Method for extracting paulownia flower polyphenol for inhibiting activity of alpha-glucosidase

Abstract

The invention discloses an extraction method of paulownia flower polyphenol capable of inhibiting alpha-glucosidase activity, and relates to the technical field of paulownia flower polyphenol extraction, paulownia flowers are crushed and sieved, then a urea solution of choline chloride is added into powder according to the material-liquid ratio of 0.1 g: 10 ml, the water content is 30%, the ultrasonic time is 5-45 min, the ultrasonic power is 200-400 W, the ultrasonic temperature is 20-60 DEG C, centrifugation and suction filtration are performed, and the paulownia flower polyphenol is obtained. And carrying out reduced pressure distillation, drying, column chromatography separation, eluent collection, concentration and drying to obtain the paulownia flower polyphenol extract. According to the polyphenol extraction method disclosed by the invention, the ultrasonic-assisted deep eutectic solvent is used for performing deep eutectic solvent extraction on the paulownia flower, the extraction filtration is combined for impurity removal, the macroporous resin adsorption purification is performed, and a series of technical means are adopted, so that the prepared polyphenol is relatively high in purity and yield, and the alpha-glucosidase activity inhibition capability is strong.

Description

Technical Field

[0001] The invention relates to the technical field of paulownia flower polyphenol extraction, and more specifically to a method for extracting paulownia flower polyphenol capable of inhibiting alpha-glucosidase activity. Background Art

[0002] Diabetes is a chronic metabolic disease that is one of the top ten causes of death worldwide. It is characterized by persistent hyperglycemia caused by the interaction of environmental and genetic factors, and is prone to cause multiple organ damage and complications. α-amylase and α-glucosidase are key enzymes that affect the digestion and absorption of major carbohydrates such as starch in the diet. Inhibiting their activity can delay the body's degradation of starch and other substances and the absorption of glucose, and to a certain extent reduce postprandial blood sugar levels.

[0003] At present, α-glucosidase inhibitors are mainly obtained through microbial fermentation or artificial synthesis, such as acarbose and miglitol, which have been used as clinical drugs for diabetic patients. However, these drugs have obvious side effects such as flatulence, diarrhea, and abdominal discomfort. It is of great significance to develop safe and effective natural enzyme inhibitors from natural plant resources. Polyphenols are secondary metabolites widely present in higher plants and have a wide range of biological activities.

[0004] Paulownia fortunei is a plant of the Paulownia genus of the Paulownia family. It is native to China and is now distributed all over the world. Paulownia flowers are rich in phytochemical active substances. In my country, Paulownia has been used as a traditional herbal medicine for the treatment of inflammatory bronchitis, tonsillitis, gonorrhea, traumatic bleeding, asthma and hypertension. Studies have found that Paulownia contains a variety of phenolic substances such as luteolin, ellagic acid, apigenin, apigenin-7-O-β-D-glucoside, quercetin, kaempferol and β-sitosterol, which have anti-inflammatory, antibacterial and antioxidant effects. However, the application research of Paulownia flower polyphenols in enzyme inhibitors is not in-depth enough. In view of this, the study and design of a Paulownia flower polyphenol extract as an α-glucosidase inhibitor can provide technical and theoretical basis for the in-depth development and utilization of Paulownia flower polyphenol functional products and related drugs, and broaden the high-value utilization of Paulownia resources. Summary of the invention

[0005] In view of this, the present invention provides a method for extracting Paulownia flower polyphenols which can inhibit the activity of α-glucosidase.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A method for extracting Paulownia flower polyphenols that inhibits α-glucosidase activity comprises the following steps:

[0008] (1) crushing raw materials: crushing and sieving paulownia flowers to obtain paulownia flower powder;

[0009] (2) Extraction: adding 1:2 (mol:mol) choline chloride urea solution to the powder at a solid-liquid ratio of 0.1 g:10 ml, with a water content of 30%, ultrasonic time of 5-45 min, ultrasonic power of 200-400 W, and ultrasonic temperature of 20-60° C. to obtain an extract;

[0010] (3) Drying: centrifugation, filtration, vacuum distillation, and drying to obtain a crude extract of Paulownia flower polyphenols;

[0011] (4) Purification: The crude extract of step (3) is separated by column chromatography, and the eluate is collected, concentrated, and dried to obtain the Paulownia flower polyphenol extract.

[0012] Preferably, in step (1), the crushing time is 2 minutes and the sieving is through a 60-mesh sieve.

[0013] Preferably, in step (3), the centrifugation is performed at 3000-6000 rpm for 10-30 min.

[0014] Preferably, in step (3), the suction filtration condition is 0.02Mpa; the reduced pressure distillation condition is 45°C; the drying is low-temperature drying, and the conditions are: sample temperature -20°C, cold trap temperature -50°C, and vacuum degree 3.2Pa.

[0015] Preferably, in step (4), the concentration is carried out at 35-50° C. for 1 h.

[0016] Preferably, in step (4), the column chromatography separation conditions are: HPD-722 macroporous resin adsorption, 70% ethanol elution for 560 min.

[0017] It can be known from the above technical scheme that the present invention discloses a method for extracting polyphenols from paulownia flowers, which relates to the technical field of plant polyphenol extraction. The extraction method comprises four steps: raw material crushing, extraction, purification, concentration and drying. The polyphenol extraction method of the present invention uses a series of technical means such as ultrasonic-assisted deep eutectic solvent extraction of paulownia flowers, combined filtration for impurity removal, and macroporous resin adsorption purification, so that the obtained polyphenols have high purity and yield, and the operation method is simple, the equipment requirements are low, the extraction cost is low, and it is suitable for large-scale production. The obtained polyphenols can significantly inhibit the activity of α-glucosidase, and its IC for inhibiting α-glucosidase is 50 The value is 219.86μg / mL. DETAILED DESCRIPTION

[0018] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0019] Example 1

[0020] A method for extracting Paulownia flower polyphenols that inhibits α-glucosidase activity comprises the following steps:

[0021] (1) Raw material grinding: Grind the paulownia flower for 2 minutes and pass through a 60-mesh sieve to obtain paulownia flower powder;

[0022] (2) Extraction: Add choline chloride urea solution to the powder at a solid-liquid ratio of 0.1 g:10 ml, with a water content of 30%, ultrasonic time of 15 min, ultrasonic power of 240 W, and ultrasonic temperature of 40°C to obtain an extract with a calculated yield of 1349.23 μg / g;

[0023] (3) Drying: centrifugation at 4000 rpm for 20 min; filtration at 0.02 MPa, vacuum distillation at 45°C, low-temperature drying at -20°C, cold trap temperature at -50°C, vacuum degree at 3.2 Pa, to obtain a crude extract of Paulownia flower polyphenols;

[0024] (4) Purification: The crude extract of step (3) is separated by column chromatography, adsorbed by HPD-722 macroporous resin, eluted with 70% ethanol, the eluate is collected, concentrated at 45° C., and dried to obtain the Paulownia flower polyphenol extract.

[0025] Example 2

[0026] A method for extracting Paulownia flower polyphenols that inhibits α-glucosidase activity comprises the following steps:

[0027] (1) Raw material grinding: Grind the paulownia flower for 2 minutes and pass through a 60-mesh sieve to obtain paulownia flower powder;

[0028] (2) Extraction: add choline chloride urea solution to the powder at a solid-liquid ratio of 0.1 g:10 mL, with a water content of 20%, ultrasonic time of 15 min, ultrasonic power of 240 W, and ultrasonic temperature of 40°C to obtain an extract with a calculated yield of 976.28 μg / g;

[0029] (3) Drying: centrifugation at 3000 rpm for 30 min, filtration at 0.02 MPa, vacuum distillation at 45° C., low-temperature drying at -20° C., cold trap temperature at -50° C., vacuum degree at 3.2 Pa, to obtain a crude extract of paulownia flower polyphenols;

[0030] (4) Purification: The crude extract of step (3) is separated by column chromatography, adsorbed by HPD-722 macroporous resin, eluted with 70% ethanol, the eluate is collected, concentrated at 45° C., and dried to obtain the Paulownia flower polyphenol extract.

[0031] Example 3

[0032] A method for extracting Paulownia flower polyphenols that inhibits α-glucosidase activity comprises the following steps:

[0033] (1) Raw material grinding: grinding the paulownia flower for 2 minutes and passing through a 60-mesh sieve to obtain paulownia flower powder;

[0034] (2) Extraction: add choline chloride urea solution to the powder at a solid-liquid ratio of 0.1 g:10 ml, with a water content of 40%, ultrasonic time of 15 min, ultrasonic power of 240 W, and ultrasonic temperature of 40°C to obtain an extract with a calculated yield of 1267.56 μg / g;

[0035] (3) Drying: centrifugation at 6000 rpm for 10 min, filtration at 0.02 MPa, vacuum distillation at 45° C., low-temperature drying at -20° C., cold trap temperature at -50° C., vacuum degree at 3.2 Pa, to obtain a crude extract of paulownia flower polyphenols;

[0036] (4) Purification: The crude extract of step (3) is separated by column chromatography, adsorbed by HPD-722 macroporous resin, eluted with 70% ethanol, the eluate is collected, concentrated at 45° C., and dried to obtain the Paulownia flower polyphenol extract.

[0037] Example 4 Enzyme activity inhibition experiment

[0038] The enzyme activity inhibition experiment was carried out on the Paulownia flower polyphenols prepared in Examples 1-3 to verify the inhibitory activity of the Paulownia flower polyphenols on α-glucosidase.

[0039] S1. Add 80 μL PBS buffer, 20 μL sample solution, and 20 μL 0.1 U / mL α-glucosidase to a 96-well plate, shake and mix, and incubate at 37°C for 20 min.

[0040] S2. After the incubation, add 20 μL of 2.5 mM nitrophenyl-β-D-pyranoglucoside (pNP G) reaction solution, shake to mix, and incubate at 37°C for 40 min;

[0041] S3. Add 80 μL 0.2 mol / L Na2CO3 to terminate the reaction;

[0042] S4. Then the absorbance of the sample well is detected at 405 nm;

[0043] S5. The blank control group used PBS buffer instead of sample and nitrophenyl-β-D-pyranoglucoside (pN PG) reaction solution; in the complete reaction group, PBS buffer was used instead of sample; the sample control group used PBS buffer instead of α-glucosidase;

[0044] S6. The calculation formula of the inhibition rate of α-glucosidase in the sample group is as follows:

[0045]

[0046] Wherein, C1 is the absorbance value of the blank control group; C2 is the absorbance value of the complete reaction group; C3 is the absorbance value of the sample reaction group; and C4 is the absorbance value of the sample control group.

[0047] The results showed that the IC of the paulownia flower polyphenols prepared in Example 1 for inhibiting α-glucosidase was 50 The value is 219.86 μg / mL. The IC value of the paulownia flower polyphenols prepared in Example 2 for inhibiting α-glucosidase is 50 The value is 303.83 μg / mL. The IC value of the paulownia flower polyphenols prepared in Example 3 for inhibiting α-glucosidase is 50 The value is 234.03μg / mL.

[0048] The Paulownia flower polyphenol extract of Example 1 was selected for experiment, and different concentrations of Paulownia flower polyphenol extract were set, namely 0, 50, 100, 150, 200, 300, 400, 500, and 600 μg / ml, and the inhibition rate of α-glucosidase was statistically analyzed, as shown in Table 1.

[0049] Table 1 Inhibitory effect of Paulownia flower polyphenol extract on α-glucosidase

[0050]

[0051] Comparative Example 1

[0052] Keeping other conditions of Example 1 unchanged, changing the ultrasonic power to 200 W, 240 W, 280 W, 320 W, and 360 W, the IC50 values ​​of the polyphenols finally obtained for α-glucosidase were 378.46 μg / mL, 302.58 μg / mL, 348 μg / mL, 352 μg / mL, and 358 μg / mL, respectively;

[0053] Comparative Example 2

[0054] Keeping other conditions of Example 1 unchanged, changing the ultrasonic time to 5min, 15min, 25min, 35min, and 45min, the IC50 values ​​of the polyphenols finally obtained for α-glucosidase were 325.58μg / mL, 312.54μg / mL, 347.31μg / mL, 356.78μg / mL, and 391.39μg / mL, respectively;

[0055] Comparative Example 3

[0056] Keeping other conditions of Example 1 unchanged, changing the ultrasonic temperature to 20°C, 30°C, 40°C, 50°C, and 60°C, the IC50 values ​​of the polyphenols finally obtained for α-glucosidase were 294.13 μg / mL, 227.18 μg / mL, 238.84 μg / mL, 243.42 μg / mL, and 267.39 μg / mL, respectively;

[0057] Comparative Example 4

[0058] Keeping other conditions of Example 1 unchanged, the solid-liquid ratio was changed to 0.05 g:10 mL, 0.1 g:10 mL, 0.15 g:10 mL, 0.2 g:10 mL, and 0.25 g:10 mL. The IC50 values ​​of the polyphenols finally obtained for α-glucosidase were 245.27 μg / mL, 228.48 μg / mL, 289.36 μg / mL, 316.48 μg / mL, and 349.92 μg / mL.

[0059] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0060] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for extracting Paulownia flower polyphenols that inhibits α-glucosidase activity, characterized in that: The following steps are involved: (1) crushing raw materials: crushing and sieving paulownia flowers to obtain paulownia flower powder; (2) Extraction: adding choline chloride urea solution with a water content of 30% to the powder at a solid-liquid ratio of 0.1 g:10 ml, ultrasonic time of 5-45 min, ultrasonic power of 200-400 W, and ultrasonic temperature of 20-60° C. to obtain an extract; (3) Drying: centrifugation, filtration, vacuum distillation, and drying to obtain a crude extract of Paulownia flower polyphenols; (4) Purification: The crude extract of step (3) is separated by column chromatography, and the eluate is collected, concentrated, and dried to obtain the Paulownia flower polyphenol extract.

2. The method for extracting Paulownia flower polyphenols that inhibits α-glucosidase activity according to claim 1, characterized in that: In step (1), the crushing time is 2 minutes, and the sieving is through a 60-mesh sieve.

3. The method for extracting Paulownia flower polyphenols that inhibits α-glucosidase activity according to claim 1, characterized in that: In step (3), the centrifugation is performed at 3000-6000 rpm for 10-30 min.

4. The method for extracting Paulownia flower polyphenols that inhibits α-glucosidase activity according to claim 1, characterized in that: In step (3), the suction filtration is performed at 0.02 MPa; the reduced pressure distillation is performed at 45°C; the drying is performed at low temperature, and the conditions are sample temperature of -20°C, cold trap temperature of -50°C, and vacuum degree of 3.2 Pa.

5. The method for extracting Paulownia flower polyphenols that inhibits α-glucosidase activity according to claim 1, characterized in that: In step (4), the concentration is carried out at 35-50° C. for 1 h.

6. The method for extracting Paulownia flower polyphenols that inhibits α-glucosidase activity according to claim 1, characterized in that: In step (4), the column chromatography separation conditions are: HPD-722 macroporous resin adsorption, 70% ethanol elution for 560 min.

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