Genistein having CD38 inhibitory activity, and preparation method and application thereof

Abstract

The application discloses genistein with CD38 inhibitory activity and a preparation method and application thereof. The preparation method comprises the following steps: crushing dried purslane, adding water and a composite enzyme into the obtained purslane powder to perform enzymolysis; adding ethanol into the obtained enzymolysis liquid to perform ultrasonic-assisted extraction, decompression concentration and organic solvent extraction; performing macroporous adsorption resin separation and elution on the obtained extraction liquid, collecting the eluate; performing gel column chromatography separation and liquid chromatography purification on the obtained eluate, and drying to obtain genistein. The preparation method is simple, the prepared genistein has high purity, and the highest purity can reach 99.05%; and the prepared genistein has good CD38 inhibitory activity, and opens up a new use of purslane genistein. + horizontal promotion of medicines, health products and food.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of natural product development, and particularly relates to a genistein with CD38 inhibitory activity and a preparation method and application thereof. BACKGROUND

[0002] Nicotinamide adenine Dinucleotide (NAD + ) is an important coenzyme in cells involved in energy metabolism, as a metabolic substrate and intracellular signaling molecule, involved in cell material metabolism, energy synthesis, DNA repair and other physiological activities. Studies have found that with increasing age, the level of NAD + in human tissues and cells shows a gradual downward trend, thereby triggering aging-related diseases, and restoring the body's NAD + level can reduce or even reverse the disease state. Targeting NAD + metabolism has become a potential functional food development strategy to delay aging, extend human healthy life and maximum life. Among the NAD + metabolic regulatory enzymes, the expression and activity of its degrading enzyme CD38 increases with age, and has a significant negative correlation with the decline of NAD + during aging. In addition, CD38 not only degrades NAD + , but also is the main enzyme for degrading NAD + precursor nicotinamide mononucleotide (NMN) in the human body, indicating that CD38 is a key target for improving age-related NAD + decline, and developing molecules with CD38 inhibitory activity has become a major research focus in recent years.

[0003] Portulaca oleracea L. Portulaca oleracea L. ), also known as Wuxingcao, Changmiancai, and Maoyacai, is listed by the World Health Organization as one of the most widely used medicinal plants in the world, and is designated by the Chinese Ministry of Health as a medicinal and edible substance. It contains bioactive components such as flavonoids, alkaloids, coumarins, and organic acids. Genistein, also known as genistein, is a natural flavonoid compound found in legume plants, various fruits and vegetables. Current research on the pharmacological activity of genistein has mainly focused on antioxidant, antitumor, antibacterial, hypolipidemic, and estrogen-like effects, but there is no reported application of genistein for CD38 inhibitory activity.

[0004] The related researches on preparation of genistein at present mainly focus on preparation from leguminous plants as sources, and the preparation processes are complex and various, for example, genistein is prepared by extraction from pagoda tree fruit, pigeon pea rhizome, big leaf flemingia, dalbergia odorifera leaf and radix sophorae tonkinensis. However, the patent for preparation of genistein from portulaca oleracea has not been reported. Therefore, an effective active ingredient of portulaca oleracea is needed to be extracted, the purity of genistein is improved, and the method for providing genistein with good activity is provided. SUMMARY

[0005] In view of the deficiencies in the prior art, the present application provides a genistein with CD38 inhibitory activity and a preparation method and application thereof. The genistein extracted from portulaca oleracea has high purity and high CD38 inhibitory activity.

[0006] To achieve the above-mentioned application purposes, the present application adopts the following technical solutions:

[0007] The present application provides a preparation method of genistein for inhibiting CD38, which comprises the following steps:

[0008] (1) The dried portulaca oleracea is crushed, water and a composite enzyme are added to the obtained portulaca oleracea powder for enzymolysis to obtain a portulaca oleracea enzymolysis liquid;

[0009] (2) Ethanol is added to the portulaca oleracea enzymolysis liquid of step (1), ultrasonic-assisted extraction is carried out, the filtrate is collected by filtration, repeated extraction and filtration are carried out, and the extraction liquid is obtained by combination;

[0010] (3) The extraction liquid of step (2) is concentrated under reduced pressure, the obtained concentrated liquid is redissolved, and then extracted and separated with an organic solvent, and the extraction liquid is obtained after repeated extraction;

[0011] (4) The extraction liquid of step (3) is separated by macroporous adsorption resin, and the eluate is collected;

[0012] (5) The eluate of step (4) is separated by a gel column and purified by liquid chromatography, and then dried to obtain genistein.

[0013] Further, the crushing in step (1) is the whole grass of portulaca oleracea.

[0014] Further, the amount of water added in step (1) is 3-6 times the volume of the portulaca oleracea powder.

[0015] Further, the composite enzyme in step (1) is a mixture of cellulase, pectinase and glucosidase, and the amount of addition is 1.5%-2% of the weight of the portulaca oleracea powder.

[0016] Further, the weight ratio of the cellulase, pectinase and glucosidase is 1:1:1.

[0017] Further, the enzymolysis conditions in step (1) are: enzymolysis temperature 35-55℃, pH = 4.5-6.0, and incubation reaction time 2-4 h.

[0018] Preferably, the enzymolysis conditions in step (1) are: enzymolysis temperature 45℃, pH = 5.5, and incubation reaction time 3 h.

[0019] Further, the concentration of ethanol in step (2) is 70-90%; the ultrasonic-assisted extraction conditions are: ultrasonic power 60-180 W, temperature 35-60℃, and time 20-50 min.

[0020] Preferably, the concentration of ethanol in step (2) is 80%; the ultrasonic-assisted extraction conditions are: ultrasonic power 120 W, temperature 50℃, and time 40 min.

[0021] Further, the temperature for reduced pressure concentration in step (3) is 40-60℃; the amount of water added for redissolution is 1-2 times the volume of the concentrated solution.

[0022] Further, the organic solvent in step (3) is at least one of ethyl acetate, butyl acetate, and petroleum ether; the amount of the organic solvent added is 2-3 times the volume of the redissolved solution.

[0023] Further, the macroporous adsorption resin in step (4) is one of AB-8, D-101, HPD-600, and SPD100 macroporous resin; the separation conditions are gradient elution with water, 45-55% ethanol solution, and 75-85% ethanol solution.

[0024] Further, the gel column in step (5) is a hydroxypropyl dextran gel column Sephadex LH-20, and the eluent is water, 60% methanol solution, and 60-80% methanol solution.

[0025] Further, the liquid chromatography conditions in step (5) are: the chromatographic column is a ZORBAX SB-C18 column, the column temperature is 30℃, the mobile phase is 1% formic acid-methanol, the flow rate is 1 mL / min, the injection volume is 500 μL, the gradient elution conditions are 0 min 30% methanol, 5 min 30% methanol, 15 min 70% methanol, 25 min 30% methanol, and the detector is an ultraviolet detector.

[0026] In summary, a preparation method of genistein for inhibiting CD38 includes the following steps:

[0027] The dried purslane is crushed, 3-6 times volume of water is added to the purslane powder, 1.5%-2% of the weight of the purslane powder of a composite enzyme composed of cellulase, pectinase and glucosidase in a weight ratio of 1:1:1 is added, the temperature is controlled at 35-55 DEG C, the pH is controlled at 4.5-6, and the reaction is incubated for 2-4 hours to obtain a purslane enzymatic hydrolysate;

[0028] Ethanol is added to the purslane enzymatic hydrolysate of step (1) to a concentration of 70%-90%, ultrasonic power is set at 60-180 W, the temperature is set at 35-60 DEG C, and the time is set at 20-50 min to perform ultrasonic-assisted extraction, the filtrate is collected by filtration, and the extraction and filtration are repeated to obtain an extract.

[0029] The extract of step (2) is concentrated under reduced pressure at 40-60 DEG C to obtain a concentrated solution, the concentrated solution is redissolved with 1-2 times volume of water to obtain a redissolved solution, and the redissolved solution is extracted with 2-3 times volume of an organic solvent to obtain an extract, and the extraction is repeated to obtain the extract.

[0030] The extract of step (3) is separated by macroporous adsorption resin, and gradient elution is performed with water, 45%-55% ethanol solution and 75%-85% ethanol solution in sequence to collect the eluate.

[0031] The eluate of step (4) is first separated by a hydroxypropyl dextran gel column and then purified by liquid chromatography, and the dye lignan is obtained after drying.

[0032] The present application also provides the dye lignan prepared by the preparation method, and the dye lignan has CD38 inhibitory activity and the purity of the dye lignan is greater than 98%.

[0033] Further, the IC 50 of the dye lignan for inhibiting CD38 is 30.58 μM.

[0034] The present application also provides the use of the dye lignan in the preparation of a CD38 inhibitor.

[0035] Further, the use concentration of the dye lignan is 20-100 μM.

[0036] The present application also provides the use of the CD38 inhibitor in the preparation of a medicine, health care product and food for promoting the NAD + level of a body and / or resisting aging.

[0037] Further, the dye lignan promotes the NAD + level of a body by inhibiting the activity of CD38.

[0038] Compared with the prior art, the present application has the following advantages and beneficial effects:

[0039] The present application uses Portulaca oleracea as raw material, adopts ultrasonic-assisted composite enzyme method for extraction, and realizes high-value utilization of Portulaca oleracea resources by high-efficiency preparation of genistein through macroporous resin adsorption, gel column chromatography and liquid chromatography separation and purification.

[0040] The genistein prepared by the present application is verified to have CD38 inhibitory activity for the first time, can be developed as a CD38 inhibitor to provide a new resource, and opens up a new use of genistein, which can be used for preparing a drug for promoting body NAD + The drug, health care product and food are horizontally promoted, and have good popularization and application value. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 The different embodiments are prepared for the inhibitory effect of genistein on CD38, wherein the abscissa is different embodiments of genistein, and the ordinate is the inhibition rate.

[0042] Figure 2 The different embodiments are prepared for the inhibitory effect of genistein on CD38 and IC 50 , wherein the abscissa is the logarithm of the concentration of genistein, and the ordinate is the inhibition rate.

[0043] Figure 3 The different embodiments are prepared for the NAD + determination experiment results, wherein the abscissa is the concentration of genistein, and the ordinate is the NAD + relative content.

[0044] Figure 4 The different embodiments are prepared for the SA-β-Gal staining experiment results of HUVEC cells treated with genistein, wherein (a) is a control group (normal cell culture), (b) is a senescence group (induced by cisplatin for 24 h), and (c) is a genistein treatment group (genistein incubated with cells for 9 h and then induced by cisplatin for 24 h). DETAILED DESCRIPTION

[0045] The present application will be further described below in combination with specific embodiments, but the scope of protection claimed by the present application is not limited to the following embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0046] Genistein purity determination: the HPLC method is used to determine the purity of genistein, and the specific method is as follows:

[0047] The column was ZORBAX SB-C18 column, column temperature 30 °C; mobile phase 1% formic acid-methanol, flow rate 1 mL / min; injection volume 20 μL; gradient elution (0 min 30% methanol, 5 min 30% methanol, 15 min 70% methanol, 30 min 30% methanol); detector was UV detector, detection wavelength was 365 nm. The mass concentration (mmol / L) of genistein standard was taken as the abscissa, and the peak area (A) was taken as the ordinate to draw the standard curve. The purity of genistein was calculated according to the standard curve.

[0048] Example 1, the effect of different enzyme species on the preparation of genistein

[0049] Basic process: 1 kg of dried Portulaca oleracea L. whole plant was crushed, 4 times the volume of water was added to the obtained Portulaca oleracea L. powder, then a composite enzyme was added, the temperature was controlled at 50 °C, pH = 5.5, and the reaction was incubated for 2 h to obtain a Portulaca oleracea L. enzyme hydrolysate. Ethanol was added to the Portulaca oleracea L. enzyme hydrolysate to a concentration of 70%, the ultrasonic power was set to 120 W, and the extraction was carried out at 50 °C for 40 min. The filtrate was collected by filtration, and the extraction was repeated twice. The filtrate was combined to obtain an extract. The extract was concentrated under reduced pressure at 45 °C to obtain a concentrated solution. The concentrated solution was dissolved in 2 times the volume of distilled water, and then 3 times the volume of ethyl acetate was added for extraction. The extract was separated and extracted twice. The extract was combined to obtain an extract. Further separation was carried out using AB-8 type macroporous adsorption resin, and gradient elution was carried out using water, 45% ethanol solution, and 75% ethanol solution in sequence. The eluate was collected. The obtained eluate was first separated by Sephadex LH-20 column chromatography, and then purified by high performance liquid chromatography. The chromatographic conditions were as follows: the column was a ZORBAX SB-C18 column, the column temperature was 30 °C, the mobile phase was 1% formic acid-methanol, the flow rate was 1 mL / min, the injection volume was 500 μL, the gradient elution conditions were 0 min 30% methanol, 5 min 30% methanol, 15 min 70% methanol, 25 min 30% methanol, and the detector was a UV detector. The eluate was dried to obtain genistein. The yield of genistein was calculated, and the purity of genistein was detected by HPLC.

[0050] On the basis of the above basic process, different formulations of composite enzymes were added, and the amount of each single enzyme was 0.5% of the weight of Portulaca oleracea L. powder. The other process conditions were unchanged. The effects of the following different enzyme species on the preparation of genistein were studied.

[0051] Composite enzyme one: cellulase, pectinase;

[0052] Composite enzyme two: cellulase, glucosidase;

[0053] Composite enzyme three: cellulase, pectinase, glucosidase;

[0054] Compound enzyme four: cellulase, pectinase, amylase;

[0055] Compound enzyme five: cellulase, amylase, glucosidase;

[0056] Compound enzyme six: cellulase, pectinase, glucosidase, amylase.

[0057] Table 1: Effect of different enzyme types on preparation of genistein

[0058]

[0059] The results are shown in Table 1: when cellulase, pectinase, and glucosidase are selected as the compound enzyme for enzymolysis, the yield and purity of genistein are the highest.

[0060] Example 2, preparation of genistein from Portulaca oleracea

[0061] Take 1 kg of dried Portulaca oleracea whole plant and crush it. Add 4 times the volume of water to the obtained Portulaca oleracea powder, and then add 1.5% of compound enzyme (cellulase, pectinase, and glucosidase) with a weight ratio of 1:1:1. Control the temperature at 55°C and the pH at 6, and incubate for 2 h to obtain the Portulaca oleracea enzymolysis solution. Add ethanol to the Portulaca oleracea enzymolysis solution until the ethanol concentration is 75%, set the ultrasonic power to 180 W, and extract at 60°C for 20 min. Collect the filtrate by filtration, repeat the extraction twice, and combine to obtain the extract. Concentrate the extract at 45°C under reduced pressure to obtain a concentrated solution. Dissolve the concentrated solution in 2 times the volume of distilled water, and then extract with 3 times the volume of ethyl acetate to separate the extract. Repeat the extraction twice, and combine to obtain the extract. Further separate the extract using AB-8 type macroporous adsorption resin, and sequentially elute with water, 45% ethanol solution, and 75% ethanol solution to collect the eluate. The obtained eluate is first separated by Sephadex LH-20 column chromatography, and then purified by liquid chromatography. The chromatography conditions are as follows: the chromatography column is a ZORBAX SB-C18 column, the column temperature is 30°C, the mobile phase is 1% formic acid-methanol, the flow rate is 1 mL / min, the sample injection amount is 500 μL, and the gradient elution conditions are 0 min 30% methanol, 5 min 30% methanol, 15 min 70% methanol, 25 min 30% methanol. The detector is an ultraviolet detector. Dry the collected eluate to obtain 4.1 g of genistein, and the yield is 0.41%. The purity of the obtained genistein monomer is 98.57%.

[0062] Example 3, preparation of genistein from Portulaca oleracea

[0063] Take 1 kg of dried Portulaca oleracea L. whole grass for crushing, add 3 times the volume of water to the obtained Portulaca oleracea L. powder, then add 1.5% of 1:1:1 composite enzyme (cellulase, pectinase, glucosidase) by weight, control the temperature at 40°C, pH=4.5, and incubate for 4 h to obtain the Portulaca oleracea L. enzymatic hydrolysate. Add ethanol to the Portulaca oleracea L. enzymatic hydrolysate until the ethanol concentration is 85%, set the ultrasonic power to 100 W, and extract at 50°C for 45 min. Filter the filtrate, repeat the extraction twice, and combine to obtain the extract. Concentrate the extract at 60°C under reduced pressure to obtain a concentrated solution. Dissolve the concentrated solution in 1 times the volume of distilled water, then add 2 times the volume of butyl acetate for extraction, separate to obtain the extract, repeat the extraction twice, and combine to obtain the extract. Further separate using D-101 macroporous adsorption resin, and sequentially elute with water, 50% ethanol solution, and 85% ethanol solution to collect the eluate. Chromatograph the eluate using a Sephadex LH-20 column, then purify using liquid chromatography. The chromatography conditions are as follows: the chromatography column is a ZORBAX SB-C18 column, the column temperature is 30°C, the mobile phase is 1% formic acid-methanol, the flow rate is 1 mL / min, the sample injection volume is 500 μL, and the gradient elution conditions are 30% methanol at 0 min, 30% methanol at 5 min, 70% methanol at 15 min, 30% methanol at 25 min, and detection by ultraviolet detector. Dry the eluate to obtain 4.3 g of genistein, and the yield is 0.43%. The purity of the obtained genistein monomer is 98.36%.

[0064] Example 4, Preparation of Genistein from Portulaca oleracea L.

[0065] Take 1 kg of dried Portulaca oleracea L. whole grass for crushing, add 5 times the volume of water to the obtained Portulaca oleracea L. powder, then add 1.5% of the weight ratio 1:1:1 of composite enzyme (cellulase, pectinase, glucosidase), control the temperature at 45℃, pH=5.5, and incubate for 3 h to obtain the Portulaca oleracea L. enzymatic hydrolysate. Add ethanol to the Portulaca oleracea L. enzymatic hydrolysate until the ethanol concentration is 80%, set the ultrasonic power to 120 W, and extract at 50℃ for 40 min. Filter the filtrate, repeat the extraction 2 times, and combine to obtain the extract. Concentrate the extract at 50℃ under reduced pressure to obtain a concentrated solution. Dissolve the concentrated solution with 1 times the volume of distilled water, then add 3 times the volume of ethyl acetate for extraction, separate to obtain the extract, repeat the extraction 2 times, and combine to obtain the extract. Further separate using a SPD100 macroporous adsorption resin, and sequentially elute with water, 45% ethanol solution, and 75% ethanol solution. Collect the eluate. Chromatograph the eluate using a Sephadex LH-20 column, and then purify using liquid chromatography. The chromatography conditions are as follows: the chromatography column is a ZORBAX SB-C18 column, the column temperature is 30℃, the mobile phase is 1% formic acid-methanol, the flow rate is 1 mL / min, the sample injection volume is 500 μL, and the gradient elution conditions are 0 min 30% methanol, 5 min 30% methanol, 15 min 70% methanol, 25 min 30% methanol. The detector is an ultraviolet detector. Dry the eluate to obtain 4.6 g of genistein, and the yield is 0.46%. The purity of the obtained genistein monomer is 99.05%.

[0066] Example 5, Preparation of Genistein from Portulaca oleracea L.

[0067] Take 1 kg of dried Portulaca oleracea L. whole grass for crushing, add 6 times the volume of water to the obtained Portulaca oleracea L. powder, then add 1.5% of 1:1:1 compound enzyme (cellulase, pectinase, glucosidase) by weight, control the temperature at 35℃, pH=5, and incubate for 4 h to obtain the Portulaca oleracea L. enzymatic hydrolysate. Add ethanol to the Portulaca oleracea L. enzymatic hydrolysate until the ethanol concentration is 90%, set the ultrasonic power to 60W, and extract at 35℃ for 50 min. Filter the filtrate, repeat the extraction twice, and combine to obtain the extract. Concentrate the extract at 40℃ under reduced pressure to obtain a concentrated solution. Dissolve the concentrated solution in 1 times the volume of distilled water, then add 4 times the volume of petroleum ether for extraction, separate to obtain the extract, repeat the extraction twice, and combine to obtain the extract. Further separate using HPD-600 macroporous adsorption resin, and sequentially elute with water, 55% ethanol solution, and 85% ethanol solution. Collect the eluate. First separate the eluate using a Sephadex LH-20 column, then purify using high performance liquid chromatography. The chromatographic conditions are as follows: the chromatographic column is a ZORBAX SB-C18 column, the column temperature is 30℃, the mobile phase is 1% formic acid-methanol, the flow rate is 1 mL / min, the sample size is 500 μL, and the gradient elution conditions are 0 min 30% methanol, 5 min 30% methanol, 15 min 70% methanol, 25 min 30% methanol. The detector is a UV detector. Dry the eluate to obtain 3.9 g of genistein, and the yield is 0.39%. The purity of the obtained genistein monomer is 98.84%.

[0068] Example 6, Effect of Different Extraction and Purification Conditions on Preparation of Genistein

[0069] Basic process: 1 kg of dried whole herb of Portulaca oleracea L. was taken and crushed. 4 times the volume of water was added to the crushed Portulaca oleracea L. and 1.5% of a composite enzyme (cellulase, pectinase, glucosidase) with a weight ratio of 1:1:1 was added. The temperature was controlled at 50°C and the pH was controlled at 5.5. The reaction was incubated for 2 hours to obtain a Portulaca oleracea L. enzymatic hydrolysate. Ethanol was added to the Portulaca oleracea L. enzymatic hydrolysate to a concentration of 75%. The ultrasonic power was set at 150 W and the extraction was carried out at 50°C for 40 min. The filtrate was collected by filtration. The extraction was repeated twice and the filtrate was combined to obtain an extract. The extract was concentrated under reduced pressure at 45°C to obtain a concentrated solution. The concentrated solution was dissolved in 2 times the volume of distilled water and then extracted with 3 times the volume of ethyl acetate. The extract was separated and the extraction was repeated twice to obtain an extract. The extract was further separated by AB-8 macroporous adsorption resin and eluted with water, 45% ethanol solution and 75% ethanol solution in sequence. The eluate was collected. The eluate was first separated by Sephadex LH-20 column chromatography and then purified by high performance liquid chromatography. The chromatographic conditions were as follows: the chromatographic column was a ZORBAX SB-C18 column, the column temperature was 30°C, the mobile phase was 1% formic acid-methanol, the flow rate was 1 mL / min, the sample size was 500 μL, the gradient elution conditions were 30% methanol at 0 min, 30% methanol at 5 min, 70% methanol at 15 min, 30% methanol at 25 min, and the detector was an ultraviolet detector. The eluate was dried to obtain genistein. The yield of genistein was calculated and the purity of genistein was detected by HPLC.

[0070] Effect of enzymolysis temperature on preparation of genistein

[0071] On the basis of the above basic process, 1.5% of a composite enzyme (cellulase, pectinase, glucosidase) with a weight ratio of 1:1:1 was added. The temperature was controlled at 25, 35, 45, 55 and 65°C, respectively. The other process conditions were unchanged. The effect of different enzymolysis temperatures on the preparation of genistein was studied.

[0072] Table 2: Effect of different enzymolysis temperatures on preparation of genistein

[0073]

[0074] The results are shown in Table 2: within the range of 25-45°C, the extraction rate and purity of genistein gradually increased with increasing temperature. Within the range of 45-65°C, the extraction rate and purity of genistein gradually decreased. The optimum extraction temperature was 45°C.

[0075] (2) Effect of ethanol concentration on preparation of genistein during ultrasonic-assisted extraction

[0076] Based on the above basic process, ethanol was added to the purslane enzymatic hydrolysate to concentrations of 55%, 65%, 75%, 85%, 90%, and 95%, respectively, while other process conditions remained unchanged, to study the effect of different ethanol concentrations on the preparation of genistein during ultrasonic-assisted extraction.

[0077] Table 3: Effect of different ethanol concentrations on the preparation of genistein

[0078]

[0079] The results are shown in Table 3: When the ethanol concentration was 80% during ultrasound-assisted extraction, the yield and purity of genistein were the highest.

[0080] (3) Effect of ultrasonic power on the preparation of genistein

[0081] Based on the above basic process, ultrasonic power was set to 0 W, 60 W, 120 W, 180 W, 240 W, and 300 W respectively, while other process conditions remained unchanged, to study the effect of different ultrasonic power on the preparation of genistein.

[0082] Table 4: Effect of different ultrasonic powers on the preparation of genistein

[0083]

[0084] The results are shown in Table 4: Without ultrasonic-assisted extraction, the yield of genistein was only 0.28%. When ultrasonic power of 120 W was used for extraction, the yield and purity of genistein were the highest.

[0085] Example 7: Inhibitory activity of genistein against CD38

[0086] In this embodiment, the following steps were used to determine the inhibitory activity of genistein on CD38 protein:

[0087] Buffer preparation: Solution A (enzyme buffer) is 100 mM HEPES-4 mM EDTA-1 mM CHAPS, pH=7.41; Solution B (substrate buffer) is 5 mM CH3COONa-1 mM CHAPS, pH=7.41.

[0088] Preparation of genistein solution: Use solution A to prepare a 100 μM solution of genistein.

[0089] CD38 enzyme solution preparation: Dilute the CD38 protein stock solution (11 μM) with solution A to 4 nM.

[0090] Take a transparent 96-well plate, first add 25 μL genistein solution to each well; then add 25 μL of prepared 4 nM CD38 to each well; incubate at room temperature for 30 min. The concentration of genistein during incubation is 50 μM, and the concentration of CD38 is 2 nM.

[0091] Substrate preparation: dilute GW323424X (mother liquor 100 mM) with B solution to 500 μM, and dilute NAD as a whole buffer gradient + to 100 μM.

[0092] Enzyme reader settings: set 405 nm, read once every 1 min within 60 min, a total of 60 cycles, shake for 5 s before the first cycle.

[0093] After the CD38 enzyme solution is incubated with genistein for 30 min, quickly add 50 μL of the substrate system to each well, and start reading.

[0094] After the cycle reading of the enzyme reader is completed, scatter plot each group of data with time as the abscissa and absorbance value as the ordinate, and use the least squares method to linearly fit to obtain a straight line equation in the form of y=bx+a, where b is the slope and a is the intercept, and b is the initial reaction rate of each group. The inhibition rate is calculated according to the following formula:

[0095] Inhibition rate (%) = (control group-experimental group) / control group x 100%.

[0096] Gradient dilution of genistein solution, PBS as blank control, according to the above method to determine the inhibition rate of sample on CD38 enzyme activity, and calculate the IC 50 value.

[0097] The CD38 inhibition activity of genistein prepared in examples 2-5 was determined respectively. The results are shown in Figure 1 The genistein prepared in the application can significantly inhibit CD38 activity, with the highest inhibition rate of 95.64% (prepared in example 4) and the lowest inhibition rate of 82.97% (prepared in example 5).

[0098] Further select the genistein prepared in example 4, and determine the inhibition activity of different concentrations of genistein on CD38. The results are shown in Figure 2 As can be seen from the figure, the IC 50 of the genistein prepared in example 4 for CD38 inhibition is 30.58 μM.

[0099] Example 8, determination of the effect of genistein on cell level NAD +

[0100] In this example, the following steps are used to determine the effect of genistein on cell level NAD + ​Effects:

[0101] HEK-293T cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS), penicillin (100 IU / mL) and streptomycin (100 μg / mL). The HEK-293T cells transfected with pEGFP-N1-CD38 plasmid were digested from a 10 cm dish with trypsin to prepare an 8 x 10 4 cell suspension, 500 μL per well was added to a 24-well transparent plate, and the plate was placed in a cell incubator (37°C, 5% CO2) for culture.

[0102] After about 24 h of culture, solution preparation was performed. Experimental group: the test compound was prepared in complete medium at the corresponding concentration, and the system in each well was 500 μL; control group: normal culture with complete medium. After 24 h of incubation, the culture solution in each well was aspirated, and 500 μL of 10 μM NAD + prepared with PBS was added to each well.

[0103] After 1 h, the NAD + content in the supernatant was determined by using the Biyun Tian NAD + / NADH detection kit (WST-8 method) and combining with the NAD standard curve.

[0104] The relative content of NAD + was calculated by using the following formula with the compound-free group as the control:

[0105] NAD + relative content = experimental group / control group mean.

[0106] The dye genistein prepared in Example 4 was selected to incubate and treat the HEK-293T cells transfected with pEGFP-N1-CD38 plasmid, and the NAD + content determination experiment was performed. The results are shown in Figure 3 , which shows that in the range of 0~100 μM, the NAD + content gradually increases with the increase of the genistein concentration, indicating that genistein can significantly inhibit the activity of CD38 at the cellular level. This conclusion further indicates that genistein has the potential to be developed as a lead compound or raw material for drugs, health products and special medical use formula foods with CD38 inhibitory activity and promotion of NAD + level in the body.

[0107] Example 9, SA-β-Gal staining experiment of HUVEC cells treated with genistein

[0108] In this embodiment, the following steps were used for the SA-β-Gal staining experiment of genistein on HUVEC cells:

[0109] Human umbilical vein endothelial cells (HUVEC) were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS), penicillin (100 IU / mL), and streptomycin (100 μg / mL). HUVEC cells with a confluence of about 80% were digested from a 10 cm dish with trypsin to prepare a cell suspension of 2 x 10 5 Each well of a 24-well transparent plate was added with 500 μL of the cell suspension, and the plate was placed in a cell culture incubator (37°C, 5% CO2) for culture.

[0110] After about 24 h of culture, solution preparation was performed. The control group was normally cultured with complete medium, and the compound group was cultured with the corresponding concentration of the test compound prepared with complete medium, with a system of 500 μL per well.

[0111] After about 9 h of incubation, 10 μM cisplatin was prepared with complete medium. The control group was normally cultured with complete medium, the aging group was induced with 10 μM cisplatin for 24 h, and the compound treatment group was induced with 10 μM cisplatin for 24 h after the test compound was incubated for 9 h.

[0112] After 24 h of cisplatin induction, the β-galactosidase staining kit was used to

[0113] perform SA-β-Gal staining.

[0114] After about 12 h of staining, the staining was observed under a microscope and photographed.

[0115] The genistein prepared in Example 4 was used to incubate HUVEC (50 μM, 9 h) and then cisplatin induction (10 μM, 24 h) was performed, and the SA-β-Gal staining experiment was performed. The results are shown in Figure 4 , which shows that the blue area of the genistein treatment group is significantly reduced compared to the HUVEC cells without genistein incubation, indicating that the β-galactosidase activity is reduced, thereby further proving that genistein has a significant anti-aging effect. This conclusion further demonstrates that genistein has the potential to be further developed as a lead compound or raw material for drugs, health products, and special medical use formula foods with CD38 inhibitory activity and anti-aging activity.

[0116] The above examples are only used to illustrate the technical solutions of the present application, but not to limit the present application; although the present application has been described in detail with reference to the foregoing examples, the technical solutions recorded in the foregoing examples can still be modified by those of ordinary skill in the art, or some technical features thereof can be replaced by equivalent replacements; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions claimed by the present application.

Claims

1. Use of genistein in the manufacture of a CD38 inhibitor, characterized in that, The use concentration of the genistein is 20 μM ~100 μM, and the preparation method of the genistein comprises the following steps: (1) dry portulaca oleracea is crushed, water and a compound enzyme are added to the obtained portulaca oleracea powder for enzymolysis, and a portulaca oleracea enzymolysis liquid is obtained; the compound enzyme is a mixture of cellulase, pectinase and glucosidase, and the weight ratio is 1:1:1, and the addition amount is 1.5%-2% of the weight of the portulaca oleracea powder; the enzymolysis temperature is 35-55 DEG C, the pH is 4.5-6.0, and the incubation reaction time is 2-4 h; (2) ethanol is added to the portulaca oleracea enzymolysis liquid in step (1), ultrasonic-assisted extraction is carried out, the filtrate is collected by filtration, repeated extraction and filtration are carried out, and an extraction liquid is obtained by combination; the concentration of the ethanol is 70%-90%; the ultrasonic-assisted extraction conditions are as follows: the ultrasonic power is 60 W-180 W, the temperature is 35-60 DEG C, and the time is 20-50 min; (3) the extraction liquid in step (2) is concentrated under reduced pressure, the obtained concentrated liquid is redissolved, and then extracted and separated with an organic solvent, and an extraction liquid is obtained after repeated extraction; (4) the extraction liquid in step (3) is separated by using a macroporous adsorption resin, and an eluent is collected; (5) the eluent in step (4) is separated by using a gel column and liquid chromatography, and then dried to obtain genistein.

2. Use according to claim 1, characterized in that, In step (3), the temperature of the concentration under reduced pressure is 40-60 DEG C; the organic solvent is at least one of ethyl acetate, butyl acetate and petroleum ether; in step (5), the liquid chromatography conditions are as follows: the chromatographic column is a ZORBAX SB-C18 column, the column temperature is 30 DEG C, the mobile phase is 1% formic acid-methanol, the flow rate is 1 mL / min, the sample injection amount is 500 μL, the gradient elution conditions are 0 min 30% methanol, 5 min 30% methanol, 15 min 70% methanol, 25 min 30% methanol, and the detector is an ultraviolet detector.

Images