Method for extracting forsythiaside A and forsythin by deep eutectic solvent method and application of forsythiaside A and forsythin

Abstract

The invention relates to a method for extracting forsythiaside A and forsythin through a deep eutectic solvent method and application of the forsythiaside A and the forsythin. The method comprises the following steps: crushing forsythia suspense leaves to 80-120 meshes, mixing the crushed forsythia suspense leaves with a betaine-levulinic acid eutectic solvent in a molar ratio of 1: 1-3: 1 according to a material-liquid ratio of 1: 10-1: 35, carrying out stirring extraction at 60-90 DEG C for 0.5-2 h, and carrying out centrifugation and D101 resin purification so as to realize efficient extraction. According to the method, the natural component is used as the solvent, the method is green and environment-friendly, the process is simple and convenient, the cost is low, the yield of forsythin is remarkably increased (1.3%-1.7%), and the yield of forsythiaside A can reach 6.7%-7.4%. The extract can be widely applied to the fields of foods, medicines and cosmetics, and has remarkable economic and social benefits.

Description

Technical Field

[0001] The present invention relates to the technical field of extraction and application of active ingredients of traditional Chinese medicine, and in particular to a method for extracting forsythiaside A and forsythiaside from Forsythia suspensa and applications of forsythiaside A and forsythiaside. Background Art

[0002] Forsythia suspensa (Thunb.) Vahl leaf is a deciduous shrub of the genus Forsythia in the family Oleaceae. Its leaves are opposite, ovate or elliptical-ovate, with serrated edges. It blooms in spring with golden yellow flowers that open before the leaves, and has high ornamental value. Forsythia is not only an ornamental plant in landscape gardens, but also an important medicinal plant in traditional Chinese medicine. Forsythiaside and forsythiaside A are important active substances in Forsythia, and are also the quality control indicator components of Forsythia specified in the National Pharmacopoeia. The latest research in the field of modern pharmacology shows that forsythiaside A has antibacterial, antiviral, antioxidant, anti-inflammatory and neuroprotective effects, and forsythiaside has multiple pharmacological activities such as anti-inflammatory, antibacterial and antioxidant. The potential application value of both has attracted widespread attention from all walks of life, especially in the fields of medicine, cosmetics and food.

[0003] Patent CN117562841A provides a method for extracting forsythiaside A and forsythin, using chitosan and organic acid as extraction reagents to achieve simultaneous extraction of forsythiaside A and forsythin. The yield of forsythiaside is 0.058-0.071%, and the yield of forsythiaside A is 6.1-7.6%. The yield of forsythiaside in the method of simultaneously extracting forsythiaside and forsythiaside A is low, resulting in a waste of forsythiaside resources. Summary of the Invention

[0004] The object of the present invention is to provide a method for extracting forsythiaside A and forsythiaside using a deep eutectic solvent method, so as to solve the technical problem of low yield of forsythiaside in the method for simultaneously extracting forsythiaside and forsythiaside A proposed in the above-mentioned background technology, and at the same time provide an application of forsythiaside A and forsythiaside.

[0005] To achieve the above object, the technical solution of the present invention is as follows:

[0006] In a first aspect, the present invention provides a method for extracting forsythiaside A and forsythiaside using a deep eutectic solvent method, comprising the following steps:

[0007] S1: crushing the forsythia leaves;

[0008] S2: preparing a binary deep eutectic solvent of betaine and levulinic acid;

[0009] (The order of S1 and S2 can be reversed)

[0010] S3: adding the crushed Forsythia suspensa leaves into a deep eutectic solvent composed of betaine and levulinic acid for stirring extraction;

[0011] S4: After the extraction is completed, the extraction mixture is centrifuged to obtain a supernatant containing forsythiaside A and forsythiaside;

[0012] S5: Purify forsythiaside and forsythiaside A using D101 resin, first elute forsythiaside A with 20-30% ethanol, then switch to 40-50% ethanol to elute forsythiaside, and dry the two eluates separately to obtain forsythiaside and forsythiaside A.

[0013] Among them, deep eutectic solvents (DESs) are binary or ternary eutectic mixtures composed of a certain stoichiometric ratio of hydrogen bond acceptors (such as quaternary ammonium salts) and hydrogen bond donors (compounds such as amides, carboxylic acids and polyols). Due to the hydrogen bonding between the components, the melting point of DESs is significantly lower than the melting point of each of its individual components, and thus presents a thick and uniform liquid state at room temperature. DESs are widely regarded as a new type of green extraction solvent due to their many advantages, such as simple preparation process, low cost, environmental friendliness, low volatility, strong solubility, and designable and biodegradable structure. Although traditional deep eutectic solvents have many advantages, some of their components are still non-natural products, and their safety still needs further verification. With the deepening of research, natural deep eutectic solvents have performed well in reducing toxicity and have gradually become the focus of research. Betaine and levulinic acid in the present invention are natural deep eutectic solvents and are a safer extraction reagent.

[0014] As a preferred technical solution of the present invention, the forsythia leaves in step S1 are crushed to 80-120 mesh;

[0015] As a preferred technical solution of the present invention, the molar ratio of betaine to levulinic acid in step S2 is in the range of 1:1-3:1;

[0016] Preferably, the preparation method of the deep eutectic solvent is to place a binary mixture of betaine and levulinic acid in a container, and heat and stir at 90-120° C. to form a uniform and transparent solution.

[0017] As a preferred technical solution of the present invention, in step S3, the material-liquid ratio of the forsythia leaves to the deep eutectic solvent composed of betaine and levulinic acid is in the range of 1:10-1:35 (g / mL);

[0018] Preferably, the temperature of the stirring extraction is 60-90°C, such as 65°C, 70°C or 80°C, but is not limited to the listed values, and other values ​​not listed within the numerical range are also applicable;

[0019] Preferably, the stirring extraction time is 0.5-2 hours, such as 1 hour or 1.5 hours, but is not limited to the listed values, and other values ​​not listed within the numerical range are also applicable;

[0020] As a preferred technical solution of the present invention, the centrifugation condition in step S4 is 3500-4500 r / min for 5-15 min to obtain a supernatant;

[0021] In a second aspect, the present invention further provides the use of forsythiaside A and forsythiaside prepared according to the method of the first aspect in food, cosmetics or medicines.

[0022] Compared with the existing technology, the beneficial effects of this technology are:

[0023] (1) Significantly improved extraction efficiency: Compared with the prior art, the present invention significantly improves the yield of forsythin by using a deep eutectic solvent composed of betaine and levulinic acid. Experimental data show that the yield of forsythin can reach 1.3%-1.7%.

[0024] (2) Green, environmentally friendly and highly safe: The betaine and levulinic acid used are both natural components, avoiding the high toxicity and environmental pollution problems of traditional organic solvents, and conforming to the concept of green chemistry.

[0025] (3) The process is simple and the cost is controllable: the preparation of low eutectic solvents only requires heating and stirring, without the need for complex equipment; the extraction process can be completed by simple stirring and centrifugation, which greatly reduces production costs.

[0026] (4) Wide range of applications: The extracts forsythiaside A and forsythiaside can be used in food (such as antioxidant beverages, preservatives), medicines (anti-inflammatory injections, wound gels) and cosmetics (acne essences, soothing masks) and other fields, with high added value. DETAILED DESCRIPTION

[0027] The specific embodiments of the present invention are further described in detail below, but the present invention is not limited to these embodiments. Any improvement or substitution on the present embodiment still falls within the scope of protection required by the claims of the present invention.

[0028] In order to solve the above problems, the present invention provides a method for extracting forsythiaside A and forsythiaside using a low eutectic solvent method, which can improve the yield of forsythiaside. At the same time, the forsythiaside A and forsythiaside of the present invention are also applied to the fields of food, medicine and cosmetics.

[0029] Specifically:

[0030] In a first aspect, the present invention provides a method for extracting forsythiaside A and forsythiaside using a deep eutectic solvent method, comprising the following steps:

[0031] S1: crushing the forsythia leaves;

[0032] S2: preparing a binary deep eutectic solvent of betaine and levulinic acid;

[0033] (The order of S1 and S2 can be reversed)

[0034] S3: adding the crushed Forsythia suspensa leaves into a deep eutectic solvent composed of betaine and levulinic acid for stirring extraction;

[0035] S4: After the extraction is completed, the extraction mixture is centrifuged to obtain a supernatant containing forsythiaside A and forsythiaside;

[0036] S5: Purify forsythiaside and forsythiaside A using D101 resin, first elute forsythiaside A with 20-30% ethanol, then switch to 40-50% ethanol to elute forsythiaside, and dry the two eluates separately to obtain forsythiaside and forsythiaside A.

[0037] As a preferred embodiment of the present invention, the forsythia leaves in step S1 are crushed to 80-120 mesh;

[0038] As a preferred embodiment of the present invention, the molar ratio of betaine to levulinic acid in step S2 is in the range of 1:1-3:1;

[0039] In some preferred embodiments, the preparation method of the deep eutectic solvent is to place a binary mixture of betaine and levulinic acid in a container, and heat and stir at 90-120° C. to form a homogeneous and transparent solution.

[0040] As a preferred embodiment of the present invention, in step S3, the material-liquid ratio of the forsythia leaf to the deep eutectic solvent composed of betaine and levulinic acid is in the range of 1:10-1:35 (g / mL);

[0041] In some preferred embodiments, the temperature of the stirring extraction is 60-90°C, such as 65°C, 70°C or 80°C;

[0042] In other preferred embodiments, the stirring extraction time is 0.5-2 hours, such as 1 hour or 1.5 hours;

[0043] As a preferred embodiment of the present invention, the centrifugation condition in step S4 is 3500-4500 r / min for 5-15 min to obtain a supernatant;

[0044] In a second aspect, the present invention further provides the use of forsythiaside A and forsythiaside prepared according to the method of the first aspect in food, cosmetics or medicines.

[0045] Example 1:

[0046] S1: Grind the Forsythia suspensa leaves and pass them through a 100-mesh sieve;

[0047] S2: preparing a binary deep eutectic solvent of betaine and levulinic acid, wherein the molar ratio of betaine to levulinic acid is 1.5:1, placing the betaine and levulinic acid in a container, and heating and stirring at 100° C. to form a homogeneous and transparent solution;

[0048] S3: adding 10 g of crushed Forsythia suspensa leaves to 200 mL of a deep eutectic solvent composed of betaine and levulinic acid for stirring extraction, wherein the solid-liquid ratio of Forsythia suspensa leaves to the deep eutectic solvent is 1:20; the stirring extraction temperature is 70° C.; and the stirring extraction time is 1 hour;

[0049] S4: After the extraction is completed, the extraction mixture is centrifuged at a speed of 3800 r / min for 10 min to obtain a supernatant containing forsythiaside A and forsythiaside;

[0050] S5: Purify forsythiaside and forsythiaside A using D101 resin, first elute forsythiaside A with 30% ethanol, then switch to 50% ethanol to elute forsythiaside, and dry the two eluates separately to obtain forsythiaside and forsythiaside A.

[0051] Example 2:

[0052] S1: Grind the Forsythia suspensa leaves and pass them through a 90-mesh sieve;

[0053] S2: preparing a binary deep eutectic solvent of betaine and levulinic acid, wherein the molar ratio of betaine to levulinic acid is 2:1, placing the betaine and levulinic acid in a container, and heating and stirring at 90° C. to form a homogeneous and transparent solution;

[0054] S3: 10 g of crushed Forsythia suspensa leaves were added to 300 mL of a deep eutectic solvent composed of betaine and levulinic acid for stirring extraction, wherein the solid-liquid ratio of Forsythia suspensa leaves to the deep eutectic solvent was 1:30; the stirring extraction temperature was 60°C; and the stirring extraction time was 1.2 hours;

[0055] S4: After the extraction is completed, the extraction mixture is centrifuged at a speed of 3500 r / min for 12 min to obtain a supernatant containing forsythiaside A and forsythiaside;

[0056] S5: Purify forsythiaside and forsythiaside A using D101 resin, first elute forsythiaside A with 20% ethanol, then switch to 40% ethanol to elute forsythiaside, and dry the two eluates separately to obtain forsythiaside and forsythiaside A.

[0057] Example 3:

[0058] S1: Grind the Forsythia suspensa leaves and pass them through an 80-mesh sieve;

[0059] S2: preparing a binary deep eutectic solvent of betaine and levulinic acid, wherein the molar ratio of betaine to levulinic acid is 1:1, placing the betaine and levulinic acid in a container, and heating and stirring at 115° C. to form a homogeneous transparent solution;

[0060] S3: adding 10 g of crushed Forsythia suspensa leaves to 100 mL of a deep eutectic solvent composed of betaine and levulinic acid for stirring extraction, wherein the solid-liquid ratio of Forsythia suspensa leaves to the deep eutectic solvent is 1:10; the stirring extraction temperature is 65° C.; and the stirring extraction time is 0.5 h;

[0061] S4: After the extraction is completed, the extraction mixture is centrifuged at a speed of 4000 r / min for 5 min to obtain a supernatant containing forsythiaside A and forsythiaside;

[0062] S5: Purify forsythiaside and forsythiaside A using D101 resin, first elute forsythiaside A with 25% ethanol, then switch to 45% ethanol to elute forsythiaside, and dry the two eluates separately to obtain forsythiaside and forsythiaside A.

[0063] Example 4:

[0064] S1: Grind the Forsythia suspensa leaves and pass them through a 120-mesh sieve;

[0065] S2: preparing a binary deep eutectic solvent of betaine and levulinic acid, wherein the molar ratio of betaine to levulinic acid is 2.5:1, placing the betaine and levulinic acid in a container, and heating and stirring at 110° C. to form a homogeneous and transparent solution;

[0066] S3: 10 g of crushed Forsythia suspensa leaves were added to 150 mL of a deep eutectic solvent composed of betaine and levulinic acid for stirring extraction, wherein the solid-liquid ratio of Forsythia suspensa leaves to the deep eutectic solvent was 1:15; the stirring extraction temperature was 90° C.; and the stirring extraction time was 1.5 hours;

[0067] S4: After the extraction is completed, the extraction mixture is centrifuged at a speed of 4500 r / min for 8 min to obtain a supernatant containing forsythiaside A and forsythiaside;

[0068] S5: Purify forsythiaside and forsythiaside A using D101 resin, first elute forsythiaside A with 25% ethanol, then switch to 45% ethanol to elute forsythiaside, and dry the two eluates separately to obtain forsythiaside and forsythiaside A.

[0069] Example 5:

[0070] S1: preparing a binary deep eutectic solvent of betaine and levulinic acid, wherein the molar ratio of betaine to levulinic acid is 3:1, placing the betaine and levulinic acid in a container, and heating and stirring at 120° C. to form a uniform and transparent solution;

[0071] S2: Grind the Forsythia suspensa leaves and pass them through a 110-mesh sieve;

[0072] S3: 10 g of crushed Forsythia suspensa leaves were added to 350 mL of a deep eutectic solvent composed of betaine and levulinic acid for stirring extraction, wherein the solid-liquid ratio of Forsythia suspensa leaves to the deep eutectic solvent was 1:35; the stirring extraction temperature was 80° C.; and the stirring extraction time was 2 hours;

[0073] S4: After the extraction is completed, the extraction mixture is centrifuged at a speed of 4200 r / min for 15 min to obtain a supernatant containing forsythiaside A and forsythiaside;

[0074] S5: Purify forsythiaside and forsythiaside A using D101 resin, first elute forsythiaside A with 25% ethanol, then switch to 40% ethanol to elute forsythiaside, and dry the two eluates separately to obtain forsythiaside and forsythiaside A.

[0075] Comparative Example 1 (deep eutectic solvent composed of betaine and glucose):

[0076] S1: Grind the Forsythia suspensa leaves and pass them through a 120-mesh sieve;

[0077] S2: preparing a binary deep eutectic solvent of betaine and glucose, wherein the molar ratio of betaine to glucose is 1:1, placing the betaine and glucose in a container, and heating and stirring at 110° C. to form a homogeneous and transparent solution;

[0078] S3: 10 g of crushed Forsythia suspensa leaves were added to 150 mL of a deep eutectic solvent composed of betaine and glucose for stirring extraction, wherein the solid-liquid ratio of Forsythia suspensa leaves to the deep eutectic solvent was 1:15; the stirring extraction temperature was 90° C.; and the stirring extraction time was 1.5 hours;

[0079] S4: After the extraction is completed, the extraction mixture is centrifuged at a speed of 4500 r / min for 8 min to obtain a supernatant containing forsythiaside A and forsythiaside;

[0080] S5: Purify forsythiaside and forsythiaside A using D101 resin, first elute forsythiaside A with 25% ethanol, then switch to 45% ethanol to elute forsythiaside, and dry the two eluates separately to obtain forsythiaside and forsythiaside A.

[0081] Comparative Example 2 (deep eutectic solvent composed of betaine and lactic acid):

[0082] S1: Grind the Forsythia suspensa leaves and pass them through a 120-mesh sieve;

[0083] S2: preparing a binary deep eutectic solvent of betaine and lactic acid, wherein the molar ratio of betaine to lactic acid is 1:1, placing the betaine and lactic acid in a container, and heating and stirring at 110° C. to form a homogeneous and transparent solution;

[0084] S3: 10 g of crushed Forsythia suspensa leaves were added to 150 mL of a deep eutectic solvent composed of betaine and lactic acid for stirring extraction, wherein the solid-liquid ratio of Forsythia suspensa leaves to the deep eutectic solvent was 1:15; the stirring extraction temperature was 90° C.; and the stirring extraction time was 1.5 hours;

[0085] S4: After the extraction is completed, the extraction mixture is centrifuged at a speed of 4500 r / min for 8 min to obtain a supernatant containing forsythiaside A and forsythiaside;

[0086] S5: Purify forsythiaside and forsythiaside A using D101 resin, first elute forsythiaside A with 25% ethanol, then switch to 45% ethanol to elute forsythiaside, and dry the two eluates separately to obtain forsythiaside and forsythiaside A.

[0087] The yields of the embodiment and the comparative example are compared, and the formula for the yield is as follows:

[0088]

[0089] Table 1 - Forsythiaside A and forsythiaside extraction rate results of Forsythia suspensa extract

[0090] category Yield of forsythiaside A (%) Forsythiaside yield (%) Example 1 6.8 1.5 Example 2 6.7 1.6 Example 3 7.1 1.6 Example 4 7.4 1.7 Example 5 7.2 1.3 Comparative Example 1 (deep eutectic solvent of betaine and glucose) 5.9 0.9 Comparative Example 2 (deep eutectic solvent of betaine and lactic acid) 6.3 0.7

[0091] Table 1 shows the yields of forsythiaside A and forsythin in the Examples and Comparative Examples. It can be seen from the table that the yield of forsythiaside A in the Examples is between 6.7% and 7.4%, and the yield of forsythin is between 1.3% and 1.7%. In Comparative Example 1, the yield of forsythiaside A extracted using a deep eutectic solvent composed of betaine and glucose is 5.9%, and the yield of forsythin is 0.9%. In Comparative Example 2, the yield of forsythiaside A extracted using a deep eutectic solvent composed of betaine and lactic acid is 6.3%, and the yield of forsythin is 0.7%. The yields of forsythiaside A and forsythin in Comparative Examples 1 and 2 are both lower than those in the Examples.

[0092] The present application takes Forsythia suspensa leaf as the research object, and successfully separates Forsythia ester glycoside A and forsythoside A by using the eutectic solvent of betaine and levulinic acid. However, the extraction method is not only limited to Forsythia suspensa leaf, but also applicable to other plant organs such as flowers and fruits of Forsythia suspensa. In addition, the present application uses dry Forsythia suspensa leaf, but the extraction method can also be used for the extraction of Forsythia ester glycoside A and forsythoside A in fresh Forsythia suspensa leaf or other plant organs such as Forsythia suspensa fruits.

[0093] Forsythoside A has anti-inflammatory, antibacterial, antioxidant, anti-inflammatory and neuroprotective effects, and can be used as an antioxidant functional beverage and a baking food modifier in the food field; can be used for preparing anti-inflammatory injection and wound healing gel in the pharmaceutical field; and can be used as an anti-photoaging essence and a soothing mask in the cosmetic field.

[0094] Forsythoside A has anti-inflammatory, antibacterial, antioxidant, anti-inflammatory and neuroprotective effects, and can be used as an antioxidant functional beverage and a baking food modifier in the food field; can be used for preparing anti-inflammatory injection and wound healing gel in the pharmaceutical field; and can be used as an anti-photoaging essence and a soothing mask in the cosmetic field.

[0095] The present application extracts Forsythia ester glycoside A and forsythoside A by using the eutectic solvent composed of betaine and levulinic acid, significantly improves the yield of forsythoside A (up to 1.3%-1.7%), and at the same time maintains a high yield of Forsythia ester glycoside A (6.7%-7.4%), which is much higher than the prior art (such as the forsythoside A yield of 0.058%-0.071% in patent CN117562841A). The method uses natural components, is green and environmentally friendly, simple in process, low in cost, and the extract can be widely used in the fields of food, medicine and cosmetics, and has significant economic and social benefits.

[0096] The above describes the preferred embodiments of the present application, but the present application is not limited to the specific details in the above embodiments, and various simple modifications can be made to the technical solutions of the present application within the technical concept of the present application, and these simple modifications all belong to the protection scope of the present application.

[0097] In addition, it should be noted that each specific technical feature described in the above specific embodiments can be combined in any appropriate manner without contradiction, and in order to avoid unnecessary repetition, the present application will not further describe various possible combination manners.

[0098] In addition, various different embodiments of the present application can also be combined in any manner, as long as they do not deviate from the technical concept of the present application, and they should also be considered as the contents invented by the present application.

Claims

1. A method for extracting forsythiaside A and forsythiaside using a deep eutectic solvent method, characterized in that: The deep eutectic solvents for extracting forsythiaside A and forsythiaside from Forsythia suspensa leaves are betaine and levulinic acid.

2. The method according to claim 1, wherein: The molar ratio of the betaine to the levulinic acid is in the range of 1:1-3:

1.

3. The method according to claim 2, wherein: The solid-liquid ratio of the forsythia leaves to the deep eutectic solvent is 1:10-1:

35.

4. The method according to claim 3, wherein: The preparation method of the deep eutectic solvent comprises placing a binary mixture of betaine and levulinic acid in a container, heating and stirring at 90-120° C. to form a uniform and transparent solution.

5. The method according to any one of claims 2 to 4, characterized in that: Before extraction, the forsythia leaves are crushed to 80-120 meshes and then stirred for extraction.

6. The method according to claim 5, characterized in that: The temperature of the stirring extraction is 60-90°C.

7. The method according to claim 6, characterized in that: The stirring extraction time is 0.5-2 hours.

8. The method according to claim 6 or 7, characterized in that: After the extraction is completed, the extraction mixture is centrifuged at 3500-4500 r / min for 5-15 min to obtain a supernatant containing forsythiaside A and forsythiaside.

9. The method according to claim 8, characterized in that: Forsythiaside and forsythiaside A were purified using D101 resin. Forsythiaside A was first eluted with 20-30% ethanol, and then switched to 40-50% ethanol to elute forsythiaside.

10. Use of forsythiaside A and forsythiaside prepared according to the method of any one of claims 1 to 9 in food, cosmetics or medicines.

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