Method for extracting ecdysterone in Cyanotis arachnoidea through eutectic solvent

Abstract

The invention discloses a method for extracting ecdysterone from cyanotis arachnoidea by using a deep-eutectic solvent, and belongs to the technical field of ecdysterone extraction. The method comprises the following steps: mixing a deep-eutectic solvent which takes choline chloride and a hydrogen bond donor material as raw materials with water, extracting ecdysterone in roots and stems of Cyanotis arachnoidea to obtain an ecdysterone crude extract, precipitating the ecdysterone crude extract by adding water, filtering, and adsorbing ecdysterone by using macroporous resin. Adding water for elution to obtain a deep-eutectic solvent-water mixed solution, and recovering under reduced pressure to obtain an extraction solvent for cyclic utilization; and eluting by using alcohol with different concentrations to obtain an ecdysterone crude extract, and purifying the crude extract to obtain high-purity ecdysterone. Compared with a conventional extraction method, the process method adopted by the invention has the advantages that the process is simple, efficient, green and environment-friendly, the used eutectic solvent can be recycled, the loss caused by heating volatilization in the extraction process is avoided, the ecdysterone extracted by the method is pure white, the content of ecdysterone can reach 90% or above after simple purification, and the method is suitable for industrial production. After re-purification, the purity of ecdysterone detected by high performance liquid chromatography is up to 98% or above.

Description

Technical Field

[0001] This invention belongs to the field of ecdysterone extraction technology, specifically relating to a method for extracting ecdysterone from dew grass using a eutectic solvent. Background Technology

[0002] Ecdysterone is a natural steroid hormone found in plants and insects. Due to its good biological activity, it is widely used in cosmetic additives and in the artificial breeding of sericulture, crustaceans, and ground beetles. Currently, industrial production of ecdysterone mainly involves extracting it from the rhizomes of *Ecdys edulis* using alcohol or water. The extract is concentrated, adsorbed onto macroporous resin, eluted with methanol, and repeatedly purified to obtain high-purity ecdysterone. Traditional extraction methods have certain problems: water extraction produces many impurities, is prone to mold growth, and consumes a lot of energy; alcohol solvents have low boiling points, resulting in significant waste and adverse environmental impact. Traditional solvent post-processing steps are cumbersome, leading to significant ecdysterone loss and extraction rates often below 80%. This invention proposes a green and efficient ecdysterone extraction technology with advantages such as simplicity, low energy consumption, and low cost. The extraction solvent can be reused after simple treatment, and the optimized extraction process can achieve an extraction rate of over 90%. Summary of the Invention

[0003] To address or partially address the problems existing in related technologies, this invention provides a method for extracting ecdysterone from *Hydrocotyle vulgaris* using a eutectic solvent.

[0004] This invention provides a method for extracting ecdysterone from *Hymenochrosite* using a eutectic solvent, comprising the following steps: (1) Mix choline chloride and hydrogen bond donor material, then heat until clear and transparent to obtain eutectic solvent, then add water to eutectic solvent to obtain DES-water extract; (2) The roots and stems of Dewgrass were crushed or crushed to obtain Dewgrass root and stem fragments, which were then added to DES-water extract for extraction. After extraction, the extract was filtered, and the extraction was repeated twice. The filtrates were then combined to obtain crude extract of ecdysterone. (3) The crude extract of ecdysterone was added to water for precipitation. After filtration, the filtrate was adsorbed through macroporous resin. Then, the macroporous resin was washed with water. The eluent was allowed to stand and precipitate. After filtration, the filtrate was obtained and evaporated under reduced pressure to obtain a eutectic solvent. (4) Elute the macroporous resin rinsed with water in step (3) with methanol or ethanol, collect the eluent, and recover the solvent under reduced pressure to obtain ecdysterone extract. The ecdysterone extract is crystallized with ethyl acetate and methanol, and then recrystallized with methanol to obtain high-purity ecdysterone.

[0005] Preferably, the molar ratio of choline chloride to the hydrogen bond donor material is 1:(1-4); the hydrogen bond donor material is lactose, D-glucose, sucrose, xylitol, octanoic acid, glacial acetic acid, boric acid, oxalic acid, citric acid, gallic acid, methylurea, urea, glycerol, 1,2-propanediol, or sodium alginate.

[0006] Eutectic solvents formulated with choline chloride can effectively disrupt the cell walls and cell membranes of *Eclipta prostrata* through swelling and osmosis, accelerating the release of ecdysterone. Ecdysterone contains a large number of hydroxyl groups and is highly polar. Eutectic solvents bind to these hydroxyl groups, reducing the lattice energy or intermolecular forces of the component, making it easier for it to transform from a solid (or bound) state to a dissolved state. Eutectic solvents can adjust polarity by using different hydrogen bond donors, forming abundant hydrogen bonds and corresponding ionic environments, thereby promoting ecdysterone release by disrupting the cell walls and cell membranes of *Eclipta prostrata* cells. On the other hand, they can also enhance the solubility of ecdysterone through hydrogen bonding and electrostatic adsorption.

[0007] Preferably, in step (1), the volume ratio of the eutectic solvent to water is 1:(1-2). If water is not added to the eutectic solvent in step (1) of the present invention, problems such as low extraction efficiency of ecdysterone and large amount of eutectic solvent used will occur.

[0008] Preferably, in step (1), the heating temperature is 30-80℃.

[0009] Preferably, the mass ratio of the crushed dewgrass rhizomes to the DES-water extraction solvent is 1:(2-40).

[0010] Preferably, in step (2), the extraction temperature is 40-70℃ and the extraction time is not less than 30 minutes.

[0011] Preferably, in step (2), the extraction method is heating, ultrasound or microwave.

[0012] Preferably, in step (3), the volume ratio of crude ecdysterone extract to water is 1:(1-8). Water molecules competitively bind to hydrogen bond acceptors and donors in the eutectic solvent, gradually breaking down the hydrogen bond network of the eutectic solvent and destroying its stability. The eutectic solvent with its unstable structure is then passed through a macroporous resin column, where the resin exerts its adsorption effect, adsorbing a large amount of the active ingredients, while some sugars and pigments are removed by elution.

[0013] Preferably, in step (3), the macroporous resin is rinsed with water at a volume of 2-10 times that of the macroporous resin.

[0014] Preferably, the water content of the dew grass rhizome is 5%-85%.

[0015] The technical solution provided by this invention has the following beneficial effects: (1) Conventional methods for extracting ecdysterone typically involve temperatures between 75-100℃, long extraction times, and significant alcohol losses during extraction. This invention utilizes a eutectic solvent to extract ecdysterone, which lowers the extraction temperature, minimizes solvent loss, and reduces energy consumption compared to conventional methods. Furthermore, eutectic solvent extraction is highly targeted; by adjusting the ratio of hydrogen bond donors and acceptors, a solvent specifically designed for ecdysterone can be formed, achieving an extraction rate of over 90%. Additionally, it minimizes the extraction of other impurities. After simple processing, the ecdysterone content in the extract can reach over 35%, higher than the approximately 21% obtained through conventional extraction using macroporous resin treatment.

[0016] (2) After extraction with eutectic solvent, the impurities and ecdysterone can be effectively separated from the solvent by water and macroporous resin treatment. The separated solvent can be reused after vacuum concentration. After five repeated extractions, the extraction efficiency of the extracted eutectic solvent for ecdysterone still reaches more than 90% of the extraction efficiency of the solvent in the first extraction. Detailed Implementation

[0017] Example 1 A method for extracting ecdysterone from *Hydrocotyle vulgaris* using a eutectic solvent includes the following steps: (1) Preparation of eutectic solvent: Choline chloride is used as hydrogen bond acceptor (HBA) and glacial acetic acid is used as hydrogen bond donor (HBD). The mixture is prepared according to a molar ratio of choline chloride to glacial acetic acid of 1:4. The mixture is heated and stirred in a water bath at 50°C until it is completely transparent. One volume of distilled water is added and the mixture is cooled to obtain the extraction solvent.

[0018] (2) Extraction of ecdysterone: Take 1 kg of dried dew grass rhizome, crush it, and add 2 kg of the extraction solvent prepared in step (1) for the first time. Stir well and then add 3 kg of the extraction solvent obtained in step 1. Heat and extract twice at 50℃, with each extraction time not less than 30 min. Combine the filtrates to obtain ecdysterone extract.

[0019] (3) Recovery of eutectic solvent: 9.883 kg of ecdysterone extract obtained in step (2) was added to 4 times the amount of distilled water, and after standing for 24 hours, it was filtered. The filtrate was used to adsorb ecdysterone with 4 kg of macroporous resin, and 8 kg of distilled water was added to wash the macroporous resin. The eluent was evaporated under reduced pressure to about 10 kg, cooled, and filtered to obtain the extraction solvent, which was reused.

[0020] (4) Purification of ecdysterone: Macroporous resin that adsorbs ecdysterone was eluted with methanol at a volume 5 times that of the macroporous resin. The eluent was concentrated under reduced pressure to obtain 73.3 g of crude ecdysterone extract with a ecdysterone content of 37% and an extraction rate of 94.3%. The crude ecdysterone extract was crystallized using a mixed solvent of methanol and ethyl acetate. The ecdysterone obtained after the first crystallization was recrystallized with methanol, and 22.3 g of ecdysterone with a purity of 96.7% was obtained after recrystallization.

[0021] Example 2 A method for extracting ecdysterone from *Hydrocotyle vulgaris* using a eutectic solvent includes the following steps: (1) Preparation of eutectic solvent: Choline chloride is used as hydrogen bond acceptor (HBA) and sodium alginate is used as hydrogen bond donor (HBD). Choline chloride: urea is mixed in a molar ratio of 1:2. The mixture is heated and stirred in a water bath at 80°C until it is completely transparent. Two times the amount of distilled water is added and cooled to obtain the extraction solvent.

[0022] (2) Extraction of ecdysterone: Take 0.5 kg of dried dew grass rhizome, crush it, and add 1 kg of the extraction solvent prepared in step (1) for the first time. Stir well and then add another 1 kg of the extraction solvent prepared in step (1). Heat and extract twice at 40℃, with each extraction time not less than 30 min. Combine the filtrates to obtain ecdysterone extract.

[0023] (3) Recovery of eutectic solvent: 3.082 kg of ecdysterone extract obtained in step (2) was added to 1 times the amount of distilled water, and after standing for 24 hours, it was filtered. The filtrate was used to adsorb ecdysterone with 5 kg of macroporous resin, and eluted with 9 kg of distilled water. The eluent was evaporated under reduced pressure to about 8 kg, cooled, and filtered to obtain the extraction solvent, which was reused.

[0024] (4) Purification of ecdysterone: Macroporous resin adsorbing ecdysterone was eluted with 15 kg of ethanol. The eluent was concentrated under reduced pressure to obtain 34.8 g of crude ecdysterone extract with a purity of 39%, and the extraction rate of ecdysterone was 94.25%. The crude ecdysterone extract was crystallized using a mixed solvent of methanol and ethyl acetate. The ecdysterone obtained after the first crystallization was recrystallized using methanol. After recrystallization, 12.1 g of ecdysterone with a purity of 95.5% was obtained.

[0025] Example 3 A method for extracting ecdysterone from *Hydrocotyle vulgaris* using a eutectic solvent includes the following steps: (1) Preparation of eutectic solvent: Choline chloride is used as hydrogen bond acceptor (HBA) and propylene glycol is used as hydrogen bond donor (HBD). Choline chloride: propylene glycol is mixed in a molar ratio of 1:1. The mixture is heated and stirred in a water bath at 30°C until it is completely transparent. One volume of distilled water is added and cooled to obtain the extraction solvent.

[0026] (2) Extraction of ecdysterone: Take 1 kg of fresh dew grass rhizome, crush it, and add 1.5 kg of the extraction solvent prepared in step (1) for the first time. Stir evenly and then add 1.5 kg of the extraction solvent obtained in step (1). Heat and extract twice at 70℃, with each extraction time not less than 30 min. Combine the filtrates to obtain ecdysterone extract.

[0027] (3) Recovery of eutectic solvent: 3.082 kg of ecdysterone extract obtained in step (2) was added to 8 times the amount of distilled water, and after standing for 24 hours, it was filtered. The filtrate was used to adsorb ecdysterone with 1 kg of macroporous resin, and 2 kg of distilled water was added for elution. The eluent was evaporated under reduced pressure to about 3 kg, cooled, and filtered to obtain the extraction solvent, which was reused.

[0028] (4) Purification of ecdysterone: Macroporous resin adsorbing ecdysterone was eluted with 2 kg of ethanol. The eluent was concentrated under reduced pressure to obtain 10.28 g of crude ecdysterone extract with a purity of 42%, and the extraction rate of ecdysterone was 95.55%. The crude ecdysterone extract was crystallized using a mixed solvent of methanol and ethyl acetate. The ecdysterone obtained after the first crystallization was recrystallized using methanol. After recrystallization, 3.2 g of ecdysterone with a purity of 96.5% was obtained.

[0029] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or improvement of the technology in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for extracting ecdysterone from *Hydrocotyle vulgaris* using a eutectic solvent, characterized in that: Includes the following steps: (1) Mix choline chloride and hydrogen bond donor material, then heat until clear and transparent to obtain eutectic solvent, then add water to eutectic solvent to obtain DES-water extract; (2) The roots and stems of Dewgrass were crushed to obtain Dewgrass root and stem fragments, which were then added to DES-water extract for extraction. After extraction, the mixture was filtered, and the extraction was repeated twice. The filtrates were then combined to obtain crude extract of ecdysterone. (3) The crude extract of ecdysterone was added to water for precipitation. After filtration, the filtrate was adsorbed through macroporous resin. Then, the macroporous resin was washed with water. The eluent was allowed to stand and precipitate. After filtration, the filtrate was obtained and evaporated under reduced pressure to obtain a eutectic solvent. (4) Elute the macroporous resin rinsed with water in step (3) with methanol or ethanol, collect the eluent, and recover the solvent under reduced pressure to obtain ecdysterone extract. The ecdysterone extract is crystallized with ethyl acetate and methanol, and then recrystallized with methanol to obtain high-purity ecdysterone.

2. The method for extracting ecdysterone from *Hydrocotyle vulgaris* using a eutectic solvent according to claim 1, characterized in that: The molar ratio of choline chloride to the hydrogen bond donor material is 1:(1-4); the hydrogen bond donor material is lactose, D-glucose, sucrose, xylitol, octanoic acid, glacial acetic acid, boric acid, oxalic acid, citric acid, gallic acid, methylurea, urea, glycerol, 1,2-propanediol, or sodium alginate.

3. The method for extracting ecdysterone from *Hydrocotyle vulgaris* using a eutectic solvent according to claim 1, characterized in that: In step (1), the volume ratio of the eutectic solvent to water is 1:(1-2).

4. The method for extracting ecdysterone from *Hydrocotyle vulgaris* using a eutectic solvent according to claim 1, characterized in that: In step (1), the heating temperature is 30-80℃.

5. The method for extracting ecdysterone from *Hydrocotyle vulgaris* using a eutectic solvent according to claim 1, characterized in that: The mass ratio of the crushed roots and stems of *Desmodium styracifolium* to the DES-water extract is 1:(2-40).

6. The method for extracting ecdysterone from *Hydrocotyle vulgaris* using a eutectic solvent according to claim 1, characterized in that: In step (2), the extraction temperature is 40-70℃ and the extraction time is not less than 30 minutes.

7. The method for extracting ecdysterone from *Hydrocotyle vulgaris* using a eutectic solvent according to claim 1, characterized in that: In step (3), the volume ratio of crude ecdysterone extract to water is 1:(1-8).

8. The method for extracting ecdysterone from *Hydrocotyle vulgaris* using a eutectic solvent according to claim 1, characterized in that: In step (3), the macroporous resin is rinsed with water at a volume of 2-10 times that of the macroporous resin.