 1 mM hydrogen peroxide.

[0103] After adding the DCFH-DA solution, the cells were treated at 37°C for 15 minutes.

[0104] After adding 10 μL of hydrogen peroxide to each well, the cells were incubated at 37°C for 1 hour.

[0105] Remove the liquid in the well plate, rinse the cells with PBS, and repeat the above steps twice.

[0106] Remove the liquid in the well plate and add 200 μL / well of 1X trypsin for 5 minutes in the dark.

[0107] The reaction was stopped with culture medium, transferred to a 1.5 mL microcentrifuge tube, and centrifuged at 400 x g for 10 minutes.

[0108] The supernatant was removed, and the cells were washed with PBS and centrifuged at 400 x g for 10 minutes.

[0109] The supernatant was removed and the cells were resuspended in 1 mL of PBS per tube.

[0110] The fluorescence signal of DCFH-DA was measured by flow cytometry (excitation light: 450-490 nm; scattered light: 510-550 nm). The results are shown in FIG4 . The antioxidant efficiency shown in the figure is presented in relative magnification.

[0111] 4-3. Test results

[0112] Refer to Figure 4. Considering the value for Control B as 100%, the relative oxidation efficiency of the experimental group was as high as 279.2%. Control A, on the other hand, had a relative oxidation efficiency of 152.9%. In other words, ginseng extract significantly boosted skeletal muscle cells' ability to resist oxidative damage caused by hydrogen peroxide, achieving a 2.7-fold improvement compared to red ginseng.

[0113] Example 5: Mitochondrial activity test

[0114] Mitochondria are crucial organelles for cellular metabolism and energy production. Low mitochondrial activity often indicates a state of cellular fatigue. This test uses flow cytometry to assess changes in mitochondrial activity in skeletal muscle C2C12 cells after treatment with various samples. The test uses JC-1 aggregation as an indicator of mitochondrial activity. In healthy mitochondria with normal membrane potential, JC-1 exists as a polymer. When the mitochondrial membrane potential decreases, JC-1 becomes a monomer, which can be used as an indicator of mitochondrial activity.

[0115] 5-1. Materials and equipment description:

[0116] Experimental cell line: Skeletal muscle cells C2C12 (hereinafter referred to as C2C12 cells) were used. C2C12 cells were purchased from the American Type Culture Collection. ) C2C12 cell line (ATCC CRL-1772 TM ).

[0117] Culture medium: Dulbecco's modified Eagle's medium was purchased from Gibco, USA, model number Gat. 11965-092, supplemented with 10% fetal bovine serum, purchased from Gibco, USA, model number Gat. 10437-028, and 1% antibiotics, purchased from Gibco, USA, model number Gat. 15240-062.

[0118] MitoScreen Flow Cytometry Mitochondrial Membrane Potential Assay Kit (BD; Cat. BDB551302) Mitochondrial Membrane Potential Assay Kit (BDTM MitoScreen (JC-1) kit, Model 551302). The Mitochondrial Membrane Potential Assay Kit contains JC-1 dye (lyophilized) and 10X assay buffer. Before use, dilute the 10X assay buffer 10-fold with 1X PBS to form 1X assay buffer. Add 130 μL of DMSO to the lyophilized JC-1 dye to form a JC-1 stock solution. Then, dilute the JC-1 stock solution with 1X assay buffer to form the JC-1 working solution (i.e., JC-1 mitochondrial-specific dye). The dilution ratio is 1:100: JC-1 stock solution to 1X assay buffer.

[0119] Trypsin: 10X Trypsin-EDTA (purchased from Gibco) was diluted 10-fold in 1X PBS solution.

[0120] Flow cytometer: Flow cytometry (purchased from Beckman).

[0121] 5-2. Testing process:

[0122] First, take a 6-well culture plate and inoculate 1×10 5 The cells were cultured at 37°C for 24 hours. The cultured cells were divided into three groups: experimental group, control group A, and control group B. The cell culture medium of each group was removed and replaced with test medium, and then incubated at 37°C for 24 hours.

[0123] The test medium for the experimental group was a cell culture medium containing 0.25% of the ginseng extract obtained in Example 1. The test medium for the control group A was a cell culture medium containing 0.25% of the white ginseng extract obtained in Example 1. The test medium for the control group B was a cell culture medium containing 0.25% of the red ginseng extract obtained in Example 1.

[0124] Remove the test medium from the culture dish and rinse twice with 1X PBS solution.

[0125] Add 200 μL of trypsin to each well and incubate in the dark for 5 minutes. After the reaction, add cell culture medium to terminate the reaction. Collect the cells and cell culture medium from each well into a corresponding 1.5 mL centrifuge tube and centrifuge the tube containing the cells and cell culture medium at 400 x g for 10 minutes.

[0126] After centrifugation, remove the supernatant and resuspend the cell pellet in 1 mL of 1X PBS or transfer the cell pellet to a 15 mL centrifuge tube to form a cell suspension.

[0127] Centrifuge the tube containing the cell suspension at 400 x g for 5 minutes.

[0128] After centrifugation, the supernatant in each centrifuge tube was removed and 100 μL of JC-1 working reagent was added to each centrifuge tube.

[0129] The cell pellet in each centrifuge tube was vortexed with JC-1 working reagent and incubated for 15 minutes in the dark.

[0130] After 15 minutes, each tube was centrifuged at 400 x g for 5 minutes.

[0131] After centrifugation, the supernatant in each centrifuge tube was removed, and the cell pellet in each centrifuge tube was re-dissolved with 1 mL of 1X PBS solution and centrifuged at 400×g for 5 minutes.

[0132] After centrifugation, the supernatant in each centrifuge tube was removed, and the cell pellet in each centrifuge tube was re-dissolved with 1 mL of 1X PBS solution and centrifuged at 400×g for 5 minutes.

[0133] After centrifugation, the supernatant in each centrifuge tube was removed, and the cells were resuspended in 500 μL of 1× PBS to obtain the cell solution to be tested.

[0134] Mitochondrial activity analysis was performed by measuring the mitochondrial membrane potential of cells in the culture medium from each well using flow cytometry. Because the experiment was performed in triplicate, the results of the triplicate experiments were averaged to obtain the mean value. The mean value of control group A was then used as 1 for relative JC-1 aggregation. The mean values ​​of the experimental and control groups B were then converted to relative JC-1 aggregation, as shown in Figure 5.

[0135] 5-3. Test results

[0136] Refer to Figure 5, assuming the value for Control A is 1. Compared to Control A, the relative mitochondrial membrane potential in the experimental group was 12.77 times higher. In contrast, the relative mitochondrial membrane potential in Control B was 6.52. In other words, ginseng extract significantly boosted skeletal muscle cells' ability to counteract the decline in mitochondrial activity caused by excessive fatigue.

[0137] Example 6: Skeletal muscle cell proliferation test

[0138] People with more muscle tissue have greater endurance and can work for longer periods of time without feeling tired.

[0139] 6-1. Materials and equipment description:

[0140] Experimental cell line: Skeletal muscle cells C2C12 (hereinafter referred to as C2C12 cells) were used. C2C12 cells were purchased from the American Type Culture Collection. ) C2C12 cell line (ATCC CRL-1772 TM ).

[0141] Culture medium: Dulbecco's modified Eagle's medium was purchased from Gibco, USA, model number Gat. 11965-092. It was supplemented with 10% FBS (fetal bovine serum), purchased from Gibco, USA, model number Gat. 10437-028, and 1% antibiotics, purchased from Gibco, USA, model number Gat. 15240-062.

[0142] Cell proliferation kit: ELISA, BrdU (purchased from Roche; model number 11647229001), including EdU reagent, FixDenat reagent, BrdU-POD reagent (Remove the antibody conjugate and rinse well three times with 200-300 μl washin g solution), washing solution, and substrate solvent.

[0143] 6-2. Test process:

[0144] First, 5,000 cells were seeded into each well of a 96-well culture plate and cultured at 37°C for 2 hours. The cultured cells were divided into five groups: experimental group, blank group, positive control group, control group A, and control group B.

[0145] Blank group: Culture medium alone was added and incubated at 37°C for 24 hours. Positive control group: Cells were supplemented with 10% FBS. Experimental group: Cell culture medium was supplemented with 0.25% of the ginseng extract obtained in Example 1. Control group A: Cell culture medium was supplemented with 0.25% of the white ginseng extract obtained in Example 1. Control group B: Cell culture medium was supplemented with 0.25% of the red ginseng extract obtained in Example 1.

[0146] Next, according to the standard test procedure of the cell proliferation kit, EdU reagent was added and cultured at 37°C for 24 hours. Then, the supernatant was removed and FixDenat reagent was added and treated at room temperature for 30 minutes. The FixDenat reagent was removed and washed once with PBS buffer. After adding BrdU-POD reagent, the cells were treated at room temperature for 90 minutes. The antibody conjugate (BrdU-POD reagent) was removed and the washing solution was rinsed 3 times. The washing solution was removed, the substrate solvent was added and treated at room temperature for 15 minutes. H2SO4 (concentration 1M) was added and treated for 10 minutes. After shaking at 300 rpm for 1 minute, the fluorescence signal of the cell fluid to be tested in each well was measured by flow cytometry (excitation light: 450 nm) and the number of cells was calculated.

[0147] The results were analyzed using Excel software to perform a student t-test to determine whether there was a statistically significant difference between the two sample groups. Figure 6 shows a "*" indicating a p-value less than 0.05, a "**" indicating a p-value less than 0.01, and a "***" indicating a p-value less than 0.001. The greater the number of "*," the more statistically significant the difference relative to the blank group.

[0148] 6-3. Test Results

[0149] Refer to Figure 6. When the blank group's skeletal muscle cell count is considered 100%, the positive control group's skeletal muscle cell count relative to the blank group is 126.22%, while the experimental group's skeletal muscle cell count relative to the blank group is 106.7%. Compared to the blank group, the experimental group has a statistically significant difference.

[0150] However, white ginseng extract (Control A) and red ginseng extract (Control B) not only failed to promote skeletal muscle growth, but actually reduced the number of skeletal muscle cells. This suggests that ginseng extract is the only one that effectively promotes skeletal muscle cell growth, thereby enhancing fatigue resistance.

[0151] Example 7: Human Experimentation

[0152] Sample: The ginseng extract prepared in Example 1 was used.

[0153] Subjects: 3 subjects, aged 20 to 55. All subjects self-reported being healthy adults with fatigue and frequent brain fog, and no underlying medical conditions.

[0154] 7-1. Test items

[0155] Items include: blood oxygen saturation concentration (SpO2, Oxygen saturation expressed in %), lipid peroxidation index (MDA, Malondialdehyde), total antioxidant capacity (TAC), judgment test (Stroop Tess), and short-term memory test (Digit Span Test).

[0156] Blood oxygen saturation refers to the ratio of oxygenated hemoglobin to total hemoglobin in the blood. It is measured using a handheld oximeter (Wetron, model TD-8255). During this test, subjects wore a mask (to control oxygen supply) throughout the hypoxic exposure period. Blood oxygen concentrations were recorded before the test (normal air, approximately 21% oxygen) and 30 minutes after exposure (7% oxygen).

[0157] Among them, the lipid peroxidation index uses malondialdehyde (MDA), one of the end products of lipid peroxidation, as an indicator to measure lipid peroxidation. Blood is used as the sample, and the entrusted testing unit is Liren Medical Laboratory.

[0158] Among them, the TPTZ redox method is used. By taking advantage of the fact that antioxidants in human serum all have reducing ability, it can promote the reduction of trivalent iron oxide chelates (Fe3+-TPTZ) into divalent reduced iron chelates (Fe2+-TPTZ) to assess the total antioxidant capacity in the blood.

[0159] The Stroop Tess (Judgment Test) uses the time it takes to answer a question to assess judgment, meaning that shorter answer times indicate higher judgment skills. Here's how the Judgment Test works: At the start of the test, a color word appears. The font color of the word may not necessarily match the color it represents. Participants are asked to correctly answer the font color, not the color represented by the word itself. This test is repeated for 40 questions, and the total time spent answering is recorded. Shorter answer times indicate higher judgment skills.

[0160] Short-term memory is assessed using the number of correct answers and the accuracy rate. The more correct answers and the higher the accuracy rate, the better the short-term memory. Here, the digit span test is used as a short-term memory test. Participants are asked to memorize a series of numbers and then, in reverse order, produce the correct string of numbers. The number of digits gradually increases until the subject can no longer recall them correctly. The accuracy rate is calculated by dividing the number of correct answers by the total number of test questions and multiplying the result by the percentage.

[0161] 7-2. Test process:

[0162] Three subjects consumed 5 mL of ginseng extract daily for four consecutive weeks. Measurements were taken using the aforementioned device before the start of the intake (week 0, also known as the control group) and after four weeks (week 4, also known as the experimental group).

[0163] 7-3. Test results:

[0164] Please refer to Figure 7. The average blood oxygen saturation of the control group dropped from 97.7% to 88.0% after 30 minutes of simulated hypoxia, with a difference of 9.7. After four weeks of daily intake of 5 mL of ginseng extract, the average blood oxygen saturation of the three subjects dropped from 97.0% to 92.3% after 30 minutes of simulated hypoxia, with a difference of 4.7. Here, the smaller the difference, the better the hypoxia tolerance. It can be seen that after only four weeks of using the ginseng extract of the present invention, even if the subjects were in hypoxia for 30 minutes, they were still able to maintain their blood oxygen saturation above 90%, indicating that their ability to tolerate hypoxia was greatly improved. In other words, daily intake of 5 mL of ginseng extract can effectively improve hypoxia tolerance.

[0165] See Figure 8. After four weeks of daily intake of 5 mL of ginseng extract, the average lipid peroxidation index of the three subjects decreased from 1.3 (week 0) to 1.11 (week 4) while maintaining their normal daily routine. This means that daily intake of 5 mL of ginseng extract can effectively improve lipid peroxidation index by 14.58%.

[0166] See Figure 9. After four weeks of daily intake of 5 mL of ginseng extract, the average total antioxidant capacity of the three subjects increased from 0.62 (week 0) to 0.67 (week 4) while maintaining their daily routine. This means that consuming 5 mL of ginseng extract daily can effectively increase total antioxidant capacity by 8.02%.

[0167] See Figure 10. After four weeks of daily intake of 5 mL of ginseng extract, the average time taken by the three subjects to answer questions decreased from 45 seconds (week 0) to 43.3 seconds (week 4) while maintaining their daily routine. This suggests that daily intake of 5 mL of ginseng extract can effectively improve judgment.

[0168] See Figure 11. After four weeks of daily intake of 5 mL of ginseng extract, the three subjects' average number of correct answers increased from 21.7 (week 0) to 25.3 (week 4) while maintaining their normal routine. This suggests that daily intake of 5 mL of ginseng extract can effectively improve short-term memory.

[0169] See Figure 12. After four weeks of daily intake of 5 mL of ginseng extract, the three subjects' average accuracy improved from 88.5 (week 0) to 89.2 (week 4) while maintaining their daily routine. This suggests that daily intake of 5 mL of ginseng extract can effectively improve short-term memory.

[0170] Industrial Applicability

[0171] In summary, the ginseng extract of any embodiment can be used to prepare a composition for fatigue relief or cognitive enhancement. The ginseng extract of any embodiment can be used to prepare a composition for promoting mitochondrial activity, promoting skeletal muscle cell proliferation, enhancing hypoxia tolerance, increasing blood oxygen saturation, increasing lipid peroxidation (MDA), increasing total antioxidant capacity (TAC), improving hypoxia tolerance, and enhancing judgment and short-term memory.

[0172] Of course, the present invention may have many other embodiments. Without departing from the spirit and essence of the present invention, those skilled in the art may make various corresponding changes and modifications based on the present invention, but these corresponding changes and modifications should all fall within the scope of protection of the claims of the present invention.

Claims

1. Use of ginseng extract for producing an anti-fatigue composition, wherein ginseng extract is obtained from Panax ginseng CA Meyer using water as an extractant.

2. The use according to claim 1, wherein the ginseng extract enhances mitochondrial activity.

3. The use according to claim 1, wherein the ginseng extract promotes the proliferation of skeletal muscle cells.

4. Use of ginseng extract for obtaining a composition for improving cognitive functions, in which the ginseng extract is obtained by aqueous extraction from Panax ginseng CA Meyer.

5. Use according to paragraph 4, wherein the ginseng extract increases resistance to hypoxia.

6. The use according to item 4, wherein the ginseng extract improves the level of oxygen saturation of the blood.

7. The use according to claim 4, wherein the ginseng extract improves lipid peroxidation by increasing the level of malondialdehyde.

8. The use according to claim 4, wherein the ginseng extract increases the total antioxidant capacity (TAC).

9. The use according to any one of claims 1 to 8, wherein the composition is a food composition and contains at least 5 ml of ginseng extract.

10. The use according to any one of claims 1 or 4, wherein the ginseng extract contains at least 13.3 mg of ginsenoside Rg3.