Blueberry polyphenol compound and application thereof in preparation of product with function of improving cell viability

Abstract

The application relates to the technical field of biological medicine, and particularly discloses a blueberry polyphenol compound and application thereof in preparation of a product with a cell vitality improving effect. The blueberry polyphenol compound has a structure shown in formula I or formula II. Research shows that the blueberry polyphenol compound has a cell vitality improving effect; and the cell vitality improving effect is significantly higher than that of a positive control drug resveratrol. Since the blueberry polyphenol compound with the structure shown in formula I or formula II has a cell vitality improving effect, the blueberry polyphenol compound is used as an effective component to prepare food, dietary supplements, functional food, skin care products or medicines with a cell vitality improving effect, and has important application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a blueberry polyphenol compound and application thereof in preparation of a product with the effect of improving cell viability. BACKGROUND

[0002] Low cell viability in the skin leads to poor self-repairing ability of the skin; for example, acne, spots, lack of moisture, and appearance of fine lines and wrinkles are closely related to cell viability in the skin. Therefore, improving cell viability in the skin helps to skin care from the root.

[0003] Blueberries have a sweet and sour taste and contain rich nutritional ingredients; research shows that they have the functions of preventing brain aging, resisting cancer, strengthening the heart, softening blood vessels, and enhancing human immunity. However, the prior art has less research on effective components in blueberries with the effect of improving cell viability.

[0004] The applicant disclosed a blueberry extract, a preparation method thereof, and application thereof in preparation of a medicine or food with the effects of lowering blood lipids and / or reducing weight in the prior application of Chinese invention patent CN114601859 A. The preparation method of the blueberry extract comprises the following steps: (1) taking fresh blueberries, then adding water at 0-5℃ to crush, to obtain a crushed mixed solution; (2) performing solid-liquid separation on the crushed mixed solution, and taking the liquid to obtain a blueberry crushed solution; (3) adding ethyl acetate to the blueberry crushed solution to perform extraction, to obtain an ethyl acetate extract solution, and drying the ethyl acetate extract solution to obtain an ethyl acetate extract; (4) further performing elution separation on the ethyl acetate extract by using a preparative liquid chromatograph, and drying the eluent to obtain the blueberry extract; research shows that it has excellent effects of lowering blood lipids and reducing weight; however, the prior art does not disclose its effect of improving cell viability. Therefore, it has important application value to develop a compound with the effect of improving cell viability from blueberries as raw materials. SUMMARY

[0005] In order to overcome at least one of the technical problems in the prior art, the present application first provides a blueberry polyphenol compound. Research shows that the blueberry polyphenol compound has the effect of improving cell viability.

[0006] The technical scheme of the present application is as follows:

[0007] The present application first provides a blueberry polyphenol compound, which has the structure shown in formula I or formula II;

[0008]

[0009] The application also provides a preparation method of blueberry polyphenols.

[0010] Preferably, the preparation method of blueberry polyphenols comprises the following steps:

[0011] (1) fresh blueberries are taken and mixed with organic solvents, and then the blueberries are crushed to obtain a crushed mixture; the mixture is centrifuged, and the supernatant is concentrated and dried to obtain a blueberry organic solvent extract;

[0012] (2) the blueberry organic solvent extract is suspended in water, and then extracted with ethyl acetate to obtain a blueberry ethyl acetate extract liquid; the blueberry ethyl acetate extract liquid is concentrated and dried to obtain a blueberry ethyl acetate extract;

[0013] (3) the ethyl acetate extract is subjected to silica gel column chromatography, and eluted with a mixed solvent composed of chloroform and acetonitrile to obtain a silica gel column elution fraction;

[0014] (4) the silica gel column elution fraction is subjected to preparative HPLC to obtain the blueberry polyphenols.

[0015] Preferably, the specific elution conditions of the silica gel column in step (3) are as follows:

[0016] The blueberry ethyl acetate extract is subjected to silica gel column chromatography, first eluted with a mixed solvent composed of chloroform and acetonitrile in a volume ratio of 100:23-15 to remove impurities; then eluted with a mixed solvent composed of chloroform and acetonitrile in a volume ratio of 100:38-40, and the eluate eluted with the mixed solvent composed of chloroform and acetonitrile in a volume ratio of 100:38-40 is concentrated and dried to obtain a silica gel column elution fraction A;

[0017] The silica gel column elution fraction A is subjected to silica gel column chromatography again, first eluted with a mixed solvent composed of chloroform and acetonitrile in a volume ratio of 100:30 to remove impurities; then eluted with a mixed solvent composed of chloroform and acetonitrile in a volume ratio of 100:35, and the eluate eluted with the mixed solvent composed of chloroform and acetonitrile in a volume ratio of 100:35 is concentrated and dried to obtain a silica gel column elution fraction B.

[0018] Most preferably, the specific elution conditions of the silica gel column in step (3) are as follows:

[0019] The blueberry ethyl acetate extract is subjected to silica gel column chromatography, first eluted with a mixed solvent composed of chloroform and acetonitrile in a volume ratio of 100:24 to remove impurities; then eluted with a mixed solvent composed of chloroform and acetonitrile in a volume ratio of 100:39, and the eluate eluted with the mixed solvent composed of chloroform and acetonitrile in a volume ratio of 100:39 is concentrated and dried to obtain a silica gel column elution fraction A;

[0020] The silica gel column elution part A is again applied to a silica gel column, first eluted with a mixed solvent of chloroform and acetonitrile with a volume ratio of 100:30 to remove impurities; then eluted with a mixed solvent of chloroform and acetonitrile with a volume ratio of 100:35, and the eluent eluted by the mixed solvent of chloroform and acetonitrile with a volume ratio of 100:35 is collected, concentrated and dried to obtain silica gel column elution part B.

[0021] Preferably, step (4) uses preparative HPLC to prepare silica gel column elution part B, thereby obtaining the blueberry polyphenol compound.

[0022] Most preferably, the preparation conditions of the blueberry polyphenol compound with the structure shown in formula I are as follows:

[0023] The chromatographic column is a C18 column, the detection wavelength is 280-330 nm, the flow rate is 5-15 mL / min, the column temperature is 20-30 DEG C, the eluent is an acetonitrile aqueous solution with a volume fraction of 18%, the eluent corresponding to the chromatographic peak with a retention time of 14.35 min is collected, and the blueberry polyphenol compound with the structure shown in formula I is obtained after concentration and drying.

[0024] Most preferably, the preparation conditions of the blueberry polyphenol compound with the structure shown in formula II are as follows:

[0025] The chromatographic column is a C18 column, the detection wavelength is 280-330 nm, the flow rate is 5-15 mL / min, the column temperature is 20-30 DEG C, the eluent is an acetonitrile aqueous solution with a volume fraction of 15%, the eluent corresponding to the chromatographic peak with a retention time of 17.47 min is collected, and the blueberry polyphenol compound with the structure shown in formula II is obtained after concentration and drying.

[0026] The application also provides a use of the above-mentioned blueberry polyphenol compound in the preparation of a product with the effect of improving cell viability.

[0027] Preferably, the cell viability refers to skin fibroblast cell viability.

[0028] Preferably, the product is food, a dietary supplement, a functional food, a skin care product or a medicine.

[0029] Beneficial effects:

[0030] (1) The application provides a brand-new blueberry polyphenol compound; research shows that the blueberry polyphenol compound has the effect of improving cell viability; and the effect of improving cell viability is significantly higher, even much higher, than that of the positive control drug resveratrol.

[0031] (2) The application also provides a novel preparation method of the blueberry polyphenol compound with the structure shown in formula I or formula II; the method first uses fresh blueberries as raw materials to prepare the blueberry polyphenol compound with the structure shown in formula I or formula II. In the preparation method of the blueberry polyphenol compound, the elution conditions of the silica gel column and the elution conditions of the preparative HPLC are very critical; if any one of the elution conditions is improperly selected, the blueberry polyphenol compound with the structure shown in formula I or formula II cannot be prepared from the blueberries. By using the method steps provided in the application, the blueberry polyphenol compound with the structure shown in formula I or formula II can be prepared only under the elution conditions of the silica gel column and the elution conditions of the preparative HPLC.

[0032] (3) Since the blueberry polyphenol compound with the structure shown in formula I or formula II has the effect of improving cell viability, it has important application value when used as an effective component to prepare food, dietary supplements, functional food, skin care products or drugs with the effect of improving cell viability. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 HR-ESI-MS spectrum of the blueberry polyphenol compound with the structure shown in formula I.

[0034] Figure 2 HR-ESI-MS spectrum of the blueberry polyphenol compound with the structure shown in formula I. 1 H NMR spectrum of the blueberry polyphenol compound with the structure shown in formula I.

[0035] Figure 3 H NMR spectrum of the blueberry polyphenol compound with the structure shown in formula I. 13 C NMR spectrum of the blueberry polyphenol compound with the structure shown in formula I.

[0036] Figure 4 H- 1 H- 1 H COSY and HMBC relationship diagram of the blueberry polyphenol compound with the structure shown in formula I.

[0037] Figure 5 H- 1 H- 1 H COSY spectrum of the blueberry polyphenol compound with the structure shown in formula I.

[0038] Figure 6 HSQC spectrum of the blueberry polyphenol compound with the structure shown in formula I.

[0039] Figure 7 HMBC spectrum of the blueberry polyphenol compound with the structure shown in formula I.

[0040] Figure 8 HR-ESI-MS spectrum of the blueberry polyphenol compound with the structure shown in formula II.

[0041] Figure 9 HSQC spectrum of blueberry polyphenols compound of formula II 1 H NMR spectrum.

[0042] Figure 10 HSQC spectrum of blueberry polyphenols compound of formula II 13 C NMR spectrum.

[0043] Figure 11 HSQC spectrum of blueberry polyphenols compound of formula II 1 H- 1 H COSY spectrum.

[0044] Figure 12 Key HMBC spectrum of blueberry polyphenols compound of formula II 1 H- 1 H COSY, HMBC and NOESY spectrum.

[0045] Figure 13 HSQC spectrum of blueberry polyphenols compound of formula II

[0046] Figure 14 HMBC spectrum of blueberry polyphenols compound of formula II

[0047] Figure 15 NOESY spectrum of blueberry polyphenols compound of formula II DETAILED DESCRIPTION

[0048] The technical solutions of the present application will be described clearly and completely below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by a person of ordinary skill in the art without any creative effort fall within the protection scope of the present application.

[0049] Example 1: Preparation of blueberry polyphenols compound

[0050] (1) Fresh blueberries were added with organic solvent mixture, and then put into a crusher for crushing for 15 min to obtain a crushed mixture; the mixture was centrifuged, and the supernatant was concentrated and dried to obtain a blueberry organic solvent extract; wherein the ratio of the amount of fresh blueberries to the amount of organic solvent was 1 g:10 mL; and the organic solvent was acetone;

[0051] (2) The blueberry organic solvent extract was suspended with water, and then extracted with ethyl acetate to obtain a blueberry ethyl acetate extract liquid; the blueberry ethyl acetate extract liquid was concentrated and dried to obtain a blueberry ethyl acetate extract; wherein the ratio of the amount of blueberry organic solvent extract to the amount of water and the amount of ethyl acetate was 1 g:25 mL:25 mL.

[0052] (3) The blueberry ethyl acetate extract is subjected to a silica gel column (the silica gel column is filled with 200-300 mesh silica gel; the weight amount of silica gel in the silica gel column is 30 times that of the blueberry ethyl acetate extract), first eluted with 4 times the column volume of a mixed solvent of chloroform and acetonitrile at a volume ratio of 100:24 to remove impurities; then eluted with 4 times the column volume of a mixed solvent of chloroform and acetonitrile at a volume ratio of 100:39, and the eluate eluted by the mixed solvent of chloroform and acetonitrile at a volume ratio of 100:39 is collected, concentrated and dried to obtain silica gel column elution part A;

[0053] The silica gel column elution part A is subjected to a silica gel column again (the silica gel column is filled with 300-400 mesh silica gel; the weight amount of silica gel in the silica gel column is 50 times that of the silica gel column elution part A), first eluted with 5 times the column volume of a mixed solvent of chloroform and acetonitrile at a volume ratio of 100:30 to remove impurities; then eluted with 5 times the column volume of a mixed solvent of chloroform and acetonitrile at a volume ratio of 100:35, and the eluate eluted by the mixed solvent of chloroform and acetonitrile at a volume ratio of 100:35 is collected, concentrated and dried to obtain silica gel column elution part B;

[0054] (4) The silica gel column elution part B is subjected to preparation by preparative HPLC to obtain the blueberry polyphenol compound;

[0055] The preparation conditions of the blueberry polyphenol compound of formula I are as follows:

[0056] The chromatographic column is a C18 column (9.4*250mm, 5μm, Agilent), the detection wavelength is 300nm, the flow rate is 10mL / min, the column temperature is 25℃, and the eluent is an acetonitrile aqueous solution with a volume fraction of 18%; the eluate corresponding to the chromatographic peak with a retention time of 14.35min is collected, concentrated and dried to obtain the blueberry polyphenol compound of formula I;

[0057] The preparation conditions of the blueberry polyphenol compound of formula II are as follows:

[0058] The chromatographic column is a C18 column (9.4*250mm, 5μm, Agilent), the detection wavelength is 300nm, the flow rate is 10mL / min, the column temperature is 25℃, and the eluent is an acetonitrile aqueous solution with a volume fraction of 15%; the eluate corresponding to the chromatographic peak with a retention time of 17.47min is collected, concentrated and dried to obtain the blueberry polyphenol compound of formula II.

[0059] The blueberry polyphenol compound of formula I; yellow powder; high-resolution mass spectrum ( Figure 1 )[HR-ESI-MS m / z: 399.1421 [M+H]+ (calcd for C 20 H 24 The molecular formula of NaO7,399.1414) is determined to be C. 20 H 24 O7 has an unsaturation degree of 9. 1 HNMR spectrum ( Figure 2 Three hydroxyl proton signals were observed in 500MHz, CDCl3[δ] H 13.91, 10.47, 8.76 (each 1H, s)], 2 unsaturated proton signals [δ H 6.10, 5.95 (each 1H, s)], 1 methoxy signal [δ H 3.99(3H,s)] and one characteristic methylene signal [δ H 3.61(2H,s)]. 13 C NMR spectrum ( Figure 3 The 2DNMR data (125 MHz, CDCl3) showed 20 carbon atom signals, which were assigned to 10 quaternary carbons, 2 methines, 5 methylenes, and 3 methyls. Further analysis of the compound's 2DNMR data... Figure 5 , 6 , 7), and its characteristic methylene signal H-7′ (δ) was found. H 3.61) respectively with C-1(δ) in the two structural segments C 104.6) / C-2(δ C 159.8) and C-1′(δ C 102.2) / C-2′(δ C 170.0) / C-6′(δ C 167.2) showed HMBC long-range correlation. The structure of the compound was ultimately identified. 1 H and 13 The C NMR data, after comprehensive analysis of 1D and 2D NMR signals, are classified as shown in Table 1.

[0060] Blueberry polyphenolic compounds with the structure shown in Formula II: yellow powder, high-resolution mass spectrometry (HPLC-MS / MS) Figure 8 HR-ESI-MS showed an ion peak of m / z 419.1709 [M+H]. + (calcd for C 22 H 27 O8,419.1700), the molecular formula is determined to be C 22 H 26 O8 has an unsaturation degree of 10. 1 H NMR spectrum ( Figure 9 Four hydroxyl proton signals were observed in 500MHz, CDCl3[δ]H 15.49, 13.84, 9.48, 8.62 (each 1H, s)], 1 aromatic proton signal [δ H 6.10 (1H, s)], 2 methoxyl signals [δ H 4.01, 3.70 (each 3H, s)], 1 characteristic methylene signal [δ H 3.77 (2H, s)], 2 singlet methyl signals [δ H 2.71, 2.08 (each 3H, s)], and 1 set of propyl signals attached to carbonyl [δ H 3.09 (2H, t, J = 7.4 Hz Z , 1.69 (m), 0.98 (3H, t, J = 7.4 Hz Z )]. 13 C NMR spectrum ( Figure 10 , 125 MHz, CDC13) showed 22 carbon signals, including 2 carbonyl (δ C 207.5, 204.5), 2 benzene ring signals (δ C 165.6, 161.8, 160.9, 160.7, 159.9, 159.4, 112.1, 109.3, 108.5, 106.9, 104.5, 92.4), and 2 methoxyl signals (δ C 61.7, 56.7). In the 2D HMBC spectrum ( Figure 14 ), 2'-OH (δ H 15.51) was observed to be correlated with C-1' (δ C 109.2) / C-2' (δ C 159.8) / C-8' (δ C 204.4), respectively, thus confirming that C-2' was hydroxyl-substituted. In the NOESY spectrum ( Figure 15 ), the methoxyl hydrogen signal H-10' (δ H 3.72) was obviously observed to be correlated with the acetyl methyl H-9' (δ H 2.73), indicating that the methoxyl group should be located at the ortho position of the carbonyl group, so that C-4' was methoxyl-substituted and C-6' was hydroxyl-substituted. The structure of the compound was finally identified. Its 1 H and 13 C NMR data were assigned by comprehensive analysis of 1D and 2D NMR signals, as shown in Table 2.

[0061] Table 1. Blueberry polyphenolic compounds of the structure shown in Formula I 1 H and 13CNMR data 1D and 2D NMR signal assignment

[0062]

[0063] 500( 1 H) and 125( 13 C) MHz, CDCl3

[0064] Table 2. Blueberry polyphenols of the structure shown in formula II 1 H and 13 CNMR data 1D and 2D NMR signal assignment

[0065]

[0066]

[0067] 500( 1 H) and 125( 13 C) MHz, CDCl3

[0068] Experimental Example 1: Cell viability test

[0069] Skin fibroblasts (1 x 10 4 cells / well) were cultured in 25 cm 2 flasks overnight, and then stimulated with paraquat (10 μM) for 6 h. Then, the cells were washed with PBS twice, and the test samples were added to the experimental groups at a concentration of 10 μg / mL for 24 h; a model group without the test sample was also set up, and a blank control group without paraquat treatment was also set up. Then, the cell viability was measured using a CCK-8 assay kit; the cell viability of the blank control group without paraquat treatment was taken as 100%, and the cell viability percentages of the experimental groups and the model group relative to the blank control group without paraquat treatment are shown in Table 3.

[0070] The test samples of the experimental groups 1-3 were blueberry polyphenols of the structure shown in formula I or formula II, and resveratrol was used as a positive control.

[0071] Table 3. Results of the blueberry polyphenols of the present application in the cell viability test

[0072]

[0073] As can be seen from the experimental data in Table 3, the blueberry polyphenols of the structure shown in formula I of the present application have a cell viability significantly higher than that of resveratrol, and have a very excellent effect of improving cell viability.

[0074] Especially, the blueberry polyphenol compound of the structure shown in formula II has a cell viability which is much higher than that of the positive control drug resveratrol, and is also much higher than that of the blueberry polyphenol compound of the structure shown in formula I; this shows that in the application, the blueberry polyphenol compounds with different structures have different effects on improving cell viability; the blueberry polyphenol compound of the structure shown in formula II has a more excellent effect on improving cell viability compared with the blueberry polyphenol compound of the structure shown in formula I.

Claims

1. A method for preparing blueberry polyphenols, the blueberry polyphenols having a structure shown in Formula I or Formula II; Formula I; Formula II; characterized in that The method comprises the following steps: (1) fresh blueberries are mixed with an organic solvent, and then the blueberries are crushed to obtain a crushed mixture;The mixture is centrifuged, and the supernatant is concentrated and dried to obtain a blueberry organic solvent extract;The organic solvent is acetone; (2) the blueberry organic solvent extract is suspended in water, and then extracted with ethyl acetate to obtain a blueberry ethyl acetate extract;The blueberry ethyl acetate extract is concentrated and dried to obtain a blueberry ethyl acetate extract; (3) the blueberry ethyl acetate extract is subjected to silica gel column chromatography, first eluted with a mixed solvent of chloroform and acetonitrile in a volume ratio of 100:23-15 to remove impurities;Then eluted with a mixed solvent of chloroform and acetonitrile in a volume ratio of 100:38-40, and the eluate eluted with the mixed solvent of chloroform and acetonitrile in a volume ratio of 100:38-40 is collected, concentrated and dried to obtain silica gel column elution fraction A; Silica gel column elution fraction A is subjected to silica gel column chromatography again, first eluted with a mixed solvent of chloroform and acetonitrile in a volume ratio of 100:30 to remove impurities;Then eluted with a mixed solvent of chloroform and acetonitrile in a volume ratio of 100:35, and the eluate eluted with the mixed solvent of chloroform and acetonitrile in a volume ratio of 100:35 is collected, concentrated and dried to obtain silica gel column elution fraction B; (4) silica gel column elution fraction B is subjected to preparative HPLC; The chromatographic column is a C18 column, the detection wavelength is 280-330 nm, the flow rate is 5-15 mL / min, and the column temperature is 20-30℃; The eluent is an aqueous acetonitrile solution with a volume fraction of 18%; The eluate corresponding to the chromatographic peak with a retention time of 14.35 min is collected, concentrated and dried to obtain the compound of Formula I; The eluent is an aqueous acetonitrile solution with a volume fraction of 15%; The eluate corresponding to the chromatographic peak with a retention time of 17.47 min is collected, concentrated and dried to obtain the compound of Formula II.

2. The method for preparing blueberry polyphenolic compounds according to claim 1, characterized in that, The specific elution conditions of the silica gel column in step (3) are as follows: The blueberry ethyl acetate extract is subjected to silica gel column chromatography, first eluted with a mixed solvent of chloroform and acetonitrile in a volume ratio of 100:23 to remove impurities;Then eluted with a mixed solvent of chloroform and acetonitrile in a volume ratio of 100:39, and the eluate eluted with the mixed solvent of chloroform and acetonitrile in a volume ratio of 100:39 is collected, concentrated and dried to obtain silica gel column elution fraction A; Silica gel column elution fraction A is subjected to silica gel column chromatography again, first eluted with a mixed solvent of chloroform and acetonitrile in a volume ratio of 100:30 to remove impurities;Then eluted with a mixed solvent of chloroform and acetonitrile in a volume ratio of 100:35, and the eluate eluted with the mixed solvent of chloroform and acetonitrile in a volume ratio of 100:35 is collected, concentrated and dried to obtain silica gel column elution fraction B.

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