A method for extracting and purifying Allium mongolicum polyphenols with high biological activity

Abstract

The present invention provides a method for extracting and purifying polyphenols of Allium sibiricum with high biological activity, comprising: pretreatment of Allium sibiricum, defatting and decolorizing of allium sibiricum leaf powder, ultrasound-assisted low-temperature extraction, preparation of AMPL of crude extract of Allium sibiricum polyphenols, solid phase extraction purification, and preparation of purified product of Allium sibiricum polyphenols. The present invention uses Allium sibiricum leaves as the main raw material, and adopts ultrasound-assisted low-temperature extraction of total polyphenols of Allium sibiricum, which significantly improves the extraction rate of Allium sibiricum polyphenols. Compared with the traditional extraction method, the extraction requires less solvent, the extraction time is shorter, the extraction efficiency is higher, and the inactivation of heat-sensitive compounds can be minimized, and the cost can be reduced while energy consumption can be reduced. The crude extract of Allium sibiricum polyphenols is purified by solid phase extraction, and impurities in the crude extract are effectively removed, the purity of active polyphenols is improved, and the effect of separation and purification is achieved. Solid phase extraction can simultaneously complete the enrichment and purification of samples, greatly improve the sensitivity of detection, and has the advantages of high efficiency, reliability, and low reagent consumption.

Description

Technical Field

[0001] The invention relates to the technical field of Chinese and Mongolian medicine extraction, and in particular to a method for extracting and purifying Allium mongolicum polyphenols with high biological activity. Background Art

[0002] Allium mongolicum Regel belongs to the angiosperms, lily family, allium genus, and its scientific name is Mongolian leek (Allium mongolicum Regel). According to the "Flora of Inner Mongolia" and "Compendium of Materia Medica", the above-ground part of Allium mongolicum can be used as medicine. According to the "Mongolian Medical Classic", Allium mongolicum also has the effects of lowering blood lipids, blood sugar and blood pressure, which is very beneficial for preventing the three high problems of modern people. In China, it is widely distributed in the desert areas of northwest my country and Inner Mongolia. It is an edible wild vegetable. This plant is a high-quality forage grass in the Inner Mongolia grassland and a valuable genetic resource to be developed. Studies have shown that as a desert plant rich in nutrients, Allium mongolicum contains higher amino acids and essential trace elements than common vegetables on the grassland. As natural plant active ingredients, Allium mongolicum and its extracts have strong antioxidant, immunomodulatory, antibacterial and anti-inflammatory activities.

[0003] At present, the main extraction methods of polyphenols from Allium sphaerocephalum include ethanol reflux extraction and enzymatic extraction. Ethanol reflux extraction separates polyphenols from plants based on the principle of "like dissolves like". The principle is simple and the operation is convenient. However, since polyphenols are easily oxidized at higher temperatures, and the boiling point of ethanol is 78.2°C at standard atmospheric pressure, in order to achieve a better reflux effect, the reflux temperature is usually set above 80°C, and the reflux time is long, which easily leads to the oxidation of some compounds in polyphenols and affects the biological activity of polyphenols. Moreover, this method is time-consuming and energy-consuming, and is not suitable for large-scale application in industry. Enzymatic extraction has mild reaction conditions, high selectivity, and the product is not easy to inactivate, but it has high requirements for experimental conditions. In order to maximize the effect of the enzyme, it is necessary to first determine the optimal temperature, pH value and most suitable action time of the enzyme through experiments, and the cost is also high. Industrial application has certain difficulties and certain limitations. Summary of the invention

[0004] Based on this, the purpose of the present invention is to provide a method for extracting and purifying allium polyphenols with high biological activity, so as to solve the problems existing in the prior art that polyphenols are easily oxidized at high temperatures, are time-consuming and energy-consuming, are costly, and allium polyphenols and impurities cannot be separated with high selectivity.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides a method for extracting and purifying Allium mongolicum polyphenols with high biological activity, comprising the following steps:

[0006] S1. Pretreatment of Allium mongolicum: clean the Allium mongolicum leaves, cut them into 1-2 cm small segments, place them in a freeze dryer, freeze dry them to absolute dryness, then place them in a grinder and grind them, pass them through an 80-mesh sieve to obtain Allium mongolicum powder;

[0007] S2. Defatting and decolorizing the whole powder of Allium mongolicum leaves: Mix the Allium mongolicum powder obtained in S1 with petroleum ether, fix it on a horizontal shaker for defatting and decolorizing for 24 h, discard the supernatant, dry the petroleum ether, obtain the defatted and decolorized Allium mongolicum powder, and store it for later use;

[0008] S3, ultrasonic-assisted low-temperature extraction: deionized water is added to an ultrasonic device, heated, and the defatted and decolorized Allium mongolicum powder obtained in S2 is mixed evenly with 75% ethanol in a reaction container according to a corresponding solid-liquid ratio, and ultrasonic extraction is performed for 10-30 minutes to obtain a crude extract of Allium mongolicum polyphenols;

[0009] S4, preparation of AMPL of crude extract of Allium mongolicum polyphenols: centrifuge the crude extract of Allium mongolicum polyphenols obtained in S3, take the supernatant, remove the solvent by rotary evaporator, spin dry into powder, obtain AMPL of crude extract of Allium mongolicum polyphenols, and store it in a -20°C refrigerator for later use;

[0010] S5, solid phase extraction purification: the crude extract of Allium mongolicum polyphenols obtained in S4 is mixed with deionized water according to the corresponding solid-liquid ratio to prepare a solution, and the solution is filtered through a 0.22 um filter membrane, and the filtrate is purified by a solid phase extraction column, and eluted with different eluents to obtain different purified product solutions;

[0011] S6. Preparation of purified Allium mongolicum polyphenol products: The different purified product solutions obtained in S5 were respectively subjected to a rotary evaporator to remove the solvent, dried to powder, and stored in a -20°C refrigerator for later use.

[0012] Preferably, the mass ratio of allium mongolicum powder to petroleum ether in S2 is 1:10.

[0013] Preferably, the deionized water in S3 is heated to 20-60°C.

[0014] Preferably, the solid-to-liquid ratio of the defatted and decolorized allium mongolicum powder to 75% ethanol in S3 is 1:10-50.

[0015] Preferably, the centrifuge speed in S4 is 12000 rpm and the time is 15 min.

[0016] Preferably, the solid-to-liquid ratio of the crude Allium mongolicum polyphenol extract to deionized water in S5 is 1:10-50.

[0017] Preferably, the filler of the solid phase extraction column in S5 is octadecyl bonded silica gel.

[0018] Preferably, the eluents in S5 are 0.01M HCl aqueous solution, ethyl acetate, and 0.1% HCl methanol solution, respectively.

[0019] Preferably, the rotary evaporation temperature of the rotary evaporator in S4 and S6 is 40°C.

[0020] The above one or more technical solutions of the present invention have the following technical effects:

[0021] The extraction and purification method of highly biologically active Allium polyphenol provided by the present invention uses Allium polyphenol leaves as the main raw material and adopts ultrasound-assisted low-temperature extraction to extract Allium polyphenol. Compared with ethanol reflux extraction and enzyme extraction, ultrasound-assisted low-temperature extraction utilizes the mechanical fragmentation and cavitation effect of ultrasound to accelerate the diffusion rate of Allium polyphenol into the extraction solvent, which is helpful for the dissolution of Allium polyphenol, thereby shortening the extraction time of Allium polyphenol and improving the extraction efficiency. In addition, ultrasound extraction has a protective effect on Allium polyphenol that is unstable when exposed to heat and is easily hydrolyzed or oxidized. However, ultrasound-assisted low-temperature extraction can only obtain a mixture of allium polyphenols, which cannot achieve the ideal separation effect. Solid phase extraction technology can achieve highly selective separation between target components and impurities by selecting different stationary phases and mobile phases for various combinations, significantly improving the extraction rate of allium polyphenols. Compared with traditional extraction methods, the extraction requires less solvent, the extraction time is shorter, the extraction efficiency is higher, and the inactivation of heat-sensitive compounds can be minimized, reducing costs while also reducing energy consumption. Solid phase extraction can be used to purify the crude extract of allium polyphenols, effectively remove impurities in the crude extract, improve the purity of the polyphenol active group components, achieve the effect of separation and purification, and obtain a more active polyphenol component group. Solid phase extraction can simultaneously complete sample enrichment and purification, greatly improving the sensitivity of detection, and has the advantages of high efficiency, reliability, and low reagent consumption. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for describing the embodiments are briefly introduced below.

[0023] Figure 1 , Flow chart of extraction and purification of Allium mongolicum polyphenols;

[0024] Figure 2 , the total polyphenol content in the crude extract of Allium mongolicum polyphenols and each purified product;

[0025] Figure 3 , standard curve of absorbance value of gallic acid changing with concentration;

[0026] Figure 4 , the total flavonoids content in the crude extract of Allium mongolicum polyphenols and each purified product;

[0027] Figure 5 , standard curve of absorbance value of rutin changing with concentration;

[0028] Figure 6 , the ability of each component of Allium polyphenol to scavenge DPPH free radicals;

[0029] Figure 7 , standard curve of DPPH free radical scavenging rate changing with Trolox concentration;

[0030] Figure 8 , the ability of each component of Allium sphaerocephalum polyphenol to scavenge ABTS free radicals;

[0031] Fig. 9 , standard curve of ABTS free radical scavenging rate changing with Trolox concentration;

[0032] Fig.10 , the scavenging ability of each component of Allium sphaerocephalum polyphenols on oxygen free radicals;

[0033] Fig.11 , standard curve of inhibition area under the curve (AUC) versus Trolox concentration;

[0034] Fig.12 , the ability of each component of Allium sphaerocephalum polyphenols to resist α-amylase;

[0035] Fig.13 , the anti-lipase ability of each component of Allium sphaerocephalum polyphenols;

[0036] Fig.14 , standard curve of lipase clearance rate changing with orlistat (OE) concentration;

[0037] Fig.15 , high performance liquid chromatogram of AMPL-C. DETAILED DESCRIPTION

[0038] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention.

[0039] Throughout the specification, unless otherwise specified, the terms used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence. Figure 1 shown.

[0040] Example 1

[0041] A method for extracting and purifying Allium mongolicum polyphenols with high biological activity comprises the following steps:

[0042] S1. Pretreatment of Allium mongolicum: clean the Allium mongolicum leaves, cut them into 1-2 cm small segments, place them in a freeze dryer, freeze dry them to absolute dryness, then place them in a grinder and grind them, pass them through an 80-mesh sieve to obtain Allium mongolicum powder;

[0043] S2. Defatting and decolorizing the whole powder of Allium mongolicum leaves: Mix the Allium mongolicum powder obtained in S1 with petroleum ether at a mass ratio of 1:10, fix it on a horizontal shaker for defatting and decolorizing for 24 h, discard the supernatant, dry the petroleum ether, obtain the defatted and decolorized Allium mongolicum powder, and store it for later use;

[0044] S3, ultrasonic-assisted low-temperature extraction: deionized water was added to an ultrasonic device and heated to 40°C. The defatted and decolorized Allium mongolicum powder obtained in S2 was mixed evenly with 75% ethanol at a solid-liquid ratio of 1:30 (mg / ml), and ultrasonic extraction was performed for 15 minutes to obtain a crude extract of Allium mongolicum polyphenols.

[0045] S4, preparation of AMPL of Allium mongolicum polyphenol crude extract: centrifuge the AMPL of Allium mongolicum polyphenol crude extract obtained in S3 at a centrifuge speed of 12000 rpm for 15 min, take the supernatant, remove the solvent by rotary evaporator at a rotary evaporation temperature of 40°C, and dry into powder to obtain AMPL of Allium mongolicum polyphenol crude extract, which is stored in a -20°C refrigerator for later use;

[0046] S5, solid phase extraction purification: the crude extract of allium polyphenols obtained in S4, AMPL, was prepared with deionized water at a solid-liquid ratio of 1:20 (mg / ml), and the filtrate was filtered through a 0.22 um filter membrane. The filtrate was purified by a solid phase extraction column filled with octadecyl bonded silica gel (C18), and eluted with 15 mL of 0.01M HCl aqueous solution to obtain a purified product AMPL-A solution, 40 mL of ethyl acetate to obtain a purified product AMPL-B solution, and 30 mL of 0.1% HCl methanol solution to obtain a purified product AMPL-C solution;

[0047] S6. Preparation of purified products of Allium mongolicum polyphenols, AMPL-A, AMPL-B and AMPL-C: The different purified product solutions obtained in S5 were respectively subjected to a rotary evaporator to remove the solvent at a temperature of 40°C. The products were dried into powder and stored in a -20°C refrigerator for later use.

[0048] Example 2

[0049] A method for extracting and purifying Allium mongolicum polyphenols with high biological activity comprises the following steps:

[0050] S1. Pretreatment of Allium mongolicum: clean the Allium mongolicum leaves, cut them into 1-2 cm small segments, place them in a freeze dryer, freeze dry them to absolute dryness, then place them in a grinder and grind them, pass them through an 80-mesh sieve to obtain Allium mongolicum powder;

[0051] S2. Defatting and decolorizing the whole powder of Allium mongolicum leaves: Mix the Allium mongolicum powder obtained in S1 with petroleum ether at a mass ratio of 1:10, fix it on a horizontal shaker for defatting and decolorizing for 24 h, discard the supernatant, dry the petroleum ether, obtain the defatted and decolorized Allium mongolicum powder, and store it for later use;

[0052] S3, ultrasonic-assisted low-temperature extraction: deionized water was added to an ultrasonic device and heated to 20°C. The defatted and decolorized Allium mongolicum powder obtained in S2 was mixed evenly with 75% ethanol at a solid-liquid ratio of 1:10 (mg / ml), and ultrasonic extraction was performed for 15 minutes to obtain a crude extract of Allium mongolicum polyphenols.

[0053] S4, preparation of AMPL of crude extract of Allium mongolicum polyphenols: centrifuge the crude extract of Allium mongolicum polyphenols obtained in S3 at a speed of 12000 rpm for 15 min, take the supernatant, remove the solvent by rotary evaporator at a rotary evaporation temperature of 40°C, and spin dry into powder to obtain the crude extract of Allium mongolicum polyphenols AMPL, which is then stored in a -20°C refrigerator for later use;

[0054] S5, solid phase extraction purification: the crude extract of allium polyphenols obtained in S4, AMPL, was prepared with deionized water at a solid-liquid ratio of 1:10 (mg / ml), and the filtrate was filtered through a 0.22 um filter membrane. The filtrate was purified by a solid phase extraction column filled with octadecyl bonded silica gel (C18), and eluted with 15 mL of 0.01M HCl aqueous solution to obtain a purified product AMPL-A solution, 40 mL of ethyl acetate to obtain a purified product AMPL-B solution, and 30 mL of 0.1% HCl methanol solution to obtain a purified product AMPL-C solution;

[0055] S6. Preparation of purified products of Allium mongolicum polyphenols, AMPL-A, AMPL-B and AMPL-C: The different purified product solutions obtained in S5 were respectively subjected to a rotary evaporator to remove the solvent at a rotary evaporation temperature of 40°C. The products were dried into powder and stored in a -20°C refrigerator for later use.

[0056] Example 3

[0057] A method for extracting and purifying Allium mongolicum polyphenols with high biological activity comprises the following steps:

[0058] S1. Pretreatment of Allium mongolicum: clean the Allium mongolicum leaves, cut them into 1-2 cm small segments, place them in a freeze dryer, freeze dry them to absolute dryness, then place them in a grinder and grind them, pass them through an 80-mesh sieve to obtain Allium mongolicum powder;

[0059] S2. Defatting and decolorizing the whole powder of Allium mongolicum leaves: Mix the Allium mongolicum powder obtained in S1 with petroleum ether at a mass ratio of 1:10, fix it on a horizontal shaker for defatting and decolorizing for 24 h, discard the supernatant, dry the petroleum ether, obtain the defatted and decolorized Allium mongolicum powder, and store it for later use;

[0060] S3, ultrasonic-assisted low-temperature extraction: deionized water was added to an ultrasonic device and heated to 60°C. The defatted and decolorized Allium mongolicum powder obtained in S2 was mixed evenly with 75% ethanol at a solid-liquid ratio of 1:50 (mg / ml), and ultrasonic extraction was performed for 30 minutes to obtain a crude extract of Allium mongolicum polyphenols.

[0061] S4, preparation of AMPL of crude extract of Allium mongolicum polyphenols: centrifuge the crude extract of Allium mongolicum polyphenols obtained in S3 at a speed of 12000 rpm for 15 min, take the supernatant, remove the solvent by rotary evaporator at a rotary evaporation temperature of 40°C, and spin dry into powder to obtain the crude extract of Allium mongolicum polyphenols AMPL, which is then stored in a -20°C refrigerator for later use;

[0062] S5, solid phase extraction purification: the crude extract of allium polyphenols obtained in S4, AMPL, was prepared with deionized water at a solid-liquid ratio of 1:50 (mg / ml), and the filtrate was filtered through a 0.22 um filter membrane. The filtrate was purified by a solid phase extraction column filled with octadecyl bonded silica gel (C18), and eluted with 15 mL of 0.01M HCl aqueous solution to obtain a purified product AMPL-A solution, 40 mL of ethyl acetate to obtain a purified product AMPL-B solution, and 30 mL of 0.1% HCl methanol solution to obtain a purified product AMPL-C solution;

[0063] S6. Preparation of purified products of Allium mongolicum polyphenols, AMPL-A, AMPL-B and AMPL-C: The different purified product solutions obtained in S5 were respectively subjected to a rotary evaporator to remove the solvent at a rotary evaporation temperature of 40°C. The products were dried into powder and stored in a -20°C refrigerator for later use.

[0064] Example 4

[0065] A method for extracting and purifying Allium mongolicum polyphenols with high biological activity comprises the following steps:

[0066] S1. Pretreatment of Allium mongolicum: clean the Allium mongolicum leaves, cut them into 1-2 cm small segments, place them in a freeze dryer, freeze dry them to absolute dryness, then place them in a grinder and grind them, pass them through an 80-mesh sieve to obtain Allium mongolicum powder;

[0067] S2. Defatting and decolorizing the whole powder of Allium mongolicum leaves: Mix the Allium mongolicum powder obtained in S1 with petroleum ether at a mass ratio of 1:10, fix it on a horizontal shaker for defatting and decolorizing for 24 h, discard the supernatant, dry the petroleum ether, obtain the defatted and decolorized Allium mongolicum powder, and store it for later use;

[0068] S3, ultrasonic-assisted low-temperature extraction: deionized water was added to an ultrasonic device and heated to 60°C. The defatted and decolorized Allium mongolicum powder obtained in S2 was mixed evenly with 75% ethanol at a solid-liquid ratio of 1:10 (mg / ml), and ultrasonic extraction was performed for 10 minutes to obtain a crude extract of Allium mongolicum polyphenols.

[0069] S4, preparation of AMPL of crude extract of Allium mongolicum polyphenols: centrifuge the crude extract of Allium mongolicum polyphenols obtained in S3 at a speed of 12000 rpm for 15 min, take the supernatant, remove the solvent by rotary evaporator at a rotary evaporation temperature of 40°C, and spin dry into powder to obtain the crude extract of Allium mongolicum polyphenols AMPL, which is then stored in a -20°C refrigerator for later use;

[0070] S5, solid phase extraction purification: the crude extract of allium polyphenols obtained in S4, AMPL, was prepared with deionized water at a solid-liquid ratio of 1:10 (mg / ml), and the filtrate was filtered through a 0.22 um filter membrane. The filtrate was purified by a solid phase extraction column filled with octadecyl bonded silica gel (C18), and eluted with 15 mL of 0.01M HCl aqueous solution to obtain a purified product AMPL-A solution, 40 mL of ethyl acetate to obtain a purified product AMPL-B solution, and 30 mL of 0.1% HCl methanol solution to obtain a purified product AMPL-C solution;

[0071] S6. Preparation of purified products of Allium mongolicum polyphenols, AMPL-A, AMPL-B and AMPL-C: The different purified product solutions obtained in S5 were respectively subjected to a rotary evaporator to remove the solvent at a temperature of 40°C. The products were dried into powder and stored in a -20°C refrigerator for later use.

[0072] Comparative Example 1

[0073] A method for extracting and purifying Allium mongolicum polyphenols with high biological activity comprises the following steps:

[0074] S1. Pretreatment of Allium mongolicum: clean the Allium mongolicum leaves, cut them into 1-2 cm small segments, place them in a freeze dryer, freeze dry them to absolute dryness, then place them in a grinder and grind them, pass them through an 80-mesh sieve to obtain Allium mongolicum powder;

[0075] S2. Defatting and decolorizing the whole powder of Allium mongolicum leaves: Mix the Allium mongolicum powder obtained in S1 with petroleum ether at a mass ratio of 1:10, fix it on a horizontal shaker for defatting and decolorizing for 24 h, discard the supernatant, dry the petroleum ether, obtain the defatted and decolorized Allium mongolicum powder, and store it for later use;

[0076] S3, ultrasonic-assisted low-temperature extraction: deionized water was added to an ultrasonic device and heated to 40°C. The defatted and decolorized Allium mongolicum powder obtained in S2 was mixed evenly with 75% ethanol at a solid-liquid ratio of 1:30 (mg / ml), and ultrasonic extraction was performed for 15 minutes to obtain a crude extract of Allium mongolicum polyphenols.

[0077] S4, preparation of AMPL of crude extract of Allium mongolicum polyphenols: centrifuge the crude extract of Allium mongolicum polyphenols obtained in S3 at a speed of 12000 rpm for 15 min, take the supernatant, remove the solvent by rotary evaporator at a rotary evaporation temperature of 40°C, and spin dry into powder to obtain the crude extract of Allium mongolicum polyphenols AMPL, which is then stored in a -20°C refrigerator for later use;

[0078] In order to verify the extraction and purification effect, the highly biologically active Allium polyphenol extracted and purified in Example 1 and Comparative Example 1 were selected for testing, and the biological activities of the crude extract of Allium polyphenol and the three purified products eluted with different eluents were evaluated in multiple dimensions using antioxidant capacity, anti-α-amylase capacity and anti-porcine pancreatic lipase capacity as indicators. The specific test results are shown in Test Examples 1 to 8.

[0079] Test Example 1

[0080] Determination of total polyphenol content of Allium mongolicum: The total polyphenol content of crude extract of Allium mongolicum polyphenol (AMPL) and purified fractions (purified products AMPL-A, AMPL-B, and AMPL-C) was determined by the Folin phenol reagent method. 20 μL of crude extract of Allium mongolicum polyphenol and purified fractions and 100 μL of 10% Folin phenol reagent were injected into a 96-well plate and incubated at 25°C for 8 min. 80 μL of 7.5% sodium carbonate solution was added to the 96-well plate and incubated at room temperature in the dark for 2 h. The absorbance at 765 nm was measured by an ELISA reader, and the test was repeated three times. A standard curve was prepared with a gallic acid concentration range (0.039-0.3125 mg / mL), and the polyphenol content of the Allium mongolicum extract sample was calculated according to the linear fitting equation obtained from the standard curve. The results were expressed as the mass of gallic acid per gram of dry mass sample (mg GAE / g DW).

[0081] Figure 2 It is the total polyphenol content in the crude extract of Allium mongolicum polyphenol and each purified product, and is used to compare the total polyphenol content in each component of Allium mongolicum polyphenol. Figure 3The standard curve of the absorbance value of gallic acid changing with the concentration was used to calculate the total polyphenol content of each component of Allium polyphenol. It can be seen that the total polyphenol content of AMPL (1811±39 mg GAE / gDW) was significantly (p<0.05) higher than that of other Allium polyphenol components after purification by solid phase extraction column filled with octadecyl bonded silica gel (C18), and the total polyphenol content of the purified component AMPL-B (5749±36 mg GAE / g DW) was 3.2 times higher than that of AMPL. The total polyphenol content of AMPL-A (1697±30 mg GAE / g DW) and the total polyphenol content of AMPL-C (1284±72.50 mg GAE / g DW) were both lower than that of the crude extract of Allium polyphenol AMPL. Therefore, the purification effect of crude Allium polyphenol by solid phase extraction column filled with octadecyl bonded silica gel (C18) and AMPL-B elution conditions was good.

[0082] Test Example 2

[0083] Determination of the total flavonoid content of Allium mongolicum: The present invention adopts the NaNO3-Al(NO3)3-NaOH method to determine the total flavonoid content of the crude extract of Allium mongolicum (AMPL) and each purified component (purified products AMPL-A, AMPL-B, AMPL-C). 30 µL of crude extract of Allium mongolicum polyphenols and each purified component, 15 µL of 5% NaNO2 solution, 15 µL of 10% Al(NO3)3 solution and 150 µL of 4% NaOH solution were added to a 96-well plate and incubated for 15 min. The absorbance value at 510 nm was determined by an ELISA instrument, and the test was repeated three times. A standard curve was prepared with a rutin concentration range (1.936-7.437 mg / mL), and the total flavonoid content in each extract of Allium mongolicum was calculated according to the linear fitting equation obtained from the standard curve. The result was expressed as the mass of rutin per gram of dry mass sample (mg RE / g DW).

[0084] Figure 4 It is the total flavonoid content in the crude extract of Allium mongolicum polyphenol and each purified product, which is used to compare the total flavonoid content in each component of Allium mongolicum polyphenol. Figure 5The standard curve of the absorbance value of rutin changes with the concentration is shown in Figure 2. The total flavonoid content of each component of Allium polyphenol is calculated by fitting the standard curve. It can be seen that the total flavonoid content of AMPL (34.41±0.59 mg RE / g DW) is significantly higher (p<0.05) than that of AMPL in the purified components AMPL-B (64.50±3.50 mgRE / g DW) and AMPL-C (79.61±1.12 mg RE / g DW) after purification by solid phase extraction column filled with octadecyl bonded silica gel (C18), which is 1.9 times and 2.3 times higher, respectively. The total polyphenol content of AMPL-A (13.74±0.26 mg RE / g DW) is the lowest. Therefore, the total flavonoid content of crude Allium polyphenol purified by solid phase extraction column filled with octadecyl bonded silica gel (C18) under the elution conditions of AMPL-B and AMPL-C is higher, and the purification effect is better.

[0085] Test Example 3

[0086] Test of DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) free radical scavenging rate: prepare 0.4 mM DPPH methanol solution, then dilute it with methanol to an absorbance of 0.700±0.020 at 515 nm to obtain the DPPH working solution required for the test. Add 50 μL of the methanol solution of the sample to be tested to a 96-well ELISA plate, then add 100 μL of the newly prepared DPPH working solution, shake and mix well, and react in the dark for 30 min. Measure the absorbance of the mixed solution at 515 nm, and test three times in parallel. A standard curve was prepared with a vitamin E (Trolox) concentration range (0.16-0.20 mM), and the total polyphenol content of each component of Allium polyphenol was calculated according to the linear fitting equation obtained from the standard curve. The results were expressed as Trolox equivalents per gram of dry mass sample (μmol TE / gDW). The DPPH free radical scavenging rate of the Allium polyphenol crude extract and each purified product was calculated as follows:

[0087]

[0088] In the formula, A s represents the absorbance value after the mixed reaction of the Allium mongolicum sample solution and the DPPH working solution. A 0 represents the absorbance value of methanol solution of Allium mongolicum sample, A b Indicates the absorbance value of DPPH working solution.

[0089] Figure 6 The ability of each component of Allium polyphenol to scavenge DPPH free radicals is used to compare the antioxidant capacity of each component of Allium polyphenol. Figure 7The standard curve of DPPH free radical scavenging rate changing with Trolox concentration was used to calculate the total polyphenol content of each component of Allium polyphenol. Then, the antioxidant ability of each component of Allium polyphenol in scavenging DPPH free radical was compared. It can be seen that under DPPH conditions, AMPL-C has the strongest antioxidant ability, with a DPPH value of 421.50±4.22 μmol TE / g DW; the DPPH value of AMPL-B is 323.40±15.98 μmol TE / g DW, both of which are 2.3-3.0 times higher than AMPL (139.90±5.64 μmolTE / g DW). AMPL-A has the lowest antioxidant ability. The scavenging rates of DPPH free radicals of each component of Allium polyphenol before and after purification were significantly different (p<0.05), indicating that purification effectively removed impurities without antioxidant activity and improved the antioxidant activity of Allium polyphenol.

[0090] Test Example 4

[0091] ABTS (2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt) free radical scavenging rate test: First, prepare ABTS at a concentration of 7 mM +· Aqueous solution (solvent is 2.45 mM K2S2O8 aqueous solution), that is, ABTS and K2S2O8 aqueous solution are reacted in the dark for 12 h to obtain ABTS +· Then dilute ABTS with deionized water +· The absorbance of the mother solution at 734 nm was 0.700 ± 0.020, and ABTS was obtained. +· The working solution was kept away from light for future use. 40 uL of the sample solution to be tested was respectively pipetted into a 96-well ELISA plate, and 160 uL of ABTS was added. +· The working solution was shaken and mixed, and then reacted in the dark for 6 min. The absorbance of the reaction solution was measured at 734 nm, and the test was repeated three times. A standard curve was prepared with a Trolox concentration range (0.05-0.3 mM). The total polyphenol content of each component sample of Allium polyphenol was calculated according to the linear fitting equation obtained from the standard curve. The results were expressed as Trolox equivalents per gram of dry mass sample (μmol TE / g DW). The scavenging rate of ABTS free radicals by the crude extract of Allium polyphenol and each purified product was calculated as follows:

[0092]

[0093] Where As represents the Allium mongolicum sample solution and ABTS +· The absorbance value of the working solution, A0 represents the absorbance value of the Allium mongolicum sample solution, A b Indicates ABTS +· The absorbance value of the working solution.

[0094] Figure 8 The ability of each component of Allium polyphenol to scavenge ABTS free radicals is used to compare the antioxidant capacity of each component of Allium polyphenol. Fig. 9 The standard curve of ABTS free radical scavenging rate changing with Trolox concentration was fitted. The total polyphenol content of each component of Allium polyphenol was calculated by fitting the standard curve, and then the antioxidant ability of each component of Allium polyphenol in scavenging ABTS free radicals was compared. Under ABTS conditions, AMPL-C had the strongest antioxidant ability, with an ABTS value of 688.80±414.08 μmol TE / gDW. The ABTS value of AMPL-B was 321.30±5.60 μmol TE / g DW, both of which were 1.7-3.7 times higher than AMPL (186.90±2.54μmol TE / gDW). AMPL-A had the lowest antioxidant ability, which was consistent with the ability of each component of Allium polyphenol to scavenge DPPH free radicals. Therefore, the scavenging rates of each component of Allium polyphenol on ABTS free radicals before and after purification were significantly different (p<0.05), and the purified Allium polyphenol can show higher antioxidant activity.

[0095] Test Example 5

[0096] Oxygen radical absorbance capacity (ORAC) assay: Allium polyphenol samples, Trolox, and sodium fluorescein were prepared with PBS buffer (75 mM, pH = 7.4) to the required concentrations. 20 μL of allium polyphenol solutions (0.0014-0.0117 mg / mL) and Trolox standard solutions (4.79-200 μM), and 200 μL of sodium fluorescein solution (81.6 nM) were added to a 96-well plate in sequence. The fluorescein solution was used as a blank control. The program of the fluorescence / chemical analyzer was set, incubated at 37°C for 20 min, automatically mixed, and 20 μL of AAPH (azobisisobutylamidine hydrochloride, 153 mM) was quickly added. In addition, the sample without AAPH and the sodium fluorescein solution were used as control controls, automatically mixed, and the assay was started. The excitation wavelength was 485 nm, the fluorescence emission wavelength was 538 nm, and the assay was performed every 1 min, for a total of 120 assays. The difference in fluorescence quenching area between the sample group and the blank group was expressed as Trolox equivalent (TE), with the unit of μmol TE / g DW, and the fluorescence intensities were recorded as f1, f2, f3, ..., f 120 , and the test was repeated three times in parallel. The area under fluorescence quenching was calculated according to the formula:

[0097]

[0098] Fig.10 In order to investigate the scavenging ability of various components of Allium polyphenols on oxygen free radicals, the ORAC value was used to compare the antioxidant capacity of various components of Allium polyphenols. Fig.11The standard curve of the inhibition area under the curve (AUC) changing with the Trolox concentration was fitted to calculate the total polyphenol content of each component of Allium polyphenols, and then the antioxidant capacity of each component of Allium polyphenols in scavenging oxygen free radicals was compared, expressed as ORAC value. AMPL-C has the strongest scavenging capacity for oxygen free radicals, with an ORAC value of 47.76±3.07mmol TE / g DW. The ORAC value of AMPL-B was 35.52±1.54 mmol TE / g DW, which was 2.5-3.4 times higher than that of AMPL (14.08±0.11mmol TE / g DW). AMPL-A had the weakest antioxidant capacity, which was consistent with the ability of various components of Allium mongolicum to scavenge DPPH and ABTS radicals. Therefore, the three antioxidant evaluation methods showed that there were significant differences in antioxidant activity among the various components of Allium mongolicum polyphenols purified by solid phase extraction column filled with octadecyl bonded silica gel (C18) (p<0.05), and the Allium mongolicum polyphenol AMPL-C had the highest antioxidant activity.

[0099] Test Example 6

[0100] α-Amylase bioactivity inhibition test: α-Amylase inhibition activity was determined by high-throughput turbidity assay. The Allium sphaerocarpon polyphenol sample was dissolved in 0.01 M PBS buffer (0.1M, pH=6.9), and acarbose was used as a positive control. In a 96-well plate, a mixture containing 20 μL of α-amylase solution (2 U / mL) and 20 μL of sample solution was incubated at 37°C for 10 min. Then, 60 μL of gelatinized starch solution (20 U / mL) was added to each test tube and read at 660 nm for 2 h using a microplate reader, with three parallel tests. The percentage inhibition rate (%) was calculated using the following formula:

[0101]

[0102] Where AUC sample is the area under the inhibition curve of Allium sphaerocephalum polyphenol sample, AUC control is the area under the curve of the control (PBS buffer).

[0103] Determination of IC from a plot of percent inhibition versus inhibitor concentration 50 The value (the concentration required for α-amylase activity when the inhibition rate is 50%) was calculated by nonlinear regression analysis based on the average inhibition value. Acarbose equivalent (AE) can be calculated using the following formula:

[0104]

[0105] The test results are shown in Table 1:

[0106] Table 1 The inhibition concentration and equivalent value of each component of Allium mongolicum polyphenols when the anti-α-amylase activity reaches 50%

[0107]

[0108] Fig.12 The α-amylase resistance of each component of Allium polyphenol is used to compare the strength of the α-amylase resistance of each component of Allium polyphenol. Fig.12 It can be seen that, except for the crude extract AMPL, the other purified components all showed inhibitory activity against α-amylase, and there was a significant difference (p<0.05). The AMPL-C component had the highest inhibitory activity against α-amylase, with an AE value of 281.10±28.01 μmol AE / g, which was 27.5 times that of AMPL-A (AE = 10.24±0.69 μmol AE / g) and 10.6 times that of AMPL-B (AE = 26.50±0.64 μmol AE / g). Therefore, the method of the present invention achieves the removal of impurity components with no antioxidant activity and no anti-α-amylase activity in the crude polyphenols of Allium sphaerocephalum, and improves the antioxidant and hypoglycemic activities of the polyphenols of Allium sphaerocephalum.

[0109] Test Example 7

[0110] Lipase (PL) bioactivity inhibition rate determination: For the selection of lipase, 4-nitrophenylbutyrate (pNPB) and sample concentration, relevant information was first collected from existing literature. A series of preliminary experiments were then carried out to determine the appropriate concentration range for use. Allium polyphenol samples, Trolox and sodium fluorescein were prepared with PBS buffer (100 mM, pH=7.2) to the required concentrations. 50 μL of sample solution and 25 μL of 5 mM p-nitrophenolate solution were added to a 96-well plate and incubated at 37°C for 5 min; then 20 uL of 50 mg / mL lipase solution and 55 μL PBS buffer were added to the 96-well plate and incubated at 37°C for 30 min. The absorbance value at 405 nm was measured using an ELISA reader. A standard curve was prepared with the orlistat (OE) concentration range (1.5-20 mg / mL), and the total polyphenol content of the Allium polyphenol extract sample was calculated based on the linear fitting equation obtained from the standard curve, expressed as micromolar OE equivalents (μmol OE / g DW). The inhibition rate of lipase from Allium mongolicum polyphenol extracts and purified products at different concentrations was calculated as follows:

[0111]

[0112] Among them, A s is the absorbance value of the sample group after the mixed reaction of Allium polyphenol components, PL and pNPB, A bis the absorbance value of the blank sample in the presence of allium polyphenol components and pNPB but without PL, A t is the absorbance value of the control group in the presence of PL and pNPB but without the polyphenol components of Allium mongolicum, and Ac is the absorbance value of the pNPB blank.

[0113] Fig.13 It is the anti-lipase ability of each component of Allium polyphenol, which is used to compare the anti-lipase ability of each component of Allium polyphenol. Fig.14 The standard curve of lipase clearance rate changing with orlistat (OE) concentration was fitted. The total polyphenol content of each component of Allium sphaerocephala polyphenol was calculated by fitting the standard curve, and then the lipase clearance ability of each component of Allium sphaerocephala polyphenol was compared. It can be seen that the crude extract of Allium sphaerocephala polyphenol and each purified component have certain lipase inhibitory activity. Among them, AMPL-C has the strongest inhibitory activity on lipase, with a PL value of 2860±0.68 μmol OE / g DW, which is more than 3 times that of AMPL. The PL value of AMPL-B is 2398±2.00 μmol OE / g DW, and the inhibitory effect of AMPL on lipase ranks third, with a PL value of 942.20±0.36 μmol OE / gDW. AMPL-A has the weakest inhibitory effect on lipase, with a PL value of 163.90±0.06 μmol OE / g DW. Therefore, there were significant differences (p<0.05) in the components of Allium mongolicum polyphenols purified by solid phase extraction column filled with octadecyl bonded silica gel (C18), and AMPL-C and AMPL-B had significantly improved anti-lipase activity.

[0114] Test Example 8

[0115] AMPL-C was subjected to LC-MS test to analyze the main components of active polyphenols and flavonoids contained in AMPL-C.

[0116] The test conditions are:

[0117] (1) A liquid chromatography column (150 mm × 46 mm × 5 μm, Phenomenex, CA, USA) filled with octadecyl bonded silica (C18) was used for HPLC-ESI-TOF-MS. 2 Chromatographic analysis was performed on an electrospray ionization (ESI) interface on an AB SCIEX 5600+, AB sciex, Foster city, CA for separation. The column temperature, sample injection volume, flow rate, and wavelength were adjusted to 25 °C, 10 μL, 1.0 mL / min, and 280 nm, respectively. MS / MS detection was performed in negative ion detection mode. The scan ranges used in the primary and secondary MS were 100–1500 m / z and 50–1500 m / z, respectively, and the collision gas was N2.

[0118] (2) The mass parameters were set as follows: ion spray voltage: −4500 V; source gas, 55 psi; curtain gas: 35 psi; declustering potential: −100 V; capillary temperature: 500 °C; collision energy: −10 V (6 primary MS) and −40 V (secondary MS); collision energy extension: 20 V (secondary MS).

[0119] (3) The mobile phase consists of 20% acetonitrile aqueous solution, 50 mM ammonium acetate aqueous solution and 200 mM acetic acid aqueous solution. The elution program is shown in Table 2:

[0120] Table 2 Chromatographic conditions of AMPL-C

[0121]

[0122] like Fig.15 As shown in the figure, 19 major polyphenols were identified from AMPL-C, of ​​which 9 were flavonoids, accounting for nearly 50%, namely, peak 2 piceatannol-3'-O-glucoside, peak 3 dehydrocorin, peak 5 mangiferin, peak 8 isoquercetin, peak 10 isorhamnetin-3-glucuronide, peak 13 irisin, peak 15 glycitin, peak 16 non-hydroxyflavonoids and peak 18 gallic quercetin. Therefore, flavonoids are an important component of the Allium polyphenol AMPL-C, and play an important role in the biological activities of anti-oxidation, hypoglycemic and antihypertensive. The specific results are shown in Table 3.

[0123] Table 3 HPLC-ESI-QTOF-MS 2 Characterization of Allium mongolicum polyphenol AMPL-C

[0124]

[0125]

[0126]

Claims

1. A method for extracting and purifying Allium mongolicum polyphenols with high biological activity, characterized in that: The steps include: S1. Pretreatment of Allium mongolicum: clean the Allium mongolicum leaves, cut them into 1-2 cm small segments, place them in a freeze dryer, freeze dry them to absolute dryness, then place them in a grinder and grind them, pass them through an 80-mesh sieve to obtain Allium mongolicum powder; S2. Defatting and decolorizing the whole powder of Allium mongolicum leaves: Mix the Allium mongolicum powder obtained in S1 with petroleum ether, fix it on a horizontal shaker for defatting and decolorizing for 24 h, discard the supernatant, dry the petroleum ether, obtain the defatted and decolorized Allium mongolicum powder, and store it for later use; S3: Ultrasonic-assisted low-temperature extraction: Deionized water is added to an ultrasonic device, heated, and the defatted and decolorized Allium mongolicum powder obtained in S2 is mixed evenly with 75% ethanol in a reaction container according to a corresponding solid-liquid ratio, and ultrasonic extraction is performed for 10-30 minutes to obtain a crude extract of Allium mongolicum polyphenols; S4, preparation of AMPL of crude extract of Allium mongolicum polyphenols: centrifuge the crude extract of Allium mongolicum polyphenols obtained in S3, take the supernatant, remove the solvent by rotary evaporator, spin dry into powder, obtain AMPL of crude extract of Allium mongolicum polyphenols, and store it in a -20°C refrigerator for later use; S5, solid phase extraction purification: the crude extract of Allium mongolicum polyphenols obtained in S4 was mixed with deionized water according to the corresponding solid-liquid ratio to prepare a solution, and the solution was filtered through a 0.22 um filter membrane. The filtrate was purified by a solid phase extraction column filled with octadecyl bonded silica gel, and eluted with 0.01M HCl aqueous solution to obtain a purified product AMPL-A solution, eluted with ethyl acetate to obtain a purified product AMPL-B solution, and eluted with 0.1% HCl methanol solution to obtain a purified product AMPL-C solution; The purified product AMPL-C solution contains 4,4'-O-dimethylellagic acid, piceatannol-3'-O-glucoside, dehydrocorin, protocatechuic acid, mangiferin, protocatechuic aldehyde, vanillic acid, isoquercetin, 3,4,5-trihydroxycinnamic acid, isorhamnetin-3-glucuronide, 4-hydroxycoumarin, 5-hydroxyferulic acid, irisin, 4-hydroxy-3-methylcinnamaldehyde, glycitin, non-hydroxyflavonoids, 4-hydroxy-6-methylcoumarin, gallic quercetin, and 3-methylellagic acid; S6. Preparation of purified Allium mongolicum polyphenol products: The different purified product solutions obtained in S5 were respectively subjected to a rotary evaporator to remove the solvent, dried to powder, and stored in a -20°C refrigerator for later use.

2. The method for extracting and purifying Allium mongolicum polyphenols with high biological activity according to claim 1, characterized in that: The mass ratio of allium mongolicum powder to petroleum ether in S2 is 1:

10.

3. The method for extracting and purifying Allium mongolicum polyphenols with high biological activity according to claim 1, characterized in that: The deionized water in S3 is heated to 20-60°C.

4. The method for extracting and purifying Allium mongolicum polyphenols with high biological activity according to claim 1, characterized in that: The solid-to-liquid ratio of the defatted and decolorized Allium mongolicum powder to 75% ethanol in S3 is 1:10-50.

5. The method for extracting and purifying Allium mongolicum polyphenols with high biological activity according to claim 1, characterized in that: The centrifuge speed in S4 is 12000 rpm and the time is 15 min.

6. The method for extracting and purifying Allium mongolicum polyphenols with high biological activity according to claim 1, characterized in that: The solid-to-liquid ratio of the crude extract of Allium mongolicum polyphenols and deionized water in S5 is 1:10-50.

7. The method for extracting and purifying Allium mongolicum polyphenols with high biological activity according to claim 1, characterized in that: The rotary evaporation temperature of the rotary evaporator in S4 and S6 is 40°C.

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