LU102716B1

LU102716B1 - Method for Extracting Anthocyanin from Perillafrutescens (L.) Britton Leaves by Using Ternary Deep Eutectic Solvent

Info

Publication number: LU102716B1
Application number: LU102716A
Authority: LU
Inventors: Yanbo Wang; Xiaoxiang Han
Original assignee: Univ Zhejiang Gongshang
Priority date: 2021-03-31
Filing date: 2021-03-31
Publication date: 2021-10-11

Abstract

The present invention discloses a method for extracting anthocyanin from Perillafrutescens (L.) Britton leaves, and particularly relates to a preparation of a deep eutectic solvent and a method for extracting anthocyanin with the aid of ultrasound-microwave-ultraviolet light. The deep eutectic solvent, a green solvent, has the advantages of diversified structure, biodegradability, environmental protection, easy preparation, regeneration, low melting point and the like. With a few addition of the solvent, the content of anthocyanin can be increased obviously, thus reducing the solvent loss. Further, the shortcoming of a single wave can be overcome through the combined use of ultrasound wave, microwave and ultraviolet light, which, as a novel technology integrating the advantages of ultrasound and microwave extraction, allows the solvent to pass through a vegetated substrate more easily and makes effective components more soluble in the solvent.

Description

DESCRIPTION Method for Extracting Anthocyanin from Perillafrutescens (L.) Britton Leaves by Using Ternary Deep Eutectic Solvent

TECHNICAL FIELD The present invention relates to the technical field of plant extraction, in particular to a method for extracting anthocyanin from Perillafrutescens (L.) Britton leaves by using a ternary deep eutectic solvent.

BACKGROUND With the development of technology and the improvement of living standard, the demand for novel green solvent and equipment for extracting natural macromolecular products is steadily on the increase.

Deep eutectic solvent (DES), an ionic-like solution and a green solvent, has the advantages of diversified structure, biodegradability, environmental protection, easy preparation, regeneration, low melting point and the like. Therefore, it has been widely used in various fields, including catalytic reactions, extraction and separation, CO2 and SO: trapping and material preparation. The physical and chemical properties of deep eutectic solvent can be adjusted by selecting different hydrogen bond receptors and donors, and binding them at different ratios.

Perillafrutescens (L.) Britton belongs to Labiatae family and is featured by the homology of medicine and food. Perillafrutescens (L.) Britton leaves contain anthocyanins such as shisonin, volatile oils such as perillaldehyde, and multiple bioactive substances such as rosmarinic acid, which make the leaves the antisepsis and anti-inflammation, anti-allergic, antipyretic and antioxidant functions. Perillafrutescens (L.) Britton is a traditional herbal medicine in China, and has been developed into more than 10 kinds of traditional Chinese medicine preparations, including Huoxiang Zhengqi Liquid and Ginseng and Perillae Decoction, which can be used for treating a variety of diseases, for example, relieving symptoms and dispersing cold, regulating qi and protecting the stomach, and bacterial and fungal infections.

Anthocyanin in Perillafrutescens (L.) Britton leaves is a strong antioxidant protecting the body from free radicals, a water-soluble natural pigment widely found in plants, and also a flavonoid compound. The basic structure of anthocyanin is a 2-phenylbenzopyrane cation (flavvhm), which makes it present different color structures in different PH environments, for example, blue in alkaline solutions with PH>11, red and stable in acidic solutions with PH=1-3, and purple in neutral solutions with PH=7-8, depending on the environment condition and different substituents on three carbon Ce-C3-Ce nuclei in two benzene rings. The anthocyanin, a water-soluble natural food pigment with 3,5, 7-trihydroxy-2-phenylbenzopyrane cationic structure, is easily soluble in methanol, ethanol, acetone and other polar solvents, and susceptible to light (possibly because anthocyanin degrades to produce chalcone in light which rapidly degrades to produce benzoic acid and other products; the presence of oxygen in air speeds up the degradation reaction of anthocyanin and chalcone, and converts anthocyanin to phenylbenzopyrane in cationic form at high temperature, which is beneficial to the rapid formation of chalcone), pH and temperature. There are more than 20 kinds of known anthocyanins, among which pelargonidin, cyaniding, delphinidin, peonidin, petunidin and malvidin are mainly present in plants.

The microwave-assisted extraction is realized by an applied physical field,

the principle of which is to make the molecules in the material collide and squeeze each other through the rapid vibration of ultra-high frequency of 2.45 billion times/s generated by the magnetron, so as to promote the leaching of effective components. Featured by high selectivity, short operation time, low solvent consumption and, high yield of effective components, the method is suitable for thermally unstable substances. However, it still has some problems such as residual organic solvents and attenuation of microwave penetrating the material. The ultrasound-assisted extraction makes use of the strong vibration, high acceleration, strong cavitation effect and stirring action produced by ultrasound waves to cause the impact of micro-jets, so as to accelerate effective components in plants into the solvent, that is, a cavitation in the liquid, destroy plant cells and membrane structure, and help the diffusion of solutes, thereby increasing the extraction rate of effective components, shortening the extraction time, and avoiding the effect on the extracted components at high temperature. Zhang Lunpei et al. studied the ultrasound-assisted extraction of flavonoid compounds in stems and leaves of Lobelia nummularia Lam., and concluded that the optimum extraction process was the ethanol concentration of 70%, the solid-liquid ratio of 1:20, the ultrasonic power of 350 W, the leaching temperature of 60 °C, and the extraction time of 30 min. Therefore, we used a combined ultrasound-microwave-ultraviolet extractor with deep eutectic solvent to extract anthocyanin from Perillafrutescens (L.) Britton leaves.

SUMMARY Aiming at the shortcomings of prior art, the present invention provides a method for extracting anthocyanin, and particularly relates to a preparation of a ternary deep eutectic solvent and a method for extracting anthocyanin from Perillafrutescens (L) Britton leaves with the aid of a combined ultrasound-microwave-ultraviolet extractor. The deep eutectic solvent, a novel green solvent, has the advantages of diversified structure, biodegradability, environmental protection, easy preparation, regeneration, low melting point and the like. Compared with other binary deep eutectic solvents, the advantages of the ternary deep eutectic solvent lie in that the amount of ethylene glycol as a hydrogen bond donor is greatly reduced, thus achieving a high utilization efficiency. Moreover, combined with the structural analysis of the extracted target component anthocyanin, together with lactic acid as an auxiliary ligand, the extraction content of anthocyanin is greatly increased. The shortcoming of a single wave can be overcome through the combined use of ultrasound wave, microwave and ultraviolet light, which, as a novel technology integrating the advantages of ultrasound and microwave extraction, allows the solvent to pass through a vegetated substrate more easily and makes effective components more soluble in the solvent. The synergistic extraction method requires simple equipment, easy process and mild working conditions, which provide some solutions for subsequent high value extraction.

The purpose of the present invention is realized by the following technical solutions: a method for extracting anthocyanin from Perillafrutescens (L.) Britton leaves by using a ternary deep eutectic solvent, comprising the following steps: (1) taking polyol as a hydrogen bond donor and sugar or carboxylic acid as an auxiliary ligand, mixing the hydrogen bond donor and the hydrogen bond receptor choline chloride (ChCl) in a molar ratio of 2-6: 1, then adding the auxiliary ligand with a molar ratio of the auxiliary ligand to ChCl being 0.1-1: 1, and mixing while heating and stirring to set off a reaction in a N2 atmosphere and an oil bath, so as to obtain a clear ternary deep eutectic solvent; (2) preparing the ternary deep eutectic solvent and distilled water into a mixed deep eutectic extracting solution, wherein the volume fraction of the ternary deep eutectic solvent is 50-90%; (3) extracting anthocyanin from a raw material Perillafrutescens (L.) Britton leaf meal to obtain an anthocyanin extracting solution; specifically, taking the mixed deep eutectic extracting solution as an extracting solvent, mixing the Perillafrutescens (L.) Britton leaf meal and the mixed deep eutectic extracting solution in a mass-volume ratio (g/ml) of 1. 4-12, and extracting anthocyanin from the Perillafrutescens (L.) Britton leaf meal under the synergistic action of a combined ultrasound-microwave-ultraviolet extractor to obtain an anthocyanin extracting solution; wherein the temperature of the combined ultrasound-microwave-ultraviolet extractor is 40-80 °C, the microwave power is 300-500 W, the ultrasonic power is 300-500 W, the action time is 10-30 min, and the ultraviolet light intensity is 10 uW/cm?; and (4) centrifuging the obtained anthocyanin extracting solution at 9000 r/min and 4 °C for 10 min, and then filtering by a 0.22 um pore filter membrane to obtain filtrate, freeze-drying the filtrate in a freeze-drying apparatus at -60 °C for 24 h to obtain anthocyanin in powder form.

Further, the polyol can be ethylene glycol or glycerol, the sugar can be fructose, glucose or sucrose, and the carboxylic acid can be oxalic acid, lactic acid and citric acid in step (1).

Further, the polyol is preferably ethylene glycol, the sugar is preferably glucose, and the carboxylic acid is preferably lactic acid.

Further, the molar ratio of the hydrogen bond donor to ChCl is preferably 3:1, and the molar ratio of the auxiliary ligand to ChCl is preferably 0.7:1 in step (1).

Further, the reaction condition of the oil bath is the heating temperature of 90 °C and the heating time of 1 h in step (1).

Further, the volume fraction of the multi-component deep eutectic solvent is preferably 80% in step (2).

Further, the mass-volume ratio (g/ml) of the Perillafrutescens (L.) Britton leaf meal to the mixed deep eutectic solvent is preferably 1:10 in step (3).

Further, the temperature of the combined ultrasound-microwave-ultraviolet extractor is preferably 60 °C, the microwave power is preferably 400 W, the ultrasonic power is preferably 400 W, and the action time is preferably 25 min in step (3).

Compared with the prior art, the advantageous technical effect of the present invention is as follows: The present invention provides a method for extracting anthocyanin from Perillafrutescens (L.) Britton leaves by using a ternary deep eutectic solvent as an extractant with the aid of a combined ultrasound-microwave-ultraviolet extractor, which shows good extraction ability. The deep eutectic solvent as the extractant is an environmentally-friendly, renewable and novel green solvent with low vapor pressure. With a few addition of the solvent to the hydrogen bond donor and the auxiliary ligand, the content of anthocyanin can be increased obviously under mild process conditions, thus reducing the solvent loss. Compared with sequential microwave-ultrasound extraction and ultrasound-microwave extraction in existing literatures, the combined ultrasound-microwave-ultraviolet extractor is a novel technology integrating the advantages of ultrasound-assisted extraction and microwave-assisted extraction, and thus can effectively make up for the shortcomings of a single wave and greatly increase the extraction rate of anthocyanin through the combined use of ultrasound wave and microwave in the ultraviolet atmosphere.

BRIEF DESCRIPTION OF THE FIGURES Fig. 1 is a flow block diagram of the present invention.

Fig. 2 is the infrared characterization of the deep eutectic solvent by software.

DESCRIPTION OF THE INVENTION In order to better illustrate the technical solution of the present invention, the present invention is further described in detail in combination with embodiments.

As shown in Fig. 1, the present invention provides a method for extracting anthocyanin from Perillafrutescens (L.) Britton leaves by using a ternary deep eutectic solvent, comprising the following steps: (1) taking polyol as a hydrogen bond donor and sugar or carboxylic acid as an auxiliary ligand, mixing the hydrogen bond donor and the hydrogen bond receptor choline chloride (ChCl) in a molar ratio of 2-6: 1, preferably 3:1, then adding the auxiliary ligand with a molar ratio of the auxiliary ligand to ChCI being 0.1-1: 1, preferably 0.7:1, and mixing while heating and stirring to set off a reaction in a Na atmosphere and an oil bath, so as to obtain a clear ternary deep eutectic solvent; wherein the polyol can be ethylene glycol or glycerol and preferably ethylene glycol, the sugar can be fructose, glucose or sucrose and preferably glucose, the carboxylic acid can be oxalic acid, lactic acid and citric acid and preferably lactic acid, and the reaction condition of the oil bath is the heating temperature of 90 °C and the heating time of 1 h.

(2) preparing the ternary deep eutectic solvent and distilled water into a mixed deep eutectic extracting solution, wherein the volume fraction of the ternary deep eutectic solvent is 50-90% and preferably 80%; (3) extracting anthocyanin from a raw material Perillafrutescens (L.) Britton leaf meal to obtain an anthocyanin extracting solution; specifically, taking the mixed deep eutectic extracting solution as an extracting solvent, mixing the Perillafrutescens (L.) Britton leaf meal and the mixed deep eutectic extracting solution in a mass-volume ratio (g/ml) of 1: 4-12, preferably 1:10, and extracting anthocyanin from the Perillafrutescens (L.) Britton leaf meal under the synergistic action of a combined ultrasound-microwave-ultraviolet extractor to obtain an anthocyanin extracting solution; wherein the temperature of the combined ultrasound-microwave-ultraviolet extractor is 40-80 °C and preferably 60 °C, the microwave power is 300-500 W and preferably 400 W, the ultrasonic power is 300-500 W and preferably 400 W, the action time is 10-30 min and preferably 25 min, and the ultraviolet light intensity is 10 HW/cem?; and (4) centrifuging the obtained anthocyanin extracting solution at 9000 r/min and 4 °C for 10 min, and then filtering by a 0.22 um pore filter membrane to obtain filtrate, freeze-drying the filtrate in a freeze-drying apparatus at -60 °C for 24 h to obtain anthocyanin in powder form, which can form a composite natural cling film with chitosan and other substances that can easily form a film.

The spoilage of aquatic products and other substances can be roughly evaluated in a sensory sense according to the color change of anthocyanin with PH.

Fig. 2 shows that the infrared characterization trends of EG, ChCI-3EG, and ChCI-3EG-0.7LA are basically the same; that is, the transition of vibrational level and rotational level in molecules has not changed obviously, indicating that there is no chemical change in the deep eutectic solvent system obtained by mixing ethylene glycol, ChCl and lactic acid, and the characteristic structure of each component is not destroyed, but a homogeneous solution is formed by through eutectic melting of hydrogen bond interaction.

In Fig. 2 (a), a strong broad peak at 3443 cm” is the hydroxyl contraction vibration mode of ethylene glycol (yC-OH), with a large number of hydrogen bonds formed inside; and the peaks at 1405 cm! and 1039 cm” are the bending vibration peak and hydroxyl contraction vibration peak of ethylene glycol methylene, respectively. In Fig. 2 (b) and (c), ethylene glycol, ChCl and lactic acid are associated by a large number of hydrogen bonds in the mixing and eutectic process in the wave number range of 3500-3000 cm”. In Fig. 2 (b), the methylene group of ethylene glycol is associated with ChCI at 1405 cm”, while C-C bond contraction vibration peak of ChCl and carbonyl contraction vibration peaks of lactic acid are at 955 cm" and 1736 cm”, respectively, which are consistent with the absorption positions of characteristic structures of ethylene glycol at 1039 cm” in Fig. 2 (a), indicating that the three components are not destroyed in the deep eutectic solvent system but associated with hydrogen bonds.

The embodiments of the present invention are specified as follows. The raw materials used in the following embodiments are commercially available.

Examples 1-6 Taking polyol as a hydrogen bond donor, mixing the hydrogen bond donor and the hydrogen bond receptor ChCl in a ratio shown in Table 1, while heating and stirring to set off a reaction in a Na atmosphere and an oil bath, with the heating temperature of 90 °C and the stirring time of 1 h, so as to obtain a clear binary deep eutectic solvent; To a three-necked flask, 5 g of Perillafrutescens (L.) Britton leaf pulp, 20 mL of deep eutectic solvent and 10 mL of distilled water were added in turn. Then, place the flask in a combined ultrasound-microwave-ultraviolet extractor, wherein the extraction temperature was 60 °C, the microwave power was 450 W, there were no ultrasonic power and ultraviolet light, and the reaction time was 20 min; After the reaction, the Perillafrutescens (L.) Britton leaf pulp and the deep eutectic solvent rich in anthocyanin were layered as follows: centrifuging at 9000 r/min and 4 °C for 10 min, filtering through 0.22 um pore filter membrane, and then detecting the content of anthocyanin by the pH differential method. The extraction content of anthocyanin is shown in Table 1.

Table 1

It can be concluded from Table 1 that in the absence of auxiliary ligands and with only hydrogen bond donors and hydrogen bond receptors, the highest anthocyanin content is observed when the ratio of ChCl to ethylene glycol is 1:3. This is due to the fact that the hydrogen bonds formed between ChCl and ethylene glycol and the covalent bonds within the molecules achieve the maximum solubility of anthocyanin when the ratio of ChCl to ethylene glycol is 1:3.

Examples 7-19 Taking ethylene glycol as a hydrogen bond donor and sugar or carboxylic acid as an auxiliary ligand, mixing the hydrogen bond donor, the hydrogen bond receptor ChCl and the auxiliary ligand in a molar ratio shown in Table 2, heating and stirring to set off a reaction in a Na atmosphere and an oil bath, with the heating temperature of 90 °C and the stirring time of 1 h, so as to obtain a clear ternary deep eutectic solvent; To a three-necked flask, 5 g of Perillafrutescens (L.) Britton leaf pulp, 20 mL of deep eutectic solvent and 10 mL of distilled water were added in turn. Then, place the flask in a combined ultrasound-microwave-ultraviolet extractor, wherein the extraction temperature was 60 °C, the microwave power was 450 W, there were no ultrasonic power and ultraviolet light, and the reaction time was 20 min; After the reaction, the Perillafrutescens (L.) Britton leaf pulp and the deep eutectic solvent rich in anthocyanin were layered as follows: centrifuging at 9000 r/min and 4 °C for 10 min, filtering through 0.22 um pore filter membrane,

and then detecting the content of anthocyanin by the pH differential method. The extraction content of anthocyanin is shown in Table 2.

Table 2 It can be concluded from Table 2 that the solubility of anthocyanin is saturated by the ternary deep eutectic solvent when the ratio of ChCl to ethylene glycol and lactic acid is 1:3:0.7. This is due to the fact that under certain acidic conditions, i.e. when the substituents on the parent nucleus are different, anthocyanin becomes stable and is not easily degraded. Compared with oxalic acid and citric acid, lactic acid is more suitable for dissolving anthocyanin in terms of the number of carboxylic acids in the structure, and adding a certain amount of lactic acid can increase the surface area of the deep eutectic solvent to a certain extent, thus increasing the contact surface with anthocyanin. When the auxiliary ligand is sugar, the ternary deep eutectic solvent is not easy to transfer mass due to a large viscosity.

Examples 20-24 Taking ethylene glycol as a hydrogen bond donor and lactic acid as an auxiliary ligand, mixing the hydrogen bond donor and the hydrogen bond receptor ChCI in a molar ratio of 3:1, then adding the auxiliary ligand with a molar ratio of the auxiliary ligand to ChCl being 0.7:1, and mixing while heating and stirring to set off a reaction in a Na atmosphere and an oil bath, with the heating temperature of 90 °C and the stirring time of 1 h, so as to obtain a clear ternary deep eutectic solvent; To a three-necked flask, 5 g of Perillafrutescens (L.) Britton leaf pulp, a certain volume of deep eutectic solvent ChCI- ethylene glycol-lactic acid (1:3:0.7) and distilled water were added, wherein the volume ratio of deep eutectic solvent to distilled water was 2:1, and the solid-liquid ratio (g/ml) was shown in Table 3. Then, place the flask in a combined ultrasound-microwave-ultraviolet extractor, wherein the extraction temperature was 60 °C, the microwave power was 450 W, there were no ultrasonic power and ultraviolet light, and the reaction time was 20 min; After the reaction, the Perillafrutescens (L.) Britton leaf pulp and the deep eutectic solvent rich in anthocyanin were layered as follows: centrifuging at 9000 r/min and 4 °C for 10 min, filtering through 0.22 um pore filter membrane, and then detecting the content of anthocyanin by the pH differential method. The extraction content of anthocyanin is shown in Table 3.

Table 3

It can be concluded from Table 3 that the highest solubility of anthocyanin is observed when the solid-liquid ratio (g/ml) of Perillafrutescens (L.) Britton leaf pulp and deep eutectic solvent is 1:10. When the solid-liquid ratio is higher than 1:10, increasing the amount of extracted solvent will cause certain solvent waste. Therefore, the best solid-liquid ratio (g/ml) is 1:10.

Examples 25-29 Taking ethylene glycol as a hydrogen bond donor and lactic acid as an auxiliary ligand, mixing the hydrogen bond donor and the hydrogen bond receptor ChCI in a molar ratio of 3:1, then adding the auxiliary ligand with a molar ratio of the auxiliary ligand to ChCl being 0.7:1, and mixing while heating and stirring to set off a reaction in a Na atmosphere and an oil bath, with the heating temperature of 90 °C and the stirring time of 1 h, so as to obtain a clear ternary deep eutectic solvent; To a three-necked flask, 5 g of Perillafrutescens (L.) Britton leaf pulp was added. If the solid-liquid ratio (g/ml) was 1:10, the total volume of ChCl-ethylene glycol-lactic acid (1:3:0.7) and distilled water should be 50 mL, wherein the volume fraction of ChCl-ethylene glycol-lactic acid (1:3:0.7) is shown in Table 4 Then, place the flask in a combined ultrasound-microwave-ultraviolet extractor, wherein the extraction temperature was 60 °C, the microwave power was 450 W, there were no ultrasonic power and ultraviolet light, and the reaction time was 20 min; After the reaction, the Perillafrutescens (L.) Britton leaf pulp and the deep eutectic solvent rich in anthocyanin were layered as follows: centrifuging at 9000 r/min and 4 °C for 10 min, filtering through 0.22 um pore filter membrane, and then detecting the content of anthocyanin by the pH differential method. The extraction content of anthocyanin is shown in Table 4.

Table 4 It can be concluded from Table 4 that in the presence of the mixed deep eutectic solvent, the maximum saturation of anthocyanin is observed when the volume fraction of DES is 80%. This is due to the fact that a certain amount of distilled water can dilute the viscosity of the deep eutectic solvent and increase its fluidity, thus making it easier to pass through a vegetated substrate.

Examples 30-35 Taking ethylene glycol as a hydrogen bond donor and lactic acid as an auxiliary ligand, mixing the hydrogen bond donor and the hydrogen bond receptor ChCI in a molar ratio of 3:1, then adding the auxiliary ligand with a molar ratio of the auxiliary ligand to ChCl being 0.7:1, and mixing while heating and stirring to set off a reaction in a Na atmosphere and an oil bath, with the heating temperature of 90 °C and the stirring time of 1 h, so as to obtain a clear ternary deep eutectic solvent; To a three-necked flask, 5 g of Perillafrutescens (L.) Britton leaf pulp, 40 mL of ChCl-ethylene glycol-lactic acid (1:3:0.7) and 10 mL of distilled water were added in turn Then, place the flask in a combined ultrasound-microwave-ultraviolet extractor, wherein the extraction temperature was 60 °C, the microwave power was 450 W, there were no ultrasonic power and ultraviolet light, and the reaction time was shown in Table 5; After the reaction, the Perillafrutescens (L.) Britton leaf pulp and the deep eutectic solvent rich in anthocyanin were layered as follows: centrifuging at 9000 r/min and 4 °C for 10 min, filtering through 0.22 um pore filter membrane, and then detecting the content of anthocyanin by the pH differential method. The extraction content of anthocyanin is shown in Table 5.

Table 5 It can be concluded from Table 5 that the extraction content of anthocyanin shows an increasing trend with the increase of time from 10 min to 25 min, but the content of anthocyanin may basically reach saturation in the presence of deep eutectic solvent by 25 min and starts to decrease gradually, which is deeply affected by external light, temperature and other environment factors.

Examples 36-40 Taking ethylene glycol as a hydrogen bond donor and lactic acid as an auxiliary ligand, mixing the hydrogen bond donor and the hydrogen bond receptor ChCI in a molar ratio of 3:1, then adding the auxiliary ligand with a molar ratio of the auxiliary ligand to ChCl being 0.7:1, and mixing while heating and stirring to set off a reaction in a Na atmosphere and an oil bath, with the heating temperature of 90 °C and the stirring time of 1 h, so as to obtain a clear ternary deep eutectic solvent; To a three-necked flask, 5 g of Perillafrutescens (L.) Britton leaf pulp, 40 mL of ChCl-ethylene glycol-lactic acid (1:3:0.7) and 10 mL of distilled water were added in turn Then, place the flask in a combined ultrasound-microwave-ultraviolet extractor, wherein the extraction temperature was shown in Table 6, the microwave power was 450 W, there were no ultrasonic power and ultraviolet light, and the reaction time was 25 min; After the reaction, the Perillafrutescens (L.) Britton leaf pulp and the deep eutectic solvent rich in anthocyanin were layered as follows: centrifuging at 9000 r/min and 4 °C for 10 min, filtering through 0.22 um pore filter membrane, and then detecting the content of anthocyanin by the pH differential method. The extraction content of anthocyanin is shown in Table 6.

Table 6 ww wm It can be concluded from Table 6 that the solubility of anthocyanin will increase with temperature, but the increase of temperature will result in the decrease of the viscosity of deep eutectic solvent and improve its fluidity to some extent. However, if the temperature is too high, anthocyanin will be degraded and deformed to a certain extent, thus reducing the extraction content of anthocyanin. The highest extraction content of anthocyanin is obtained when the extraction temperature is 60 °C.

Examples 41-45 Taking ethylene glycol as a hydrogen bond donor and lactic acid as an auxiliary ligand, mixing the hydrogen bond donor and the hydrogen bond receptor ChCI in a molar ratio of 3:1, then adding the auxiliary ligand with a molar ratio of the auxiliary ligand to ChCl being 0.7:1, and mixing while heating and stirring to set off a reaction in a Na atmosphere and an oil bath, with the heating temperature of 90 °C and the stirring time of 1 h, so as to obtain a clear ternary deep eutectic solvent; To a three-necked flask, 5 g of Perillafrutescens (L.) Britton leaf pulp, 40 mL of ChCl-ethylene glycol-lactic acid (1:3:0.7) and 10 mL of distilled water were added in turn Then, place the flask in a combined ultrasound-microwave-ultraviolet extractor, wherein the microwave power was shown in Table 7, there were no ultrasonic power and ultraviolet light, the extraction temperature was 60 °C, and the reaction time was 25 min; After the reaction, the Perillafrutescens (L.) Britton leaf pulp and the deep eutectic solvent rich in anthocyanin were layered as follows: centrifuging at 9000 r/min and 4 °C for 10 min, filtering through 0.22 um pore filter membrane, and then detecting the content of anthocyanin by the pH differential method. The extraction content of anthocyanin is shown in Table 7.

Table 7 It can be concluded from Table 7 that when the microwave power is 400 W, the polar components in the Perillafrutescens (L.) Britton leaves will cause strong polar oscillation under the action of microwave, which will easily lead to the rupture of cell membrane structure, thus promoting the leaching of the contents in the matrix into a deep eutectic solvent; as a result, the deep eutectic solvent will absorb more microwave energy and have higher molecular activity. However, the higher the microwave power is, the more likely is to produce a local overheating phenomenon, causing anthocyanin to be easily degraded.

Examples 46-50 Taking ethylene glycol as a hydrogen bond donor and lactic acid as an auxiliary ligand, mixing the hydrogen bond donor and the hydrogen bond receptor ChCI in a molar ratio of 3:1, then adding the auxiliary ligand with a molar ratio of the auxiliary ligand to ChCl being 0.7:1, and mixing while heating and stirring to set off a reaction in a Na atmosphere and an oil bath, with the heating temperature of 90 °C and the stirring time of 1 h, so as to obtain a clear ternary deep eutectic solvent; To a three-necked flask, 5 g of Perillafrutescens (L.) Britton leaf pulp, 40 mL of ChCl-ethylene glycol-lactic acid (1:3:0.7) and 10 mL of distilled water were added in turn Then, place the flask in a combined ultrasound-microwave-ultraviolet extractor, wherein the extraction temperature was 60 °C, the microwave power was 400 W, the ultrasonic power was shown in Table 8, the ultraviolet intensity was 10uW/cm?, and the reaction time was min; After the reaction, the Perillafrutescens (L.) Britton leaf pulp and the deep eutectic solvent rich in anthocyanin were layered as follows: centrifuging at 9000 r/min and 4 °C for 10 min, filtering through 0.22 um pore filter membrane, and then detecting the content of anthocyanin by the pH differential method. The extraction content of anthocyanin is shown in Table 8.

Table 8 It can be concluded from Table 8 that the ultrasound-microwave-assisted extraction achieves a high extraction content of anthocyanin, due to the fact that microwave can produce thermal effect; while ultrasound wave can make the same effect for all points in the system, increase the frequency and speed of molecular movement in the system, and improve the leaching of anthocyanin when the system is kept at a certain temperature. The highest extraction content of anthocyanin is obtained when the ultrasonic power is 400 W.

Examples 51-55 Taking ethylene glycol as a hydrogen bond donor and lactic acid as an auxiliary ligand, mixing the hydrogen bond donor and the hydrogen bond receptor ChCI in a molar ratio of 3:1, then adding the auxiliary ligand with a molar ratio of the auxiliary ligand to ChCl being 0.7:1, and mixing while heating and stirring to set off a reaction in a Na atmosphere and an oil bath, with the heating temperature of 90 °C and the stirring time of 1 h, so as to obtain a clear ternary deep eutectic solvent; To a three-necked flask, 5 g of Perillafrutescens (L.) Britton leaf pulp, 40 mL of ChCl-ethylene glycol-lactic acid (1:3:0.7) and 10 mL of distilled water were added in turn Then, place the flask in a combined ultrasound-microwave-ultraviolet extractor, wherein the extraction temperature was 60 °C, the microwave power was 400 W, the ultrasonic power was 400 W, the ultraviolet intensity was 10 uW/cm?, and the reaction time was shown in Table 9: After the reaction, the Perillafrutescens (L.) Britton leaf pulp and the deep eutectic solvent rich in anthocyanin were layered as follows: centrifuging at 9000 r/min and 4 °C for 10 min, filtering through 0.22 um pore filter membrane, and then detecting the content of anthocyanin by the pH differential method. The extraction content of anthocyanin is shown in Table 9.

Table 9 It can be concluded from Table 9 that under the action of ultrasonic-microwave, the anthocyanin content can be improved more effectively in the same time, compared with Table 5. Similarly, the highest extraction content of anthocyanin is obtained when the extraction time is 25 min.

Examples 56-60 Taking ethylene glycol as a hydrogen bond donor and lactic acid as an auxiliary ligand, mixing the hydrogen bond donor and the hydrogen bond receptor ChCI in a molar ratio of 3:1, then adding the auxiliary ligand with a molar ratio of the auxiliary ligand to ChCl being 0.7:1, mixing, and setting off a heating and stirring reaction in Na atmosphere and an oil bath, with the heating temperature of 90 °C and stirring time of 1 h, so as to obtain a clear ternary deep eutectic solvent after the reaction; To a three-necked flask, 5 g of Perillafrutescens (L.) Britton leaf pulp, 40 mL of ChCl-ethylene glycol-lactic acid (1:3:0.7) and 10 mL of distilled water were added in turn Then, place the flask in a combined ultrasound-microwave-ultraviolet extractor, wherein the extraction temperature was shown in Table 10, the microwave power was 400 W, the ultrasonic power was 400 W, the ultraviolet intensity was 10uW/cm?, and the reaction time was min; After the reaction, the Perillafrutescens (L.) Britton leaf pulp and the deep eutectic solvent rich in anthocyanin were layered as follows: centrifuging at 9000 r/min and 4 °C for 10 min, filtering through 0.22 um pore filter membrane, and then detecting the content of anthocyanin by the pH differential method. The extraction content of anthocyanin is shown in Table 10.

Table 10 wm w= It can be concluded from Table 10 that when the extraction temperature is the same, the solubility of anthocyanin is greatly increased under the action of ultrasonic-microwave, compared with Table 6. Similarly, the highest extraction content of anthocyanin is obtained when the extraction temperature is 60 °C.

After the above reaction, the deep eutectic solvent can be recycled. After repeating the experiment four times, the content of anthocyanin in Perillafrutescens (L.) Britton leaves was fluctuated within 5%.

The above examples show that the extraction of anthocyanin from Perillafrutescens (L.) Britton leaves by using a ternary deep eutectic solvent with the aid of ultrasound-microwave-ultraviolet light has good extraction ability, and can reduce solvent loss and cost.

The foregoing is only a preferred embodiment of the present for invention and is not intended to limit the technical features of the patent for invention.

Any change or modification made by a person skilled in the pertinent art within the field of the present invention shall fall within the protection scope of the patent for invention.

Claims

Claims

1 A method for extracting anthocyanin from Perillafrutescens (L.) Britton leaves by using a ternary deep eutectic solvent, characterized by comprising the following steps:

(1) taking polyol as a hydrogen bond donor and sugar or carboxylic acid as an auxiliary ligand, mixing the hydrogen bond donor and the hydrogen bond receptor choline chloride (ChCl) in a molar ratio of 2-6: 1, then adding the auxiliary ligand with a molar ratio of the auxiliary ligand to ChCl being 0.1-1: 1, and mixing while heating and stirring to set off a reaction in a N2 atmosphere and an oil bath, so as to obtain a clear ternary deep eutectic solvent;

(2) preparing the ternary deep eutectic solvent and distilled water into a mixed deep eutectic extracting solution, wherein the volume fraction of the ternary deep eutectic solvent is 50-90%;

(3) extracting anthocyanin from a raw material Perillafrutescens (L.) Britton leaf meal to obtain an anthocyanin extracting solution; specifically, taking the mixed deep eutectic extracting solution as an extracting solvent, mixing the Perillafrutescens (L.) Britton leaf meal and the mixed deep eutectic extracting solution in a mass-volume ratio (g/ml) of 1. 4-12, and extracting anthocyanin from the Perillafrutescens (L.) Britton leaf meal under the synergistic action of a combined ultrasound-microwave-ultraviolet extractor to obtain an anthocyanin extracting solution; wherein the temperature of the combined ultrasound-microwave-ultraviolet extractor is 40-80 °C, the microwave power is 300-500 W, the ultrasonic power is 300-500 W, the action time is 10-30 min, and the ultraviolet light intensity is 10 uW/cm?; and

(4) centrifuging the obtained anthocyanin extracting solution at 9000 r/min and 4 °C for 10 min, and then filtering by a 0.22 um pore filter membrane to obtain filtrate, freeze-drying the filtrate in a freeze-drying apparatus at -60 °C for 24 h to obtain anthocyanin in powder form.
2 The preparation method of a deep eutectic solvent according to claim 1, characterized in that the polyol can be ethylene glycol or glycerol, the sugar can be fructose, glucose or sucrose, and the carboxylic acid can be oxalic acid, lactic acid and citric acid in step (1).
3 The preparation method of a deep eutectic solvent according to claim 2, characterized in that the polyol is preferably ethylene glycol, the sugar is preferably glucose, and the carboxylic acid is preferably lactic acid.
4 The preparation method according to claim 1, characterized in that the molar ratio of the hydrogen bond donor to ChCl is preferably 3:1, and the molar ratio of the auxiliary ligand to ChCl is preferably 0.7:1 in step (1).
The preparation method according to claim 1, characterized in that the reaction condition of the oil bath is the heating temperature of 90 °C and the heating time of 1 h in step (1).
6 The preparation method according to claim 1, characterized in that the volume fraction of the multi-component deep eutectic solvent is preferably 80% in step (2).
7 The extraction method according to claim 1, characterized in that the mass-volume ratio (g/ml) of the Perillafrutescens (L.) Britton leaf meal to the mixed deep eutectic solvent is preferably 1:10 in step (3).
8 The extraction method according to claim 1, characterized in that the temperature of the combined ultrasound-microwave-ultraviolet extractor is preferably 60 °C, the microwave power is preferably 400 W, the ultrasonic power is preferably 400 W, and the action time is preferably 25 min in step (3).