5,7-Dihydroxychromone for inhibiting CD38 activity, preparation method and application thereof

Abstract

The present invention provides a 5,7-dihydroxychromone for inhibiting CD38 activity, and a preparation method and application thereof. The preparation method comprises the following steps: drying, pulverizing and sieving raw materials to obtain an ultrafine powder with a particle size greater than 600 mesh; adding water and a complex enzyme to the ultrafine powder for enzymatic hydrolysis to obtain an enzymatic hydrolysate; performing alcohol extraction and filtration on the enzymatic hydrolysate, collecting the filtrate to obtain an extract; concentrating the extract under reduced pressure, extracting with an organic solvent, and separating to obtain an extract; concentrating the extract under reduced pressure, purifying by column chromatography and liquid chromatography, and drying to obtain 5,7-dihydroxychromone. The 5,7-dihydroxychromone prepared by the present invention has high purity and strong CD38 inhibitory activity, so it can be used as a new source of CD38 inhibitors to improve skin aging-related problems benefited from the reduction of CD38 activity, and has broad application prospects in skin care products or cosmetics.

Description

Technical Field

[0001] The present invention belongs to the field of natural product development, and particularly relates to a 5,7-dihydroxy chromone for inhibiting CD38 activity, its preparation method and application. Background Art

[0002] Nicotinamide adenine dinucleotide (NAD + ) is one of the most important biomolecules, directly or indirectly affecting cell metabolism, DNA repair, cell senescence, and immune cell function, etc. A decrease in intracellular NAD + level is a characteristic of aging. Increasing the NAD + level through the NAM pathway is a basic step for the irreversible restoration of NAD + levels mediated by different types of NAD + - consuming enzymes (including CD38, CD157, SARM1, Sirtuins, and PARPs). Cluster differentiation 38 (CD38) with a molecular weight of 45 kDa is a type II transmembrane protein that is widely present in mammalian immune cells and various tissues. CD38 is the main NAD + consumer in cells, and its enzyme activity is manifested as catalyzing the conversion of NAD + into metabolites such as ADPR, cADPR, and NAM. Developing safe and efficient compounds that inhibit CD38 activity and target NAD + metabolism, thereby alleviating or reversing many aging - related diseases, including cognitive decline, cancer, metabolic diseases, sarcopenia, and frailty, has become a potential therapeutic method for extending human healthspan.

[0003] Flavonoids are widely present in plants and are secondary metabolites in plants. They have physiological activities such as antioxidant, anti - inflammatory and antibacterial, free radical scavenging, and anti - aging effects, and are widely used in industries such as food, cosmetics, and agricultural and sideline products. Currently, natural flavonoids such as luteolin, quercetin, apigenin, and resveratrol have been reported to inhibit intracellular CD38 activity and promote an increase in NAD + levels in in vitro experiments or animal experiments. 5,7 - dihydroxy chromone is also a natural flavonoid. Since its content in each plant is not the most abundant component, it has not been taken seriously. Currently, the research on the activity of 5,7 - dihydroxy chromone mainly focuses on aspects such as antioxidant, antibacterial, and neuroprotective effects, and there is no report on its research for inhibiting CD38 activity.

[0004] The prior art shows that Niu Dandan (Natural Product Research and Development, 2011) et al. refluxed peanut shell powder with 70% ethanol three times, purified it with LSA-30 macroporous resin, freeze-dried it, and extracted it with an organic solvent (n-hexane:ethyl acetate:methanol:water:glacial acetic acid = 5:3:3.5:5:0.25, v / v). Then, 5.2 mg of 5,7-dihydroxy chromone with a purity of 96% was purified from 70 mg of the extract by high-speed counter-current chromatography. Kiene (Molecules, 2023) et al. repeatedly extracted 58 g of peanut shells with 80% aqueous ethanol three times for 24 h each time. After the extract was concentrated under reduced pressure and freeze-dried, 4 g of the crude extract was obtained. 1000 mg of the crude extract was separated by high-speed counter-current chromatography (the volume ratio of the solvent system was n-hexane:ethyl acetate:methanol:water = 1.0:1.0:1.0:1.5) and an RP-18 semi-preparative column to obtain 3 mg of 5,7-dihydroxy chromone with a purity of 99%. Du (Journal of Liquid Chromatography & Related Technologies, 2004) et al. extracted willow bark with 80% aqueous ethanol, concentrated it under reduced pressure, spray-dried it, and extracted it with ethyl acetate to obtain a flavonoid mixture. 1 g of the mixture was purified by high-speed counter-current chromatography (the volume ratio of the solvent system was water:methanol:ethyl acetate:n-hexane = 3:2:2:2) to obtain 29.5 mg of 5,7-dihydroxy chromone. These preparation methods cannot achieve the large-scale preparation of 5,7-dihydroxy chromone. CN109734693B discloses a method for purifying 5,7-dihydroxy chromone from peanut shells by removing flavonoid compounds with copper salt complexation, and the purity of the product is only 90%. Currently, there are only the above four reports on the targeted preparation of 5,7-dihydroxy chromone from plants. Other reports mainly focus on the discovery that 5,7-dihydroxy chromone is contained in a certain plant (such as coffee beans, Selaginella doederleinii, Wikstroemia chamaedaphne, Adina pilulifera, Rhododendron dauricum, Drynaria fortunei, Cnidium monnieri, etc.) when studying the chemical constituents of the plant. Therefore, it is necessary to prepare high-purity and highly active 5,7-dihydroxy chromone from plants in a targeted and high-extraction-rate manner. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the present invention provides a 5,7-dihydroxy chromone that inhibits CD38 activity, as well as its preparation method and application. The present invention prepares 5,7-dihydroxy chromone from Ophiopogon japonicus, Polygonum multiflorum Thunb, Caulis Polygoni Multiflori, and Cimicifuga foetida. The product has a high extraction rate, high purity, and good inhibitory effect on CD38 activity, and can significantly improve the NAD + level.

[0006] To achieve the above invention purposes, the present invention adopts the following technical solutions to be realized:

[0007] The present invention provides a preparation method of 5,7-dihydroxy chromone for inhibiting CD38 activity, comprising the following steps:

[0008] (1) After drying, ultrafine pulverization and sieving of plant raw materials, ultrafine powder is obtained;

[0009] (2) Water and a complex enzyme are added to the ultrafine powder in step (1) for enzymatic hydrolysis to obtain an enzymatic hydrolysate;

[0010] (3) An ethanol aqueous solution is added to the enzymatic hydrolysate in step (2) for extraction to obtain an extract;

[0011] (4) The extract in step (3) is concentrated under reduced pressure, and after extraction with an organic solvent, an extractant is obtained;

[0012] (5) The extractant in step (4) is concentrated under reduced pressure, purified by fractionation and liquid chromatography, and dried to obtain 5,7-dihydroxy chromone.

[0013] Further, the plant raw materials in step (1) include Ophiopogon japonicus, Polygonum multiflorum, Caulis Polygoni Multiflori, and Cimicifuga foetida.

[0014] Preferably, the raw materials in step (1) are Polygonum multiflorum and Caulis Polygoni Multiflori.

[0015] Further, the complex enzyme in step (2) is composed of cellulase, hemicellulase, rhamnosidase, and acidic protease, and its addition amount is 1.5% - 2.0% of the weight of the raw materials.

[0016] Further, the mass ratio of cellulase, hemicellulase, rhamnosidase, and acidic protease is 3 - 5:2 - 3:1:1.

[0017] Preferably, the complex enzyme in step (2) is composed of cellulase, hemicellulase, rhamnosidase, and acidic protease mixed in a mass ratio of 3:3:1:1, and its addition amount is 1.5% of the weight of the raw materials.

[0018] Further, the addition amount of water in step (2) is 10 - 20 times the weight of the raw materials.

[0019] Preferably, the addition amount of water in step (2) is 15 times the weight of the raw materials.

[0020] Further, the enzymatic hydrolysis conditions in step (2) are: enzymatic hydrolysis temperature 45°C - 55°C, pH 5.5 - 6.5, and enzymatic hydrolysis time 3h - 5h.

[0021] Preferably, the enzymatic hydrolysis conditions in step (2) are: enzymatic hydrolysis temperature 45°C, pH 6.5, and enzymatic hydrolysis time 3h.

[0022] Further, in the step (3), the extraction is carried out by adding 2.2 - 3.5 times the volume of 95% ethanol aqueous solution to the enzymatic hydrolysate, stirring at room temperature for 2 - 3 h, and filtering to obtain filtrate I; then adding 1 - 2 times the volume of 65% ethanol aqueous solution to the residue for repeated extraction, filtering to obtain filtrate II, and combining filtrate I and filtrate II to obtain the extract.

[0023] Further, in the step (4), the volume of the organic solvent is 5 - 8 times the volume of the extract obtained after vacuum concentration; the organic solvent is a mixture of ethyl acetate, n-butanol and water.

[0024] Further, the volume ratio of ethyl acetate, n-butanol and water is 6 - 8:3 - 5:5.

[0025] Further, in the step (5), the fractionation is divided into coarse fractionation and fine fractionation. The condition for the coarse fractionation is that the stationary phase is polyamide resin and the eluent is 40% - 60% ethanol aqueous solution; the condition for the fine fractionation is that the stationary phase is Cross-inked dextran gel LH-20 and the eluent is 50% - 70% methanol aqueous solution.

[0026] Further, the conditions for the liquid chromatography in the step (5) are that the chromatographic column is C18ME (10 mm×250 mm, 10μm), the column temperature is 25°C, the injection volume is 500 μL, the flow rate is 5 mL / min, mobile phase A is 0.1% trifluoroacetic acid aqueous solution, mobile phase B is methanol solution, the eluent is 40% B, and the detector is a DAD detector.

[0027] In summary, a preparation method of 5,7-dihydroxy chromone for inhibiting CD38 activity includes the following steps:

[0028] (1) Dry Ophiopogon japonicus, Polygonum multiflorum, Caulis Polygoni multiflori or Rhizoma Cimicifugae, ultrafinely pulverize, and sieve to obtain an ultrafine powder with a particle size larger than 600 mesh;

[0029] (2) Add 10 - 20 times the volume of water to the ultrafine powder in the step (1), and then add a composite enzyme accounting for 1.5% - 2.0% of the raw material weight. The composite enzyme is composed of cellulase, hemicellulase, rhamnosidase and acidic protease mixed in a mass ratio of 3 - 5:2 - 3:1:1. Control the enzymatic hydrolysis temperature at 45°C - 55°C, pH 5.5 - 6.5, and time 3 - 5 h to obtain an enzymatic hydrolysate;

[0030] (3) Add 2.2 - 3.5 times the volume of 95% aqueous ethanol to the enzymatic hydrolysate obtained in step (2), stir and extract at room temperature for 2 - 3 h, filter to obtain filtrate I, then use 1 - 2 times the volume of 65% aqueous ethanol to stir and extract the residue at room temperature for 2 - 3 h, filter to obtain filtrate II, and combine filtrate I and filtrate II to obtain the extract;

[0031] (4) Concentrate the extract obtained in step (3) under reduced pressure at 55 °C - 65 °C to obtain an extract, extract with 5 - 8 times the volume of an ethyl acetate - n - butanol - water mixture (volume ratio 6 - 8:3 - 5:5), separate to obtain the upper layer liquid I, repeat the extraction, separate to obtain the upper layer liquid II, and combine upper layer liquid I and upper layer liquid II to obtain the extraction solution;

[0032] (5) Concentrate the extraction solution obtained in step (4) under reduced pressure, first perform rough fractionation through polyamide resin, collect the eluate with 40% - 60% aqueous ethanol, concentrate under reduced pressure, then perform fine fractionation through Cross - linked dextran gel LH - 20, collect the eluate with 50% - 70% aqueous methanol, concentrate under reduced pressure, and finally purify through liquid chromatography, collect the eluate, and obtain 5,7 - dihydroxy chromone after drying.

[0033] The present invention also provides the application of 5,7 - dihydroxy chromone prepared by the described preparation method in the preparation of CD38 inhibitors.

[0034] Further, the usage concentration of the 5,7 - dihydroxy chromone is 5 μM - 150 μM.

[0035] Further, the purity of the 5,7 - dihydroxy chromone is greater than 97%.

[0036] Further, the IC 50 of the 5,7 - dihydroxy chromone for inhibiting CD38 activity is 54.83 μM.

[0037] The present invention also provides the application of 5,7 - dihydroxy chromone prepared by the described preparation method in the preparation of anti - aging skin care products or cosmetics.

[0038] The present invention also provides the application of 5,7 - dihydroxy chromone prepared by the described preparation method in the preparation of drugs for preventing and treating aging - related diseases.

[0039] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0040] The present invention adopts the method of enzymatic hydrolysis-assisted alcohol extraction, polyamide resin coarse fractionation, gel column fine fractionation, and liquid chromatography purification to efficiently prepare 5,7-dihydroxychromone from Radix Ophiopogonis, Radix Polygoni Multiflori, Radix Polygoni Multiflori, and Rhizoma Cimicifugae. The product extraction rate is as high as 0.279%, and the purity is greater than 97%, and can reach up to 99.13%. In addition, the present invention verifies for the first time that 5,7-dihydroxychromone has a CD38 inhibitory effect, and the inhibition rate is greater than 70%. It can be used as a CD38 inhibitor, thereby effectively improving NAD + Content, targeted regulation of NAD + The metabolic level of 5,7-dihydroxychromone can be used to prepare skin care products or cosmetics with anti-aging effects, as well as to prepare drugs for preventing and treating aging-related diseases. It effectively expands the application scope of 5,7-dihydroxychromone and has broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The figure shows the inhibitory effect of 5,7-dihydroxychromone prepared in Examples 2 to 6 on CD38, wherein the horizontal axis represents the 5,7-dihydroxychromone prepared in different examples, and the vertical axis represents the inhibition rate.

[0042] Figure 2 The inhibitory effect of 5,7-dihydroxychromone prepared in Example 5 on CD38 and IC 50 , where the horizontal axis is the logarithm of the concentration of 5,7-dihydroxychromone and the vertical axis is the inhibition rate.

[0043] Figure 3 The 5,7-dihydroxychromone prepared in Examples 2 to 6 has an effect on the cellular level of NAD + The influence of the content, wherein the horizontal axis is 5,7-dihydroxychromone prepared in different examples, and the vertical axis is NAD + Relative content.

[0044] Figure 4 The effect of 5,7-dihydroxychromone prepared in Example 5 on the cellular level of NAD + The horizontal axis is the concentration of 5,7-dihydroxychromone and the vertical axis is the concentration of NAD + Relative content.

[0045] Figure 5 These are the results of SA-β-Gal staining experiments after the 5,7-dihydroxychromone prepared in Example 5 acted on HUVEC cells, where (a) is the control group (normal cell culture), (b) is the aging group (cisplatin-induced for 24 h), and (c) is the 5,7-dihydroxychromone group (cells were incubated with 5,7-dihydroxychromone for 9 h and then induced with cisplatin for 24 h). DETAILED DESCRIPTION

[0046] The technical solution of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0047] The purity of 5,7-dihydroxy chromone was determined by high performance liquid chromatography, and the specific method was as follows:

[0048] The chromatographic column was ZORBAX SB C18 (4.6 mm×250 mm, 5 μm), the column temperature was 25 °C, the injection volume was 10 μL, the flow rate was 1 mL / min, the mobile phase A was 0.2% formic acid aqueous solution, the mobile phase B was methanol solution, and the elution conditions were 20% B at 0 min, 20% at 5 min, 60% B at 25 min, 20% B at 30 min. The detector was an ultraviolet detector, and the detection wavelength was 365 nm. The mass concentration (mM) of the 5,7-dihydroxy chromone standard was taken as the abscissa, and the peak area (A) was taken as the ordinate to plot the standard curve. The purity of 5,7-dihydroxy chromone was calculated according to the standard curve.

[0049] Example 1. Influence of enzyme type on the preparation of 5,7-dihydroxy chromone

[0050] Take 0.1 kg of Polygoni Multiflori Caulis, dry it, ultrafinely crush it, and sieve it to obtain an ultrafine powder with a particle size larger than 600 mesh. Add 15 times the volume of water and a certain mass of enzyme to the ultrafine powder, control the temperature at 45 °C, pH = 6.5, and the time at 3 h to obtain an enzymolysis solution. Add 3 times the volume of 95% ethanol aqueous solution to the enzymolysis solution, stir and extract at room temperature for 2 h, filter to obtain filtrate I, then add 1 time the volume of 65% ethanol aqueous solution to the residue for repeated extraction, filter to obtain filtrate II, and combine filtrate I and filtrate II to obtain an extraction solution. Concentrate the extraction solution under reduced pressure at 65 °C into an extract, extract it with 6 times the volume of an organic solvent (the volume ratio of ethyl acetate - n-butanol - water is 8:4:5), separate to obtain the upper layer liquid I, repeat the extraction, separate to obtain the upper layer liquid II, and combine the upper layer liquid I and the upper layer liquid II to obtain an extraction solution. Concentrate the extraction solution under reduced pressure at 65 °C into an extract, first perform rough fractionation with polyamide resin, collect the eluate of 60% ethanol aqueous solution with 10 column volumes (BV), concentrate it under reduced pressure, then perform fine fractionation with Cross-inked dextran gel LH-20, collect the eluate of 70% methanol aqueous solution with 10 column volumes (BV), concentrate it under reduced pressure, and finally purify it by liquid chromatography. The chromatographic conditions are as follows: the chromatographic column is C18ME (10 mm × 250 mm, 10 μm), the column temperature is 25 °C, the flow rate is 5 mL / min, mobile phase A is 0.1% trifluoroacetic acid aqueous solution, mobile phase B is methanol solution, the elution concentration is 40%B, the injection volume is 500 μL, and the detector is a DAD detector. Collect the eluate and dry it to obtain the monomer of 5,7-dihydroxy chromone. Calculate the extraction rate of 5,7-dihydroxy chromone and detect the purity of 5,7-dihydroxy chromone.

[0051] The types, addition amounts and ratios of the enzymes added are detailed as follows to study the effect of enzyme types on the preparation of 5,7-dihydroxy chromone:

[0052] ①: Without adding enzyme

[0053] ②: Cellulase, 1%

[0054] ③: Hemicellulase, 1%

[0055] ④: Pectinase, 1%

[0056] ⑤: Amylase, 1%

[0057] ⑥: Acid protease, 1%

[0058] ⑦: Cellulase - rhamnosidase, 2% (1:1, w / w)

[0059] ⑧: Hemicellulase - rhamnosidase, 2% (1:1, w / w)

[0060] ⑨: Cellulase - hemicellulase - rhamnosidase, 3% (1:1:1, w / w)

[0061] ⑩: Cellulase - hemicellulase - rhamnosidase - acid protease, 4% (1:1:1:1, w / w)

[0062] Table 1: Effects of different enzyme types on the preparation of 5,7 - dihydroxy chromone

[0063]

[0064] The results are shown in Table 1: Enzymes can significantly improve the purity and extraction rate of 5,7 - dihydroxy chromone monomer. Moreover, the enzymatic hydrolysis effect of the composite enzyme is better than that of the single enzyme. Among them, when cellulase - hemicellulase - rhamnosidase - acid protease is compounded, the effect of preparing 5,7 - dihydroxy chromone is the best.

[0065] Example 2: Preparation of 5,7 - dihydroxy chromone from Ophiopogon japonicus

[0066] Take 1.5 kg of Ophiopogon japonicus, dry, ultrafinely pulverize, and sieve it to obtain an ultrafine powder with a particle size greater than 600 mesh. Add 10 times the volume of water and 1.5% of a composite enzyme (cellulase - hemicellulase - rhamnosidase - acid protease mass ratio 5:3:1:1) to the Ophiopogon japonicus powder, control the temperature at 50 °C, pH = 6.0, and the time for 4 h to obtain an enzymolysis solution of Ophiopogon japonicus. Add 2.5 times the volume of 95% ethanol aqueous solution to the enzymolysis solution of Ophiopogon japonicus, stir and extract at room temperature for 2 h, filter to obtain filtrate I, then add 1 times the volume of 65% ethanol aqueous solution to the Ophiopogon japonicus residue for repeated extraction, filter to obtain filtrate II, and combine filtrate I and filtrate II to obtain an extraction solution of Ophiopogon japonicus. Concentrate the extraction solution of Ophiopogon japonicus under reduced pressure to an extract at 55 °C, extract with 5 times the volume of an organic solvent (ethyl acetate - n - butanol - water volume ratio 6:5:5), separate to obtain the upper layer liquid I, repeat the extraction, separate to obtain the upper layer liquid II, and combine upper layer liquid I and upper layer liquid II to obtain an extraction solution. Concentrate the extraction solution under reduced pressure to an extract at 55 °C, first perform rough fractionation with polyamide resin, collect 4 times the column volume (BV) of 60% ethanol aqueous solution eluate, concentrate under reduced pressure, then perform fine fractionation with Cross - linked dextran gel LH - 20, collect 4 times the column volume (BV) of 60% methanol aqueous solution eluate, concentrate under reduced pressure, and finally purify by liquid chromatography. The chromatographic conditions are: the chromatographic column is C18ME (10 mm × 250 mm, 10 μm), the column temperature is 25 °C, the flow rate is 5 mL / min, mobile phase A is 0.1% trifluoroacetic acid aqueous solution, mobile phase B is methanol solution, the elution concentration is 40%B, the injection volume is 500 μL, and the detector is a DAD detector. Collect the eluate, dry to obtain 1.875 g of 5,7 - dihydroxy chromone monomer, the extraction rate of 5,7 - dihydroxy chromone is 0.125%, and the purity is 97.88%.

[0067] Example 3: Preparation of 5,7 - dihydroxy chromone from Polygonum multiflorum

[0068] Take 1.0 kg of Polygonum multiflorum, dry it, ultrafinely pulverize it, and sieve it to obtain an ultrafine powder with a particle size larger than 600 mesh. Add 15 times the volume of water and 1.8% of a composite enzyme (cellulase - hemicellulase - rhamnosidase - acid protease mass ratio 3:3:1:1) to the Polygonum multiflorum powder, control the temperature at 55 °C, pH = 5.5, and the time for 3 h to obtain a Polygonum multiflorum enzymatic hydrolysate. Add 2.2 times the volume of 95% ethanol aqueous solution to the Polygonum multiflorum enzymatic hydrolysate, stir and extract at room temperature for 2.5 h, filter to obtain filtrate I, then add 1.5 times the volume of 65% ethanol aqueous solution to the Polygonum multiflorum residue for repeated extraction, filter to obtain filtrate II, and combine filtrate I and filtrate II to obtain a Polygonum multiflorum extract. Concentrate the Polygonum multiflorum extract under reduced pressure to an extract at 60 °C, extract with 7 times the volume of an organic solvent (ethyl acetate - n - butanol - water volume ratio 8:3:5), separate to obtain the upper layer liquid I, repeat the extraction, separate to obtain the upper layer liquid II, and combine the upper layer liquid I and the upper layer liquid II to obtain an extraction solution. Concentrate the extraction solution under reduced pressure to an extract at 60 °C, first perform rough fractionation with polyamide resin, collect the eluate of a 40% ethanol aqueous solution with 8 column volumes (BV), concentrate under reduced pressure, then perform fine fractionation with Cross - linked dextrangel LH - 20, collect the eluate of a 50% methanol aqueous solution with 8 column volumes (BV), concentrate under reduced pressure, and finally purify by liquid chromatography. The chromatographic conditions are: the chromatographic column is C18ME (10 mm×250 mm, 10 μm), the column temperature is 25 °C, the flow rate is 5 mL / min, mobile phase A is a 0.1% trifluoroacetic acid aqueous solution, mobile phase B is a methanol solution, the elution concentration is 40%B, the injection volume is 500 μL, and the detector is a DAD detector. Collect the eluate and dry it to obtain 2.610 g of 5,7 - dihydroxy chromone monomer, and the extraction rate of 5,7 - dihydroxy chromone is 0.261%, and the purity is 98.13%.

[0069] Example 4: Preparation of 5,7 - dihydroxy chromone from Polygonum multiflorum

[0070] Take 0.5 kg of Polygonum multiflorum, dry, ultrafinely pulverize, and sieve it to obtain an ultrafine powder with a particle size larger than 600 mesh. Add 20 times the volume of water and 2% of a composite enzyme (cellulase - hemicellulase - rhamnosidase - acid protease mass ratio 4:2:1:1) to the Polygonum multiflorum powder, control the temperature at 45 °C, pH = 6.5, and the time at 5 h to obtain a Polygonum multiflorum enzymatic hydrolysate. Add 3 times the volume of 95% ethanol aqueous solution to the Polygonum multiflorum enzymatic hydrolysate, stir and extract at room temperature for 3 h, filter to obtain filtrate I, then add 2 times the volume of 65% ethanol aqueous solution to the Polygonum multiflorum residue for repeated extraction, filter to obtain filtrate II, and combine filtrate I and filtrate II to obtain a Polygonum multiflorum extract. Concentrate the Polygonum multiflorum extract under reduced pressure to an extract at 65 °C, extract with 6 times the volume of an organic solvent (ethyl acetate - n - butanol - water volume ratio 6:3:5), separate to obtain upper layer liquid I, repeat the extraction, separate to obtain upper layer liquid II, and combine upper layer liquid I and upper layer liquid II to obtain an extraction solution. Concentrate the extraction solution under reduced pressure to an extract at 65 °C, first perform rough fractionation with polyamide resin, collect the eluate of 50% ethanol aqueous solution with 6 column volumes (BV), concentrate under reduced pressure, then perform fine fractionation with Cross - linked dextran gel LH - 20, collect the eluate of 55% methanol aqueous solution with 6 column volumes (BV), concentrate under reduced pressure, and finally purify by liquid chromatography. The chromatographic conditions are: the chromatographic column is C18ME (10 mm × 250 mm, 10 μm), the column temperature is 25 °C, the flow rate is 5 mL / min, mobile phase A is 0.1% trifluoroacetic acid aqueous solution, mobile phase B is a methanol solution, the elution concentration is 40%B, the injection volume is 500 μL, and the detector is a DAD detector. Collect the eluate, dry to obtain 1.235 g of 5,7 - dihydroxy chromone monomer, the extraction rate of 5,7 - dihydroxy chromone is 0.247%, and the purity is 98.94%.

[0071] Example 5: Preparation of 5,7 - dihydroxy chromone from the vine of Polygonum multiflorum

[0072] Take 0.2 kg of Polygonum multiflorum Thunb. vine, dry it, ultrafinely pulverize it, and sieve it to obtain an ultrafine powder with a particle size greater than 600 mesh. Add 12 times the volume of water and 1.6% of a composite enzyme (cellulase - hemicellulase - rhamnosidase - acid protease mass ratio 5:2:1:1) to the Polygonum multiflorum Thunb. vine powder, control the temperature at 48 °C, pH = 5.8, and the time at 4.5 h to obtain a Polygonum multiflorum Thunb. vine enzymatic hydrolysate. Add 3.5 times the volume of 95% ethanol aqueous solution to the Polygonum multiflorum Thunb. vine enzymatic hydrolysate, stir and extract at room temperature for 2.5 h, filter to obtain filtrate I, then add 1 time the volume of 65% ethanol aqueous solution to the Polygonum multiflorum Thunb. vine residue for repeated extraction, filter to obtain filtrate II, and combine filtrate I and filtrate II to obtain a Polygonum multiflorum Thunb. vine extract. Concentrate the Polygonum multiflorum Thunb. vine extract under reduced pressure to an extract at 58 °C, extract it with 5.5 times the volume of an organic solvent (ethyl acetate - n - butanol - water volume ratio 7:4:5), separate to obtain the upper layer liquid I, repeat the extraction, separate to obtain the upper layer liquid II, and combine the upper layer liquid I and the upper layer liquid II to obtain an extraction solution. Concentrate the extraction solution under reduced pressure to an extract at 58 °C, first perform rough fractionation with polyamide resin, collect the eluate of 55% ethanol aqueous solution with 10 column volumes (BV), concentrate it under reduced pressure, then perform fine fractionation with Cross - inked dextran gel LH - 20, collect the eluate of 70% methanol aqueous solution with 10 column volumes (BV), concentrate it under reduced pressure, and finally purify it by liquid chromatography. The chromatographic conditions are: the chromatographic column is C18ME (10 mm×250 mm, 10 μm), the column temperature is 25 °C, the flow rate is 5 mL / min, mobile phase A is 0.1% trifluoroacetic acid aqueous solution, mobile phase B is methanol solution, the elution concentration is 40%B, the injection volume is 500 μL, and the detector is a DAD detector. Collect the eluate, dry it to obtain 0.558 g of 5,7 - dihydroxy chromone monomer, and the extraction rate of 5,7 - dihydroxy chromone is 0.279%, and the purity is 99.13%.

[0073] Example 6. Preparation of 5,7 - dihydroxy chromone from Cimicifuga foetida L.

[0074] Take 2.0 kg of Cimicifuga foetida, dry, ultrafinely pulverize, and sieve to obtain an ultrafine powder with a particle size greater than 600 mesh. Add 18 times the volume of water and 1.9% of a composite enzyme (cellulase - hemicellulase - rhamnosidase - acid protease mass ratio 4:3:1:1) to the Cimicifuga foetida powder, control the temperature at 52 °C, pH = 6.3, and the time for 3.5 h to obtain the Cimicifuga foetida enzymatic hydrolysate. Add 2.8 times the volume of 95% aqueous ethanol solution to the Cimicifuga foetida enzymatic hydrolysate, stir and extract at room temperature for 3 h, filter to obtain filtrate I, then add 2 times the volume of 65% aqueous ethanol solution to the Cimicifuga foetida residue for repeated extraction, filter to obtain filtrate II, and combine filtrate I and filtrate II to obtain the Cimicifuga foetida extract. Concentrate the Cimicifuga foetida extract under reduced pressure to an extract at 60 °C, extract with 8 times the volume of an organic solvent (ethyl acetate - n - butanol - water volume ratio 7:5:5), separate to obtain the upper layer liquid I, repeat the extraction, separate to obtain the upper layer liquid II, and combine upper layer liquid I and upper layer liquid II to obtain the extraction solution. Concentrate the extraction solution under reduced pressure to an extract at 60 °C, first perform rough fractionation with polyamide resin, collect the eluate of 45% aqueous ethanol solution with 5 column volumes (BV), concentrate under reduced pressure, then perform fine fractionation with Cross - linked dextran gel LH - 20, collect the eluate of 65% aqueous methanol solution with 5 column volumes (BV), concentrate under reduced pressure, and finally purify by liquid chromatography. The chromatographic conditions are: the chromatographic column is C18ME (10 mm×250 mm, 10 μm), the column temperature is 25 °C, the flow rate is 5 mL / min, mobile phase A is 0.1% trifluoroacetic acid aqueous solution, mobile phase B is methanol solution, the elution concentration is 40%B, the injection volume is 500 μL, and the detector is a DAD detector. Collect the eluate, dry to obtain 2.840 g of 5,7 - dihydroxy chromone monomer, the extraction rate of 5,7 - dihydroxy chromone is 0.142%, and the purity is 98.01%.

[0075] Example 7. Influence of Process Conditions on the Preparation of 5,7 - Dihydroxy Chromone

[0076] Basic process: Take 0.5 kg of Polygonum multiflorum Thunb. vine, dry, ultrafinely pulverize, and sieve to obtain an ultrafine powder with a particle size larger than 600 mesh. Add 15 times the volume of water and 1.5% of a composite enzyme (cellulase - hemicellulase - rhamnosidase - acid protease mass ratio 3:3:1:1) to the ultrafine powder, control the temperature at 45°C, pH = 6.5, and the time for 3 h to obtain an enzymolysis solution. Add 3 times the volume of 95% ethanol aqueous solution to the enzymolysis solution, stir and extract at room temperature for 2 h, filter to obtain filtrate I, then add 1 time the volume of 65% ethanol aqueous solution to the residue for repeated extraction, filter to obtain filtrate II, and combine filtrate I and filtrate II to obtain an extraction solution. Concentrate the extraction solution under reduced pressure to an extract at 65°C, extract with 6 times the volume of an organic solvent (ethyl acetate - n - butanol - water volume ratio 8:4:5), separate to obtain the upper layer solution I, repeat the extraction, separate to obtain the upper layer solution II, and combine upper layer solution I and upper layer solution II to obtain an extraction solution. Concentrate the extraction solution under reduced pressure to an extract at 65°C, first perform rough fractionation with polyamide resin, collect the eluate of 60% ethanol aqueous solution with 10 column volumes (BV), concentrate under reduced pressure, then perform fine fractionation with Cross - linked dextran gel LH - 20, collect the eluate of 70% methanol aqueous solution with 10 column volumes (BV), concentrate under reduced pressure, and finally purify by liquid chromatography. The chromatographic conditions are: the chromatographic column is C18ME (10 mm×250 mm, 10 μm), the column temperature is 25°C, the flow rate is 5 mL / min, mobile phase A is 0.1% trifluoroacetic acid aqueous solution, mobile phase B is a methanol solution, the elution concentration is 40%B, the injection volume is 500 μL, and the detector is a DAD detector. Collect the eluate and dry to obtain the 5,7 - dihydroxy chromone monomer. Calculate the extraction rate of 5,7 - dihydroxy chromone and detect the purity of 5,7 - dihydroxy chromone.

[0077] (1)Effect of raw materials on the preparation of 5,7 - dihydroxy chromone

[0078] On the basis of the above basic process, select Ophiopogon japonicus, Polygonum multiflorum Thunb., Polygonum multiflorum Thunb. vine, and Cimicifuga foetida as raw materials, and keep other process conditions unchanged to study the effect of different raw materials on the preparation of 5,7 - dihydroxy chromone.

[0079] Table 2: Effect of different raw materials on the preparation of 5,7 - dihydroxy chromone

[0080]

[0081] The results are shown in Table 2: When using Polygonum multiflorum Thunb. and Polygonum multiflorum Thunb. vine as raw materials, the purity of the prepared 5,7 - dihydroxy chromone is above 98%, and the extraction rate is also relatively high. Among them, the purity and extraction rate of the 5,7 - dihydroxy chromone monomer prepared from Polygonum multiflorum Thunb. vine are the best. Therefore, Polygonum multiflorum Thunb. vine is the optimal raw material choice.

[0082] (2)Effect of enzyme dosage on the preparation of 5,7 - dihydroxy chromone

[0083] On the basis of the above basic process, compound enzymes with mass ratios of 0%, 0.25%, 0.5%, 0.75%, 1.0%, 1.25%, 1.5%, 1.75%, 2.0%, 2.25%, and 2.5% were added respectively, and other process conditions remained unchanged to study the effect of different enzyme addition amounts on the preparation of 5,7-dihydroxy chromone.

[0084] Table 3: Effect of different enzyme addition amounts on the preparation of 5,7-dihydroxy chromone

[0085]

[0086] The results are shown in Table 3: When no enzyme was used for assisted extraction, the purity of the 5,7-dihydroxy chromone monomer was only 85.21%, and the extraction rate was only 0.089%, both of which were relatively low. When enzyme-assisted extraction was used, the purity of the 5,7-dihydroxy chromone monomer was above 90%, and the extraction rate also increased significantly. When the enzyme addition amount was 0.25% - 1.5%, both the purity and extraction rate of the 5,7-dihydroxy chromone monomer increased with the increase of the enzyme addition amount. When the enzyme addition amount was 1.5% - 2.5%, the extraction rate of the 5,7-dihydroxy chromone monomer remained almost unchanged with the increase of the enzyme addition amount, but the purity decreased with the increase of the enzyme addition amount. Considering comprehensively, the enzyme addition amount of 1.5% - 2.0% was selected.

[0087] (3) Effect of enzymatic hydrolysis temperature on the preparation of 5,7-dihydroxy chromone

[0088] On the basis of the above basic process, the enzymatic hydrolysis temperature was controlled at 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, and 60°C respectively, and other process conditions remained unchanged to study the effect of different enzymatic hydrolysis temperatures on the preparation of 5,7-dihydroxy chromone.

[0089] Table 4: Effect of different enzymatic hydrolysis temperatures on the preparation of 5,7-dihydroxy chromone

[0090]

[0091] The results are shown in Table 4: Within the range of 30°C - 55°C, both the purity and extraction rate of the 5,7-dihydroxy chromone monomer increased with the increase of the enzyme addition amount. Within the range of 55°C - 65°C, both the purity and extraction rate of the 5,7-dihydroxy chromone monomer decreased with the increase of the enzyme addition amount. Within the range of 45°C - 55°C, both the purity and extraction rate of the 5,7-dihydroxy chromone monomer remained at a relatively high level. For the enzymatic hydrolysis reaction, both the optimal temperature of the enzyme and the temperature stability of the compounds in the enzymatic hydrolysis system need to be considered. Considering comprehensively, the enzymatic hydrolysis temperature of 45°C - 55°C was selected.

[0092] (4) Effect of methanol elution concentration in liquid chromatography on the preparation of 5,7-dihydroxy chromone

[0093] On the basis of the above basic process, during the purification by liquid chromatography, elution was carried out with methanol solutions at concentrations of 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, and 60% respectively, while other process conditions remained unchanged, to study the effect of different methanol elution concentrations on the preparation of 5,7-dihydroxy chromone.

[0094] Table 5: Effect of different methanol elution concentrations on the preparation of 5,7-dihydroxy chromone

[0095]

[0096] The results are shown in Table 5: During the purification by high-performance chromatography, when the methanol concentration was 40%, the purity and extraction rate of the 5,7-dihydroxy chromone monomer were both the highest. Either too high or too low methanol concentration was not conducive to the dissociation of 5,7-dihydroxy chromone from the chromatographic column.

[0097] Example 8, Inhibitory activity of 5,7-dihydroxy chromone against CD38

[0098] In this example, the following steps were used to determine the inhibitory activity of 5,7-dihydroxy chromone against CD38:

[0099] (1) Solution preparation:

[0100] Buffer A: 100 mM HEPES, 4 mM EDTA, 1 mM CHAPS, pH = 7.41.

[0101] Buffer B: 5 mM CH3COONa, 1 mM CHAPS, pH = 7.41.

[0102] 5,7-dihydroxy chromone solution: Prepared with Buffer A, with a concentration of 100 μM.

[0103] CD38 enzyme solution: Prepared with Buffer A, with a concentration of 4 nM.

[0104] GW323424X solution: Prepared with Buffer B, with a concentration of 500 μM.

[0105] NAD + solution: Prepared with the GW323424X solution, with a concentration of 100 μM.

[0106] (2) Measurement steps:

[0107] Add 25 μL of the 5,7-dihydroxy chromone solution and 25 μL of CD38 to each well of a 96-well plate, and incubate at room temperature for 30 min. Add 50 μL of NAD +The solution was shaken in an enzyme-labeled instrument for 5 s, and the readings were taken at 405 nm. The readings were taken once every 1 min within 60 min, for a total of 60 cycles. For each set of data, a scatter plot was made with time as the abscissa and absorbance value as the ordinate, and linear fitting was performed using the least squares method to calculate the initial reaction rate of each group. The inhibition rate was calculated according to the following formula:

[0108] Inhibition rate (%) = (control group - experimental group) / (control group - blank group) × 100%.

[0109] (3)The 5,7-dihydroxy chromone solution was serially diluted, with PBS used as the blank control. The inhibition rate of the sample against CD38 was measured according to the above method, and the IC 50 value was calculated.

[0110] The inhibitory effects of the 5,7-dihydroxy chromone prepared in Examples 2 to 6 against CD38 were measured respectively. The results are as Figure 1 shown. The 5,7-dihydroxy chromone prepared in Examples 2 to 6 could significantly inhibit the activity of CD38, and the inhibition rates were 75.64%, 70.35%, 82.67%, 85.96%, and 79.22% respectively. Among them, the 5,7-dihydroxy chromone prepared in Example 5 had the highest inhibition rate and the strongest inhibitory activity.

[0111] The 5,7-dihydroxy chromone prepared in Example 5 was selected to study the inhibitory activity of different concentrations of 5,7-dihydroxy chromone against CD38. The results are as Figure 2 shown. The IC 50 of 5,7-dihydroxy chromone against CD38 inhibition was 54.83 μM. The results showed that the 5,7-dihydroxy chromone prepared in the present invention had high purity, low use concentration, and good inhibitory effect on CD38, and had the potential to be further developed as a CD38 inhibitor as a lead compound or raw material.

[0112] Example 9, Effect of 5,7-dihydroxy chromone on NAD at the cellular level + Effect

[0113] In this example, the following steps were used to measure the effect of 5,7-dihydroxy chromone on NAD at the cellular level + Effect:

[0114] ((1) HEK-293T cells were cultured in DMEM containing fetal bovine serum (10%), penicillin (100 IU / mL), and streptomycin (100 μg / mL). When the cells adhered and the density reached about 80%, the pEGFP-N1-CD38 plasmid was transfected.

[0115] (2) Trypsinize the transfected and untransfected HEK-293T cells from 10 cm dishes, and prepare a cell suspension of 8×10 4 cells / mL. Add 500 μL to each well of a 24-well transparent plate and place it in a cell culture incubator (37 °C, 5% CO2) for culture.

[0116] (3) After 24 h, add 500 μL of the test compound prepared with complete medium to the experimental group; add 500 μL of complete medium to the control group. Set 6 replicate wells for each group.

[0117] (4) After 24 h, aspirate the culture medium in the wells, and then add 500 μL of 10 μM NAD + prepared with PBS to each well.

[0118] (5) After 1 h, use the NAD + / NADH detection kit (WST-8 method) from Beyotime to measure the NAD + content in the supernatant of each group, and calculate the NAD+ content according to the NAD standard curve. Use the group without compound as the control, and calculate the relative content of NAD + in each group according to the following formula:

[0119] Relative content of NAD + = mean of experimental group / mean of control group.

[0120] Measure the effects of 5,7-dihydroxy chromone prepared in Examples 2-6 on NAD + at the cellular level respectively. The results are as Figure 3 shown. The 5,7-dihydroxy chromone prepared in Examples 2-6 can significantly improve the NAD + content at the cellular level. The NAD + contents are all higher than those of the control group, which are 2.32 times, 1.94 times, 2.43 times, 2.87 times, and 2.38 times that of the control group respectively. Among them, the 5,7-dihydroxy chromone prepared in Example 5 has the best effect on improving the NAD + content at the cellular level.

[0121] Select the 5,7-dihydroxy chromone prepared in Example 5 to study the effects of different concentrations of 5,7-dihydroxy chromone on NAD + at the cellular level. The results are as Figure 4 shown. When the concentration of 5,7-dihydroxy chromone is 50 μM, 75 μM, and 100 μM, the NAD + contents are 2.56 times, 2.87 times, and 2.87 times that of the control group respectively. The results show that the 5,7-dihydroxy chromone prepared in the present invention can effectively improve the NAD + content by inhibiting the activity of CD38 and targetedly regulate NAD+ Metabolic levels have become potential therapeutic methods for alleviating or reversing many age-related diseases.

[0122] Example 10, Anti-aging effect of 5,7-dihydroxy chromone on HUVEC

[0123] In this example, the following steps were used to determine the anti-aging effect of 5,7-dihydroxy chromone on HUVEC cells:

[0124] (1) Human umbilical vein endothelial cells (HUVEC) were cultured in RPMI 1640 containing fetal bovine serum (10%), penicillin (100 IU / mL), and streptomycin (100 μg / mL). When the cells adhered and reached a density of about 80%, they were digested with trypsin and prepared into a cell suspension of 2×10 5 cells / mL. 500 μL of the cell suspension was added to each well of a 24-well transparent plate and placed in a cell culture incubator (37°C, 5% CO2) for culture.

[0125] (2) After 24 h, the culture medium in the wells was aspirated. In the control group, 500 μL of complete medium was added; in the senescence group, 500 μL of complete medium was added; and in the 5,7-dihydroxy chromone group, 500 μL of 50 μM 5,7-dihydroxy chromone prepared with complete medium was added. After incubation for 9 h, the culture medium in the wells was aspirated. In the control group, complete medium was added; in the senescence group, 10 μM cisplatin was added; and in the 5,7-dihydroxy chromone group, 10 μM cisplatin was added.

[0126] (3) After 24 h of cisplatin induction, a SA-β-Gal staining experiment was performed using the Beyotime Cell Senescence β-Galactosidase Staining Kit; after staining for 12 h, the staining situation was observed under a microscope and photographed for recording.

[0127] The 5,7-dihydroxy chromone prepared in Example 5 was selected to study the anti-aging effect of 5,7-dihydroxy chromone on HUVEC through the SA-β-Gal staining experiment. The results are as Figure 5 shown. SA-β-Gal is a widely used cell senescence marker. During the process of cell senescence, the morphological enlargement and the increase in lysosome size will both lead to an increase in the activity of SA-β-Gal, resulting in an increase in the blue area. Compared with the compound group, the blue area in the 5,7-dihydroxy chromone group was significantly reduced, indicating that the addition of 5,7-dihydroxy chromone effectively reduced the activity of β-galactosidase in cells, proving that the 5,7-dihydroxy chromone prepared in this invention has an obvious anti-aging effect. The four raw materials used to prepare 5,7-dihydroxy chromone in this invention have large yields, low prices, and are all cosmetic raw materials, and can be used to develop skin care products, cosmetics and other products with skin anti-aging effects.

[0128] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, for those of ordinary skill in the art, it is still possible to modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. A use of 5,7-dihydroxychromone in the preparation of a CD38 inhibitor, characterized in that: The preparation method of the 5,7-dihydroxychromone comprises the following steps: (1) The plant raw materials are dried, ultrafinely crushed, and sieved to obtain ultrafine powder; (2) adding water and a complex enzyme to the ultrafine powder of step (1) for enzymatic hydrolysis to obtain an enzymatic hydrolysis solution; (3) adding an ethanol aqueous solution to the enzymatic hydrolyzate of step (2) for extraction to obtain an extract; (4) Concentrating the extract obtained in step (3) under reduced pressure and extracting with an organic solvent to obtain an extract; (5) The extract of step (4) is concentrated under reduced pressure, subjected to classification, purified by liquid chromatography, and dried to obtain 5,7-dihydroxychromone.

2. The use according to claim 1, characterized in that The plant raw materials in step (1) include Radix Ophiopogonis, Radix Polygoni Multiflori, Radix Polygoni Multiflori and Rhizoma Cimicifugae.

3. The use according to claim 1, characterized in that In step (2), the complex enzyme is a mixture of cellulase, hemicellulase, rhamnosidase and acid protease, and the added amount is 1.5% to 2.0% of the weight of the raw materials.

4. The use according to claim 3, characterized in that The mass ratio of the cellulase, hemicellulase, rhamnosidase and acid protease is 3-5:2-3:1:

1.

5. The use according to claim 1, characterized in that The enzymatic hydrolysis conditions in step (2) are: enzymatic hydrolysis temperature 45°C to 55°C, pH 5.5 to 6.5, and enzymatic hydrolysis time 3 h to 5 h.

6. The use according to claim 1, wherein The liquid chromatography conditions in step (5) are as follows: the chromatographic column is C18ME, the column temperature is 25°C, the injection volume is 500 μL, the flow rate is 5 mL / min, the mobile phase A is 0.1% trifluoroacetic acid aqueous solution, the mobile phase B is methanol solution, the eluent is 40% B, and the detector is a DAD detector.

7. The use according to claim 1, characterized in that The 5,7-dihydroxychromone is used at a concentration of 5 µM to 150 µM.

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