Prunus sibirica polyphenol extracting solution and preparation method thereof

Abstract

The invention relates to a prunus sibirica polyphenol extracting solution and a preparation method thereof, and belongs to the technical field of plant extraction. The method comprises the following steps: firstly, selecting fresh and mature padus nakai fresh fruits, cleaning, removing kernels, drying at 53-57 DEG C, crushing, sieving with a 60-mesh sieve, taking the sieved product, and storing at 4 DEG C to obtain padus nakai fruit powder; the preparation method comprises the following steps: carrying out ultrasonic extraction on prunus sibirica fruit powder at 68-72 DEG C for 38-42 minutes by adopting ethanol with the volume concentration of 28%-32% to obtain a prunus sibirica polyphenol extracting solution; according to the prunus sibirica polyphenol extracting solution prepared by the invention, the IC50 value of the DPPH free radical scavenging rate is 0.246 mu g / mL, the IC50 value of the hydroxyl free radical scavenging rate is 33.594 mu g / mL, and the prunus sibirica polyphenol extracting solution and the prunus sibirica polyphenol extracting solution have better effects than ascorbic acid; the nitrite clearance rate IC50 value is 7.409 [mu] g / mL. The invention provides theoretical basis and reference for the development of functional active products of Prunus cerasifera.

Description

Technical Field

[0001] The invention belongs to the technical field of plant extraction, and particularly relates to a polyphenol extract of Prunus thunbergii and a preparation method thereof. Background Art

[0002] Padus napaulensis belongs to the genus Padus in the Rosaceae family. It is commonly known as rouge fruit, mountain plum and ghost eye fruit. It is planted in large areas in Yunnan, Guizhou, Shaanxi and other places as well as Myanmar, Nepal and other countries. Padus napaulensis, as a nectar source and ornamental tree species, is planted in Europe and North Asia. Its leaf color changes from purple-red green to purple-red as the temperature rises, and turns red in autumn. It has a high ornamental value; the fruit is not only high in oil content, but also can be used as industrial oil after extraction and refining, and has many potential nutritional and commercial values. It is eaten as a fruit in Fengqing and other places in Yunnan. It is sweet and sour and can be eaten fresh or made into rock sugar mountain plums. Because its juice is fresh in color and rich in anthocyanins, locals usually take it to make wine, which has appetizing and health-promoting effects.

[0003] Polyphenols are compounds found in plant foods that have the potential to promote health. They exist in some common plant foods, have strong antioxidant effects, can scavenge free radicals, have antibacterial and anti-cardiovascular and cerebrovascular disease physiological functions, and are a class of natural antioxidants worth developing. Polyphenols are important secondary metabolites formed by plants themselves through various biosynthetic pathways. They exist in the roots, skins, stems, leaves and fruits of many edible crops and Chinese herbal medicines. As the second largest chemical group in the plant kingdom, their antibacterial and antioxidant properties make them one of the research hotspots in related fields such as antibacterial agent development and food storage. Carrying out the evaluation of the antibacterial activity of plant polyphenols, finding the structure of highly efficient and low-toxic plant polyphenols, and clarifying their antibacterial mechanism of action are of great significance to the development of green antibacterial agents and the application of polyphenols in the food storage industry.

[0004] Although there are reports that Prunus truncatus has certain antioxidant effects, there are no reports on the effects of the gastrointestinal environment on the polyphenol components and antioxidant activity of Prunus truncatus in the existing literature. However, the structure, content and composition of polyphenols may change under the action of various digestive enzymes and acid-base environment in the body, affecting their physiological activity. Due to the differences in the morphology and types of polyphenols in different foods, the changes in their gastrointestinal digestion are also inconsistent. Therefore, how to overcome the shortcomings of the existing technology is a problem that needs to be solved urgently in the field of plant extraction technology. Summary of the invention

[0005] The purpose of the present invention is to solve the deficiencies of the prior art and provide a Prunus thunbergii polyphenol extract and a preparation method thereof.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] The preparation method of the polyphenol extract of Prunus thunbergii comprises the following steps:

[0008] Step (1), preparation of thick-stemmed chrysanthemum fruit powder: select fresh and ripe thick-stemmed chrysanthemum fruits, clean them, remove the cores, dry them at 53-57° C., crush them and pass them through a 60-mesh sieve, take the sieved material, and store it at 4° C. to obtain thick-stemmed chrysanthemum fruit powder;

[0009] Step (2), preparation of a polyphenol extract of Prunus thunbergii: taking Prunus thunbergii fruit powder, using ethanol with a volume concentration of 28% to 32%, and ultrasonically extracting at 68 to 72° C. for 38 to 42 minutes to obtain a polyphenol extract of Prunus thunbergii;

[0010] The volume ratio of ethanol to the mass ratio of the Prunus armeniaca fruit powder is 34-36:1 mL / g.

[0011] Furthermore, preferably, in step (1), the drying temperature is 55°C.

[0012] Furthermore, preferably, in step (2), ethanol having a volume concentration of 30% is used for ultrasonic extraction at 70° C. for 40 minutes to obtain the Prunus thunbergii polyphenol extract.

[0013] Furthermore, preferably, in step (2), the volume ratio of ethanol to the mass ratio of the Prunus armeniaca fruit powder is 35:1 mL / g.

[0014] The present invention also provides a Prunus cerasifera polyphenol extract obtained by the preparation method of the Prunus cerasifera polyphenol extract, wherein the polyphenol content of the Prunus cerasifera polyphenol extract is 66.398±0.411 mg / g.

[0015] The third aspect of the present invention provides the use of the polyphenol extract of Prunus thunbergii in the preparation of a drug for scavenging DPPH free radicals.

[0016] The fourth aspect of the present invention provides the use of the polyphenol extract of Prunus thunbergii in the preparation of drugs for scavenging hydroxyl free radicals.

[0017] The fifth aspect of the present invention provides the use of the polyphenol extract of Prunus thunbergii in the preparation of a nitrite-scavenging drug.

[0018] The sixth aspect of the present invention provides the use of the above-mentioned Prunus thunbergii polyphenol extract in the preparation of antibacterial drugs.

[0019] A seventh aspect of the present invention provides the use of the polyphenol extract of Prunus thunbergii in the preparation of antioxidant drugs for gastrointestinal use.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention provides a method for preparing a polyphenol extract of Prunus thunbergii, which is simple and reliable. The polyphenol content of the obtained polyphenol extract of Prunus thunbergii is as high as 66.398±0.411 mg / g. According to research, the IC value of the scavenging rate of DPPH free radicals of the polyphenol extract of Prunus thunbergii prepared by the present invention is 50 The scavenging rate of hydroxyl radicals was 0.246 μg / mL. 50 The value was 33.594 μg / mL, which was better than ascorbic acid; the reducing power was lower than ascorbic acid; the nitrite clearance rate IC 50 The value is 7.409 μg / mL, which is less than ascorbic acid. The antibacterial test shows that the antibacterial zone of the thick-stemmed cherry polyphenol against Escherichia coli is 16.00±0.10 mm, and the antibacterial zone diameter against Staphylococcus aureus is 18.05±0.05 mm. The present invention provides a reference for the development of functional active products of thick-stemmed cherry.

[0022] At the same time, in the simulated digestion process; the antioxidant capacity of the polyphenols of Prunus chinensis was slightly weak during the gastric digestion period, but after intestinal digestion, the antioxidant capacity measurements showed an obvious growth trend, and the reducing capacity, hydroxyl radical scavenging capacity, and DPPH radical scavenging capacity reached the highest at 2h (1.099±0.030) of intestinal digestion, 1h (99.306±0.434%) of intestinal digestion, and 0h (93.115±0.628%) of intestinal digestion. Through data analysis, there is a certain correlation between the various indicators. In summary, Prunus chinensis is a good source of active substances, and the present invention can provide a certain experimental basis and new ideas for exploring the changes and action rules of active substances in Prunus chinensis polyphenols during human digestion. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the result graph of the effect of ethanol concentration on the amount of polyphenols extracted from Prunus thunbergii;

[0024] Figure 2 This is the result graph of the effect of ultrasound time on the extraction amount of polyphenols from Prunus thunbergii;

[0025] Figure 3 This is the result graph of the effect of extraction temperature on the amount of polyphenols extracted from Prunus thunbergii;

[0026] Figure 4 This is the result graph of the effect of the solid-liquid ratio on the extraction amount of polyphenols from Prunus thunbergii;

[0027] Figure 5 This is the result diagram of the scavenging ability of Prunus thunbergii polyphenols on DPPH;

[0028] Figure 6 This is the result diagram of the scavenging ability of polyphenols of Prunus thunbergii for hydroxyl radicals;

[0029] Figure 7 This is the result of the determination of reducing power of Prunus thunbergii polyphenols;

[0030] Figure 8 This is the result of the test on the nitrite scavenging ability of Prunus thunbergii polyphenols;

[0031] Fig. 9 This is the result of the antibacterial effect of Prunus thunbergii polyphenols on Escherichia coli;

[0032] Fig.10 This is the result of the inhibition of Staphylococcus aureus. DETAILED DESCRIPTION

[0033] The present invention is further described in detail below in conjunction with embodiments.

[0034] Those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the art or the product specifications are used. If the manufacturer of the materials or equipment used is not specified, they are all conventional products that can be purchased.

[0035] Example 1

[0036] The method for preparing the polyphenol extract of Prunus thunbergii is characterized by comprising the following steps:

[0037] Step (1), preparation of thick-stemmed chrysanthemum fruit powder: select fresh and ripe thick-stemmed chrysanthemum fruits, clean them, remove the cores, dry them at 53° C., grind them and pass them through a 60-mesh sieve, take the sieved material, and store it at 4° C. to obtain thick-stemmed chrysanthemum fruit powder;

[0038] Step (2), preparation of a polyphenol extract of Prunus truncatus: taking Prunus truncatus fruit powder, using ethanol with a volume concentration of 28%, and ultrasonically extracting at 68° C. for 38 minutes to obtain a polyphenol extract of Prunus truncatus;

[0039] The volume ratio of ethanol to the mass ratio of Prunus armeniaca fruit powder is 34:1 mL / g.

[0040] Example 2

[0041] The method for preparing the polyphenol extract of Prunus thunbergii is characterized by comprising the following steps:

[0042] Step (1), preparation of thick-stemmed chrysanthemum fruit powder: select fresh and ripe thick-stemmed chrysanthemum fruits, clean them, remove the cores, dry them at 57° C., grind them and pass them through a 60-mesh sieve, take the sieved material, and store it at 4° C. to obtain thick-stemmed chrysanthemum fruit powder;

[0043] Step (2), preparation of a polyphenol extract of Prunus truncatus: taking Prunus truncatus fruit powder, using ethanol with a volume concentration of 32%, and ultrasonically extracting at 72° C. for 42 minutes to obtain a polyphenol extract of Prunus truncatus;

[0044] The volume ratio of ethanol to the mass ratio of Prunus armeniaca fruit powder is 36:1 mL / g.

[0045] Example 3

[0046] The method for preparing the polyphenol extract of Prunus thunbergii is characterized by comprising the following steps:

[0047] Step (1), preparation of thick-stemmed chrysanthemum fruit powder: select fresh and ripe thick-stemmed chrysanthemum fruits, clean them, remove the cores, dry them at 55° C., grind them and pass them through a 60-mesh sieve, take the sieved material, and store it at 4° C. to obtain thick-stemmed chrysanthemum fruit powder;

[0048] Step (2), preparation of a polyphenol extract of Prunus thunbergii: taking Prunus thunbergii fruit powder, using ethanol with a volume concentration of 30%, and ultrasonically extracting at 70° C. for 40 minutes to obtain a polyphenol extract of Prunus thunbergii;

[0049] The volume ratio of ethanol to the mass ratio of Prunus armeniaca fruit powder is 35:1 mL / g.

[0050] The polyphenol content in the polyphenol extract of Prunus thunbergii is 66.398±0.411 mg / g.

[0051] Application Examples

[0052] 1 Materials and methods

[0053] 1.1 Materials and reagents

[0054] Thick-stemmed plum fruit powder: Select ripe thick-stemmed plums, wash them, remove the core, dry them, grind them, pass them through a 60-mesh sieve, and store them in a sealed container.

[0055] Gallic acid standards (HPLC), pepsin, trypsin, and porcine bile salts were from Beijing Solebow Technology Co., Ltd.

[0056] 1.2 Instruments and Equipment

[0057] Model 754 UV-visible spectrophotometer: Shanghai Sunny Hengping Scientific Instrument Co., Ltd.; 3k15 desktop high-speed refrigerated centrifuge: Sigma, Germany.

[0058] 1.3 Methods

[0059] 1.3.1 Sample pretreatment

[0060] Fresh and ripe Prunus thunbergii fruits are selected, cleaned, the cores are removed, dried in a 55°C oven, crushed through a 60-mesh sieve, and sealed and stored in a 4°C refrigerator to obtain Prunus thunbergii fruit powder.

[0061] 1.3.2 Determination of polyphenol content

[0062] The polyphenol content in Prunus truncatula was determined by the Folin-Ciocalteu method. The regression equation of the standard curve was obtained: y = 9.5229x + 0.0232, R 2 = 0.9947. The results are expressed as mg gallic acid / g dry basis (mg / g).

[0063] 1.3.3 Single factor experiment for optimization of extraction process conditions

[0064] 1.000 g of Prunus schrenkiana fruit powder was weighed and ultrasonically treated at a power of 260 w. The extraction amount of polyphenols from Prunus schrenkiana was used as the evaluation index. The effects of ethanol concentration (30%, 40%, 50%, 60%, 70%, 80%), ultrasonic time (15, 30, 60, 90, 120 min), ultrasonic extraction temperature (20, 30, 40, 50, 60, 70 ℃) and liquid-to-solid ratio (5:1, 10:1, 20:1, 30:1, 40:1 mL / g) on ​​the extraction amount of polyphenols from Prunus schrenkiana were investigated.

[0065] 1.3.4 Experiment on scavenging DPPH free radicals by polyphenol extract of Prunus thunbergii

[0066] (1) Reagents used: 0.1mmol / L DPPH, L-ascorbic acid

[0067] (2) Measurement method

[0068] Take out 3 test tubes and add the following reagents in sequence:

[0069] 0.4 mL of Prunus serrata polyphenol extract and 2.0 mL of 0.1 mmol / L DPPH methanol solution;

[0070] 0.4 mL of Prunus thunbergii polyphenol extract and 2.0 mL of methanol;

[0071] 0.4 mL of distilled water and 2.0 mL of 0.1 mmol / L DPPH methanol solution;

[0072] Mix these reagents evenly, place them in a dark place for 30 minutes, adjust to zero with 0.4mL distilled water and 2.0mL methanol, and take the supernatant from the above-mentioned fully mixed and light-proof reagents when measuring the absorbance, and perform accurate measurements at a wavelength of 517nm. The absorbance values ​​of the three different reaction solutions are recorded as A1, A2, and A0, respectively. As a positive control, Vc was used, and each sample was tested three times in parallel. According to the calculation formula, the scavenging ability of the sample to be tested for DPPH free radicals is calculated:

[0073]

[0074] 1.3.5 Hydroxyl free radical scavenging test

[0075] (1) Reagents: 9.0 mmol / L FeSO4 solution, 6 mmol / L H2O2 solution, 9.0 mmol / L salicylic acid ethanol solution, L-ascorbic acid

[0076] (2) Measurement method

[0077] Prepare three centrifuge tubes and add reagents in the following order:

[0078] Group A1: first add 1.0 mL of Prunus thunbergii polyphenol extract, then add 0.3 mL of FeSO4 and 0.25 mL of H2O2, mix well, let stand at room temperature for 10 minutes, and then add 1.0 mL of salicylic acid solution.

[0079] Group A2: first add 1.0 mL of Prunus thunbergii polyphenol extract and 0.3 mL of FeSO4, then add 0.25 mL of H2O2, mix thoroughly, place at room temperature for 10 min, and finally add 1.0 mL of distilled water.

[0080] Group A0: First, 1.0 mL of distilled water, 0.3 mL of FeSO4 and 0.25 mL of H2O2 were mixed, and then placed at room temperature for 10 min, and then 1.0 mL of salicylic acid solution was added.

[0081] Mix the reagents in the centrifuge tube thoroughly and place it in a 37℃ water bath for 30 minutes. Take it out, cool it with running water, and add 0.45mL of distilled water to each centrifuge tube. Centrifuge it at 6000r / min for 10 minutes. Finally, measure and record the absorbance values ​​of the three reaction solutions, marked as A1, A2 and A0 respectively. Before the measurement, use distilled water to adjust the zero value and ensure that the absorbance value is measured at a wavelength of 510nm.

[0082] V c As a positive control, three parallels were made for each sample, and the scavenging rate of the sample for hydroxyl free radicals was calculated according to the following formula:

[0083]

[0084] 1.3.6 Determination of reducing power

[0085] (1) Reagents: 0.2 mol / L PBS phosphate buffer (pH 6.6), 1% (m / v) potassium ferrocyanide solution, 10% (m / v) trichloroacetic acid, 0.1% (m / v) ferric chloride, L-ascorbic acid; Note: The unit of mass m in m / v is g, and the unit of volume v is mL.

[0086] (2) Measurement method

[0087] Sample: 1.0mL extract

[0088] Zero adjustment: 1.0mL distilled water,

[0089] Take 1.0mL of 0.2mol / L PBS phosphate buffer (pH 6.6), add 1.0mL of 1% potassium ferricyanide solution, and place the mixture in a 50°C water bath to react for 20 minutes. Subsequently, the reaction solution was quickly cooled to ice water temperature, and 1.0mL of 10% trichloroacetic acid (TCA) was added and mixed evenly. After that, centrifuge at 10000r / min for 5 minutes. After centrifugation, take 1.0mL of supernatant, add 1.0mL of distilled water and 0.2mL of 0.1% ferric chloride, mix again, and let it react for 10 minutes. Distilled water was used instead of the extract for zeroing as above, and the absorbance of each sample at a wavelength of 700nm was measured. Vc was used as a positive control, and three parallels were made for each sample.

[0090] 1.3.7 Determination of nitrite removal capacity

[0091] (1) Reagents: 200 mg / mL sodium nitrite solution, 5 μg / mL NaNO2 standard working solution, 0.4% (m / v) p-aminobenzenesulfonic acid, 20% hydrochloric acid (mass percentage concentration), 0.2% (m / v) naphthylethylenediamine hydrochloride

[0092] Take four 25mL graduated test tubes and add the following reagents in sequence: A1: 5.0mL distilled water

[0093] +5.0mL NaNO2 (5μg / mL) standard working solution - incubate at room temperature for 30 minutes - immediately add 2mL 0.4% p-aminobenzenesulfonic acid - shake to mix and let stand for 3-5 minutes, then add 1mL 0.2% naphthylethylenediamine hydrochloride.

[0094] Zeroing tube procedure for A1 test: Use 5.0mL of distilled water instead of NaNO2 standard solution and mix with another 5.0mL of distilled water. Also let the reaction stand at room temperature for 30 minutes. Quickly add 2mL of 0.4% p-aminobenzenesulfonic acid solution. Shake thoroughly to mix and let stand for 3 to 5 minutes. Add 1mL of 0.2% naphthylethylenediamine hydrochloride solution.

[0095] Step A2: Mix 5.0 mL of the extract with 5.0 mL of 5 μg / mL NaNO2 standard solution. Let the reaction stand at room temperature for 30 minutes. Quickly add 2 mL of 0.4% p-aminobenzenesulfonic acid solution. Shake thoroughly to mix, and let stand for 3 to 5 minutes. Add 1 mL of 0.2% naphthaleneethylenediamine hydrochloride solution.

[0096] Zeroing tube procedure for A2 test: Use 5.0mL of extract solution instead of NaNO2 standard solution and mix with another 5.0mL of distilled water. Let the reaction stand at room temperature for 30 minutes. Quickly add 2mL of 0.4% p-aminobenzenesulfonic acid solution. Shake thoroughly to mix and let stand for 3 to 5 minutes. Add 1mL of 0.2% naphthaleneethylenediamine hydrochloride solution. Finally, shake all samples and make up to 25mL. After standing for 15 minutes, perform zeroing and measure its absorbance at 540nm.

[0097]

[0098] Where, A1 is the absorbance without extract;

[0099] A2——absorbance of added extract;

[0100] 1.3.8 Half inhibitory concentration (IC 50 )calculate

[0101] The different mass concentrations of Prunus thunbergii polyphenols were plotted against the reduction capacity, hydroxyl radical (·OH), DPPH radical, and nitrite scavenging capacity, and linear fitting was performed. According to the linear curve of the fitting, the mass concentration of polyphenols corresponding to the scavenging rate of 50% was the IC 50 , with IC 50 The value is used as an indicator of the antioxidant capacity of thick stem polyphenols. The lower the value, the stronger the antioxidant activity.

[0102] 1.3.9 Determination of antibacterial activity of polyphenols from Prunus truncatula

[0103] (1) Preparation of test bacterial solution

[0104] The formula of LB solid culture medium is as follows: add 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and 15 g of agar to 950 mL of distilled water, adjust the pH to 7.0, make up to 1 L with distilled water, sterilize at 121°C for 20 min, culture at 37°C for 24 h, and check for contamination.

[0105] LB liquid culture medium formula: add 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride to 950 mL of distilled water, adjust the pH to 7.0, make up to 1 L with distilled water, sterilize at 121°C for 20 min, culture at 37°C for 24 h, and check for bacterial contamination.

[0106] Activated bacteria: Inoculate the test bacteria preservation solution (Escherichia coli, Staphylococcus aureus) on the LB solid culture medium in a "Z" shape. After culturing the test bacteria in a constant temperature incubator at 37°C for 24 hours, pick out a typical, well-growing single colony from the cultured culture dish, inoculate it into 5 mL of LB liquid culture medium, place it in a shaker at a speed of 160 r / min and a temperature of 37°C for constant temperature cultivation, and set the cultivation time to 20 hours.

[0107] Prepare bacterial suspension: transfer 4 mL of cultured bacterial solution into a 15 mL centrifuge tube, and then centrifuge it in a centrifuge at 10,000 r / min and 4°C for 1 min. Next, remove the supernatant and wash the precipitate with 5 mL of sterile saline. After vortexing, centrifuge again at 10,000 r / min and 4°C for 1 min. After centrifugation, discard the supernatant into the waste liquid tank, and repeat this step twice (this step specifically refers to washing the precipitate with 5 mL of sterile saline, vortexing, and centrifuging again at 10,000 r / min and 4°C for 1 min). Finally, resuspend the bacteria with 5 mL of saline, and evenly disperse the bacteria using a vortex shaker for subsequent use.

[0108] (2) Determination of antibacterial rate

[0109] Aseptic operation was performed in a clean bench. 5 mL of the polyphenol extract of Prunus thunbergii was drawn with a sterile syringe and filtered through a water-based microporous filter membrane with a diameter of 0.22 μm to obtain a sterile polyphenol extract of Prunus thunbergii for the determination of antibacterial activity.

[0110] (3) Determination of inhibition zone diameter

[0111] With tea polyphenol standard as positive control and sterile distilled water as negative control, 0.1mL bacterial solution of Escherichia coli and Staphylococcus aureus diluted at a ratio of 1:50 were evenly spread on LB solid culture medium by the punching method. A 6mm hole was punched on the LB solid culture medium, and 0.10mL of sterile Prunus thunbergii polyphenol extract was added. After diffusion for 3min at room temperature, the medium was placed in a constant temperature incubator at 37℃ and cultured for 24h. The diameter (mm) of the inhibition zone was observed and measured.

[0112] 1.3.10 Simulated in vitro gastrointestinal digestion of Prunus thunbergii polyphenols

[0113] (1) Simulated gastric digestion

[0114] 6 mL of Prunus thunbergii polyphenol extract was measured, 12 mL of 9 mg / mL NaCl solution and 12 mL of 4 mg / mL pepsin solution were added, the pH was adjusted to 2.0 with 0.1 mol / L HCl, and the mixture was placed in a constant temperature shaking box at 37°C for digestion for 1 h to obtain the Prunus thunbergii polyphenol extract after gastric digestion.

[0115] (2) Simulated intestinal digestion

[0116] Pipette 5 portions of 2.0 mL each of the polyphenol extract of Prunus serrata (pre-adjusted to pH 7.0) digested in the stomach for 1 h into 5 10.0 mL stoppered test tubes, add 4.0 mL of a mixture of pancreatic juice and bile (2 mg / mL pancreatic juice and 12 mg / mL bile mixed in a volume ratio of 1:1) and place them in a constant temperature shaking box at 37°C for digestion for 4 h. Take out the sample solution at different time periods for storage.

[0117] The formula of 2 mg / mL pancreatic juice is to accurately weigh 0.2 g of trypsin, dissolve it in 0.1 mol / L NaHCO3, and dilute to 100 mL and shake well.

[0118] The formula of 12mg / mL bile is to accurately weigh 0.12g bile salt, dissolve it in 0.1mol / L NaHCO3 and dilute to 100mL and shake well.

[0119] 1.3.11 Study on the antioxidant activity of polyphenols from gastrointestinal digestion samples of Prunus thunbergii

[0120] The existing methods were used to determine the reducing ability, hydroxyl radical scavenging ability, and DPPH radical scavenging ability. For details, please refer to Yang Bingxin, Liu Xiaoli. Study on the extraction and antioxidant activity of total polyphenols from Phyllanthus emblica[J]. Food Industry Science and Technology, 2019, 40(16): 151-155162.

[0121] 1.3.12 Data Statistical Analysis

[0122] The results were expressed as mean ± standard deviation, and variance analysis was performed using SPSS 22.0 software, with p < 0.05 used for significance analysis.

[0123] 2 Results and Analysis

[0124] 2.1 Single factor test results

[0125] 2.1.1 Effect of ethanol concentration on the extraction of polyphenols from Prunus truncatula

[0126] Depend on Figure 1 It can be seen that when the ethanol concentration increases from 30% to 70%, the polyphenol extraction amount shows a significant downward trend. When the ethanol concentration is 30%, the polyphenol extraction amount is the largest, and the polyphenol extraction amount of the thick-stemmed plum is 31.251±1.460mg / g. When the ethanol concentration continues to increase, the polarity of polyphenols and solvents is quite different, and the protein denatures, resulting in a decrease in the solubility of polyphenols. At the same time, the dissolution of alcohol-soluble impurities and pigment components will also affect the extraction effect, thereby reducing the polyphenol yield. Therefore, 30% is the optimal ethanol concentration.

[0127] 2.1.2 Effect of ultrasonic time on the extraction of polyphenols from Prunus truncatula

[0128] Depend on Figure 2 It can be seen that when the ultrasonic time is 60 minutes, the polyphenol extraction amount reaches a maximum value of 64.99±2.20 mg / g, and then begins to decline. There is no significant difference when the ultrasonic extraction time is 60 minutes, 90 minutes, and 120 minutes, but it is significantly higher than 15 minutes and 30 minutes (p<0.05). Considering the time cost saving, 60 minutes is selected as the optimal ultrasonic extraction time.

[0129] 2.1.3 Effect of extraction temperature on the amount of polyphenols extracted from Prunus truncatula

[0130] Depend on Figure 3 It can be seen that starting from the extraction temperature of 30℃, with the increase of ultrasonic temperature, the polyphenol extraction amount of Prunus serrata first increased, then slowly decreased and then increased again. At 50℃, the polyphenol extraction amount decreased. When the ultrasonic temperature was 60℃, the polyphenol extraction amount reached a maximum of 57.15±5.01mg / g, and then began to decrease. At high extraction temperature, ethanol in the solvent will evaporate and other impurities will also dissolve, so the polyphenol extraction amount will decrease. There is no significant difference between the extraction temperature of 60℃ and 70℃, but there is a significant difference between 30℃, 40℃, and 50℃ (p<0.05). Considering the energy cost saving, 60℃ was selected as the optimal ultrasonic extraction temperature.

[0131] 2.1.4 Effect of extraction material-liquid ratio on the extraction amount of polyphenols from Prunus truncatula

[0132] Depend on Figure 4It can be seen that with the increase of solvent, polyphenols have been almost completely dissolved, and impurities will be dissolved afterwards. The polyphenol extraction amount is the largest when the liquid-to-solid ratio is 40:1, and the polyphenol extraction amount of Prunus serrata at this time is 55.50±0.22mg / g. There is no significant difference between the liquid-to-solid ratio of 30:1 and 40:1, but it is significantly different from 5:1, 10:1, and 20:1 (p<0.05), so the best liquid-to-solid ratio is selected as 40:1.

[0133] However, in the research process of the present invention, it was creatively found that the optimal process conditions were 30% ethanol concentration, 40 minutes ultrasonic time, 35:1 liquid-to-solid ratio, and 70°C extraction temperature. The polyphenol extraction amount of Prunus serrata reached 66.398±0.411 mg / g.

[0134] 2.2 Determination of antioxidant activity of polyphenol extracts from Prunus truncatula

[0135] 2.2.1 DPPH free radical scavenging test

[0136] When evaluating the antioxidant capacity of a substance, the DPPH free radical scavenging test is a key evaluation method. It aims to accurately measure the scavenging efficiency of DPPH free radicals, thereby efficiently, conveniently and sensitively evaluating the performance and activity of natural antioxidants with good reproducibility. Figure 5 It can be seen that the scavenging capacity of Prunus chinensis polyphenols for DPPH free radicals at the same mass concentration is better than ascorbic acid. Starting from a concentration of 0.005 mg / mL, the scavenging capacity of free radicals is better than ascorbic acid and is constantly increasing. This proves that the Prunus chinensis polyphenol extract has good scavenging capacity. When evaluating the scavenging rate of DPPH free radicals, when the scavenging rate reaches 50%, the corresponding polyphenol mass concentration is IC 50 After calculation, the IC 50 The value is 0.246 μg / mL, while the IC 50 The value is 0.774μg / mL. 50 The value is used as an indicator to measure the antioxidant capacity of Prunus chinensis polyphenols. The lower the value, the stronger the antioxidant activity. This conclusion is very prominent when the clearance rate is 50%. The calculation formula of the clearance rate of Prunus chinensis and ascorbic acid obtains the concentration of the corresponding substance. In the concentrations calculated from Prunus chinensis polyphenols and ascorbic acid, the concentration of ascorbic acid is higher than the concentration corresponding to Prunus chinensis polyphenols, which means that when measuring the clearance rate of DPPH, the antioxidant activity of Prunus chinensis polyphenols is higher than that of ascorbic acid, and the antioxidant activity of Prunus chinensis polyphenols is high.

[0137] 2.2.2 Hydroxyl free radical scavenging test

[0138] Through research, it was found that plant polyphenols can effectively scavenge hydroxyl free radicals, thereby reducing their production and further reducing the absorbance at a wavelength of 510nm. This mechanism helps prevent DNA chain breaks and base modifications, thereby preventing the occurrence of diseases such as tumors. By measuring the hydroxyl free radical scavenging rate of the test substance at different concentrations, its potential protective effect can be further evaluated. By measuring the hydroxyl free radical scavenging rate of the test substance at different concentrations, a curve of the relationship between the scavenging rate and the concentration of Prunus thunbergii polyphenols and ascorbic acid is drawn to evaluate the strength of its antioxidant properties. Figure 6 It can be known that the scavenging rate of hydroxyl free radicals by Prunus oleraceus polyphenols is dependent. The scavenging rate of free radicals by Prunus oleraceus polyphenols increases with the increase of polyphenol concentration. When the polyphenol concentration exceeds 10 μg / mL, the growth rate of the scavenging rate slows down, but it is still increasing. Figure 6 It can also be observed that at the same concentration, the scavenging rate of hydroxyl free radicals by Prunus paniculata polyphenols is significantly better than that by ascorbic acid, and the scavenging capacity increases with the increase of concentration. When evaluating the antioxidant capacity, the half inhibition concentration is calculated, that is, the mass concentration of polyphenols required when the scavenging rate reaches 50%. IC 50 The value was used as a key indicator to measure the antioxidant capacity of Prunus thunbergii polyphenols, among which the lower IC 50 The value represents a higher antioxidant activity. According to the corresponding formulas for the clearance rate of Prunus paniculata polyphenol and ascorbic acid in the figure, when the clearance rate is 50%, the concentrations calculated by the corresponding formulas of Prunus paniculata polyphenol and ascorbic acid are different. When the hydroxyl radical clearance rate is 50%, the corresponding Prunus paniculata polyphenol concentration calculated is 33.594μg / mL, and the ascorbic acid concentration is 94.456μg / mL. The ascorbic acid concentration is higher than the concentration corresponding to Prunus paniculata polyphenol, which means that when measuring the clearance rate of hydroxyl radical scavenging ability, the antioxidant activity of Prunus paniculata polyphenol is higher than that of ascorbic acid, and Prunus paniculata polyphenol has high antioxidant activity.

[0139] 2.2.3 Determination of reducing power

[0140] Samples with significant reducing power show their ability to supply electrons, stabilize free radicals, and effectively terminate free radical chain reactions. In addition, they can prevent the formation of peroxides, thereby exerting excellent antioxidant effects. The principle of measuring the reducing power of polyphenols is based on the fact that the reducing properties of polyphenols can reduce some oxidants. The reducing power of polyphenols can be reflected by measuring the absorbance through the color change of the oxidant. Figure 7It can be found that the reducing power of Prunus chinensis polyphenol is higher than that of ascorbic acid at a concentration below 40 μg / mL, and lower than that of ascorbic acid at a concentration above 40 μg / mL. However, in general, when evaluating the reducing power, it is found that the reducing power of Prunus chinensis polyphenol is comparable to that of ascorbic acid. With the gradual increase of concentration, its reducing power also shows a significant increasing trend, indicating that Prunus chinensis polyphenol is an effective electron donor.

[0141] 2.2.4 Determination of nitrite removal capacity

[0142] Plant polyphenols, due to their good hydrogen supply capacity, can react chemically with nitrite in an acidic environment, effectively reducing the nitrite content in food. By comparing the key indicator of nitrite clearance rate, the effects of Prunus schrenkiana polyphenol extract and ascorbic acid in inhibiting nitrosation reactions were analyzed. Using nitrite clearance rate as an indicator, the effects of Prunus schrenkiana polyphenol extract and ascorbic acid on nitrosation reactions were compared. Figure 8 The nitrite scavenging rates of Prunus schrenkiana polyphenols and ascorbic acid at the same concentration can be obtained. Prunus schrenkiana polyphenols continue to have lower nitrite scavenging capacity than ascorbic acid, but the observation results show that with the increase of concentration, the nitrite scavenging rates of both show an increasing trend. When calculating the half inhibition concentration, the corresponding polyphenol mass concentration when the scavenging rate is 50% was determined, and this value was used as a key indicator to measure the antioxidant capacity of Prunus schrenkiana polyphenols. The lower the index, the stronger the antioxidant activity. According to the corresponding formulas for nitrite clearance rates of Prunus aviculare polyphenols and ascorbic acid in the figure, when the clearance rate is 50%, the corresponding concentration of Prunus aviculare polyphenols is 7.409 μg / mL and the corresponding concentration of ascorbic acid is 4.412 μg / mL. The concentrations calculated by the corresponding formulas are different. The concentration of ascorbic acid is lower than that of Prunus aviculare polyphenols. This means that when measuring the clearance rate of nitrite scavenging ability, the antioxidant activity of Prunus aviculare polyphenols is lower than that of ascorbic acid. This means that when measuring the activity of nitrite clearance rate, the antioxidant activity of ascorbic acid is high.

[0143] 2.3 Determination of antibacterial activity of polyphenols from Prunus truncatula

[0144] from Fig. 9 and Fig.10 It can be seen that, whether it is Escherichia coli or Staphylococcus aureus, each culture dish has four holes, three of which are Prunus schrenkiana polyphenol extracts, and one hole is sterile distilled water. From the figure, it can be seen that the antibacterial ability of polyphenols against Escherichia coli and Staphylococcus aureus is stronger than that of sterile distilled water. The diameter of the antibacterial circle of polyphenols in the culture dish is compared with the diameter of the antibacterial circle of sterile distilled water. The diameter of sterile distilled water is significantly smaller than the diameter of the antibacterial circle of Prunus schrenkiana polyphenol extract. It can be seen that Prunus schrenkiana polyphenols have antibacterial ability.

[0145] Table 1 The diameter of the inhibition zone of Prunus thunbergii polyphenols against Escherichia coli

[0146]

[0147] Table 2 The diameter of the inhibition zone of Prunus thunbergii polyphenols against Staphylococcus aureus

[0148]

[0149]

[0150] From several parallel tests in Tables 1 and 2, it can be seen that the measured diameters of the three polyphenol holes in the culture dishes of the parallel tests of the two bacterial species Escherichia coli and Staphylococcus aureus are different. From this, it can be seen that the antibacterial ability of polyphenols in different culture media in parallel tests is also different. When comparing the two bacterial species, when observing the parallel test data of the two bacterial species, it can be found that the diameter of the inhibition zone formed by the polyphenols of Prunus thunbergii on Staphylococcus aureus is significantly larger than the corresponding value on Escherichia coli. This discovery reveals that under the same culture environment, the antibacterial effect of the polyphenols of Prunus thunbergii on Staphylococcus aureus is more significant than that on Escherichia coli.

[0151] 2.4 Study on the antioxidant activity of polyphenols from gastrointestinal digestion samples of Prunus thunbergii

[0152] 2.4.1 Determination of reducing capacity

[0153] As shown in Tables 3 and 4, the polyphenols of Prunus chinensis have a certain reducing ability after gastrointestinal digestion, which generally shows a trend of first increasing and then decreasing. The reducing ability value reaches 1.099±0.030 at 2h of intestinal digestion. This may be because the trypsin in the simulated intestinal fluid hydrolyzes the ester bond formed by polyphenols and proteins or polysaccharides inside and outside the cells, and the bound polyphenols are released, resulting in increased reducing ability. Although ascorbic acid has not been tested for gastric digestion and intestinal digestion, by comparing with ascorbic acid, the polyphenols of Prunus chinensis still show reducing ability after simulated gastrointestinal digestion.

[0154] Table 3 Reduction capacity of sample solution at different time periods

[0155]

[0156] Table 4 Reducing ability of ascorbic acid at different concentrations

[0157] <![CDATA[V C Concentration]]> 0.1mg / mL 1mg / mL Restore capability 1.463±0.0655 2.129±0.061

[0158] 2.4.2 Hydroxyl free radical scavenging ability determination

[0159] As shown in Tables 5 and 6, the polyphenols of Prunus chinensis have good hydroxyl radical scavenging ability after gastrointestinal digestion, and generally show a trend of first increasing and then decreasing. The hydroxyl radical reduction ability value reached a maximum of 99.306±0.434% at 1h of intestinal digestion, proving that after gastrointestinal digestion, the polyphenols of Prunus chinensis can be further released due to the action of digestive enzymes. Although ascorbic acid has not been tested for gastric digestion and intestinal digestion, by comparing with ascorbic acid, the polyphenols of Prunus chinensis still show hydroxyl radical scavenging ability after simulated gastrointestinal digestion.

[0160] Table 5 Hydroxyl radical scavenging capacity of sample solution at different time periods

[0161]

[0162] Table 6 Hydroxyl radical scavenging ability of ascorbic acid at different concentrations

[0163] <![CDATA[V C Concentration]]> 0.1mg / mL 1mg / mL Hydroxyl radical scavenging ability 16.062±3.312％ 95.666±3.525％

[0164] 2.3.3 DPPH free radical scavenging ability determination

[0165] As shown in Tables 7 and 8, the scavenging ability of Prunus chinensis after gastrointestinal digestion is certain, and the overall trend is to increase first and then decrease. The scavenging ability is weak during the gastric digestion period, indicating that the gastric acid environment and pepsin may reduce the scavenging ability of Prunus chinensis polyphenols DPPH free radicals, but at 1h of intestinal digestion, the DPPH free radical scavenging rate is the largest. This is due to the action of digestive enzymes in the intestinal digestive juice, which decomposes the bound phenols to release free phenols. In the subsequent intestinal digestion, the DPPH free radical scavenging rate dropped significantly and then increased. It may be because the polyphenols that are easily oxidized in the reaction system are degraded or transformed, thereby reducing the DPPH free radical scavenging rate. Later, the data fluctuated due to the unstable decomposition of the reaction polyphenols. Although ascorbic acid has not been tested for gastric digestion and intestinal digestion, by comparing with ascorbic acid, Prunus chinensis polyphenols still show DPPH free radical scavenging ability after simulated gastrointestinal digestion.

[0166] Table 7 DPPH free radical scavenging ability of sample solutions at different time periods

[0167]

[0168]

[0169] Table 8 DPPH free radical scavenging ability of ascorbic acid at different concentrations

[0170] <![CDATA[V C Concentration]]> 0.1mg / mL 1mg / mL DPPH free radical scavenging ability 50.398±3.863％ 65.026±1.696％

[0171] 3 Conclusion

[0172] The present invention explores the antioxidant activity of the polyphenol extract of Prunus thunbergii. When comparing the antioxidant activity of the polyphenol extract of Prunus thunbergii with that of ascorbic acid, the effects of extracts at different concentrations on the reducing power, DPPH free radical scavenging ability, hydroxyl free radical scavenging ability and nitrite scavenging ability were studied. The results showed that the polyphenol of Prunus thunbergii showed higher activity in DPPH free radical scavenging rate, and its IC 50 The value was 0.246μg / mL, which was lower than ascorbic acid’s 0.774μg / mL. In terms of hydroxyl radical scavenging rate, Prunus thunbergii polyphenols also showed stronger ability, with its IC 50 The value was 33.594 μg / mL, which was much lower than 94.456 μg / mL of ascorbic acid. 50 The value is 7.409μg / mL, which is higher than 4.412μg / mL of ascorbic acid, indicating that its antioxidant activity in this regard is slightly lower than that of ascorbic acid. The results show that the polyphenols of Prunus truncatus have good scavenging effects on DPPH, reducing power and hydroxyl radicals, and the scavenging ability of Prunus truncatus polyphenols in nitrite scavenging is not as good as that of ascorbic acid. Prunus truncatus is rich in antioxidant active substances polyphenols and exhibits excellent antioxidant properties. The study of antibacterial activity showed that the polyphenol extract of Prunus truncatus has a good inhibitory effect on Escherichia coli and Staphylococcus aureus. Comparing the two strains, it can be seen from the diameter of the inhibition zone that the polyphenols of Prunus truncatus have a stronger inhibitory effect on Staphylococcus aureus and a more obvious antibacterial effect. These findings provide valuable reference data for the in-depth exploration of Prunus truncatus and its derivatives.

[0173] The present invention studies the antioxidant activity and change law of the thick-stemmed cherry after artificially simulating gastrointestinal digestion, and uses ascorbic acid of the same concentration as a positive control. It is found that the antioxidant components of the thick-stemmed cherry polyphenol extract are unstable in the gastric digestion environment, and are easily degraded or transformed, resulting in weak antioxidant capacity; and in the intestinal digestion process, although the antioxidant capacity fluctuates, it is better than the gastric digestion period as a whole. However, the thick-stemmed cherry polyphenols in different digestion periods have certain antioxidant activity. Among them, the reduction ability determination (1.099±0.030), the hydroxyl radical scavenging ability determination (99.306±0.434%), and the DPPH radical scavenging ability determination (93.115±0.628%) all reach the highest value during the intestinal digestion period. Therefore, the present invention provides a new idea for the development and utilization of thick-stemmed cherry resources in the health care field. By comparing with ascorbic acid, it is found that the thick-stemmed cherry polyphenols still show antioxidant activity after simulating gastrointestinal digestion.

[0174] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for preparing a polyphenol extract of Prunus thunbergii, characterized in that: The steps include: Step (1), preparation of thick-stemmed chrysanthemum fruit powder: select fresh and ripe thick-stemmed chrysanthemum fruits, clean them, remove the cores, dry them at 53-57°C, grind them and pass them through a 60-mesh sieve, take the sieved material, and store it at 4°C to obtain thick-stemmed chrysanthemum fruit powder; Step (2), preparation of a polyphenol extract of Prunus thunbergii: taking Prunus thunbergii fruit powder, using ethanol with a volume concentration of 28%-32%, and ultrasonically extracting at 68-72° C. for 38-42 minutes to obtain a polyphenol extract of Prunus thunbergii; The volume ratio of ethanol to the mass ratio of the Prunus armeniaca fruit powder is 34-36:1 mL / g.

2. The method for preparing the polyphenol extract of Prunus thunbergii according to claim 1, characterized in that: In step (1), the drying temperature is 55°C.

3. The method for preparing the polyphenol extract of Prunus thunbergii according to claim 1, characterized in that: In step (2), ethanol with a volume concentration of 30% is used for ultrasonic extraction at 70° C. for 40 minutes to obtain a polyphenol extract of Prunus thunbergii.

4. The method for preparing the polyphenol extract of Prunus thunbergii according to claim 1, characterized in that: In step (2), the volume ratio of ethanol to the mass ratio of the Prunus armeniaca fruit powder is 35:1 mL / g.

5. The polyphenol extract of Prunus thunbergii obtained by the method for preparing the polyphenol extract of Prunus thunbergii according to any one of claims 1 to 4, characterized in that: The polyphenol content in the polyphenol extract of Prunus thunbergii is 66.398±0.411 mg / g.

6. Use of the polyphenol extract of Prunus thunbergii according to claim 5 in the preparation of drugs for scavenging DPPH free radicals.

7. Use of the polyphenol extract of Prunus thunbergii as claimed in claim 5 in the preparation of drugs for scavenging hydroxyl free radicals.

8. Use of the polyphenol extract of Prunus thunbergii as claimed in claim 5 in the preparation of nitrite-scavenging drugs.

9. Use of the polyphenol extract of Prunus thunbergii according to claim 5 in the preparation of antibacterial drugs.

10. Use of the polyphenol extract of Prunus thunbergii according to claim 5 in the preparation of antioxidant drugs for gastrointestinal use.

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