Method for extracting and purifying flavone in litsea coreana through ultrasonic-assisted eutectic solvent

Abstract

The invention discloses a method for extracting and purifying flavone in litsea coreana through an ultrasonic-assisted eutectic solvent, and belongs to the technical field of green processing of natural products. The invention provides a method for efficiently preparing litsea coreana flavone by integrating computer prediction, green solvent extraction and resin purification technologies. The method comprises the following steps: firstly, calculating and screening a deep eutectic solvent system with the highest affinity with the litsea coreana flavone on the basis of a COSMO-RS theoretical model, then coupling the optimized DES with an ultrasonic-assisted extraction (UAE) technology, and through the synergistic effect of the DES and the UAE technology, remarkably improving the extraction rate of the litsea coreana flavone and furthest keeping the biological activity of a target component; then, a macroporous resin dynamic adsorption-desorption purification process is linked, so that high-selectivity enrichment and impurity removal of the flavonoid compounds in the crude extract are realized, a high-purity product is obtained, and a complete process route from intelligent screening, efficient extraction to precise purification is finally formed.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of green processing of natural products, and particularly relates to a method for extracting and purifying flavones in Litsea chrysocarpa by using ultrasonic-assisted deep eutectic solvent. BACKGROUND

[0002] Litsea chrysocarpa (tender leaves and leaf buds of Litsea chrysocarpa) is rich in flavones such as isoquercitrin and quercetin, has excellent biological activities such as antioxidant, antibacterial, anti-inflammatory and prevention of chronic diseases, and has great application potential in the fields of functional food and medicine. At present, the extraction of plant flavones still heavily relies on organic solvent (such as methanol and ethanol) extraction, hot reflux and other technologies. These methods generally have problems such as high solvent toxicity, serious environmental pollution, high temperature leading to degradation of active ingredients and the like. Although supercritical fluid and microwave-assisted extraction can improve the efficiency to some extent, they still face the industrialization bottlenecks of high equipment cost and limited process adaptability.

[0003] In recent years, deep eutectic solvent (DES) has become a green extraction medium to replace traditional organic solvents due to its low toxicity, biodegradability and friendliness to heat-sensitive substances. Ultrasonic-assisted extraction (UAE) technology can use cavitation effect to efficiently destroy plant cell walls, significantly improve solute release rate and reduce extraction temperature. The combination of DES and UAE can greatly improve the extraction efficiency of flavones while taking into account environmental friendliness, and has become a research hotspot in the field of green extraction of natural products. However, the combination modes of hydrogen bond donors and acceptors of DES are extremely diverse, and the traditional "trial and error" experimental screening method has large workload and high cost, which seriously restricts the development and application promotion of the technology.

[0004] In addition, the subsequent purification of the extract is also a key link to realize the high-value utilization of flavones. Macroporous adsorption resin has been widely used in the separation and purification of natural products due to its high adsorption capacity, customizable polarity and pore size, and reusability. Developing a resin purification process that efficiently connects with the DES extraction process is of great significance to realize the green, efficient and large-scale preparation of Litsea chrysocarpa flavones. SUMMARY

[0005] In view of the above prior art, the application provides a method for extracting and purifying flavones in Litsea chrysocarpa by using ultrasonic-assisted deep eutectic solvent, which solves the problems of large solvent toxicity, low extraction efficiency, blind screening and difficult purification in the existing extraction technology of Litsea chrysocarpa flavones.

[0006] In order to achieve the above purpose, the technical scheme adopted by the application is as follows: a method for extracting and purifying flavones in Litsea chrysocarpa by using ultrasonic-assisted deep eutectic solvent is provided, which comprises the following steps: S1: calculating and screening a deep eutectic solvent DES system suitable for the extraction of Litsea chrysocarpa flavones based on a COSMO-RS theoretical model, and preparing a deep eutectic solvent DES system according to the screening result; S2: mixing the raw material of the eagle tea with the DES system, performing ultrasonic-assisted extraction, and obtaining the crude extract of the eagle tea flavonoids after centrifugation; S3: loading the crude extract into a macroporous adsorption resin column, performing dynamic adsorption and elution, and collecting the desorption liquid rich in flavonoids; S4: concentrating and drying the desorption liquid to obtain the eagle tea flavonoids product.

[0007] Further, the DES system screened in S1 includes a hydrogen bond acceptor and a hydrogen bond donor, the hydrogen bond acceptor is carnitine, betaine, choline chloride or glycerol, and the hydrogen bond donor is propylene glycol, 1,3-propanediol, acetic acid, 1,2-butanediol, levulinic acid, urea, 1,4-butanediol, ethylene glycol or citric acid; the preparation of the DES system includes the following steps: mixing the hydrogen bond acceptor and the hydrogen bond donor and heating to 80℃, and stirring to form a homogeneous transparent liquid, and then adding water to obtain the DES system.

[0008] Further, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:2.

[0009] Further, the proportion of water in the DES system is 30% (w / w).

[0010] Further, the solid-liquid ratio of the eagle tea raw material to the DES system is 1g:20mL.

[0011] Further, the ultrasonic-assisted extraction is performed under the conditions of ultrasonic power 250-300 W and temperature 40-60℃ for 30 min.

[0012] Further, the macroporous adsorption resin is HPD600 resin.

[0013] Further, the loading flow rate of dynamic adsorption is 1.0-2.0 BV / h.

[0014] Further, the elution is performed by using 20%-30% (v / v) ethanol aqueous solution to elute impurities, and then using 85%-95% (v / v) ethanol aqueous solution to elute and collect flavonoids.

[0015] The beneficial effects of the present application are: the method provided by the present application establishes an intelligent solvent screening strategy based on COSMO-RS molecular simulation prediction combined with experimental verification, quickly and accurately constructs the optimal deep eutectic solvent (DES) system for old eagle tea flavonoids, and fundamentally reduces the large resource and time consumption caused by the traditional "trial and error method". Then, the DES selected is coupled with the ultrasonic assisted extraction (UAE) technology, through the synergistic effect of the two, the extraction rate of old eagle tea flavonoids is significantly improved, and the biological activity of the target components is maximized. Then, the macroporous resin dynamic adsorption-desorption purification process is connected to realize the high selectivity enrichment and impurity removal of flavonoids in the crude extract, and high-purity products are obtained, and finally a complete process route from intelligent screening, efficient extraction to accurate purification is formed. The present application integrates the technical chain of "intelligent screening-green extraction-accurate purification", and the whole process is environmentally friendly, simple to operate, stable and reliable, easy to scale up and continuous production, which is not only suitable for old eagle tea, but also can provide a complete and popularized solution for the large-scale green preparation of flavonoids in other plant resources. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The sigma surface (1A), sigma profile (1B) and sigma potential (1C) of five flavonoids, carnitine and 1,4-butanediol; Figure 2 The solubility thermogram of hyperoside in DES predicted by the COSMO-RS model; Figure 3 The solubility thermogram of trilobatin in DES predicted by the COSMO-RS model; Figure 4 The solubility thermogram of kaempferol-3-rutinoside in DES predicted by the COSMO-RS model; Figure 5 The solubility thermogram of isoquercitrin in DES predicted by the COSMO-RS model; Figure 6 The solubility thermogram of astragalin in DES predicted by the COSMO-RS model; Figure 7 The column chart of old eagle tea flavonoid content of examples 1-16 and comparative examples 1-3. DETAILED DESCRIPTION

[0017] The specific embodiments of the present application will be described in detail below in combination with examples.

[0018] Example 1 A method for ultrasonic assisted deep eutectic solvent extraction and purification of flavonoids in old eagle tea, comprising the following steps: S1: calculate and screen the deep eutectic solvent DES system (carnitine and 1,4-butanediol) based on the COSMO-RS theoretical model; S2: mix carnitine and 1,4-butanediol at a molar ratio of 1:2 and heat to 80°C and stir for 1h until a homogeneous transparent liquid is formed, then add water after cooling to obtain the deep eutectic solvent DES system screened in S1, wherein the proportion of water in the deep eutectic solvent DES system is 30% w / w; S3: take 0.2g of old eagle tea powder in a 10mL centrifuge tube, add 4mL of deep eutectic solvent DES system, vortex mix uniformly, ultrasonic treatment under the condition of ultrasonic power 300W and temperature 50℃ for 30min, obtain the mixed liquid, collect the supernatant after centrifugation of the mixed liquid at 5000rpm for 10min, repeat the extraction of tea residue with equal volume of deep eutectic solvent DES system for 1 time, and combine the supernatant to obtain the old eagle tea flavonoid crude extract; S4: dilute the old eagle tea flavonoid crude extract to 0.75mg / mL, load to HPD600 macroporous resin (flow rate 1.5BV / h), elute with 20% ethanol to remove impurities, and then elute with 90% ethanol to collect the desorption liquid rich in flavonoids; S5: concentrate the desorption liquid at 40℃ by rotary evaporation, then freeze-dry to obtain the old eagle tea flavonoid product.

[0019] Example 2 A method for ultrasonic-assisted deep eutectic solvent extraction and purification of flavonoids in old eagle tea, comprising the following steps: S1: calculate and screen the deep eutectic solvent DES system (betaine and 1,3-propanediol) based on the COSMO-RS theoretical model; S2: mix betaine and 1,3-propanediol at a molar ratio of 1:2 and heat to 80°C and stir for 1h until a homogeneous transparent liquid is formed, then add water after cooling to obtain the deep eutectic solvent DES system screened in S1, wherein the proportion of water in the deep eutectic solvent DES system is 30% (w / w); S3: take 0.2g of old eagle tea powder in a 10mL centrifuge tube, add 4mL of deep eutectic solvent DES system, vortex mix uniformly, ultrasonic treatment under the condition of ultrasonic power 250W and temperature 40℃ for 30min, obtain the mixed liquid, collect the supernatant after centrifugation of the mixed liquid at 5000rpm for 10min, repeat the extraction of tea residue with equal volume of deep eutectic solvent DES system for 1 time, and combine the supernatant to obtain the old eagle tea flavonoid crude extract; S4: dilute the old eagle tea flavonoid crude extract to 0.75mg / mL, load to HPD600 macroporous resin (flow rate 1.0BV / h), elute with 30% ethanol to remove impurities, and then elute with 95% ethanol to collect the desorption liquid rich in flavonoids; S5: The desorbed liquid is concentrated by rotary evaporation at 40°C, and then freeze-dried to obtain the eagle tea flavonoid product.

[0020] Example 3 A method for extracting and purifying flavonoids from Eagle Tea using ultrasound-assisted eutectic solvent extraction includes the following steps: S1: Based on the COSMO-RS theoretical model, the DES system (betaine and propylene glycol) was calculated and screened out as a low eutectic solvent. S2: Mix betaine and propylene glycol in a molar ratio of 1:2 and heat to 80°C and stir for 1 hour until a homogeneous transparent liquid is formed. After cooling, add water to obtain the eutectic solvent DES system screened in S1, wherein the proportion of water in the eutectic solvent DES system is 30% (w / w). S3: Take 0.2g of Eagle Tea powder into a 10mL centrifuge tube, add 4mL of eutectic solvent DES system, vortex mix evenly, and sonicate for 30min under ultrasonic power of 300W and temperature of 60℃ to obtain a mixture. Centrifuge the mixture at 5000rpm for 10min and collect the supernatant. Then use an equal volume of eutectic solvent DES system to extract the tea residue once more. Combine the supernatants to obtain the crude extract of Eagle Tea flavonoids. S4: Dilute the crude extract of flavonoids from Eagle Tea to 0.75 mg / mL, load it onto HPD600 macroporous resin (flow rate 2.0 BV / h), elute with 30% ethanol to remove impurities, and then elute with 85% ethanol to collect the flavonoid-rich eluent. S5: The desorbed liquid is concentrated by rotary evaporation at 40°C, and then freeze-dried to obtain the eagle tea flavonoid product.

[0021] Example 4 A method for extracting and purifying flavonoids from Eagle Tea using ultrasound-assisted eutectic solvent extraction differs from Example 1 in that the hydrogen bond acceptor in the eutectic solvent DES system is carnitine, and the hydrogen bond donor is propylene glycol.

[0022] Example 5 A method for extracting and purifying flavonoids from Eagle Tea using ultrasound-assisted eutectic solvent extraction differs from Example 1 in that the hydrogen bond acceptor in the eutectic solvent DES system is betaine, and the hydrogen bond donor is urea.

[0023] Example 6 A method for extracting and purifying flavonoids from Eagle Tea using ultrasound-assisted eutectic solvent extraction differs from Example 1 in that the hydrogen bond acceptor in the eutectic solvent DES system is betaine, and the hydrogen bond donor is 1,2-butanediol.

[0024] Example 7 A method for extracting and purifying flavonoids from Eagle Tea using ultrasound-assisted eutectic solvent extraction differs from Example 1 in that the hydrogen bond acceptor in the eutectic solvent DES system is carnitine, and the hydrogen bond donor is urea.

[0025] Example 8 A method for extracting and purifying flavonoids from Eagle Tea using ultrasound-assisted eutectic solvent extraction differs from Example 1 in that the hydrogen bond acceptor in the eutectic solvent DES system is carnitine, and the hydrogen bond donor is 1,2-butanediol.

[0026] Example 9 A method for extracting and purifying flavonoids from Eagle Tea using ultrasound-assisted eutectic solvent extraction differs from Example 1 in that the hydrogen bond acceptor in the eutectic solvent DES system is betaine, and the hydrogen bond donor is acetic acid.

[0027] Example 10 A method for extracting and purifying flavonoids from Eagle Tea using ultrasound-assisted eutectic solvent extraction differs from Example 1 in that the hydrogen bond acceptor in the eutectic solvent DES system is carnitine, and the hydrogen bond donor is acetic acid.

[0028] Example 11 A method for extracting and purifying flavonoids from Eagle Tea using ultrasound-assisted eutectic solvent extraction differs from Example 1 in that the hydrogen bond acceptor in the eutectic solvent DES system is betaine, and the hydrogen bond donor is 1,4-butanediol.

[0029] Example 12 A method for extracting and purifying flavonoids from Eagle Tea using ultrasound-assisted eutectic solvent extraction differs from Example 1 in that the hydrogen bond acceptor in the eutectic solvent DES system is betaine, and the hydrogen bond donor is levulinic acid.

[0030] Example 13 A method for extracting and purifying flavonoids from Eagle Tea using ultrasound-assisted eutectic solvent extraction differs from Example 1 in that the hydrogen bond acceptor in the eutectic solvent DES system is carnitine, and the hydrogen bond donor is 1,3-propanediol.

[0031] Example 14 A method for extracting and purifying flavonoids from Eagle Tea using ultrasound-assisted eutectic solvent extraction differs from Example 1 in that the hydrogen bond acceptor in the eutectic solvent DES system is carnitine, and the hydrogen bond donor is levulinic acid.

[0032] Example 15 A method for extracting and purifying flavonoids from Eagle Tea using ultrasound-assisted eutectic solvent extraction differs from Example 1 in that the hydrogen bond acceptor in the eutectic solvent DES system is choline chloride, and the hydrogen bond donor is ethylene glycol.

[0033] Example 16 A method for extracting and purifying flavonoids from Eagle Tea using ultrasound-assisted eutectic solvent extraction differs from Example 1 in that the hydrogen bond acceptor in the eutectic solvent DES system is glycerol, and the hydrogen bond donor is citric acid.

[0034] Comparative Example 1 A method for extracting and purifying flavonoids from Eagle Tea using ultrasound-assisted eutectic solvent extraction includes the following steps: S1: Take 0.2g of Lao Ying tea powder into a 10mL centrifuge tube, add 4mL of 80% ethanol aqueous solution, vortex mix evenly, and sonicate for 30min under ultrasonic power of 300W and temperature of 50℃ to obtain a mixture. Centrifuge the mixture at 5000rpm for 10min and collect the supernatant. Then, extract the tea residue once again with an equal volume of eutectic solvent DES system. Combine the supernatants to obtain crude extract of Lao Ying tea flavonoids. S2: Dilute the crude extract of flavonoids from Eagle Tea to 0.75 mg / mL, load it onto HPD600 macroporous resin (flow rate 1.5 BV / h), elute with 20% ethanol to remove impurities, and then elute with 90% ethanol to collect the flavonoid-rich eluent. S3: The desorbed liquid is concentrated by rotary evaporation at 40°C, and then freeze-dried to obtain the eagle tea flavonoid product.

[0035] Comparative Example 2 A method for extracting and purifying flavonoids from Eagle Tea using ultrasound-assisted eutectic solvent extraction includes the following steps: S1: Take 0.2g of Lao Ying tea powder into a 10mL centrifuge tube, add 4mL of 80% methanol aqueous solution, vortex mix evenly, and sonicate for 30min under ultrasonic power of 300W and temperature of 50℃ to obtain a mixture. Centrifuge the mixture at 5000rpm for 10min and collect the supernatant. Then, extract the tea residue once again with an equal volume of eutectic solvent DES system. Combine the supernatants to obtain crude extract of Lao Ying tea flavonoids. S2: Dilute the crude extract of flavonoids from Eagle Tea to 0.75 mg / mL, load it onto HPD600 macroporous resin (flow rate 1.5 BV / h), elute with 20% ethanol to remove impurities, and then elute with 90% ethanol to collect the flavonoid-rich eluent. S3: The desorbed liquid is concentrated by rotary evaporation at 40°C, and then freeze-dried to obtain the eagle tea flavonoid product.

[0036] Comparative Example 3 A method for extracting and purifying flavonoids from Eagle Tea using ultrasound-assisted eutectic solvent extraction includes the following steps: S1: Take 0.2g of Lao Ying tea powder into a 10mL centrifuge tube, add 4mL of distilled water, vortex mix evenly, and sonicate for 30min under ultrasonic power of 300W and temperature of 50℃ to obtain a mixture. Centrifuge the mixture at 5000rpm for 10min and collect the supernatant. Then, extract the tea residue once again with an equal volume of eutectic solvent DES system. Combine the supernatants to obtain crude extract of Lao Ying tea flavonoids. S2: Dilute the crude extract of flavonoids from Eagle Tea to 0.75 mg / mL, load it onto HPD600 macroporous resin (flow rate 1.5 BV / h), elute with 20% ethanol to remove impurities, and then elute with 90% ethanol to collect the flavonoid-rich eluent. S3: The desorbed liquid is concentrated by rotary evaporation at 40°C, and then freeze-dried to obtain the eagle tea flavonoid product.

[0037] Experimental Example 1 DES system screening experiment: COSMO-RS was used to predict the σ-surface, σ-profile, and σ-potential curves of five flavonoid compounds and DES under conditions of 50℃, a hydrogen bond acceptor-hydrogen bond donor molar ratio of 1:2, and infinite dilution. Figure 1 Significant differences were observed; further, the solubility of five flavonoid compounds in 390 DES samples was predicted using COSMO-RS, and the results are as follows. Figures 2-6 Sixteen DES systems were selected (i.e., Examples 1 to 16).

[0038] Experiment Example 2 Flavonoid content detection: The flavonoids extracted in Examples 1-16 and Comparative Examples 1-3 were detected. The specific steps were as follows: the absorbance of the crude extract was measured at 510 nm using the sodium nitrite-aluminum nitrate colorimetric method. The flavonoid content was calculated according to the formula and finally expressed as rutin equivalent RT (standard curve: y=1.0554x-0.032).

[0039] ; Where c is the flavonoid yield of the extract (mg / g), v is the volume of the extract (mL), N is the dilution factor, and m is the sample mass.

[0040] The results are as follows Figure 7 As shown, there are significant differences in extraction efficiency among different combinations of HBA and HBD. Except for acidic DES, the extraction efficiency of the others is better than that of traditional organic solvents. Among them, carnitine-1,4-butanediol has the highest extraction efficiency, which is 685.02 mg·RE / g·DW.

[0041] Although specific embodiments of the present invention have been described in detail with reference to examples, they should not be construed as limiting the scope of protection of this patent. Various modifications and variations that can be made by those skilled in the art without inventive effort within the scope described in the claims are still within the scope of protection of this patent.

Claims

1. A method for extracting and purifying flavonoids from Eagle Tea using ultrasound-assisted eutectic solvent extraction, characterized in that, Includes the following steps: S1: Based on the COSMO-RS theoretical model, the DES system suitable for the extraction of flavonoids from eagle tea was calculated and screened, and the DES system was prepared according to the screening results. S2: Mix the raw material of Eagle Tea with the eutectic solvent DES system, perform ultrasonic-assisted extraction, and centrifuge to obtain crude extract of Eagle Tea flavonoids; S3: Load the crude extract onto a macroporous adsorption resin column for dynamic adsorption and elution, and collect the flavonoid-rich eluent. S4: Concentrate and dry the desorption solution to obtain the eagle tea flavonoid product.

2. The method for extracting and purifying flavonoids from Eagle Tea using ultrasound-assisted eutectic solvent extraction according to claim 1, characterized in that, The eutectic solvent DES system screened by S1 includes hydrogen bond acceptors and hydrogen bond donors. The hydrogen bond acceptors are carnitine, betaine, choline chloride, or glycerol, and the hydrogen bond donors are propylene glycol, 1,3-propanediol, acetic acid, 1,2-butanediol, levulinic acid, urea, 1,4-butanediol, ethylene glycol, or citric acid. The preparation of the eutectic solvent DES system includes the following steps: The hydrogen bond acceptor and hydrogen bond donor are mixed and heated to 80°C. The mixture is stirred until a homogeneous transparent liquid is formed. After cooling, water is added to obtain the eutectic solvent DES system.

3. The method for extracting and purifying flavonoids from Eagle Tea using ultrasound-assisted eutectic solvent extraction according to claim 2, characterized in that: The molar ratio of hydrogen bond acceptor to hydrogen bond donor is 1:

2.

4. The method for extracting and purifying flavonoids from Eagle Tea using ultrasound-assisted eutectic solvent extraction according to claim 2, characterized in that: The mass ratio of water in the eutectic solvent DES system is 30%.

5. The method for extracting and purifying flavonoids from Eagle Tea using ultrasound-assisted eutectic solvent extraction according to claim 1, characterized in that: The ratio of the raw material of Eagle Tea to the eutectic solvent DES system is 1g:20mL.

6. The method for extracting and purifying flavonoids from Eagle Tea using ultrasound-assisted eutectic solvent extraction according to claim 1, characterized in that: The ultrasound-assisted extraction was performed by ultrasonic treatment for 30 minutes at an ultrasonic power of 250-300W and a temperature of 40-60℃.

7. The method for extracting and purifying flavonoids from Eagle Tea using ultrasound-assisted eutectic solvent extraction according to claim 1, characterized in that: The macroporous adsorption resin is HPD600 type resin.

8. The method for extracting and purifying flavonoids from Eagle Tea using ultrasound-assisted eutectic solvent extraction according to claim 1, characterized in that: The loading flow rate for the dynamic adsorption is 1.0~2.0 BV / h.

9. The method for extracting and purifying flavonoids from Eagle Tea using ultrasound-assisted eutectic solvent extraction according to claim 1, characterized in that: The elution process involves eluting impurities with a 20%–30% aqueous ethanol solution, followed by elution with an 85%–95% aqueous ethanol solution to collect the flavonoids.

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