Preparation method and application of coix seed green seedling source polyphenol composition with anti-fatigue effect

Abstract

The invention discloses a preparation method and application of a coix seed green seedling source composition with an anti-fatigue effect, and belongs to the field of food. The method comprises the following steps: identifying the components of the purified polyphenol of the pearl barley green seedlings by using a Q-Orbitrap high-resolution liquid chromatography-mass spectrometry technology; screening potential anti-fatigue active components by utilizing network pharmacology and molecular docking; the anti-fatigue effects of the active components and the corresponding composition are verified in vitro and in vivo, so that the coix seed green seedling source polyphenol composition for fatigue regulation is obtained, and the composition consists of luteolin, 4-coumaric acid, caffeic acid and isoferulic acid. The coix seed green seedling anti-fatigue polyphenol source composition is obtained for the first time, and the anti-fatigue effect is remarkable and superior to that of a green seedling polyphenol purified product. The raw materials are green, safe and easy to obtain, and good application value is achieved.

Description

Technical Field

[0001] The invention belongs to the field of food, and particularly relates to a preparation method and application of a coix seedling-derived polyphenol composition with anti-fatigue effect. Background Art

[0002] Fatigue is a complex physiological and biochemical process that occurs when the human body's mental or physical activities reach a certain stage. It can be divided into physical fatigue and mental fatigue. The former refers to the fatigue of the body and the decline of muscle movement ability; while the latter refers to the phenomenon of mental fatigue that occurs when the body is in a state of tension for a long time. The causes of fatigue mainly include the consumption of energy substances, the accumulation of metabolic byproducts, the excessive production of reactive oxygen, the accumulation or reduction of neurotransmitters, etc. The generation of fatigue can affect a person's mental health and body function, and is often accompanied by symptoms such as headache, dizziness, nausea, and fatigue. Fatigue can cause slow brain response, and severe cases can lead to diseases related to the immune system and biological regulation, such as mental illness, musculoskeletal and joint diseases, respiratory diseases, etc. Traditional anti-fatigue substances are mainly ephedrine and caffeine, but most of these drugs have addiction dependence and toxic side effects, and are not suitable for long-term use. Therefore, it is necessary to use modern science and technology to develop a green and safe anti-fatigue functional component from natural products.

[0003] Job's tears green shoots are popular because of their rich bioactive ingredients and nutritional value. Developing anti-fatigue functional foods from natural products is one of the main ways to find anti-fatigue substances. In previous studies, the inventors found that the ethanol crude extract of Job's tears green shoots has significant antioxidant and radiation protection effects, but its anti-fatigue function is unclear, and the active ingredients in the crude extract are unclear, and the utilization rate of the effective anti-fatigue components is low. The compound combination can synergistically act on multiple targets to achieve better anti-fatigue effects. Therefore, it is necessary to further develop a combination of green shoot-derived polyphenols to achieve a synergistic effect.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a preparation method and application of a coix seedling-derived polyphenol composition having an anti-fatigue effect.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for preparing a coix seedling-derived polyphenol composition having an anti-fatigue effect comprises the following steps:

[0008] Step 1, extracting with ethanol solution and purifying with macroporous resin to obtain purified coix seedling polyphenols;

[0009] Step 2, identifying the active components of the purified coix seedling polyphenols by Q-Orbitrap high-resolution liquid chromatography-mass spectrometry technology;

[0010] Step 3: Using network pharmacology and molecular docking to screen out active components with potential anti-fatigue function in the purified coix seedling polyphenols;

[0011] Step 4: Use cell models and animal models to evaluate the anti-fatigue effects of the potential anti-fatigue active components in the coix seedling polyphenol purification and the coix seedling polyphenol composition, and determine the coix seedling polyphenol composition with anti-fatigue effect.

[0012] Preferably, in step one, the crushed fresh coix seedlings are mixed with an ethanol aqueous solution with a mass fraction of 60%-80%, with a solid-liquid ratio of (1-3): (20-40), and the mixture is heated in a 50-80°C water bath for extraction for 2-3h, and a macroporous resin is used as a separation medium to purify the extract to obtain a purified coix seedling polyphenol product.

[0013] Preferably, in step 1, the macroporous resins used for the purification of the coix seedling polyphenol purified product are D101 and S-8, and the conditions for the primary purification of D101 are: the sample volume is 30-40mL, the sample polyphenol concentration is 2-4mg / mL, and the eluent is a 30%-70% ethanol aqueous solution by mass fraction; the conditions for the secondary purification of S-8 are: the sample volume is 30-50mL, the sample polyphenol concentration is 2-4mg / mL, and the eluent is a 30%-70% ethanol aqueous solution by mass fraction, and gradient elution is performed. Preferably, the mass fraction of the eluent is 50% ethanol aqueous solution.

[0014] Preferably, in step 2, the conditions for Q-Orbitrap high-resolution liquid-mass spectrometry are: the mass spectrometry condition is an electrospray ionization source (ESI), a positive and negative ion switching scanning detection mode, a scanning range of 100.0 to 1500.0 m / z, an electrospray voltage of 3.8 kV, a capillary temperature of 300 ° C, a collision gas of high-purity argon, a sheath gas of nitrogen, and a data acquisition time of 30.0 min; the chromatographic column of the chromatographic conditions is AQ-C18, 150×2.1 mm, 1.8 μm, the aqueous phase is a formic acid aqueous solution with a mass fraction of 0.1%, the organic phase is methanol, the column oven temperature is 35 ° C, and the injection volume is 5.00 μL.

[0015] More preferably, in step 2, the active components of the purified coix seedling polyphenols are identified by Q-Orbitrap high-resolution liquid chromatography-mass spectrometry technology to include rutin, naringin, 7-hydroxycoumarin, luteolin, 4-coumaric acid, caffeic acid and isoferulic acid.

[0016] Preferably, in step three, the steps of network pharmacology are as follows: the active components in the purified coix seedling polyphenols are screened as luteolin, 4-coumaric acid, caffeic acid and isoferulic acid by intestinal absorbability and drug-like index; then the potential targets (CP) of the four active components of luteolin, 4-coumaric acid, caffeic acid and isoferulic acid and the fatigue target (FA) with "fatigue" as the keyword are obtained through the online database, and then the candidate targets of the compound and fatigue (CP-FA) are obtained by intersection; the candidate targets are introduced into the STRING platform to make a protein interaction network and analyze it, and the key targets are screened. After that, the binding ability of the active components with the fatigue targets is verified by molecular docking, and the molecular docking software is AutoDock software, and the docked fatigue targets are PI3K, PIK3CA, AKT1, PIK3R1, PIK3CB, SRC, and STAT3.

[0017] More preferably, in step three, the network pharmacology and molecular docking screen out active components with potential anti-fatigue function as luteolin, 4-coumaric acid, caffeic acid and isoferulic acid.

[0018] Preferably, in step 4, the composition is composed of luteolin, 4-coumaric acid, caffeic acid and isoferulic acid in a mass ratio of (6-10):(1-3):(1-3):(1-3).

[0019] Preferably, in step 4, the cell model is a C2C12 mouse myoblast oxidative damage model. The animal model is a mouse exhaustive swimming model.

[0020] The composition prepared by the preparation method is used in food, health products or medicines.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention has found that the composition of luteolin, 4-coumaric acid, caffeic acid and isoferulic acid in coix seedling polyphenols has a significant regulating effect on fatigue mice, and has a better effect than the purified coix seedling polyphenols. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The figure shows the results of the determination of the purity of flavonoids in the purified coix seedling polyphenols obtained by using eluents of different mass fractions;

[0024] Figure 2 The total ion current spectrum of the purified coix seedling polyphenols is shown in Figure 1; A is the total ion current chromatogram in the positive ion mode; B is the total ion current chromatogram in the negative ion mode.

[0025] Figure 3The intersection targets of luteolin, 4-coumaric acid, caffeic acid, isoferulic acid and fatigue and their PPI diagrams; A is the intersection target of luteolin, 4-coumaric acid, caffeic acid, isoferulic acid and fatigue; B is the PPI network diagram of the intersection target of luteolin, 4-coumaric acid, caffeic acid, isoferulic acid and fatigue;

[0026] Figure 4 Graphs showing the effects of luteolin, 4-coumaric acid, caffeic acid and isoferulic acid on the viability of C2C12 mouse myoblasts, respectively; A is a graph showing the effect of luteolin on the viability of C2C12 mouse myoblasts; B is a graph showing the effect of 4-coumaric acid on the viability of C2C12 mouse myoblasts; C is a graph showing the effect of caffeic acid on the viability of C2C12 mouse myoblasts; D is a graph showing the effect of isoferulic acid on the viability of C2C12 mouse myoblasts;

[0027] Figure 5 The figure shows the effect of luteolin, 4-coumaric acid, caffeic acid and isoferulic acid combination on the viability of C2C12 mouse myoblasts;

[0028] Figure 6 The graph shows the effects of the combination of luteolin, 4-coumaric acid, caffeic acid and isoferulic acid, the purified coix seedling polyphenols and luteolin on the exhaustive swimming time of mice. DETAILED DESCRIPTION

[0029] The technical solution of the present invention will be clearly and completely described below in conjunction with the drawings and embodiments. Obviously, the described embodiments are only part of the embodiments of the invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0030] The present invention prepares and identifies components of coix seedling polyphenol purified material (PPCSS), and verifies the anti-fatigue active components in the coix seedling polyphenol purified material and the anti-fatigue effect of the composition by combining network pharmacology and cell experiments. The present invention embodiment experiments found that the active components of the composition consist of luteolin, 4-coumaric acid, caffeic acid and isoferulic acid, and the composition has obvious anti-fatigue effect and is superior to the coix seedling polyphenol purified material.

[0031] Embodiment 1:

[0032] Step 1: Preparation of Purified Polyphenols from Coix Seedlings (PPCSS)

[0033] 1. Take fresh coix seedlings after picking, add 60%-80% ethanol aqueous solution, solid-liquid ratio (1-3): (20-40), heat in a water bath at 50-80°C for extraction for 2-3h, and filter the extract.

[0034] 2. Use D101 macroporous resin as separation medium for primary purification. The sample volume is 30-40mL. After adsorption for 1-2h, rinse with distilled water to remove unadsorbed polyphenols and impurities, then elute with 30%-70% ethanol solution, collect the subsequent eluate and concentrate the eluate.

[0035] 3. Use S-8 macroporous resin as separation medium for secondary purification. Adjust the sample volume to 30-50 mL, rinse with distilled water to remove unadsorbed polyphenols and impurities after adsorption for 1-2 hours, then use 30%-70% ethanol solution for gradient elution until colorless, collect the polyphenol eluate and concentrate it to obtain the purified coix seedling polyphenol (PPCSS).

[0036] Purity determination of purified polyphenols from coix seedlings (PPCSS)

[0037] The NaNO2-AlCl3-NaOH method was used to determine the flavonoid purity of the coix seedling polyphenol purification obtained in step one by eluting with 30%, 50% and 70% ethanol solutions.

[0038] The results are as follows Figure 1 As shown, the purity of flavonoids in the purified coix seedling polyphenols eluted with 50% ethanol solution is as high as 75.05%.

[0039] Step 2: Identification of the components of purified coix seedling polyphenols (PPCSS)

[0040] The purified coix seedling polyphenols were eluted with 50% ethanol solution in step 1, and the supernatant was centrifuged for Q-Orbitrap high-resolution liquid-mass spectrometry analysis. The mass spectrometry conditions were electrospray ionization source (ESI), positive and negative ion switching scanning. The chromatographic conditions were AQ-C18 column, 150×2.1 mm, 1.8 μm, the aqueous phase was 0.1% formic acid aqueous solution, the organic phase was methanol, and the column oven temperature was 35°C.

[0041] The results are as follows Figure 2 As shown, the active components of the purified coix seedling polyphenols were identified as seven polyphenol flavonoids, including caffeic acid, 4-coumaric acid, rutin, naringin, luteolin, isoferulic acid, and 7-hydroxycoumarin.

[0042] Intestinal absorption characteristics and drug-like properties of active components from purified coix seedling polyphenols (PPCSS)

[0043] The database SwissADME (http: / / www.swissadme.ch / ) was used to confirm the intestinal absorption characteristics and drug-like properties of the 7 active components of the coix seedling polyphenol purification identified by Q-Orbitrap high-resolution liquid chromatography-mass spectrometry, and the screening indicators were set as GI absorption as High and 4 Yes in Druglikeness.

[0044] The results are shown in Table 1 , which shows the seven polyphenols identified by mass spectrometry of coix seedling polyphenols, and screened out luteolin, 4-coumaric acid, caffeic acid and isoferulic acid based on their intestinal absorbability and drug-like properties.

[0045] Table 1 is a comparison of intestinal absorption and drug-like properties of each active ingredient

[0046]

[0047]

[0048] Step 3: Network pharmacology of luteolin, 4-coumaric acid, caffeic acid, and isoferulic acid and fatigue

[0049] The molecular targets of four polyphenolic compounds (CP), namely luteolin, 4-coumaric acid, caffeic acid and isoferulic acid, were determined using Pubchem (https: / / pubchem.ncbi.nlm.nih.gov / ) and SwissTargetPrediction (http: / / www.swisstargetprediction.ch / ) databases. The "fatigue" disease targets (FA) were collected using the GeneCards (https: / / www.genecards.org / ) database, and the potential common targets of CP-FA were screened using the Venny2.1.0 (https: / / bioinfogp.cnb.csic.es / tools / venny / ) website. The CP-FA intersection genes were imported into the STRING

[0050] (https: / / cn.string-db.org / ) database, and set the species to “Homo sapiens” to obtain the PPI data of potential therapeutic targets of compounds CP and FA. The data were further visualized in Cytoscape 3.7.2 (http: / / cytoscape.org / ).

[0051] The results are as follows Figure 3 As shown, Figure 3 A total of 1,892 fatigue targets and 88 compound targets were obtained, and 63 candidate targets were screened. Figure 3The nodes in B are arranged by degree value. The larger the node, the darker the color, indicating that the number of genes acting on it is greater and the correlation is greater. Among them, PI3K, PIK3CA, AKT1, PIK3R1, PIK3CB, SRC, and STAT3 may play an important role in the protein interaction network.

[0052] Molecular docking validation

[0053] AutoDock software was used to perform molecular docking between the three-dimensional structures of luteolin, 4-coumaric acid, caffeic acid and isoferulic acid and the structures of the corresponding proteins of the above targets to analyze the binding activity. When the binding energy was <-5 Kcal / mol, it indicated that the ligand bound well to the target.

[0054] The results are shown in Table 2. The binding energies of the four key components, luteolin, 4-coumaric acid, caffeic acid and isoferulic acid, predicted by network pharmacology with the above targets are all less than -5 Kcal / mol, indicating that luteolin, 4-coumaric acid, caffeic acid and isoferulic acid have potential anti-fatigue effects.

[0055] Table 2 Molecular docking of luteolin, 4-coumaric acid, caffeic acid and isoferulic acid with fatigue targets

[0056]

[0057] Step 4: Effects of luteolin, 4-coumaric acid, caffeic acid and isoferulic acid on the viability of C2C12 mouse myoblasts

[0058] After differentiation, C2C12 mouse myoblasts were cultured at 5×10 3The cells were seeded at a density of 100 μg / well in a 96-well culture dish, and DMEM medium (containing 10% fetal bovine serum) was added and incubated for 24 hours. Luteolin (concentrations were 0.3125 μg / mL, 0.625 μg / mL, 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL), 4-coumaric acid (concentrations were 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL), and caffeic acid (concentrations were 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL) were supplemented to the cells. ) and isoferulic acid solution (concentrations were 12.5μg / mL, 25μg / mL, 50μg / mL, 100μg / mL, 200μg / mL, 400μg / mL) were incubated for 12 hours, and then 0.75mM H2O2 (experimentally confirmed that the viability of C2C12 mouse myoblasts decreased to less than 50% after treatment with this concentration of H2O2) was continued to be incubated for 4 hours, with the cell group without the composition but treated with H2O2 as the model group, and the cell group without H2O2 and the composition as the blank group, and then replaced with fresh culture medium containing CCK-8 solution. After incubation for 2 hours, the absorbance of each well was detected by enzyme labeling (Bio-Rad model 1680, USA), and the cell viability was calculated.

[0059] The results are as follows Figure 4 As shown, the cell viability after treatment with luteolin, 4-coumaric acid, caffeic acid and isoferulic acid was improved to varying degrees compared with the model group.

[0060] Cell experiments of the composition

[0061] The selected active components, luteolin, 4-coumaric acid, caffeic acid and isoferulic acid, were prepared into a composition at a mass ratio of 10:1:1:1. 3The cells were inoculated at a density of 100 / well in a 96-well culture dish, and DMEM medium (containing 10% fetal bovine serum) was added and incubated for 24 hours. The cells were supplemented with the composition solution (concentrations were 0.3125μg / mL, 0.625μg / mL, 1.25μg / mL, 2.5μg / mL, 5μg / mL, and 10μg / mL, respectively) and incubated for 12 hours, and then incubated with 0.75mM H2O2 (experimentally confirmed that the cell viability of C2C12 decreased to less than 50% after treatment with this concentration of H2O2) for another 4 hours. The cell group without the composition but treated with H2O2 was used as the model group, and the cell group without H2O2 and the composition was used as the blank group, and then replaced with fresh culture medium containing CCK-8 solution. After incubation for 2 hours, the absorbance of each well was detected with an enzyme marker (Bio-Rad model 1680, USA), and the cell viability was calculated.

[0062] The results are as follows Figure 5 As shown, compared with the model group, the cell viability of the pre-treated cells with the composition was significantly improved. And the protective effect of the composition on C2C12 mouse myoblasts was better than that of luteolin, 4-coumaric acid, caffeic acid and isoferulic acid used alone.

[0063] Animal testing of the composition

[0064] Forty healthy male Kunming mice (SPF grade, weight 18-20g, 6-8 weeks old) were randomly divided into five groups, 8 mice in each group: normal control group (NC), model group (MC), positive control group (PC), purified coix seedling polyphenols group (PPCSS group), luteolin group (LU), and combination group. The mice were given the corresponding dose of the test substance according to their body weight every day, with an intragastric dose of 0.01mL / g·bw, once a day, for 13 consecutive days. The NC group and the MC group were intragastrically administered with an equal amount of normal saline, the PC group was intragastrically administered with 500mg / kg / d of Rhodiola rosea capsules, the PPCSS group was intragastricly administered with 200mg / kg / d of purified coix seedling polyphenols, the LU group was intragastricly administered with 50mg / kg / d of luteolin, and the combination group was intragastricly administered with 50mg / kg / d of a combination of luteolin, 4-coumaric acid, caffeic acid, and isoferulic acid (10:1:1:1). On the 13th day, 30 minutes after the last administration, mice in all groups except the NC group were subjected to an exhaustive swimming test. The tail of the mice was loaded with a lead segment of 5% of their body weight, and they swam in a water tank (50cm×50cm×40cm) at a water temperature of 25±2℃ and a water depth of 35cm, and stirred with a glass rod to ensure that the mice kept moving. The time taken by the mice from the beginning of swimming to the time when their heads were submerged in the water for 7s and could not float to the surface was recorded, which was the exhaustive swimming time of the mice.

[0065] The results are as follows Figure 6As shown, the purified coix seedling polyphenols, luteolin and the combination can improve the anti-fatigue ability of mice. At the same time, it can be seen that the combination of luteolin, 4-coumaric acid, caffeic acid and isoferulic acid has the best effect.

[0066] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for preparing a coix seedling-derived polyphenol composition having an anti-fatigue effect, characterized in that: The following steps are involved: Step 1, extracting with ethanol solution and purifying with macroporous resin to obtain purified coix seedling polyphenols; Step 2, identifying the active components of the purified coix seedling polyphenols by Q-Orbitrap high-resolution liquid chromatography-mass spectrometry technology; Step 3: Using network pharmacology and molecular docking to screen out active components with potential anti-fatigue function in the purified coix seedling polyphenols; Step 4: Use cell models and animal models to evaluate the anti-fatigue effects of each active component and its combination, and determine the coix seedling-derived polyphenol composition with anti-fatigue effect.

2. The preparation method according to claim 1, characterized in that: In step 1, the crushed fresh coix seedlings are mixed with a 60%-80% ethanol solution in a solid-liquid ratio of (1-3): (20-40), and the mixture is heated in a 50-80°C water bath for extraction for 2-3 hours, and then purified using a macroporous resin as a separation medium to obtain a purified coix seedling polyphenol product.

3. The preparation method according to claim 1 or 2, characterized in that: In step 1, the macroporous resins used for purifying the coix seedling polyphenol purified product are D101 and S-8, and the conditions for the primary purification of D101 are: the sample volume is 30-40 mL, the sample polyphenol concentration is 2-4 mg / mL, and the eluent is an ethanol aqueous solution with a mass fraction of 30%-70%; the conditions for the secondary purification of S-8 are: the sample volume is 30-50 mL, the sample polyphenol concentration is 2-4 mg / mL, and the eluent is an ethanol aqueous solution with a mass fraction of 30%-70%, and gradient elution is performed.

4. The preparation method according to claim 1, characterized in that: In step 2, the conditions of the Q-Orbitrap high-resolution liquid-mass spectrometry are as follows: the mass spectrometry conditions are an electrospray ionization source, positive and negative ion switching scanning, a scanning range of 100.0 to 1500.0 m / z, an electrospray voltage of 3.8 kV, a collision gas of high-purity argon, a sheath gas of nitrogen, and a data acquisition time of 30.0 min; the chromatographic conditions are a chromatographic column of AQ-C18, 150×2.1 mm, 1.8 μm, an aqueous phase of 0.1% formic acid aqueous solution by mass fraction, an organic phase of methanol, a column oven temperature of 35° C., and an injection volume of 5.00 μL.

5. The preparation method according to claim 1, characterized in that: In step 2, the active components of the purified coix seedling polyphenols are identified by Q-Orbitrap high-resolution liquid chromatography-mass spectrometry technology, including rutin, naringin, 7-hydroxycoumarin, luteolin, 4-coumaric acid, caffeic acid and isoferulic acid.

6. The preparation method according to claim 1, characterized in that: In step three, the steps of network pharmacology are as follows: the active components in the purified coix seedling polyphenols are screened as luteolin, 4-coumaric acid, caffeic acid and isoferulic acid through intestinal absorbability and drug-like index; then, the potential targets of the four active components of luteolin, 4-coumaric acid, caffeic acid and isoferulic acid and the fatigue target with "fatigue" as the keyword are obtained through an online database, and then the candidate targets of the compounds and fatigue are obtained by intersection; the candidate targets are imported into the STRING platform to prepare a protein interaction network and analyze it, and the key targets are screened; then, the binding ability of the active components with the fatigue targets is verified by molecular docking, and the molecular docking software is AutoDock software, and the docked fatigue targets are PI3K, PIK3CA, AKT1, PIK3R1, PIK3CB, SRC, and STAT3.

7. The preparation method according to claim 1 or 6, characterized in that: In step three, the network pharmacology and molecular docking screened out active components with potential anti-fatigue function, namely, luteolin, 4-coumaric acid, caffeic acid and isoferulic acid.

8. The preparation method according to claim 7, characterized in that: In step 4, the composition is composed of luteolin, 4-coumaric acid, caffeic acid and isoferulic acid in a mass ratio of (6-10):(1-3):(1-3):(1-3).

9. The preparation method according to claim 1, characterized in that: In step 4, the cell model is a C2C12 mouse myoblast oxidative damage model, and the animal model is a mouse exhaustive swimming model.

10. An application of a composition prepared by the preparation method according to any one of claims 1 to 9, characterized in that: The composition is used in food, health products or medicines.

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