CN115569158A - Method for extracting total flavonoids from guava leaves by using ultrasonic-assisted eutectic solvent extraction technology - Google Patents

Abstract

The invention provides an extraction method for extracting total flavonoids from guava leaves by utilizing an ultrasonic-assisted eutectic solvent extraction technology, which comprises the following steps: (1) drying and crushing guava leaves into powder and sieving the powder; (2) Leaching by an ultrasonic-assisted eutectic solvent extraction technology; (3) centrifuging the filtrate to obtain a supernatant; (4) And adsorbing and enriching the supernatant by using macroporous adsorption resin, and concentrating and drying the eluent to obtain the total flavone extract of the guava leaves. The preparation method of the invention extracts the total flavonoids in the guava leaves by utilizing the cavitation of ultrasonic waves and the solubilization of a low co-dissolving reagent on polar and non-polar compounds, accelerates the diffusion and dissolution of the flavonoids compounds, is simple and easy to implement, avoids the complicated process of long-time concentration treatment before the measurement by the traditional organic solvent extraction method, is green and environment-friendly, has high extraction rate, and has the total flavonoids content of more than 80 percent in the extract after the purification by macroporous resin.

Description

Extraction method for extracting total flavonoids from guava leaves by using ultrasonic-assisted eutectic solvent extraction technology

Technical Field

The invention belongs to the field of plant processing, and particularly relates to an extraction method for extracting total flavonoids from guava leaves by using an ultrasonic-assisted eutectic solvent extraction technology.

Background

Guava leaf, name of traditional Chinese medicine. Is leaf of Psidium guajava Linn (Psidium guajava) belonging to Myrtaceae. Is distributed or cultivated in Fujian, taiwan, guangdong, guangxi, sichuan and Yunnan provinces of China. Has the effects of eliminating dampness, strengthening spleen, clearing away heat and toxic materials. It is commonly used for diarrhea, dysentery, abdominal distention, gingival swelling and pain, rheumatalgia, furuncle, swelling and pain, traumatic hemorrhage, snake and insect bite. The chemical component research of the guava leaves shows that the guava leaves contain protein, polysaccharide, triterpenes, flavonoids and the like.

The flavonoid compound is a large class of natural products, widely exists in the plant world, and is an effective component of a plurality of Chinese herbal medicines. The most common in nature are flavones and flavonols, others include dihydroflavons (alcohols), isoflavones, biflavones, flavanols, chalcones, aurones, anthocyanins, neoflavonoids, and the like. Phytochemical research shows that the flavonoids compounds in the guava leaves have the effects of reducing blood sugar, reducing blood fat, resisting bacteria and viruses, promoting blood circulation to remove blood stasis and the like. Therefore, the guava leaf total flavonoids as the medicinal active parts have great clinical application value and market development value and are widely researched at present.

The extraction method of flavonoid components comprises solvent extraction, microwave extraction, supercritical extraction, enzyme immersion extraction, ultrasonic extraction, reflux, soxhlet extraction, decoction, percolation, etc. The traditional extraction method has high cost, the obtained extract has low content of total flavone, the extraction rate of the total flavone calculated by rutin is 1.5-5%, and the extraction rate after purification by macroporous resin is usually below 70%. At present, the extraction of the total flavonoids in the guava leaves mainly comprises the following methods: the water decoction method has long time consumption and low extraction efficiency, and partial active substances are easily inactivated by high-temperature water decoction; the heating reflux extraction method has long-term heat effect, which easily causes thermal decomposition and oxidation of flavone, thus causing damage and loss of effective components; the invention patent CN111228334A (a preparation method for extracting total flavonoids from guava leaves) discloses a method for extracting total flavonoids by using ultrasonic-assisted ethanol, but the extraction rate of flavonoids can be improved by single ultrasonic-assisted traditional solvent extraction, but the improvement of the extraction rate is not obvious; the invention patent CN107115382A (a method for extracting total flavone from guava leaf) discloses a technology for extracting total flavone from guava leaf by using surfactant and microwave-ultrasonic wave to assist, and uses aqueous solution to replace organic solvent, and uses wetting and solubilizing actions of surfactant to extract flavonoid substances, but 4 nonionic surfactants and 2 anionic surfactants are added according to different proportions in the extraction process, so the operation is more complicated and residues are easy to cause in the subsequent operation; the invention patent CN107252093A (a guava leaf rich in soluble polyphenol and flavonoid aglycone and a preparation method and application) discloses a method for degrading the flavonoid glycoside component of the guava leaf into the flavonoid aglycone component by an enzymatic hydrolysis method so as to improve the extraction rate.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides an extraction method for extracting total flavonoids from guava leaves by utilizing an ultrasonic-assisted eutectic solvent extraction technology. The extraction method provided by the invention can effectively solve the defect of decomposition of effective substances caused by high temperature, and can avoid the problems of impurity residue and the like.

The invention provides an extraction method for extracting total flavonoids from guava leaves by utilizing an ultrasonic-assisted eutectic solvent extraction technology, which comprises the following steps:

(1) Drying and crushing guava leaves to obtain guava leaf powder;

(2) Leaching guava leaf powder by using an extraction technology of an ultrasonic-assisted eutectic solvent (DESs) to obtain a filtrate;

(3) Centrifuging the filtrate to obtain a supernatant;

(4) Purifying the supernatant to obtain an eluent, and concentrating and drying the eluent to obtain the total flavonoids.

Preferably, the particle size of the guava leaf powder in the step (1) is 40-mesh sieve.

Preferably, the drying temperature in the step (1) is 50 ℃.

Preferably, in the step (2), the ultrasonic-assisted eutectic solvent extraction technology involves using an aqueous solution of a eutectic solvent comprising a hydrogen bond acceptor, a hydrogen bond donor and water;

the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:1;

in the water solution of the eutectic solvent, the water content is 25-40% in terms of mass fraction;

the hydrogen bond receptor comprises choline chloride;

the hydrogen bond donor includes: lactic acid or glucose or glycerol or butanediol or ethylene glycol.

In the present invention, the synthesis of the DESs preferably comprises the following steps: choline chloride (ChCl) has strong water absorption and needs to be dried in a vacuum drying oven at 80 deg.C for 24 hr for use. Mixing the dried ChCl and different hydrogen bond donors according to a proper proportion, placing the mixture in a round bottom flask, heating and stirring the mixture in a constant temperature water bath kettle at 90 ℃ until a uniform and stable transparent system is formed. Cooling the obtained DESs to room temperature, sealing and placing in a dryer with P ₂ O ₅ Drying for at least 2 weeks;

the preparation method of the aqueous solutions of the DESs preferably comprises the following steps: dissolving target DESs in deionized water according to mass fraction, preparing eutectic aqueous solution with certain water content (0%, 25%, 40% and 55%), fully shaking up, and standing to obtain uniform and transparent DESs aqueous solution.

Preferably, in the step (2), the specific operation step of ultrasonic assistance comprises the following steps:

mixing guava leaf powder with an aqueous solution of a eutectic solvent and then leaching;

the feed-liquid ratio of the guava leaf powder to the aqueous solution of the eutectic solvent is 1 (20-30) g/mL;

the ultrasonic power during the leaching is 600w, the leaching temperature is 25-30 ℃, and the leaching time is 45-60 min.

Preferably, in the step (3), the frequency of centrifugation is 4500r/min, and the time of centrifugation is 15min.

Preferably, in the step (4), the purification method comprises purification by using an adsorption resin;

the adsorption resin is nonpolar HPD100 type macroporous adsorption resin;

the adsorption resin also comprises pretreatment before use: removing impurities and screening out shrivelled particles, soaking the particles in ethanol for 3-4 h, repeatedly washing the particles after soaking until 3 times of water is added into outlet washing liquid and the outlet washing liquid is not turbid, and fully washing the particles by using clear water until no obvious ethanol smell exists to obtain the activated adsorption resin.

Preferably, in the step (4), the purification comprises the following steps:

the method comprises the following steps of (1) loading activated adsorption resin to a column by a wet method at a flow rate of 2BV/h, loading a supernatant at a flow rate of 2BV/h for adsorption, washing the chromatographic column by ultrapure water at a flow rate of 2BV/h after adsorption is finished, eluting by using ethanol with a volume ratio of 70% at a flow rate of 2BV/h, collecting eluent until the effluent eluent is colorless, wherein the volume of the ultrapure water is 50 mL.

In the invention, the content of the total flavone is determined by the following method: using NaNO ₂ -Al (NO ₃ ) ₃ And (3) colorimetric determination, namely determining a light absorption value at the wavelength of 510nm, drawing a standard curve by taking the rutin mass concentration as a horizontal coordinate and the light absorption value as a vertical coordinate, and obtaining a regression equation: y =3.9184x +0.0043. Preparing a sample solution with a certain concentration, and determining the light absorption value of the sample solution in a colorimetric tube. Calculating the concentration of flavone according to the standard curve, and calculating the content of total flavone according to the formula (1).

Total flavone content (%) = (C × V)/(M × 1000) × 100%

C: total flavone concentration calculated from the standard curve, V: sample liquid volume, M: guava leaf powder quality

Deep Eutectic Solvents (DESs), most of which are Eutectic salts formed by the interaction of hydrogen bonds, exist in liquid form at a lower temperature range and have a melting point lower than any component constituting the Eutectic Solvents, and belong to a novel class of ionic liquids. The DESs have good solubility for various substances, both polar and nonpolar compounds have solubilization effect, and the solubility of some metabolites in a eutectic solvent is obviously better than that of water. The polarity of the DESs changes upon addition of water, thereby changing its solubility properties. For example, DESS have a relatively high solubility for non-polar compounds, while DESS with a certain water content has a relatively good solubility for compounds of medium polarity. The research shows that the solubility of rutin, quercetin, DNA, starch and the like in DESs is 18-460000 times of that in water; the solubility of the drugs such as benzoic acid, griseofulvin, danazol, itraconazole and the like with poor solubility in water in the DESs is 5-22000 times that of the drugs in water, which shows that the synergistic effect between the two components promotes the enhancement of the dissolving capacity of the drugs on the substances when the DESs are formed. At present, the DESs are used as green solvents to extract and separate polyphenol, flavonoid, terpene, polysaccharide, protein and other compounds with different polarities from natural products, and have great potential in the field of extraction and separation of traditional Chinese medicines.

Ultrasonic-assisted extraction, which utilizes the cavitation effect of ultrasonic waves to promote a solvent to penetrate into cells to a greater extent, and stimulates intracellular glands continuously to increase the mass transfer rate; meanwhile, secondary effects of the plant cell wall breaking agent such as mechanical effect, mechanical vibration, emulsification, diffusion, crushing, chemical effect and the like are utilized to break the cell wall so as to accelerate the diffusion and release of the flavonoid compound in the plant, and the heat-sensitive substance is effectively prevented from being damaged in a high-temperature environment.

The invention improves various defects of flavone extraction in the prior art by combining ultrasonic-assisted extraction and DESS.

Drawings

FIG. 1 is a comparison of the extraction rate of total flavonoids from guava leaves by the thermal reflux extraction method (A) provided in comparative example 2, the ultrasonic assisted organic reagent extraction method (B) provided in comparative example 1, the ultrasonic assisted aqueous surfactant extraction method (C) provided in comparative example 4, and the ultrasonic assisted DESS extraction method (D) provided in example 1;

FIG. 2 is a comparison of the extraction rates of total flavonoids from guava leaves with different kinds of DESs at different water contents;

FIG. 3 shows the total flavone extraction rate of the same DESS (ChCl: EG) and guava leaf powder under different material-to-liquid ratios;

FIG. 4 shows the extraction rate of total flavonoids from guava leaves after purification with different types of macroporous resins.

Detailed Description

The reagents and instruments used in the following description are not specifically described in terms of techniques or conditions, and are performed under the conditions of the conventional experiments, or under the conditions suggested in the specification. The reagents or instruments used are conventional products which are commercially available, and are not indicated by manufacturers.

Example 1

(1) Cleaning guava leaves, removing impurities such as branch, stalk, silt and the like, drying the guava leaves in a 50 ℃ oven, crushing the guava leaves, and sieving the crushed guava leaves with a 40-mesh sieve;

(2) Leaching guava leaf powder, wherein the selected DESS combination is as follows: the hydrogen bond acceptor is choline chloride (ChCl), the hydrogen bond donor is Ethylene Glycol (EG), and the molar ratio of the ChCl to the hydrogen bond donor is 1:1,DESS aqueous solution has a water content of 25%. The material-liquid ratio of the guava leaf powder to the DESs aqueous solution is 1.

(3) Vacuum filtering to remove residue, collecting filtrate, and centrifuging at 4500r/min for 15min.

(4) Taking the supernatant after centrifugation, adopting NaNO ₂ -Al(NO ₃ ) ₃ The method is used for measuring the content of the total flavone.

Example 2

The present embodiment is different from embodiment 1 in that:

the DESS selected in the step (2) is combined as follows: the hydrogen bond acceptor is choline chloride (ChCl), the hydrogen bond donor is Ethylene Glycol (EG), and the molar ratio of the choline chloride to the hydrogen bond donor is 1:1, the water content of the aqueous solutions of the DESs is 40%, and the feed-liquid ratio of the guava leaf powder to the aqueous solutions of the DESs is 1. The rest is the same as in embodiment 1.

Example 3

The present embodiment is different from embodiment 1 in that:

the DESS selected in the step (2) is combined as follows: the hydrogen bond acceptor is choline chloride (ChCl), the hydrogen bond donor is Butanediol (BDO), and the molar ratio of the choline chloride to the hydrogen bond donor is 1:1, the water content of the DES aqueous solution is 25%, and the feed-liquid ratio of the guava leaf powder to the DES aqueous solution is 1. The rest is the same as in embodiment 1.

Example 4

The present embodiment is different from embodiment 1 in that:

the DESS selected in the step (2) is combined as follows: the hydrogen bond acceptor is choline chloride (ChCl), the hydrogen bond donor is Glycerol (GLY), and the molar ratio of the choline chloride to the hydrogen bond donor is 1:1, the water content of the aqueous solutions of the DESs is 25%, and the feed-liquid ratio of the guava leaf powder to the extraction of the aqueous solutions of the DESs is 1. The rest is the same as in embodiment 1.

Example 5

The present embodiment is different from embodiment 1 in that:

the DES combination selected in the step (2) is as follows: the hydrogen bond acceptor is choline chloride (ChCl), the hydrogen bond donor is Lactic Acid (LA), and the molar ratio of the choline chloride to the hydrogen bond donor is 1:1, the water content of the DES aqueous solution is 25%, and the feed-liquid ratio of the guava leaf powder to the DES aqueous solution is 1. The rest is the same as in embodiment 1.

Example 6

The present embodiment is different from embodiment 1 in that:

the DES combination selected in the step (2) is as follows: the hydrogen bond acceptor is choline chloride (ChCl), the hydrogen bond donor is Glucose (GLU), and the molar ratio of the choline chloride to the hydrogen bond donor is 1:1, the water content of the aqueous solutions of the DESs is 40%, and the feed-liquid ratio of the guava leaf powder to the aqueous solutions of the DESs is 1. The rest is the same as in embodiment 1.

Example 7

In this example, on the basis of example 1, the purification steps of a macroporous resin column are added as follows:

(1) Adding the centrifuged guava leaf extract supernatant into the activated HPD100 type macroporous resin at the flow rate of 2 BV/h; after adsorption, the chromatographic column is washed by ultrapure water at the flow rate of 2BV/h, the volume of the washed chromatographic column is 50mL, then the elution is carried out by 70 percent ethanol at the flow rate of 2BV/h, and the eluent is collected.

(2) Concentrating the eluate by rotary evaporator to obtain extract, and dissolving in 70% ethanol to obtain 1mg/mL total flavone extract of fructus Psidii Guajavae Immaturus.

(3) Using NaNO ₂ -Al(NO ₃ ) ₃ Measuring the aboveThe content of total flavone in the extractive solution.

The following comparative examples aim to clarify the improvement of the extraction effect of the ultrasonic-assisted EDSs on the total flavonoids in the guava leaves compared with other extraction methods.

Comparative example 1

(1) Cleaning guava leaves, removing impurities such as branch, stalk, silt and the like, drying the guava leaves in a 50 ℃ oven, crushing the guava leaves, and sieving the crushed guava leaves with a 40-mesh sieve;

(2) An ultrasonic-assisted organic reagent extraction method is adopted, wherein an extraction reagent is 70% ethanol, the material-liquid ratio is 1.

(3) Vacuum filtering to remove residue, collecting filtrate, and concentrating with rotary evaporator to obtain extract.

(4) Re-dissolving the extract in anhydrous ethanol to obtain 1mg/mL total flavone crude extract, and extracting with NaNO ₂ -Al(NO ₃ ) ₃ Measuring the content of total flavonoids in the guava leaves in the extracting solution by the method.

Comparative example 2

This comparative example differs from comparative example 1 in that:

in the step (2), a heating reflux extraction method is adopted, wherein an extraction reagent is 70% ethanol, the material-liquid ratio is 1. The others are the same as in comparative example 1.

Comparative example 3

This comparative example differs from comparative example 1 in that:

in the step (2), a Soxhlet extraction method is adopted, wherein an extraction reagent is 70% ethanol, the material-liquid ratio is 1. The rest is the same as in comparative example 1.

Comparative example 4

The present comparative example differs from comparative example 1 in that:

in the step (2), an ultrasonic-assisted surfactant aqueous solution extraction method is adopted, the mass ratio of the surfactant sodium dodecyl sulfate to the Tween-80 is 2: 1, the mixed surfactant accounts for 2% of the mixed surfactant aqueous solution by mass, the material-liquid ratio is 1. The others are the same as in comparative example 1.

Comparative example 5

(1) Cleaning guava leaves, removing impurities such as branch, stalk, silt and the like, drying the guava leaves in a 50 ℃ oven, crushing the guava leaves, and sieving the crushed guava leaves with a 40-mesh sieve;

(2) Leaching guava leaf powder, wherein the selected DESS combination is as follows: the hydrogen bond acceptor is choline chloride (ChCl), the hydrogen bond donor is Ethylene Glycol (EG), and the molar ratio of the choline chloride to the hydrogen bond donor is 1: the water content of the 1,DESS aqueous solution was 25%. The feed-liquid ratio of guava leaf powder to DESs aqueous solution is 1.

(3) Vacuum filtering to remove residue, collecting filtrate, centrifuging at 4500r/min for 15min, and collecting supernatant to obtain crude extractive solution of folium Psidii Guajavae.

(4) Adding coarse guava leaf extract at a flow rate of 2BV/h by using ADS-7 type macroporous resin; after adsorption, the chromatographic column is washed by ultrapure water at the flow rate of 2BV/h, the volume of the washed chromatographic column is 50mL, then the elution is carried out by 70 percent ethanol at the flow rate of 2BV/h, and the eluent is collected.

(5) Concentrating the eluate with rotary evaporator, dissolving in 70% ethanol, and making into 1mg/mL total flavone extractive solution.

(6) Using NaNO ₂ -Al(NO ₃ ) ₃ Measuring the content of total flavonoids in the guava leaves in the extracting solution by the method.

Comparative example 6

This comparative example differs from comparative example 5 in that:

the model of the macroporous resin adopted in the step (4) is D101, and the rest is the same as that of the comparative example 4.

Comparative example 7

The comparative example is different from comparative example 5 in that

The model of the macroporous resin adopted in the step (4) is AB-8, and the rest is the same as that of the macroporous resin adopted in the comparative example 4.

Comparative example 8

This comparative example differs from comparative example 5 in that:

the model of the macroporous resin used in step (4) was DM-130, and the rest was the same as in comparative example 4.

Application example 1

The extraction rate of total flavonoids was counted for the extraction methods provided in example 1, comparative example 2 and comparative example 4, and the statistical results are shown in fig. 1.

As can be seen from fig. 1, the extraction method provided in embodiment 1 of the present invention can greatly increase the extraction rate of total flavonoids.

Application example 2

According to the DESS with different components in the table 1, the extraction rates of the total flavonoids in the leaves of the DESS guava with different water contents are compared, and the statistical results are shown in the figure 2.

Extraction conditions are as follows: the material-liquid ratio is 1.

TABLE 1 eutectic solvents (DESS) for different compositions

As shown in FIG. 2, the combination of ChCl and EG can effectively improve the extraction rate of total flavonoids in guava leaves.

Application example 3

The statistical results of the comparison of the extraction rates of total flavonoids under different feed-to-liquid ratios are shown in fig. 3. Extraction conditions are as follows: the extraction solvent is choline chloride (ChCl) and Ethylene Glycol (EG) mixed according to a molar ratio of 1.

Application example 4

Comparing the effect of purifying total flavonoids of guava leaves by different types of macroporous resin in example 7 and comparative examples 5 to 8. The comparison results are shown in fig. 4.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that several modifications and improvements can be made to the inventor without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Claims (8)

1. An extraction method for extracting total flavonoids from guava leaves by utilizing an ultrasonic-assisted eutectic solvent extraction technology is characterized by comprising the following steps of:

(1) Drying and crushing guava leaves to obtain guava leaf powder;

(2) Leaching guava leaf powder by using an extraction technology of an ultrasonic-assisted eutectic solvent to obtain a filtrate;

(3) Centrifuging the filtrate to obtain a supernatant;

(4) Purifying the supernatant to obtain an eluent, and concentrating and drying the eluent to obtain the total flavonoids.

2. The extraction method according to claim 1, wherein the guava leaf powder in the step (1) has a particle size of 40 mesh.

3. The extraction method according to claim 1, wherein the drying temperature in the step (1) is 50 ℃.

4. The extraction method according to claim 1, wherein in the step (2), the ultrasonic-assisted eutectic solvent extraction technique involves using an aqueous solution of a eutectic solvent including a hydrogen bond acceptor, a hydrogen bond donor and water;

the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:1;

in the aqueous solution of the eutectic solvent, the water content is 25-40% by mass;

the hydrogen bond receptor comprises choline chloride;

the hydrogen bond donor includes: lactic acid or glucose or glycerol or butanediol or ethylene glycol.

5. The extraction method according to claim 1, wherein in the step (2), the specific operation step of ultrasonic wave assistance comprises the following steps:

mixing guava leaf powder with an aqueous solution of a eutectic solvent, and leaching;

the feed-liquid ratio of the guava leaf powder to the aqueous solution of the eutectic solvent is 1 (20-30) g/mL;

the ultrasonic power during the leaching is 600w, the leaching temperature is 25-30 ℃, and the leaching time is 45-60 min.

6. The extraction method according to claim 1, wherein in the step (3), the centrifugation frequency is 4500r/min, and the centrifugation time is 15min.

7. The extraction method according to claim 1, wherein in the step (4), the purification method comprises purification by using an adsorption resin;

the adsorption resin is nonpolar HPD100 type macroporous adsorption resin;

the adsorption resin also comprises pretreatment before use: removing impurities and screening out shriveled particles, soaking the particles for 3 to 4 hours by using ethanol, repeatedly washing the particles after soaking until the outlet washing liquid is not turbid after 3 times of water is added, and fully leaching the particles by using clear water until no obvious ethanol smell exists to obtain the activated adsorption resin.

8. The extraction method according to claim 1, characterized in that, in the step (4), the purification comprises the following steps:

the method comprises the following steps of (1) loading activated adsorption resin to a column by a wet method at a flow rate of 2BV/h, loading a supernatant at a flow rate of 2BV/h for adsorption, washing the chromatographic column by ultrapure water at a flow rate of 2BV/h after adsorption is finished, eluting by using ethanol with a volume ratio of 70% at a flow rate of 2BV/h, collecting eluent until the effluent eluent is colorless, wherein the volume of the ultrapure water is 50 mL.

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