Method for extracting alligator oil by using hydrophobic deep-eutectic solvent

Abstract

The invention discloses a method for extracting alligator oil by using a hydrophobic deep-eutectic solvent. According to the method provided by the invention, the hydrophobic deep-eutectic solvent HDES is prepared by adopting a specific hydrogen bond acceptor and a hydrogen bond donor, the alligator oil obtained by mixing the alligator fat and the HDES, heating, carrying out ultrasonic extraction and centrifuging has a relatively high extraction rate, active substances in the alligator oil can be well reserved, and the contents of EPA and DHA are increased. Compared with a traditional extraction method, the method is simple, convenient and high in efficiency, more active ingredients are reserved, and better anti-inflammatory and anti-aging effects are achieved; the adopted HDES has the advantages of being non-toxic, simple to prepare, easy to degrade, green, environment-friendly, recoverable and the like, and in cooperation with ultrasonic-assisted extraction, release and dissolution of the alligator oil are well promoted, and the extraction rate of the alligator oil is increased. The method for extracting the alligator oil by adopting the HDES is simple to operate, mild in condition, low in pollution, non-toxic and high in extraction rate, and a new thought is provided for efficiently extracting the alligator oil.

Description

Technical Field

[0001] The invention belongs to the technical field of oil extraction and preparation, and more specifically, relates to a method for extracting crocodile oil using a hydrophobic low eutectic solvent. Background Art

[0002] In the development and utilization of biological resources, the efficient extraction of valuable substances has always been the goal of researchers' continuous exploration. Crocodile oil is an oil extracted from the crocodile body. It has good permeability and is easily accepted and absorbed by the human body. Crocodile oil has a strong antioxidant effect, can remove chloasma, and also has the functions of promoting blood circulation and removing blood stasis, activating cell regeneration, and accelerating metabolism. It plays a special role in regulating the metabolic activity of skin cells, can enhance the skin's immune function, efficient penetration, multi-layer nourishment, moisturizing and locking water, and comprehensive and balanced nutrition. It has the effects of moisturizing, wrinkle removal, repair, whitening, and anti-aging. As a substance with multiple potential medicinal and cosmetic values, the improvement of its extraction method is crucial. Traditional crocodile oil extraction methods often have the disadvantage of low extraction rate, which greatly limits the wide application of crocodile oil.

[0003] Traditional methods for extracting crocodile oil include dry boiling, organic solvent extraction, enzymatic hydrolysis, and supercritical fluid extraction. In existing studies, Lin Jiahao et al. used supercritical fluid extraction and dry boiling to extract crocodile oil. The results showed that the extraction rate of supercritical fluid extraction was low, at 32.6%. Although the extraction rate of dry boiling was higher than that of supercritical fluid extraction, it was only 60%, and the high-temperature heating conditions of dry boiling would destroy the structure of the oil. Qian Junqing et al. studied the process of extracting fish oil from yellow catfish viscera by autolysis and enzymatic hydrolysis. The results showed that the enzymatic hydrolysis method had a poor effect on the extraction rate of fish oil, with an extraction rate of only 56.42%. FOLCH et al. used organic solvents to extract crocodile oil. This method is limited to small-scale preparation in the laboratory and is not suitable for mass production. In addition, the refining of crocodile oil mostly uses organic solvents and repeated washing with ethanol or acid and alkali. This process will not only leave residual organic solvents in the crocodile oil, bringing more risks to the application of crocodile oil in the cosmetics and pharmaceutical fields, but also repeated ethanol, acid and alkali washing will cause the loss of core components in crocodile oil, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The current maximum extraction rate is about 79%, and the finished product yield is low. The content of EPA and DHA in crocodile oil can only reach about 0.1%-0.2%.

[0004] The traditional crocodile oil extraction method requires the use of a large amount of organic solvents, and the extraction rate of crocodile oil is not high. It is not only unfriendly to the environment, but also the subsequent solvent removal process is relatively complicated, and it is very easy to leave residual organic solvents, which brings certain risks to the use of crocodile oil; and repeated extraction with alcohol solvents will also cause the loss of active ingredients in crocodile oil, and it is impossible to fully extract the effective ingredients in crocodile fat to meet the high demand for crocodile oil today, which will also lead to waste of resources. At present, there is a lack of efficient methods for extracting crocodile oil, and there is no method for extracting crocodile oil using low eutectic solvents. Therefore, it is urgent to develop more green, efficient and safe methods for extracting crocodile oil, which can better retain its active ingredients, reduce loss, avoid the use of alcohol solvents, and improve the extraction rate of crocodile oil. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the existing crocodile oil extraction method, such as low extraction rate, loss of active ingredients and waste of resources, and to provide a method for extracting crocodile oil using a hydrophobic low eutectic solvent.

[0006] The invention aims to provide a method for extracting crocodile oil by using a hydrophobic deep eutectic solvent.

[0007] Another object of the present invention is to provide an application of the method for extracting crocodile oil using a hydrophobic deep eutectic solvent.

[0008] Another object of the present invention is to provide a crocodile oil.

[0009] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0010] The present invention provides a method for extracting crocodile oil using a hydrophobic deep eutectic solvent, comprising the following steps:

[0011] S1. A hydrophobic low eutectic solvent is prepared by using a hydrogen bond acceptor and a hydrogen bond donor, and its water content is controlled to be 30-60%; the hydrogen bond acceptor is selected from tetrabutylammonium bromide, menthol or tributyl phosphate; the hydrogen bond donor is selected from 10-hydroxydecanoic acid, decanoic acid or laurel;

[0012] S2. crocodile fat and a hydrophobic low eutectic solvent are mixed at a solid-liquid ratio of 1:(10-60) g / mL, heated, ultrasonically extracted, centrifuged, and the supernatant oily liquid is collected to obtain crocodile oil.

[0013] The present invention aims at the defect of low extraction rate of crocodile oil in traditional methods, and adopts hydrophobic low eutectic solvent (HDES) to extract crocodile oil by ultrasound-assisted extraction, which has a high extraction rate, can well retain unsaturated fatty acids in crocodile oil, and increase the content of EPA and DHA. Compared with the existing organic solvent extraction method, the extraction method using hydrophobic low eutectic solvent is simple and convenient, with high efficiency, and more active substance components are retained; at the same time, compared with the existing ionic liquid, HDES has the advantages of non-toxic, simple preparation, easy degradation, and recyclability, and cooperates with ultrasound-assisted extraction to promote the release and dissolution of crocodile oil, improve the extraction rate of crocodile oil, and the extraction steps are simple, and volatile organic reagents are not used, which is green and environmentally friendly. The present invention uses hydrophobic low eutectic solvent to extract crocodile oil, which brings new ideas for extracting crocodile oil, not only improves the extraction rate of crocodile oil, reduces resource waste, and further increases the content of active ingredients in crocodile oil. At the same time, the crocodile oil provided can be better applied to the field of cosmetics, and also provides a safer source for crocodile oil.

[0014] Preferably, the hydrogen bond acceptor is tetrabutylammonium bromide, and the hydrogen bond donor is 10-hydroxydecanoic acid or decanoic acid.

[0015] Preferably, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is (1-2):(1-3).

[0016] More preferably, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is (1-2):3.

[0017] Preferably, the water content of the hydrophobic deep eutectic solvent is 30-50%.

[0018] Preferably, the heating condition in S2 is 30-80°C.

[0019] Preferably, the ultrasonic conditions in S2 are power 80-100 W and time 20-90 min.

[0020] The invention provides application of a method for extracting crocodile oil by using a hydrophobic low eutectic solvent in improving the content of active components in the crocodile oil.

[0021] The invention provides crocodile oil, which is prepared by the method.

[0022] The present invention provides application of the crocodile oil in preparing anti-inflammatory and anti-aging products.

[0023] The invention also provides a product containing the crocodile oil.

[0024] Furthermore, the crocodile oil needs to be deacidified, degummed, dehydrated, decolorized and deodorized by conventional methods in the art.

[0025] The present invention has the following beneficial effects:

[0026] The present invention provides a method for extracting crocodile oil using a hydrophobic low eutectic solvent. The crocodile oil is extracted using a hydrophobic low eutectic solvent, which has a high extraction rate, can well retain unsaturated fatty acids in the crocodile oil, and can further increase the content of EPA and DHA. Compared with the existing organic solvent extraction method, the extraction method of crocodile oil using a hydrophobic low eutectic solvent is simple, convenient, efficient, and retains more active substance components; at the same time, compared with the existing ionic liquid, HDES has the advantages of being non-toxic, simple to prepare, easy to degrade, and recyclable. It is combined with ultrasound-assisted extraction to promote the release and dissolution of crocodile oil, improve the extraction rate of crocodile oil, and the extraction steps are simple. No volatile organic reagents are used, which is green and environmentally friendly. The present invention uses a hydrophobic low eutectic solvent to extract crocodile oil, which brings new ideas for extracting crocodile oil.

[0027] Compared with the existing extraction methods, the traditional crocodile oil extraction method may have a low extraction rate due to insufficient affinity between the extractant and the crocodile oil, or failure to fully penetrate into the crocodile tissue; the hydrophobic low eutectic solvent used in the present invention has good solubility and permeability, can fully contact with the crocodile oil, and effectively extract it from the tissue. At the same time, the ultrasonic auxiliary effect can further enhance the interaction between the solvent and the crocodile tissue, and destroy the cell structure of the crocodile tissue through the cavitation effect and mechanical vibration of the ultrasound, so that the crocodile oil is more easily released, thereby significantly improving the extraction rate; and the conditions of the traditional extraction method (light alkaline hydrolysis method) will have an adverse effect on the quality of the crocodile oil and destroy the active ingredients therein, while the hydrophobic low eutectic solvent used in the present invention can be extracted at normal pressure, avoiding the damage of high pressure to the oil, and the lower pressure conditions also make the extraction process safer and more reliable. At the same time, compared with common ionic liquids, HDES has the advantages of mild operating conditions, low pollution, non-toxicity, and high extraction rate. Combined with ultrasound-assisted extraction, it can promote the release and dissolution of crocodile oil and improve the extraction rate of crocodile oil. The application of the extracted crocodile oil in the subsequent preparation of cosmetics is simple, fast, green, safe, and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 GC-MS analysis of crocodile oil primary extract.

[0029] Figure 2 GC-MS analysis of refined crocodile oil. DETAILED DESCRIPTION

[0030] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0031] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0032] Example 1

[0033] (1) Preparation of a hydrophobic deep eutectic solvent (HDES): accurately weigh hydrogen bond acceptor (HBA) tetrabutylammonium bromide and hydrogen bond donor (HBD) 10-hydroxydecanoic acid in a molar ratio of 2:3 in a round-bottom flask, add 50% water, and stir in a constant temperature water bath at 80°C for 2 h until a uniform transparent liquid is formed to obtain HDES;

[0034] (2) Extraction of crocodile oil: crocodile fat and HDES were mixed at a solid-liquid ratio of 1:50 g / mL, treated with ultrasound at a temperature of 80°C for 60 min and an ultrasound power of 100 W. The mixture was centrifuged in a TDZ5WS centrifuge for 8 min to obtain layered HDES and crocodile oil.

[0035] Example 2

[0036] (1) Preparation of HDES: HBA tetrabutylammonium bromide and HBD decanoic acid in a molar ratio of 2:3 were accurately weighed and placed in a round-bottom flask, 50% water was added, and the mixture was stirred at 80°C in a constant temperature water bath for 2 h until a uniform transparent liquid was formed to obtain HDES;

[0037] (2) Extraction of crocodile oil: crocodile fat and HDES were mixed at a solid-liquid ratio of 1:50 g / mL, treated with ultrasound at 80 °C for 60 min with an ultrasound power of 100 W, and centrifuged in a TDZ5WS centrifuge for 8 min to obtain layered HDES and crocodile oil.

[0038] Example 3

[0039] (1) Preparation of HDES: HBA tetrabutylammonium bromide and HBD lauric acid in a molar ratio of 2:3 were accurately weighed and placed in a round-bottom flask, 50% water was added, and the mixture was stirred at 80°C in a constant temperature water bath for 2 h until a uniform transparent liquid was formed to obtain HDES;

[0040] (2) Extraction of crocodile oil: crocodile fat and HDES were mixed at a solid-liquid ratio of 1:30 g / mL, treated with ultrasound at a temperature of 80°C for 60 min and an ultrasound power of 100 W. The mixture was centrifuged in a TDZ5WS centrifuge for 8 min to obtain layered HDES and crocodile oil.

[0041] Example 4

[0042] (1) Preparation of HDES: HBA menthol and HBD 10-hydroxydecanoic acid in a molar ratio of 2:3 were accurately weighed and placed in a round-bottom flask, 40% water was added, and the mixture was stirred at 80°C in a constant temperature water bath for 2 h until a uniform transparent liquid was formed to obtain HDES;

[0043] (2) Extraction of crocodile oil: crocodile fat and HDES were mixed at a solid-liquid ratio of 1:30 g / mL, treated with ultrasound at a temperature of 80°C for 60 min and an ultrasound power of 100 W. The mixture was centrifuged in a TDZ5WS centrifuge for 8 min to obtain layered HDES and crocodile oil.

[0044] Example 5

[0045] (1) Preparation of HDES: HBA menthol and HBD lauric acid in a molar ratio of 2:3 were accurately weighed and placed in a round-bottom flask, 50% water was added, and the mixture was stirred at 80°C in a constant temperature water bath for 2 h until a uniform transparent liquid was formed to obtain HDES;

[0046] (2) Extraction of crocodile oil: crocodile fat and HDES were mixed at a solid-liquid ratio of 1:30 g / mL, and treated with ultrasound at a temperature of 50°C for 60 min with an ultrasonic power of 100 W. The mixture was centrifuged in a TDZ5WS centrifuge for 8 min to obtain layered HDES and crocodile oil, which were then separated to obtain crocodile oil.

[0047] Example 6

[0048] (1) Preparation of HDES: HBA tributyl phosphate and HBD decanoic acid in a molar ratio of 2:3 were accurately weighed and placed in a round-bottom flask, 50% water was added, and the mixture was stirred at 80°C in a constant temperature water bath for 2 h until a uniform transparent liquid was formed to obtain HDES;

[0049] (2) Extraction of crocodile oil: crocodile fat and HDES were mixed at a solid-liquid ratio of 1:30 g / mL, treated with ultrasound at a temperature of 80°C for 90 min and an ultrasonic power of 100 W. The mixture was centrifuged in a TDZ5WS centrifuge for 8 min to obtain layered HDES and crocodile oil.

[0050] Example 7

[0051] (1) Preparation of HDES: HBA tributyl phosphate and HBD lauric acid in a molar ratio of 2:3 were accurately weighed and placed in a round-bottom flask, 50% water was added, and the mixture was stirred at 80°C in a constant temperature water bath for 2 h until a uniform transparent liquid was formed to obtain HDES;

[0052] (2) Extraction of crocodile oil: crocodile fat and HDES were mixed at a solid-liquid ratio of 1:30 g / mL, treated with ultrasound at a temperature of 80°C for 60 min and an ultrasonic power of 90 W. The mixture was centrifuged in a TDZ5WS centrifuge for 8 min to obtain layered HDES and crocodile oil.

[0053] Comparative Example 1: Extraction of crocodile oil using a hydrophilic deep eutectic solvent

[0054] In this comparative example, a hydrophilic deep eutectic solvent is used to extract crocodile oil. The hydrophilic deep eutectic solvent is made of choline chloride and ethylene glycol. The specific method is as follows:

[0055] (1) Preparation of DES: Choline chloride and ethylene glycol in a molar ratio of 2:3 were accurately weighed and placed in a round-bottom flask, 50% of water was added, and the mixture was stirred at 80°C in a constant temperature water bath for 2 h until a uniform transparent liquid was formed to obtain DES;

[0056] (2) Extraction of crocodile oil: crocodile fat and DES were mixed at a solid-liquid ratio of 1:50 g / mL, treated with ultrasound at a temperature of 80°C for 60 min and an ultrasound power of 100 W. The mixture was centrifuged in a TDZ5WS centrifuge for 8 min to obtain layered DES and crocodile oil.

[0057] Comparative Example 2: Extraction of crocodile oil using a hydrophilic deep eutectic solvent

[0058] In this comparative example, a hydrophilic deep eutectic solvent is used to extract crocodile oil. The hydrophilic deep eutectic solvent is made of betaine and D-glucosamine hydrochloride. The specific method is as follows:

[0059] (1) Preparation of DES: Betaine and D-glucosamine hydrochloride in a molar ratio of 2:3 were accurately weighed and placed in a round-bottom flask, 50% water was added, and the mixture was stirred at 80° C. in a constant temperature water bath for 2 h until a uniform transparent liquid was formed to obtain DES;

[0060] (2) Extraction of crocodile oil: crocodile fat and DES were mixed at a solid-liquid ratio of 1:50 g / mL, treated with ultrasound at a temperature of 80°C for 60 min and an ultrasound power of 100 W. The mixture was centrifuged in a TDZ5WS centrifuge for 8 min to obtain layered DES and crocodile oil.

[0061] Comparative Example 3: Extraction of crocodile oil by conventional method (light alkali hydrolysis method)

[0062] Weigh 20 g of crushed crocodile fat into a conical flask, add distilled water at a solid-liquid ratio of 1:1 g / mL, adjust the pH value of the crocodile fat mixture to 9 with 1 mol / L NaOH solution, put it into a 60°C water bath and stir for 200 min. During this period, add 4% NaCl solution to hydrolyze for 15 min, and finally centrifuge at 5000 r / min for 15 min to separate the crocodile oil.

[0063] Test Example 1 Determination of crocodile oil extraction rate

[0064] 1. Effect of preparation conditions on extraction rate

[0065] On the basis of Example 1, a single factor variable was performed, and after adjusting the condition parameters in the crocodile oil extraction method, crocodile oil was prepared respectively. The specific condition parameters were set as shown in Table 1 below, and the extraction rate of each group of crocodile oil was calculated.

[0066] Crocodile oil extraction rate = crocodile oil mass / (crocodile fat mass × crude fat content in crocodile storage fat) × 100%

[0067] The measurement results are shown in Table 1 below, which shows that when crocodile oil is prepared using a hydrophobic low eutectic solvent, the molar ratio of HBA to HBD will affect the extraction rate. From the extraction effects of HDES1-3 groups, it can be seen that the extraction rate is highest when the molar ratio of HBA to HBD is 2:3; the solid-liquid ratio will affect the extraction rate. From the extraction effects of HDES1, 4, and 5 groups, it can be seen that the extraction rate is highest when the solid-liquid ratio is 1:50 g / mL; the DES water content will affect the extraction rate. From the extraction effects of HDES1, 6, and 7 groups, it can be seen that the extraction rate is highest when the DES water content is 50%; the extraction temperature will affect the extraction rate. From the extraction effects of HDES1, 8, and 9 groups, it can be seen that the extraction rate is highest when the extraction temperature is 80°C; the extraction rate will also be affected by changing the ultrasonic time and power. From the extraction effects of HDES1, 10-13 groups, it can be seen that the extraction rate is highest when the ultrasonic time and ultrasonic power are 60 min and 100 W.

[0068] Table 1 Effect of preparation conditions on extraction rate

[0069]

[0070]

[0071] 2. Extraction rate of crocodile oil in different embodiments and comparative examples

[0072] The crocodile oils prepared in the above Examples 1-7 and Comparative Examples 1-3 were further statistically analyzed. The extraction results of the crocodile oils in each group were calculated and shown in Table 2 below. The crocodile oil extraction rate in Example 1 was the highest. By comparing the extraction rates of seven hydrophobic deep eutectic solvents and two hydrophilic deep eutectic solvents, it was found that the effect of extracting crocodile oil by the hydrophobic deep eutectic solvent was significantly better than that by the hydrophilic deep eutectic solvent. The extraction effect of the crocodile oil by the light alkali hydrolysis method in Comparative Example 3 was also weaker than that by the hydrophobic deep eutectic solvent.

[0073] Table 2 Effect of different extraction methods on crocodile oil extraction rate

[0074] Case Crocodile oil extraction rate / % Example 1 85.85 Example 2 75.12 Example 3 70.87 Example 4 65.68 Example 5 70.87 Example 6 60.98 Example 7 60.78 Comparative Example 1 45.86 Comparative Example 2 55.90 Comparative Example 3 40.96 <![CDATA[Dry boiling method a > 60.00 <![CDATA[Supercritical fluid extraction method b > 32.60

[0075] Note: The extraction rates of dry boiling method and supercritical fluid extraction method in the table are from the existing technical records: a and b are the extraction rates obtained by Lin Jiahao et al. using supercritical CO2 fluid extraction method and dry boiling method to extract Siamese crocodile oil.

[0076] Test Example 2 Determination of EPA and DHA Contents of Crocodile Oil Active Substances

[0077] The EPA and DHA content in the extracted crocodile oil was determined by gas chromatography internal standard method. The specific method is:

[0078] 1. Preparation of reference solution

[0079] The docosahexaenoic acid methyl ester, eicosapentaenoic acid methyl ester and undecanoic acid methyl ester reference substances were prepared with n-heptane into reference substance stock solutions with mass concentrations of 0.695, 0.557 and 0.975 mg / mL, respectively, and stored in a refrigerator below -10°C; the undecanoic acid triglyceride reference substance was prepared with methanol into a reference substance stock solution with a mass concentration of 4.028 mg / mL and refrigerated in a refrigerator.

[0080] 2. Preparation of test solution (ester exchange method)

[0081] Weigh 60.0 mg of crocodile oil into a stoppered test tube, add 2.0 mL of 4.028 mg / mL undecanoic acid triglyceride internal standard solution; then add 4 mL of isooctane, slightly heat to dissolve the sample, then add 200 μL of potassium hydroxide methanol solution, cover with a glass stopper, shake vigorously for 30 seconds, and let stand until clear; finally, add 1 g of sodium bisulfate, shake vigorously to neutralize the potassium hydroxide; centrifuge, take the supernatant and transfer it to the upper machine bottle for testing.

[0082] 3. Selection and determination of chromatographic conditions

[0083] An Agilent HP-88 chromatographic column (100m×0.25mm×0.2μm) was used, with an inlet temperature of 270°C, a FID temperature of 280°C, a nitrogen carrier gas, a column flow rate of 1mL / min, and screening was performed with a programmed heating rate and different split ratios. The chromatographic conditions required to obtain the theoretical plate number n) were at least 2000 / m and a separation R of at least 1.25. 1.0μL of the reference substance series solution and the test solution were drawn and injected into a gas chromatograph for qualitative analysis using the retention time and quantitative analysis using the chromatographic peak area.

[0084] 4. Linear relationship investigation

[0085] The reference stock solutions of docosahexaenoic acid methyl ester, eicosapentaenoic acid methyl ester and undecanoic acid methyl ester were diluted with n-heptane to prepare a series of working solutions. The corresponding mass concentrations of eicosapentaenoic acid methyl ester and docosahexaenoic acid methyl ester were 5.57, 11.14, 27.85, 55.70, 278.50 μg / mL and 6.95, 13.90, 34.75, 69.50, 347.50 μg / mL, respectively. The mass concentration of undecanoic acid methyl ester was 97.45 μg / mL. Mix well and set aside. Accurately pipette 1.0 μL of each working solution and inject it into gas chromatograph for determination.

[0086] 5. Precision experiment

[0087] Accurately pipette reference solutions with mass concentrations of 55.70, 69.50 and 97.45 μg / mL of docosahexaenoic acid methyl ester, eicosapentaenoic acid methyl ester and undecanoic acid methyl ester, respectively, and inject 6 times in succession, 1.0 μL each time, record the peak area of ​​the target substance and the peak area of ​​the internal standard, and calculate the response factor.

[0088] 6. Repeatability and stability experiments

[0089] Take 6 samples from the same batch and process them according to the method in step 2 above, with an injection volume of 1.0 μL, and calculate the contents of EPA and DHA; take 6 samples from the same batch and inject them at 0, 2, 4, 8, 16, and 24 h, and calculate the contents of EPA and DHA.

[0090] EPA and DHA content calculation formula: X = (P × 1.1 × 20 × F FAΜE-FA ) / m

[0091] Wherein, X is the content of EPA and DHA; P is the content of fatty acid methyl ester; FFAΜE-FA is the conversion factor between fatty acid methyl esters and fatty acids, and the F FAME-FA =0.9557, F of DHA FAME-FA =0.9590, m is the mass of crocodile oil.

[0092] The results of the determination are shown in Table 3, showing that the EPA and DHA contents in the crocodile oil extracted by the low eutectic solvent are higher than those in the traditional supercritical fluid extraction method, the dry boiling method and the enzymolysis method of the prior art, wherein the EPA and DHA contents in the crocodile oil prepared by the methods of Embodiments 2-3 are higher. In Comparative Example 3, the contents of EPA and DHA are not low after the crocodile oil is extracted by the light alkali hydrolysis method, because the light alkali hydrolyzate can hydrolyze the protein in the crocodile fat protein, so that the grease bound to the protein is dissociated, and the pH value of the hydrolyzate of the light alkali hydrolysis method is 8-9, which is conducive to the extraction of crocodile oil, but the crocodile oil extracted at the same time may be hydrolyzed and saponified to destroy the active ingredient, and its extraction rate will decrease, and its unsaturated fatty acid effective ingredients will be reduced, which further brings the deficiency of product efficacy, showing instability in the cosmetic formula, and the wastewater is subjected to fine treatment after the light alkali hydrolysis method is extracted, and the cost is relatively large.

[0093] Table 3 Effect of different extraction methods on the content of EPA and DHA in crocodile oil

[0094] Case EPA content / % DHA content / % Example 1 2.07 2.04 Example 2 2.57 3.54 Example 3 2.25 3.25 Example 4 2.04 2.68 Example 5 2.24 2.32 Example 6 2.92 2.43 Example 7 2.85 2.02 Comparative Example 1 1.32 1.41 Comparative Example 2 1.53 1.67 Comparative Example 3 0.94 1.06 <![CDATA[Supercritical fluid extraction method a > 0.52 0.19 <![CDATA[Dry boiling method b > 0.52 0.23 <![CDATA[Enzymatic hydrolysis c1 > 0.59 1.83 <![CDATA[Enzymatic hydrolysis c2 > 0.61 1.94

[0095] Note: The data of superscript a, b, c in the table are from the existing technical records: a and b are the extraction effects obtained by Lin Jiahao et al. using supercritical CO2 fluid extraction and dry boiling to extract Siamese crocodile oil; c1 and c2 are the EPA and DHA contents obtained by Dong Helei et al. by optimizing the enzymatic extraction of crocodile oil using the response surface methodology.

[0096] Test Example 3: Reuse rate of low eutectic solvent

[0097] Since the low eutectic solvent has the advantages of being non-toxic, easily degradable, green and environmentally friendly, and recyclable, it can be recycled and reused after the crocodile oil is extracted, and multiple extractions are performed. The low eutectic solvent prepared after the crocodile oil extraction is recovered and extracted multiple times, and the EPA and DHA contents of the extracted crocodile oil are determined by the detection method in Test Example 2.

[0098] The results of EPA and DHA contents after repeated use of the same low eutectic solvent for different times are shown in Table 4, which shows that the same low eutectic solvent still has excellent effect on the EPA and DHA contents of the extracted crocodile oil after repeated use for 5 times. This is attributed to the chemical stability of HDES. It will not decompose after repeated use and can still effectively extract the oil in the crocodile tissue. In addition, the conditions for extracting crocodile oil with the low eutectic solvent are mild, and unsaturated fatty acids such as EPA and DHA are not easily destroyed. Therefore, after repeated use of the same low eutectic solvent for many times, the EPA and DHA contents remain stable or even increase.

[0099] Table 4 Extraction effect of repeated use of deep eutectic solvent

[0100]

[0101] Test Example 4 Determination of the anti-inflammatory and anti-aging effects of crocodile oil

[0102] The biological activity (in terms of anti-inflammatory and anti-aging) of the crocodile oil extracted by the present invention is detected, and the anti-inflammatory and anti-aging efficacy is detected using an animal model. The anti-inflammatory and anti-aging biological activity is detected by relevant experimental methods such as immunofluorescence (ROS) and Elisa (anti-inflammatory IL-6, TNF-α), so as to verify the active effect of the crocodile oil extracted by the present invention.

[0103] 1. Detection of IL-6 and TNF-α in mouse skin inflammation model

[0104] The same concentration and amount of crocodile oil (Examples 1-7, Comparative Examples 1-3) were applied to the ear epidermis of mice with skin inflammation for 7 days. After 7 days, the ear epidermal tissue of the mice was collected by dissection and the serum was collected. The TNF-α (tumor necrosis factor α) and IL-6 (interleukin-6) contents were detected according to the method in the instructions of the ELISA kit.

[0105] 2. Detection of ROS average fluorescence value

[0106] After the epidermal tissue of the inflammatory model mouse was rinsed with pure water, it was wiped dry and frozen in liquid nitrogen. After the frozen sections were restored to temperature, they were ensured to be dry and water-free. The peripheral area of ​​the tissue was marked with a histochemical pen and an autofluorescence inhibitor was added, and then the staining was performed. Then it was placed in a light-proof incubator and kept at 37°C for 30 minutes. DAPI was used to re-stain the cell nucleus, and DAPI dye was added. It was placed in an indoor environment without sunlight for 10 minutes to achieve the best effect. The anti-fluorescence quenching sealing agent completed the encapsulation process. After capturing the image with a fluorescence microscope, ImageJ was used for image analysis to obtain the total regional fluorescence intensity I and area A.

[0107] Average fluorescence value of ROS = I / A

[0108] The measurement results are shown in Table 5, which show that the IL-6 and TNF-α levels of Example 1 are low, both lower than those of the other example groups, indicating that there is no obvious inflammation or immune response in the mice. The average fluorescence value of ROS is 8.85, and compared with other examples and the control group, the level of active oxygen in the mouse endometrial tissue is low, and the level of oxidative stress is also low, which is an ideal state. High concentrations of ROS can cause lipid peroxidation, destroy cell membranes, damage proteins and DNA, and then induce cell apoptosis or necrosis.

[0109] Table 5 Anti-inflammatory and anti-aging effects of crocodile oil

[0110] IL-6 / μg / mL TNF-α / ng / L Average fluorescence value of ROS Example 1 2.89 18.95 8.85 Example 2 3.85 19.33 9.12 Example 3 2.45 20.95 9.25 Example 4 3.81 21.65 9.67 Example 5 3.85 22.93 9.72 Example 6 3.84 24.93 9.33 Example 7 3.83 20.90 9.28 Comparative Example 1 14.43 21.93 9.83 Comparative Example 2 13.86 20.95 9.83 Comparative Example 3 12.85 34.90 9.78

[0111] Test Example 5 GC-MS Analysis of Alligator Oil

[0112] The crocodile oil (primary extract) obtained by extraction in Example 1 is further refined and extracted, that is, the crocodile oil obtained after deacidification, degumming, dehydration, decolorization, deodorization and other processes on the basis of the primary extract is refined crocodile oil, and the active substance components and contents in the two crocodile oils are analyzed by GC-MS.

[0113] 1. Methylation

[0114] Accurately measure 0.3 mL of crocodile oil primary extract and refined crocodile oil in 10 mL centrifuge tubes, add 1 mL of KOH-methanol solution, heat in a 50°C water bath for 30 min, shake well, add 0.9 mL of n-hexane, shake thoroughly, and add water to the 10 mL mark. After sufficient stratification, take the supernatant in a 5 mL EP tube, store in a refrigerator filled with nitrogen, and wait for use.

[0115] 2. GC-MS analysis

[0116] After methyl esterification, the oil was mixed with chromatographic grade n-hexane at a ratio of 1:100 in a brown injection bottle and qualitatively and quantitatively detected and analyzed using a Shimadzu gas chromatograph-mass spectrometer (GCMS-QP2010 Plus).

[0117] The GC-MS conditions were as follows: injector temperature: 250°C; column oven temperature: 60°C-240°C; heating rate: 3°C / min; carrier gas: helium; flow rate: 36.5 cm / s (1.0 mL / min); split ratio: 20:1; ionization energy: 70 eV; interface temperature: 250°C; ion source temperature: 200°C; scanning range: 35-400 m / z.

[0118] Data processing: The retention index of each compound was calculated using normal alkane standards (C8-C40). The calculated retention index (RI) was compared with the retention index (Exp. RI) in the reference (Ref.) by comparing the mass spectra of each chromatographic peak in the NIST library to identify the compounds, and the peak area normalization method was used to quantify each component.

[0119] After GC-MS analysis, Figure 1 and Figure 2As shown, there are 18 kinds of total fatty acid types in crude crocodile oil, among which hexadecanoic acid, i.e. palmitic acid, has the highest content, reaching 31.74%, followed by octadecenoic acid (commonly known as oleic acid), with a content of 9.62%. There are 17 kinds of total fatty acid types in refined crocodile oil, among which fatty acids with 16 carbon atoms and 18 carbon atoms account for the main components. Among them, the fatty acid with 18 carbon atoms has the highest content, and the unsaturated fatty acids in the refined crocodile oil account for the main part, removing trans oleic acid and tetracosanoic acid, and adding erucic acid to the composition.

[0120] The specific components are shown in Table 6 and Table 7. Before and after crocodile oil is refined, the percentage of 9 fatty acids increases, and the percentage of 7 fatty acids decreases. Among them, trans oleic acid and tetradecanoic acid are removed after refining, and erucic acid is added to the refined crocodile oil. The main fatty acids contained in crude crocodile oil are C 16：0 , C 18：1 and C 18：2 The main fatty acids in refined crocodile oil are C 18 . The metal element content in crocodile oil was analyzed by atomic spectral absorption analyzer. Crocodile oil contains Cu, Fe, Zn, Se, Mn, Co, Ni, Li, As, K, Na, Ca, Mg and other elements, among which Cu, Fe, Zn, Se, Mn, Co and Ni have been confirmed as essential trace elements for the human body. It was found that the metal elements beneficial to the human body are as follows from high to low: Ca, Na, K, Fe, Mg and Zn, and the content of heavy metals harmful to the human body is much lower than the safe dose of the national food inspection standard.

[0121] Comparison of the unsaturated fatty acid content in crudely extracted crocodile oil and refined crocodile oil shows that the EPA and DHA contents of crocodile oil extracted with a hydrophobic low eutectic solvent are significantly improved after refining, while crudely extracted crocodile oil has many impurities, including phospholipids, pigments, proteins, etc., with a slight fishy smell and peculiar smell, and the surface is doped with unremoved low eutectic solvents. Therefore, the necessity of crocodile oil refining is highlighted. Crudely extracted crocodile oil cannot be used directly, and refined crocodile oil must be obtained through deacidification, decolorization, deodorization and other steps for further application. If the crude extract is directly applied, it will cause harm to the human body, and the refined crocodile oil should be obtained through decolorization, surface cleaning, deodorization and other steps. It can play a better role in the field of cosmetics, achieving better anti-inflammatory, anti-aging, anti-oxidation, soothing the skin, and improving the skin barrier.

[0122] Table 6 Retention index (RI) and relative content (%) of each compound in crudely extracted crocodile oil

[0123]

[0124]

[0125] Table 7 Retention index (RI) and relative content (%) of each compound in refined crocodile oil

[0126]

[0127] In summary, the present invention provides a method for extracting crocodile oil with a hydrophobic low eutectic solvent, which uses a hydrophobic low eutectic solvent to extract crocodile oil, has a high extraction rate, can well retain the unsaturated fatty acids in crocodile oil, and can further increase the EPA and DHA content in crocodile oil. Compared with the existing organic solvent extraction method, the extraction of crocodile oil with a hydrophobic low eutectic solvent is simple and convenient, with high efficiency, and retains more active substance components; at the same time, compared with the existing ionic liquids, HDES has the advantages of non-toxicity, simple preparation, easy degradation, and recyclability. It cooperates with ultrasound-assisted extraction to promote the release and dissolution of crocodile oil, improve the extraction rate of crocodile oil, and the extraction steps are simple. No volatile organic reagents are used, which is green and environmentally friendly. The present invention uses a hydrophobic low eutectic solvent to extract crocodile oil, which brings new ideas for extracting crocodile oil.

[0128] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for extracting crocodile oil using a hydrophobic deep eutectic solvent, characterized in that: The following steps are involved: S1. A hydrophobic low eutectic solvent is prepared by using a hydrogen bond acceptor and a hydrogen bond donor, and its water content is controlled to be 30-60%; the hydrogen bond acceptor is selected from tetrabutylammonium bromide, menthol or tributyl phosphate; the hydrogen bond donor is selected from 10-hydroxydecanoic acid, capric acid or lauric acid; S2. crocodile fat and a hydrophobic low eutectic solvent are mixed at a solid-liquid ratio of 1:(10-60) g / mL, heated, ultrasonically extracted, centrifuged, and the supernatant oily liquid is collected to obtain crocodile oil.

2. The method according to claim 1, characterized in that: The hydrogen bond acceptor is tetrabutylammonium bromide, and the hydrogen bond donor is 10-hydroxydecanoic acid or decanoic acid.

3. The method according to claim 1 or 2, characterized in that: The molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is (1-2):(1-3).

4. The method according to claim 3, characterized in that: The heating condition in S2 is 30-80°C.

5. The method according to claim 3, characterized in that: The ultrasonic conditions in S2 are power 80-100 W and time 20-90 min.

6. Application of the method according to claims 1 to 5 in increasing the content of eicosapentaenoic acid and docosahexaenoic acid, active ingredients in crocodile oil.

7. A crocodile oil, characterized in that The invention is prepared by the method according to any one of claims 1 to 5.

8. Use of the crocodile oil according to claim 7 in the preparation of anti-inflammatory and anti-aging products.

9. A product, characterized in that Containing the crocodile oil as claimed in claim 8.

10. The product according to claim 9, characterized in that: The crocodile oil needs to be deacidified, degummed, dehydrated, decolored and deodorized.

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