A white birch betulin-pectin nanoparticle loaded with peony polyphenol, a preparation method and application thereof

Abstract

The present application relates to a kind of preparation methods of white birch alcohols-pectin nanoparticles loaded with peony polyphenol: 1) preparation peony polyphenol: 2) preparation pectin: 3) preparation white birch alcohols-pectin nanoparticles: white birch alcohols and peony seed pectin are dissolved in DMSO, N,N-dimethylaminopyridine is added as catalyst, reaction is carried out at room temperature for 8-16h, dialysis, freeze-drying is obtained white birch alcohols-pectin nanoparticles;4) white birch alcohols-pectin nanoparticles and peony polyphenol are dissolved in appropriate amount of DMSO according to proportion, then drop into distilled water under stirring, dialysis, freeze-drying, white birch alcohols-pectin nanoparticles loaded with peony polyphenol are obtained.The present application is connected by ester bond using carboxyl on pectin and hydroxyl on white birch alcohols, forms nano delivery carrier, then with peony polyphenol, forms water-soluble white birch alcohols-pectin / peony polyphenol nanoparticles, which can be used for preparing antitumor or hypoglycemic drug.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of natural products and nanotechnology, and particularly relates to a white birch resin alcohol-pectin nanoparticle loaded with peony polyphenol, a preparation method and applications thereof. BACKGROUND

[0002] Diabetes, as a public health problem after tumor, cardiovascular and cerebrovascular diseases, seriously threatens human health, and its incidence and mortality are rapidly increasing worldwide. According to the International Diabetes Federation, the number of global diabetes patients in 2019 was 463 million, and the number of diabetes patients in China reached 116 million, ranking first in the world.

[0003] In the 2017 edition of the guidelines for the prevention and treatment of type 2 diabetes in China, alpha-glucosidase inhibitors are recommended as the first alternative drug for the treatment of type 2 diabetes. Some artificially synthesized alpha-glucosidase inhibitors, such as acarbose and miglitol, have been used in clinical treatment, and long-term use can cause a series of gastrointestinal discomfort, liver poisoning and other adverse reactions, and their therapeutic effect on complications is very limited. Natural products have the advantages of small toxic and side effects, stable efficacy, multi-pathway and multi-target, etc., so it is of great scientific significance to obtain alpha-glucosidase inhibitors from abundant natural products to control postprandial blood glucose and prevent and treat diabetes.

[0004] Nanotechnology can solve the problems of poor water solubility, insufficient targeting ability, non-specific distribution, high systemic toxicity and low therapeutic index of conventional therapeutic preparations during drug delivery. Because of its simple preparation, good physical stability, low viscosity, good biocompatibility and other characteristics, it is often used as a carrier for liposoluble drugs and water-sensitive drugs, and has been widely used as an effective carrier for difficult-to-dissolve traditional Chinese medicines.

[0005] Among them, polysaccharide materials have obvious advantages in drug loading research, such as: wide source, safety and non-toxic, good biocompatibility, etc. Therefore, the research on nanometer drug loading system using polysaccharide as the matrix carrier is attracting great interest.

[0006] Paeonia suffruticosa seed shell is a byproduct in the production process of paeonia suffruticosa seed oil. Studies have found that the weight of paeonia suffruticosa seed shell accounts for more than 31% of the seed mass. In recent years, with the increasing demand for paeonia suffruticosa seed oil on the market, paeonia suffruticosa seed shells are discarded in large quantities, causing serious resource waste and environmental problems. Paeonia suffruticosa seed shells contain flavonoids, polyphenols, melanin, oligomeric stilbenes and other components, and it is necessary to develop and utilize paeonia suffruticosa seed shell resources.

[0007] The present application studies peony polyphenol, the peony polyphenol is not soluble in water, and is easy to decompose under high temperature, light and the like, the present application intends to adopt the carboxyl on pectin and the hydroxyl on betulin ester bond connection, forms a nano delivery carrier, and then forms a water-soluble betulin-pectin / peony polyphenol nanoparticle with the peony polyphenol, which opens up a new way for the development of peony polyphenol into medicines and health products. SUMMARY

[0008] The present application aims at overcoming the defects of the prior art, and provides a betulin-pectin nanoparticle loaded with peony polyphenol, which adopts the carboxyl on pectin and the hydroxyl on betulin ester bond connection to form a nano delivery carrier, and then forms a water-soluble betulin-pectin / peony polyphenol nanoparticle with the peony polyphenol, and can be used for preparing an antitumor or hypoglycemic drug.

[0009] The present application also provides a preparation method of the above-mentioned betulin-pectin nanoparticle loaded with peony polyphenol and application thereof.

[0010] To achieve the above-mentioned purpose, the present application adopts the following technical solutions.

[0011] A preparation method of a betulin-pectin nanoparticle loaded with peony polyphenol, comprising the following steps:

[0012] 1) Preparation of peony polyphenol: peony seed shell is crushed, 75-85% ethanol is added for ultrasonic extraction, solid-liquid separation is performed, the filter residue is dried, NaOH solution is added, and the mixture is shaken on a shaking bed at room temperature for 4-6 h, centrifugation is performed, the supernatant is collected, the pH value is adjusted to 1-3, and D101 macroporous resin column is used for elution, 2-4 column volumes of water are used for elution to remove impurities, and then 2-4 column volumes of 50-70% ethanol are used for elution, and the eluate is concentrated to dryness under reduced pressure, to obtain peony seed shell combined polyphenol, which is referred to as peony polyphenol;

[0013] 2) Preparation of pectin: peony seed meal is crushed, 75-85% ethanol is added for ultrasonic extraction (generally 4-5 times of ultrasonic extraction, 0.5-2 h each time), solid-liquid separation is performed, the filter residue is dried to remove ethanol, acid water is added for extraction at 70-90℃, the extraction liquid is concentrated under reduced pressure, alcohol precipitation is performed with 75-85% ethanol, and the mixture is allowed to stand for 12-24 h, and the Sevage method is used to remove protein, DEAE-Cellulose chromatography column is used for elution, water and 0.4-0.6M NaCl are used for elution in sequence, the 0.4-0.6M NaCl elution part is taken, dialysis is performed, and freeze-drying is performed, to obtain peony seed pectin;

[0014] 3) Preparation of betulin-pectin nanoparticles: betulin and the pectin prepared in step 2) are dissolved in DMSO solvent, 10-20% of the mass of the pectin of the tree peony seed is added as a catalyst, and the reaction is carried out at room temperature for 8-16 hours. After the reaction is completed, the product is obtained by dialysis in distilled water and freeze-drying to obtain betulin-pectin nanoparticles;

[0015] 4) Preparation of betulin-pectin nanoparticles loaded with polyphenols of tree peony: betulin-pectin nanoparticles and polyphenols of tree peony are dissolved in an appropriate amount of DMSO, and then distilled water is added dropwise under stirring. After dialysis in distilled water and freeze-drying, betulin-pectin nanoparticles loaded with polyphenols of tree peony are obtained.

[0016] Specifically, in step 1), the tree peony seed shell is crushed into 60-80 mesh powder, and 10-20 times the mass of 75-85% ethanol is added for ultrasonic extraction for 0.5-2 hours, and the extraction is repeated 4-5 times.

[0017] Further, in step 1), 3-5 mol / L NaOH solution is added according to the solid-liquid ratio of 1g:2-5 mL.

[0018] Specifically, in step 2), the pH of the acidic water is 2-3, and the amount of the acidic water added is 15-25 times the mass of the dried tree peony seed meal residue. The extraction is carried out for 1.5-3 hours and repeated 1-3 times. The tree peony seed meal is a byproduct after the tree peony seed is pressed for oil and can be purchased directly or prepared by conventional techniques in the art. Since the preparation is not the innovation of the present application, it will not be described here.

[0019] Specifically, in step 3), the mass ratio of betulin to the pectin prepared in step 2) is 1:1-3. The amount of DMSO added is 50-100 times the volume (g / mL) of the mass of the pectin.

[0020] Further, in step 3), the betulin is prepared by the following steps: 8-15 times the mass of 90-95% ethanol is added to forsythia leaves, and the forsythia leaves are refluxed at 70-90°C for 1-3 times, each time for 0.5-2 hours. The extraction liquid is combined and concentrated under reduced pressure until there is no alcohol smell. The product is eluted on a D101 macroporous adsorption resin, and then eluted with water, 40% ethanol, and 90% ethanol, respectively. The 90% ethanol elution fraction is taken and eluted on a silica gel column with petroleum ether-ethyl acetate (20:1 by volume) to obtain crystals. The crystals are recrystallized with ethyl acetate to obtain betulin monomers.

[0021] Specifically, in step 4), the mass ratio of betulin-pectin nanoparticles to polyphenols of tree peony is 4-1:1. The amount of DMSO added is 50-100 times the volume (g / mL) of the mass of the polyphenols of tree peony, and the amount of distilled water added is 2-4 times the volume of DMSO.

[0022] Further preferably, in steps 2), 3), 4), the dialysis bag used for dialysis has a molecular weight cut-off of 3500-5000 d.

[0023] The present application provides a peony polyphenol prepared by the above preparation method.

[0024] The present application provides a white birch resin alcohol-pectin nanoparticle loaded with peony polyphenol prepared by the above preparation method.

[0025] The present application also provides the use of the above peony polyphenol and / or white birch resin alcohol-pectin nanoparticle loaded with peony polyphenol in the preparation of an antitumor drug (especially esophageal cancer and the like) or an alpha-glucosidase inhibitor (especially a hypoglycemic drug).

[0026] Compared with the prior art, the present application has the following advantages:

[0027] 1) The peony polyphenol of the present application is obtained from the residue after extracting free polyphenols from peony seed shells, and belongs to bound polyphenols, which fully exploits peony seed shell resources;

[0028] 2) The peony polyphenol is not soluble in water, and the present application prepares it into nanoparticles, which has the characteristics of simple preparation, good physical stability, improved solubility and stability of peony polyphenol, and expanded use range;

[0029] 3) Pectin material has the advantages of wide source, safety, non-toxicity, good biocompatibility, etc. in the research of nano drug loading, and the present application fully utilizes peony resources by using pectin in peony seeds and white birch resin alcohol to prepare nanoparticles;

[0030] 4) The peony polyphenol and white birch resin alcohol-pectin nanoparticle loaded with peony polyphenol prepared by the present application have good antitumor and alpha-glucosidase inhibitory effects. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 Figure 4 is a graph showing the change in alpha-glucosidase inhibitory activity of different concentrations of peony polyphenol and white birch resin alcohol-pectin nanoparticles loaded with peony polyphenol;

[0032] Figure 2 Figure 5 is a graph showing the change in cell survival rate of esophageal cancer cell KYSE 70 after 24h and 48h of treatment with different concentrations of peony polyphenol and white birch resin alcohol-pectin nanoparticles loaded with peony polyphenol;

[0033] Figure 3 Figure 6 is a graph showing the change in cell survival rate of esophageal cancer cell KYSE 150 after 24h and 48h of treatment with different concentrations of peony polyphenol and white birch resin alcohol-pectin nanoparticles loaded with peony polyphenol;

[0034] Figure 4 A scanning electron microscope image of the betulin-pectin nanoparticles loaded with peony polyphenols obtained in Example 1;

[0035] Figure 5 An HPLC chart of peony polyphenols;

[0036] Figure 6 An HPLC chart of peony seed shell free polyphenols. DETAILED DESCRIPTION

[0037] The technical solutions of the present application are further described in detail below in conjunction with examples, but the scope of protection of the present application is not limited thereto.

[0038] In the following examples, the raw materials used are all ordinary commercially available products that can be directly purchased unless otherwise specified. The ethanol concentration not mentioned in detail refers to the volume percentage. In the preparation process, the dialysis bag selected for dialysis has a molecular weight cut-off of 3500-5000 d.

[0039] Example 1

[0040] For comparison, a preparation method of peony seed shell free polyphenols is provided as follows:

[0041] The peony seed shell is crushed into 80-mesh powder, 80% ethanol is added at 15 times the mass of the peony seed shell, and ultrasonic extraction is performed for 1 h. Filtration is performed, and the extraction is performed a total of 4 times. The ethanol extract is concentrated at 55°C under reduced pressure until no alcohol taste is present. The pH value is adjusted to 2, and the D101 macroporous resin column is used for elution. Two column volumes of water are used for elution to remove impurities, and then four column volumes of 80% ethanol are used for elution. The eluate is concentrated to dryness under reduced pressure to obtain the peony seed shell free polyphenols.

[0042] A preparation method of betulin-pectin nanoparticles loaded with peony polyphenols, which comprises the following steps:

[0043] 1) Preparation of peony polyphenols: The peony seed shell is crushed into 80-mesh powder, 80% ethanol is added at 15 times the mass of the peony seed shell, and ultrasonic extraction is performed for 1 h. Filtration is performed, and the extraction is performed a total of 4 times. The filter residue is dried at low temperature (50°C) to remove ethanol. A 4 mol / L NaOH solution is added at a solid-liquid ratio of 1 g:3 mL (m / V), and the mixture is shaken on a shaker at room temperature for 4 h. Centrifugation is performed at 4000 rpm / min for 10 min, and the supernatant is collected. The pH value is adjusted to 3 using concentrated HCl, and the 101 macroporous resin column is used for elution. Four column volumes of water are used for elution to remove impurities, and then four column volumes of 50% ethanol are used for elution. The 50% ethanol eluate is concentrated to dryness at 50°C under reduced pressure to obtain the peony seed shell bound polyphenols, which are referred to as peony polyphenols for short. The extraction yield is 2%, and the polyphenol content is 2.64 mg / g.

[0044] 2) Pectin preparation: The peony seed meal was crushed, 80% ethanol was added for ultrasonic extraction for 4 times, 1 h each time, solid-liquid separation, the peony seed meal residue was dried at 50°C to remove ethanol, 20 times the mass of the dried peony seed meal residue was added to pH = 2 acidic (sulfuric acid) water, 80°C extraction for 2 h, a total of 2 times, the extract was combined, concentrated under reduced pressure to about one tenth of the volume of the extract, 95% ethanol was added to the solution under rapid stirring until the ethanol concentration was 80%, alcohol precipitation, overnight (12 h) standing, Sevage method to remove protein, DEAE-Cellulose column chromatography, eluted with water and 0.5M NaCl in turn, 0.5M NaCl elution fraction was taken, dialysis bag dialysis to remove small molecules, freeze-drying to obtain peony seed pectin.

[0045] 3) Preparing betulin: Forsythia leaves, 10 times the mass of the forsythia leaves was added to 95% ethanol, 80°C reflux extraction for 2 times, 1 h each time, the extract was combined, concentrated under reduced pressure to remove alcohol, D101 macroporous adsorption resin was used, eluted with water, 40% ethanol, 90% ethanol in turn, 90% ethanol elution fraction was taken, silica gel column was used, eluted with petroleum ether-ethyl acetate solvent with a volume ratio of 20:1, 20:1 elution fraction was crystallized after concentration, the crystal was recrystallized with ethyl acetate to obtain betulin monomer, the purity reached 98%.

[0046] 4) Preparation of betulin-pectin nanoparticles: 10 mg of betulin and 10 mg of peony seed pectin were dissolved in 1 mL of DMSO solvent, 2 mg of N,N-dimethylaminopyridine (DMAP) was added as a catalyst, and the reaction was carried out at room temperature for 12 h. The reaction solution was placed in a dialysis bag and repeatedly dialyzed with distilled water to remove unreacted small molecule solvents, and freeze-dried (cold trap temperature -55~ -58°C, time 72-96 h, same below) to obtain betulin-pectin nanoparticles.

[0047] 5) Preparation of betulin-pectin / peony polyphenol drug-loaded nanoparticles. 15 mg of betulin-pectin nanoparticles and 15 mg of peony polyphenol were dissolved in 1 mL of DMSO, and then the dissolved solution was slowly added dropwise into 2 mL of distilled water under stirring, and the obtained solution was moved into a dialysis bag and repeatedly dialyzed with distilled water until the small molecule substances were completely removed, and freeze-dried to obtain betulin-pectin nanoparticles loaded with peony polyphenol.

[0048] Figure 4 The scanning electron microscope image of the betulin-pectin nanoparticles loaded with peony polyphenol is given. Figure 4 It can be seen that: the betulin-pectin nanoparticles loaded with peony polyphenol are spherical, and the particle size range is 50-200 nm.

[0049] Figure 5 and Figure 6The HPLC chart of the free polyphenol in peony seed shell and the peony polyphenol under the same detection condition is shown in two charts, and it can be seen from the two charts that the components of the free polyphenol in peony seed shell and the peony polyphenol are basically different, and the content of each component is quite different. The free polyphenol in peony seed shell is the polyphenol in the extraction process commonly seen in the literature at present, and the peony polyphenol is prepared by the method of the present application Figure 5 and Figure 6 The peony polyphenol in the present application and the peony seed shell polyphenol commonly seen in the existing literature have different preparation methods, so that their effective components are also different. Therefore, it is illustrated that even if the pharmacological activity is the same, it cannot be obviously deduced from the pharmacological activity of the free polyphenol in peony seed shell, because the effective components of the two are quite different.

[0050] The content of the peony polyphenol = the total phenol mass (mg) in the peony polyphenol extract / the weight (dry weight) of each gram of peony seed shell;

[0051] The total phenol mass in the peony polyphenol extract is detected by the Folin phenol method, and the specific steps are as follows: gallic acid is used as a standard to draw a standard curve. Draw the standard curve: accurately weigh 0.05 g of gallic acid standard, dissolve and dilute to 50 mL with water to obtain a 1 g / L gallic acid standard solution, and respectively transfer 2, 3, 4, 5, 6, 7 mL of the above gallic acid standard solution to a 100 mL volumetric flask, and dilute to 100 mL with water to prepare 20, 30, 40, 50, 60, 70 µg / mL standard solutions. Respectively, accurately measure 1 mL of the above standard solution and distilled water (blank) in a 10 mL stoppered colorimetric tube, add 0.5 mL of 1N Folin phenol reagent, 2 mL of 7.5% sodium carbonate solution, and 6.5 mL of distilled water, shake well, place in the dark for 1 h, and measure the absorbance at 765 nm. Perform 3 sets of parallel tests, take the gallic acid content as the abscissa and the absorbance as the ordinate, and draw a standard curve. The peony polyphenol extract is diluted to 25 mL with methanol, and the measurement method is the same as that of gallic acid. According to the standard curve drawn, the total phenol mass in the peony polyphenol extract is calculated, and the content of the peony polyphenol in the sample is calculated according to the total phenol mass in the peony polyphenol extract, and the result is expressed as mg / g of peony seed shell weight (dry weight).

[0052] Example 2

[0053] A preparation method of a white birch betulin-pectin nanoparticle loaded with peony polyphenol, comprising the following steps:

[0054] 1) Preparation of peony polyphenol: peony seed shell was crushed into 60 mesh powder, 80% ethanol was added at 15 times the mass of peony seed shell, and ultrasonic extraction was performed for 1 h. Filtration was performed, and a total of 4 times of extraction was performed. The filtrate was dried at low temperature (40°C) to remove ethanol, 4 mol / L NaOH solution was added at a solid-liquid ratio of 1 g:4 mL (m / V), and the mixture was shaken on a shaker at room temperature for 6 h. Centrifugation was performed at 4000 rpm / min for 10 min, and the supernatant was collected. The pH value was adjusted to 2 using concentrated HCl, and the mixture was subjected to D101 macroporous resin column. Water was used for elution to remove impurities, and then 60% ethanol was used for elution for 3 column volumes. The 60% ethanol eluate was concentrated to dryness at 50°C under reduced pressure to obtain peony polyphenol. The extraction amount was 1.5%, and the polyphenol content was 2.62 mg / g.

[0055] 2) Pectin preparation: peony seed meal was crushed, 80% ethanol was added, and ultrasonic extraction was performed for 4 times, 1 h each time. Solid-liquid separation was performed, and the peony seed meal residue was dried at 50°C to remove ethanol. Acidic water (6 mol / L hydrochloric acid) was added at 18 times the mass of the dried peony seed meal residue, and extraction was performed at 80°C for 2 h. A total of 2 times of extraction was performed, the extraction liquid was combined, and the volume was reduced to about one-ninth of the original volume under reduced pressure. 95% ethanol was added to the solution to a concentration of 80% under rapid stirring, and alcohol precipitation was performed. After standing overnight (24 h), Sevage method was used to remove protein, and DEAE-Cellulose chromatography column was used for elution. Water and 0.5 M NaCl were used for elution in sequence, and the 0.5 M NaCl elution part was taken. Dialysis bag dialysis was performed to remove small molecular substances, and freeze-drying was performed to obtain peony seed pectin.

[0056] 3) Preparing betulin: forsythia leaves were added with 95% ethanol at 12 times the mass of forsythia leaves, and reflux extraction was performed at 85°C for 2 times, 1.5 h each time. The extraction liquid was combined, and the alcohol was removed under reduced pressure. D101 macroporous adsorption resin was used for elution, and water, 40% ethanol, and 90% ethanol were used for elution in sequence. The 90% ethanol elution part was taken, and a silica gel column was used for elution with petroleum ether-ethyl acetate solvent at a volume ratio of 20:1. Crystals were precipitated after concentration of the 20:1 elution part, and the crystals were recrystallized with ethyl acetate to obtain betulin monomers with a purity of 98.2%.

[0057] 4) Preparation of betulin-pectin nanoparticles: 10 mg of betulin and 20 mg of peony seed pectin were dissolved in 1.2 mL of DMSO, 4 mg of N,N-dimethylaminopyridine (DMAP) was added as a catalyst, and the mixture was reacted at room temperature for 12 h. The reaction solution was placed in a dialysis bag and repeatedly dialyzed with distilled water to remove unreacted small molecular solvents. Freeze-drying was performed to obtain betulin-pectin nanoparticles.

[0058] 5) Preparation of betulinol-pectin / peony polyphenol drug-loaded nanoparticles. 20 mg of betulinol-pectin nanoparticles and 10 mg of peony polyphenol were dissolved in 0.8 mL of DMSO. The resulting solution was then slowly added dropwise to 3 mL of rapidly stirred distilled water while stirring. The solution was transferred to a dialysis bag and repeatedly dialyzed with distilled water until all small molecules were removed. The solution was then freeze-dried to obtain betulinol-pectin nanoparticles loaded with peony polyphenol.

[0059] The following experiments were conducted using the betulinol-pectin nanoparticles loaded with peony polyphenols prepared in Example 1 as an example, and betulinol-pectin nanoparticles without peony polyphenols loaded (hereinafter referred to as "betulinol-pectin nanoparticles") were used as a comparison.

[0060] Experiment 1 showed inhibitory activity against α-glucosidase.

[0061] Accurately weigh the sample, add DMSO to aid dissolution, and directly dissolve the betulin-pectin nanoparticles loaded with peony polyphenols in water. Adjust the volume to 25 mL, and then dilute with distilled water to a series of concentrations (15.625, 31.25, 62.5, 125, 250 μg / mL). Using a 96-well plate method, add phosphate-buffered saline (PBS) buffer, sample solution, and 2 U / mL α-glucosidase solution sequentially, mix well, and incubate at a pre-stabilized 37°C for 10 min. Remove from the plate, add 20 μL of 2.5 mmol / L PNPG solution, mix well, and incubate at 37°C for 30 min. Terminate the reaction by adding 0.5 mol / L sodium carbonate solution. Measure the absorbance (A value) at 405 nm using a microplate reader and calculate the α-glucosidase activity inhibition rate. Results are shown in [Figure Number]. Figure 1 .

[0062] The formula for calculating the inhibition rate of α-glucosidase is as follows:

[0063] Enzyme activity inhibition rate (%) = [1 - (sample group - blank group) / (control group - blank group)] × 100%.

[0064] Table 1. Amount and order of addition of various reactants

[0065]

[0066] Depend on Figure 1 It can be seen that both peony polyphenols and betulin-pectin nanoparticles loaded with peony polyphenols (abbreviated as peony polyphenol nanoparticles in the figure, the same below) have good inhibitory activity against α-glucosidase. The IC50 of the inhibition rate of α-glucosidase by both is shown in the figure. 50The values are 33.92 ± 2.16 μg / mL, 24.74 ± 1.92 μg / mL, respectively, which are better than the positive control acarbose. The inhibition activity of betulin-pectin nanoparticles on α-glucosidase is poor (because its activity is too low, Figure 1 The inhibition rate of betulin-pectin nanoparticles on α-glucosidase is only 6.21% when its concentration is 1 mg / mL, which shows that the influence of betulin-pectin nanoparticles on the efficacy of betulin-pectin nanoparticles loaded with peony polyphenols is very low.

[0067] Test 2 Anti-tumor activity

[0068] 1. Cell culture

[0069] When the esophageal cancer cells KYSE 70, KYSE 150 grow to more than 80%, passaging is performed, the old culture solution is poured out, washed with PBS for 2-3 times, and trypsin (Beijing Solaybao Technology Co., Ltd.) is added for digestion (about 1 mL), so as to cover the entire bottom of the dish, and the room temperature is digested for 2-3 min, 2 mL of RPMI1640 culture solution is added to terminate the digestion, and it is gently blown off and transferred to a BD tube, centrifuged at 800 rpm for 3 min, the upper culture solution is poured out, and 1:2 or 1:3 is selected for passaging according to the number of cells, and placed in a CO2 incubator for cell culture under the culture conditions of 37°C and 5% CO2.

[0070] 2. Cell toxicity detection (CCK8 experiment)

[0071] The tumor cells are inoculated on a 96-well plate at 5×10 3 cells / well, cultured for 24 h, 100 uL of RPMI1640 culture solution containing different test samples is added, acted for 24 h, 100 uL of RPMI1640 culture solution containing 10% CCK-8 is added in the form of liquid exchange, and incubated for 2 h. The OD value at 450 nm wavelength is measured by an enzyme marker, the normal cells are used as a control, and the cell viability % is calculated according to the following formula:

[0072] Cell viability % = [(As-Ac) / (Ab-Ac)]x100%, wherein:

[0073] As is the absorbance of the test hole (containing cell culture medium, CCK-8, and the drug to be tested);

[0074] Ab is the absorbance of the control hole (containing cell culture medium, CCK-8, and no drug to be tested);

[0075] Ac is the absorbance of the blank hole (culture medium without cells and the drug to be tested, containing CCK-8).

[0076] 3 Test results

[0077] Figure 2 The changes of cell survival rates of esophageal cancer cells KYSE 70 after 24h and 48h of the action of different concentrations of peony polyphenols and white birch alcohol-pectin nanoparticles loaded with peony polyphenols are given. Figure 3 The changes of cell survival rates of esophageal cancer cells KYSE 150 after 24h and 48h of the action of different concentrations of peony polyphenols and white birch alcohol-pectin nanoparticles loaded with peony polyphenols are given. Figure 2 and Figure 3 It can be known that: the peony polyphenols and the white birch alcohol-pectin nanoparticles loaded with peony polyphenols have good inhibitory activity on esophageal cancer cells KYSE 70 and KYSE 150, and when the concentration is 31.25 μg / mL and the time is 24h, the cell survival rates of KYSE 70 are 14.95 ± 1.71% and 10.26 ± 3.26% respectively, and the cell survival rates of KYSE 150 are 25.51 ± 4.39% and 21.48 ± 1.29% respectively, which are obviously reduced, indicating that the inhibitory activity of the peony polyphenols and the white birch alcohol-pectin nanoparticles loaded with peony polyphenols on esophageal cancer cells KYSE 70 and KYSE 150 is more significant when the concentration is higher. In addition, under the same concentration, the inhibitory activity of the white birch alcohol-pectin nanoparticles loaded with peony polyphenols on esophageal cancer cells KYSE 70 and KYSE 150 is slightly higher than that of the peony polyphenols.

[0078] The inhibitory activity of the white birch alcohol-pectin nanoparticles on esophageal cancer cells KYSE 70 and KYSE 150 is poor (because the activity is too low, Figure 2 and 3 not shown in the table), and when the concentration is 1 mg / mL, the cell survival rates of KYSE 70 and KYSE 150 still reach 100%, indicating that the white birch alcohol-pectin nanoparticles have little effect on the drug efficacy of the white birch alcohol-pectin nanoparticles loaded with peony polyphenols.

[0079] In summary, the peony polyphenols and the white birch alcohol-pectin nanoparticles loaded with peony polyphenols have good α-glucosidase inhibitory activity and anti-tumor activity, and can be used as anti-tumor drugs (especially esophageal cancer and the like) or hypoglycemic drugs, and have great application prospects.

Claims

1. A method for preparing betulinol-pectin nanoparticles loaded with peony polyphenols, characterized in that, Includes the following steps: 1) Preparation of peony polyphenols: Peony seed shells are crushed, and extracted with 75-85% ethanol by ultrasonic extraction. Solid-liquid separation is performed, the filter residue is dried to remove ethanol, NaOH solution is added, and the mixture is shaken on a shaker at room temperature for 4-6 hours. After centrifugation, the supernatant is collected, the pH is adjusted to 1-3, and the mixture is loaded onto a D101 macroporous resin column. First, impurities are removed by elution with water, and then elution is performed with 50-70% ethanol. The eluent is concentrated to dryness under reduced pressure to obtain peony polyphenols. 2) Preparation of pectin: Peony seed meal is pulverized, and extracted with 75-85% ethanol by ultrasonic extraction. Solid-liquid separation is performed, the filter residue is dried to remove ethanol, acidic water is added and extracted at 70-90℃, the extract is concentrated under reduced pressure, precipitated with 75-85% ethanol, allowed to stand, and protein is removed by the Sevage method. The sample is then loaded onto a DEAE-Cellulose chromatography column and eluted sequentially with water and 0.4-0.6M NaCl. The 0.4-0.6M NaCl eluent is collected, dialyzed, and freeze-dried to obtain peony seed pectin. 3) Preparation of betulinol-pectin nanoparticles: betulinol and the peony seed pectin obtained in step 2) were dissolved in DMSO solvent, and 10-20% of N,N-dimethylaminopyridine by weight of peony seed pectin was added as a catalyst. The reaction was carried out at room temperature for 8-16 hours. After the reaction was completed, the betulinol-pectin nanoparticles were obtained by dialysis and freeze drying. 4) Preparation of betulinol-pectin nanoparticles loaded with peony polyphenols: dissolve betulinol-pectin nanoparticles and peony polyphenols in an appropriate amount of DMSO in a certain proportion, then add them dropwise to distilled water while stirring. After dialysis and freeze drying, betulinol-pectin nanoparticles loaded with peony polyphenols are obtained. In step 3), betulin is prepared by the following steps: Forsythia leaves are added to 8-15 times their weight of 90-95% ethanol and refluxed at 70-90℃ for 1-3 times, each time for 0.5-2 hours. The extracts are combined and concentrated under reduced pressure until there is no alcohol odor. The extracts are then loaded onto D101 macroporous adsorption resin and eluted sequentially with water, 40% ethanol, and 90% ethanol. The 90% ethanol eluent is loaded onto a silica gel column and eluted with a petroleum ether-ethyl acetate gradient at a volume ratio of 20:

1. The 20:1 eluent is concentrated and crystals precipitate. The crystals are recrystallized from ethyl acetate to obtain betulin monomer.

2. The method for preparing betulinol-pectin nanoparticles loaded with peony polyphenols as described in claim 1, characterized in that, In step 1), the peony seed shells are crushed into 60-80 mesh powder, and then ultrasonically extracted with 75-85% ethanol at 10-20 times the weight of the peony seed shells for 0.5-2 hours, and the extraction is repeated 4-5 times.

3. The method for preparing betulinol-pectin nanoparticles loaded with peony polyphenols as described in claim 1, characterized in that, In step 1), add 3-5 mol / L NaOH solution at a material-to-liquid ratio of 1g:2-5mL.

4. The method for preparing betulinol-pectin nanoparticles loaded with peony polyphenols as described in claim 1, characterized in that, In step 2), the pH of the acidic water is 2-3, and the amount of acidic water added is 15-25 times the mass of the dried peony seed meal residue; the extraction time is 1.5-3 hours, and the extraction is repeated 1-3 times.

5. The method for preparing betulinol-pectin nanoparticles loaded with peony polyphenols as described in claim 1, characterized in that, In step 3), the mass ratio of betulin to the peony seed pectin prepared in step 2) is 1:1-3.

6. The method for preparing betulinol-pectin nanoparticles loaded with peony polyphenols as described in claim 1, characterized in that, In step 4), the mass ratio of betulinol-pectin nanoparticles to peony polyphenols is 4-1:

1.

7. The method for preparing betulinol-pectin nanoparticles loaded with peony polyphenols as described in claim 1, characterized in that, In steps 2), 3), and 4), the molecular weight cutoff of the dialysis bag used for dialysis is 3500-5000 d.

8. Betulinol-pectin nanoparticles loaded with peony polyphenols prepared by any one of the preparation methods described in claims 1 to 7.

9. The use of the betulinol-pectin nanoparticles loaded with peony polyphenols as described in claim 8 in the preparation of anti-esophageal cancer drugs or hypoglycemic drugs.

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