A green preparation method for hyperoside derived from lotus leaves and its application

Abstract

This invention provides a green preparation method for hyperoside derived from lotus leaves and its application, belonging to the field of natural product separation and purification technology. Addressing the shortcomings of existing preparation methods, this invention, for the first time, uses abundant and inexpensive lotus leaves and their extracts as raw materials, employing green solvents to establish a green, efficient, high-purity, simple, rapid, and scalable method for preparing hyperoside. This provides raw materials for the development of hyperoside series products and therefore has significant practical application value.

Description

Technical Field

[0001] This invention belongs to the field of natural product separation and purification technology, specifically relating to a green preparation method of hyperoside derived from lotus leaves and its application. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Hypericin belongs to the flavonol glycoside class of compounds and has a wide range of pharmacological effects. Literature reports that hypericin has significant local analgesic effects, stronger than aspirin but weaker than morphine, and without dependence; it also has significant protective effects on the cardiovascular and cerebrovascular systems, showing good efficacy against myocardial and cerebral ischemia-reperfusion disorders and cerebral infarction; hypericin has a strong inhibitory effect on ocular aldose reductase, and has potential applications in the prevention of diabetic cataracts. In addition, hypericin also possesses various physiological activities such as anti-inflammatory, antispasmodic, diuretic, antitussive, antihypertensive, cholesterol-lowering, and protein-assimilating effects, making it of significant application and development value in the food, pharmaceutical, and cosmetic fields.

[0004] Currently, hyperoside is mainly derived from extracts of Hypericaceae plants, which presents challenges such as high organic solvent consumption, complex processes, the need for specialized equipment, and high costs. Chemical synthesis is also difficult, and there are no reports of preparation using lotus leaves and their extracts, especially using green processes. The applicant previously developed a method for simultaneously separating and purifying lotus leaf flavonoids and polysaccharides (patent number ZL201510150815.2). This method involves extracting lotus leaves with an alkaline aqueous solution to obtain an extract, adsorbing the lotus leaf flavonoids and polysaccharides from the extract using a macroporous resin composition, and then sequentially rinsing with water and eluting with ethanol to obtain lotus leaf flavonoids. To further develop the application of lotus leaf flavonoids in food additives, cosmetics, and pharmaceutical intermediates, it is necessary to develop a method for preparing hyperoside. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a green preparation method for hyperoside derived from lotus leaves and its applications. This invention, for the first time, utilizes abundant and inexpensive lotus leaves and their extracts as raw materials, employing green solvents to establish a green, efficient, high-purity, simple, rapid, and scalable method for preparing hyperoside. This method provides raw materials for the development of hyperoside-based products and therefore has significant practical application value.

[0006] To achieve the above-mentioned technical objectives, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides a green method for preparing hyperoside derived from lotus leaves, the method comprising the following steps:

[0008] S1. Dissolve lotus leaf flavonoids in the first solvent, filter, and dry to obtain an extract;

[0009] S2. Dissolve the extract obtained in step S1 using a second solvent and filter to obtain a filtrate.

[0010] S3. Add the third solvent to the filtrate obtained in step S2, filter, and obtain the filtrate.

[0011] S4. Cool the filtrate obtained in step S3 to allow it to crystallize.

[0012] A second aspect of this invention provides the application of the above-described method in the industrial production of hyperoside. Because the above-described preparation and purification method is green and pollution-free, simple to operate, low in cost, and yields a high product, it is very suitable for industrial production.

[0013] The beneficial technical effects of one or more of the above technical solutions are as follows:

[0014] The above-mentioned hyperoside preparation and purification process uses green solvents for impurity removal, generating very little waste. The purification process uses only conventional acids and bases, and the final separation is achieved through crystallization. The operation is relatively simple, and the hyperoside product does not introduce impurities such as inorganic salts.

[0015] The hyperoside preparation method involved in the above technical solution is low-cost, efficient and rapid, and has a high product yield, thus it has good prospects for practical industrial application. Attached Figure Description

[0016] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0017] Figure 1 The chromatogram is of the hyperoside product prepared in Example 1 of this invention.

[0018] Figure 2 Hyperoside product prepared in Example 1 of this invention 1 H-NMR spectrum. Detailed Implementation

[0019] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0020] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0021] The present invention will now be further illustrated with specific examples. These examples are for illustrative purposes only and do not limit the scope of the invention. Unless otherwise specified, experimental conditions not explicitly stated in the examples are generally performed under conventional conditions or as recommended by the reagent company. Unless otherwise specified, all reagents and consumables used in the following examples are commercially available.

[0022] In a typical embodiment of the present invention, a green preparation method for hyperoside derived from lotus leaves is provided, the method comprising the following steps:

[0023] S1. Dissolve lotus leaf flavonoids in the first solvent, then separate the solid and liquid components, and dry the solid separated product to obtain an extract;

[0024] S2. Dissolve the extract obtained in step S1 using a second solvent, and separate the solid and liquid to obtain a liquid.

[0025] S3. Add a third solvent to the liquid obtained in step S2, and perform solid-liquid separation to obtain the liquid.

[0026] S4. Cool the liquid obtained in step S3 to allow it to crystallize.

[0027] In another specific embodiment of the present invention, the lotus leaf flavonoids in step S1 are not specifically limited, as long as the total flavonoids are obtained through lotus leaf extraction. In one specific embodiment of the present invention, the lotus leaf flavonoids are obtained using the preparation method provided by ZL201510150815.2.

[0028] The first solvent can be any one or more of ethanol, acetone, and water;

[0029] The ethanol can be high-concentration ethanol, specifically, the ethanol is not less than 80%, such as 80%, 85%, 90%, 95% or anhydrous ethanol.

[0030] The mass-to-volume ratio of lotus leaf flavonoids to the first solvent is 1:5-30 (g / ml);

[0031] The drying process can be carried out by evaporation drying.

[0032] In step S2, the second solvent is an alkaline solution, and the alkaline is selected from any one or more of sodium bicarbonate, sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, triethylamine, pyridine, and ammonia; more preferably, the alkaline is sodium hydroxide or potassium hydroxide.

[0033] The concentration of the alkali solution is controlled to be 0.01 to 1 M; in one specific embodiment of the present invention, the concentration of the alkali solution is 0.1 M.

[0034] The mass-to-volume ratio of lotus leaf flavonoids to the second solvent is 1:5-30 (g / ml); more preferably 1:10-30 (g / ml);

[0035] In step S3, the third solvent is an acid solution, and the acid is selected from any one or more of hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, and trichloroacetic acid. More preferably, the acid is hydrochloric acid.

[0036] The concentration of the acid solution is controlled to be 0.01–1 M; in one specific embodiment of the present invention, the concentration of the acid solution is 0.1 M.

[0037] The mass-to-volume ratio of lotus leaf flavonoids to the third solvent is 1:0.5-30 (g / ml); more preferably 1:10-30 (g / ml).

[0038] In steps S1-S3, solid-liquid separation can be performed by filtration, centrifugation, sedimentation, etc., preferably by filtration. Specifically, the filtration is microfiltration. In one specific embodiment of the present invention, the microfiltration is performed using a microporous membrane with a pore size of 0.22 μm.

[0039] In step S4, the cooling crystallization process is specifically carried out at a temperature of -4℃ to 6℃, and the duration of the cooling crystallization is controlled to be 20-60 minutes; then it is placed at room temperature.

[0040] The method further includes separating the crystals or precipitates obtained in step S4 from the mother liquor and drying them. The crystals or precipitates obtained by the above method are high-purity hyperoside.

[0041] In another specific embodiment of the present invention, the application of the above method in the industrial production of hyperoside is provided. Because the above preparation and purification method is green and pollution-free, simple to operate, low in cost, and yields a high product, it is very suitable for industrial production.

[0042] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention. The following examples are experimental methods with specific conditions specified, and are generally carried out under conventional conditions. In the following examples and comparative examples, microfiltration was performed using 0.22 μm microporous membranes.

[0043] Example 1

[0044] Take 0.1g of lotus leaf flavonoids (prepared according to the method of Example 1 of ZL201510150815.2), dissolve in 3.0ml of anhydrous ethanol, microfilter, evaporate the microfiltrate to dryness to obtain a brownish-yellow extract; dissolve the extract in 2.5ml of 0.1M sodium hydroxide solution, microfilter, add 2.8ml of 0.1M hydrochloric acid to the filtrate while shaking, refrigerate at 4℃ for 20min, let stand at room temperature overnight, dry the pale yellow precipitate to obtain 40mg of hyperoside product with a product content of 93.7%.

[0045] Example 2

[0046] Take 0.5g of lotus leaf flavonoids (prepared according to the method of Example 1 of ZL201510150815.2), dissolve in 2.5ml of anhydrous ethanol, microfilter, evaporate the microfiltrate to dryness, and obtain a brownish-yellow extract; dissolve the extract in 5ml of 0.01M potassium hydroxide solution, microfilter, add 5ml of 0.005M sulfuric acid to the filtrate while shaking, refrigerate at 4℃ for 60min to cool and crystallize, let stand at room temperature overnight, dry the pale yellow precipitate, and obtain 105mg of hyperoside product with a product content of 95.5%.

[0047] Example 3

[0048] Take 0.1g of lotus leaf flavonoids (prepared according to the method of Example 2 of ZL201510150815.2), dissolve in 2.5ml of 80% ethanol, microfilter, evaporate the microfiltrate to dryness to obtain a brownish-yellow extract; dissolve the extract in 2.5ml of 0.1M sodium hydroxide solution, microfilter, add 2.8ml of 0.1M hydrochloric acid to the filtrate while shaking, refrigerate at 4℃ for 20min, let stand at room temperature overnight, dry the pale yellow precipitate to obtain 40mg of hyperoside product with a product content of 92.6%.

[0049] Example 4

[0050] Take 0.1g of lotus leaf flavonoids (prepared using the method of Example 3, ZL201510150815.2), dissolve in 2.5ml of acetone, microfilter, and evaporate the microfiltrate to dryness to obtain a brownish-yellow extract; dissolve the extract in 2.5ml of 0.05M sodium carbonate solution, microfilter, add 2.8ml of 0.1M hydrochloric acid to the filtrate while shaking, refrigerate at 4℃ for 20min, leave at room temperature overnight, dry the pale yellow precipitate to obtain 41mg of hyperoside product with a product content of 91.8%.

[0051] Example 5

[0052] Take 0.1g of lotus leaf flavonoids (prepared using the method of Example 4, ZL201510150815.2), dissolve in 2.5ml of ethanol, microfilter, and evaporate the microfiltrate to dryness to obtain a brownish-yellow extract; dissolve the extract in 4.0ml of water and 1.0ml of 0.1M sodium hydroxide solution, microfilter, add 55μl of 1.0M hydrochloric acid to the filtrate while shaking, refrigerate for 20min, leave at room temperature overnight, dry the pale yellow precipitate to obtain 43mg of hyperoside product with a product content of 95.7%.

[0053] Example 6

[0054] Take 0.1g of lotus leaf flavonoids (prepared using the method in Example 5 of ZL201510150815.2), dissolve in 2.5ml of water, microfilter, and evaporate the microfiltrate to dryness to obtain a brownish-yellow extract; dissolve the extract in 2.5ml of 0.1M sodium hydroxide solution, microfilter, add 2.8ml of 0.1M hydrochloric acid to the filtrate while shaking, refrigerate at 4℃ for 20min, leave at room temperature overnight, dry the pale yellow precipitate to obtain 40mg of hyperoside product with a product content of 89.8%.

[0055] Example 7

[0056] Take 0.1g of lotus leaf flavonoids (prepared according to the method of Example 1 of ZL201510150815.2), dissolve in 2.5ml of anhydrous ethanol, centrifuge at 3000rpm for 10min, evaporate the supernatant to dryness, and obtain a brownish-yellow extract; dissolve the extract in 2.5ml of 0.1M sodium hydroxide solution, centrifuge at 8000rpm for 5min, add 2.6ml of 0.1M hydrochloric acid to the supernatant while shaking, refrigerate at 4℃ for 60min, let stand at room temperature overnight, dry the pale yellow precipitate, and obtain 42mg of hyperoside product with a product content of 92.8%.

[0057] Comparative Example 1

[0058] Take 0.1g of lotus leaf flavonoids (prepared according to the method of Example 1 of ZL201510150815.2), dissolve in 2.5ml of anhydrous ethanol, centrifuge at 3000rpm for 10min, evaporate the supernatant to dryness to obtain a brownish-yellow extract; dissolve the extract in 2.5ml of 0.1M sodium hydroxide solution, centrifuge at 8000rpm for 5min, add 2.0ml of 0.1M hydrochloric acid to the supernatant while shaking, refrigerate at 4℃ for 60min, and leave at room temperature overnight, no obvious precipitate is formed.

[0059] Comparative Example 2

[0060] Take 0.1g of lotus leaf extract (specification 10:1, Kepler Biotechnology), dissolve it in 2.5ml of anhydrous ethanol, centrifuge at 3000rpm for 10min, evaporate the supernatant to dryness to obtain a brownish-yellow extract; dissolve the extract in 2.5ml of 0.1M sodium hydroxide solution, centrifuge at 8000rpm for 5min, add 2.6ml of 0.1M hydrochloric acid to the supernatant while shaking, refrigerate at 4℃ for 60min, let stand at room temperature overnight, dry the pale yellow precipitate to obtain 17.4mg of hyperoside product, with a product content of 53.6%.

[0061] Experimental Example 1: HPLC Analysis of Hyperoside

[0062] The purity of hyperoside was determined under given conditions by high performance liquid chromatography (Appendix VI D, Part I of the 2020 Pharmacopoeia).

[0063] 1) Instruments, reagents and chromatographic conditions

[0064] Instruments: Shimadzu LC-20A high performance liquid chromatograph; CBM-20Alite system controller; LC-20AT infusion pump; CT0-20A column oven; SIL-20A autosampler; 10A-RF UV detector; LC solution workstation.

[0065] Reagents: Methanol (chromatographic grade, Tedia); Acetonitrile (chromatographic grade, Tedia); Phosphoric acid (chromatographic grade, Tedia); Water was ultrapure water.

[0066] Chromatographic conditions: C 18 The chromatographic column (250*4.6mm, 5μm) was used at 30℃. The mobile phase consisted of methanol (A) and 0.1% phosphoric acid water (B). The linear gradient elution was as follows: 0–18 min, 14–28% A; 18–30 min, 28–35% A; 30–55 min, 35–69% A; 55–65 min, 69%–100% A. The injection volume was 10 μl, the flow rate was 1.0 mL / min, and the detection wavelength was 360 nm.

[0067] 2) Preparation of reference solution

[0068] Take an appropriate amount of hyperoside reference standard, accurately weigh it, and add methanol to prepare a reference solution containing 360 μg hyperoside per 1 ml.

[0069] 3) Preparation of the test solution

[0070] An appropriate amount of the hyperoside product prepared in Example 1 was accurately weighed and placed in a 100ml volumetric flask. 60% ethanol was added to dissolve and dilute to the mark to prepare a hyperoside sample solution with a concentration of 0.92mg / ml. The solution was then filtered through a microporous membrane to obtain the final product.

[0071] The results showed that the hyperoside product in Example 1 had a purity of 93.7%. The extraction and purification technology involved in this invention can effectively remove coexisting impurities of hyperoside. The chromatogram is shown below. Figure 1 .

[0072] Experimental Example 2 Hyperoside 1 H-NMR test:

[0073] The hyperoside product prepared in Example 1, 1 H-NMR see Figure 2 Spectral analysis revealed the following: δ 4.293-5.285 represents the hydrogen atoms of the substituted glycosyl -OH group; δ 6.076-6.702 ppm represents the hydrogen atoms of the 2-substituted benzene ring; δ 7.398-7.555 ppm represents the two hydrogen atoms of the flavonoid parent benzene ring; δ 9.030 ppm represents the hydrogen atoms of the 2-substituted benzene ring -OH group; δ 9.605 ppm represents the hydrogen atoms of the 2-substituted benzene ring -OH group; δ 10.741 ppm represents the hydrogen atoms of the -OH group at position 7 of the benzene ring; and δ 12.512 ppm represents the hydrogen atoms of the -OH group at position 5 of the benzene ring.

[0074]

[0075] Experimental Example 3: Antioxidant Activity of Hypericin (DPPH Method):

[0076] 1) Solution preparation

[0077] DPPH-ethanol solution: Weigh 9 mg of DPPH and dissolve it in 200 mL of anhydrous ethanol. Dissolve by sonication to prepare a 0.0045% DPPH-ethanol solution.

[0078] Sample solution: Prepare an appropriate amount of hyperoside (or lotus leaf flavonoids) in the example, dissolve it in 60% ethanol and make up to volume to prepare a solution with a concentration of 0.06 mg / mL.

[0079] 2) Detection

[0080] Measure 2.0 mL of DPPH-ethanol solution and add it to the sample solution in small, repeated additions while shaking. The volume added when the solution color has essentially faded is the maximum volume of sample solution (0.8 mL). Subsequent additions of 0.05, 0.2, 0.35, 0.5, and 0.65 mL are then used. The same method is used to prepare five additional volumes of lotus leaf flavonoid solution: 0.05, 0.2, 0.35, 0.5, and 0.65 mL.

[0081] Add a certain amount of sample solution to 2.0 mL of DPPH-ethanol solution, mix well, and react for 0.5 h. Use anhydrous ethanol as a blank control, measure absorbance A at 517 nm, and calculate the scavenging rate of hyperoside on DPPH free radicals.

[0082] The results showed that the scavenging rate of hyperoside solution against DPPH free radicals in the concentration range of 0.001–0.01 mg / mL was positively correlated with its concentration, with a linear equation of I = 4.33 + 2.539x (r = 0.9973), indicating that the prepared hyperoside has a strong DPPH free radical scavenging ability.

[0083] Matters not covered in this invention are common knowledge.

[0084] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A process for the green preparation of hyperoside derived from lotus leaves, characterized in that, The method comprises the following steps: S1, dissolving lotus leaf flavone into a first solvent, then solid-liquid separation, drying the solid separation to obtain an extract; the first solvent is any one or more of ethanol, acetone, water; S2, dissolving the extract obtained in step S1 using a second solvent, solid-liquid separation to obtain a liquid; the second solvent is an alkali liquor, the alkali is selected from any one or more of sodium bicarbonate, sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, triethylamine, pyridine, ammonia; the concentration of the alkali liquor is controlled to be 0.01-1M; S3, adding a third solvent to the liquid obtained in step S2, solid-liquid separation to obtain a liquid; The third solvent is an acid liquor, the acid is selected from any one or more of hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, trichloroacetic acid; the concentration of the acid liquor is controlled to be 0.01-1M; S4, performing a cooling treatment to crystallize the liquid obtained in step S3 to obtain a crystal or precipitate; The process of cooling treatment to crystallize is specifically performed at a temperature of-4℃-6℃, and the duration of cooling crystallization is controlled to be 20-60min; then it is placed at room temperature.

2. The method of claim 1, wherein, In step S1, the ethanol is high-concentration ethanol.

3. The method of claim 2, wherein, In step S1, the ethanol is not less than 80% ethanol.

4. The method of claim 1, wherein, In step S1, the mass-volume ratio of lotus leaf flavone to the first solvent is 1:5-30, g / ml; The drying is performed by evaporation drying.

5. The method of claim 1, wherein, In step S2, the alkali is sodium hydroxide or potassium hydroxide; the concentration of the alkali liquor is 0.1M; The mass-volume ratio of lotus leaf flavone to the second solvent is 1:5-30, g / ml.

6. The method of claim 5, wherein, In step S2, the mass-volume ratio of lotus leaf flavone to the second solvent is 1:10-30, g / ml.

7. The method of claim 1, wherein, In step S3, further, the acid is hydrochloric acid; the concentration of the acid solution is 0.1M; The mass-volume ratio of lotus leaf flavone to the third solvent is 1:0.5-30, g / ml.

8. The method of claim 7, wherein, In step S3, the mass-volume ratio of lotus leaf flavone to the third solvent is 1:10-30, g / ml.

9. The method of claim 1, wherein, In steps S1-S3, the solid-liquid separation is performed by filtration, centrifugation, or sedimentation.

10. The method of claim 9, wherein, In steps S1-S3, the solid-liquid separation is performed by filtration, and the filtration is specifically microfiltration.

11. The method of claim 10, wherein, In steps S1-S3, the microfiltration is performed using a microporous filter membrane, and the pore size of the microporous filter membrane is 0.22μm.

12. The method of claim 1, wherein, The method further comprises the steps of separating the crystal or precipitate obtained in step S4 from the mother liquor and drying.

13. Use of the method of any one of claims 1-12 in the industrial production of hyperoside.

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