A method for extracting chlorogenic acid from eucommia ulmoides oliver

Abstract

The application relates to a method for extracting chlorogenic acid from eucommia ulmoides, which comprises the following steps: collecting fresh eucommia ulmoides leaves, beating the eucommia ulmoides leaves by a beater to obtain eucommia ulmoides leaf pulp; adding a ternary eutectic solvent to the eucommia ulmoides leaf pulp for ultrasonic extraction; filtering the eucommia ulmoides leaf pulp after the extraction is completed to obtain an extraction liquid; adding a flocculating agent to the extraction liquid, carrying out centrifugation after flocculation, and obtaining supernatant; concentrating the supernatant to obtain a concentrated liquid; passing the concentrated liquid through a modified nano-hydroxyapatite filled adsorption column, carrying out elution with an aqueous solution to remove impurities, then eluting chlorogenic acid with an eluent, and obtaining an eluate; performing vacuum concentration on the eluate, vacuum drying the concentrated liquid, and obtaining a chlorogenic acid product. In the application, betaine, acetylpropionic acid and ascorbic acid are used as the eutectic solvent to form a strong hydrogen bond network structure, which can better promote the dissolution of chlorogenic acid and thus improve the extraction rate of chlorogenic acid.

Description

Technical Field

[0001] This application relates to the field of plant extraction technology, and in particular to a method for extracting chlorogenic acid from Eucommia ulmoides. Background Technology

[0002] The chemical name of chlorogenic acid from Eucommia ulmoides is 3-caffeoylquinic acid, and its molecular formula is C1. 16 H 18 O9 molecular weight: 354.30. It is the main functional component of Eucommia ulmoides leaves. Eucommia ulmoides chlorogenic acid has pharmacological effects such as antibacterial, anti-inflammatory, detoxifying, choleretic, hypotensive and white blood cell raising, as well as significantly increasing gastrointestinal motility and promoting gastric juice secretion. It has a strong inhibitory effect on Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae and viruses. It has obvious curative effects on acute pharyngitis and skin diseases. Clinically, it is used to treat acute bacterial infections and leukopenia caused by radiotherapy and chemotherapy. It has a good hemostatic effect on menorrhagia and dysfunctional uterine bleeding. The main pharmacological effects of Eucommia ulmoides chlorogenic acid are: (1) hypotensive effect; (2) antitumor effect; (3) kidney tonification and immune enhancement effect; (4) antioxidant, anti-aging and anti-musculoskeletal aging effect; (5) antibacterial and antiviral effect; (6) other effects: diuretic, choleretic, lipid-lowering and pregnancy-preserving effects.

[0003] Chlorogenic acid is widely found in higher dicotyledonous plants and ferns, primarily in plants of the *Lonicera* genus (Caprifoliaceae) and *Artemisia* genus (Asteraceae). Plants with relatively high chlorogenic acid content include honeysuckle, eucommia, sunflower, cocoa tree, coffee, and sea buckthorn. Honeysuckle has the highest chlorogenic acid content, followed by eucommia leaves. Currently, chlorogenic acid is mainly extracted from honeysuckle and eucommia. The extraction process from honeysuckle is relatively mature, and its low lipid and gum content contributes to good extraction efficiency. However, the price of honeysuckle has increased significantly in recent years, raising costs. Eucommia leaves contain relatively high levels of lipids and eucommia gum, resulting in a low extraction rate and higher impurity content, leading to low purity chlorogenic acid. Therefore, further improving the extraction process to increase the extraction rate and purity of chlorogenic acid from eucommia is crucial. Summary of the Invention

[0004] To improve the extraction rate and purity of chlorogenic acid, this application provides a method for extracting chlorogenic acid from Eucommia ulmoides.

[0005] Firstly, this application provides a method for extracting chlorogenic acid from Eucommia ulmoides, employing the following technical solution:

[0006] A method for extracting chlorogenic acid from Eucommia ulmoides includes the following steps:

[0007] S1: Fresh Eucommia ulmoides leaves are collected and pulped using a pulping machine to obtain Eucommia ulmoides leaf pulp;

[0008] S2: Add a ternary eutectic solvent to the Eucommia ulmoides leaf pulp in step S1 and perform ultrasonic extraction; after extraction, filter and the filtrate is the extract; wherein the ternary eutectic solvent is composed of betaine, levulinic acid and ascorbic acid in a mass ratio of 1:(0.8~1.6):(0.6~0.9);

[0009] S3: Add flocculant to the extract from step S2. After flocculation, centrifuge to obtain supernatant and flocculated precipitate. Concentrate the supernatant to obtain concentrated solution.

[0010] S4: The concentrated solution is passed through an adsorption column filled with modified nano-hydroxyapatite. After treatment, impurities are removed by elution with an aqueous solution. Then, chlorogenic acid is eluted with an eluent to obtain an eluent. The eluent is concentrated under reduced pressure and dried under vacuum to obtain the chlorogenic acid product.

[0011] By adopting the above technical solution, this application uses a ternary eutectic solvent as the extractant. Betaine acts as the hydrogen acceptor, while levulinic acid and ascorbic acid act as hydrogen donors. When blended, these three components form a strong hydrogen bond network, resulting in a mixture with a certain viscosity. When used for extraction, this solvent exhibits a strong dissolving ability for chlorogenic acid in Eucommia ulmoides leaf pulp, thus promoting the release of chlorogenic acid and increasing its extraction rate. The eutectic solvent in this application includes ascorbic acid, which has strong reducing properties and can protect chlorogenic acid from oxidation, improving its stability during the extraction process.

[0012] This application uses modified nano-hydroxyapatite as an adsorption column. Firstly, chlorogenic acid has a strong complexing effect on calcium ions. Moreover, the modified hydroxyapatite has chlorogenic acid imprints on its surface and a large pore structure. Therefore, the modified hydroxyapatite in this application has a strong specific adsorption of chlorogenic acid, which can effectively purify chlorogenic acid and improve its purity.

[0013] Preferably, in step S2, the preparation method of the ternary eutectic solvent includes the following steps: mixing betaine, levulinic acid and ascorbic acid in a certain proportion, heating to 130~150℃, melting, and cooling to obtain a viscous liquid; then adding 1~1.5 times the total mass of deionized water to the viscous liquid, stirring and mixing to obtain the ternary eutectic solvent.

[0014] By adopting the above technical solution, when preparing the ternary eutectic solvent in this application, the three components are first mixed and melted, which is beneficial to the formation of a hydrogen bond network structure. However, the viscosity of the three components after melting is relatively high, which will lead to a slow mass transfer rate in the system during extraction, thereby affecting the extraction effect. Therefore, water needs to be added for dilution to reduce the viscosity. However, the amount of water added should not be too much, as excessive dilution will reduce the hydrogen bond network structure between the three components and also reduce the extraction effect.

[0015] Preferably, in step S2, during ultrasonic extraction, the amount of the ternary eutectic solvent added is 4 to 6 times that of the Eucommia ulmoides leaf pulp; the ultrasonic extraction temperature is 40 to 60°C; and the ultrasonic extraction time is 1 to 2 hours.

[0016] By adopting the above technical solution, the process parameters of the extraction process in this application can extract chlorogenic acid from Eucommia ulmoides leaf pulp as much as possible, which helps to improve the extraction rate of chlorogenic acid.

[0017] Preferably, in step S3, the flocculant is cationic polyacrylamide, and the amount of flocculant added is 4-6% of the total amount of extract; the flocculation time is 20-40 min.

[0018] By adopting the above technical solution, this application uses cationic polyacrylamide as a flocculant, which can form hydrogen bonds with levulinic acid and ascorbic acid in the extract, thereby causing flocculation and removing most of the levulinic acid and ascorbic acid; reducing the impact of levulinic acid and ascorbic acid on the subsequent purification process; cationic polyacrylamide flocculant can also remove some lipids and colloids, improving the effect of the subsequent purification process.

[0019] Preferably, in step S4, the method for preparing modified nano-hydroxyapatite includes the following steps:

[0020] 4-1: Prepare porous nano-hydroxyapatite; then modify the porous hydroxyapatite with hydrophobicity; and disperse it in an organic solvent to obtain a hydrophobic hydroxyapatite dispersion.

[0021] 4-2: Chlorogenic acid, 4-vinylpyridine, 4-vinylphenylboronic acid, ethylene glycol dimethacrylate and an initiator were added to the hydrophobic hydroxyapatite dispersion in step 4-1 to carry out a polymerization reaction. After the reaction was completed, the mixture was filtered, and then chlorogenic acid was eluted. After washing and drying, the modified nano-hydroxyapatite was obtained.

[0022] By adopting the above technical solution, the modified nano-hydroxyapatite of this application possesses porous properties and certain adsorption capacity, and a chlorogenic acid-imprinted layer is prepared on the outside of the hydroxyapatite; thus, the modified hydroxyapatite nano-hydroxyapatite is obtained. When packed into a column, the calcium element of the hydroxyapatite firstly forms a complex with chlorogenic acid, which can adsorb chlorogenic acid in the pores. Moreover, the molecularly imprinted layer for chlorogenic acid is prepared on the outside, which can improve the recognition and adsorption of chlorogenic acid, thereby better improving the purification effect of chlorogenic acid.

[0023] Preferably, in step 4-1, the hydrophobic modification treatment of porous hydroxyapatite specifically involves: dispersing porous hydroxyapatite in ethanol, then adding a silane coupling agent, followed by heating the reaction, and after the reaction is complete, filtering and washing to obtain hydrophobically modified hydroxyapatite; dispersing it in acetonitrile to prepare a dispersion with a concentration of 0.03~0.04 g / mL.

[0024] Preferably, the silane coupling agent is one of 3-(isobutenoyloxy)propyltrimethoxysilane and 3-(methacryloyloxy)propyltrimethoxysilane; the mass-to-volume ratio of porous hydroxyapatite to the silane coupling agent is 1g:0.2~1mL; the heating temperature is 60~80℃, and the reaction time is 3~5h.

[0025] By adopting the above technical solution, this application grafts a silane coupling agent onto the surface of hydroxyapatite. The silane coupling agent contains acryloyloxy or butenoyloxy groups, which can react with the reactants in the subsequent polymerization reaction, thereby better forming an imprint layer on the surface.

[0026] Preferably, in step 4-2, the mass ratio of hydrophobic hydroxyapatite, chlorogenic acid, 4-vinylpyridine, 4-vinylphenylboronic acid, ethylene glycol dimethacrylate, and initiator is 10:(0.5~0.8):(4~5):(2~3):(15~20):(0.03~0.05); the initiator is azobisisobutyronitrile, the polymerization temperature is 40~60℃, and the time is 10~16h; during elution, chlorogenic acid is eluted with acetic acid and ethanol solution.

[0027] By adopting the above technical solution, this application requires strict control over the relationship between the components in the polymerization reaction. This ensures that while an imprinted layer forms on the surface of cyanoapatite, the imprinted layer does not become excessive, as this would significantly reduce the porosity of the hydroxyapatite and decrease its adsorption effect. Preparing an imprinted layer on the surface of hydroxyapatite also reduces the nanoscale effect of the hydroxyapatite, thus allowing for better packing into the purification column.

[0028] Preferably, in step S4, the flow rate of the concentrate treated with modified nano-hydroxyapatite as the adsorption column is 0.1~0.5 mL / min; the eluent is a solution of acetic acid and ethanol with a volume ratio of (3~4):(6~7).

[0029] By employing the above technical solutions, controlling the flow rate of the concentrate can better facilitate the adsorption of chlorogenic acid onto the modified nano-hydroxyapatite, thereby improving purity while reducing the loss rate of chlorogenic acid. Using acetic acid and ethanol as eluents can further desorb chlorogenic acid from the modified nano-hydroxyapatite, further reducing the loss rate of chlorogenic acid.

[0030] In summary, this application includes at least one of the following beneficial technical effects:

[0031] 1. In this application, betaine, levulinic acid, and ascorbic acid are used as eutectic solvents to form a strong hydrogen bond network structure, which can better promote the dissolution of chlorogenic acid and thus improve the extraction rate of chlorogenic acid. Then, cationic flocculants are used to remove most of the acetoacetic acid, ascorbic acid, lipids, and colloids, thereby reducing impurities in the solution and helping to improve the purification effect of subsequent steps.

[0032] In this application, modified nano-hydroxyapatite is used as the adsorption column in the separation and purification process. First, the presence of calcium ions can play a role in complexing chlorogenic acid. After modification, there is also an imprinted layer on the surface, which can further improve the selectivity for chlorogenic acid, thereby improving the purification effect of chlorogenic acid. Detailed Implementation

[0033] Preparation of porous hydroxyapatite: High-porosity porous nano-hydroxyapatite microspheres were prepared using the preparation method in Example 1 of patent publication number CN110538346A (without subsequent drug coating or other steps).

[0034] Preparation Example 1: Preparation of Modified Nano-hydroxyapatite

[0035] 5g of porous hydroxyapatite was dispersed in anhydrous ethanol, and then 2.5mL of 3-(isobutenoyloxy)propyltrimethoxysilane was added. The mixture was heated to 70℃ and stirred for 4h. After the reaction was completed, the mixture was filtered and washed to obtain hydrophobically modified hydroxyapatite (approximately 6.48g). The hydroxyapatite was then dispersed in acetonitrile to prepare a dispersion with a concentration of 0.04g / mL.

[0036] 0.39 g chlorogenic acid, 2.6 g 4-vinylpyridine, 1.62 g 4-vinylphenylboronic acid, 11.67 g ethylene glycol dimethacrylate and 0.025 g azobisisobutyronitrile were added to the dispersion, and the mixture was heated to 50 °C for 12 h for polymerization. After the reaction was completed, the mixture was filtered, and the precipitate was eluted with a mixed solution of acetic acid and ethanol (volume ratio of 1:2) until no chlorogenic acid was detected in the eluent. After elution, the precipitate was obtained by washing and vacuum drying to obtain the modified nano-hydroxyapatite.

[0037] Preparation Example 2

[0038] 5g of porous hydroxyapatite was dispersed in anhydrous ethanol, and then 4mL of 3-(methacryloyloxy)propyltrimethoxysilane was added. The mixture was heated to 60℃ and stirred for 5h. After the reaction was completed, the mixture was filtered and washed to obtain hydrophobically modified hydroxyapatite (approximately 7.15g). The hydroxyapatite was then dispersed in acetonitrile to prepare a dispersion with a concentration of 0.03g / mL.

[0039] 0.57 g chlorogenic acid, 3.58 g 4-vinylpyridine, 2.15 g 4-vinylphenylboronic acid, 14.30 g ethylene glycol dimethacrylate and 0.036 g azobisisobutyronitrile were added to the dispersion, and the mixture was heated to 60 °C for 14 h for polymerization. After the reaction was completed, the mixture was filtered, and the precipitate was eluted with a mixed solution of acetic acid and ethanol (volume ratio of 1:2) until no chlorogenic acid was detected in the eluent. After elution, the precipitate was obtained by washing and vacuum drying to obtain the modified nano-hydroxyapatite.

[0040] Preparation Example 3

[0041] 5g of porous hydroxyapatite was dispersed in anhydrous ethanol, and then 1.5mL of 3-(isobutenoyloxy)propyltrimethoxysilane was added. The mixture was heated to 80℃ and stirred for 3h. After the reaction was completed, the mixture was filtered and washed to obtain hydrophobically modified hydroxyapatite (approximately 5.97g). The hydroxyapatite was then dispersed in acetonitrile to prepare a dispersion with a concentration of 0.04g / mL.

[0042] 0.30 g chlorogenic acid, 2.39 g 4-vinylpyridine, 1.19 g 4-vinylphenylboronic acid, 8.96 g ethylene glycol dimethacrylate and 0.024 g azobisisobutyronitrile were added to the dispersion, and the mixture was heated to 40 °C for polymerization for 16 h. After the reaction was completed, the mixture was filtered, and the precipitate was eluted with a mixed solution of acetic acid and ethanol (volume ratio of 1:2) until no chlorogenic acid was detected in the eluent. After elution, the precipitate was obtained by washing and vacuum drying to obtain the modified nano-hydroxyapatite.

[0043] Example 1

[0044] Wash 50g of fresh Eucommia ulmoides leaves and pulp them at room temperature to obtain Eucommia ulmoides liquid pulp.

[0045] Betaine, levulinic acid, and ascorbic acid were mixed in a mass ratio of 1:1.2:0.7, heated to 150°C, melted, and then cooled to obtain a viscous liquid. Deionized water with a mass ratio of 1.0 to the total mass of the viscous liquid was then added and stirred to obtain a ternary eutectic solvent.

[0046] Add 250 mL of ternary eutectic solvent to the Eucommia ulmoides extract and extract ultrasonically at 50 °C for 1.5 h. After extraction, filter and remove the residue to obtain the extract.

[0047] Add 5% polyimide relative to the mass of the extract to the extract and allow it to flocculate for 30 minutes. After flocculation, filter the solution to obtain the supernatant. Concentrate the supernatant to half its original volume and recover the solvent to obtain the concentrate.

[0048] 5g of the modified nano-hydroxyapatite prepared in Preparation Example 1 was moistened with 10mL of ethanol solution and then packed into a 15mL chromatographic column. The top and bottom of the packing material were covered with polypropylene sieve plates. After packing, the column was rinsed with 10mL of pure water. 60mL of the concentrated solution was loaded onto the column at a flow rate of 0.5mL / min. Elution was first performed with 40mL of pure water to remove impurities (mainly betaine), followed by elution with 70mL of a mixed solvent of acetic acid and ethanol (3:7, v / v). The eluent was collected, concentrated under reduced pressure, and then vacuum dried to obtain chlorogenic acid powder.

[0049] Comparative Example 1

[0050] It is basically the same as Example 1, except that a pure aqueous solution is used instead of the ternary eutectic solvent.

[0051] Comparative Example 2

[0052] The method is basically the same as in Example 1, except that an equal mass of levulinic acid is used instead of ascorbic acid, that is, the mass ratio of betaine to levulinic acid is 1:1.9.

[0053] Comparative Example 3

[0054] The method is basically the same as in Example 1, except that an equal mass of ascorbic acid is used instead of levulinic acid, i.e., the mass ratio of betaine to ascorbic acid is 1:1.9.

[0055] Comparative Example 4

[0056] The method is basically the same as in Example 1, except that an equal mass of chlorylcholine is used instead of betaine, that is, chlorylcholine, levulinic acid and ascorbic acid are in a mass ratio of 1:1.2:0.7.

[0057] Comparative Example 5

[0058] It is basically the same as Example 1, except that the modified nano-hydroxyapatite is replaced with macroporous adsorption resin HP-20.

[0059] Comparative Example 6

[0060] It is basically the same as Example 1, except that porous hydroxyapatite is used instead of the modified nano-hydroxyapatite.

[0061] The purity and recovery rate of chlorogenic acid prepared in Example 1 and Comparative Examples 1-6 are shown in Table 1.

[0062]

[0063] As can be seen from the data in Table 1, although the purity of Example 1 was slightly improved compared with Comparative Example 1, its total recovery rate decreased significantly. This indicates that the ultrasonic extraction using pure water was not effective, resulting in a significant decrease in the total recovery rate.

[0064] Compared with Comparative Examples 2 and 3, the main difference in Example 1 is that Comparative Example 2 did not contain ascorbic acid, and Comparative Example 3 did not contain levulinic acid. The results show that the purity changes in Comparative Examples 2 and 3 were very slight, but their total recovery rates both showed a significant decrease. This is because different substances have their optimal composition and ratio of eutectic solvents, and the ratio in Example 1 is more favorable for the leaching of chlorogenic acid compared to Comparative Examples 2 and 3.

[0065] Compared with Comparative Example 4, Example 1 replaced betaine with chloracylcholine. The data shows that the total recovery rate in Comparative Example 4 decreased significantly. This may be because different hydrogen acceptors have different extraction effects on different extractable substances. According to the data in this application, under the condition of extracting chlorogenic acid with a ratio of levulinic acid and ascorbic acid, betaine is more helpful in improving the extraction effect.

[0066] Compared with Comparative Example 5, Comparative Example 5 used macroporous resin. Due to the problem with the extraction method of this application, the extract contained more betaine and other impurities. Therefore, the purification effect of macroporous resin was poor, and the purity and recovery rate of chlorogenic acid were also reduced accordingly.

[0067] Compared with Comparative Example 6, Example 1 uses unmodified porous hydroxyapatite. Although porous hydroxyapatite has a certain complexing and adsorption effect on chlorogenic acid, its porous structure adsorbs certain impurities. However, Example 1 has an imprinted layer, which can recognize chlorogenic acid to a certain extent. Therefore, the purity of Example 1 is higher.

[0068] Example 2

[0069] It is basically the same as Example 1, except that the modified hydroxyapatite used in Preparation Example 2 is used.

[0070] Example 3

[0071] It is basically the same as Example 1, except that the modified hydroxyapatite used in Preparation Example 2 is used.

[0072] The extraction and purification effects of Examples 2 and 3 are shown in Table 2.

[0073]

[0074] Compared with Example 1, Example 2 differs only in the type of modified hydroxyapatite. In terms of preparation method, the imprinted layer in Example 2 is thicker, resulting in a higher recognition effect of chlorogenic acid and thus an improvement in its purity. However, due to the slightly thicker coating, some pores are blocked, which reduces the adsorption effect and leads to a slight decrease in the total recovery rate.

[0075] Compared with Example 1, Example 3 has a thinner imprinted layer, resulting in a lower recognition rate of chlorogenic acid and thus a lower purity of chlorogenic acid. However, the pores are more abundant, leading to a greater adsorption capacity and a slightly higher overall recovery rate.

[0076] Example 4

[0077] It is basically the same as Example 1, except that the mass ratio of betaine, levulinic acid and ascorbic acid is 1:0.8:0.9.

[0078] Example 5

[0079] It is basically the same as Example 1, except that the mass ratio of betaine, levulinic acid and ascorbic acid is 1:1.4:0.6.

[0080] The purification and extraction effects of Examples 4 and 5 are shown in Table 3.

[0081]

[0082] In Example 4, the purity of chlorogenic acid was slightly improved compared to Example 1, but its recovery rate decreased. The improvement in purity may be due to the smaller amount of ascorbic acid and levulinic acid added, which is more conducive to purification. The low recovery rate may be due to the poor extraction effect caused by the ratio of the ingredients.

[0083] In Example 5, the purity and total recovery rate of chlorogenic acid were both lower than those in Example 1. The lower purity may be due to the addition of more ascorbic acid and levulinic acid, which is not conducive to purification. The lower recovery rate may be due to the poor extraction effect caused by the ratio of the ingredients.

[0084] Example 6

[0085] Wash 50g of fresh Eucommia ulmoides leaves and pulp them at room temperature to obtain Eucommia ulmoides liquid pulp.

[0086] Betaine, levulinic acid, and ascorbic acid were mixed in a mass ratio of 1:1.3:0.8, heated to 140°C, melted, and then cooled to obtain a viscous liquid. Deionized water with a mass ratio of 1.0 to the total mass of the viscous liquid was then added and stirred to obtain a ternary eutectic solvent.

[0087] Add 200 mL of ternary eutectic solvent to the Eucommia ulmoides extract and extract by ultrasonication at 60 °C for 1 h. After extraction, filter and remove the residue to obtain the extract.

[0088] Add 6% cationic polyimide relative to the mass of the extract to the extract and allow it to flocculate for 20 minutes. After flocculation, filter the solution to obtain the supernatant. Concentrate the supernatant to half its original volume and recover the solvent to obtain the concentrate.

[0089] 5g of the modified nano-hydroxyapatite prepared in Preparation Example 1 was moistened with 10mL of ethanol solution and then packed into a 15mL chromatographic column. The top and bottom of the packing material were covered with polypropylene sieve plates. After packing, the column was rinsed with 10mL of pure water. 60mL of the concentrated solution was loaded onto the column at a flow rate of 0.6mL / min. Elution was first performed with 40mL of pure water to remove impurities (mainly betaine), followed by elution with a 70mL mixture of acetic acid and ethanol (4:6, v / v). The eluent was collected, concentrated under reduced pressure, and then vacuum dried to obtain chlorogenic acid powder.

[0090] Example 7

[0091] Wash 50g of fresh Eucommia ulmoides leaves and pulp them at room temperature to obtain Eucommia ulmoides liquid pulp.

[0092] Betaine, levulinic acid, and ascorbic acid were mixed in a mass ratio of 1:1.6:0.6, heated to 130°C, melted, and then cooled to obtain a viscous liquid. Deionized water with a mass ratio of 1.5 times the total mass of the viscous liquid was then added to the viscous liquid and stirred until homogeneous to obtain a ternary eutectic solvent.

[0093] Add 300 mL of ternary eutectic solvent to the Eucommia ulmoides extract and extract by ultrasonication at 60 °C for 1 h. After extraction, filter and remove the residue to obtain the extract.

[0094] Add 4% cationic polyimide relative to the mass of the extract to the extract and allow it to flocculate for 40 minutes. After flocculation, filter the solution to obtain the supernatant. Concentrate the supernatant to half its original volume and recover the solvent to obtain the concentrate.

[0095] 5g of the modified nano-hydroxyapatite prepared in Preparation Example 1 was moistened with 10mL of ethanol solution and then packed into a 15mL chromatographic column. The top and bottom of the packing material were covered with polypropylene sieve plates. After packing, the column was rinsed with 10mL of pure water. 60mL of the concentrated solution was loaded onto the column at a flow rate of 0.6mL / min. Elution was first performed with 40mL of pure water to remove impurities (mainly betaine), followed by elution with 70mL of a mixed solvent of acetic acid and ethanol (3:7, v / v). The eluent was collected, concentrated under reduced pressure, and then vacuum dried to obtain chlorogenic acid powder.

[0096] The purification and extraction effects of Examples 6 and 7 are shown in Table 4.

[0097]

[0098] The chlorogenic acid extracted in Examples 6 and 7 both had high purity and relatively high total recovery rate. The change of parameters caused a certain degree of change in both purity and total recovery rate, but overall, they both showed good results.

[0099] The above are all preferred embodiments of this application, and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the structure, shape and principle of this application should be covered within the scope of protection of this application.

Claims

1. A method for extracting chlorogenic acid from Eucommia ulmoides, comprising the following steps: S1: Fresh Eucommia ulmoides leaves are collected and pulped using a pulping machine to obtain Eucommia ulmoides leaf pulp; S2: Add a ternary eutectic solvent to the Eucommia ulmoides leaf pulp in step S1 and perform ultrasonic extraction; after extraction, filter and the filtrate is the extract; wherein the ternary eutectic solvent is composed of betaine, levulinic acid and ascorbic acid in a mass ratio of 1:(0.8~1.6):(0.6~0.9); S3: Add flocculant to the extract from step S2, and after flocculation, centrifuge to obtain supernatant and flocculated precipitate; concentrate the supernatant to obtain concentrated solution. S4: The concentrated solution from step S3 is passed through an adsorption column filled with modified nano-hydroxyapatite. After treatment, impurities are removed by elution with an aqueous solution. Then, chlorogenic acid is eluted with an eluent to obtain an eluent. The eluent is concentrated under reduced pressure and dried under vacuum to obtain the chlorogenic acid product. In step S2, the preparation method of the ternary eutectic solvent includes the following steps: betaine, levulinic acid and ascorbic acid are mixed in proportion, heated to 130~150℃, melted and cooled to obtain a viscous liquid; then, 1~1.5 times the total mass of deionized water is added to the viscous liquid, and after stirring and mixing, the ternary eutectic solvent is obtained. In step S4, the method for preparing modified nano-hydroxyapatite includes the following steps: 4-1: Prepare porous nano-hydroxyapatite; then modify the porous hydroxyapatite with hydrophobicity; and disperse it in an organic solvent to obtain a hydrophobic hydroxyapatite dispersion. 4-2: Chlorogenic acid, 4-vinylpyridine, 4-vinylphenylboronic acid, ethylene glycol dimethacrylate and an initiator were added to the hydrophobic hydroxyapatite dispersion in step 4-1 to carry out a polymerization reaction. After the reaction was completed, the mixture was filtered, and then chlorogenic acid was eluted. After washing and drying, the modified nano-hydroxyapatite was obtained.

2. The method for extracting chlorogenic acid from Eucommia ulmoides according to claim 1, characterized in that, In step S2, during ultrasonic extraction, the amount of ternary eutectic solvent added is 4 to 6 times that of Eucommia ulmoides leaf pulp; the ultrasonic extraction temperature is 40 to 60°C, and the ultrasonic extraction time is 1 to 2 hours.

3. The method for extracting chlorogenic acid from Eucommia ulmoides according to claim 1, characterized in that, In step S3, the flocculant is cationic polyacrylamide, and the amount of flocculant added is 4-6% of the total amount of extract; the flocculation time is 20-40 min.

4. The method for extracting chlorogenic acid from Eucommia ulmoides according to claim 1, characterized in that, In step 4-1, the hydrophobic modification treatment of porous hydroxyapatite specifically involves: dispersing porous hydroxyapatite in ethanol, then adding a silane coupling agent, followed by heating the reaction, and after the reaction is complete, filtering and washing to obtain hydrophobically modified hydroxyapatite; dispersing it in acetonitrile to prepare a dispersion with a concentration of 0.03~0.04 g / mL.

5. The method for extracting chlorogenic acid from Eucommia ulmoides according to claim 4, characterized in that, The silane coupling agent is one of 3-(isobutenoyloxy)propyltrimethoxysilane and 3-(methacryloyloxy)propyltrimethoxysilane; the mass-volume ratio of porous hydroxyapatite to silane coupling agent is 1g:0.2~1mL; the heating temperature is 60~80℃, and the reaction time is 3~5h.

6. The method for extracting chlorogenic acid from Eucommia ulmoides according to claim 1, characterized in that, In step 4-2, the mass ratio of hydrophobic hydroxyapatite, chlorogenic acid, 4-vinylpyridine, 4-vinylphenylboronic acid, ethylene glycol dimethacrylate, and initiator is 10:(0.5~0.8):(4~5):(2~3):(15~20):(0.03~0.05); the initiator is azobisisobutyronitrile, the polymerization temperature is 40~60℃, and the time is 10~16h; during elution, chlorogenic acid is eluted with acetic acid and ethanol solution.

7. The method for extracting chlorogenic acid from Eucommia ulmoides according to claim 1, characterized in that, In step S4, the flow rate of the concentrate treated with modified nano-hydroxyapatite as an adsorption column is 0.1~0.5 mL / min; the eluent is a solution of acetic acid and ethanol with a volume ratio of (3~4):(6~7).

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