CN110917248B

CN110917248B - Method for extracting and separating active substances of nigella damascena seed oil

Info

Publication number: CN110917248B
Application number: CN201911376281.XA
Authority: CN
Inventors: 刘学; 伏琳玥; 杨成
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2019-12-27
Filing date: 2019-12-27
Publication date: 2021-10-26
Anticipated expiration: 2039-12-27
Also published as: CN110917248A

Abstract

The invention discloses a method for extracting and separating active substances of nigella sativa seed oil, which is to prepare a eutectic solvent by using choline chloride and levulinic acid and heat the eutectic solvent for later use; adding the eutectic solvent into the nigella sativa seed oil, and performing ultrasonic extraction; centrifuging and taking a DES phase. The method for extracting the nigella sativa seed oil by using the eutectic solvent has the advantages that other organic solvents do not have, so that the good extraction effect can be achieved, the biological activity is reserved, the advantages of low toxicity, difficulty in volatilization, greenness, easiness in degradation and good biological activity of the solvent can be realized, and the method has a wide application prospect.

Description

Method for extracting and separating active substances of nigella damascena seed oil

Technical Field

The invention belongs to the technical field of extraction and separation, and particularly relates to a method for extracting and separating active substances from nigella sativa oil.

Background

Nigella sativa is an annual herbaceous plant of the family ranunculaceae, the seeds of which are often referred to as nigella sativa seeds. The plant is native to southwest Asia, North Africa and south Europe, and it is also grown in many Mediterranean and middle east countries, such as Iran, Pakistan, India, Saudi Arabia, Syria and Turkey, and is also distributed in Xinjiang in China. The plants and seeds are widely used as an edible herb and flavoring. It is also used for medical purposes. Nigella sativa oil is widely used in the traditional treatment of diseases such as rheumatism, hemorrhoids, jaundice, dyspepsia, helminthiasis and skin disorders. Animal studies have shown that the oil has antihypertensive, antidiabetic, antibacterial, anticancer, diuretic, analgesic, anti-inflammatory and antioxidant effects. Therefore, the method is very important for the efficient and high-purity separation and extraction of the active substances of the nigella sativa oil.

The research on the extraction and separation method for extracting the nigella sativa seed oil from the nigella sativa seeds mainly comprises the optimization of the extraction process of the seed oil or volatile oil by the traditional squeezing method, the steam distillation method, the supercritical fluid extraction method and the like, but the extraction efficiency and the biological activity of the extract cannot be guaranteed.

Disclosure of Invention

This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.

The present invention has been made in view of the above-mentioned technical drawbacks.

Therefore, as one aspect of the invention, the invention overcomes the defects in the prior art and provides a method for extracting and separating the active substances of the nigella sativa oil.

In order to solve the technical problems, the invention provides the following technical scheme: a method for extracting and separating active substances from semen Sojae Atricolor oil comprises preparing eutectic solvent, and heating for use; adding the eutectic solvent into the nigella sativa seed oil, and performing ultrasonic extraction; centrifuging and taking a DES phase.

The preferable scheme of the method for extracting and separating the active substances of the nigella sativa oil is as follows: the eutectic solvent is obtained by compounding choline chloride with one or more of levulinic acid, glycerol, 1, 2-propylene glycol and malic acid.

The preferable scheme of the method for extracting and separating the active substances of the nigella sativa oil is as follows: the mass ratio of the nigella sativa oil to the eutectic solvent is 1 (1-12); the mol ratio of the choline chloride to the levulinic acid is 1 (1-12).

The preferable scheme of the method for extracting and separating the active substances of the nigella sativa oil is as follows: the heating is carried out to 60-90 ℃; the ultrasonic extraction time is 10-50 min, and the extraction temperature is 40-80 ℃; the extraction is performed by oscillating for 0.5-2 min every 5-15 min; and centrifuging for 5-20 min at 4000-5000 r/min.

The preferable scheme of the method for extracting and separating the active substances of the nigella sativa oil is as follows: the mass ratio of the nigella sativa oil to the eutectic solvent is 1 (4-8); the mol ratio of the choline chloride to the levulinic acid is 1 (4-8).

The preferable scheme of the method for extracting and separating the active substances of the nigella sativa oil is as follows: the heating is carried out to 70-80 ℃; the ultrasonic extraction time is 10-50 min, and the extraction temperature is 50-60 ℃; the extraction is performed by oscillating for 1min every 5 min; and centrifuging for 5-20 min at 4000-5000 r/min.

In one aspect of the present invention, the present invention overcomes the disadvantages of the prior art, and provides a black grass seed oil active substance obtained by a black grass seed oil active substance extraction and separation method, wherein: extracting the mixture by using an extracting agent for 10-50 min;

the extractant comprises one or more of petroleum ether and ethyl acetate.

The preferable scheme of the black grass seed oil active substance is as follows: including thymoquinone; after the active substances of the nigella sativa oil are diluted by 500 times, the antioxidant capacity is 105.57 mu mol/L Trolox equivalent concentration and 110.93 mu mol/L ferrous sulfate equivalent concentration.

In one aspect of the present invention, the present invention overcomes the disadvantages of the prior art, and provides a method for extracting and separating black grass seed oil active substances, which comprises: preparing a eutectic solvent from choline chloride and levulinic acid, and heating for later use; adding the eutectic solvent into the nigella sativa seed oil, and carrying out ultrasonic treatment or stirring.

The preferable scheme of the method for extracting and separating the active substances of the nigella sativa oil is as follows: the mass ratio of the black grass seed oil to the eutectic solvent is 1: 4; the mol ratio of the choline chloride to the levulinic acid is 1:4, and the heating is carried out to 60 ℃; the ultrasonic extraction time is 10min and the extraction temperature is 50 ℃ in the ultrasonic treatment.

The invention has the beneficial effects that:

the method for extracting the nigella sativa seed oil by using the eutectic solvent has a plurality of advantages which are not possessed by other organic solvents, can achieve a good extraction effect, retain the biological activity, realize the advantages of low toxicity, difficult volatilization, environmental friendliness, easy degradation and good biological activity of the solvent, and has a wide application prospect:

firstly, DES has high viscosity and is not beneficial to extraction, solvents such as water or ethanol are generally added to reduce the viscosity, and the invention adopts a heating mode to reduce the viscosity;

DDPH indicates that the antioxidant activity is insufficient, and antioxidant methods such as DPPH, ABTS, FRAP and the like are comprehensively considered to evaluate the antioxidant activity;

the DES solvent has certain oxidation resistance, and the invention optimizes the extraction process for extracting and separating the antioxidant active substances from the nigella sativa oil under the influence of removing the DES solvent.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:

FIG. 1 shows the DPPH radical scavenging rate of the eutectic solvent;

FIG. 2 is a data diagram of an extraction effect experiment of a eutectic solvent;

FIG. 3 is a data diagram of the eutectic solvent extraction effect experiment;

FIG. 4 is an experiment of the extraction effect of the eutectic solvent;

FIG. 5 shows different eutectic solvent extraction experiments;

FIG. 6 is a graph showing the effect of extraction time on extraction efficiency;

FIG. 7 is a Trolox ABTS anti-oxidation experiment standard curve;

FIG. 8 is a standard curve of ferrous sulfate FRAP anti-oxidation experiment;

FIG. 9 is a UV spectral scan;

FIG. 10 is a fragment of an ultraviolet spectral scan;

FIG. 11 is a thymoquinone UV spectrum scan;

FIG. 12 is a thin layer chromatogram of each part of DES extract after petroleum ether extraction, wherein 1 is thymoquinone reference substance; 2 is petroleum ether extract; 3 is DES residual phase.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.

Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.

Raw materials and reagents

Example 1: extraction of antioxidant active substance from semen nigellae oil

(1) Preparation of eutectic solvent

The choline chloride and the levulinic acid are placed in a centrifuge tube with a cover according to the molar ratio of 1:4, the centrifuge tube is vibrated, heated in a water bath to 70 ℃, and continuously shaken until uniform and stable colorless clear transparent liquid is formed. After preparation, the liquid can be kept for later use and is still liquid at room temperature.

(2) Eutectic solvent extraction

And heating the eutectic solvent to 50-60 ℃ in a water bath to reduce the viscosity of the eutectic solvent and improve the extraction efficiency. In order to save energy and arrange more reasonably, the prepared eutectic solvent can be cooled to 50-60 ℃ and directly applied to extraction. A1.5 mL centrifuge tube was charged with nigella sativa oil 0.5mL followed by eutectic solvent 0.5 mL. After sufficient shaking, sonication was carried out at 60 ℃ for 10min, followed by extraction in a water bath at 60 ℃ for 1 h. During the extraction, the mixture was shaken for half a minute every 15 min. The mixture was then centrifuged at 5000r/min for 10 min. The DES phase (lower layer) was removed and the volume was adjusted to 2.5mL with absolute ethanol.

(3) 80% methanol/ethanol extraction

A1.5 mL centrifuge tube was charged with nigella sativa oil 0.5mL followed by 80% methanol/ethanol 0.5 mL. After sufficient shaking, sonication was carried out at 60 ℃ for 10min, followed by extraction in a water bath at 60 ℃ for 1 h. During the extraction, the mixture was shaken for half a minute every 15 min. The mixture was then centrifuged at 5000r/min for 10 min. The 80% methanol/ethanol phase (upper layer) was removed and the volume was adjusted to 2.5mL with absolute ethanol.

Example 2: preparation of different eutectic solvents

Choline chloride is used as a hydrogen bond acceptor and is prepared into different eutectic solvents with different hydrogen bond donors according to a certain proportion, wherein most of the solvents cannot be used as solvents at an extraction temperature due to overhigh viscosity or overhigh freezing point, such as choline chloride-sorbitol, other substances are also selected as the hydrogen bond acceptor, such as betaine hydrochloride, but the eutectic solvents prepared from the betaine hydrochloride have overhigh freezing point and cannot be used as the extraction solvents, and therefore, the choline chloride is selected as the hydrogen bond acceptor and is prepared into different eutectic solvents for screening.

Finally, a eutectic solvent prepared from choline chloride-levulinic acid in a ratio of 1:4, a eutectic solvent prepared from choline chloride-1, 2-propanediol in a ratio of 1:4, a eutectic solvent prepared from choline chloride-malic acid in a ratio of 1:1 and a eutectic solvent prepared from choline chloride-glycerol in a ratio of 1:2 are selected as a comparison. The treatment method of the 4 eutectic solvents comprises the following steps:

adding choline chloride-levulinic acid into a centrifugal tube according to a molar ratio of 1:4, heating in a water bath to 80 ℃ after shaking, and continuously shaking until uniform and stable colorless clear transparent liquid is formed;

adding choline chloride and 1, 2-propylene glycol into a centrifugal tube according to a molar ratio of 1:4, heating in a water bath to 80 ℃ after shaking, and continuously shaking until uniform and stable colorless clear transparent liquid is formed;

adding choline chloride and glycerol (Gly) into a beaker according to the molar ratio of 1:2, heating in a water bath to 80 ℃, adding a stirrer, and stirring in the water bath at 80 ℃ for 6 hours to form uniform and stable colorless clear transparent liquid;

choline chloride and malic acid (Mali) are added into a beaker according to the molar ratio of 1:1, heated to 80 ℃ in a water bath, added with a stirrer and stirred in the water bath at 80 ℃ for 6 hours to form uniform and stable colorless clear transparent liquid.

The specific procedure for DPPH free radical scavenging experiments was as follows:

(1) 858 μ L of concentrated hydrochloric acid (HCl) was made up to 100mL with deionized water to make up 0.1mol/L HCl. (2) Tris (tris) buffer solution was prepared by weighing 1.514g tris (hydroxymethyl) aminomethane (tris) and dissolving in HCl solution to a constant volume of 250mL, and determining pH 7.4. (3) Accurately weighing 0.008g of DPPH, dissolving in 10mL of absolute ethanol solution, performing ultrasonic treatment for 30s, and fixing the volume to 100mL after complete dissolution. (4) Prior to the experiment, the DPPH ethanol solution was mixed with tris buffer solution 1:1 to make a 0.1mmol/L DPPH solution. (5) mu.L of the Sample and 300. mu.L of the DPPH solution were mixed, the mixed solution was a Sample group, 300. mu.L of the Sample and 300. mu.L of the DPPH solvent (tris solvent: absolute ethanol ═ 1:1) were mixed, the mixed solution was a Blank group, and 300. mu.L of the DPPH solution and the Sample solvent (absolute ethanol) were mixed, the mixed solution was a Control group. After mixing thoroughly, the mixture was reacted for 30min in the dark, and the absorbance at 517nm was measured.

The experimental results are shown in fig. 5 and the following table, and show that the choline chloride-levulinic acid still has the best extraction effect, which is about twice of the extraction effect of choline chloride-1, 2 propanediol, the extraction effect of choline chloride-1, 2 propanediol is similar to that of choline chloride-glycerol, and the extraction effect of choline chloride-malic acid is the worst. The poor extraction effect of choline chloride-malic acid is probably due to poor mass transfer effect caused by high viscosity at the extraction temperature, and the extraction effect is reduced under the same extraction conditions. Therefore, choline chloride-levulinic acid is selected as the eutectic solvent for extraction, and the best extraction effect is achieved.

DPPH free radical scavenging experiment

The DPPH radical scavenging experiment was performed using a eutectic solvent in which choline chloride and levulinic acid were arranged at a molar ratio of 1:4, and the experimental results are shown in fig. 1.

When DPPH free radical scavenging experiment is carried out, the extraction solvent is found to have DPPH free radical scavenging activity in the process of carrying out a large number of preliminary experiments, and then the measurement experiment of the deep eutectic solvent scavenging activity is carried out, and the experimental result shows that the deep eutectic solvent has DPPH free radical scavenging activity. The result shows that the eutectic solvent has certain antioxidant capacity and relatively high antioxidant activity, and the eutectic solvent still has a DPPH clearance rate of 12.43% after being diluted by 100 times, which means that a set of negative controls must be made to eliminate the influence of the antioxidant property of the eutectic solvent in the process of using the DPPH free radical clearance experiment as a detection method for the extraction effect of antioxidant active substances in an experiment for extracting the nigella sativa oil from the eutectic solvent, and the detection result has accuracy. In the negative control experiment, the concentration of the eutectic solvent is the same as that of the eutectic solvent in the sample, and the clearance rate of the negative control is calculated and subtracted from the original formula. Each change in the concentration of a eutectic solvent will also increase the number of negative control experiments.

The choline chloride-levulinic acid eutectic solvent extract and an 80% methanol extract were subjected to DPPH free radical scavenging experiments, and the antioxidant activities were compared, and the results are shown in fig. 2 and 3.

The results show that the antioxidant activity of the eutectic solvent extract is higher than that of the 80% methanol extract. The DPPH free radicals are completely eliminated when the eutectic solvent extract is diluted 50 times and 100 times, i.e. 3.66g/L and 1.83g/L of nigella sativa oil is extracted. Wherein the eutectic solvent extract is diluted about 350 times to achieve half inhibitory concentration when extracting nigella sativa oil 0.52g/L, and the methanol extract 80% is diluted about 80 times to achieve half inhibitory concentration when extracting nigella sativa oil 2.29 g/L.

The results of diluting the three extraction solvents by the same factor are shown in fig. 4, and the results show that the 80% methanol extraction effect is better than the 80% ethanol extraction effect, and the clearance rate under the same extraction conditions is twice of that of the 80% ethanol extraction solution. The extraction effect of the eutectic solvent is better than that of an extraction solution of 80% methanol and 80% ethanol, and the clearance rates are three times and six times of that of the extraction solution of 80% methanol and 80% ethanol respectively. The result shows that the extraction effect of the eutectic solvent is better than that of a solution of 80% methanol and 80% ethanol.

The experimental result also indicates that the nigella sativa oil has quite excellent antioxidant capacity, and the extract of the eutectic solvent can better extract antioxidant active substances in the nigella sativa oil, so that the extraction effect of the antioxidant capacity of the nigella sativa oil extracted by the eutectic solvent is far higher than that of 80% methanol and 80% ethanol, and the research on the optimal process of the extraction by the eutectic solvent has practical significance.

Example 3: eutectic solvent extraction process optimization experiment

Screening an optimal extraction process by adopting a single-factor experimental method, and inspecting different eutectic solvents (the molar ratio of choline chloride to levulinic acid is 1:4, the molar ratio of choline chloride to 1, 2-propylene glycol is 1:4, the molar ratio of choline chloride to glycerol is 1:2, and the molar ratio of choline chloride to malic acid is 1: 1); the ratio of the eutectic solvent (the mol ratio of choline chloride to levulinic acid is 1:2, 1:4, 1:6, 1:8 and 1: 10); ultrasonic extraction time (10, 20, 30, 40, 50 min); the extraction temperature (30, 40, 50, 60, 70 ℃); the effect of the ratio of the extracted oil to the eutectic solvent (1:1, 1:2, 1:4, 1:6, 1: 8; 4:1, 2:1, 1:2, 1:4) on the extraction efficiency of the eutectic solvent, with DPPH radical scavenging activity as an indicator.

When a certain factor is taken as a variable, the fixed values of other factors are eutectic solvent of choline chloride and levulinic acid in a molar ratio of 1:8, the extraction temperature is 50 ℃, the ultrasonic extraction time is 30min, and the ratio of the extraction oil to the eutectic solvent is 1: 1. During the ultrasonic extraction, the mixture was shaken every 5min for 1min, and after the ultrasonic extraction was completed, the mixture was centrifuged at 5000r/min for 10 min. The DES phase (lower layer) was removed and the volume was adjusted to 2.5mL with absolute ethanol.

Extraction effect of eutectic solvents with different proportions

The fixed extraction temperature is 50 ℃, the ultrasonic extraction time is 30min, the ratio of the extraction oil to the eutectic solvent is 1:1, the extraction effects of different eutectic solvents prepared by the choline chloride and the levulinic acid in the molar ratios of 1:2, 1:4, 1:6, 1:8 and 1:10 are considered, the extraction is carried out according to the extraction effects, the DPPH free radical scavenging experiment is carried out according to the method in 2.3.2, and the experiment results are shown in the following table:

the results show that the extraction effect is not obviously different (non-linearity) among the eutectic solvents with different molar ratios, wherein the extraction effect of the eutectic solvent with the molar ratio of 1:8 is the best. The higher the proportion of hydrogen bond donor (levulinic acid), the lower the viscosity of the prepared eutectic solvent. The difference in scavenging activity of the solvent itself is also not significant between the negative controls corresponding to different molar ratios of eutectic solvent. Therefore, the extraction with choline chloride-levulinic acid molar ratio of 1:8 as the eutectic solvent has the best extraction effect.

Eutectic solvent extraction effect at different extraction times

The mol ratio of the fixed choline chloride to the levulinic acid is 1:8, the extraction temperature is 50 ℃, the ratio of the extraction oil to the eutectic solvent is 1:1, the extraction effects of the extraction time of 10min, 20min, 30min, 40min and 50min are respectively considered, and the experimental result is shown in figure 6.

The experimental result shows that the extraction effect of the eutectic solvent is almost the same after the operation error is ignored, which indicates that the extraction time does not obviously influence the extraction effect, and the saturated extraction effect can be achieved in ten minutes. Therefore, a relatively short time should be selected as optimal, i.e. an extraction time of 10 min.

Eutectic solvent extraction effect at different extraction temperatures

Fixing eutectic solvent of choline chloride and levulinic acid in a molar ratio of 1:8, carrying out ultrasonic extraction for 30min, wherein the ratio of extraction oil to the eutectic solvent is 1:1, and examining the extraction effect at the extraction temperature of 30 ℃, 40 ℃, 50 ℃, 60 ℃ and 70 ℃, wherein the experimental results are shown in the following table:

the experimental result shows that the extraction temperature has certain influence on the extraction effect, but the influence is small, and the method is wireless. The saturated extraction temperature is reached at about 60 ℃. Thus 60 ℃ was chosen as the extraction temperature.

Extraction effect of different extraction oil and eutectic solvent ratios

(1) Adding the same volume of eutectic solvent into a centrifugal tube, observing and adding black grass seed oil in different proportions to perform an extraction experiment, fixing the eutectic solvent of choline chloride and levulinic acid in a molar ratio of 1:8, wherein the extraction temperature is 50 ℃, the ultrasonic extraction time is 30min, and the experimental results are shown in the following table:

the experimental result shows that the extraction effect of the eutectic solvent with the same volume is in a linear relation with the proportion of oil, the higher the proportion of the eutectic solvent is, the better the effect of extracting active substances is, but the extraction efficiency is relatively reduced.

(2) Adding the same volume of nigella sativa seed oil into a centrifugal tube, observing and adding different proportions of eutectic solvents for extraction experiments, fixing the eutectic solvent of choline chloride and levulinic acid in a molar ratio of 1:8, wherein the extraction temperature is 50 ℃, the ultrasonic extraction time is 30min, and the experimental results are shown in the following table.

The experimental result shows that when the ratio of the nigella damascena oil to the eutectic solvent is 1:4, the active ingredients in the nigella damascena oil are nearly completely extracted, and when the extraction ratio is 1:6, the maximum extraction effect is achieved. Therefore, when the volume of the eutectic solvent is six times of the volume of the black grass seed oil, the active ingredients in the black grass seed oil can be completely extracted.

By means of process optimization and considering the cost, the suggested industrial process for extracting the active substances of the nigella sativa oil is as follows: the mass ratio of the black grass seed oil to the eutectic solvent is 1: 4; the mol ratio of choline chloride to levulinic acid is 1:4, and the heating temperature is 60 ℃; the extraction time is 10min, the extraction temperature is 60 ℃, the ultrasonic or high-speed stirring (800-2000 rmp) is carried out, if the ultrasonic extraction is carried out, the ultrasonic extraction time is 10min, and the extraction temperature is 60 ℃, so that the cost and the process period can be saved to the maximum extent under the condition of obtaining high active substance extraction rate.

Example 4 antioxidant capacity test

ABTS antioxidant capacity test

(1) Preparation of ABTS (2, 2-biazonitrogen-bis (3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt) stock solution: 6.6mg of potassium persulfate was added to a volume of 10mL to prepare a 2.45mmol/L potassium persulfate solution. (2) 7.7mg of the ABTS concentrated solution was dissolved in 2mL of potassium persulfate solution and left for 12 to 16 hours as an ABTS stock solution. (3) Preparing an ABTS working solution: an appropriate amount of ABTS stock solution was taken and diluted with deionized water to an absorbance of 0.7. + -. 0.02(734 nm). (4) 10. mu.L of the sample solution and 190. mu.L of the ABTS working solution were placed in a 96-well plate in this order for 15min, and the absorbance was measured at 734 nm.

Inhibition rate calculation formula:

inhibition ratio%

FRAP antioxidant capacity test

(1) Preparing an acetate buffer solution: sodium acetate trihydrate 3.1g was mixed with glacial acetic acid 16mL, and 1000mL of water was added to prepare a 0.3mmol/L acetate buffer solution with PH 3.6. (2) Preparation of TPTZ solution: adding a certain amount of water into 31.2mg of TPTZ (2,4, 6-tripyridyltriazine), adding a certain amount of concentrated hydrochloric acid to dissolve the TPTZ, and carrying out constant volume treatment to 10 mL. (3) Preparing a ferric trichloride solution: 32.4mg of ferric chloride was made up to 10 mL. (4) Preparing TPTZ working solution: adding 25mL of acetate buffer solution, 2.5mL of TPTZ solution and 2.5mL of ferric trichloride solution into a beaker to prepare TPTZ working solution. (now ready for use) (5) preparation of ferrous sulfate standard solution: 1000 mu mol/L, 2000 mu mol/L, 3000 mu mol/L, 4000 mu mol/L and 5000 mu mol/L are respectively prepared to be used as ferrous sulfate standard solution. And (5) measuring the absorbance of the ferrous sulfate standard solution at 593nm, and drawing a ferrous sulfate standard curve.

The experimental steps are as follows: sequentially adding 10 μ L of sample and 190 μ L of FRAP solution into 96-well plate, shaking with microplate reader for 10s, holding at 37 deg.C for 10min, and measuring absorbance at 593 nm. The antioxidant capacity of the sample is expressed in terms of equivalent concentration of ferrous ions in μmol/L.

ABTS antioxidant capacity experimental results

Diluting the eutectic solvent extract under the optimal extraction process by 500 times, diluting the eutectic solvent by corresponding times to the corresponding concentration, performing ABTS free radical oxidation resistance experiment, and simultaneously making a group of Trolox standard curves as positive control. As a result, as shown in FIG. 7, the antioxidant capacity of the sample was measured to be 105.57. mu. mol/L Trolox equivalent concentration. The antioxidant capacity of the sample measured by the sample solvent (negative control) is 52.99 mu mol/L Trolox equivalent concentration, and the result still shows that the sample and the sample solvent both have ABTS free radical scavenging capacity, and the eutectic solvent extract has excellent antioxidant capacity.

Experimental results of FRAP antioxidant ability

Diluting the eutectic solvent extract by 500 times under the optimal extraction process, diluting the eutectic solvent by corresponding times to the corresponding concentration, performing FRAP oxidation resistance experiment, and simultaneously making a group of ferrous sulfate standard curves as positive control. The results are shown in FIG. 8, and the antioxidant capacity of the sample is 110.93 μmol/L ferrous sulfate equivalent concentration. The antioxidant capacity of the sample as measured by the sample solvent (negative control) was 38.47. mu. mol/L ferrous sulfate equivalent concentration. The results still show that both the sample and the sample solvent have FRAP antioxidant ability, and the eutectic solvent extract has excellent antioxidant ability.

Example 5

5mL of nigella sativa L.oil was extracted with an optimum extraction process (when the extraction effect was optimum when the ratio of the nigella sativa L.oil to the eutectic solvent was 1: 6) at 60 ℃ for 10min using an eutectic solvent prepared with choline chloride-levulinic acid in a molar ratio of 1: 8), with an extraction rate of 20.09%, and the extract was extracted with petroleum ether, with an extraction yield of 11.31%. In the development process, ethyl acetate is used as an extract, the extraction yield is similar to that of petroleum ether, but turbidity is easy to occur in the extraction process and is not beneficial to further separation, and the DPPH free radical scavenging capacity of the obtained ethyl acetate extract is lower than that of the petroleum ether extract, so that the ethyl acetate as the extract is not suitable for the invention.

The nigella sativa oil, the petroleum ether extract and the DES residue after petroleum ether extraction were diluted to a certain concentration, respectively, and subjected to spectral scanning at a wavelength ranging from 200nm to 900nm, with the results shown in fig. 9 and 10.

The experiment result shows that the petroleum ether extract has a maximum absorption peak at the 251nm wavelength, and the result is shown in figure 12 by combining literature data research and an ultraviolet spectrogram (figure 11) of a thymoquinone reference substance, preliminarily supposing that the petroleum ether extract contains thymoquinone, and then carrying out thin layer chromatography analysis by taking the thymoquinone as the reference substance. The same spot was present at the same location as the thymoquinone control, and therefore thymoquinone can be essentially inferred to be one of the major active ingredients in DES extracts.

It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.

Claims

1. A method for extracting and separating active substances of nigella damascena seed oil is characterized by comprising the following steps: comprises the steps of (a) preparing a mixture of a plurality of raw materials,

preparing a eutectic solvent, and heating for later use; the eutectic solvent is obtained by compounding choline chloride and levulinic acid, and the molar ratio of the choline chloride to the levulinic acid is 1 (4-8); the heating temperature is 60 ℃;

adding the eutectic solvent into the nigella sativa seed oil, and performing ultrasonic extraction; the mass ratio of the nigella sativa oil to the eutectic solvent is 1 (4-8); the ultrasonic extraction time is 10min, and the extraction temperature is 60 ℃; the extraction is performed by oscillating for 1min every 5 min;

centrifuging and taking a DES phase.

2. The method for extracting and separating active substances from nigella sativa oil as claimed in claim 1, wherein: and centrifuging for 5-20 min at 4000-5000 r/min.

3. An active substance of nigella sativa oil obtained by the extraction and separation method of the active substance of nigella sativa oil according to claim 1 or 2, wherein:

the nigella sativa oil active comprises thymoquinone;

after the active substances of the nigella sativa oil are diluted by 500 times, the antioxidant capacity is 105.57 mu mol/L Trolox equivalent concentration and 110.93 mu mol/L ferrous sulfate equivalent concentration.

4. An active substance of nigella sativa oil, which is characterized in that: the black grass seed oil active substance is obtained by the method for extracting and separating the black grass seed oil active substances according to claim 1 or 2, and then is extracted by an extracting agent for 10-50 min; the extractant is petroleum ether.