CN117357576A - Lotus seed husk polyphenol extract with blood sugar reducing effect, and preparation method and application thereof - Google Patents
Lotus seed husk polyphenol extract with blood sugar reducing effect, and preparation method and application thereof Download PDFInfo
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- CN117357576A CN117357576A CN202311473866.XA CN202311473866A CN117357576A CN 117357576 A CN117357576 A CN 117357576A CN 202311473866 A CN202311473866 A CN 202311473866A CN 117357576 A CN117357576 A CN 117357576A
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- lotus seed
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- polyphenol extract
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Classifications
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- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
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- Medicines Containing Plant Substances (AREA)
Abstract
The invention belongs to the technical field of extraction of plant active ingredients, and particularly relates to a lotus seed shell polyphenol extract with a blood sugar reducing effect, and a preparation method and application thereof. The preparation method comprises the following steps: (1) Crushing lotus seed shells, and mixing with the solution A to obtain a mixed solution; (2) Ultrasonic extracting the mixed solution for 1-4 times, centrifuging, mixing the supernatant, and concentrating to obtain concentrated solution; (3) Filtering the concentrated solution, eluting with D101 macroporous resin and ethanol solution, and purifying to obtain lotus seed shell polyphenol extract. The invention treats the wastes in food processing, reduces the pollution to the environment, and obtains an active ingredient with the inhibition effect of alpha-amylase and alpha-glucosidase, which can be applied to functional foods.
Description
Technical Field
The invention belongs to the technical field of extraction of plant active ingredients, and particularly relates to a lotus seed shell polyphenol extract with a blood sugar reducing effect, and a preparation method and application thereof.
Background
Diabetes mellitus (diabetes mellitus, DM) is a disease of the endocrine system with a more complex pathogenesis, which is responsible for the metabolic disorders of sugar, fat and protein. Hyperglycemia and long-term metabolic disorders in DM exacerbate the disease progression, damage to systemic tissue and organs, leading to serious complications including coronary artery disease, stroke, peripheral arterial disease, retinopathy, nephropathy and neuropathy, which are one of the leading causes of increased mortality from diabetes.
Studies have shown that both alpha-amylase and alpha-glucosidase are involved in the breakdown of dietary carbohydrates, increasing glucose absorption, and thus causing elevated blood glucose levels. Postprandial hyperglycemia may increase the risk of type II diabetes and subsequent complications. Thus, one of the strategies to control hyperglycemia is to inhibit the activity of the carbohydrate hydrolases α -amylase and α -glucosidase. Currently most diabetes drugs belong to the class of alpha-amylase and alpha-glucosidase inhibitors, such as acarbose (acarbose), voglibose (voglibose) and miglitol (miglitol), but these drugs also have many side effects, including diarrhea, abdominal pain, abdominal distension and vomiting, etc., and thus it is of great importance to obtain low side effects of alpha-amylase and alpha-glucosidase inhibitors from natural plants.
The lotus seed shell is a byproduct in lotus seed processing production, is hard in texture and not edible, can be used as fertilizer when being burnt or when being used as firewood in a small amount, but is largely abandoned on the field, and only is naturally spoiled, so that the environment is polluted.
Polyphenols are a generic name of polyphenols-containing substances, which are one of the secondary metabolites of plants and are widely distributed in nature. The polyphenols have strong antioxidation, are widely applied to daily chemicals, foods, medicines and other industries, especially in the medical production, and are reported to have the effects of resisting tumor, resisting inflammation, resisting bacteria, reducing blood pressure, reducing blood sugar and the like.
The existing polyphenol extraction process comprises a solvent extraction method, an ion precipitation method and the like, wherein the solvent extraction method is stable and reliable, but has low extraction rate, low purity of products, easy oxidation of the products in the extraction process, and the ion precipitation method reduces the solvent consumption, but has larger loss and low extraction efficiency.
The Chinese patent application CN112472726A discloses a method for extracting brown algae polyphenol stock solution, which comprises the following steps: sequentially cleaning, soaking, chopping and dehydrating fresh brown algae; adding dehydrated brown algae into an extractant for extraction, and then removing residues through a filtering procedure to obtain an extract; centrifuging the extract, collecting supernatant, centrifuging the concentrated solution, collecting supernatant, concentrating again by low temperature evaporation to make the concentration of extractant in the solution below 5%, crystallizing at low temperature, centrifuging the concentrated solution after low temperature crystallization again, and collecting supernatant as brown algae polyphenols stock solution; the extraction, centrifugation and low-temperature evaporation concentration processes are all carried out in an environment with the temperature of 18-20 ℃, and the process is complex and takes a long time.
The Chinese patent application CN109303798A discloses a lotus seed shell extract with the function of reducing blood sugar, which is prepared by the following method: firstly, lotus seed shell powder and water or hydrophilic organic solvent are mixed and stirred uniformly, and then the obtained product is subjected to ultrasonic leaching and freeze-drying. However, the extract obtained by the method has low polyphenol content and low purity, and is not suitable for direct application.
In view of this, there is a need to develop an extraction method of lotus seed husk polyphenol extract, which can rapidly extract high purity polyphenol.
Disclosure of Invention
The invention provides a lotus seed shell polyphenol extract with a blood sugar reducing effect, a preparation method and application thereof, and aims to overcome the defects of low polyphenol purity, high price of blood sugar reducing medicines and large side effects in the existing extract. The lotus seed hull polyphenol extract prepared by the invention can obviously inhibit the activities of alpha-amylase and alpha-glucosidase.
In order to achieve the above purpose of the present invention, the present invention adopts the following specific technical scheme:
a preparation method of lotus seed hull polyphenol extract with blood sugar reducing effect comprises the following steps:
(1) Crushing lotus seed shells, and mixing with the solution A to obtain a mixed solution;
(2) Ultrasonic extracting the mixed solution for 1-4 times, centrifuging, mixing the supernatant, concentrating, and filtering to obtain concentrated solution;
(3) And (3) eluting and purifying the concentrated solution by using D101 macroporous resin and ethanol solution to obtain the lotus seed shell polyphenol extract.
Preferably, the crushed lotus seed shells in the step (1) have a particle size of 50-70 meshes, and the solution A consists of 1-butyl-3-methylimidazole bromide, ammonium sulfate and ethanol.
Preferably, the solution A consists of 5-20% of brominated 1-butyl-3-methylimidazole, 10-50% of ammonium sulfate and 40-70% of ethanol in percentage by mass, and the mass-volume ratio of lotus seed shells to the solution A is 1g:8-20mL.
Preferably, the frequency of the ultrasonic wave in the step (2) is 90-110KHz, the times of ultrasonic leaching are 2-3 times, and the time of each ultrasonic wave is 25-35min.
Preferably, the centrifugation in step (2) is carried out at a rotational speed of 5000-7000rpm for a period of 10-20min, the concentration is carried out at a temperature of 35-45℃and the filtration is carried out with a filter membrane of 0.4-0.5. Mu.m.
Preferably, the mass concentration of the ethanol solution in the step (3) is 25% -35%.
Preferably, the step of purifying in step (3) comprises: by adopting a wet loading method, eluting 1-2 column volumes with distilled water, discarding eluent, eluting 2-3 column volumes with ethanol solution, collecting eluent, concentrating, and drying to obtain lotus seed shell polyphenol extract.
Further preferably, the temperature of concentration in step (3) is 35-45 ℃, the drying is freeze-drying, and the drying temperature is-40 to-20 ℃.
The invention also relates to the lotus seed hull polyphenol extract prepared by the preparation method.
Preferably, the total polyphenol content of the lotus seed hull polyphenol extract is 500-700mg Gallic Acid Equivalent (GAE)/g.dry weight (DW), and the total flavone content is 300-500mg Rutin Equivalent (RE)/g.dry weight (DW).
The invention also relates to application of the lotus seed hull polyphenol extract in preparation of hypoglycemic drugs.
Compared with the prior art, the invention has the beneficial effects that:
(1) The extraction process is simple and easy to operate, and the content of polyphenol obtained by extraction is increased from about 20% to more than 60%;
(2) The polyphenol extracted by the invention has excellent blood sugar reducing effect, and the half inhibition concentration of alpha-amylase is reduced from 165.12 mug/mL to 121.31 mug/mL; the half-inhibition concentration of alpha-glucosidase is reduced from 3.5 mug/mL to 0.99 mug/mL;
(3) The lotus seed shell polyphenol extract can inhibit the digestibility of starch.
Drawings
FIG. 1 is a graph showing the effect of lotus seed husk polyphenol extraction on corn starch digestibility in examples 1-3 and comparative examples 1-4.
Detailed Description
The technical solutions of the embodiments of the present invention are further clearly described, and the described embodiments are only a part of the present invention, which are used to explain the present invention, but not to limit the present invention, so that other embodiments obtained by other persons skilled in the art without creative efforts fall within the protection scope of the present invention.
Acarbose, LOT: S25M11X109783; alpha-amylase (derived from porcine pancreas), LOT: j08HS184244; alpha-glucosidase (from saccharomyces cerevisiae), LOT: n25HS202422; p-nitrophenyl- α -D-glucopyranoside (PNPG), LOT: j15HS188691; all purchased from Shanghai Source leaf Biotechnology Co.
Starch, LOT:20210322 from national pharmaceutical group chemical company, ltd.
The reagents not specifically described below are those commonly used in the art and are commercially available.
Example 1
The extraction method of lotus seed hull polyphenol extract with blood sugar reducing effect comprises the following steps:
(1) Crushing the dried lotus seed shells to 60 meshes to obtain lotus seed shell powder; mixing lotus seed shell powder with a solution A (the solution A comprises 5% of brominated 1-butyl-3-methylimidazole by mass percent, 60% of ethanol and 35% of ammonium sulfate) according to a ratio of 1:10 (g/mL);
(2) Extracting with ultrasound at 25deg.C for 2 times, each time for 30min, and centrifuging at 6000rpm for 15min; mixing the centrifuged supernatants, and concentrating by rotary evaporation at 40deg.C to obtain concentrated solution of lotus seed shell extract;
(3) Filtering the concentrated solution of lotus seed hull extract with 0.45 μm filter membrane, loading on D101 macroporous resin column, adsorbing the sample with resin, eluting with 1 volume of pure water at a flow rate of 2.5mL/min to remove impurities, eluting with 30% ethanol water solution at a flow rate of 2.5mL/min to 2 volumes, collecting 30% ethanol eluate, concentrating by rotary evaporation, and lyophilizing at-30deg.C to obtain lotus seed hull polyphenol extract.
Example 2
The extraction method of lotus seed hull polyphenol extract with blood sugar reducing effect comprises the following steps:
(1) Crushing the dried lotus seed shells to 50 meshes to obtain lotus seed shell powder; mixing lotus seed shell powder with a solution A (the solution A comprises 10% of brominated 1-butyl-3-methylimidazole by mass percent, 40% of ethanol and 50% of ammonium sulfate) according to a ratio of 1:12 (g/mL);
(2) Ultrasonic extracting at room temperature for 3 times for 30min each time, centrifuging at 6000rpm for 15min after each leaching; mixing the supernatants, and concentrating at 45deg.C to obtain concentrated solution of lotus seed shell extract;
(3) Filtering the concentrated solution of lotus seed hull extract with 0.45 μm filter membrane, loading on D101 macroporous resin column, adsorbing the sample with resin, eluting with 1 column volume pure water at 2.5mL/min to remove impurities, eluting with 20% ethanol water solution at 2.5mL/min to 3 column volumes, collecting 20% ethanol eluate, concentrating by rotary evaporation, and lyophilizing at-40deg.C to obtain lotus seed hull polyphenol extract.
Example 3
The extraction method of lotus seed hull polyphenol extract with blood sugar reducing effect comprises the following steps:
(1) Crushing the dried lotus seed shells to 70 meshes to obtain lotus seed shell powder; mixing lotus seed shell powder with a solution A (the solution A comprises 20% of brominated 1-butyl-3-methylimidazole by mass percent, 70% of ethanol and 10% of ammonium sulfate) according to a ratio of 1:10 (g/mL);
(2) Ultrasonic extracting at room temperature for 2 times for 30min each time, centrifuging at 6000rpm for 15min after each leaching; combining the supernatants of each extraction, and concentrating by rotary evaporation at 35 ℃ to obtain lotus seed shell extract concentrate;
(3) Filtering the concentrated solution of lotus seed hull extract with 0.45 μm filter membrane, loading on D101 macroporous resin column, adsorbing the sample with resin, eluting with 2 times of pure water at a flow rate of 2.5mL/min, eluting with 50% ethanol water solution at a flow rate of 2.5mL/min for 2 times of column volume, collecting 50% ethanol eluate, concentrating by rotary evaporation, and lyophilizing at-20deg.C to obtain lotus seed hull polyphenol extract.
Comparative example 1
The extraction method of the lotus seed shell polyphenol extract comprises the following steps:
(1) Crushing the dried lotus seed shells to 60 meshes to obtain lotus seed shell powder; mixing lotus seed shell powder with a solution A (the solution A comprises 5% of brominated 1-butyl-3-methylimidazole by mass percent, 60% of ethanol and 35% of ammonium sulfate) according to a ratio of 1:10 (g/mL);
(2) Ultrasonic extracting at room temperature for 2 times for 30min, mixing the supernatants, concentrating by rotary evaporation at 40deg.C, and lyophilizing to obtain semen Nelumbinis polyphenol extract.
Comparative example 2
The extraction method of the lotus seed husk polyphenol extract is the same as that of the embodiment 1, and the difference is that: in the step (1), lotus seed shell powder and a solution A (the solution A consists of 60% of ethanol and 5% of brominated 1-butyl-3-methylimidazole in mass percent) are mixed according to the proportion of 1:10 (g/mL).
Comparative example 3
The extraction method of the lotus seed husk polyphenol extract is the same as that of the embodiment 1, and the difference is that: in the purification in the step (3), XDA-8 resin was used instead of D101 resin, and the column packing amount was the same as in example 1.
Comparative example 4
The extraction method of the lotus seed husk polyphenol extract is the same as that of the embodiment 1, and the difference is that: and (3) purifying by eluting with 60% ethanol to obtain lotus seed hull polyphenol extract.
Effect testing
Test example 1 substance identification
(1) Taking 5mg of lotus seed hull polyphenol extract in example 1, adding 50 mu L of methanol, uniformly mixing by ultrasonic, centrifuging at 12000rpm and 4 ℃ for 10min, and taking supernatant and loading into a machine.
(2) Liquid chromatography conditions: chromatographic column: sepax GP-C18 Column (1.8 μm)2.1mm x 150 mm); column temperature: 40 ℃; mobile phase a:0.1% formic acid; mobile phase B:100% acn; flow rate: 0.3mL/min; sample analysis time: 21min; the elution procedure is shown in table 1:
table 1 liquid chromatography elution procedure
(3) Mass spectrometry conditions: electrospray ionization (ESI) positive and negative ion modes were used for detection, respectively. The ESI source conditions were as follows: ion Source Gas1 (Gas 1): 50,Ion Source Gas2 (Gas 2): 50, curtain Gas (CUR): 25,Source Tempreture:500 ℃ (positive ion) and 450 ℃ (negative ion), ion Sapary Voltage Floating (ISVF) 5500V (positive ion) and 4400V (negative ion), TOF MS scan range:100-1200Da,product ion scan range:50-1000Da,TOF MS scan accumulation time 0.2s,product ion scan accumulation time 0.01s, and secondary mass spectrometry using Information Dependent Acquisition (IDA) and high sensitivity mode, declustering Potential (DP):.+ -. 60V,Collision Energy:35.+ -. 15eV.
(4) Database retrieval: the mass spectrum collected wiff file is preprocessed by software MS-DIAL 4.70 (MS-DIAL: data independent MS/MS deAvolution for comprehensive metabolome analysis (Nature Methods,12,523-526,2015) and comprises peak extraction, noise removal, deconvolution, peak alignment, and derivation of a three-dimensional data matrix (original data matrix) in CSV format.
57 phenolic compounds, 13 phenolic acids and derivatives thereof, 34 flavonoid compounds, 5 coumarins and derivatives thereof, 4 lignans and 1 gingerol were identified from lotus seed shell extract by UPLC-Q/TOF-MS, and the identification list of lotus seed shell extract substances is shown in Table 2.
TABLE 2 identification of lotus seed husk extract
(5) Determination of total polyphenol and total flavone content in lotus seed shell extract
1) Method for testing Total Polyphenol (Folin-Phenolmethod)
Determining a standard curve: accurately weighing gallic acid 10mg, dissolving with distilled water to constant volume of 100mL to obtain gallic acid standard solution with concentration of 0.1mg/mL, respectively placing 0.1mg/mL gallic acid standard solution of 0, 150, 200, 250, 300, 350, 400, 450, 500 μL into 5mL centrifuge tube, adding 2mLNa 2 CO 3 Solution (10%), after 2min, 0.9mL of Fu Lin Fen reagent is added into the mixture, and after light-shielding reaction is carried out for 1h, the absorbance value is measured at 750 nm; and drawing a standard curve by taking the mass concentration of gallic acid as an abscissa and the absorbance as an ordinate.
Sample measurement: and (3) properly sucking the sample liquid and diluting to ensure that the absorbance is between 0.2 and 0.8, and measuring the absorbance value by using the operation steps and a standard curve method, thereby obtaining the gallic acid equivalent content, namely the total polyphenol content by using a regression equation.
2) Method for testing total flavone (sodium nitrite-aluminium nitrate method)
Determining a standard curve: weighing rutin control 10mg to 50mL volumetric flask, adding 95% ethanol, diluting to scale, and obtaining rutin control solution of 0.2 mg/mL; accurately measuring rutin control solution 0.0, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0mL in 15mL test tube, and adding 5% NaNO 2 0.5mL of solution, mixing well, standing for 6min, adding 10% Al (NO) 3 ) 3 0.5mL of solution, shaking up, standing for 6min, adding 5mL of 4% NaOH solution, adding water until the system is 12.5mL, mixing uniformly, and standing for 15min. The absorbance at 510nm was measured, and a standard curve was drawn with the mass concentration X as the abscissa and the absorbance A as the ordinate Y.
Determination of the samples: and (3) properly sucking the sample liquid, diluting to ensure that the absorbance is between 0.2 and 0.8, placing the sample liquid in a 15mL test tube, measuring the absorbance A according to a standard curve method, and calculating the total flavone content in the sample according to a standard curve equation. The results of the total polyphenol and total flavone content tests are shown in table 3.
TABLE 3 Total Polyphenol and Total Flavonoids content
Test example 2 determination of alpha-amylase inhibitory Activity of lotus seed husk polyphenol extract
1. Solution preparation
7U/mL of alpha-amylase solution: precisely weighing 7mg of alpha-amylase (derived from pig pancreas), and adding 10mL of PBS solution (pH 6.8, 0.1M) to prepare 7U/mL of alpha-amylase solution;
1% starch aqueous solution: 0.1g of soluble starch was weighed and 10mL of distilled water was added to prepare a 1% starch aqueous solution.
2. Enzyme inhibition assay
(1) Sample group: the lotus seed husk extracts obtained in examples 1 to 3 and comparative examples 1 to 4 were diluted to 6 different concentrations, respectively, 20. Mu.L of the sample was pipetted and mixed with 40. Mu.L of PBS and 20. Mu.L of amylase solution, respectively, and the mixture was taken out in a 37℃water bath for 30min, then 40. Mu.L of a 1% aqueous starch solution was pipetted, reacted at 37℃for 10min, taken out, 80. Mu.L of DNS was added, developed in a 100℃water bath for 8min, and absorbance was measured at 540nm in 200. Mu.L to 96 well plates.
(2) Sample blank: 20. Mu.L of the sample was mixed with 60. Mu.L of PBS by pipetting, respectively, in a water bath at 37℃for 30min, taken out, then 40. Mu.L of a 1% aqueous starch solution was pipetted, reacted at 37℃for 10min, taken out, 80. Mu.L of DNS was added, developed in a water bath at 100℃for 8min, and absorbance was measured at 540nm in 200. Mu.L to 96 well plates.
(3) Control group: 60. Mu.L of PBS was pipetted and mixed with 20. Mu.L of amylase solution, the mixture was taken out in a water bath at 37℃for 30min, then 40. Mu.L of 1% aqueous starch solution was pipetted, reacted at 37℃for 10min, taken out, 80. Mu.L of DNS was added, color development was performed in a water bath at 100℃for 8min, and absorbance was measured at 540nm by pipetting 200. Mu.L to 96 well plates.
(4) Blank group: 80. Mu.L of PBS was pipetted in a water bath at 37℃for 30min, removed, then 40. Mu.L of 1% aqueous starch solution was pipetted, reacted at 37℃for 10min, removed, 80. Mu.L of DNS was added, color developed in a water bath at 100℃for 8min, and absorbance was measured at 540nm in 200. Mu.L to 96 well plates.
(5) Acarbose group: acarbose is diluted to 7 different concentrations, 20 mu L of the acarbose is respectively sucked by a pipette and mixed with 40 mu L of PBS and 20 mu L of amylase solution, the mixture is taken out in a water bath at 37 ℃ for 30min, then 40 mu L of 1% starch aqueous solution is sucked, the mixture is reacted for 10min at 37 ℃, 80 mu L of DNS is taken out, the mixture is developed in a water bath at 100 ℃ for 8min, and the absorbance of the mixture is measured at 540nm by sucking 200 mu L of the mixture to a 96-well plate.
The parameters and condition settings for the different experimental groups are shown in table 4, three replicates were run for each group.
TABLE 4 parameter and condition settings for different experimental groups
3. Test results
As shown in Table 5, the lotus seed husk polyphenol extract of example 1 has a better inhibitory effect on alpha-amylase than the lotus seed husk polyphenol extract of comparative examples 1 to 4 and the positive control acarbose.
TABLE 5 half inhibitory concentration of lotus seed husk extract for alpha-amylase (. Mu.g/mL)
Test example 3 determination of alpha-glucosidase inhibitory Activity of Lian Zingiber Fabricius polyphenol extract
1. Solution preparation
0.265U/mL of alpha-glucosidase solution: precisely weighing 1mg of alpha-glucosidase (from Saccharomyces cerevisiae), and adding 10mL of PBS solution (pH 6.8, 0.1M) to prepare 0.265U/mL of alpha-glucosidase solution;
10mmol/L p-nitrophenyl-alpha-D-glucopyranoside (PNPG): 0.1506g of the sample was weighed out precisely, and a 50mL of PNPG solution was prepared by adding 50mL of the PNBS solution.
0.2mol/L Na 2 CO 3 Solution: 1.06g of anhydrous sodium carbonate is precisely weighed, dissolved by adding distilled water and fixed to 50mL.
2. Enzyme inhibition assay
(1) Sample group: the lotus seed husk extracts obtained in examples 1 to 3 and comparative examples 1 to 4 were used and diluted to 5 piecesDifferent concentrations of 20. Mu.L of sample are respectively sucked by a pipette and mixed with 20. Mu.L of PBS and 20. Mu.L of alpha-glucosidase solution, the mixture is taken out in a water bath with the temperature of 37 ℃ for 10min, then 10mmol/L PNPG solution is sucked for 40. Mu.L, the mixture is reacted for 20min with the temperature of 37 ℃, the mixture is taken out, and 100. Mu.L of Na is added 2 CO 3 The reaction was stopped and the absorbance was measured at 405 nm.
(2) Sample blank: respectively pipetting 20. Mu.L of sample with 40. Mu.L of PBS, mixing with water bath at 37deg.C for 10min, taking out, pipetting 10mmol/L PNPG solution 40. Mu.L, reacting at 37deg.C for 20min, taking out, adding 100. Mu.L Na 2 CO 3 The reaction was stopped and the absorbance was measured at 405 nm.
(3) Control group: mixing 40. Mu.L PBS with 20. Mu.L alpha-glucosidase solution by a pipette, taking out in a water bath at 37 ℃ for 10min, then sucking 10mmol/L PNPG solution 40. Mu.L, reacting for 20min at 37 ℃, taking out, adding 100. Mu.L Na 2 CO 3 The reaction was stopped and the absorbance was measured at 405 nm.
(4) Blank group: sucking 60 μL of PBS with a pipette, taking out in a water bath at 37 ℃ for 10min, then sucking 40 μL of PNPG solution at 10mmol/L, reacting for 20min at 37 ℃, taking out, adding 100 μL of Na 2 CO 3 The reaction was stopped and the absorbance was measured at 405 nm.
(5) Acarbose group: diluting acarbose to 5 different concentrations, respectively sucking 20 μL with 40 μL PBS and 20 μL alpha-glucosidase solution by a pipette, mixing in a water bath at 37deg.C for 10min, taking out, then sucking 10mmol/L PNPG solution 40 μL, reacting at 37deg.C for 20min, taking out, adding 100 μL Na 2 CO 3 The reaction was stopped and the absorbance was measured at 405 nm.
The parameters and condition settings for the different experimental groups are shown in table 6, three replicates were run for each group.
TABLE 6 parameter and condition settings for different experimental groups
3. Test results
As shown in Table 7, the lotus seed husk polyphenol extract of example 1 has a better inhibitory effect on α -glucosidase than the lotus seed husk polyphenol extract of comparative examples 1 to 4 and the positive control acarbose.
TABLE 7 half inhibitory concentration of lotus seed husk extract for alpha-glucosidase (. Mu.g/mL)
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Test example 4 Effect of lotus seed husk polyphenol extract on corn starch digestibility
1. Solvent preparation
5mM CaCl 2 0.0555g of anhydrous calcium chloride is precisely weighed, 100mL of distilled water is added to prepare 5mmol/L CaCl 2 A solution;
mixed enzyme solution 0.15g of alpha-amylase (derived from porcine pancreas) was precisely weighed, 2077. Mu.L of PBS solution (pH 6.8, 0.1M) was added, 923. Mu.L of amyloglucosidase (derived from Aspergillus niger) was added, and 3mL of mixed enzyme solution (porcine pancreas alpha-amylase: amyloglucosidase=500:80U/mL) was prepared.
2. Digestibility measurement
Mixing 0.5g corn starch with 0.025g of the lotus seed hull polyphenol extracts of examples 1-3 and comparative examples 1-4, respectively, adding 25mL acetic acid buffer (100 mM,5mM CaCl) 2 pH 5.2) and gelatinized for 20min with stirring (2000 rpm) in a water bath at 90 ℃. After the gelatinized liquid is cooled to 37 ℃, 3mL of mixed enzyme liquid preheated at 37 ℃ is immediately added for enzymolysis for 180min at 37 ℃. During this period, samples (0.5 mL) were taken at 20, 40, 60, 90, 120, 180min, respectively, absolute ethanol (4.5 mL) was added to inactivate enzymes, centrifuged (2000 (. Times.g), 10 min), and a mixture of 0.5mL supernatant and 1mL DNS was incubated in boiling water for 8min, and then cooled to room temperature. The mixture was adjusted to a final volume of 10mL with deionized water and absorbance was measured at 540 nm. The absorbance of the reaction product of the enzymolysis product at 0min is used as a blank to eliminate the interference of flavonoid compounds and pigments. Measuring the content of glucose produced in the supernatant by DNS method, and calculating starch enzymolysis rate (SH), mass percent of fast-digestible starch (RDS), mass percent of fast-digestible starch (SDS) and the like according to the formulaResistant Starch (RS) mass percent. The calculation formulas of SH, RDS, SDS and RS are as follows:
SH(％)＝G t ×0.9/TS×100％；
RDS(％)＝(G 20 -G 0 )×0.9/TS×100％；
SDS(％)＝(G 120 -G 20 )×0.9/TS×100％；
RS(％)＝(1-RDS-SDS)×100％；
wherein G is t The content of glucose produced in the enzymolysis process is mg; g 20 For 20min of enzymolysis, the content of glucose produced is mg; g 120 For the glucose content produced by the enzymolysis for 120min, mg; TS is the total starch content in the sample, mg.
3. Test results
As shown in fig. 1, the lotus seed husk polyphenol extract can inhibit the digestibility of starch. After the addition of 5% lotus seed husk polyphenol extract, the corn starch hydrolysis rate of examples 1-3 was significantly lower than that of comparative examples 1-4, with the best effect of inhibiting starch hydrolysis with example 1. In addition, as shown in table 8, after adding 5% lotus seed husk polyphenol extract, the proportion of fast-digested starch in corn was significantly reduced (p < 0.05), and the proportion of slow-digested starch and resistant starch was increased (p < 0.05).
TABLE 8 Effect of lotus seed husk polyphenol extract on corn starch digestion
The foregoing detailed description is directed to one of the possible embodiments of the present invention, which is not intended to limit the scope of the invention, but is to be accorded the full scope of all such equivalents and modifications so as not to depart from the scope of the invention.
Claims (10)
1. The preparation method of the lotus seed hull polyphenol extract with the blood sugar reducing effect is characterized by comprising the following steps of:
(1) Crushing lotus seed shells, and mixing with the solution A to obtain a mixed solution;
(2) Ultrasonic extracting the mixed solution for 1-4 times, centrifuging, mixing the supernatant, concentrating, and filtering to obtain concentrated solution;
(3) And (3) eluting and purifying the concentrated solution by using D101 macroporous resin and ethanol solution to obtain the lotus seed shell polyphenol extract.
2. The preparation method according to claim 1, wherein the crushed lotus seed hulls in the step (1) have a particle size of 50-70 meshes, and the solution a consists of 1-butyl-3-methylimidazole bromide, ammonium sulfate and ethanol.
3. The preparation method according to claim 2, wherein the solution A consists of 5-20% of brominated 1-butyl-3-methylimidazole, 10-50% of ammonium sulfate and 40-70% of ethanol in percentage by mass, and the mass-volume ratio of lotus seed shells to the solution A is 1g:8-20mL.
4. The method according to claim 1, wherein the frequency of the ultrasonic waves in the step (2) is 90-110KHz, the number of ultrasonic leaching is 2-3, and the time of each ultrasonic wave is 25-35min.
5. The method according to claim 1, wherein the centrifugation in step (2) is performed at 5000-7000rpm for 10-20min, the concentration temperature is 35-45 ℃, and the filtration is performed with a filter membrane of 0.4-0.5 μm.
6. The method according to any one of claims 1 to 5, wherein the step of purifying in step (3) comprises: by adopting a wet loading method, eluting 1-2 column volumes with distilled water, discarding eluent, eluting 2-3 column volumes with ethanol solution, collecting eluent, concentrating, and drying to obtain lotus seed shell polyphenol extract, wherein the mass concentration of the ethanol solution is 25% -35%.
7. The method according to claim 6, wherein the concentration in the step (3) is carried out at a temperature of 35 to 45 ℃, the drying is freeze-drying, and the drying temperature is-40 to-20 ℃.
8. A lotus seed hull polyphenol extract prepared by the method of any one of claims 1-7.
9. The lotus seed husk polyphenol extract according to claim 8, wherein the total polyphenol content of the lotus seed husk polyphenol extract is 500-700mg GAE/g-DW.
10. Use of the lotus seed hull polyphenol extract of claim 8 in the preparation of a hypoglycemic agent.
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