A chestnut shell extract having an inhibitory effect on alpha-glucosidase activity, and a preparation method and application thereof

Abstract

The application discloses a Chinese chestnut peel extract with alpha-glucosidase inhibitory activity, a preparation method and application thereof, and belongs to the technical field of natural extract preparation. First, Chinese chestnut peel powder is mixed with water, the pH is adjusted to 4.5-5.5, and a Viscozyme L composite enzyme preparation is added for enzymolysis, then choline chloride-sucrose eutectic solvent is added to the enzymolysis system, the liquid-solid ratio is 20:1-40:1, heating and stirring extraction is carried out at 50-80 DEG C for 1-3 hours, an extract is obtained, then centrifugation and reduced-pressure concentration are carried out, a concentrated solution is finally obtained, and finally, the concentrated solution is subjected to freeze-drying or vacuum drying to obtain Chinese chestnut peel extract powder. In vitro alpha-glucosidase inhibition experiments show that the Chinese chestnut peel extract prepared by the application has an alpha-glucosidase inhibition rate of 95% at a concentration of 0.1 mg / mL, and can be used as an effective component of a hypoglycemic drug.

Description

Technical Field

[0001] This invention belongs to the field of natural extract preparation technology, and particularly relates to a chestnut seed coat extract that inhibits α-glucosidase activity, its preparation method and application. Background Technology

[0002] Chestnuts are deciduous trees belonging to the genus *Castanea* in the family Fagaceae. Their seed coat is a major byproduct of chestnut processing. The chestnut seed coat, also known as the "astringent skin," is the thin layer surrounding the chestnut kernel. Located between the involucre and the kernel, it is the inner layer of the chestnut fruit connecting the hard shell to the edible portion. Traditionally, chestnut seed coats were mostly treated as waste or used as fuel.

[0003] Alpha-glucosidase is an important glycosidic hydrolase that specifically hydrolyzes α-glycosidic bonds in carbohydrates such as starch and glycogen. In the human body, it is primarily responsible for breaking down disaccharides and oligosaccharides into glucose, and is a key enzyme in regulating postprandial blood glucose. Patents CN103948654A and CN115414398A disclose the application of extracts obtained from chestnut shells and chestnut kernels in lowering blood glucose. However, during chestnut production and processing, the byproduct chestnut seed coat is often discarded as waste, failing to realize its high-value application. This invention uses inexpensive chestnut seed coat as a raw material to obtain a chestnut extract that inhibits α-glucosidase. Summary of the Invention

[0004] This invention overcomes the shortcomings of the prior art and provides a chestnut seed coat extract with α-glucosidase activity, its preparation method and application, aiming to solve the problem that existing chestnut seed coats are often discarded as waste and cannot achieve high-value applications.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A method for preparing a chestnut seed coat extract with inhibitory α-glucosidase activity, specifically comprising:

[0007] (1) Preparation of choline chloride-sucrose eutectic solvent: Mix choline chloride and sucrose in a molar ratio of (1-5):1, add 20%-50% of deionized water by total mass, heat and stir in a water bath at 60-90℃ until a clear, transparent and homogeneous liquid is formed, and then cool for later use.

[0008] (2) Enzymatic hydrolysis pretreatment: Mix chestnut seed coat powder with water, adjust the pH to 4.5-5.5, add Viscozyme L complex enzyme preparation, and hydrolyze at 40-60℃ for 30-90 minutes, then heat to inactivate the enzyme. The amount of Viscozyme L added is 0.5%-2.0% of the mass of chestnut seed coat powder;

[0009] (3) Extraction of effective components: Add the eutectic solvent prepared in step (1) to the enzymatic hydrolysis system to make the liquid-solid ratio 20:1 to 40:1 (mL:g), heat and stir at 50 to 80°C for 1 to 3 hours to obtain the extract, then centrifuge and concentrate under reduced pressure to finally obtain the concentrate;

[0010] (4) The concentrate is freeze-dried or vacuum-dried to obtain chestnut seed coat extract powder.

[0011] In step (1) of this invention, the method for preparing the chestnut seed coat powder is as follows: fresh chestnut seed coats are washed, dried at 60°C to reduce their moisture content to 5% or less, and then pulverized through a 40-mesh sieve to obtain chestnut seed coat powder.

[0012] In step (2) of this invention, the enzymatic hydrolysis process is carried out in a shaker, and the shaker is shaken once every 15 minutes.

[0013] In step (2) of this invention, the enzymatic hydrolysis system is placed in a boiling water bath and heated for 10 minutes to inactivate the enzyme.

[0014] In step (3) of this invention, the extract is centrifuged to separate the supernatant, and then the collected supernatant is concentrated under reduced pressure at 45-60°C to 1 / 5-1 / 10 of its original volume to obtain a concentrated solution. The centrifugation conditions are: rotation speed 5000-8000 r / min, time 10-20 minutes; and vacuum degree of reduced pressure concentration is 0.09-0.1 MPa.

[0015] The chestnut seed coat extract can inhibit α-glucosidase activity and can be used as an effective ingredient in hypoglycemic drugs. The preparation of hypoglycemic drugs is as follows: chestnut seed coat extract is mixed with 0.5% sodium carboxymethyl cellulose aqueous solution to form a suspension.

[0016] The present invention has the following beneficial effects:

[0017] (1) The raw materials are widely available and inexpensive. Chestnut seed coats are usually discarded or used as fuel. This invention uses them to extract active ingredients, which reduces the cost of raw materials and realizes the high-value utilization of agricultural waste, thus achieving both economic and environmental benefits.

[0018] (2) Green and simplified extraction process. The present invention uses choline chloride-sucrose eutectic solvent as the extraction medium. This solvent is composed of food-grade raw materials, is biodegradable and has low toxicity, thus avoiding the use and residue problems of organic solvents.

[0019] (3) The active ingredients are well preserved. The present invention uses enzymatic hydrolysis-assisted extraction, and the extraction temperature is controlled at 50-70℃. The extraction process is mild, with only brief heating in the enzyme inactivation step, so the overall impact on the active ingredients is small.

[0020] (4) Efficacy verification is sufficient. In vitro α-glucosidase inhibition experiments showed that the chestnut seed coat extract prepared in this invention inhibited α-glucosidase by 95% at a concentration of 0.1 mg / mL, which was significantly better than traditional water extraction, alcohol extraction, and single DES extract. Animal experiments showed that the chestnut seed coat extract prepared by the method of this invention had a hypoglycemic effect in STZ-induced diabetic mouse models. Compared with the model group, the high-dose group showed a p-value of approximately 0.057, which was marginally significant and showed a dose-dependent trend. These results provide sufficient experimental evidence for the application of the extract of this invention in hypoglycemic drugs or functional foods. Attached Figure Description

[0021] Figure 1 This is a comparison chart of the inhibition rates of chestnut seed coat extracts obtained by different extraction methods on α-glucosidase in Example 2 of the present invention.

[0022] Figure 2 This is a line graph showing the effect of chestnut seed coat extract on fasting blood glucose in diabetic mice in Example 3 of the present invention.

[0023] Figure 3 This is a line graph showing the effect of chestnut seed coat extract on the fasting body weight of diabetic mice in Example 3 of the present invention.

[0024] Figure 4 The figure shows the results of the oral glucose tolerance test for each group of mice in Example 3 of the present invention.

[0025] Figure 5 The graph shows the experimental results of the area under the blood glucose curve (AUC) of each group of mice in Example 3 of this invention. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Example 1: Extraction of chestnut seed coat polyphenols by enzymatic hydrolysis of choline chloride-sucrose-DES

[0028] (1) Raw material pretreatment

[0029] Take fresh chestnut seed coats and wash off surface impurities with clean water. Place the washed chestnut seed coats in an oven and dry them at 60℃ for 12 hours until the moisture content drops below 5%. Grind the dried chestnut seed coats into powder using a grinder, pass the powder through a 40-mesh sieve, and store it in a desiccator in an airtight container for later use.

[0030] (2) Preparation of choline chloride-sucrose eutectic solvent

[0031] Weigh 27.9 g (0.2 mol) of choline chloride and 34.2 g (0.1 mol) of sucrose in a molar ratio of 2:1 into a round-bottom flask. Add 24.8 g of deionized water (equivalent to 40% of the total mass of choline chloride and sucrose) and stir until homogeneous. Place the flask in an 80°C water bath and stir magnetically for 1.5 hours until a clear, transparent, and homogeneous liquid is formed. Remove from the heat, cool to room temperature, and seal for later use.

[0032] (3) Enzymatic pretreatment

[0033] Weigh 2.00 g of chestnut seed coat powder and place it in a 150 ml Erlenmeyer flask with a stopper. Add 10 mL of deionized water and stir well. Adjust the pH to 5.0 with 1 mol / L HCl or NaOH. Add 1% (by weight of the seed coat powder) of Viscozyme L complex enzyme preparation and mix well. Place the Erlenmeyer flask in a constant temperature water bath shaker at 50°C for 60 minutes, shaking every 15 minutes. After enzymatic hydrolysis, place the Erlenmeyer flask in a boiling water bath for 10 minutes to inactivate the enzyme, and then cool to room temperature.

[0034] (4) Extraction with eutectic solvent

[0035] Add 40 mL of the choline chloride-sucrose eutectic solvent prepared in step (2) to the above enzymatic hydrolysis system, and add deionized water to bring the total volume to about 60 mL (liquid-solid ratio 30:1). After mixing, place the Erlenmeyer flask in a magnetic stirrer and stir at 70°C for 2 hours.

[0036] (5) Solid-liquid separation

[0037] After extraction, the mixture was transferred to centrifuge tubes and centrifuged at 6000 r / min for 15 minutes. The supernatant was collected. The filter residue was extracted again under the same conditions (adding DES, adding water to a liquid-to-solid ratio of 30:1, and stirring at 70°C for 2 hours). The supernatants from both extractions were combined.

[0038] (6) Reduced pressure concentration

[0039] The combined supernatant was transferred to a rotary evaporator and concentrated under reduced pressure at 50°C and 0.1 MPa until the volume of the concentrated liquid was about 1 / 8 to 1 / 10 of the original volume.

[0040] (7) Freeze-drying

[0041] The concentrate was transferred to a freeze-drying bottle and pre-frozen in a -80°C freezer for 12 hours. Then it was transferred to a freeze dryer and dried at -50°C and a vacuum of less than 20 Pa for 48 hours to obtain a light brown fluffy powder, which is the chestnut seed coat extract.

[0042] Comparative Example 1: Water Extraction Group

[0043] This comparative example obtained chestnut seed coat extract through raw material pretreatment, water extraction, solid-liquid separation, vacuum concentration, and freeze drying. Except for water extraction and solid-liquid separation, all other steps were the same as in Example 1. Specifically, water extraction was performed as follows: 2.00 g of the chestnut seed coat powder obtained in step (1) was weighed and placed in a 150 ml stoppered Erlenmeyer flask. 60 mL of deionized water was added at a liquid-to-solid ratio of 30:1 (mL:g). After mixing, the Erlenmeyer flask was placed in a magnetic stirrer and stirred at 70°C for 2 hours. After extraction, the mixture was transferred to a centrifuge tube and centrifuged at 6000 r / min for 15 minutes. The supernatant was collected. The residue was extracted again under the same conditions, and the two supernatants were combined.

[0044] Comparative Example 2: Alcohol Extraction Group

[0045] This comparative example obtained chestnut seed coat extract through raw material pretreatment, alcohol extraction, solid-liquid separation, vacuum concentration, and freeze drying. Except for alcohol extraction and solid-liquid separation, all other steps were the same as in Example 1. Specifically, alcohol extraction involved preparing a 70% (v / v) ethanol aqueous solution as the extraction solvent. 2.00 g of the chestnut seed coat powder obtained in step (1) was weighed and placed in a 150 ml stoppered Erlenmeyer flask. 60 mL of 70% ethanol solution was added at a liquid-to-solid ratio of 30:1 (mL:g). After mixing, the flask was placed in a magnetic stirrer and stirred at 70°C for 2 hours. After extraction, the mixture was transferred to a centrifuge tube and centrifuged at 6000 r / min for 15 minutes. The supernatant was collected. The residue was extracted again under the same conditions, and the two supernatants were combined.

[0046] Comparative Example 3: Single DES Group

[0047] This comparative example involved raw material pretreatment, preparation of a choline chloride-sucrose eutectic solvent, eutectic solvent extraction, solid-liquid separation, vacuum concentration, and freeze-drying to obtain chestnut seed coat extract. Except for the eutectic solvent extraction and solid-liquid separation, all other steps were the same as in Example 1. Specifically, the eutectic solvent extraction was performed as follows: 2.00 g of chestnut seed coat powder obtained in step (1) was placed in a 150 ml stoppered Erlenmeyer flask. Approximately 40 mL of the choline chloride-sucrose eutectic solvent prepared in step (2) was added, and deionized water was added to bring the total volume to approximately 60 mL (liquid-to-solid ratio 30:1). After mixing, the Erlenmeyer flask was placed in a magnetic stirrer and stirred at 70°C for 2 hours. After extraction, the mixture was transferred to a centrifuge tube and centrifuged at 6000 r / min for 15 minutes. The supernatant was collected. The residue was extracted again under the same conditions, and the two supernatants were combined.

[0048] Comparative Example 4: Single Enzyme Digestion Group

[0049] This comparative example obtained chestnut seed coat extract through a series of pretreatment steps: raw material pretreatment, enzymatic hydrolysis pretreatment, solid-liquid separation, vacuum concentration, and freeze drying. Except for the enzymatic hydrolysis pretreatment and solid-liquid separation, all other steps were the same as in Example 1. The enzymatic hydrolysis pretreatment was as follows: 2.00 g of the chestnut seed coat powder obtained in step (1) was weighed and placed in a 150 ml stoppered Erlenmeyer flask. 10 mL of deionized water was added, and the mixture was stirred until homogeneous. The pH was adjusted to 5.0 using 1 mol / L HCl or NaOH. 1% (by weight of the seed coat powder) of Viscozyme L complex enzyme preparation was added and mixed thoroughly. The Erlenmeyer flask was placed in a constant temperature water bath shaker and hydrolyzed at 50°C for 60 minutes, shaking every 15 minutes. After hydrolysis, the Erlenmeyer flask was placed in a boiling water bath for 10 minutes to inactivate the enzyme, and then cooled to room temperature. Deionized water was then added to a total volume of approximately 60 mL. After extraction, the mixture was transferred to a centrifuge tube and centrifuged (6000 r / min, 15 min), collecting the supernatant. The filter residue was extracted again under the same conditions, and the two supernatants were combined.

[0050] Example 2: In vitro α-glucosidase inhibitory activity assay

[0051] A. Reagent Preparation

[0052] 0.1 U / mL α-glucosidase solution (from Saccharomyces cerevisiae, Sigma-Aldrich, prepared with 0.2 M phosphate buffer, pH 6.8), 10 mmol / L p-nitrophenyl-α-D-glucopyranoside pNPG substrate solution (prepared with phosphate buffer), 0.2 mol / L Na2CO3 solution, and phosphate buffer containing 7% methanol.

[0053] B. Sample Preparation

[0054] Take the chestnut seed coat extracts prepared in Example 1 and Comparative Examples 1-4 respectively, dissolve them in 70% methanol and dilute them to 1 mg / mL for later use.

[0055] C. Experimental Methods

[0056] In the reaction system, 200 μL of sample solution was mixed with 200 μL of 0.1 U / mL α-glucosidase solution and reacted at 37℃ for 5 min. Then, 200 μL of 10 mmol / L pNPG substrate solution was added, and the volume was brought up to 2 mL with phosphate buffer. The mixture was then incubated at 37℃ for 40 min. After the reaction was completed, 2 mL of 0.2 mol / L Na2CO3 solution was added to terminate the reaction. The absorbance was measured at 405 nm using a spectrophotometer. Blank group, control group, sample group, and sample background group were set up during the process, as detailed below:

[0057] Blank group (OD) A This is a mixed solution of 200 μL of phosphate buffer containing 7% methanol and 200 μL of phosphate buffer.

[0058] control group (OD) B The solution is a mixture of 200 μL of phosphate buffer containing 7% methanol and 200 μL of α-glucosidase solution (0.1 U / mL).

[0059] Sample group (OD) C The solution is a mixture of 200 μL of sample solution and 200 μL of α-glucosidase solution (0.1 U / mL);

[0060] Sample background group (OD) D This is a mixture of 200 μL of sample solution and 200 μL of phosphate buffer;

[0061] Each experiment was repeated three times, and the average value was taken. The inhibition rate was calculated using the following formula:

[0062] α-glucosidase inhibition rate (%) = (OD B - OD C + OD D ) / (OD B - OD A ) × 100%

[0063] D. Experimental Results

[0064] At a concentration of 0.1 mg / mL, the α-glucosidase inhibition rate of each sample was... Figure 1As shown, the inhibition rate of the DES+enzyme hydrolysis group extract of this invention reached 95.22%, significantly higher than that of the water extraction group, alcohol extraction group, single DES group, and single enzyme hydrolysis group. This result indicates that the chestnut seed coat extract prepared by the method of this invention possesses excellent α-glucosidase inhibitory activity.

[0065] Example 3: Hypoglycemic Efficacy Test of Chestnut Seed Coat Extract

[0066] A. Establishment of animal models

[0067] Male C57BL / 6J mice (specific pathogen-free) weighing 20-25g were randomly divided into a normal control group and a model group after one week of acclimatization. Mice in the model group were fed a high-fat diet (60% kcal from fat) for 6 months to establish an insulin resistance background. Subsequently, streptozotocin (STZ, 40 mg / kg) was injected intraperitoneally for 5 consecutive days to induce partial damage to pancreatic β-cells, establishing a type 2 diabetes model. The normal control group received an equal volume of citrate buffer. On day 7 post-modeling, fasting blood glucose was measured by tail blood collection. Mice with fasting blood glucose ≥11.1 mmol / L were selected for subsequent experiments.

[0068] B. Drug Preparation

[0069] Preparation of physiological saline: Dissolve 0.9 g of sodium chloride in distilled water and bring the volume to 100 mL. Shake well to obtain physiological saline.

[0070] 0.5% Sodium Carboxymethyl Cellulose Aqueous Solution: Add 0.5g of sodium carboxymethyl cellulose powder to distilled water at 65°C, stir until completely dissolved and clear, and after cooling, add water to a final volume of 100ml. It can be stored for several days at 4°C.

[0071] Chestnut seed coat extract solution: Take a few grams of chestnut seed coat extract and add it to a 0.5% sodium carboxymethyl cellulose aqueous solution. Shake well and mix thoroughly. Prepare fresh before use.

[0072] C. Animal grouping, drug administration, and blood glucose measurement

[0073] Diabetic mice that had successfully developed the model were randomly divided into a model control group, a positive drug group, a low-dose group, and a high-dose group, with six mice in each group. A normal control group of six mice was also included. The drug administration regimen is as follows:

[0074] Normal control group (blank group): administered an equal volume of physiological saline by gavage;

[0075] Model control group: administered an equal volume of physiological saline by gavage;

[0076] Positive drug group: Metformin 200 mg / kg administered by gavage;

[0077] Low-dose group: 100 mg / kg of chestnut seed coat extract prepared in Example 1 was administered by gavage;

[0078] High-dose group: 300 mg / kg of chestnut seed coat extract prepared in Example 1 was administered by gavage.

[0079] Each group received the medication once daily via gavage at a set time each evening for three consecutive weeks. Patients were allowed free access to food and water during the treatment period.

[0080] After fasting for eight hours starting at 9 a.m., blood is drawn from the tail tip to measure fasting blood glucose and weight, and the data are recorded.

[0081] D. Oral glucose tolerance test (OGTT)

[0082] After drug administration, mice in each group were fasted for 12 hours and then administered glucose solution (2 g / kg) by gavage. Blood samples were collected from the tail tip at 0, 30, 60, and 120 minutes after gavage, and blood glucose levels were measured using a glucometer. The area under the blood glucose curve (AUC) was calculated using the trapezoidal method, as shown in the following formula:

[0083] AUC=(C0+C1)×0.5 / 2+(C1+C2)×0.5 / 2+(C2+C3)×0.5 / 2+(C3+C4)×0.5 / 2

[0084] In the formula: C0, C1, C2, C3, and C4 are the blood glucose values ​​at 0, 30, 60, 90, and 120 minutes, respectively.

[0085] Experimental Results

[0086] Compared with the model control group, the area under the blood glucose curve in the high-dose group mice was reduced (P≈0.057), which was marginally significant and showed a dose-dependent trend.

[0087] Animal experiments demonstrated that, after 3 weeks of oral administration of chestnut seed coat extract (300 mg / kg), fasting blood glucose levels in STZ- and high-fat diet-induced type 2 diabetic mice decreased from 19.33 mmol / L to 14.48 mmol / L, and the area under the oral glucose tolerance test curve decreased (P≈0.057). Furthermore, the weight loss (-1.6 g) was significantly less than that in the model group (-3.7 g). These results indicate that the chestnut seed coat extract of this invention has hypoglycemic and diabetic waste-improving effects, and can be used to prepare hypoglycemic drugs or functional foods.

[0088] Based on the preferred embodiments of the present invention described above, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A method for preparing a chestnut seed coat extract with inhibitory α-glucosidase activity, characterized in that, Specifically, it includes: (1) Preparation of choline chloride-sucrose eutectic solvent: Mix choline chloride and sucrose in a molar ratio of (1-5):1, add 20%-50% of the total mass of choline chloride and sucrose in deionized water, heat and stir in a water bath at 60-90℃ until a clear, transparent and homogeneous liquid is formed, and then cool for later use. (2) Enzymatic hydrolysis pretreatment: Mix chestnut seed coat powder with water, adjust the pH to 4.5-5.5, add Viscozyme L complex enzyme preparation for enzymatic hydrolysis, and finally heat to inactivate the enzyme; (3) Extraction of effective components: Add the eutectic solvent prepared in step (1) to the enzymatic hydrolysis system to make the liquid-solid ratio 20:1 to 40:1, heat and stir at 50 to 80°C to obtain the extract, then centrifuge and concentrate under reduced pressure to finally obtain the concentrate; (4) The concentrate is freeze-dried or vacuum-dried to obtain chestnut seed coat extract powder.

2. The method for preparing chestnut seed coat extract with α-glucosidase inhibitory activity according to claim 1, characterized in that, Fresh chestnut seed coats are washed, dried at 60°C to reduce their moisture content to 5% or less, and then pulverized through a 40-mesh sieve to obtain chestnut seed coat powder.

3. The method for preparing chestnut seed coat extract with α-glucosidase inhibitory activity according to claim 1, characterized in that, The enzymatic hydrolysis is carried out in a shaker at 40-60℃ for 30-90 minutes, with shaking every 15 minutes. The amount of Viscozyme L added is 0.5%-2.0% of the mass of chestnut seed coat powder.

4. The method for preparing chestnut seed coat extract with α-glucosidase inhibitory activity according to claim 1, characterized in that, The enzyme hydrolysis system was heated in a boiling water bath for 10 minutes to inactivate the enzyme.

5. The method for preparing chestnut seed coat extract with α-glucosidase inhibitory activity according to claim 1, characterized in that, The extract was centrifuged and the supernatant was collected. The collected supernatant was then concentrated under reduced pressure at 45–60°C to 1 / 5–1 / 10 of its original volume to obtain a concentrated solution. The centrifugation conditions were: 5000–8000 r / min for 10–20 minutes; and the vacuum degree of the reduced pressure concentration was 0.09–0.1 MPa.

6. A chestnut seed coat extract with α-glucosidase inhibitory activity prepared by any one of the methods of claims 1-5.

7. The use of the chestnut seed coat extract according to claim 6 in hypoglycemic drugs.

8. The application of the chestnut seed coat extract according to claim 7 in hypoglycemic drugs, characterized in that, Chestnut seed coat extract was mixed with 0.5% sodium carboxymethyl cellulose aqueous solution to prepare a suspension.

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