CN110526804B - Method for extracting hydroxytyrosol by using eutectic solvent - Google Patents

Abstract

The invention discloses a method for extracting hydroxytyrosol by using a eutectic solvent, which comprises the steps of preparing the eutectic solvent by different combinations of various food-grade raw materials, mixing olive oil or olive leaves with the eutectic solvent, and carrying out constant-temperature oscillation or ultrasonic extraction to obtain an extracting solution; centrifuging the extracting solution to obtain a centrifugal clear solution, namely a hydroxytyrosol extracting solution, and detecting the hydroxytyrosol extracting solution by HPLC to obtain the content of hydroxytyrosol. The method has the advantages of higher efficiency of extracting the hydroxytyrosol by adopting the eutectic solvent, environmental friendliness and easiness in operation, and can realize large-scale industrial production.

Description

Method for extracting hydroxytyrosol by using eutectic solvent

Technical Field

The invention relates to the technical field of compound extraction, in particular to a method for extracting natural active substance hydroxytyrosol from olive oil, olive leaves and olive oil extraction residues through a eutectic solvent.

Background

The olive oil is oil directly cold-pressed from fresh olive fruits, retains natural nutritional ingredients due to no heating and chemical treatment, is considered as the oil most suitable for human nutrition in the oil discovered so far, and is known as 'liquid gold', 'vegetable oil queen', 'Mediterranean nectar'. At present, researches consider that unsaturated fatty acids and antioxidant components in olive oil may have better biological activity, wherein hydroxytyrosol is one of the most main antioxidant components in olive oil and has better pharmacological value.

Hydroxytyrosol is an oleuropein hydrolysate, mainly exists in virgin olive oil, is a natural polyphenol compound, has various biological activities and pharmacological activities, and has antibacterial effect, antioxidant effect, blood sugar reducing activity, retina epithelial cell protection, cardiovascular disease prevention and other visible hydroxytyrosol which play important roles in medicine, pharmacology and other aspects.

At present, hydroxytyrosol is mainly extracted and separated from olive fruits, olive leaves, and residues and waste water generated in the olive oil preparation process, oleuropein is mostly extracted by adopting alcohol-water, a high-purity olive leaf extract is hydrolyzed by an acid-base chemical method, or processing waste water of olive oil is used as a raw material, and hydroxytyrosol is prepared and extracted by ion exchange resin, macroporous adsorption resin and silica gel chromatography, but the purity and the extraction efficiency of hydroxytyrosol obtained by an organic solvent are low, the method is complicated, and a large amount of organic solvent is used, so certain influence is caused on the environment.

Therefore, the problem to be solved by those skilled in the art is how to provide a simple, efficient and environmentally friendly method for extracting hydroxytyrosol.

Disclosure of Invention

In view of this, the invention provides a method for extracting hydroxytyrosol by using a eutectic solvent by using the principle of similar phase dissolution, which has the advantages of simple operation, high extraction rate, biodegradable solvent and recycling, and can realize large-scale industrial production.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for extracting hydroxytyrosol from a eutectic solvent is characterized by comprising the following steps:

(1) Mixing a hydrogen bond donor and a hydrogen bond acceptor, heating until a uniform and stable mixed solution system is formed, adding water, and uniformly stirring to obtain a eutectic solvent;

(2) Mixing olive oil or leaves with the eutectic solvent obtained in the step (1) in proportion, and performing constant-temperature oscillation or ultrasonic extraction to obtain an extracting solution;

(3) And (3) centrifuging the extracting solution obtained in the step (2), and obtaining a centrifugal clear liquid, namely the hydroxytyrosol extracting solution.

Preferably, in the above method for extracting hydroxytyrosol from a eutectic solvent, the hydrogen bond acceptor in step (1) includes, but is not limited to choline chloride, malic acid, lactic acid.

Preferably, in the above method for extracting hydroxytyrosol by using a eutectic solvent, the hydrogen bond donor in step (1) includes, but is not limited to, citric acid, glycerol, tartaric acid, propylene glycol, ethylene glycol, erythritol, xylitol, and sorbitol.

Preferably, in the above method for extracting hydroxytyrosol from a eutectic solvent, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor in step (1) is 1.

The beneficial effects of the above technical scheme are: hydroxytyrosol can be extracted when the molar ratio of hydrogen bond acceptor to hydrogen bond donor is 1.

Preferably, in the above method for extracting hydroxytyrosol from a eutectic solvent, the amount of water added in step (1) is 10-30%, more preferably 30% of the mass of the mixed liquid system.

The beneficial effects of the above technical scheme are: the hydroxytyrosol can be extracted within the range, the content of the hydroxytyrosol extracted is highest when the water addition amount is 30%, hydrogen bonds in the eutectic solvent can be influenced when the water content exceeds 40%, subsequent extraction is not facilitated, the hydrogen bonds of the mixed solution can be damaged when the water addition amount is too much, and therefore the optimal water addition amount is 30%.

Preferably, in the above method for extracting hydroxytyrosol by eutectic solvent, the heating in step (1) is carried out in an oil bath pan at 80-120 ℃.

The beneficial effects of the above technical scheme are: the temperature of the oil bath can exceed 100 ℃, which cannot be achieved by a common water bath; meanwhile, the oil bath kettle can be magnetically stirred, and the formation of the eutectic solvent can be accelerated more quickly after the rotor is added.

Preferably, in the above method for extracting hydroxytyrosol by using a eutectic solvent, the volume ratio of the mass of the olive oil to the eutectic solvent in the step (2) is 1-1.

Preferably, in one of the above methods for extracting hydroxytyrosol from eutectic solvent, the volume ratio of the mass of the olive leaves to the eutectic solvent in step (2) is 1.

Preferably, in the method for extracting hydroxytyrosol by using the eutectic solvent, the constant-temperature shaking extraction in the step (2) is carried out at the temperature of 25-40 ℃, the rotating speed is 100-500r/min, the extraction time is 0.5-2 h, the ultrasonic extraction frequency is 20-40kHz, and the ultrasonic extraction time is 5-15min.

Further preferably, the temperature of the constant-temperature shaking extraction is 35 ℃, the rotating speed is 280r/min, the extraction time is 2h, the frequency of the ultrasonic extraction is 40kHz at room temperature, and the ultrasonic extraction time is 15min.

The beneficial effects of the above technical scheme are: the structural property of the target object can be influenced by the overhigh temperature of the constant temperature oscillation extraction, and the extraction speed can be controlled by controlling the rotating speed.

Preferably, in the above method for extracting hydroxytyrosol from the eutectic solvent, the rotation speed of the centrifugation in the step (3) is 5000-12000r/min, and the centrifugation time is 5-15min;

further preferably, the rotation speed of the centrifugation in the step (3) is 8000r/min, and the centrifugation time is 15min.

According to the technical scheme, the invention discloses a method for extracting hydroxytyrosol by using a eutectic solvent, and compared with the prior art, the method has the following advantages: compared with the common organic solvent method, the method adopts food-grade raw materials to prepare various eutectic solvents in different combinations, does not generate impurities during preparation of the eutectic solvents, can be degraded by microorganisms, can be recycled after extraction, is more economical, green and safe, and is an environment-friendly and efficient green solvent;

in addition, the invention can extract olive oil, olive leaves and olive pomace, has strong universality and wide application range; the method is simple to operate, the extraction rate of the prepared product is high, the extraction rate of the method (the eutectic solvent is choline chloride-glycerol) is 38.97 percent higher than that of a common organic solvent (methanol), and the extraction rate of the olive leaves is 38.69 percent higher than that of the common organic solvent (methanol).

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a schematic diagram of a process for extracting hydroxytyrosol from a eutectic solvent according to the present invention;

FIG. 2 is a liquid chromatogram of hydroxytyrosol standard of the present invention;

FIG. 3 is a liquid chromatogram of a hydroxytyrosol sample obtained in example 2 of the present invention;

FIG. 4 is a diagram showing the effect of different eutectic solvents on hydroxytyrosol content according to the present invention;

FIG. 5 is a graph showing the effect of different eutectic solvents on hydroxytyrosol content according to the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.

Example 1

(1) Accurately weighing 1.396g of choline chloride and 0.921g of glycerol, putting the choline chloride and the glycerol into a closed small bottle, putting the small bottle into an oil bath kettle at 110 ℃, heating and mixing until a uniform and stable system is formed, adding 0.695g of water, and uniformly stirring to obtain a eutectic solvent for later use;

(2) Mixing 0.4g of olive oil with 0.8ml of the eutectic solvent prepared in the step (1), and extracting in a constant temperature shaking table for 2h, wherein the constant temperature shaking table has the parameters of 35 ℃ and 280r/min of rotation speed;

(3) Centrifuging the extracting solution in the step (2) at a speed of 8000r/min for 10min to obtain a centrifugal clear solution, namely a hydroxytyrosol extracting solution;

(4) HPLC detection of the hydroxytyrosol extract solution shows that the content of hydroxytyrosol is 9.9838 + -0.2494 mg/Kg.

Comparative example 1

The amount of hydroxytyrosol extracted by methanol under the same conditions as in example 1 was 7.1842. + -. 0.2546mg/Kg.

Wherein, the HPLC detection conditions are as follows:

c18 chromatographic column

Detection wavelength: 280nm;

gradient of elution of olive oil: 90% A and 10% B (0-5 min), 60% A and 40% B (5-30 min), 40% A and 60% B (30-45 min), 40% A and 60% B (45-50 min), 90% A and 10% B (50-60 min), wherein A is water and B is methanol;

gradient elution of olive leaves: 95% A and 5% B (0-5 min), 90% A and 10% B (5-10 min), 82% A and 18% B (10-20 min), 72% A and 28% B (20-50 min), 95% A and 5% B (50-65 min), 95% A and 5% B (55-60 min), wherein A is water, B is methanol;

flow rate: 1ml/min.

With reference to fig. 4-5, the present invention also examined the content of hydroxytyrosol extracted from olive oil by changing the hydrogen bond acceptor and the hydrogen bond donor of the eutectic solvent in example 1, see table 1 specifically:

TABLE 1 olive oil extraction data

Due to different hydrogen bond donors and hydrogen bond acceptors of the eutectic solvent, the viscosity, polarity and number of hydrogen bonds of different solvents are different, thereby influencing the extraction rate of hydroxytyrosol.

Example 2

(1) Accurately weighing 1.396g of choline chloride and 0.921g of glycerol, putting the choline chloride and the glycerol into a closed small bottle, putting the small bottle into an oil bath kettle at 110 ℃, heating and mixing until a uniform and stable system is formed, adding 0.695g of water, and uniformly stirring to obtain a eutectic solvent for later use;

(2) Mixing 0.1g of olive leaves with 1ml of the eutectic solvent prepared in the step (1), and putting the mixture into ultrasonic-assisted extraction for 15min, wherein the ultrasonic parameter is 40kHz;

(3) Centrifuging the extracting solution in the step (2) at 10000r/min, and centrifuging for 10min to obtain a centrifugal clear solution, namely the hydroxytyrosol extracting solution;

(4) And (3) detecting the extracting solution in the step (3) by HPLC (high performance liquid chromatography), wherein the detection conditions are the same as those in example 1, and the content of the hydroxytyrosol is 189.01 +/-2.57 mg/kg.

Comparative example 2

The amount of hydroxytyrosol extracted by 80% methanol under the same conditions as in example 2 was 136.28. + -. 45.42mg/Kg.

The invention also takes choline chloride-glycerol as an example for extracting hydroxytyrosol in olive leaves, researches the influence of the molar ratio between a hydrogen bond acceptor and a hydrogen bond donor, the water addition amount and the olive leaf ultrasonic extraction time in the step (2) on the content of the extracted hydroxytyrosol, and concretely refers to the following tables 2-4:

TABLE 2 influence of the molar ratio between hydrogen bond acceptor and hydrogen bond donor on the hydroxytyrosol content

Molar ratio of hydrogen bond acceptor to hydrogen bond donor	1:2	1:1	2:1
				Content (mg/kg)	72.79±10.43	189.01±2.57	Without formation of eutectic solvent

TABLE 3 Effect of water addition on hydroxytyrosol content

Amount of added water	10％	20％	30％
				Content (mg/kg)	7.48±4.60	70.41±0.69	189.01±2.57

TABLE 4 Effect of ultrasonic extraction time of Olive leaves on hydroxytyrosol content

In the present specification, the embodiments are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other. The device disclosed by the embodiment corresponds to the method disclosed by the embodiment, so that the description is simple, and the relevant points can be referred to the method part for description.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (1)

1. A method for extracting hydroxytyrosol by using a eutectic solvent is characterized by comprising the following steps of:

(1) Accurately weighing 1.396g of choline chloride and 0.921g of glycerol, putting the choline chloride and the glycerol into a closed small bottle, putting the small bottle into an oil bath kettle at 110 ℃, heating and mixing until a uniform and stable system is formed, adding 0.695g of water, and uniformly stirring to obtain a eutectic solvent for later use;

(2) Mixing 0.1g of olive leaves with 1ml of the eutectic solvent prepared in the step (1), and putting the mixture into ultrasonic-assisted extraction for 15min, wherein the ultrasonic parameter is 40kHz;

(3) Centrifuging the extracting solution in the step (2) at 10000r/min, and centrifuging for 10min to obtain a centrifugal clear solution, namely the hydroxytyrosol extracting solution;

(4) And (4) detecting the extracting solution in the step (3) by HPLC to obtain the content of hydroxytyrosol.

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