CN114053754A

CN114053754A - Two-phase deep eutectic solvent for simultaneously separating and extracting multiple active ingredients from waste ginkgo nut testa and extraction method thereof

Info

Publication number: CN114053754A
Application number: CN202111447037.5A
Authority: CN
Inventors: 苏二正; 董其惠; 吴蓉; 张芳; 曹福亮; 汪贵斌; 王佳宏
Original assignee: Nanjing Forestry University
Current assignee: Nanjing Forestry University
Priority date: 2021-11-30
Filing date: 2021-11-30
Publication date: 2022-02-18
Anticipated expiration: 2041-11-30
Also published as: CN114053754B

Abstract

The invention discloses a preparation and extraction method of a two-phase deep eutectic solvent for simultaneously separating and extracting a plurality of active ingredients from waste ginkgo nut testa and an obtained product thereof. The hydrophobic phase is a hydrophobic deep eutectic solvent prepared from menthol, hexanol and lauric acid; the hydrophilic phase is formed by mixing a hydrophilic deep eutectic solvent and water, and the hydrophilic deep eutectic solvent is prepared from choline chloride, citric acid and ethylene glycol. The two-phase deep eutectic solvent prepared by the invention is a green solvent, and has the characteristics of simple preparation, stable hydrogen bond, easy biodegradation, safety, no pollution and the like; can be used for simultaneously separating and extracting hydrophobic active ingredients, namely ginkgolic acid and hydrophilic active ingredients, namely ginkgetin, terpene lactone and procyanidine from the waste ginkgo biloba sarcotesta, and has the advantages of high extraction rate, low cost, simple operation and safe use.

Description

Two-phase deep eutectic solvent for simultaneously separating and extracting multiple active ingredients from waste ginkgo nut testa and extraction method thereof

Technical Field

The invention belongs to high-value utilization of agricultural and forestry waste biological resources, and particularly relates to a two-phase deep eutectic solvent for simultaneously separating and extracting multiple active ingredients from waste ginkgo biloba sarcotesta and an extraction method thereof.

Background

Ginkgo biloba (Ginkgo biloba L.) is a plant of the genus Ginkgo of the family Ginkgoaceae. Gingko is a rare tree species of middle-age wiggles, which is a special product in China and only exists in a wild state in Tianmu mountain of Zhejiang. The gingko is a fast-growing and precious tree species, and can be used for buildings, furniture, interior decoration, engraving, drawing boards and the like. The semen Ginkgo can be used for food and medicine. The ginkgo leaves can be used as medicine and pesticide, and also can be used as feed and fertilizer.

Modern medical research on ginkgo biloba originated in 1928, in which ginkgo biloba leaves have been developed by Schwabe, germany, as a variety of drugs for treating cardiovascular diseases, such as EGb 761. However, the ginkgo nut, especially the testa thereof, has not been well developed and utilized. At present, a large amount of dry ginkgo nut testa is discarded every year, which not only causes resource waste, but also pollutes the environment. In recent years, many studies have shown that ginkgo biloba testa also has the same active ingredients as ginkgo biloba leaves, such as ginkgetin, terpene lactones, procyanidins, ginkgolic acids, and the like.

Researches show that the ginkgo flavonoid compound has certain functions of resisting oxidation, protecting nerves, improving cognition, inhibiting bacteria and the like. Ginkgo terpene lactones are unique components of ginkgo biloba and are not found in other plants at present. The ginkgolides belong to terpenoids, and mainly comprise ginkgolide A, B, C, J, M and bilobalide, wherein ginkgolide A, B, C, J, M belongs to diterpenoids, and bilobalide is sesquiterpene compound. A large number of researches show that the ginkgolide has a protective effect on nerve functions and cerebral ischemia injury. The procyanidin has the effects of resisting hypertension, endothelium-dependent vasodilation activity, ischemia-reperfusion injury, atherosclerosis, platelet aggregation and the like, and also has a remarkable protection effect on acute renal failure.

Ginkgolic Acid (GAs) belongs to urushiol substances, and is a 6-alkyl or 6-alkenyl salicylic acid derivative, the side chain is usually 13-17 carbon atoms, the number of double bonds of the side chain is 0-2, and the Ginkgolic acid mainly comprises five components: ginkgolic acids (C13:0), ginkgolic acids (C15:1), hydroginkgolic acids (C15:0), heptadecadienergenic ginkgolic acids (C17:2) and heptadecadienergenic ginkgolic acids (C17: 1). At present, ginkgolic acid is an adverse component for ginkgo biloba preparations, and can cause cytotoxicity, embryotoxicity, sensitization, enzyme system inhibition, slight neurotoxicity and the like. However, in recent years, a great deal of research shows that the ginkgolic acid has certain pharmacological effects, such as bactericidal effect, anti-tumor effect, anti-virus effect, anti-oxidation effect and the like, and can be used in the fields of pesticides, cosmetics and the like.

The ginkgetin, terpene lactone and procyanidin have hydrophilicity, while the ginkgolic acid has hydrophobicity. Therefore, the conventional extraction methods use a large amount of different organic solvents to extract the hydrophilic component and the hydrophobic component separately or stepwise, which not only increases the complexity of the extraction step and operation, but also causes the waste of the active component and increases the extraction cost. In addition, the traditional organic solvent is easy to volatilize and leave, and causes harm to the environment and human bodies. The prior art discloses a two-phase deep eutectic solvent (CN107789376A) for extracting active ingredients of ginkgo leaves, which takes the ginkgo leaves as a raw material and adopts a specific two-phase deep eutectic solvent to extract a plurality of active ingredients of the ginkgo leaves, including flavone, terpene lactone, procyanidine and poly (isopentenol) acetate. However, there is no report in the prior art on how to extract various active ingredients from the waste ginkgo biloba sarcotesta, so that it is very necessary to find a green, safe, efficient method for simultaneously separating and extracting the active ingredients from the ginkgo biloba sarcotesta.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides a two-phase deep eutectic solvent for simultaneously extracting a plurality of active ingredients in the episperm of the waste ginkgo nut, wherein a hydrophobic ingredient, namely ginkgolic acid, is enriched in an upper phase hydrophobic phase, and hydrophilic ingredients, namely ginkgetin, terpene lactone and procyanidine, are enriched in a lower phase hydrophilic phase.

The two-phase deep eutectic solvent provided by the invention has the characteristics of simple preparation, stable hydrogen bond, easy biodegradation, safety, no pollution and the like as a green solvent, can synchronously extract and separate ginkgolic acid, flavone, terpene lactone and procyanidine from the ginkgo biloba exocarp in a green, safe and efficient manner, and solves a series of problems of large usage amount of organic solvent, complex operation, high cost, low efficiency and the like in the existing method.

The invention also provides a preparation method of the two-phase deep eutectic solvent and a method for extracting active ingredients of the seed coats of the waste ginkgo nuts by using the two-phase deep eutectic solvent.

The technical scheme is as follows: in order to achieve the purpose, the invention discloses a two-phase deep eutectic solvent for simultaneously separating and extracting multiple active ingredients from waste ginkgo nut testa, which consists of a hydrophobic phase and a hydrophilic phase according to the volume ratio of 1: 9-9: 1; the hydrophobic phase is a hydrophobic deep eutectic solvent DES-U prepared from menthol, hexanol and lauric acid according to a molar ratio of 2.5:0.8: 0.2-1: 2: 0.5; the hydrophilic phase consists of DES-D and water, wherein the water content is 40-70% (mass percent), and the DES-D is a hydrophilic deep eutectic solvent prepared from choline chloride, citric acid and ethylene glycol in a molar ratio of 1:1: 1-3: 2: 1.

Preferably, the volume ratio of the hydrophobic phase to the hydrophilic phase is 2: 8-8: 2.

Further, the volume ratio of a hydrophobic phase to a hydrophilic phase in the two-phase deep eutectic solvent is 8:2, the DES-U is prepared from menthol, hexanol and lauric acid according to the molar ratio of 1:1.6:0.4, the DES-D is prepared from choline chloride, citric acid and ethylene glycol according to the molar ratio of 3:2:1, the DES-D in the hydrophilic phase accounts for 55% of the total mass, and water accounts for 45% of the total mass.

The preparation method of the two-phase deep eutectic solvent comprises the following steps:

(1) weighing menthol, hexanol and lauric acid according to a molar ratio, uniformly mixing, heating and stirring until a uniform transparent liquid is formed, namely a hydrophobic deep eutectic solvent DES-U serving as a hydrophobic phase of a two-phase deep eutectic solvent;

(2) weighing choline chloride, citric acid and ethylene glycol according to a molar ratio, uniformly mixing, heating and stirring until a uniform transparent liquid is formed to be used as DES-D in a hydrophilic phase, and mixing the DES-D and water to be used as a hydrophilic phase of a two-phase deep eutectic solvent;

(3) mixing the hydrophobic phase and the hydrophilic phase according to the volume ratio, standing and layering to obtain the two-phase deep eutectic solvent.

The method for simultaneously separating and extracting the active ingredients in the waste ginkgo nut testa by using the two-phase deep eutectic solvent comprises the following steps:

(1) placing the two-phase deep eutectic solvent in an extraction container, accurately weighing ginkgo nut testa dry powder, adding into the two-phase deep eutectic solvent in the extraction container, and mixing uniformly;

(2) mixing, heating for extraction, centrifuging, separating two phase system with upper phase as hydrophobic phase and lower phase as hydrophilic phase, and detecting the contents of ginkgolic acid, terpene lactone, flavone and procyanidin in hydrophobic phase and hydrophilic phase respectively; the mass-volume ratio of the waste ginkgo nut testa dry powder in the step (1) to the two-phase deep eutectic solvent is 1: 10-1: 50 g/mL.

The preferred mass to volume ratio is 1:30 g/mL.

Wherein the heating extraction in the step (2) is oscillation heating extraction or stirring heating extraction.

Wherein the rotation speed of the oscillation or stirring in the step (2) is 100-300 rpm, the temperature is 25-50 ℃, and the time is 5-55 min.

The preferred speed is 220rpm, temperature is 30 ℃ and time is 40 min.

Wherein, the centrifugation temperature in the step (2) is room temperature, the centrifugation force is 2000-6000 g, and the centrifugation time is 3-7 min.

Preferably, the centrifugal force is 3000g and the centrifugation time is 5 min.

Wherein, the ginkgolic acid is recovered from the hydrophobic phase after extraction, and the terpene lactone, the flavone and the procyanidin are recovered from the hydrophilic phase by one or more of liquid-liquid extraction method, macroporous resin adsorption method or anti-solvent method. Preferably, macroporous resin adsorption is used.

Wherein the product recovered from the hydrophobic phase is ginkgolic acid, and the product recovered from the hydrophilic phase is terpene lactone or flavone or procyanidin or a mixture of terpene lactone, flavone and procyanidin.

The raw material of the invention is waste ginkgo nut testa which is different from ginkgo leaf in a plurality of ways: firstly, the tissue structure is different from the main components, the ginkgo leaves are the nutritive organs of the ginkgo trees, the tissue structure is compact and firm, the main components contain more cellulose, hemicellulose and lignin, the ginkgo episperm is one part of the fruits of the reproductive organs of the ginkgo trees, the tissue structure of the mature fruit episperm is soft, and the main components contain more pectin and crude fat; secondly, the contained bioactive components are different, although the testa is approximately the same as the hydrophilic active components of the ginkgo leaves, the hydrophobic active components are different, the hydrophobic active components in the testa of the ginkgo fruits are mainly ginkgolic acid, and the ginkgo leaves are mainly polyprenol acetate.

The two-phase deep eutectic solvent in the prior art can not be used for simultaneously extracting the effective components in the seed coats of the waste ginkgo nuts. The main reason is that: (1) the hydrophobic phase in the prior art is prepared by directional design according to the need of extracting the polyprenol acetate, the polyprenol acetate is an ester of long-chain fatty alcohol, the ginkgolic acid to be separated in the invention is an aromatic acid, the structure of the ginkgolic acid is different from that of the polyprenol acetate, the hydrophobic phase in the prior art cannot be simply used for two-phase system construction, and the directional design needs to be carried out according to the need of extracting the ginkgolic acid; (2) in the prior art, swelling and dissolving of the deep eutectic solvent to cellulose, hemicellulose and lignin are considered more during two-phase construction, and dissolving of pectin and lipid in ginkgo nut testa is considered more in the patent. Therefore, it is not practical to use the two-phase deep eutectic solvent in the prior art directly for extracting the active ingredients from the testa of the waste ginkgo nut.

The invention mainly aims to construct a two-phase deep eutectic solvent system and simultaneously extract a plurality of active ingredients in the ginkgo nut testa. In order to achieve the purpose, when the two-phase deep eutectic solvent is designed, the disintegration or dissolution capacity of the components selected for preparing the deep eutectic solvent on the main components of pectin and crude fat of the ginkgo biloba sarcotesta is considered, and the selective directional enrichment capacity on ginkgolic acid, terpene lactones, flavone and procyanidine is also considered. In the optimization of an actual experiment, by component screening and molar ratio optimization of the deep eutectic solvent, the hydrophobic deep eutectic solvent and the hydrophilic deep eutectic solvent which can simultaneously and efficiently extract a plurality of active ingredients in the ginkgo nut testa are obtained, and the directional enrichment of the hydrophilic active ingredients and the hydrophobic active ingredients is realized.

According to the invention, the hydrophobic phase of the hydrophobic-hydrophilic two-phase deep eutectic solvent has a dissolving effect on hydrophobic crude fat in the testa, and the hydrophilic phase has a disintegrating effect on hydrophilic pectin in the testa; the hydrophobic phase and the hydrophilic phase of the hydrophobic-hydrophilic two-phase deep eutectic solvent achieve similar compatibility of hydrophobic phase with respect to ginkgolic acid and similar compatibility of hydrophilic phase with respect to terpene lactone, flavone and procyanidine to the maximum extent through component screening, molar ratio optimization and two-phase proportion optimization, thereby realizing directional enrichment and efficient separation of hydrophilic active ingredients and hydrophobic active ingredients.

The final purpose of the invention is to simultaneously separate and extract a plurality of active ingredients in the ginkgo biloba sarcotesta, wherein the hydrophobic ingredient ginkgolic acid is enriched in the hydrophobic phase, and the hydrophilic ingredients terpene lactones, flavones and procyanidins are enriched in the hydrophilic phase. The invention designs a hydrophobic-hydrophilic two-phase deep eutectic solvent, which can efficiently dissolve or disintegrate the ginkgo nut testa tissue structure and is beneficial to the free release of active ingredients, thereby achieving high-efficiency extraction, and can maximally achieve similar compatibility of hydrophobic and hydrophilic active ingredients, thereby realizing high-efficiency separation and directional enrichment.

Has the advantages that: compared with the prior art, the invention has the following advantages:

(1) the two-phase deep eutectic solvent prepared by the invention mostly adopts compounds with natural sources as raw materials, and has the advantages of environmental protection, safety, biodegradability and the like.

(2) The two-phase deep eutectic solvent prepared by the invention is a green solvent, has chemical properties similar to those of ionic liquid, and has the advantages of simple preparation method, green and safety and the like.

(3) The hydrophobic phase of the hydrophobic-hydrophilic two-phase deep eutectic solvent prepared by the invention has a dissolving effect on hydrophobic crude fat in the testa, the hydrophilic phase has a disintegrating effect on hydrophilic pectin in the testa, and the dissolution of the crude fat and the disintegration of the pectin are beneficial to the free release of intracellular bioactive components, so that the aim of high-efficiency extraction is fulfilled.

(4) The two-phase deep eutectic solvent prepared by the invention has the advantages that the hydrophobic phase consists of menthol, hexanol and lauric acid, the hydrophilic phase consists of choline chloride, citric acid, ethylene glycol and water, the polarity of the two phases is adjusted by optimizing the molar ratio of substances in the two phases, so that the polarity of the hydrophobic phase is closer to ginkgolic acid, and the polarity of the hydrophilic phase is closer to terpene lactone, flavone and procyanidine, thereby achieving the purpose that ginkgolic acid is enriched in the hydrophobic phase and ginkgolic acid, terpene lactone and procyanidine are enriched in the hydrophilic phase.

(5) The two-phase deep eutectic solvent prepared by the method can achieve the purpose of simultaneously separating and extracting various active ingredients in the ginkgo nut testa, is simple to operate, high in extraction efficiency, green and safe, provides a new idea for development and utilization of the ginkgo nut testa, reduces waste of the ginkgo nut testa and protects the environment.

Drawings

FIG. 1 is a high performance liquid chromatogram of ginkgolic acids.

FIG. 2 is a state diagram of ten two-phase deep eutectic solvents prepared by preliminary screening.

FIG. 3 is a diagram showing the effect of ten two-phase deep eutectic solvents prepared by preliminary screening on the extraction and separation of active ingredients from the ginkgo nut testa.

FIG. 4 is a graph showing the relationship between the effect of different alcohol components in the hydrophobic deep eutectic solvent on the extraction and separation of active ingredients from the ginkgo nut testa.

FIG. 5 is a graph showing the effect of different molar ratios of HBA/HBD in a hydrophobic deep eutectic solvent on the extraction and separation effect of active ingredients in the ginkgo nut testa.

FIG. 6 is a graph showing the relationship between the effect of different alcohol components in the hydrophilic deep eutectic solvent on the extraction and separation of active ingredients from the ginkgo nut testa.

FIG. 7 is a graph showing the relationship between the effect of different acid components in the hydrophilic deep eutectic solvent on the extraction and separation of active ingredients from the ginkgo nut testa.

FIG. 8 is a graph showing the effect of different molar ratios of HBA/HBD in a hydrophilic deep eutectic solvent on the extraction and separation effect of active ingredients in the ginkgo nut testa.

FIG. 9 is a graph showing the effect of different molar ratios of HBD1/HBD2 in a hydrophilic deep eutectic solvent on the extraction and separation effect of active ingredients in the ginkgo nut testa.

FIG. 10 is a graph showing the relationship between the effect of different water contents in the hydrophilic deep eutectic solvent on the extraction and separation of active ingredients from the ginkgo nut testa.

Fig. 11 is a graph showing the influence of different volume ratios of hydrophobic DES/hydrophilic DES on the extraction and separation effect of active ingredients in ginkgo nut testa in a two-phase deep eutectic solvent system.

FIG. 12 is a graph showing the relationship between the volume of two-phase deep eutectic solvent and the effect of extracting and separating active ingredients from the ginkgo nut testa powder in comparison with different feed liquids.

FIG. 13 shows the effect of different extraction temperatures on the extraction and separation of active ingredients from the ginkgo biloba sarcotesta.

FIG. 14 shows the effect of different extraction times on the extraction and separation of active ingredients from the ginkgo nut testa.

Detailed Description

The experimental methods described in the examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.

Experimental raw materials and reagents:

the experimental ginkgo nut testa is collected from the city of herba Metaplexis in Jiangsu province, dried to constant weight at 40 ℃, crushed into powder by a solid crusher, and sieved by a 40-mesh sieve to obtain ginkgo nut testa powder below 40 meshes; the ginkgolic acid standard is purchased from Sichuan Vickqi Biotech limited; the total ginkgolic acid standard and the ginkgolide A standard are purchased from Nanjing Dierger pharmaceutical science and technology Limited; the rutin standard is purchased from Shanghai leaf Biotech limited. In the experiment, glacial acetic acid and methanol are chromatographically pure, and other reagents are analytically pure. Other starting materials are commercially available.

The experimental apparatus is shown in Table 1.

TABLE 1 Experimental instruments List

HPLC detection of ginkgolic acid:

determination of the conditions of the liquid phase

In this embodiment, an HPLC isocratic elution method is adopted to determine the content of ginkgolic acid, and finally, the determined HPLC analysis conditions are as follows: the chromatographic column is an Elite SinoChrom ODS-BP 5 μm (4.6 mm. times.250 mm), the mobile phase is methanol-4% glacial acetic acid (94:6, v/v), the detection wavelength is 310nm, the column temperature is 36 ℃, the flow rate is 1mL/min, and the sample injection amount is 10 μ L. The liquid phase diagram of the standard under the conditions is shown in figure 1. The ginkgolic acid comprises the following components: peak emergence time of neoginkgolic acid (C13:0) 13.190min, peak emergence time of ginkgolic acid (C15:1) 14.077min, peak emergence time of heptadecadienylginkgolic acid (C17:2) 15.870min, peak emergence time of hydroginkgolic acid (C15:0) 19.332min and peak emergence time of heptadecadienylginkgolic acid (C17:1) 20.497 min. As can be seen from FIG. 1, the five components of phenolic acid have good peak patterns, are completely separated and can be used for the detection of the invention.

Preparing standard solution with neoacid standard substance of semen Ginkgo, diluting to corresponding times, with concentration gradient of 0.0000, 0.0108, 0.108, 0.216, 0.324, 0.432, 0.540, 0.648mg/mL, and drawing standard curve after HPLC detection. The standard curve of phenolic acid detection is that y is 690.3131x-1.2774, R²The detection range is 0.9999, and the detection range is 0-0.648 mg/mL.

The flavone detection method comprises the following steps:

the flavone detection adopts a rutin method of an ultraviolet spectrophotometer, and the specific detection method comprises the following steps: taking 0.5mL of sample to be detected, adding 0.3mL of 5% NaNO₂The solution was allowed to stand for 6min, 0.3mL of 10% Al (NO) was added₃)₃Standing the solution for 6min, adding 4mL of 4% NaOH solution and 4.5mL of 70% ethanol solution, mixing, standing for 20min, and detecting the light absorption value at 510 nm.

Preparing standard solution with rutin standard substance, diluting to corresponding times, with concentration gradient of 0.000, 0.0161, 0.161, 0.322, 0.644, 0.966, 1.288, and 1.610mg/mL, detecting after developing color, and drawing standardA quasi-curve. The standard curve of flavone detected by rutin method is y-0.2963 x-0.0081, R²0.9991, detection range 0-0.610 mg/mL.

The terpene lactone detection method comprises the following steps:

the terpene lactone is detected by adopting a spectrophotometry, and the specific detection method comprises the following steps: taking 0.5mL of sample to be tested, adding 0.4mL of alkaline hydroxylamine mixed solution (13.9% hydroxylamine hydrochloride aqueous solution-12.3% sodium hydroxide solution (1:2) for mixing, standing for 5min, adding 0.4mL of 3mol/L HCl solution and 6% FeCl₃0.2mL of the solution is mixed evenly, 3mL of 70% ethanol solution is added and mixed evenly, and the light absorption value is detected at 517 nm.

Preparing standard solution with ginkgolide A standard substance, diluting to corresponding times, detecting after color development, and drawing a standard curve, wherein the concentration gradient is 0.000, 0.085, 0.425, 0.850, 1.700, 2.550, 3.400 and 4.250 mg/mL. The terpene lactone standard curve is that y is 0.071x +0.003, R²0.9945, detection range 0-4.250 mg/mL.

The procyanidine detection method comprises the following steps:

4-Dimethylcinnamaldehyde (DMAC) spectrophotometry is selected for determination of procyanidine content, and the specific detection method comprises the following steps: accurately measuring 12.5mL of concentrated hydrochloric acid and 12.5mL of water, and diluting to 100mL with absolute ethyl alcohol to obtain acidic ethyl alcohol which needs to be prepared for use. 50mg of DMAC is made into a color developing agent by diluting to 50mL with acidic ethanol. Adding 3mL of color developing agent into 1mL of sample solution, standing at room temperature for 10min for color development reaction, and detecting at 644nm after reaction.

Preparing standard solution with standard substance of procyanidin from folium Ginkgo, diluting to corresponding times, with concentration gradient of 0.0129, 0.0258, 0.0387, 0.0516, 0.0645, 0.0774, 0.0903, 0.1032, 0.1161, and 0.129mg/mL, detecting after developing color, and drawing standard curve. The standard curve of procyanidine is that y is 4.8401x +0.0357, R²The detection range is 0.9994, and the detection range is 0-0.129 mg/mL.

Example 1

(1) Weighing menthol, hexanol and lauric acid according to the molar ratio of 1:0.4:0.1, uniformly mixing, and heating at 80 ℃ until a transparent liquid is formed, namely a deep cosolvent DES-U serving as a hydrophobic phase of a two-phase deep cosolvent;

(2) weighing choline chloride, citric acid and ethylene glycol according to a molar ratio of 2:2:1, uniformly mixing, heating at 80 ℃ until a transparent liquid is formed, namely a deep eutectic solvent DES-D, and mixing the DES-D and water according to a mass ratio of 7:3 to obtain a hydrophilic phase of the two-phase deep eutectic solvent;

(3) mixing the hydrophobic phase and the hydrophilic phase according to the volume ratio of 9:1, standing and layering to obtain the two-phase deep eutectic solvent.

Example 2

(1) Weighing menthol, hexanol and lauric acid according to a molar ratio of 1:0.8:0.2, uniformly mixing, and heating at 80 ℃ until a transparent liquid is formed, namely a deep cosolvent DES-U serving as a hydrophobic phase of a two-phase deep cosolvent;

(2) weighing choline chloride, citric acid and ethylene glycol according to a molar ratio of 2.5:1.5:1, uniformly mixing, heating at 80 ℃ until a transparent liquid is formed, namely a deep eutectic solvent DES-D, and mixing the DES-D and water according to a mass ratio of 4:6 to obtain a hydrophilic phase of the two-phase deep eutectic solvent;

(3) mixing the hydrophobic phase and the hydrophilic phase according to the volume ratio of 1:9, standing and layering to obtain the two-phase deep eutectic solvent.

Example 3

(1) Weighing menthol, hexanol and lauric acid according to a molar ratio of 1:1.6:0.4, uniformly mixing, and heating at 80 ℃ until a transparent liquid is formed, namely a deep cosolvent DES-U serving as a hydrophobic phase of a two-phase deep cosolvent;

(2) weighing choline chloride, citric acid and ethylene glycol according to a molar ratio of 3:2:1, uniformly mixing, heating at 80 ℃ until a transparent liquid is formed, namely a deep eutectic solvent DES-D, and mixing the DES-D and water according to a mass ratio of 4.5:5.5 to obtain a hydrophilic phase of the two-phase deep eutectic solvent;

(3) mixing the hydrophobic phase and the hydrophilic phase according to the volume ratio of 8:2, standing and layering to obtain the two-phase deep eutectic solvent.

Example 4

Example 4 was prepared in the same manner as example 3, except that the hydrophobic phase was mixed with the hydrophilic phase in a volume ratio of 7: 3.

Example 5

Example 5 was prepared in the same manner as example 3, except that the hydrophobic phase was mixed with the hydrophilic phase in a volume ratio of 6: 4.

Example 6

Example 6 was prepared in the same manner as example 3, except that the hydrophobic phase was mixed with the hydrophilic phase at a volume ratio of 5:5.

Example 7

(1) Accurately weighing 0.2g of ginkgo nut testa powder in a glass bottle with a plug, adding 2mL of the two-phase deep eutectic solvent system prepared in the example 1, and uniformly mixing;

(2) and (2) oscillating and extracting the mixture obtained in the step (1) at the constant temperature of 50 ℃ and 300rpm for 5min, centrifuging at 2000g for 7min after extraction is finished, and measuring the contents of ginkgolic acid, terpene lactone, flavone and procyanidine in the two phases by respectively taking a hydrophobic phase and a hydrophilic phase after centrifugation.

(3) After extraction, active ingredients in the hydrophobic phase and the hydrophilic phase of the two-phase system are respectively recovered by adopting a macroporous resin adsorption method.

Example 8

(1) Accurately weighing 0.2g of ginkgo nut testa powder in a glass bottle with a plug, adding 5mL of the two-phase deep eutectic solvent system prepared in the example 2, and uniformly mixing;

(2) and (2) oscillating and extracting the mixture obtained in the step (1) at the constant temperature of 25 ℃ and 100rpm for 55min, centrifuging at 6000g for 3min after extraction is finished, and measuring the contents of ginkgolic acid, terpene lactone, flavone and procyanidine in the two phases by respectively taking a hydrophobic phase and a hydrophilic phase after centrifugation.

(3) After extraction, an anti-solvent method is adopted to respectively recover active ingredients in the hydrophobic phase and the hydrophilic phase of the two-phase system.

Example 9

(1) Accurately weighing 0.2g of ginkgo nut testa powder in a glass bottle with a plug, adding 6mL of the two-phase deep eutectic solvent system prepared in the embodiment 3, and uniformly mixing;

(2) oscillating and extracting the mixture obtained in the step (1) at constant temperature of 30 ℃ and 220rpm for 40min, centrifuging at 3000g for 5min after extraction is finished, and measuring the contents of ginkgolic acid, terpene lactone, flavone and procyanidine in the two phases by respectively taking a hydrophobic phase and a hydrophilic phase after centrifugation.

Example 10

(1) Accurately weighing 0.2g of ginkgo nut testa powder in a glass bottle with a plug, adding 8mL of the two-phase deep eutectic solvent system prepared in the embodiment 4, and uniformly mixing;

(2) oscillating and extracting the mixture obtained in the step (1) at the constant temperature of 25 ℃ and 200rpm for 50min, centrifuging at 3000g for 5min after extraction is finished, and measuring the contents of ginkgolic acid, terpene lactone, flavone and procyanidine in the two phases by respectively taking a hydrophobic phase and a hydrophilic phase after centrifugation.

(3) After extraction, a liquid-liquid extraction method is adopted to respectively recover active ingredients in the hydrophobic phase and the hydrophilic phase of the two-phase system.

Example 11

(1) Accurately weighing 0.2g of ginkgo nut testa powder in a glass bottle with a plug, adding 9mL of the two-phase deep eutectic solvent system prepared in the example 5, and uniformly mixing;

(2) oscillating and extracting the mixture obtained in the step (1) at 35 ℃ and 200rpm for 40min, centrifuging at 3000g for 5min after extraction is finished, and measuring the contents of ginkgolic acid, terpene lactone, flavone and procyanidine in the two phases by respectively taking a hydrophobic phase and a hydrophilic phase after centrifugation.

Example 12

(1) Accurately weighing 0.2g of ginkgo nut testa powder in a glass bottle with a plug, adding 10mL of the two-phase deep eutectic solvent system prepared in the embodiment 6, and uniformly mixing;

(2) oscillating and extracting the mixture obtained in the step (1) at 35 ℃ and 220rpm for 40min, centrifuging at 3000g for 5min after extraction is finished, and measuring the contents of ginkgolic acid, terpene lactone, flavone and procyanidine in the two phases by respectively taking a hydrophobic phase and a hydrophilic phase after centrifugation.

Test example 1

Influence of two-phase deep eutectic solvent composed of different hydrophobic phases and hydrophilic phases on extraction and separation effects of the target substance.

The invention first prepares two hydrophobic DES and five hydrophilic DES, the preparation method refers to example 1, the Hydrogen Bond Donor (HBD), the Hydrogen Bond Acceptor (HBA), the molar ratio of HBA and HBD and the water content of the hydrophilic deep eutectic solvent are shown in Table 2. Wherein DES1-2 is hydrophobic phase, and DES3-7 is hydrophilic phase.

TABLE 2 hydrophobic DES and hydrophilic DES prepared

Two-phase deep eutectic solvent systems were prepared by combining the hydrophobic DES and hydrophilic DES in table 2 in pairs, with reference to the procedure of example 6 (figure 2). The effect of extraction and separation of active ingredients from the ginkgo nut testa was examined with reference to the method of example 9. Experimental results show that the two-phase deep eutectic solvent consisting of different hydrophobic deep eutectic solvents and hydrophilic deep eutectic solvents has different extraction effects on active ingredients of the ginkgo nut testa. As shown in FIG. 3, it can be seen that the ginkgolic acid content in the hydrophobic phase is higher and the content in the hydrophilic phase is almost none in the 10 two-phase deep eutectic solvents. In the longitudinal view of the five-group two-phase system consisting of the hydrophobic deep eutectic solvent DES2 and other hydrophilic deep eutectic solvents, although the total extraction amount of terpene lactones is higher than that of the two-phase system consisting of DES1, the distribution ratio of terpene lactones in the hydrophobic phase is higher, which is not in line with the purpose of enriching terpene lactones in the hydrophilic phase, and the extraction effect of ginkgolic acid is not as good as that of the two-phase system consisting of DES 1. The two-phase systems based on DES5 have lower extraction effect of the four active ingredients than the two-phase systems based on other hydrophilic deep eutectic solvents, and therefore, are not suitable for the application purpose of the invention. Flavone and procyanidin are not considered primarily because of their relatively low content. In conclusion, two-phase deep eutectic solvent 1-3(DES1: DES3 volume ratio 1:1) was selected for subsequent experiments.

Test example 2

Influence of the hydrophobic HBD component on the extraction and separation effects of active ingredients in the ginkgo nut testa.

The hydrogen bond donor components hexanol and lauric acid in the hydrophobic phase deep eutectic solvent are replaced by components with longer carbon chains, so that the hydrophobicity of the hydrophobic phase deep eutectic solvent is improved, and a better extraction and separation effect is expected to be achieved. The hexanol was first replaced with the other components and the hydrophobic deep eutectic solvents were prepared as shown in table 3.

TABLE 3 hydrophobic deep eutectic solvents for different HBDs

The method of reference example 6 was used to prepare two-phase deep eutectic solvent systems by combining different hydrophobic DES and hydrophilic DES3 of table 3, and the extraction and separation effect of active ingredients from the ginkgo nut testa was examined by the method of reference example 9. The results are shown in fig. 4, and show that there is no significant difference in the extraction effect of ginkgolic acid. With the increase of the length of the carbon chain, the total extraction amount of the ginkgolide shows a descending trend, the partition rates of the upper phase and the lower phase are not obviously different, the extraction effects of the flavone and the procyanidine are improved, and the partition rate of the lower phase is higher. According to the result analysis, the increase of the hydrophobicity of the carbon chain is not beneficial to the extraction of the lactone, the acid component accounts for a small proportion in the hydrophobic DES, and in addition, the lauric acid belongs to the acid with a longer carbon chain, so the optimization of the acid HBD is not performed. In summary, hexanol and lauric acid were selected as the optimal HBD for the hydrophobic deep eutectic solvent.

Test example 3

Influence of the hydrophobic eutectic solvent HBA/HBD molar ratio on the extraction and separation effect of the active ingredients of the ginkgo biloba sarcotesta.

The deep eutectic solvent with the same component composition has different component molar ratios, so that the property of the deep eutectic solvent is changed, and the extraction effect is influenced. Preparing a hydrophobic eutectic solvent according to the HBA/HBD molar ratio of 2.5:1, 2:1, 1.5:1, 1:1.5, 1:2 and 1:2.5, preparing a two-phase deep eutectic solvent system by referring to the method of example 6, and inspecting the extraction and separation effects of active ingredients in the ginkgo nut testa by referring to the method of example 9. The results are shown in fig. 5, and show that the total amount of ginkgolic acid extracted is higher when the molar ratio is 1:1.5, 1:2 and 1:2.5, and the total amount of ginkgolide extracted is higher when the molar ratio is 1:2, so that the molar ratio of HBA/HBD is 1:2, namely menthol: hexanol: lauric acid 1:1.6:0.4 is the best hydrophobic eutectic solvent and is numbered DES 14.

Test example 4

Influence of hydrophilic deep eutectic solvent alcohol component on extraction and separation effects of active ingredients in ginkgo nut testa.

The components with hydrophilicity larger than that of the malonic acid, namely the logP value smaller than that of the malonic acid, choline chloride and the malonic acid are selected to prepare the ternary DES, and the preparation conditions are shown in Table 4.

TABLE 4 hydrophilic deep eutectic solvents prepared from different alcohols HBD

Two-phase deep eutectic solvent systems were prepared using the best hydrophobic DES14 from experimental example 3 in combination with the different hydrophilic DES from table 4, according to the method of reference example 6, and the extraction and separation effect of the active ingredients from the ginkgo nut testa was examined according to the method of reference example 9. The results are shown in fig. 6, and indicate that the extraction effect of ginkgolic acid is firstly reduced and improved with the increase of the hydrophilicity of the alcohol, and the extraction effect of phenolic acid is the worst when the alcohol component is glycerol. The lactone of the hydrophilic phase is increased and then reduced along with the increase of the hydrophilicity of the alcohol component, and the extraction effect of the lactone is best when the alcohol component is ethylene glycol and glycerol. For flavones and procyanidins, as the hydrophilicity of the alcoholic component increases, the extraction effect of the hydrophilic phase decreases; the extraction effect of the hydrophobic phase has no obvious difference. In summary, ethylene glycol was chosen as the best alcohol component for the hydrophilic deep eutectic solvent.

Test example 5

Influence of hydrophilic deep eutectic solvent acid components on the extraction and separation effects of active ingredients in the ginkgo nut testa.

The acid with hydrophilicity larger than that of the malonic acid, namely the logP value smaller than that of the malonic acid, choline chloride and ethylene glycol are selected to prepare the deep eutectic solvent, and the preparation conditions are shown in Table 5.

TABLE 5 hydrophilic deep eutectic solvents prepared from different acids HBD

Two-phase deep eutectic solvent systems were prepared using the best hydrophobic DES14 from experimental example 3 in combination with the different hydrophilic DES from table 5, according to the method of example 6, and the extraction and separation of active ingredients from the ginkgo nut testa was examined according to the method of example 9. The results are shown in FIG. 7. The result shows that when the glycolic acid is used as an acid component, the extraction effect of the ginkgolic acid is low, and other components have no significant difference. When citric acid is used as an acid component, the extraction effect of the ginkgolide is higher, and the lower phase distribution rate is also higher. The deep eutectic solvent prepared from flavone and procyanidine and long-chain acids has low extraction effect. In summary, citric acid is selected as the optimal acid component of the hydrophilic deep eutectic solvent. Test example 6

Influence of the hydrophilic eutectic solvent HBA/HBD molar ratio on the extraction and separation effect of active ingredients in the ginkgo nut testa.

Hydrophilic deep eutectic solvents are prepared according to HBA/HBD molar ratios of 0:1, 0.25:1, 0.5:1, 0.75:1 and 1:1 (wherein HBD1/HBD2 is citric acid/ethylene glycol molar ratio is 1:1), the prepared hydrophilic deep eutectic solvent is combined with the optimal hydrophobic deep eutectic solvent DES14 obtained in test example 3 to prepare a two-phase deep eutectic solvent system according to the method of example 6, and the extraction and separation effects of active ingredients in the ginkgo nut testa are examined according to the method of example 9, and the results are shown in FIG. 8. The result shows that the extraction effect of the ginkgolic acid has no significant difference on the whole. With the increase of the HBA/HBD molar ratio, the extraction effect of the ginkgolide hydrophilic phase is gradually increased, and the hydrophobic phase has no significant difference. For flavone and procyanidine, the total extraction effect of the two is increased to a certain extent along with the increase of the HBA/HBD molar ratio. Therefore, an HBA/HBD molar ratio of 1:1 was selected as the optimum molar ratio.

Test example 7

Influence of the hydrophilic eutectic solvent HBD1/HBD2 molar ratio on the extraction and separation effects of active ingredients in the ginkgo nut testa.

On the basis that the mole ratio of the hydrophilic phase deep eutectic solvent HBA to HBD is 1:1, the mole ratio between two HBDs is changed to examine the influence of the HBD on the extraction and separation effects of active ingredients in the ginkgo nut testa. A two-phase deep eutectic solvent system is prepared by preparing a hydrophilic deep eutectic solvent from HBD1 (citric acid)/HBD 2 (ethylene glycol) in a molar ratio of 0:1, 0.5:1, 0.75:1, 1:1, 1.25:1, 1.5:1 and 2:1 and combining the prepared hydrophilic deep eutectic solvent with the optimal hydrophobic deep eutectic solvent DES14 obtained in the test example 3 by the method of the reference example 6, and the extraction and separation effects of active ingredients in the ginkgo nut testa are examined by the method of the reference example 9, and the results are shown in FIG. 9. The results show that the extraction effect of the ginkgo flavone and the procyanidin is not obviously changed. The total extraction effect of ginkgolide is gradually improved with the increase of the molar ratio of HBD1/HBD2, but the molar ratio of HBD1/HBD2 is selected to be the optimal molar ratio of 2:1 because the extraction effect of ginkgolic acid is lower when the molar ratio of HBD1/HBD2 is 1.5: 1. Finally, the optimal hydrophilic deep eutectic solvent component is determined to be choline chloride: citric acid: the molar ratio of ethylene glycol is 3:2: 1.

Test example 8

The influence of the water content of the hydrophilic phase on the extraction and separation effects of active ingredients in the ginkgo nut testa.

The extraction method of example 9 was employed, wherein the extraction conditions: the extraction time is 45 min; the influence of the water content of the hydrophilic phase in the two-phase deep eutectic solvent prepared in example 6 of the present invention on the extraction and separation effects of the active ingredients in the ginkgo nut testa is examined. The water content of the hydrophilic phase was selected to be 40%, 45%, 50%, 55%, 60%, 65%, 70% respectively, and the results are shown in fig. 10. The result shows that the change of the water content of the hydrophilic phase has no obvious influence on the extraction effect of the ginkgolic acid, the flavone and the procyanidine. With the increase of the water content of the hydrophilic phase, the total extraction amount of terpene lactones of ginkgo is increased and then reduced, and when the water content is 45%, terpene lactones have the highest extraction effect, and the distribution ratio of the hydrophobic phase and the hydrophilic phase is smaller, and most terpene lactones are enriched in the hydrophilic phase. Therefore, the optimum hydrophilic phase water content is 45%.

Test example 9

Influence of volume ratio of hydrophobic phase DES/hydrophilic phase DES on extraction and separation effect of active ingredients in Ginkgo biloba testa.

The method of example 9 was employed, wherein the extraction conditions: the extraction time is 45 min; the influence of the volume ratio of the hydrophobic phase DES/hydrophilic phase DES in the two-phase deep eutectic solvent prepared in example 6 of the invention on the extraction and separation effects of the active ingredients in the ginkgo nut testa is examined. The hydrophobic DES/hydrophilic DES volume ratios were selected to be 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, with the results shown in figure 11. According to the results, the total extraction effect of the ginkgolic acid and the ginkgolide is almost the same when the volume ratio is 5:5 and the volume ratio is 8:2, but the extraction effect of the ginkgetin and the procyanidine is obviously higher than the volume ratio of 5:5 when the volume ratio is 8: 2. Therefore, the hydrophobic phase DES/hydrophilic phase DES volume ratio of 8:2 was chosen as the optimal volume ratio.

Test example 10

The effect of the ginkgo nut testa powder and the feed liquid of the two-phase deep eutectic solvent on the extraction and separation effect of active ingredients in the ginkgo nut testa is influenced.

By adopting the extraction method of example 9, wherein the volume ratio of the hydrophobic phase to the hydrophilic phase is 8:2, the influence of the feed liquid ratio of the ginkgo biloba exocarp powder and the two-phase deep eutectic solvent prepared in example 3 of the invention on the extraction and separation effect of the active ingredients in the ginkgo biloba exocarp is examined, and the feed liquid ratio in the step (1) is selected to be 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40 and 1:50g/mL, respectively, and the result is shown in fig. 12. The results show that the extraction effects of the ginkgolic acid and the procyanidin are almost not different under different material-liquid ratios. With the increase of the dosage of the deep eutectic solvent, the extraction effect of the ginkgolide and the flavone is gradually improved, and the balance is achieved when the feed-liquid ratio is 1:30 g/mL. Therefore, the optimal feed-liquid ratio is selected to be 1:30 g/mL.

Test example 11

The extraction temperature has influence on the extraction and separation effect of active ingredients in the ginkgo nut testa.

The method of example 9, in which the volume ratio of the hydrophobic phase to the hydrophilic phase is 8:2, was adopted to examine the effect of the two-phase deep eutectic solvent of example 3 of the present invention on the extraction and separation effect of the active ingredients in the ginkgo nut testa at different temperatures, and the temperatures selected in step (2) were 25, 30, 35, 40, 45 and 50 (deg.c), respectively, and the results are shown in fig. 13. From the results analysis, it was found that the temperature does not greatly affect the extraction effect of 4 active ingredients, and 30 ℃ was selected as the optimum extraction temperature in consideration of the fact that a certain temperature is advantageous for reducing the viscosity of the deep eutectic solvent.

Test example 12

The extraction time influences the extraction and separation effects of active ingredients in the ginkgo nut testa.

Using the method of example 9, wherein the volume ratio of the hydrophobic phase to the hydrophilic phase is 8:2, the effect of the two-phase deep eutectic solvent prepared in example 3 of the present invention on the extraction and separation effect of the active ingredients in the ginkgo nut testa is examined under different shaking extraction times, and the time selected in step (2) is 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, and 55(min), respectively, and the results are shown in fig. 14. As can be seen from the figure, the extraction effect of the ginkgolic acid and the ginkgolide basically shows a gradually improved trend along with the increase of time, and the balance is basically achieved within 40 min. Therefore, the oscillation extraction time of 40min was selected as the optimum extraction time.

In conclusion, the two-phase deep eutectic solvent prepared by the invention is extracted at 220rpm for 40min at 30 ℃, and when the material-liquid ratio is 1:30g/mL, the extraction effect of the ginkgolic acid, the terpene lactone, the flavone and the procyanidine is higher. And the hydrophobic component ginkgolic acid is enriched in the upper hydrophobic DES, and the hydrophilic components terpene lactones, flavones and procyanidine are mainly enriched in the lower hydrophilic phase. Under the optimal conditions, the extraction rate of the ginkgolic acid in the hydrophobic phase is 162.43 +/-1.54 mg/g, the extraction rate of the total flavone in the hydrophilic phase is 14.79 +/-0.62 mg/g, the extraction rate of the terpene lactone is 246.96 +/-4.83 mg/g, and the extraction rate of the procyanidine is 3.28 +/-0.03 mg/g. Therefore, the two-phase deep eutectic solvent prepared by the invention can be used for simultaneously extracting and separating a plurality of active ingredients in the ginkgo nut testa.

Claims

1. A two-phase deep eutectic solvent for simultaneously separating and extracting multiple active ingredients from waste ginkgo nut testa is characterized by comprising a hydrophobic phase and a hydrophilic phase according to a volume ratio of 1: 9-9: 1; the hydrophobic phase is a hydrophobic deep eutectic solvent DES-U prepared from menthol, hexanol and lauric acid according to a molar ratio of 2.5:0.8: 0.2-1: 2: 0.5; the hydrophilic phase consists of DES-D and water, wherein the water content is 40-70% by mass, and the DES-D is a hydrophilic deep eutectic solvent prepared from choline chloride, citric acid and ethylene glycol in a molar ratio of 1:1: 1-3: 2: 1.

2. The two-phase deep eutectic solvent of claim 1, wherein the volume ratio of the hydrophobic phase to the hydrophilic phase is 2:8 to 8: 2.

3. A method for preparing the two-phase deep eutectic solvent according to claim 1, comprising the steps of:

(1) weighing menthol, hexanol and lauric acid according to a molar ratio, uniformly mixing, heating and stirring until a uniform transparent liquid is formed and is used as a hydrophobic phase DES-U of a two-phase deep eutectic solvent;

4. A method for simultaneously separating and extracting active ingredients in the testa of the waste ginkgo nut by using the two-phase deep eutectic solvent as defined in claim 1, which is characterized by comprising the following steps:

(2) mixing, heating for extraction, centrifuging, separating two phase system with upper phase as hydrophobic phase and lower phase as hydrophilic phase, and detecting the contents of ginkgolic acid, terpene lactone, flavone and procyanidin in the hydrophobic phase and hydrophilic phase respectively.

5. The method for simultaneously separating and extracting active ingredients from the rind of the waste ginkgo nuts by using the two-phase deep eutectic solvent as claimed in claim 4, wherein the mass-to-volume ratio of the dried powder of the rind of the waste ginkgo nuts and the two-phase deep eutectic solvent in the step (1) is preferably 1:10 to 1:50 g/mL.

6. The method for simultaneously separating and extracting active ingredients from the testa of the waste ginkgo nut by using the two-phase deep eutectic solvent as claimed in claim 4, wherein the heating extraction in the step (2) is vibration heating extraction or stirring heating extraction.

7. The method for simultaneously separating and extracting active ingredients from the testa of the waste ginkgo nut by using the two-phase deep eutectic solvent as claimed in claim 4, wherein the rotation speed of the oscillation or stirring in the step (2) is 100-300 rpm, the temperature is 25-50 ℃, and the time is 5-55 min.

8. The method for simultaneously separating and extracting active ingredients from the testa of the waste ginkgo nut by using the two-phase deep eutectic solvent as claimed in claim 4, wherein the centrifugation temperature in the step (2) is room temperature, the centrifugation force is 2000-6000 g, and the centrifugation time is 3-7 min.

9. The method for separating and extracting active ingredients from the testa of the ginkgo biloba according to claim 4, wherein the step (2) of recovering the ginkgolic acid from the hydrophobic phase and recovering the terpene lactones, flavones and procyanidins from the hydrophilic phase is carried out by one or more of liquid-liquid extraction, macroporous resin adsorption or anti-solvent extraction.

10. The method as claimed in claim 9, wherein the product recovered from the hydrophobic phase is ginkgolic acid and the product recovered from the hydrophilic phase is terpene lactones or flavonoids or procyanidins or a mixture of terpene lactones, flavonoids and procyanidins.