TW202110466A - Buckwheat sprout extract, use thereof for preparing composition for enhancing cellular antioxidant and/or keeping liver health, and preparing method thereof - Google Patents

Abstract

A use of buckwheat sprout extract is for preparing a composition. The composition has one or more of the following functions: enhancing cellular antioxidant activity, enhancing metabolic activity, and enhancing intracellular glutathione (GSH) synthesis.

Description

Buckwheat seedling extract, its use for preparing cell antioxidant composition and/or liver health care composition and preparation method thereof

The present invention relates to the use of the extract of buckwheat seedlings, in particular to the use of the buckwheat seedlings to prepare a composition for improving cell antioxidant and/or liver health care.

Since the rise of the concept of organic and natural diets, biotech companies and food companies have actively invested in the research and development of products related to natural plants. In order to make plant-related products have a basis for scientific verification of health benefits, plant active ingredient analysis and efficacy evaluation have become key items in product development.

Among many natural plants, buckwheat can grow in poor, acidic soil without excessive nutrients. Common uses of buckwheat are, for example, making buckwheat flour, making wine, or making tea beverages. If the other effects of buckwheat can be developed and further effective use of buckwheat, it will be beneficial to the health and well-being of the whole people.

In one embodiment, a buckwheat seedling extract is used to prepare a composition for enhancing the antioxidant activity of cells.

In one embodiment, a use of the extract of buckwheat sprouts is to prepare a liver health care composition.

In some embodiments, the buckwheat seedling extract is used to enhance the antioxidant and metabolic activities of cells.

In some embodiments, the aforementioned buckwheat seedling extract is used to enhance the synthesis of glutathione (GSH) in cells.

In some embodiments, the aforementioned buckwheat seedling extract is obtained by solvent extraction of buckwheat seedlings, and the buckwheat seedlings are seedlings that grow for 6 to 12 days after the buckwheat seeds germinate.

In some embodiments, the aforementioned solvent is water, the weight ratio of the solvent to the buckwheat seedlings is 2-5:1, the extraction temperature of the buckwheat seedlings is between 80°C and 90°C, and the extraction time of the buckwheat seedlings is 120°C. minute.

In some embodiments, the aforementioned buckwheat sprouts extract is 4.7°Bx to 5.3°Bx.

In some embodiments, the flavonoid content of the aforementioned buckwheat seedling extract is about 815 μg/mL.

In some embodiments, the aforementioned buckwheat seedling extract contains at least one of the following biologically active substances: Guanosine, Vitexin, Isovitexin, Rutin , different Oriental hormone (Isoorientin) and luteolin-8-C- glucoside (luteolin-8- C -glucoside).

In some embodiments, the aforementioned guanosine is used to enhance the synthesis of glutathione in the cell.

In one embodiment, a method for preparing a buckwheat seedling extract includes: extracting water with a weight ratio of 2-5:1 and a buckwheat seedling at a temperature of 80 degrees to 90 degrees for 60 minutes to 180 minutes to obtain An extracting stock solution; and filtering the extracting stock solution to obtain a buckwheat seedling extract.

In some embodiments, the buckwheat seedlings used for extraction are seedlings grown for 6 to 12 days after the buckwheat seeds germinate.

In some embodiments, the preparation method of the buckwheat seedling extract further includes: concentrating the buckwheat seedling extract under reduced pressure until the degree Brix of the buckwheat seedling extract is 4.7 to 5.3 (ie, 4.7°Bx To 5.3°Bx) to obtain a concentrated buckwheat seedling extract.

In some embodiments, the aforementioned concentration step under reduced pressure is performed at 50°C to 60°C.

In some embodiments, the total flavonoid content of the concentrated buckwheat seedling extract is 815 μg/mL.

In some embodiments, the buckwheat seedling extract obtained after filtration includes at least one of the following biologically active substances: Guanosine, Vitexin, Isovitexin, Rutin (Rutin), iso-East hormone (Isoorientin) and luteolin-8-C- glucoside (luteolin-8- C -glucoside).

In summary, in any embodiment, the use of the buckwheat seedling extract for preparing the cell antioxidant composition and/or the liver health care composition and the preparation method of the buckwheat seedling extract are suitable for providing buckwheat seedlings Leaching liquid or its composition. Wherein, the provided buckwheat seedling extract or its composition has one or more of the following functions: enhancing cellular antioxidant activity, enhancing metabolic activity, and enhancing intracellular glutathione (GSH) synthesis.

Regarding the concentration symbol "wt%" used in this article usually refers to weight percent concentration, and the concentration symbol "vol%" usually refers to volume percent concentration. Regarding the term "extraction" mentioned in this article, it refers to the method of immersing the plant (solid) in a solvent (liquid) to extract certain components of the plant, and the term "extracting solution" refers to the method of extracting certain components of the plant (Solid) The sap produced by immersing certain components of plants in a solvent (liquid). It should be clear that the term "extraction" mentioned in this article can be used interchangeably with the term "extraction", and the term "biologically active substance" mentioned in this article can be used interchangeably with the term "compound".

In one embodiment, a buckwheat seedling extract is used to prepare a composition for enhancing the antioxidant activity of cells.

In one embodiment, a use of the extract of buckwheat sprouts is to prepare a liver health care composition.

In some embodiments, the buckwheat seedling extract is used to enhance the antioxidant and metabolic activities of cells.

In some embodiments, the buckwheat seedling extract is used to enhance the synthesis of glutathione (GSH) in cells.

In some embodiments, the buckwheat seedling extract contains at least one of the following biologically active substances: Guanosine, Vitexin, Isovitexin, Rutin, East hormone (Isoorientin) and luteolin-8-C- glucoside (luteolin-8- C -glucoside).

Among them, guanosine can be used to enhance the synthesis of glutathione in cells.

In some embodiments, the structural formula of guanosine is as shown in Formula 1.

式1

Formula 1

In some embodiments, the structural formula of vitexin is as shown in Formula 2.

式2

Formula 2

In some embodiments, the structural formula of isovitexin is as shown in Formula 3.

式3

Formula 3

In some embodiments, the structural formula of rutin is as shown in Formula 4.

式4

Formula 4

In some embodiments, the structural formula of isoorientin is as shown in Formula 5.

式5

Formula 5

In some embodiments, the structural formula of luteolin 8-C-glucoside is the following formula 6.

式6

Formula 6

In some embodiments, the buckwheat seedling extract can be obtained from buckwheat (Fagopyrum esculentum) seedlings (hereinafter referred to as buckwheat seedlings). Wherein, the preparation method of buckwheat sprouts extract includes: extracting buckwheat sprouts with water at a weight ratio of 2-5:1 at a temperature of 80°C to 90°C for 60 minutes to 120 minutes to obtain an extract stock solution; and Filter the extract to obtain a buckwheat seedling extract (ie filtrate).

In some embodiments of the extraction step, the buckwheat seedling is a seedling that grows for 6 to 12 days after the buckwheat seed germinates. In some embodiments of the extraction step, the buckwheat seedlings may preferably be seedlings grown for 9 days after the buckwheat seeds germinate. In some embodiments of the extraction step, the buckwheat seedlings may be Fagopyrum tataricum. In some embodiments of the extraction step, the buckwheat seedlings may include the cotyledons of the seedlings, the hypocotyls of the seedlings, and the roots of the seedlings. In some embodiments of the extraction step, the weight ratio of water to buckwheat sprouts may be 3:1.

In some embodiments, the aforementioned filtering step may be filtering the leaching stock solution obtained in the previous step with a 400-mesh filter screen to remove fine solids.

In some embodiments, the preparation method of the buckwheat seedling extract may further include: concentrating the buckwheat seedling extract under reduced pressure until the degree Brix (°Bx) of the buckwheat seedling extract is 4.7 to 5.3 to obtain Concentrated buckwheat seedling extract (ie concentrated solution). Among them, the reduced pressure concentration step can be carried out at 50°C to 60°C.

In some embodiments, the total flavonoid content of the concentrated buckwheat seedling extract may be 815 μg/mL.

In some embodiments, the buckwheat sprouts extract can be the filtrate obtained in the filtration step or the concentrated solution obtained in the reduced-pressure concentration step according to actual needs. In some embodiments, the aforementioned composition may be a pharmaceutical product. In other words, this medicine contains an effective content of buckwheat seedling extract.

In some embodiments, the aforementioned medicines can be manufactured to be suitable for enteral, parenterally, orally, or topically using techniques well known to those skilled in the art. Dosage type.

In some embodiments, the dosage form for enteral or oral administration may be, but is not limited to, a tablet, a troche, a lozenge, a pill, or a capsule. , Dispersible powder or granule, solution, suspension, emulsion, syrup, elixir, slurry or similar. In some embodiments, the dosage form for parenteral or topical administration may be, but is not limited to, injection, sterile powder, external preparation, or the like. In some embodiments, the injection method may be subcutaneous injection, intraepidermal injection, intradermal injection, or intralesional injection.

In some embodiments, the aforementioned pharmaceutical products may further include a pharmaceutically acceptable carrier that is widely used in pharmaceutical manufacturing technology. In some embodiments, the pharmaceutically acceptable carrier may be one or more of the following carriers: solvent (solvent), buffer (buffer), emulsifier (emulsifier), suspending agent (suspending agent), decomposing agent ( decomposer, disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent, glue Gelling agent, preservative, wetting agent, lubricant, absorption delaying agent, liposome, and the like. Regarding the type and quantity of the selected carrier, it falls within the scope of professionalism and routine technology of those who are familiar with this technology. In some embodiments, the solvent as a pharmaceutically acceptable carrier may be water, normal saline (normal saline), phosphate buffered saline (PBS), or alcohol containing aqueous solution (alcohol containing aqueous solution). ).

In some embodiments, the aforementioned composition may be an edible composition. In other words, the edible composition contains a specific content of buckwheat sprouts extract. In some embodiments, the aforementioned edible composition may be a food product or a food additive. In some embodiments, the food product may be, but is not limited to: beverages, fermented foods, bakery products, health foods, or dietary supplements.

In some embodiments, the aforementioned edible composition may be administered to the recipient orally. Among them, the form of the edible composition can be powder, granule, solution, colloid or paste.

In some embodiments, the aforementioned composition may be cosmetics or skin care products. In other words, cosmetics or skin care products contain a specific content of buckwheat sprouts extract.

In some embodiments, the aforementioned cosmetics or skin care products can be any of the following types: toner, gel, jelly film, mud mask, lotion, cream, lipstick, foundation, powder, powder, makeup remover, makeup remover Milk, facial cleanser, body wash, shampoo, hair conditioner, sunscreen, hand cream, nail polish, perfume, essence and facial mask. In some embodiments, the aforementioned cosmetics or skin care products may further include externally acceptable ingredients as needed. In some embodiments, the external product acceptable ingredient may be, for example, an emulsifier, a penetration enhancer, a softener, a solvent, an excipient, an antioxidant, or a combination thereof.

Example 1: Preparation of buckwheat seedling extract

Experimental steps:

1. Obtain ungerminated tartary buckwheat seeds (i.e. 0 days of growth) and buckwheat seedlings grown on the 3rd, 6th, 9th, 12th, 15th and 18th day after germination of the tartary buckwheat seeds, a total of seven groups of raw materials.

2. Extract the raw materials obtained in the previous step with water at a temperature of 85°C for 120 minutes to obtain each group of solid-containing leaching stock solutions. Among them, the weight ratio of water to raw materials is 3:1.

3. Filter the seven groups of extracts obtained in the previous step with a 400-mesh filter to remove fine solids to obtain each group of buckwheat seedling extracts.

4. The seven groups of buckwheat seedling extract obtained in the previous step are concentrated under reduced pressure with a concentrator (brand: BUCHI-Rotavapor R-100) at 55°C to the Brix value of the buckwheat seedling extract ( Degrees Brix, °Bx) is 5 to obtain the concentrated buckwheat seedling extract of each group.

In the following, the concentrated buckwheat seedling extract obtained by using the ungerminated seeds as the raw material is called the extract 1. The concentrated buckwheat seedling extract obtained by using the buckwheat seedlings grown for 3 days as the raw material is called Extract 2. The concentrated buckwheat seedling extract obtained from the buckwheat seedlings grown for 6 days is called the extract liquid 3. The concentrated buckwheat seedling extract obtained from the buckwheat seedlings grown for 9 days as the raw material The extract is called extract 4. The concentrated buckwheat seedling extract obtained from buckwheat seedlings grown for 12 days is called extract 5. The concentrated buckwheat seedling extract obtained from buckwheat seedlings grown for 15 days is the raw material. The buckwheat seedling extract is called extract 6 and the concentrated buckwheat seedling extract obtained from buckwheat seedlings grown for 18 days is called extract 7.

Example 2: Analysis of the components of the extract of buckwheat seedlings

Take the extracts 1 to 7 obtained in Example 1, and analyze the fingerprints of the extracts 1 to 7 by liquid chromatography-mass spectrometry (LC-MS) technology.

(1) Fingerprint analysis.

Experimental steps:

1. Concentrate the extract 1 to 7 obtained in Example 1 to remove the solvent to obtain solids 1 to 7. The solids 1 to 7 were prepared into samples 1 to 7 of 10 milligrams per milliliter (mg/mL) with water as the solvent respectively, for a total of 7 samples.

2. Take 10μL of each sample in the previous step and use High Performance Liquid Chromatography (HPLC) (column used is Mightysil RP-18 GP 250, 250 × 10mm, 5 μm, purchased from Kanto, Japan Chemical company) for analysis. Among them, the pump system of the high-performance liquid chromatograph is Hitachi L-2310 series, the detector model is Hitachi L-2420 UV-VIS, and the data processing software is D-2000 Elite.

In this step, the flow rate of the mobile phase is controlled to 1 mL/min, and the detection wavelength is set to 280 nm, and gradient extraction is performed. The gradient changes of the mobile phase are shown in Table 1: Mobile phase A is 0.1 vol% formic acid (configured in water), and mobile phase B is 0.1 vol% formic acid (configured in methanol).

Table I Preparation time (minutes) Mobile phase A (%) Mobile phase B (%) 0 98 2 10 98 2 40 30 70 50 0 100 60 0 100 62 98 2 70 98 2

Experimental results:

The HPLC fingerprints of extract 1 to 7 are shown in FIG. 1. Please refer to FIG. 1, the fingerprints of extract 3 to 5 have obvious peaks at time points A, B, C, D, E, F, and G. Calculate and calculate the area of each wave peak at time points A, B, C, D, E, F, and G in Fig. 1 with data processing software integration. Then, according to the obtained wave peak area, calculate the ratio of the maximum wave front surface base to the minimum wave peak area, and the calculation results for each sample are shown in Table 2 below. Referring to Figure 1 and Table 2, the growth metabolites (hereinafter referred to as metabolites) separated from extracts 3 to 5 at time points A, B, C, D, E, F, and G are significantly higher than those of other extracts. Extraction (that is, there are obvious peaks). Among them, extract 4 (that is, the extract of buckwheat seedlings obtained from buckwheat seedlings grown for 9 days after germination of buckwheat seeds), the content of metabolites separated at time points A, D, F, and G, relative to Compared with the buckwheat seedling extract obtained from the buckwheat seedlings grown for other days after germination, it has the highest value (that is, the peak of the wave peak is the highest). In addition, the content of metabolites separated in extract 4 at time points B, C, and E is only less than extract 5 (the extract of buckwheat seedlings obtained from buckwheat seedlings grown for 12 days after germination liquid). Therefore, it can be known that the buckwheat seedling extract obtained from buckwheat seedlings grown for 6 to 12 days after germination has high content of metabolites, and the buckwheat seedlings grown for 9 days after germination are used as raw materials. The buckwheat seedling extract obtained from the raw material has a higher content of metabolites than the buckwheat seedling extract obtained from buckwheat seedlings grown for other days after germinating.

Table II A E G F D C B Extract 1 1.01 1.00 1.00 - - - - Extract 2 1.95 1.80 1.89 1.38 1.37 1.39 1.46 Extract 3 3.20 6.33 8.82 16.47 10.98 15.35 46.49 Extract 4 3.25 8.04 11.99 23.54 15.27 21.47 54.69 Extract 5 2.63 8.78 12.69 21.46 15.74 20.29 21.21 Extract 6 1.29 1.43 1.49 1.20 1.14 1.16 1.31 Extract 7 1.00 1.26 1.28 1.00 1.00 1.00 1.00

(2) Metabolite analysis

The separated liquids collected at time points A, C, D, E, F, and G in the aforementioned experiment (1) were used with a nuclear magnetic resonance spectrometer (NMR) (model: Ascend 400 MHz, Bruker Co) The analysis was performed to obtain the proton spectra of the biologically active substance 01 to the biologically active substance 06, as shown in Figs. 2-7. Next, determine the chemical structure and chemical name of each biologically active substance with the obtained hydrogen spectrum, as shown in Table 3 below. The codes, hydrogen spectra and chemical names of the separated and purified biologically active substances are shown in Table 3 below.

          圖2 生物活性物質02 E 牡荊素

          圖3 生物活性物質03 G 異牡荊素

        圖4 生物活性物質04 F 芸香苷

              圖5 生物活性物質05 D 異東方素

        圖6 生物活性物質06 C 木犀草素8-C-葡萄糖苷

          圖7 Table Three Point in time Ingredient name Chemical structure Schema Bioactive substance 01 A Guanosine

figure 2 Biologically active substance 02 E Vitexin

image 3 Bioactive substance 03 G Isovitexin

Figure 4 Bioactive substance 04 F Rutin

Figure 5 Bioactive substance 05 D Isoorientin

Figure 6 Biologically active substance 06 C Luteolin 8-C-glucoside

Figure 7

Example 3: Determination of total flavonoids

Here, rutin equivalent is used as the expression of the relative content of total flavonoids. Use Rutin (purchased from ChromaDex) and water to prepare Rutin standard solutions of 0μg/mL, 400μg/mL, 600μg/mL, 1000μg/mL and 1200μg/mL. Then, for each test tube, take 200μL of the rutin standard solution from the test tube to another new test tube, and then add 200μL of 5 wt% sodium citrate aqueous solution to these new test tubes, mix well and let stand for 6 minutes. . Then, add 200μL of 10 wt% aluminum nitrate aqueous solution (brand: Alfa Aesar 12360), mix well and let stand for 6 minutes, then add 2 mL of 4 wt% sodium hydroxide aqueous solution (brand: Macron 7708-10 ) To make it evenly mixed. Finally, add 1.4 mL of water to the test tube and mix well to obtain the reaction solution. Take 200 μL of the reaction solution in the test tube and transfer it to the 96-well reaction plate, and use the ELISA (enzyme-linked immunosorbent assay, enzyme-linked immunosorbent assay) to read the instrument ( Brand: Thermo Fisher Scientific) to detect absorbance at a wavelength of 500 nm. The absorbance values measured in the same way with different concentrations of rutin standard solutions are plotted as calibration lines.

Dilute the extract 1 and the extract 4 obtained in Example 1 with water 10 times according to their volume, and then take 200 μL into a test tube. Next, add 200 μL of 5 wt% sodium citrate aqueous solution, mix well, and let stand for 6 minutes to react. Next, add 200 μL of 10 wt% aluminum nitrate aqueous solution, mix well and let stand to react for 6 minutes, and then add 2 mL of 4 wt% sodium hydroxide aqueous solution to make it evenly mixed. Finally, add 1.4 mL of water to the test tube and mix evenly to obtain a reaction solution. Take 200 μL of the reaction solution in the test tube into a 96-well reaction plate, and detect the absorbance with an ELISA reader at a wavelength of 500 nm.

Then, using the calibration curve, the absorbance value of the diluted extract 4 is calculated as the total flavonoid content of the extracted extract 4 before dilution. From this, it can be calculated that the total flavonoid content of the extract 4 obtained in Example 1 is about 815 μg/mL, and the total flavonoid content of the extract 1 obtained in Example 1 is about 432 μg/mL.

Example 4: Cell test-liver cell anti-oxidation

The experiment will be divided into experimental group 1 (addition of extract 1 obtained in Example 1 and treated with hydrogen peroxide) and experimental group 2 (addition of extract 4 obtained in Example 1 and treated with hydrogen peroxide) The four groups were conducted in four groups: control group (no extraction solution added, and no hydrogen peroxide treatment group), and control group (no extraction solution added, but hydrogen peroxide treatment group).

Experimental steps:

1. Inoculate human liver cancer cells HepG2 (hereinafter referred to as HepG2 cells) (purchased from the American Type Culture Collection ATCC; Cat. HB-8065) in the form of 2×10 ⁵ cells per well in a 2 ml medium per well. 6-well culture dish.

Among them, cell culture medium: DMEM containing 10 vol% fetal bovine serum (FBS) (purchased from Gibco, Cat.10437-028) and 1 vol% penicillin/streptomycin (purchased from Gibco, Cat. 15140122) (Purchased from GIBCO Company, Cat. 11965-092).

2. Place the above-mentioned culture plate at 37°C and incubate for 24 hours.

3. Remove the cell culture medium from each well of the culture plate.

4. Add experimental medium to each group and react the cells of each group at 37°C for 1 hour. The experimental medium of each group is as follows: Experimental group 1: Add 2 mL of cell culture medium containing 1 mg/ml extract 1 to each well. Experimental group 2: Add 2 mL of cell culture medium containing 1 mg/ml extract 4 to each well. Control group: Add 2 mL of cell culture medium to each well (without extract 4). Control group: Add 2 mL of cell culture medium to each well (without any extract).

5. Add 2μL of cell culture medium containing DCFH-DA solution at a concentration of 5μg/ml to each well to allow DCFH-DA to treat the cells for 15 minutes. After treatment, the experimental group and the control group were added with 1 mM hydrogen peroxide (H ₂ O ₂ ) (purchased from Sigma), and then each group was incubated at 37°C for 1 hour.

Among them, the DCFH-DA solution of 5μg/ml is a solution of 2,7-dichloro-dihydro-fluorescein diacetate (DCFH-DA; purchased from Sigma; Cat. SI-D6883-50MG). ) It is prepared by dissolving in dimethyl sulfoxide (DMSO, purchased from Sigma, Cat. D2650).

6. Each well in each group was rinsed once with 1 mL of 1XPBS (phosphate buffered saline) (purchased from Gibco, Cat. 14200-075) solution.

7. For each group, add 200 μL of trypsin (purchased from Sigma; Cat.59427C) to each well and react in the dark for 5 minutes. After the reaction, add 0.6 mL of cell culture medium to each well.

8. Collect the cells and cell culture medium in each well of each group into the corresponding 15mL centrifuge tube, and centrifuge the tube containing the cells and cell culture medium at 400 xg for 5 minutes.

9. After centrifugation of each group, the supernatant of each group was removed, and the cell pellet was re-dissolved with PBS solution as a cell suspension. The cell suspension of each group was centrifuged again at 400 xg for 10 minutes.

10. After centrifugation of each group, the supernatant of each group was removed again, and the cell pellet was re-dissolved with 1XPBS solution as the cell liquid to be tested.

11. Use a flow cytometer (Beckman brand; purchased from BD Accuri) to detect the fluorescent signal of DCFH-DA in the cell liquid to be tested in each well. The excitation wavelength for fluorescence detection is 450 nm-490 nm, and the emission wavelength is 510 nm-550 nm. As DCFH-DA enters the cell, it will be firstly hydrolyzed to DCFH (dichlorodihydroluciferin), and then oxidized by reactive oxygen species into DCF (dichloroluciferin) that emits green fluorescence, and is treated by DCFH-DA The fluorescence intensity of the cells can reflect the content of reactive oxygen species in the cells, and by this means the ratio of the number of cells with highly expressed reactive oxygen species to the number of original cells can be known. Since the experiment is performed twice, the measurement results of the two repeated experiments in each group are averaged to get the average value, and then the average value of the control group is taken as 100% of the relative ROS generation amount, and the control group and the experimental group are averaged The value is converted to relative ROS generation, as shown in Figure 8.

Experimental results:

Please refer to Figure 8. From the results of the control group and the control group, it can be seen that after hydrogen peroxide treatment, the proportion of cells with high ROS performance (high fluorescence performance) in the control group increased significantly, indicating that blue light irradiation does cause the production of reactive oxygen species in the cells. It then causes subsequent damage to human liver cancer cells (HepG2 cells).

Among them, the ROS production of the control group was about 12.54 times that of the control group, while the ROS production of the experimental group 1 was about 4.41 times that of the control group, and the ROS production of the experimental group 2 was about 4.37 times that of the control group. It can be seen that when the cells are treated with Extract 1 and Extract 4, compared with the control group, the experimental group can significantly reduce the production of ROS. That is, the buckwheat seedling extract can effectively reduce the production or accumulation of reactive oxygen species in the cells, and can be used as a scavenger of reactive oxygen species, and the buckwheat seedling extract can reduce the content of reactive oxygen species in the cell and reduce the exposure of cells. For oxidative damage, here, especially the extract 4 has the best effect.

Example 5: Cell test-GSH content detection

The experiment will be divided into experimental group 1 (addition of extract 1 from Example 1), experimental group 2 (addition of extract 4 from Example 1) and control group (without adding any buckwheat seedling extract).

Experimental steps:

1. Inoculate human liver cancer cells HepG2 (hereinafter referred to as HepG2 cells) (purchased from the American Type Culture Collection ATCC; Cat. HB-8065) in the form of 2×10 ⁵ cells per well in 2 mL cells per well Culture medium in a 6-well culture dish.

Among them, cell culture medium: add additional components to the minimum essential medium (MEM, purchased from Gibco) to contain 10 vol% fetal bovine serum (FBS, purchased from Gibco; Cat.10437-028), 1 mM sodium pyruvate (purchased from Gibco), 1.5 g/L sodium bicarbonate (purchased from Gibco), and 0.1 mM non-essential amino acids (purchased from Gibco) ).

2. Incubate each group at 37°C for 24 hours. Among them, each group is processed as follows before cultivation: Experimental group 1: Add 2 mg/ml extract 1 to each well. Experimental group 2: Add extract 4 containing 2 mg/ml to each well. Control group: No extract was added.

3. Collect the cells in each well of each group. Specifically, each group was rinsed once with 1 mL of 1XPBS (phosphate buffered saline) (purchased from Gibco; Cat. 14200-075) solution, and then 200 μL of trypsin (purchased from Sigma; Cat.59427C) was added. ) Add to each well and react in the dark for 5 minutes. After the reaction, add 0.6 mL of cell culture medium to each well, and collect the cells and cell culture medium in each well of each group into the corresponding centrifuge tube.

4. Each group was rinsed once with PBS solution. Specifically, centrifuge each centrifuge tube at 400 xg for 5 minutes, remove the supernatant after centrifugation, add PBS solution to re-dissolve, then centrifuge at 400 xg for 5 minutes, and remove the supernatant after centrifugation to obtain cells Precipitate.

5. In each group, the cell pellets were re-dissolved in PBS solution as cell suspensions.

6. Dilute the GSH detection dye (purchased from abcam, model Ab112132) by 1000 times to obtain the GSH detection solution, and stain each group with the GSH detection solution for 15 minutes.

7. Each group was rinsed once with PBS solution. Specifically, each group was centrifuged at 400 xg for 5 minutes, the supernatant was removed after centrifugation, re-dissolved by adding PBS solution, then centrifuged at 400 xg for 5 minutes, and the supernatant was removed after centrifugation to obtain a cell pellet Things.

8. In each group, the cell pellet was re-dissolved with 200 μL of PBS solution as the cell liquid to be tested.

9. Use a flow cytometer (Beckman; BD Accuri) to detect the fluorescent signal of the non-fluorescent Green Dye in the cell sap of each group. The excitation wavelength for fluorescence detection is 490 nm, and the emission wavelength is 520 nm.

Experimental results:

Please refer to Figure 9. If the control group is used as 100% glutathione (GSH) production, the conversion of experimental group 1 and experimental group 2 to the control group's glutathione production is shown in Figure 9. It can be seen that the glutathione production of experimental group 1 is 34% longer than the control composition, and the glutathione production of experimental group 2 is 93% longer than the control composition. This means that the 0-day and 9-day buckwheat seedling extracts can promote the production of glutathione in liver cells, and the 9-day buckwheat seedling extract can promote glutathione production better than 0-day buckwheat seedlings. Extract.

Glutathione is a tripeptide composed of glutamine, cysteine and glycine. Its thiol group (G-SH) is easily combined with free radicals and has an antioxidant function. It can be seen from Example 5 that on the 9th, the buckwheat seedling extract can increase the production of glutathione in liver cells and has a good antioxidant effect.

Example 6: Reducing power test

The main principle of the determination of reducing power is to reduce red blood salt [K ₃ Fe(CN) ₆ ] into yellow blood salt [K ₄ Fe(CN) ₆ ] (the following formula 7), and the yellow blood salt is formed _{by reusing Fe 3} ⁺ Prussian blue (Equation 8 below) uses the change in absorbance at 700nm to detect the reducing power. The higher the absorbance, the stronger the reducing power.

K ₃ Fe(CN) ₆ + sample → K ₄ Fe(CN) ₆ + sample-oxide Equation 7

Fe ₃ ^＋ ＋K ₄ Fe(CN) ₆ →Fe ₄ [Fe(CN) ₆ ] ₃ (Prussian blue) Equation 8

Configuration solution:

Configure phosphate buffer solution: weigh 1.34g of anhydrous sodium dihydrogen phosphate (NaH ₂ PO ₄ ) (brand: JTBaker 3828-01), 1.26g of disodium hydrogen phosphate (Na ₂ HPO ₄ ) (brand: Sigma) 04270) Put it in a quantitative bottle, then _{dissolve it with pure water (H 2} O) and quantify to 100 mL.

Configure 1 vol% red blood salt solution: weigh 1 g of red blood salt (K ₃ Fe(CN)) and place it in a quantitative bottle, then _{dissolve it with pure water (H 2} O) and quantify it to 100 mL. (It needs to be stored away from light, and the shelf life is two weeks and stored at 4°C).

Prepare 10 vol% Trichloroacetic acid (TCA) solution: Weigh 10g of trichloroacetic acid (CCl ₃ COOH) and place it in a quantitative bottle, then _{dissolve it with pure water (H 2} O) and quantify it to 100 mL.

Configure a 0.1 vol% ferric chloride (FeCl ₃ ) solution: weigh 0.1 g of ferric chloride and place it in a quantitative flask, then _{dissolve it with pure water (H 2} O) and quantify it to 100 mL. (Keep away from light, configure on the day and store at 4℃)

Prepare 1 mg/mL vitamin C (Vit C) solution: weigh 10 mg of L-Ascorbic acid (L-Ascorbic acid) into a quantitative bottle, then _{dissolve it with pure water (H 2} O) and quantify it to 10 mL. (Need to configure on the day)

Experimental steps:

Take 1 mL of 1 mg/mL vitamin C solution and place it in a quantitative bottle, then add water (H ₂ O) and quantify to 10 mL to obtain 100 μg/mL vitamin C solution.

Prepare standard solutions of 0μg/mL, 20μg/mL, 40μg/mL, 60μg/mL, 80μg/mL and 100μg/mL in glass test tubes with 100μg/ml vitamin C solution. The preparation method is shown in Table 4.

Table Four 100 μg/mL Vitamin C solution (μL) Water (μL) Standard solution (μg/mL) 0 100 0 10 90 10 20 80 20 40 60 40 60 40 60 80 20 80 100 0 100

Next, take 250 μL of standard solutions (ie, samples) of various concentrations in different test tubes, add 250 μL of phosphate buffer solution to each test tube, and vortex to mix the solutions evenly. Then, add 250 μL of red blood salt solution (1vol%) to each test tube, and vortex to mix the solution evenly. Next, each test tube was placed in a water bath at 50°C for 20 minutes. Then, add 250 μL of trichloroacetic acid solution (10 vol%) to each test tube, and vortex to mix the solution evenly. Then, it was centrifuged at 3000 g for 10 minutes. Next, take 30 μL of the supernatant from each test tube and add 300 μL of pure water, and then add 120 μL of ferric chloride solution (0.1 vol%), and vortex the solution to mix the solution uniformly and react for 10 minutes to get the reaction liquid. Take 200μL of the reaction solution into a 96-well reaction plate, and use an ELISA reader (brand: Thermo Fisher Scientific) to detect the absorbance at a wavelength of 700 nm. The absorbance values of six different concentrations of vitamin C solutions measured in the same way are plotted as a calibration curve.

Take 250 μL of extract 1 (specimen) obtained in Example 1 and 250 μL of extract 4 (specimen) obtained in Example 1 into different test tubes. Add 250 μL of phosphate buffer solution to each test tube, and add 250 μL of phosphate buffer solution to each test tube. Vortex (vortex) to mix the solution evenly. Then, add 250 μL of red blood salt solution (1vol%) to each test tube, and vortex to mix the solution evenly. Next, each test tube was placed in a water bath at 50°C for 20 minutes. Then, add 250 μL of trichloroacetic acid solution (10 vol%) to each test tube, and vortex to mix the solution evenly. Next, each test tube was centrifuged at 3000 g for 10 minutes. Next, take 30 μL of the supernatant from each test tube and add 300 μL of water, and then add 120 μL of ferric chloride solution (0.1 vol%), and vortex the solution to mix the solution uniformly and react for 10 minutes to obtain a reaction solution . Take 200 μL of the reaction solution in the test tube into a 96-well reaction plate, and detect the absorbance value with an ELISA reader at a wavelength of 700 nm.

Experimental results:

Please refer to Figure 10, using the calibration curve to convert the absorbance values of the diluted 9-day buckwheat seedling extract and 0-day buckwheat seedling extract into the reducing power generated by the corresponding vitamin C content. It can be seen that the reducing power of the 0-day buckwheat seedling extract before dilution (ie the extract 1 obtained in Example 1) is equivalent to that of vitamin C (L-Ascorbic Acid Sodium Salt) with a content of about 437μg/mL The reducing power of the buckwheat seedling extract on the 9th day before dilution (ie the extract 4 obtained in Example 1) is equivalent to the reducing power of vitamin C with a content of about 1752μg/mL, so it can be inferred The reducing power of extract 4 is significantly higher than that of extract 1, that is, compared to ungerminated seeds, the buckwheat seedling extract obtained from buckwheat seedlings grown for 9 days has stronger reducing power.

Example 7: Cell test-GSH content detection

Here, six biologically active substances are obtained after removing the solvent from the separated liquid collected from the extract 4 at time points A, C, D, E, F and G in the experiment (1) of Example 2 (Table 5 below) Perform a cell test.

Table 5 Point in time Ingredient name Bioactive substance 01 A Guanosine Biologically active substance 02 E Vitexin Bioactive substance 03 G Isovitexin Bioactive substance 04 F Rutin Bioactive substance 05 D Isoorientin Biologically active substance 06 C Luteolin 8-C-glucoside

The experiment will be divided into experimental group 1 (addition of biologically active substance 01), experimental group 2 (addition of biologically active substance 02), experimental group 3 (addition of biologically active substance 03), experimental group 4 (addition of biologically active substance 04), experiment Group 5 (with biologically active substance 05), experimental group 6 (with biologically active substance 06) and control group (without biologically active substance).

Experimental steps:

1. Inoculate human liver cancer cells HepG2 (hereinafter referred to as HepG2 cells) (purchased from the American Type Culture Collection ATCC; Cat. HB-8065) in the form of 2×10 ⁵ cells per well in 2 mL cells per well Culture medium in a 6-well culture dish.

Among them, cell culture medium: DMEM containing 10 vol% fetal bovine serum (FBS) (purchased from Gibco, Cat.10437-028) and 1 vol% penicillin/streptomycin (purchased from Gibco, Cat. 15140122) (Purchased from GIBCO Company, Cat. 11965-092).

2. Incubate each group at 37°C for 24 hours. Among them, each group is treated as follows before cultivation: Experimental group 1: Add 2 mg/ml of biologically active substance 01 to each well. Experimental group 2: Add 2 mg/ml of biologically active substance 02 to each well. Experimental group 3: Add 2 mg/ml of biologically active substance 03 to each well. Experimental group 4: Add biologically active substance 04 containing 2 mg/ml to each well. Experimental group 5: Add 2 mg/ml of biologically active substance 05 to each well. Experimental group 6: Add 2 mg/ml of biologically active substance 06 to each well. Control group: No biologically active substance was added to each well, that is, only 2 mL of cell culture medium.

3. Collect the cells in each well of each group. Specifically, each group was rinsed once with 1 mL of 1XPBS (phosphate buffered saline) (purchased from Gibco; Cat. 14200-075) solution, and then 200 μL of trypsin (purchased from Sigma; Cat.59427C) was added. ) Add to each well and react in the dark for 5 minutes. After the reaction, add 0.6 mL of cell culture medium to each well, and collect the cells and cell culture medium in each well of each group into the corresponding centrifuge tube.

4. Each group was rinsed once with PBS solution. Specifically, each group was centrifuged at 400 xg for 5 minutes, the supernatant was removed after centrifugation, re-dissolved by adding PBS solution, then centrifuged at 400 xg for 5 minutes, and the supernatant was removed after centrifugation to obtain a cell pellet Things.

5. In each group, the cell pellets were re-dissolved in PBS solution as cell suspensions.

6. Dilute the GSH detection dye (purchased from abcam, model Ab112132) by 1000 times to obtain the GSH detection solution, and stain each group with the GSH detection solution for 15 minutes.

7. Each group was rinsed once with PBS solution. Specifically, each group was centrifuged at 400 xg for 5 minutes, the supernatant was removed after centrifugation, re-dissolved by adding PBS solution, then centrifuged at 400 xg for 5 minutes, and the supernatant was removed after centrifugation to obtain a cell pellet Things.

8. In each group, 200 μL of PBS solution was used to re-dissolve the cell pellet as the cell liquid to be tested.

9. Use a flow cytometer (Beckman; BD Accuri) to detect the fluorescent signal of the non-fluorescent Green Dye in the cell sap of each group. The excitation wavelength for fluorescence detection is 490 nm, and the emission wavelength is 520 nm.

Experimental results:

Please refer to Figure 11, where HepG2 cells treated with biologically active substance 01 (guanosine) produced 1.6 times the amount of glutathione in the control group; HepG2 cells treated with biologically active substance 02 (vitexin), The production of glutathione (GSH) was 0.6 times that of the control group; HepG2 cells treated with the biologically active substance 03 (isovitexin) produced 0.4 times that of the control group ; HepG2 cells treated with biologically active substance 04 (rutin), the production of glutathione is 0.6 times that of the control group; hepatocytes treated with biologically active substance 05 (isoorientin), its glutathione The amount of glutathione produced by HepG2 cells treated with the biologically active substance 06 (luteolin 8-C-glucoside) was 0.4 times that of the control group.

Glutathione is a tripeptide composed of glutamine, cysteine and glycine. Its thiol group (G-SH) is easily combined with free radicals and has an antioxidant function. It can be seen from Fig. 11 that the biologically active substance 01 separated from the extract 4 can increase the production of glutathione in liver cells, and thus has a good antioxidant effect.

In summary, in any embodiment, the use of the buckwheat seedling extract for preparing the cell antioxidant composition and/or the liver health care composition and the preparation method of the buckwheat seedling extract are suitable for providing buckwheat seedlings Leaching liquid or its composition. Wherein, the provided buckwheat seedling extract or its composition has one or more of the following functions: enhancing cellular antioxidant activity, enhancing metabolic activity, and enhancing intracellular glutathione (GSH) synthesis.

A, B, C, D, E, F, G: point in time

Figure 1 is the HPLC fingerprint of the buckwheat seedling extract obtained from buckwheat seedlings grown for 0, 3, 6, 9, 12, 15, and 18 days after germinating. Figure 2 is a hydrogen spectrum of the biologically active substance 01. Figure 3 is a hydrogen spectrum of the biologically active substance 02. Figure 4 is a hydrogen spectrum of the biologically active substance 03. Figure 5 is a hydrogen spectrum of the biologically active substance 04. Figure 6 is a hydrogen spectrum of the biologically active substance 05. Figure 7 is a hydrogen spectrum of the biologically active substance 06. FIG. 8 is a graph of the experimental results of the relative production amount of ROS in the control group, the control group, the experimental group 1 and the experimental group 2. Figure 9 is a graph showing the experimental results of the relative production of glutathione in the control group, experimental group 1 and experimental group 2. Figure 10 is a graph showing the experimental results of the reducing power of the buckwheat seedling extract. Figure 11 is a graph of the experimental results of the relative production of glutathione in the control group, experimental group 1, experimental group 2, experimental group 3, experimental group 4, experimental group 5, and experimental group 6.

Claims (18)

A use of the extract of buckwheat seedlings is used to prepare a composition for enhancing the antioxidant activity of cells. A use of the buckwheat seedling extract is used to prepare a liver health-care composition. The use of the extract of buckwheat seedlings according to claim 2, wherein the extract of buckwheat seedlings is used to enhance the antioxidant and metabolic activities of cells. The use of the buckwheat seedling extract of claim 3, wherein the buckwheat seedling extract is used to enhance the synthesis of glutathione (GSH) in cells. The use of the buckwheat seedling extract according to claim 1 or 2, wherein the buckwheat seedling extract is obtained by extracting a buckwheat seedling with a solvent, wherein the buckwheat seedling grows for 6 days to 12 days after germination of buckwheat seedlings. Seedlings. The use of the buckwheat sprouts extract according to claim 5, wherein the solvent is water, the weight ratio of the solvent to the buckwheat sprouts is 2:1 to 5:1, and the extraction temperature of the buckwheat sprouts ranges from 80°C to Between 90°C and the extraction time of the buckwheat seedlings is 120 minutes. The use of the extract of buckwheat seedlings according to claim 6, wherein the extract of buckwheat seedlings is 4.7°Bx to 5.3°Bx. The use of the buckwheat seedling extract according to claim 6, wherein the flavonoid content of the buckwheat seedling extract is about 815 μg/mL. The use of the buckwheat seedling extract according to claim 1 or 2, wherein the buckwheat seedling extract contains at least one of the following biologically active substances: Guanosine, Vitexin, and Isoma Jing Su (Isovitexin), rutin (rutin), iso-East hormone (Isoorientin) and luteolin-8-C- glucoside (luteolin-8- C -glucoside). The use of the extract of buckwheat seedlings according to claim 9, wherein the guanosine is used to enhance the synthesis of glutathione (GSH) in cells. A preparation method of buckwheat seedling extract, including: Leaching water and a buckwheat seedling at a temperature of 80°C to 90°C for 60 minutes to 180 minutes to obtain an extraction stock solution, wherein the weight ratio of the water to the buckwheat seedling is 2:1 to 5:1; and The leaching stock solution is filtered to obtain a buckwheat seedlings leaching solution. The preparation method according to claim 11 further includes: The buckwheat seedling extract is concentrated under reduced pressure until the buckwheat seedling extract is 4.7°Bx to 5.3°Bx to obtain the concentrated buckwheat seedling extract. The preparation method according to claim 11, wherein the step of concentration under reduced pressure is performed at 50°C to 60°C. The preparation method according to claim 11, wherein the total flavonoid content of the concentrated buckwheat seedling extract is 815 μg/mL. The preparation method according to claim 11, wherein the buckwheat seedling is a seedling that grows from 6 to 12 days after the buckwheat seed germinates. The preparation method according to claim 11, wherein the buckwheat seedling extract contains at least one of the following biologically active substances: Guanosine, Vitexin, Isovitexin, Ruta glycoside (Rutin), iso-East hormone (Isoorientin) and luteolin-8-C- glucoside (luteolin-8- C -glucoside). The preparation method according to claim 11, wherein the guanosine is used to enhance the synthesis of glutathione (GSH) in the cell. A buckwheat seedling extract containing at least one of the following biologically active substances: Guanosine, Vitexin, Isovitexin, Rutin, Isoorientin ) and luteolin-8-C- glucoside (luteolin-8- C -glucoside).

Images