Lotus seed flavone extract, extraction method and application thereof

Abstract

The application discloses a lotus seed flavone extract and an extraction method and application thereof, and the extraction method comprises the following steps: crushing dried lotus seed peels, placing the crushed lotus seed peels in an ethanol solution for reflux extraction, filtering, and collecting the extract; concentrating the extract, adsorbing and eluting impurities by using a macroporous adsorption resin, eluting by using an ethanol solution with a volume fraction of 60-80%, and collecting the eluate; and concentrating, drying and crushing the eluate to obtain the lotus seed flavone extract; the combination process of the extraction method, i.e., the combination process of ethanol hot reflux extraction and macroporous resin purification, can effectively enrich flavone components in the lotus seed peels, and the obtained extract has significant inhibitory activity on alpha-glucosidase.

Description

Technical Field

[0001] This invention relates to the field of flavonoid extract technology, specifically to a lotus seed flavonoid extract, its extraction method, and its applications. Background Technology

[0002] Alpha-glucosidases are key enzymes located on the brush border membrane of the small intestine, responsible for hydrolyzing oligosaccharides and disaccharides (such as sucrose and maltose) in food into glucose for absorption in the small intestine. Inhibiting alpha-glucosidase activity can slow down the rate of carbohydrate digestion and absorption, thereby effectively reducing postprandial blood glucose spikes. Based on this principle, alpha-glucosidase inhibitors have become one of the first-line drugs for the clinical treatment of type 2 diabetes.

[0003] Currently, commonly used α-glucosidase inhibitors in clinical practice mainly include acarbose, voglibose, and miglitol. Among them, acarbose is the most widely used α-glucosidase inhibitor. Its mechanism of action is to competitively inhibit α-glucosidase on the brush border of the small intestine, delaying the breakdown of carbohydrates and the absorption of glucose. However, long-term use of these drugs is often accompanied by a series of adverse reactions. The most common adverse reactions of acarbose are gastrointestinal symptoms, including bloating, diarrhea, flatulence, and abdominal pain, with an incidence rate of 30% to 50%. The mechanism of these adverse reactions is that undigested carbohydrates are fermented by intestinal flora in the colon, producing a large amount of gas, thus leading to gastrointestinal discomfort. In addition, some patients may also experience systemic side effects such as elevated liver enzymes. These adverse reactions seriously affect patients' medication adherence and quality of life. Therefore, the development of novel α-glucosidase inhibitors that are highly effective, low in toxicity, and naturally derived has significant clinical significance and market value. Summary of the Invention

[0004] The purpose of this invention is to provide a lotus seed flavonoid extract, its extraction method, and its application. The extraction method of this invention uses a combination of ethanol hot reflux extraction and macroporous resin purification, which can effectively enrich the flavonoid components of lotus seed peel. The obtained extract has significant inhibitory activity against α-glucosidase.

[0005] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0006] The first aspect of this invention provides a method for extracting lotus seed flavonoids, the extraction method comprising the following steps:

[0007] (a) After crushing the dried lotus seed peel, it was placed in an ethanol solution for reflux extraction, filtered, and the extract was collected;

[0008] (b) After concentrating the extract, adsorb and elute it with macroporous adsorption resin to remove impurities, and then elute it with 60%~80% ethanol solution by volume, and collect the eluent;

[0009] (c) The eluent is concentrated, dried and pulverized to obtain the lotus seed flavonoid extract.

[0010] Preferably, in step (a), the volume fraction of the ethanol solution is 50% to 70%.

[0011] Preferably, in step (a), the reflux extraction temperature is 60~80℃ and the time is 1~3h.

[0012] Preferably, in step (a), the mass-to-volume ratio of lotus seed peel to ethanol solution is 1:(15~25).

[0013] Preferably, in step (b), the macroporous adsorption resin is a D101 type or an AB-8 type macroporous adsorption resin.

[0014] Preferably, in step (b), the extract is concentrated to 0.2 to 0.3 times its original volume.

[0015] Preferably, in step (b), the elution and impurity removal includes elution with distilled water and 20%~40% ethanol solution in sequence.

[0016] Preferably, in step (b), the adsorption loading flow rate is 1.2~1.8 BV / h.

[0017] A second aspect of the present invention provides a lotus seed flavonoid extract obtained by the above extraction method.

[0018] A third aspect of the present invention provides the application of lotus seed flavonoid extract obtained by the above extraction method as an α-glucosidase inhibitor.

[0019] Compared with the prior art, the beneficial effects of the present invention include at least the following:

[0020] The IC50 of lotus seed flavonoid extract against α-glucosidase in this invention 50 The concentration was ≤0.5 mg / mL, which is significantly better than the α-glucosidase inhibitory effect of lotus seed extract in the prior art; experiments have confirmed that at a concentration of 2.0 mg / mL, the inhibition rate of lotus seed flavonoid extract against α-glucosidase is more than 85%.

[0021] This invention employs a combined process of ethanol hot reflux extraction and macroporous resin purification, avoiding the use of complex equipment such as high-speed countercurrent chromatography. The method is simple to operate, low in cost, and suitable for industrial production. In the macroporous resin purification step, gradient elution effectively removes water-soluble impurities, low-polarity impurities, and flavonoids, achieving targeted enrichment of flavonoids. The total flavonoid content of the lotus seed flavonoid extract of this invention is ≥60%, significantly superior to existing technologies. Furthermore, lotus seed peel is a byproduct of lotus seed processing and is abundant. The implementation of this invention can provide a new avenue for the high-value utilization of lotus seed peel. Attached Figure Description

[0022] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to scale.

[0023] Figure 1 The curve showing the α-glucosidase inhibition rate of the lotus seed flavonoid extract extracted with 40% ethanol in the experimental examples of this invention;

[0024] Figure 2 The curve showing the α-glucosidase inhibition rate of the lotus seed flavonoid extract extracted with 50% ethanol in the experimental examples of this invention;

[0025] Figure 3 The curve showing the α-glucosidase inhibition rate of the lotus seed flavonoid extract extracted with 60% ethanol in the experimental examples of this invention.

[0026] Figure 4 The curve showing the α-glucosidase inhibition rate of the lotus seed flavonoid extract extracted with 70% ethanol in the experimental examples of this invention;

[0027] Figure 5 The curve showing the α-glucosidase inhibition rate of the lotus seed flavonoid extract extracted with 80% ethanol in the experimental examples of this invention;

[0028] Figure 6 The curve of α-glucosidase inhibition rate of lotus seed flavonoid extract extracted with 90% ethanol in the experimental example of this invention. Detailed Implementation

[0029] The embodiments of the technical solution of the present invention will be described in detail below with reference to the examples. The following embodiments are only used to illustrate the technical solution of the present invention more clearly, and are therefore only examples, and should not be used to limit the scope of protection of the present invention.

[0030] It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0031] This invention provides a method for extracting lotus seed flavonoids, the extraction method comprising the following steps:

[0032] (a) After crushing the dried lotus seed peel, it was placed in an ethanol solution for reflux extraction, filtered, and the extract was collected;

[0033] (b) After concentrating the extract, adsorb and elute it with macroporous adsorption resin to remove impurities, and then elute it with 60%~80% ethanol solution by volume, and collect the eluent;

[0034] (c) The eluent is concentrated, dried and pulverized to obtain the lotus seed flavonoid extract.

[0035] In one embodiment, in step (a), the volume fraction of the ethanol solution is 50% to 70%.

[0036] In one embodiment, in step (a), the reflux extraction temperature is 60~80℃ and the time is 1~3h.

[0037] In one embodiment, in step (a), the mass-to-volume ratio of lotus seed peel to ethanol solution is 1:(15~25).

[0038] In one embodiment, in step (b), the macroporous adsorption resin is a D101 type or an AB-8 type macroporous adsorption resin.

[0039] In one embodiment, in step (b), the extract is concentrated to 0.2 to 0.3 times its original volume.

[0040] In one embodiment, step (b) involves elution with distilled water and a 20% to 40% ethanol solution in sequence.

[0041] In one embodiment, in step (b), the adsorption loading flow rate is 1.2~1.8 BV / h.

[0042] Another embodiment of the present invention provides a lotus seed flavonoid extract obtained by the above extraction method.

[0043] In another embodiment of the present invention, the lotus seed flavonoid extract obtained by the above extraction method is used as an α-glucosidase inhibitor.

[0044] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0045] Example

[0046] This embodiment describes a method for extracting lotus seed flavonoids, which includes the following steps:

[0047] (a) The dried lotus seed peel was crushed and placed in a 60% ethanol solution for reflux extraction at a material-to-liquid ratio of 1:20 (w / v). The extract was filtered and collected. The reflux extraction temperature was 70°C and the time was 2 hours.

[0048] (b) The extract was concentrated to 0.2 times its original volume and adsorbed on an AB-8 macroporous adsorption resin column (column volume 500 ml) (sample loading flow rate 1.5 BV / h). The extract was eluted with distilled water (3 BV) and 30% ethanol solution (3 BV) to remove impurities, and then eluted with 70% ethanol solution (5 BV) and collected.

[0049] (c) The eluent is concentrated, dried and pulverized at 60°C to obtain the lotus seed flavonoid extract.

[0050] Experimental Example

[0051] 1. Using the total flavonoid extract yield as an indicator, the ethanol volume fraction, solid-liquid ratio, extraction temperature, and extraction time were optimized according to the methods in the examples.

[0052] Ethanol volume fraction: 40%, 50%, 60%, 70%, 80%;

[0053] Material-to-liquid ratio: 1:10, 1:15, 1:20, 1:25, 1:30;

[0054] Extraction temperatures: 50℃, 60℃, 70℃, 80℃, 90℃;

[0055] Extraction time: 0.5h, 1h, 2h, 3h, 4h;

[0056] The total flavonoid yield under different conditions is shown in Tables 1 to 4.

[0057] Table 1

[0058] ethanol volume fraction 40% 50% 60% 70% 80% Yield (mg / g) 82.4 98.6 115.7 103.2 89.5

[0059] Table 2

[0060] Material-liquid ratio 1∶10 1∶15 1∶20 1∶25 1∶30 Yield (mg / g) 87.3 102.8 115.2 114.6 113.1

[0061] Table 3

[0062] Extraction temperature 50℃ 60℃ 70℃ 80℃ 90℃ Yield (mg / g) 79.5 97.2 114.9 105.3 92.4

[0063] Table 4

[0064] Extraction time 0.5h 1h 2h 3h 4h Yield (mg / g) 68.7 93.4 115.5 109.8 101.6

[0065] As shown in Tables 1-4:

[0066] The optimal extraction conditions are: 60% ethanol (volume fraction), 1:20 solid-liquid ratio, 70℃ extraction temperature, and 2h extraction time.

[0067] 2. Using the α-glucosidase inhibitory activity of lotus seed flavonoid extract as an indicator, the effect of the volume fraction concentration of ethanol solution (40%, 50%, 60%, 70%, 80%, 90%) in the elution step (b) of the method in the example on the efficacy of the extracted lotus seed flavonoid extract was optimized.

[0068] The reaction system consisted of 870 μl of 67 mmol / L potassium phosphate buffer (37℃, pH 6.8), 25 μl of 1 mg / ml glutathione solution, 35 μl of 2.74 U / ml α-glucosidase solution, and 20 μl of lotus seed flavonoid extract (0.1, 0.5, 1.0, 1.5, 2.0, 2.5 mg / ml). After incubating at 37℃ for 10 min, 70 μl of 15 mmol / L pNPG solution was added, and the mixture was incubated in a water bath for 10 min. Then, 4 ml of 0.1 mol / L Na2CO3 solution was added. The system was zeroed using a blank tube, and the absorbance (A) was measured at 405 nm.

[0069] The formula for calculating the α-glucosidase inhibition rate is as follows: Enzyme activity inhibition rate (%) = [1 - (A sample - A background)] / (A enzyme - A blank) × 100%. Where, A sample = enzyme solution + reduced glutathione solution + sample solution + PNPG solution, A background = enzyme solution + reduced glutathione solution + sample solution, A enzyme = enzyme solution + reduced glutathione solution + PNPG solution, A blank = enzyme solution + reduced glutathione solution, and the volumes of all solutions are made up with buffer solution.

[0070] The experimental results are as follows: Figures 1-6 As shown, Figure 1 The inhibition rate of lotus seed extract against α-glucosidase was prepared using an ethanol solution with a volume fraction concentration of 40%. Figure 2 The inhibition rate of lotus seed extract against α-glucosidase was prepared using an ethanol solution with a volume fraction concentration of 50%. Figure 3 The inhibition rate of lotus seed extract against α-glucosidase was prepared using an ethanol solution with a volume fraction concentration of 60%. Figure 4 The inhibition rate of lotus seed extract against α-glucosidase was prepared using an ethanol solution with a volume fraction concentration of 70%. Figure 5 The inhibition rate of lotus seed extract against α-glucosidase was prepared using an ethanol solution with a volume fraction concentration of 80%. Figure 6 The inhibition rate of lotus seed extract against α-glucosidase was prepared using an ethanol solution with a volume fraction concentration of 90%.

[0071] Depend on Figures 1-6 It can be seen that lotus seed flavonoid extract has a significant inhibitory effect on α-glucosidase, and the lotus seed flavonoid extract prepared with an ethanol solution volume fraction of 70% showed the best effect (IC50). 50 =0.32 mg / mL).

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. 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. A method for extracting lotus seed flavonoids, characterized in that, The extraction method includes the following steps: (a) After crushing the dried lotus seed peel, it was placed in an ethanol solution for reflux extraction, filtered, and the extract was collected; (b) After concentrating the extract, adsorb and elute it with macroporous adsorption resin to remove impurities, and then elute it with 60%~80% ethanol solution by volume, and collect the eluent; (c) The eluent is concentrated, dried and pulverized to obtain the lotus seed flavonoid extract.

2. The extraction method according to claim 1, characterized in that, In step (a), the volume fraction of the ethanol solution is 50% to 70%.

3. The extraction method according to claim 1, characterized in that, In step (a), the reflux extraction temperature is 60~80℃ and the time is 1~3h.

4. The extraction method according to claim 1, characterized in that, In step (a), the mass-to-volume ratio of lotus seed peel to ethanol solution is 1:(15~25).

5. The extraction method according to claim 1, characterized in that, In step (b), the macroporous adsorption resin is a D101 type or an AB-8 type macroporous adsorption resin.

6. The extraction method according to claim 1, characterized in that, In step (b), the extract is concentrated to 0.2 to 0.3 times its original volume.

7. The extraction method according to claim 1, characterized in that, In step (b), the elution and impurity removal includes elution with distilled water and 20%~40% ethanol solution in sequence.

8. The extraction method according to claim 1, characterized in that, In step (b), the adsorption loading flow rate is 1.2~1.8 BV / h.

9. The lotus seed flavonoid extract obtained by the extraction method according to any one of claims 1 to 8.

10. The use of the lotus seed flavonoid extract obtained by the extraction method according to any one of claims 1 to 8 as an α-glucosidase inhibitor.

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