Use of coffee silver bark extract in the preparation of metabolic enzyme inhibitors

Abstract

The invention discloses a new use of a coffee silver peel extract, namely, the use of the coffee silver peel extract in preparing natural inhibitors of alpha-glycosidase, alpha-amylase and xanthine oxidase. The coffee silver peel extract is prepared by freeze-drying, grinding and screening the coffee silver peel, defatting with petroleum ether, centrifuging and removing the supernatant, extracting the precipitate with a methanol-acetone aqueous solution, collecting the supernatant by centrifugation, concentrating and removing the solvent by rotary evaporation, adjusting the pH of the supernatant with hydrochloric acid, extracting with an ether-ethyl acetate mixed solution until the organic phase is colorless and clear, collecting and merging the organic phases, rotary evaporating and removing the organic reagent, and freeze-drying. The coffee silver peel extract has inhibitory activity on the activities of alpha-glycosidase, alpha-amylase and xanthine oxidase in vitro, and the coffee silver peel extract has good development and utilization prospects as a natural inhibitor of metabolic enzymes.

Description

Technical Field

[0001] The invention belongs to the field of preparation and application of coffee silver bark extract, and particularly relates to the use of coffee silver bark extract as a natural inhibitor of α-glycosidase, α-amylase and xanthine oxidase. The invention provides an application scheme for preventing, improving or treating type 2 diabetes and hyperuricemia. Background Art

[0002] Blood sugar control is considered an effective method for type 2 diabetes. α-Glucosidase and α-amylase can catalyze the digestion of carbohydrates by hydrolyzing α-1,4-glucosidic bonds. Therefore, the use of α-glycosidase and α-amylase inhibitors has been widely considered in recent years as an effective way to improve type 2 diabetes. However, some clinically used α-glycosidase and α-amylase inhibitors, such as acarbose and voglibose, are not only expensive, but also accompanied by many side effects, such as diarrhea, bloating, liver disease, and abdominal cramps. Therefore, it is necessary to develop some low-cost, easy-to-obtain, and side-free natural plant extracts as α-glycosidase and α-amylase inhibitors. The typical manifestation of hyperuricemia is increased uric acid content, which can lead to gout, chronic kidney disease, hypertension, diabetes, hemolytic diseases, atherosclerosis, etc. Uric acid is produced by the key enzyme xanthine oxidase catalyzing hypoxanthine and xanthine. Therefore, inhibiting the activity of xanthine oxidase is one of the main methods to alleviate hyperuricemia. Although the xanthine oxidase inhibitors currently used in clinic, such as allopurinol and febuxostat, can effectively inhibit the activity of xanthine oxidase in the body and reduce the uric acid content in the body, long-term use can cause side effects such as diarrhea, rash, and dizziness. Therefore, it is necessary to develop new, safe, natural plant-derived xanthine oxidase inhibitors.

[0003] Coffee silver skin is the last layer of skin covering coffee beans, which will fall off during roasting. Studies have found that coffee silver skin has good antibacterial and antioxidant activity. During the roasting process of coffee, a large amount of industrial waste is generated, especially coffee silver skin. Therefore, it is necessary to develop potential applications of food processing by-products and improve their economic value. At present, there are no reports on the inhibition of coffee silver skin extract on α-glucosidase, α-amylase and xanthine oxidase. Summary of the invention

[0004] The present invention provides a new use of a coffee silver bark extract, namely, the use of the coffee silver bark extract in the preparation of natural inhibitors of metabolic enzymes such as α-glycosidase, α-amylase and xanthine oxidase. Experiments have shown that the coffee silver bark extract obtained by the extraction method of the present invention can effectively inhibit the activities of α-glycosidase, α-amylase and xanthine oxidase. The present invention provides a new approach for further development and utilization of coffee silver bark.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] 1. Preparation of coffee silver bark extract

[0007] The coffee silver skin is freeze-dried and crushed, and the powder is sieved through a 40-mesh sieve. Petroleum ether is added to the coffee silver skin powder for defatting. The powder is centrifuged at 800g-1200g, and the precipitate is collected. The precipitate is extracted with a methanol-acetone aqueous solution, and the powder is centrifuged at 500g-1500g. The supernatant is collected, and after the solvent is concentrated and removed, the pH value of the supernatant is adjusted to 1-4 with a hydrochloric acid solution, and the powder is extracted with a mixture of ether and ethyl acetate until the organic phase is colorless and clear, and the organic phase is collected and combined, and the mixture is rotary evaporated and freeze-dried to obtain the product;

[0008] The methanol-acetone aqueous solution is prepared by mixing a methanol aqueous solution with a volume concentration of 50-70% and an acetone aqueous solution with a volume concentration of 50-70% in a ratio of 1:2-2:1; the ether-ethyl acetate mixed solution is prepared by mixing ether and ethyl acetate in a ratio of 1:2-2:1; the concentration of the hydrochloric acid solution for adjusting the pH is 2-6 mol / L;

[0009] Petroleum ether degreasing is to add petroleum ether and then ultrasonicate for 15-30min;

[0010] 2. Activity test of coffee silver bark extract

[0011] The present invention studies the inhibition of the in vitro α-glucosidase, α-amylase and xanthine oxidase activities by the coffee silver bark extract, and the experimental results show that the coffee silver bark extract prepared by the method of the present invention can significantly inhibit the activities of α-glucosidase, α-amylase and xanthine oxidase;

[0012] The purpose of the coffee silver bark extract of the present invention is to use the coffee silver bark extract as a natural inhibitor of α-glycosidase, α-amylase and xanthine oxidase for preparing medicine or food, or as a raw material.

[0013] The component (or active ingredient) of the metabolic enzyme inhibitor described in the present invention is coffee silver bark extract, and one or more drug or food acceptable excipients can also be added to improve the absorption effect of the drug or food or facilitate use, so as to prepare a suitable dosage form, such as capsules or pills, powders, tablets, granules, oral liquids and injections.

[0014] Compared with the prior art, the present invention has the following advantages:

[0015] 1. The coffee silver peel extract prepared by the present invention has an inhibitory effect on the activities of α-glycosidase, α-amylase and xanthine oxidase in vitro, providing a new application field for coffee silver peel;

[0016] 2. Coffee silver skin is a by-product of coffee roasting. The raw materials are easy to obtain. Coffee silver skin comes from natural plants and is highly safe. Coffee silver skin extract comes from edible natural plant raw materials, has a simple preparation process, has considerable market prospects, and contains good social and economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The inhibitory effect of different concentrations of coffee silver bark extract on α-glucosidase;

[0018] Figure 2 The inhibitory effect of coffee silver bark extracts at different concentrations on α-amylase;

[0019] Figure 3 The inhibitory effect of different concentrations of coffee silver bark extract on xanthine oxidase;

[0020] Different letters in the figure indicate significant differences (p<0.05). Implementation

[0021] The technical scheme of the present invention is further described below in conjunction with the accompanying drawings and embodiments, but these embodiments shall not be used to interpret the limitation of the present invention; the experimental methods in the following embodiments are conventional methods unless otherwise specified, and the experimental materials used in the following embodiments are commercially available unless otherwise specified; after reading the description of the present invention, various equivalent changes, modifications and modifications made by technical personnel in the field shall all fall within the scope defined by the claims of the present invention;

[0022] Example 1: Preparation of coffee silver bark extract

[0023] The silver bark of coffee collected from Baoshan City, Yunnan Province was freeze-dried and powdered, and passed through a 60-mesh sieve to obtain silver bark powder of coffee. 250 mL of petroleum ether was added to 50 g of silver bark powder of coffee, and the mixture was defatted by ultrasonic treatment for 30 min. The mixture was centrifuged at 800 g for 10 min, and the precipitate was collected. A methanol-acetone aqueous solution (a mixture of a 70% methanol aqueous solution and a 70% acetone aqueous solution in a volume ratio of 1:1) was added to the precipitate for extraction for 30 min. The mixture was centrifuged at 800 g for 10 min, and the supernatant was collected. After concentrating at 40 °C to remove the organic reagent in the supernatant, the pH was adjusted to 2 with 6 mol / L hydrochloric acid, and an ether-ethyl acetate mixture (a mixture of ether and ethyl acetate in a volume ratio of 1:1) was added to the solution after the pH was adjusted for extraction until the organic phase was colorless and clear. The organic phases were collected and combined, and the concentrate was rotary evaporated at 40 °C. The concentrate was freeze-dried to obtain 1.17 g of silver bark extract of coffee, and the acquisition rate was 2.34%.

[0024] Example 2: Experimental study on the inhibitory effect of coffee silver bark extract on α-glucosidase, α-amylase and xanthine oxidase

[0025] 1. Determination of α-glucosidase activity

[0026] The coffee silver bark extract prepared in Example 1 was used as an experimental sample, and was diluted with a phosphate buffered saline solution with a pH of 6.8 and a concentration of 0.2 mol / L to prepare sample solutions with concentrations of 20, 30, 40, 50, and 60 μg / mL;

[0027] Prepare 1U / mL α-glycosidase solution, and use phosphate buffered saline (PBS) with a pH of 6.8 and a concentration of 0.05mol / L to prepare 5mmol / L p-nitrophenyl-α-D-pyranoglucoside (PNPG) solution as the substrate reaction solution;

[0028] Set up blank group, blank control group, sample group, and sample control group;

[0029] The blank group was 60 μL PBS solution + 20 μL PNPG solution; the blank control group was 40 μL PBS solution + 20 μL α-glycosidase solution + 20 μL PNPG solution; the sample group was 20 μL PBS solution + 20 μL sample solution + 20 μL α-glycosidase solution + 20 μL PNPG solution; the sample control group was 40 μL PBS solution + 20 μL sample solution + 20 μL PNPG solution; 3 replicate wells were set for each group, the entire reaction system was incubated at 37°C for 30 min, and the absorbance was measured at 405 nm; the specific calculation formula is as follows:

[0030] Inhibition rate % = ((B 1 -B 2 ) / (A 2 -A 1 ))×100

[0031] In the formula, A 1 represents the absorbance value of the blank group, A 2 represents the absorbance value of the blank control group, B 1 Represents the absorbance value of the sample group, B 2 represents the absorbance value of the sample control group;

[0032] Results Figure 1 ,It can be seen from the figure that with the increase of the concentration of coffee silver peel extract, its inhibitory effect on α-glucosidase gradually increased, and the IC50 value of coffee silver peel extract on α-glucosidase was 40.28±1.40μg / mL.

[0033] 2. Determination of α-amylase activity

[0034] The coffee silver bark extract prepared in Example 1 was used as an experimental sample, and was diluted with a phosphate buffered saline solution with a pH of 6.9 and a concentration of 0.2 mol / L to prepare sample solutions with concentrations of 40, 80, 120, 160, and 200 μg / mL;

[0035] Prepare 2U / mL α-amylase solution and use phosphate buffered saline (PBS) with a pH of 6.9 and a concentration of 0.2 mol / L to prepare a 1% starch solution as the substrate reaction solution;

[0036] Set up blank group, blank control group, sample group, and sample control group;

[0037] The blank group was 50 μL PBS solution + 50 μL starch solution; the blank control group was 25 μL PBS solution + 25 μL α-amylase solution + 50 μL starch solution; the sample group was 25 μL sample solution + 25 μL α-amylase solution + 50 μL starch solution; the sample control group was 25 μL PBS solution + 25 μL sample solution + 50 μL starch solution; 3 replicate wells were set for each group, and the entire reaction system was incubated at 37°C for 10 min, then 200 μL of dinitrosalicylic acid solution was added, and kept in a boiling water bath for 15 min. After cooling to room temperature, the absorbance was measured at 540 nm; the specific calculation formula is as follows:

[0038] Inhibition rate % = ((B 1 -B 2 ) / (A 2 -A 1 ))×100

[0039] In the formula, A 1 represents the absorbance value of the blank group, A 2 represents the absorbance value of the blank control group, B 1 Represents the absorbance value of the sample group, B 2 represents the absorbance value of the sample control group;

[0040] Results Figure 2 ,It can be seen from the figure that with the increase of the concentration of coffee silver peel extract, its inhibitory effect on α-amylase gradually increased, and the IC50 value of coffee silver peel extract on α-amylase was 114.52±3.62μg / mL.

[0041] 3. Determination of xanthine oxidase activity

[0042] The coffee silver bark extract prepared in Example 1 was used as an experimental sample, and was diluted with a phosphate buffered saline solution with a pH of 7.5 and a concentration of 0.2 mol / L to prepare sample solutions with concentrations of 25, 50, 100, 150, and 200 μg / mL;

[0043] Prepare 0.1U / mL xanthine oxidase solution, and use phosphate buffered saline (PBS) with a pH of 7.5 and a concentration of 0.2mol / L to prepare 0.8mmol / L xanthine solution as substrate reaction solution;

[0044] Set up blank group, blank control group, sample group, and sample control group;

[0045] The blank group was 300 μL PBS solution + 100 μL xanthine solution; the blank control group was 200 μL PBS solution + 100 μL xanthine oxidase solution + 100 μL xanthine solution; the sample group was 100 μL PBS solution + 100 μL sample solution + 100 μL xanthine oxidase solution + 100 μL xanthine solution; the sample control group was 200 μL PBS solution + 100 μL sample solution + 100 μL xanthine solution; 3 replicate wells were set for each group, the entire reaction system was incubated at 37°C for 30 min, and the absorbance was measured at 295 nm; the specific calculation formula is as follows:

[0046] Inhibition rate % = ((B 1 -B 2 ) / (A 2 -A 1 ))×100

[0047] In the formula, A 1 represents the absorbance value of the blank group, A 2 represents the absorbance value of the blank control group, B 1 Represents the absorbance value of the sample group, B 2 Represents the absorbance value of the sample control group.

[0048] Results Figure 3 , It can be seen from the figure that with the increase of the concentration of coffee silver peel extract, its inhibitory effect on xanthine oxidase gradually increased, and the IC50 value of coffee silver peel extract on xanthine oxidase was 34.05±3.10μg / mL;

[0049] It can be seen that coffee silver bark extract has a good inhibitory effect on the activities of α-glycosidase, α-amylase and xanthine oxidase.

Claims

1. Application of a coffee silver bark extract in the preparation of metabolic enzyme inhibitors; The coffee silver peel extract is prepared by freeze-drying, grinding and screening the coffee silver peel, adding petroleum ether to the coffee silver peel powder for defatting, centrifuging, collecting the precipitate, extracting the precipitate with a methanol-acetone aqueous solution, centrifuging, collecting the supernatant, concentrating and removing the solvent, adjusting the pH of the supernatant to 1-4 with a hydrochloric acid solution, extracting with an ether-ethyl acetate mixed solution until the organic phase is colorless and clear, collecting and merging the organic phases, and rotary evaporating and freeze-drying; wherein the methanol-acetone aqueous solution is prepared by mixing a methanol aqueous solution with a volume concentration of 40-70% and an acetone aqueous solution with a volume concentration of 40-70% in a ratio of 1:2-2:1; and the ether-ethyl acetate mixed solution is prepared by mixing ether and ethyl acetate in a ratio of 1:2-2:1; The metabolic enzyme is one of α-glycosidase, α-amylase and xanthine oxidase.

2. The use according to claim 1, characterized in that: Petroleum ether degreasing is carried out by adding petroleum ether and then ultrasonically treating for 20-30 minutes.

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