Application of ginseng extract in preparation of composition for resisting fatigue or improving cognitive function performance

Abstract

The invention relates to application of a ginseng extract, in particular to application of the ginseng extract to preparation of a composition for resisting fatigue or improving cognitive function performance. Wherein the ginseng extract is extracted from the root of the ginseng (Panax ginseng C.A.Meyer) by taking water as a solvent, and the ginseng extract is prepared from the root of the ginseng (Panax ginseng C.A.Meyer).

Description

Technical Field

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

[0002] Fresh ginseng just dug out of the soil contains a lot of water and is not easy to preserve. It is rarely seen on the market and is only occasionally used in cooking near the production area. Ginseng usually undergoes different processing steps to make fresh ginseng into red ginseng, white ginseng or Tai Chi ginseng.

[0003] Although it is generally believed that red ginseng, white ginseng and Taiji ginseng have better effects and higher saponin content than fresh ginseng, red ginseng, white ginseng and Taiji ginseng have different characteristics and applicability, and the quality of red ginseng, white ginseng and Taiji ginseng varies due to different processing methods. Summary of the invention

[0004] 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, which can be used to prepare an anti-fatigue or cognitive function performance enhancement composition.

[0005] In some embodiments, a use of a ginseng extract is for preparing an anti-fatigue composition, wherein the ginseng extract is extracted from ginseng (Panax ginseng CA Meyer) using water as a solvent.

[0006] In some embodiments, ginseng extract promotes mitochondrial activity.

[0007] In some embodiments, ginseng extract promotes skeletal muscle cell proliferation.

[0008] In some embodiments, a ginseng extract is used to prepare a composition for improving cognitive function performance, wherein the ginseng extract is extracted from ginseng (Panax ginseng CA Meyer) using water as a solvent.

[0009] In some embodiments, ginseng extract is used to enhance hypoxia tolerance.

[0010] In some embodiments, ginseng extract is used to increase blood oxygen saturation.

[0011] In some embodiments, the ginseng extract increases the lipid peroxidation marker MDA.

[0012] In some embodiments, ginseng extract increases total antioxidant capacity (TAC).

[0013] 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.

[0014] In some embodiments, the ginseng extract has at least 13.3 mg of ginsenoside Rg3 per 100 ml.

[0015] In summary, the ginseng extract of any embodiment can be used to prepare a composition for anti-fatigue or improving cognitive function performance. The ginseng extract of any embodiment can be used to prepare a composition for promoting mitochondrial activity, promoting skeletal muscle cell proliferation, improving hypoxia tolerance, improving blood oxygen saturation, improving lipid peroxidation index MDA, improving total antioxidant capacity TAC, improving hypoxia tolerance, improving judgment and short-term memory. The ginseng extract of any embodiment has at least 13.3 mg of ginsenoside Rg3 per 100 ml. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is the fingerprint analysis result diagram.

[0017] Figure 2 This is a graph showing the test results for the relative ginsenoside Rg3 content.

[0018] Figure 3 This is a graph showing the relative total alkaloid content test results.

[0019] Figure 4 This is a graph showing the relative antioxidant efficiency test results.

[0020] Figure 5 This is a graph showing the relative mitochondrial membrane potential test results.

[0021] Figure 6 This is a graph of relative bone cell proliferation test results.

[0022] Figure 7 This is a graph showing the test results of blood oxygen saturation concentration in a human trial of ginseng extract.

[0023] Figure 8 This is a graph showing the test results of lipid peroxidation indicators in human trials of ginseng extract.

[0024] Fig. 9 This is a graph showing the test results of the total antioxidant capacity of ginseng extract in human trials.

[0025] Fig.10 This is a graph showing the time spent answering questions in a human trial of ginseng extract.

[0026] Fig.11 This is a graph showing the number of correct answers in a human trial of ginseng extract.

[0027] Fig.12 This is a graph showing the accuracy of answers in a human trial of ginseng extract.

[0028] Wherein, the reference numerals are:

[0029] Rg3: Ginsenoside Rg3 DETAILED DESCRIPTION

[0030] 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.

[0031] As used herein, "ginseng" refers to the fleshy root of fresh ginseng (scientific name: Panax ginseng), also known as Korean ginseng. In some embodiments, ginseng refers to the root of ginseng that has not been dried or cooked. In some embodiments, ginseng is a root that has been frozen or refrigerated within seven days of harvest, which may further include roots that are whole, chopped, diced, ground, milled, or otherwise processed to affect the size and physical integrity of the raw material.

[0032] In some embodiments, the ginseng is ginseng produced in Northeast China. In some embodiments, the ginseng is ginseng that has been artificially cultivated for less than five years.

[0033] In some embodiments, ginseng extract is obtained by extracting ginseng using water as a solvent. In some embodiments, ginseng extract is obtained by heating ginseng using water as a solvent. In some embodiments, heating extraction refers to heating the mixed solution after 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 may be 85°C and the extraction time may be 60 minutes. Here, extraction is to soak ginseng in heated water.

[0034] 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.

[0035] In some embodiments, the ginseng extract is obtained by extracting ginseng with water as a solvent to obtain a primary extract, and then filtering out solids in the primary extract through a sieve. For example, the sieve can be a 400 mesh sieve.

[0036] In some embodiments, the ginseng extract has at least 13.3 mg of ginsenoside Rg3 per 100 ml.

[0037] 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. That is, ginseng extract can promote muscle cell force conversion.

[0038] In some embodiments, ginseng extract can be used to prepare a composition for improving cognitive function performance. In some embodiments, ginseng extract improves cognitive function by improving hypoxia tolerance. In some embodiments, ginseng extract improves cognitive function by improving blood oxygen saturation. In some embodiments, ginseng extract improves cognitive function by improving lipid peroxidation index MDA. In some embodiments, ginseng extract improves cognitive function by improving total antioxidant capacity TAC.

[0039] In some embodiments, the composition is a food composition, and the composition comprises at least 5 mL of ginseng extract.

[0040] In some embodiments, the aforementioned composition may be a medicine. In other words, the medicine comprises an effective amount of ginseng extract.

[0041] 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. These 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.

[0042] 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 via a parenteral route selected from the group consisting of subcutaneous injection, intraepidermal injection, intradermal injection, and intralesional injection.

[0043] In some embodiments, the pharmaceutical product may further include a pharmaceutically acceptable carrier widely used in drug manufacturing technology. For example, the pharmaceutically acceptable carrier may include one or more of the following agents: solvent, buffer, emulsifier, suspending agent, decomposer, disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent, gelling agent, preservative, wetting agent, lubricant, absorption delaying agent, liposome and the like. The selection and amount of these agents are within the professional qualities and routine technical scope of ordinary technicians in the field.

[0044] 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.

[0045] In some embodiments, the aforementioned composition may be an edible composition for non-medical purposes. In some embodiments, the edible composition may be made into a food product or may be a food additive, which means that the food product is prepared by adding the composition to the food material during preparation by a conventional method, or is added during the production process of the food product. Here, the food product may be a product prepared with an edible material for human or animal consumption.

[0046] 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.

[0047] Example 1: Preparation of test samples

[0048] 1-1. Preparation of ginseng extract: The whole fleshy root of ginseng (artificially cultivated for less than five years, fresh ginseng frozen within seven days after harvest) is crushed into powder, and then sieved with a 12mm mesh screen to obtain ginseng powder with a size of less than 12mm. Then, using water as a solvent, the ginseng powder and water are mixed in a weight ratio of 1:5 and extracted at 85°C for 1 hour to obtain a primary extract. Subsequently, the extracted primary extract is passed through a 400 mesh screen to obtain a ginseng extract.

[0049] 1-2. Preparation of white ginseng extract: After the white ginseng is crushed into powder, it is sieved with a 12mm mesh screen to obtain white ginseng powder with a size of less than 12mm. Then, using water as a solvent, the white ginseng powder is mixed with water in a weight ratio of 1:5 and extracted at 85°C for 1 hour to obtain a primary extract. Subsequently, the extracted primary extract is passed through a 400 mesh screen to obtain a white ginseng extract. Here, the white ginseng is obtained by peeling fresh ginseng and drying it with hot air at 45°C to 50°C.

[0050] 1-3. Preparation of red ginseng extract: After the red ginseng is crushed into powder, it is sieved with a 12mm mesh screen to obtain red ginseng powder with a size of less than 12mm. Then, 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 obtain a primary extract. Subsequently, the extracted primary extract is passed through a 400 mesh screen to obtain a 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.

[0051] Example 2: Component Analysis of Ginseng Extract

[0052] In order to understand the differences in the components of ginseng compared to white ginseng and red ginseng obtained by different processing methods, the content of ginsenoside Rg3 in ginseng, white ginseng and red ginseng and their fingerprints are analyzed below.

[0053] 2-1. Equipment, instrumentation, setting methods and equipment sources are described as follows:

[0054] Nuclear Magnetic Resonance Spectrometer (NMR) was used for 1D and 2D spectra. Ascend 400 MHz, Bruker Co., Germany, chemical shift is expressed as δ, and the unit is ppm.

[0055] Mass Spectrometer (MS) tandem mass spectrometry-two-dimensional ion trap tandem Fourier transform mass spectrometry and ESI-MS / MS: measured using Bruker amaZon SL system, the unit is m / z.

[0056] Medium pressure liquid chromatography (MPLC): R +,Teledyne ISCO,Lincoln,NE,High Performance Liquid Chromatography (HPLC): High Performance Liquid Chromatography (HPLC) is Agilent 1200 series; Degasser is Agilent Vacuum Degasser 1322A; Flushing solvent delivery is Agilent Quaternary Pump G1311A; Multiple Wavelength Detector (MWD) is Agilent G1314B; Diode Array Detector (DAD) is Agilent 1260 Infinity DAD VL G1315D, detection wavelengths are 210nm, 280nm, 320nm, 365nm (Agilent Germany).

[0057] Analytical column: μm C18(2) (250 x 10 mm, Phenomenex, USA).

[0058] Column chromatography filling materials: Sephadex LH-20 (Pharmacia, Piscataway, NJ, USA). Diaion HP-20 (Mitsubishi Chemical Co., Japan) Merck Kieselgel 60 (40-63um, Art. 9385) Merck RP-18(40-63um,Art.0250).

[0059] Thin-layer chromatography (TLC) was performed using TLC aluminium sheets (Silica gel 60F254, 0.25 mm, Merck, Germany) and TLC aluminium sheets (RP-18F254-S, 0.25 mm, Merck, Germany).

[0060] Solvents used and their sources: n-hexane, ethylacetate, 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%), dimethyl sulfoxide-d6 (deuteration degree>99.9%) (Merck, Taiwan).

[0061] 2-2. Analysis process:

[0062] First, the ginseng extract, white ginseng extract and red ginseng extract prepared in Example 1 were analyzed by high performance liquid chromatography (HPLC) to obtain their respective fingerprints, such as Figure 1 shown.

[0063] The relevant settings of the above liquid chromatography are: the solvents used are methanol (A) and water (B), and 0.1% formic acid is additionally added, 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 with a 0.22 μm polyvinylidene fluoride membrane before injection.

[0064] The stripping conditions were as follows: 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.

[0065] At the same time, quantitative analysis of ginsenoside Rg3 was also performed on ginseng extract, red ginseng extract and white ginseng extract. First, a standard curve was prepared using ginsenoside Rg3 standard compound. Here, ginsenoside Rg3 standard compound was purchased from Merck, Taiwan, China (CAS No. 14197-60-5, purity above 98%).

[0066] Preparation of standard curve: 10 mg of ginsenoside Rg3 was weighed accurately and diluted to a 10 ml volumetric flask with methanol / water = 1 / 1 (volume ratio), which is a 100 mg / 100 ml ginsenoside Rg3 standard solution. Ginsenoside Rg3 calibration curve: Ginsenoside Rg3 solutions with concentrations of 40, 20, 10, 5, and 1 mg / 100 ml were prepared respectively, HPLC analysis column: Mightysil RP-18GP 250 (250x4.6mm, 5μm, Kanto, Tokyo, Japan), detection wavelength 210nm. The peak areas of 40, 20, 10, 5, and 1 mg / 100 ml were 2206.5, 1108.1, 477.7, 230.4, and 48.1, respectively. Linear regression gave a standard curve of ginsenoside Rg3 concentration (x-axis) - peak area (y-axis): y = 43.84x + 16.275 (r^2 = 0.9998).

[0067] According to the above fingerprint, the peak areas of ginseng extract, red ginseng extract and white ginseng extract are 599.4, 208.7 and 92.1 respectively. Through the standard curve and calculation, it can be known that the content of ginsenoside Rg3 in each extract is 13.3 mg / 100 ml, the content of ginsenoside Rg3 in red ginseng extract is 4.389 mg / 100 ml, and the content of ginsenoside Rg3 in white ginseng extract is 1.792 mg / 100 ml. After conversion to relative percentage, Figure 2 shown.

[0068] 2-3. Test results

[0069] Reference Figure 1 The peak at 27 minutes represents the bioactive substance, which is ginsenoside Rg3. Figure 1 It is clear that the peak of ginseng extract at 27 minutes is the highest, which means that the ginseng extract has the highest content of ginsenoside Rg3. The fingerprint of red ginseng extract also has a small peak near 27 minutes, which means that although red ginseng extract also contains ginsenoside Rg3, its content is less than that of ginseng extract. White ginseng extract also has a small peak near 27 minutes, which means that its ginsenoside Rg3 is not much.

[0070] Reference Figure 2 The value of ginseng extract (experimental group) was regarded as 100%. Compared with ginseng extract, the relative content of ginsenoside Rg3 in red ginseng extract (control group B) was 13.0%, and the relative content of ginsenoside Rg3 in white ginseng extract (control group A) was 33.0%.

[0071] Example 3: Total alkaloid content test

[0072] 3-1. Material description:

[0073] 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).

[0074] Standard bismuth nitrate (Bi(NO3)3.5H2O) solution: Dissolve 10 mg of bismuth nitrate in 5 mL of concentrated nitric acid, and then dilute to 100 mL with distilled water.

[0075] 3% thiourea solution: prepared by dissolving 3 g of thiourea in 100 ml of distilled water.

[0076] 1% disodium sulfide solution: prepared by dissolving 1 g of disodium sulfide in 100 mL of distilled water.

[0077] 3-2. Preparation of standard curve:

[0078] First, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL and 9 mL of standard bismuth nitrate solution were transferred into individual 10 mL standard flasks and diluted to 10 mL with distilled water to dilute the standard bismuth nitrate solution to 9 different concentrations of bismuth nitrate solutions as standard samples. 1 mL was taken from each of the 9 different concentrations of bismuth nitrate solutions (standard samples), and 5 mL of thiourea solution was added to each standard sample to form 9 yellow standard samples. The absorbance values ​​of these 9 yellow standard samples were then measured at 435 nm relative to the colorless reagent blank.

[0079] 3-3. Test process:

[0080] 5 mL of sample was taken from each group, and HCl was added to maintain the pH value at 2 to 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.

[0081] Each group was added with 2 mL of DR solution and centrifuged to form a centrifuge with precipitation. At the same time, the centrifuge was checked by adding DR solution to the centrifuge to see if the centrifuge was completely precipitated.

[0082] After centrifugation, the liquid of the centrifuge was decanted, and the precipitate was taken out and further washed with alcohol.

[0083] 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 2 more drops of disodium sulfide.

[0084] The residue was dissolved in 2 mL of concentrated nitric acid, with warming if necessary to achieve complete dissolution.

[0085] This solution was diluted with distilled water to 10 mL in a standard flask. Then, 1 mL was taken out, 5 mL of thiourea solution was added thereto, and the absorbance was measured at 435 nm.

[0086] The absorbance of the sample is compared with the standard curve to obtain the total alkaloid content of the sample. The results are displayed on Figure 3 .

[0087] 3-4. Test results

[0088] Continued reference Figure 3 The values ​​of the experimental group were regarded as 100%. Compared with 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%.

[0089] Example 4: Antioxidant efficiency test

[0090] 4-1. Materials and equipment description:

[0091] Cell line: Skeletal muscle cells C2C12 (hereinafter referred to as C2C12 cells) were used. C2C12 cells were purchased from the American Type Culture Collection (American Type Culture Collection, ) of C2C12 cell line (ATCC CRL-1772 TM ).

[0092] Cell culture medium: Minimum essential medium (MEM, hereinafter referred to as MEM medium) (Gibco, No. 11095080) supplemented with 10% fetal bovine serum (FBS; Gibco, No. 10437-028), 1% penicillin-streptomycin (Antibiotic-Antimycotic) (Gibco, Cat. 15140122) and 1 mM sodium pyruvate (sodium pyruvate, Gibco, No. 11360-070).

[0093] Phosphate buffered saline (PBS solution): purchased from Gibco, product number 10437-028.

[0094] DCFH-DA solution: 2,7-dichloro-dihydro-fluoresceindiacetate (DCFH-DA; product number SI-D6883, purchased from Sigma) was dissolved in dimethylsulfoxide (DMSO, purchased from Sigma, product number SI-D6883-50MG) to prepare a 5 μg / mL DCFH-DA solution.

[0095] Flow cytometry: BD Accuri C6 Plus.

[0096] Hydrogen peroxide (H2O2): purchased from Sigma-Aldrich, product model 95299-1L.

[0097] Trypsin-EDTA: 10X Trypsin-EDTA (purchased from Gibco) was diluted 10 times with 1X PBS solution.

[0098] 4-2. Test process:

[0099] First, take 2×10 5 C2C12 cells were seeded into a 6-well cell culture dish at a density of 1000 cells / mL, 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.

[0100] After the C2C12 cells adhered to the bottom of the cell culture dish, the C2C12 cells were divided into an experimental group, a control group A, and a control group B. The cell culture medium of each group was removed and replaced with 500 μl / well test culture medium, and then placed at 37° C. for 1 hour.

[0101] The test medium of the experimental group was a cell culture medium containing 0.25% of the ginseng extract obtained in Example 1 and 1 mM hydrogen peroxide. The test medium of the control group A was a cell culture medium containing 0.25% of the white ginseng extract obtained in Example 1 and 1 mM hydrogen peroxide. The test medium of the control group B was a cell culture medium containing 0.25% of the red ginseng extract obtained in Example 1 and 1 mM hydrogen peroxide.

[0102] After adding the DCFH-DA solution, the cells were treated at 37°C for 15 minutes.

[0103] After adding 10 μL of hydrogen peroxide to each well, the cells were treated at 37°C for 1 hour.

[0104] Remove the liquid in the well plate, rinse the cells with PBS, and repeat the above steps twice.

[0105] Remove the liquid in the well plate, add 200 μL / well 1X trypsin and incubate for 5 minutes in the dark.

[0106] The reaction was terminated with culture medium, transferred to a 1.5 mL microcentrifuge tube, and centrifuged at 400 x g for 10 minutes.

[0107] The supernatant was removed, the cells were washed with PBS, and centrifuged at 400 x g for 10 minutes.

[0108] Remove the supernatant and resuspend the cells in 1 mL of PBS per tube.

[0109] The fluorescence signal of DCFH-DA was measured by flow cytometry (set as excitation light: 450-490nm; scattered light: 510-550nm). Figure 4 As shown, the antioxidant efficiency shown in the figure is presented in relative ratios.

[0110] 4-3. Test results

[0111] See also Figure 4 . The value of control group B is regarded as 100%. Compared with control group B, the relative oxidation efficiency of the experimental group is as high as 279.2%. The relative oxidation efficiency of control group A is 152.9%. In other words, ginseng extract can significantly promote skeletal muscle cells to resist the oxidative damage caused by hydrogen peroxide, and it is significantly improved by 2.7 times compared with red ginseng.

[0112] Example 5: Mitochondrial activity test

[0113] Mitochondria are important organelles for cell metabolism and energy supply. Low mitochondrial activity usually means that the cell is in a state of fatigue. This test uses flow cytometry to evaluate the changes in mitochondrial activity of skeletal muscle cells C2C12 after being treated with different samples. In the test, the aggregation ability of JC-1 is used as an indicator of mitochondrial activity. In healthy mitochondria with normal membrane potential, JC-1 exists in the form of polymers. When the mitochondrial membrane potential decreases, JC-1 will become a monomer, which can be used as an indicator for evaluating mitochondrial activity.

[0114] 5-1. Materials and equipment description:

[0115] Experimental cell line: Skeletal muscle cells C2C12 (hereinafter referred to as C2C12 cells) were used. C2C12 cells were purchased from American Type Culture Collection (American Type Culture Collection, ) of C2C12 cell line (ATCC CRL-1772 TM ).

[0116] Culture medium: Dulbecco's modified Eagle's medium was used, purchased from Gibco, USA, model Gat.11965-092. 10% fetal bovine serum was added, purchased from Gibco, USA, model Gat.10437-028. 1% antibiotics were added, purchased from Gibco, USA, model Gat.15240-062.

[0117] MitoScreen Flow Cytometry Mitochondrial Membrane Potential Detection Kit (BD; Cat. BDB551302) Mitochondrial Membrane Potential Detection Kit (BDTM MitoScreen (JC-1) kit, Model 551302). The mitochondrial membrane potential detection kit contains JC-1 dye (lyophilized) and 10X assay buffer. Before use, dilute the 10X assay buffer 10 times with 1X PBS to form a 1X assay buffer. Add 130μL DMSO to the JC-1 dye (lyophilized) to form a JC-1 stock solution. Then, dilute the JC-1 stock solution with 1X assay buffer to form a JC-1 working solution (i.e., JC-1 mitochondrial specific dye). The dilution ratio is 1:100 for the JC-1 stock solution to the 1X assay buffer.

[0118] Trypsin: 10X Trypsin-EDTA (purchased from Gibco) was diluted 10 times in 1X PBS solution.

[0119] Flow cytometer: Flow cytometry (purchased from Beckman).

[0120] 5-2. Test process:

[0121] First, take a 6-well culture plate and inoculate each well with 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 the test medium, and then placed at 37°C for 24 hours.

[0122] The test medium of the experimental group was a cell culture medium containing 0.25% of the ginseng extract obtained in Example 1. The test medium of the control group A was a cell culture medium containing 0.25% of the white ginseng extract obtained in Example 1. The test medium of the control group B was a cell culture medium containing 0.25% of the red ginseng extract obtained in Example 1.

[0123] Remove the test medium from the culture dish and rinse twice with 1X PBS solution.

[0124] 200 μL of trypsin was added to each well and reacted in the dark for 5 minutes. After the reaction, cell culture medium was added to terminate the reaction. The cells and cell culture medium in each well were collected into corresponding 1.5 mL centrifuge tubes, and the centrifuge tubes containing cells and cell culture medium were centrifuged at 400 x g for 10 minutes.

[0125] After centrifugation, remove the supernatant and resuspend the cells in 1 mL of 1X PBS or transfer the cell pellet to a 15 mL centrifuge tube to form a cell suspension.

[0126] Centrifuge the tube containing the cell suspension at 400 x g for 5 min.

[0127] After centrifugation, the supernatant in each centrifuge tube was removed and 100 μL of JC-1 working reagent was added to each centrifuge tube.

[0128] The cell pellet in each centrifuge tube was vortexed with JC-1 working reagent and incubated for 15 minutes in the dark.

[0129] After 15 minutes, each tube was centrifuged at 400 x g for 5 minutes.

[0130] 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.

[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 cells were resuspended in 500 μL of 1X PBS to obtain the cell solution to be tested.

[0133] The membrane potential of the mitochondria in the test fluid of each well was measured by flow cytometry to analyze the mitochondrial activity. Since the experiment was repeated three times, the results of the three repeated experiments in each group were averaged to obtain the average value. Then, the average value of the control group A was taken as 1 for the relative JC-1 aggregation, and the average values ​​of the experimental group and the control group B were converted into the relative JC-1 aggregation, such as Figure 5 shown.

[0134] 5-3. Test results

[0135] See also Figure 5 , the value of control group A is regarded as 1. Compared with control group A, the relative mitochondrial membrane potential of the experimental group is as high as 12.77 times. The relative mitochondrial membrane potential of control group B is 6.52. In other words, ginseng extract can significantly promote skeletal muscle cells to resist the decline in mitochondrial activity caused by excessive fatigue.

[0136] Example 6: Skeletal muscle cell proliferation test

[0137] People with more muscle tissue have greater endurance and can work longer without feeling tired.

[0138] 6-1. Materials and equipment description:

[0139] Experimental cell line: Skeletal muscle cells C2C12 (hereinafter referred to as C2C12 cells) were used. C2C12 cells were purchased from American Type Culture Collection (American Type Culture Collection, ) of C2C12 cell line (ATCC CRL-1772 TM ).

[0140] Culture medium: Dulbecco's modified Eagle's medium was used, purchased from Gibco, USA, model Gat.11965-092. 10% FBS fetal bovine serum was added, purchased from Gibco, USA, model Gat.10437-028. 1% antibiotics were added, purchased from Gibco, USA, model Gat.15240-062.

[0141] Cell proliferation kit: ELISA, BrdU (purchased from Roche; model 11647229001). It includes EdU reagent, FixDenat reagent, BrdU-POD reagent (Remove the antibody conjugate and rinse well three times with

[0142] 200-300μl washing solution), washing solution, substrate solvent.

[0143] 6-2. Test process:

[0144] First, 5000 cells were inoculated 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: only culture medium was added and cultured at 37°C for 24 hours. Positive control group: additionally 10% FBS fetal bovine serum was added. Experimental group: cell culture medium containing 0.25% of the ginseng extract obtained in Example 1 was added. Control group A: cell culture medium containing 0.25% of the white ginseng extract obtained in Example 1 was added. Control group B: cell culture medium containing 0.25% of the red ginseng extract obtained in Example 1 was added.

[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 the FixDenat reagent was added and treated at room temperature for 30 minutes. The FixDenat reagent was removed and washed once with PBS buffer. BrdU-POD reagent was added and 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 300rpm for 1 minute, the fluorescence signal of the cell fluid to be tested in each well was measured by flow cytometry (excitation light: 450nm) and the number of cells was calculated.

[0147] The results were used to perform student t-test using Excel software to determine whether there was a statistically significant difference between the two sample groups. Figure 6 As shown, "*" represents a p value less than 0.05, "**" represents a p value less than 0.01, and "***" represents a p value less than 0.001. When there are more "*", it means that the difference relative to the blank group is more significant.

[0148] 6-3. Test results

[0149] refer to Figure 6 When the number of skeletal muscle cells measured in the blank group is regarded as 100%, the number of skeletal muscle cells in the positive control group is 126.22% relative to the blank group, while the number of skeletal muscle cells in the experimental group is 106.7% relative to the blank group. Compared with the blank group, the experimental group has a statistically significant difference.

[0150] However, white ginseng extract (control group A) and red ginseng extract (control group B) not only did not have the effect of skeletal muscle hyperplasia, but also reduced the number of skeletal muscle cells. It can be seen that ginseng extract can effectively promote the growth of skeletal muscle cells, thereby improving the anti-fatigue efficiency of individuals.

[0151] Example 7: Human Experimentation

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

[0153] Subjects: 3 subjects. The age of each subject was between 20 and 55 years old. The subjects self-reported that they were easy to fatigue, often had brain fog, and were healthy adults with no other underlying diseases.

[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] Among them, blood oxygen saturation concentration refers to the ratio of oxygenated hemoglobin to total hemoglobin in the blood, which is detected using a blood oximeter (Wetron handheld, model TD-8255). During this test, the subjects wore masks (to control the oxygen supply) throughout the hypoxia exposure, and recorded the blood oxygen concentration before the start (normal air, oxygen content of about 21%) and 30 minutes after exposure to hypoxia (oxygen content of 7%).

[0157] Among them, the lipid peroxidation index uses Malondialdehyde (MDA), one of the end products of lipid peroxidation, as a pointer to measure lipid peroxidation. Blood is used as a sample, and the entrusted inspection 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 have reducing ability, the trivalent iron oxide chelate (Fe3+-TPTZ) can be reduced to a divalent reduced iron chelate (Fe2+-TPTZ) to evaluate the total antioxidant capacity in the blood.

[0159] Among them, the judgment test (Stroop Tess) uses the time it takes to answer questions for evaluation, which means that the shorter the time it takes to answer questions, the higher the judgment. Here, the judgment test method test method: After the test begins, a color word will appear, but the font color of the word may not match the color it represents. The subject needs to correctly answer the font color, not the color represented by the word itself. The test is continuous for 40 questions, and the total time spent on answering is recorded. The shorter the answering time, the higher the judgment.

[0160] Among them, the short-term memory test uses the number of correct answers and the accuracy of the answers to evaluate, that is, the more correct answers, the higher the accuracy of the answers, the better the short-term memory. Here, the digit span test is used as a short-term memory test. The subjects need to remember multiple numbers and then answer the correct string of numbers in reverse order according to the display order. The number of digits will gradually increase until the subject can no longer recall correctly. In addition, the number of correct answers is divided by the total number of test questions and multiplied by the percentage, and the average is calculated to get the accuracy of the answers.

[0161] 7-2. Test process:

[0162] Three subjects were asked to take 5 mL of ginseng extract every day for four weeks. Measurements were performed using the above equipment before the start of intake (i.e., week 0, also known as the control group) and after four weeks of drinking (i.e., week 4, also known as the experimental group).

[0163] 7-3. Test results:

[0164] See also Figure 7 . The average blood oxygen saturation of the control group dropped from 97.7% to 88.0% after 30 minutes of simulated hypoxia, and the difference between the two was 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, and the difference between the two was 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 could still maintain their blood oxygen saturation above 90%, indicating that their hypoxia tolerance was greatly improved. That is, daily intake of 5 mL of ginseng extract can effectively improve hypoxia tolerance.

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

[0166] See also Fig. 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. In other words, daily intake of 5 mL of ginseng extract can effectively increase the total antioxidant capacity by 8.02%.

[0167] See also Fig.10 After taking 5 mL of ginseng extract daily for four weeks, 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 means that taking 5 mL of ginseng extract daily can effectively improve judgment.

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

[0169] See also Fig.12After taking 5 mL of ginseng extract daily for four weeks, the average accuracy of the three subjects increased from 88.5 (week 0) to 89.2 (week 4) while maintaining their daily routine. This means that taking 5 mL of ginseng extract daily can effectively improve short-term memory.

[0170] In summary, the ginseng extract of any embodiment can be used to prepare a composition for anti-fatigue 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, improving hypoxia tolerance, improving blood oxygen saturation, improving lipid peroxidation index MDA, improving total antioxidant capacity TAC, improving hypoxia tolerance, and improving judgment and short-term memory.

[0171] Of course, the present invention may have many other embodiments. Without departing from the spirit and essence of the present invention, technicians familiar with the field may make various corresponding changes and deformations based on the present invention, but these corresponding changes and deformations should all fall within the scope of protection of the claims of the present invention.

Claims

1. A use of a ginseng extract for preparing an anti-fatigue composition, wherein the ginseng extract is extracted from ginseng (Panax ginseng CA Meyer) using water as a solvent.

2. The use as claimed in claim 1, wherein the ginseng extract promotes mitochondrial activity.

3. The use as claimed in claim 1, wherein the ginseng extract promotes skeletal muscle cell proliferation.

4. A use of a ginseng extract for preparing a composition for improving cognitive function performance, wherein the ginseng extract is extracted from ginseng (Panax ginseng CA Meyer) using water as a solvent.

5. The use as claimed in claim 4, wherein the ginseng extract is used to enhance the ability to tolerate hypoxia.

6. The use as claimed in claim 4, wherein the ginseng extract is used to increase blood oxygen saturation.

7. The use as claimed in claim 4, wherein the ginseng extract increases the lipid peroxidation index MDA.

8. The use as claimed in claim 4, wherein the ginseng extract enhances 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 the composition comprises at least 5 mL of the ginseng extract.

10. The use according to claim 1 or 4, wherein the ginseng extract contains 13.3 mg of ginsenoside Rg3 per 100 ml.

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