Application of banana peel melanin in preparation of medicine for treating diabetes

Abstract

The invention discloses application of banana peel melanin to preparation of a medicine for treating diabetes mellitus, and belongs to the technical field of medicines. The melanin is separated from the banana peel through the technologies of ethanol extraction, AB-8 macroporous adsorption resin purification, infrared spectrum identification and the like, the melanin has remarkable inhibitory activity on alpha-glucosidase, the median inhibitory concentration (IC50) is as low as 3.04 mu g / mL, the inhibitory effect is superior to that of a positive control drug acarbose (IC50 is 11.10 mu g / mL), and the melanin has a good application prospect. And a high-quality natural raw material is provided for developing a novel medicine for treating diabetes. In addition, enzyme kinetic research shows that the banana peel melanin inhibits the activity of alpha-glucosidase through an anti-competitive inhibition mechanism, can effectively delay decomposition and absorption of starch substances in intestinal tracts and reduce the postprandial blood sugar peak value, and can be used for preparing safe and efficient blood sugar reducing drugs; meanwhile, high-value utilization of the banana peel waste is achieved, and the method has important economic value and environmental protection significance.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the use of banana peel melanin in the preparation of drugs for treating diabetes. Background Technology

[0002] Diabetes mellitus is a global chronic metabolic disease characterized by persistent hyperglycemia. Long-term illness can lead to progressive damage to multiple organs and tissues, including the eyes, kidneys, nervous system, heart, and blood vessels, seriously threatening human health. Type 2 diabetes is the most common type, often accompanied by insulin resistance and progressive loss of pancreatic beta cell function, requiring long-term medication to control blood sugar.

[0003] Currently used oral medications for diabetes mainly include sulfonylureas, biguanides, and thiazolidinediones; however, long-term use often leads to side effects and drug resistance. Alpha-glucosidase inhibitors, as a new class of diabetes medications, reduce postprandial blood glucose spikes by slowing the rate at which starch is broken down by alpha-glucosidase in the intestine, exhibiting a unique hypoglycemic mechanism and becoming an important direction in diabetes treatment. However, existing alpha-glucosidase inhibitors still have certain side effects, making the search for novel, natural, and low-toxicity alpha-glucosidase inhibitors of significant clinical importance.

[0004] Bananas are a widely consumed fruit globally, with their peels accounting for approximately 30% of the fruit's total weight. Despite their enormous production volume, bananas are utilized at a very low rate; indiscriminate disposal not only wastes resources but also causes environmental problems. Research has found that banana peels are rich in physiologically active components such as polysaccharides, polyphenols, and organic acids, possessing potential pharmacological effects including antioxidant, antibacterial, and hypoglycemic properties. However, the key active components in banana peels that lower blood sugar and their mechanisms of action remain unclear, limiting the development and utilization of banana peel resources. Summary of the Invention

[0005] This invention aims to at least partially solve one of the technical problems in related technologies. Therefore, the main objective of this invention is to provide the use of banana peel melanin in the preparation of drugs for treating diabetes, to identify the key active ingredient in banana peel that inhibits α-glucosidase, to provide new raw materials and ideas for developing novel drugs for treating diabetes, and to achieve high-value utilization of banana peel waste.

[0006] The objective of this invention is achieved through the following technical solution: Application of banana peel melanin in the preparation of drugs for treating diabetes.

[0007] In some specific embodiments, the banana peel melanin inhibits α-glucosidase activity through an anti-competitive inhibition mechanism.

[0008] In some specific embodiments, the method for preparing banana peel melanin includes the following steps: 1) Preparation of crude extract: Banana peel and ethanol were mixed at a material-to-liquid ratio of 1:(0.5-10) (g / mL), and extracted by ultrasonication and soaking overnight. After filtration, the ethanol was recovered by rotary evaporation. The extraction and soaking process was repeated. The mixture was concentrated under reduced pressure at 40-60℃ until no ethanol was found to obtain the crude extract of banana peel. 2) Separation and purification: The aqueous suspension of the crude banana peel extract was loaded onto the pretreated AB-8 macroporous adsorption resin column and eluted with an ethanol-water gradient. The 80% and 95% ethanol eluent fractions were collected, combined, and concentrated under reduced pressure. 3) Refining: Dissolve the concentrate in ethanol, collect the insoluble substances, wash with ethanol multiple times, and dry at 40-60℃ to obtain banana peel melanin.

[0009] In some specific embodiments, during the ethanol-water gradient elution process described in step 2), water, 20%, 40%, 60%, 80%, and 95% ethanol solutions are used as eluents in sequence.

[0010] Preferably, the drug further includes a pharmaceutically acceptable carrier, which includes (but is not limited to): diluents, buffers, suspensions, emulsions, granules, encapsulation agents, excipients, fillers, binders, sprays, transdermal absorbents, humectants, disintegrants, absorption enhancers, surfactants, colorants, flavoring agents, or adsorbents.

[0011] The drug of the present invention can be prepared into various dosage forms as needed, including but not limited to tablets, solutions, granules, patches, ointments, capsules, aerosols or suppositories.

[0012] The administration route of the drug of the present invention is not limited, as long as it can achieve the desired therapeutic or preventive effect, including but not limited to oral, intravenous, intramuscular, subcutaneous, sublingual, rectal, nasal spray, oral spray, local or systemic transdermal administration.

[0013] The drug of this invention can also be used in combination with other drugs for treating diabetes. The combined use of multiple drugs can greatly improve the success rate of treatment.

[0014] Compared with the prior art, the present invention has at least the following advantages: 1) The application of banana peel melanin provided by this invention in the preparation of drugs for treating diabetes: Melanin was isolated from banana peel using techniques such as ethanol extraction, purification with AB-8 macroporous adsorption resin, and infrared spectroscopy identification. This melanin exhibits significant inhibitory activity against α-glucosidase, with a half-maximal inhibitory concentration (IC50) of [missing value]. 50 The concentration was as low as 3.04 μg / mL, showing better inhibitory effects than the positive control drug acarbose (IC50).50 (11.10 μg / mL), providing high-quality natural raw materials for the development of novel drugs for the treatment of diabetes; 2) The enzyme kinetics study of this invention shows that banana peel melanin inhibits α-glucosidase activity through an anti-competitive inhibition mechanism, which can effectively delay the decomposition and absorption of starch in the intestine, reduce postprandial blood glucose peak, and banana peel melanin is a natural product with low toxicity, few side effects, and higher safety, and can be used to prepare safe and effective hypoglycemic drugs. 3) The raw material in this invention is banana peel, which realizes the high-value utilization of banana peel waste. It transforms the originally discarded banana peel into active ingredients with important medicinal value, which not only reduces environmental pollution but also improves resource utilization, and has significant economic value and environmental significance. Moreover, the preparation method of banana peel melanin provided by this invention is simple, feasible, low-cost, and easy to industrialize, laying the foundation for its large-scale application. Attached Figure Description

[0015] To more clearly illustrate the specific embodiments of the present invention, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below.

[0016] Figure 1 The infrared spectrum of the product obtained by the preparation method provided in Example 1 of this invention; Figure 2 This is the dose-response curve of banana peel melanin inhibiting α-glucosidase in this invention; Figure 3 The Lineweaver-Burk diagram of banana peel melanin inhibiting α-glucosidase in this invention. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in further detail below with reference to the accompanying drawings and embodiments, but this should not be construed as limiting the scope of protection of this invention. Any formal equivalent modifications made based on the concept of this invention should be considered within the scope of this invention.

[0018] This invention provides a general and / or specific description of the materials and experimental methods used in the experiments. Unless otherwise specified, all experimental or testing methods are conventional methods; all reagents or instruments used, unless otherwise specified, are commercially available conventional products prepared or used using conventional methods.

[0019] Example 1: Preparation and Identification of Melanin from Banana Peel This embodiment provides a method for preparing banana peel melanin, which includes the following steps: 1) Preparation of crude extract: Take 5 kg of fresh banana peels, wash and chop them, put them in a plastic bucket, add 15 L of ethanol, extract by ultrasonication for 30 minutes, and let them stand and soak overnight; the next day, filter by vacuum filtration flask, collect the filtrate, recover the ethanol by rotary evaporation, add the recovered ethanol back to the plastic bucket to soak the filter residue, repeat the operation of ultrasonic extraction for 30 minutes and soaking overnight; combine the filtrates from the two extractions, concentrate under reduced pressure at 50℃ until there is no ethanol odor, and obtain about 120 g of crude banana peel extract; 2) Separation and purification: AB-8 macroporous adsorption resin was soaked in 95% ethanol for 24 hours, then rinsed with deionized water until no ethanol odor was detected, and packed into a 5cm×50cm chromatographic column; the crude banana peel extract was dissolved in 500mL of deionized water to prepare a suspension, which was then slowly loaded into the resin column; gradient elution was performed sequentially with 2L of deionized water, 2L of 20% ethanol, 2L of 40% ethanol, 2L of 60% ethanol, 2L of 80% ethanol, and 2L of 95% ethanol, collecting one fraction per 1L, labeled as W, B2-1, B2-2, B4-1, B4-2, B6-1, B6-2, B8-1, B8-2, B95-1, and B95-2, respectively; 3) Activity screening: The inhibitory activity of each fraction on α-glucosidase was determined; the results showed that the inhibition rates of fractions B8-1, B8-2, and B95-1 were 80.75%, 76.45%, and 70.68%, respectively, all exceeding 70%, while the inhibition rates of the remaining fractions were lower; fractions B8-1, B8-2, and B95-1 were combined and concentrated under reduced pressure to a volume of about 50 mL to obtain the concentrate; 4) Refining: Add 100mL of ethanol to the concentrate, stir for 10 minutes, let stand for 30 minutes, and centrifuge to collect the precipitate; wash the precipitate 3 times with ethanol, 50mL each time, centrifuge to collect the precipitate, and dry the precipitate in a 50℃ oven to obtain 2.3g of banana peel melanin.

[0020] This application uses infrared spectroscopy to analyze the product prepared in this embodiment to determine the possible functional groups and chemical bond types. The results are as follows: Figure 1 As shown in the figure, the infrared spectrum is similar to the infrared spectral data of melanin in black sesame and black fungus described in existing literature, which is consistent with the typical infrared spectral characteristics of melanin. It is also completely consistent with the infrared spectrum in the literature on banana peel melanin. It can be inferred that the final active substance obtained is banana peel melanin.

[0021] Test Example 1: Determination of the inhibitory activity of banana peel melanin on α-glucosidase This test example provides a method for determining the inhibitory activity of banana peel melanin on α-glucosidase, including the following test steps: 1. Solution preparation: (1) Phosphate buffer (PBS, pH 6.8, 0.1M): Weigh 35.814g Na2HPO4·12H2O and 13.799g NaH2PO4·2H2O, and dissolve them separately in 100mL of deionized water to prepare 1M solutions. Mix 49.7mL of 1M Na2HPO4 solution and 50.3mL of 1M NaH2PO4 solution, and bring the volume to 1000mL with deionized water. Verify the pH with a pH meter to be 6.8. Store at 4℃ for later use. (2) α-glucosidase solution: Accurately weigh 1 mg of 76.4 U / mg α-glucosidase, add 1 mL of PBS to dissolve it, and prepare a 76.4 U / mL stock solution. Take 20 μL of the stock solution and add it to 744 μL of PBS to dilute it to a 2.0 U / mL working solution. (3) Substrate solution: Accurately weigh 2.2594 mg pNPG, add 5 mL PBS to dissolve, and prepare a 1.5 mmol / L pNPG solution; (4) Termination solution: Weigh 1.06g of sodium carbonate, add 10mL of deionized water to dissolve it, and prepare a 1.0M Na2CO3 solution; (5) Sample solution: Accurately weigh 10 mg of banana peel melanin prepared in Example 1, dissolve it in 200 μL DMSO, add 800 μL methanol to prepare a 10 mg / mL stock solution, and then serially dilute it to a series of concentration solutions of 5, 2.5, 1.25, 0.625, 0.3125, 0.156, and 0.078 mg / mL; (6) Positive control solution: Accurately weigh 10 mg of acarbose, dissolve it in PBS to prepare a 10 mg / mL stock solution, and then serially dilute it to obtain a series of solutions of the corresponding concentrations.

[0022] 2. Inhibition Activity Assay: A 96-well plate was used for the assay, with three replicates for each group: blank group, normal reaction group, sample group, sample blank group, and positive control group. Reagents were added according to Table 1, and after thorough mixing, the mixture was incubated at 37°C for 10 min. Then, 20 μL of pNPG solution was added to each well, and the mixture was incubated at 37°C for 15 min. Finally, 100 μL of Na₂CO₃ solution was added to terminate the reaction. The absorbance (OD) value of each well was measured at 405 nm, and the inhibition rate was calculated as follows: Inhibition rate (%) = [(OD normal reaction group - OD blank group) - (OD sample group - OD sample blank group)] / (OD normal reaction group - OD blank group) × 100%.

[0023] 3. Results: Regression analysis was performed with the inhibition rate on the ordinate and the logarithm of the inhibitor concentration on the abscissa, yielding the fitted equation y = 34.521x + 33.311 (R²). 2 =0.9795 (Appendix) Figure 2 IC is calculated 50 The value was 3.04 μg / mL. The IC50 value for the positive control acarbose was... 50 The value was 11.10 μg / mL, indicating that the inhibitory activity of banana peel melanin on α-glucosidase was significantly better than that of acarbose.

[0024] Test Example 2: Determination of the type of melanin inhibition in banana peels This test example provides a method for determining the inhibition type of melanin in banana peels, specifically: 1. Experimental design: Banana peel melanin at concentrations of 2, 4, and 6 μg / mL was selected as inhibitors, and the substrate pNPG concentrations were set at 12.5, 10, 7.5, 5, and 2.5 mmol / L, respectively. A control group without inhibitors was also set up.

[0025] 2. Measurement method: The enzyme-catalyzed reaction rate was measured at different substrate and inhibitor concentrations according to the inhibition activity measurement method in Example 2.

[0026] 3. Data processing: Plot a Lineweaver-Burk plot with the reciprocal of the enzyme reaction rate (1 / v) on the ordinate and the reciprocal of the substrate concentration (1 / [S]) on the abscissa.

[0027] 4. Results: Lineweaver-Burk plot (attached) Figure 3 The results show that the lines corresponding to different concentrations of inhibitors are parallel to each other, indicating that the inhibition of α-glucosidase by banana peel melanin is an anti-competitive inhibition, that is, melanin only binds to the enzyme-substrate complex, thereby inhibiting the enzymatic reaction.

[0028] Test Example 3: Banana peel melanin inhibits postprandial blood glucose elevation in mice This test example provides a method for testing the effect of banana peel melanin on blood sugar, specifically: Six-week-old female C57BL / 6J mice weighing 16-20g were used. A 12-hour light-dark cycle was maintained at 22±1℃, and mice were fed standard pelleted feed and water. After one week of acclimatization, the mice were randomly divided into three groups (n=8 per group). Sucrose or maltose, along with inhibitors (banana peel melanin and acarbose), were dissolved in 0.5% sodium carboxymethyl cellulose (CMC-Na) solution. Banana peel melanin was tested at a dose of 30 mg / kg body weight (BW), while acarbose was evaluated at a dose of 50 mg / kg BW. Depending on their mechanism of action, acarbose was administered 15 minutes prior to meal (before a meal), while banana peel melanin was administered concurrently with the glucose (with a meal). Glucose administration was performed after a 16-hour fast. Blood samples were collected from the tail vein at 0, 30, 60, and 120 minutes after maltose loading, and blood glucose levels were measured using an Accu-Chek glucometer (Roche, Germany).

[0029] The results showed that after oral administration of maltose, the blood glucose level in the control group rapidly increased from 4.2 mM to a maximum of 20.3 mM within 30 minutes, and then returned to the pre-treated level at 120 minutes. Compared with the control group, banana peel melanin significantly inhibited the increase in blood glucose levels at 30 and 60 minutes, and the inhibitory effect was better than that of acarbose at lower dose levels. According to the area under the blood glucose curve from 0 to 120 minutes after a meal, the banana peel melanin treatment reduced blood glucose levels by 35% compared with the control group, which was better than the 18.5% reduction achieved by acarbose.

[0030] Banana peel melanin has superior α-glucosidase inhibitory activity compared to acarbose, and it also showed a significant effect in inhibiting the rise in postprandial blood glucose levels in mice. Therefore, banana peel melanin can be used as an α-glucosidase inhibitor to treat diabetes and control postprandial blood glucose.

[0031] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. Application of banana peel melanin in the preparation of drugs for treating diabetes.

2. The application according to claim 1, characterized in that, The banana peel melanin inhibits α-glucosidase activity through an anti-competitive inhibition mechanism.

3. The application according to claim 1, characterized in that, The dosage form of the drug includes, but is not limited to, tablets, capsules, granules, oral liquids, suspensions, or injections.

4. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable carriers or excipients.

5. The application according to claim 4, characterized in that, The method for preparing banana peel melanin includes the following steps: 1) Preparation of crude extract: Banana peel and ethanol were mixed at a material-to-liquid ratio of 1:(0.5-10) (g / mL), and extracted by ultrasonication and soaking overnight. After filtration, the ethanol was recovered by rotary evaporation. The extraction and soaking process was repeated. The mixture was concentrated under reduced pressure at 40-60℃ until no ethanol was found to obtain the crude extract of banana peel. 2) Separation and purification: The aqueous suspension of the crude banana peel extract was loaded onto the pretreated AB-8 macroporous adsorption resin column and eluted with an ethanol-water gradient. The 80% and 95% ethanol eluent fractions were collected, combined, and concentrated under reduced pressure. 3) Refining: Dissolve the concentrate in ethanol, collect the insoluble substances, wash with ethanol multiple times, and dry at 40-60℃ to obtain banana peel melanin.

6. The application according to claim 1, characterized in that, In step 2), during the ethanol-water gradient elution process, water, 20%, 40%, 60%, 80%, and 95% ethanol solutions are used as eluents in sequence.

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