NL2028630A

NL2028630A - Method for improving purity of batyl alcohol in shark liver oil

Info

Publication number: NL2028630A
Application number: NL2028630A
Authority: NL
Inventors: Shen Qing; Wang Jiabin; Dai Zhiyuan; Song Gongshuai; Zheng Ping'an; Zhu Beiwei
Original assignee: Univ Zhejiang Gongshang
Priority date: 2020-07-06
Filing date: 2021-07-06
Publication date: 2022-02-22
Also published as: NL2028630B1; CN111848368A

Abstract

Disclosed is a method for improving purity of batyl alcohol in shark liver oil. The method includes the following steps: adding a potassium hydroxide-ethanol solution with a concentration of 1 mol/L into shark liver oil and conducting a reaction at a temperature of (70i10)°C for (50i10) min; post-treating an obtained reaction solution to obtain an unsaponifiable matter; selecting a neutral alumina chromatographic column; and dissolving the unsaponifiable matter with dichloromethane, and loading the dissolved unsaponifiable matter to enable it to pass through the column, wherein 2.5-3.5 g of the unsaponifiable matter is loaded, an eluent is a mixed solution of dichloromethane and methanol in a volume ratio of 95:05 to 8.5: 1.5, an elution flow rate is 1.5-2.5 mL/min, and an obtained eluate contains enriched batyl alcohol. The neutral alumina chromatographic column is utilized, such that the purity of the batyl alcohol in the shark liver oil is obviously improved.

Description

-1-

METHOD FOR IMPROVING PURITY OF BATYL ALCOHOL IN SHARK LIVER OIL TECHNICAL FIELD

[01] The present disclosure belongs to the technical field of oil processing and in particular, relates to a method for obviously improving purity of batyl alcohol in shark liver oil.

BACKGROUND ART

[02] Shark liver oil is an important source of bioactive substances such as alkylglycerols, squalene and the like, and has a good physiological health-care function. The alkylglycerols are a kind of ether lipid compounds and mainly classified into batyl alcohol (1-0-octadecylglycerol), selachyl alcohol (1-O-octadec-9-enyl glycerol) and chimyl alcohol (1-O-hexadecyl-rac-glycerol) according to the saturation degree and length of an alkyl chain connected to a glycerol backbone. The batyl alcohol is an important component of alkylglycerols, has effects on resisting hematopoietic system inhibition caused by cytotoxic drugs or benzene poisoning, resisting and preventing cancers, promoting leucocyte proliferation, resisting radioactive rays and the like, and is an important immunostimulating factor. However, an enrichment technology of the batyl alcohol is relatively immature and different raw materials have different contents of batyl alcohol. Currently, most of batyl alcohol products sold on the market are chemically synthesized except for the batyl alcohol- enriched shark liver oil, while the chemically synthesized batyl alcohol has low biological activity and efficacy.

[03] At present, the batyl alcohol is generally separated and purified by means of: supercritical fluid extraction, molecular distillation, ester exchange, thin layer chromatography (TLC), etc. The supercritical fluid extraction and molecular distillation have a high equipment investment, relatively complex operations and a high requirement on operators. However, the batyl alcohol prepared by the traditional ester exchange and TLC fails to have the purity that can meet an actual industrial production requirement.

SUMMARY

-2-

[04] The present disclosure aims to solve the above technical problems by providing a method for obviously improving purity of batyl alcohol in shark liver oil.

[05] To solve the above-mentioned technical problems, the present disclosure provides a method for improving purity of batyl alcohol in shark liver oil, which includes the following steps:

[06] 1) extraction of unsaponifiable matter

[07] adding | mol/L of a potassium hydroxide-ethanol solution to shark liver oil and reacting at (70+10)°C for (50+10) min,

[08] where a ratio of the shark liver oil to the potassium hydroxide-ethanol solution islg:{(2.5-3.5) ml; and

[09] post-treating obtained reaction liquid to obtain the unsaponifiable matter; and

[10] 2) column chromatography

[11] selecting a neutral alumina chromatographic column; and

[12] dissolving the unsaponifiable matter in dichloromethane and loading an obtained sample and passing it through the column, where 2.5-3.5 g of the unsaponifiable matter is loaded, an eluent is a mixed solution of dichloromethane and methanol at a volume ratio of 9.5:0.5 to 8.5:1.5, an elution flow rate is 1.5-2.5 mL/min,

[13] and an obtained eluate contains enriched batyl alcohol.

[14] As an improvement of the method for improving purity of batyl alcohol in shark liver oil of the present disclosure, the post-treating of step 1) may specifically include:

[15] adding water into the obtained reaction liquid, conducting an extraction with n- hexane and washing (with a 10% ethanol-water solution) and rotary-evaporating an obtained extract to obtain the unsaponifiable matter.

[16] As an further improvement of the method for improving purity of batyl alcohol in shark liver oil of the present disclosure, a preparation method of the chromatographic column of step 2) may specifically include:

[17] mixing neutral alumina with a particle size of 100-200 meshes and distilled water at a ratio of 1 g:(5+1) ml, adjusting a pH to 1.5-2.5 with hydrochloric acid, standing for 8-12 min and washing the treated alumina (with distilled water until an eluate is neutral ); and

-3-

[18] placing the washed alumina at 105°C for activation for (24+1) h, packing the column by a dry method, eluting with dichloromethane to remove impurities in the column and conducting an equilibration for 2 h.

[19] As an further improvement of the method for improving purity of batyl alcohol in shark liver oil of the present disclosure, in step 2):

[20] 3 g of the unsaponifiable matter is loaded, the eluent is a mixed solution of the dichloromethane and the methanol at a volume ratio of 9:1, the elution flow rate is 2 mL/min, every 40 mL is taken as one collection unit and 20 collection units are obtained; and preferably the 7th collection unit is selected.

[21] In the present disclosure, a chromatographic column has a loading capacity about 0.012 g/g.

[22] In the present disclosure, thin layer chromatography (TLC) rapid qualitative analysis and a gas chromatographic quantitative analysis are used together for tracking. The details are as follows:

[23] A. TLC analysis:

[24] after drying and activating a silica gel plate at 103°C for 1 h and placing itin a desiccator for cooling to a room temperature for later use; respectively using pure batyl alcohol and pure squalene as samples; dissolving 100 mg of the samples in 1 mL of dichloromethane, spotting the samples on the TLC silica gel plate by using a capillary, using a mixed solution of n-hexane:diethyl ether:acetic acid (85:15:1, v/v/v) as a developing solution, and developing color with iodine vapor as a standard; and

[25] analyzing an obtained eluate in the present disclosure by the TLC according to the above method to obtain strips of batyl alcohol and squalene respectively; and therefore, it can be proved that the neutral alumina chromatographic column has an obvious effect on separating and purifying the batyl alcohol and can effectively separate the batyl alcohol and squalene in shark liver oil;

[26] B. GC analysis of batyl alcohol:

[27] analyzing the relative content of the batyl alcohol by using a peak area normalization method;

[28] dissolving and diluting a batyl alcohol standard substance with n-hexane and conducting a detection according to the following gas chromatographic conditions: HP-5 capillary chromatographic column (30 m=0.32 mm, 0.25 um); heating program: keeping an initial temperature at 200°C for 5 min and heating to 280°C at 5°C/min

-4- and keeping it for 20 min; and a temperature of an injection port at 250°C, a splitless injection with an injection volume of 1 pL and a carrier gas flow rate at 1 mL/min;

[29] scraping the corresponding strip of the batyl alcohol on the TLC silica gel plate, adding 500 pL of a silanization reagent (BSTFA:TMCS=99:1), conducting water bath at 65°C for 30 min, cooling and purge with nitrogen, removing excess solvent, adding 2 mL of n-hexane for redissolving, and conducting a filtration and a detection according to the above-mentioned gas chromatographic conditions;

[30] detecting the obtained eluate in the present disclosure according to the above- mentioned gas chromatographic conditions; and [BI] respectively obtaining the content of the batyl alcohol in the strip of the batyl alcohol and in the eluate by using the peak area normalization method,

[32] where BSTFA is N,O-bistrimethylsilyl trifluoroacetamide and TMCS is trimethylchlorosilane; and

[33] C. GC analysis of squalene:

[34] dissolving a squalene standard substance with n-hexane and conducting a detection according to the following gas chromatographic conditions Test: HP-5 capillary chromatographic column (30 m=0.32 mm, 0.25 um); heating program: an initial temperature at 160°C , heating to 220°C at 15°C /min and keep it for 2 min; heating to 280°C at 5°C /min and keeping it for 20 min; heating to 300°C at 5°C/min and keeping it for 2 min; a temperature of an injection port at 250°C and a split ratio at 1:10; a temperature of an injection port at 300°C, an injection amount at 1 uL and a flow rate of carrier at 1 ml/min; repeating testing each group of the sample for 3 times

[35] to obtain a corresponding standard curve; and

[36] detecting the unsaponifiable matter (pre-enriched squalene) and the eluate (post-enriched squalene) according to the above-mentioned gas chromatographic conditions and substituting into the standard curve to obtain the content of the pre- and post-enriched squalene in shark liver oil.

[37] In the present disclosure, the following experimental groups are designed for column chromatography and each group of samples is tested repeatedly 3 times.

Results of purity of the batyl alcohol in the eluate are shown in Table 1.

[38] Table 1 Experimental design and results No. | A: Fluent |B: Loading C: Elution flow rate Purity of batyl

-5- Cone we Ee]

[39] Through an analysis of the above experimental results, when an eluent ratio (dichloromethane:anhydrous methanol) is 9:1, the loading amount is 3 g, an elution flow rate is 2 mL/min, and the purity of the batyl alcohol is the highest; therefore, it is a preferred solution.

[40] The present disclosure has the following beneficial effects:

[41] 1. A neutral alumina chromatographic column is utilized in the present disclosure and can obviously improve purity of batyl alcohol in shark liver oil. Compared with a traditional supercritical fluid extraction, a molecular distillation, an ester exchange, TLC, etc, the method of the present disclosure is simpler and more convenient to operate, has a low equipment requirement, can significantly improve the purity and is expected to be used in industrial production.

[42] 2. The present disclosure further determines content of squalene in shark liver oil after a neutral alumina column chromatography to study an effect of the neutral alumina column chromatography on the squalene. The squalene is a highly unsaturated straight-chain triterpenoid formed by connecting 6 isoprenes and light yellow or colorless oily liquid at a normal temperature. The squalene has biological activities of improving anoxia tolerance, inhibiting microorganism growth, resisting microorganisms, relieving inflammation, and regulating cholesterol metabolism.

Therefore, the squalene is widely used in food, medicine and cosmetics industries.

-6- Shark liver oil mainly contains the squalene, alkyl glycerols and a small amount of EPA, DHA, vitamin A and vitamin E and other nutrients needed by human body, and is one of high-quality fish liver oil. The neutral alumina column chromatography enriches the batyl alcohol and only causes a small loss of the content of the squalene.

[43] In the present disclosure, an organic solvent in an eluate is removed to obtain a high-purity batyl alcohol product. The present disclosure provides a basis for an industrial preparation of a high-purity batyl alcohol product.

BRIEF DESCRIPTION OF DRAWINGS

[44] The specific implementations of the disclosure will be further described in detail below with reference to the accompanying drawings.

[45] FIG. 11s a flowchart of a method of the present disclosure;

[46] FIG. 2 shows results of thin layer chromatography analysis;

[47] FIG. 3 is a graph showing an influence of different eluents on content of batyl alcohol;

[48] FIG. 4 shows gas chromatograms before and after separation and purification of batyl alcohol;

[49] In FIG. 4, the left is the gas chromatogram before separation and purification of batyl alcohol and the right is the gas chromatogram after separation and purification of Dbatyl alcohol;

[50] FIG. 5 shows a gas chromatogram of a squalene standard substance; [S1] In FIG. 5, the left is a gas chromatogram of a squalene standard substance and the right is a standard curve of squalene;

[52] FIG. 6 is a graph showing an influence of different sample loading amount on content of batyl alcohol; and

[53] FIG. 7 is a graph showing an influence of different elution flow rates on content of batyl alcohol.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[54] To make the foregoing objective, features, and advantages of the present disclosure clearer and more comprehensible, the present disclosure is further described in detail below with reference to the examples, but the protection scope required by the present disclosure is not limited to the scope described in the examples.

-7-

[55] Shark liver oil is provided by Zhejiang Hailisheng Pharmaceutical Co., Ltd. Batyl alcohol has a purity about 9.4% and squalene has a purity about 31.5%.

[56] Example 1 A method for improving purity of batyl alcohol in shark liver oil (a method for separating and purifying a batyl alcohol product by a neutral alumina column chromatography) included the following steps:

[57] 1) preparation of chromatographic column: 100-200 mesh neutral alumina was weighed, the weighed neutral alumina was mixed with distilled water at a use amount ratio of 1 g:5 ml, concentrated hydrochloric acid was added, a pH of an obtained solution was adjusted to about 2 and kept for 10 min, and distilled water was used for washing until an eluate was neutral; the neutral alumina was placed at 105°C for an activation for 24 h; and a column was packed by a dry method until a neutral alumina interface no longer dropped, dichloromethane was used for eluting to remove impurities in the column and an equilibration was conducted for 2 h (the dichloromethane was added to the column and kept for 2 h, and the dichloromethane was drained); and standing was conducted for later use;

[58] 2) extraction of unsaponifiable matter:

[59] 50 g of the shark liver oil was weighed into a 500-mL three-necked flask, 150 mL of 1 mol/L potassium hydroxide-ethanol solution was added, and reaction was conducted at 70°C for 50 min;

[60] after the reaction was over (preset reaction time was reached), about 50 mL of distilled water was added from a top part of a condenser tube to cool to a room temperature, an obtained product was poured into a 500-mL separatory funnel and extracted 3 times with n-hexane (the total use amount of the 3 times extraction with the n-hexane was about 200 mL), and extracts were combined; a combined extract was washed with an ethanol-water solution at a volume concentration of 10% until an eluate was neutral, and if turbidity occurred during the washing process, a small amount of absolute ethanol must be added for demulsification; the use amount of absolute ethanol only needed to ensure that the turbidity disappeared; and

[61] the extract was dehydrated by anhydrous sodium sulfate, a rotary evaporation (at a rotary evaporation temperature of 65°C and a pressure of 0.1 Pa) was conducted until constant weight to obtain an unsaponifiable matter, and after nitrogen filling, the unsaponifiable matter was stored at a low temperature for later use;

-8-

[62] 3) column chromatography: 5 groups of unsaponifiable matter samples were weighed and passed through the column;

[63] the unsaponifiable matter was dissolved in dichloromethane, an obtained sample was loaded and passed through the column, where the loading amount of the unsaponifiable matter was 3 g, an elution flow rate was 2 mL/min, an eluent is a mixed solution of dichloromethane and methanol at a volume ratio of 10:0, 9:1, 8:2, 7:3 and 6:4; each 40 mL was taken as one collection unit (i.e., every 40 mL was collected into one tube) and a TLC rapid qualitative analysis and a gas chromatography quantitative analysis were combined for tracking; and

[64] each group of the unsaponifiable matter sample received 20 collection units;

[65] 4) TCL analysis: after a silica gel plate was dried and activated at 103°C for 1 h, the silica gel plate was placed in a desiccator for cooling to a room temperature for later use; pure batyl alcohol and pure squalene were respectively used as samples; 100 mg of the samples were dissolved in 1 mL of dichloromethane, the samples were spotted on the TLC silica gel plate by using a capillary, a mixed solution of n- hexane: diethyl ether:acetic acid (85:15:1, v/v/v) was used as a developing solution, and color developing with iodine vapor was conducted as a standard;

[66] an obtained product in a 7th collection unit of step 3) was used as an enriched sample, the TLC analysis was conducted according to the above method, a strip at a lower position was the batyl alcohol and a strip at a higher position was squalene;

[67] the unsaponifiable matter was used as a pre-enriched sample and the TLC analysis was conducted according to the above method;

[68] obtained results were shown in FIG. 2; according to FIG. 2, it can be seen that the neutral alumina chromatographic column has an obvious effect on separating and punfying the batyl alcohol and can effectively separate the batyl alcohol and squalene in the shark liver oil; and

[69] the above 5 eluents can effectively separate the batyl alcohol and squalene in the shark liver oil.

[70] 5) GC analysis of batyl alcohol:

[71] the corresponding strip (about 10 mg) of the batyl alcohol was scraped on the TLC silica gel plate, 500 pL of a silanization reagent (BSTFA:TMCS=99:1) was added, water bath was conducted at 65°C for 30 min, cooling and purge with nitrogen were conducted, excess solvent was removed, 2 mL of n-hexane was added for

-9- redissolving, and a filtration (through a filter membrane with a filter diameter of 0.22 um) was conducted for detection; a purpose of adding the silanization reagent was to carry out derivatization, that is, to reduce a boiling point of the batyl alcohol and make the batyl alcohol easier to separate;

[72] gas chromatographic conditions were as follows: HP-5 capillary chromatographic column (30 m~0.32 mm, 0.25 um); heating program: an initial temperature was kept at 200°C for 5 min and heated to 280°C at 5°C/min, the temperature was kept for 20 min, a temperature of an injection port was 250°C, a splitless injection with an injection volume of 1 uL was conducted and a carrier gas flow rate was 1 mL/min;

[73] after a batyl alcohol standard substance was dissolved and diluted with n- hexane, it was detected according to the above-mentioned gas chromatographic conditions;

[74] the relative content of the batyl alcohol was analyzed by using a peak area normalization method; each group of the sample was repeatedly tested for 3 times; results were shown in FIG. 3;

[75] it can be seen from FIG. 3 that the purity of the batyl alcohol increased first and decreased as the content of the dichloromethane in the eluent decreased; when the eluent was selected, a solvent with a smaller polarity was generally firstly chosen and the polarity was successively increased according to a separation effect; polarity of methanol in the experiment was greater than that of dichloromethane; when only the dichloromethane was selected as the eluent, the separation effect of the dichloromethane was weaker than that of a combination of the dichloromethane and methanol (9:1, v/v); a reason may be that the polarity of the dichloromethane was weak, thus the batyl alcohol cannot be very well separated; however, with an increase in a ratio of the methanol, the purity of the batyl alcohol continued to decrease, this may due to the fact that polarity of the compound eluent was too strong and some impurities were mixed into the batyl alcohol; therefore, the eluent with the ratio of (dichloromethane: anhydrous methanol, v/v) at 9:1 was selected for subsequent optimization;

[76] when the eluent consisted of the dichloromethane and methanol at the ratio of 9:1 (v/v), the corresponding collection unit was detected according to the above

-10- method, the 7th collection unit (the 7th tube) had the highest purity of the batyl alcohol, which was about 18%;

[77] the unsaponifiable matter obtained in step 2 was used as “a sample before separation and purification” and the 7th collection unit component obtained in step 3 was used as “a sample after separation and purification”, detections were conducted respectively according to the above-mentioned gas chromatographic conditions, obtained results were shown in FIG. 4;

[78] FIG. 4 showed gas chromatograms before and after separation and purification of the batyl alcohol; the result of the gas chromatogram detection of the unsaponifiable matter of the shark liver oil was shown in the left of FIG. 4, the unsaponifiable matter mainly contained two active substances, batyl alcohol and squalene, the relative content of the batyl alcohol was 9.42% and the relative content of the squalene was

69.14% according to the area normalization method; under conditions that the eluent with the ratio of (dichloromethane:anhydrous methanol, v/v) at 9:1, a sample loading amount of 3 g and an elution flow rate of 2 mL/min, the chromatographic analysis results after the batyl alcohol was separated and purified by the neutral alumina chromatographic column were shown in the right of the FIG. 4 and the content of the batyl alcohol was significantly increased; from the chromatograms alone, a gap between the content of the batyl alcohol and squalene was shortening, the batyl alcohol accounted for 18%, while the squalene became 60.32%; experiment results proved that the neutral alumina column chromatography technology can effectively separate and purify the batyl alcohol from shark liver oil;

[79] in conclusion, the neutral alumina column chromatography can effectively separate and purify the batyl alcohol and had a screening function on the squalene in the unsaponifiable matter of the shark liver oil; and in a future research, the unsaponifiable matter from which the squalene was removed can be used as a raw material for enriching and purifying the batyl alcohol product;

[80] 6) gas chromatographic analysis of squalene standard substance:

[81] the squalene standard substance was weighed and dissloved in n-hexane to prepare standard working solutions with concentrations of 20 pg/mL, 50 pg/mL, 100 ug/mL, 200 pg/mL, 400 pg/mL and 800 pg/mL; a mass concentration was used as an x-coordinate and a peak area was used as a y-coordinate, and a standard curve of the squalene was drawn;

-11-

[82] gas chromatographic conditions were set as follows: HP-5 capillary chromatographic column (30 m*0.32 mm, 0.25 pm); heating program: an initial temperature was 160°C, the temperature was heated to 220°C at 15°C/min and kept for 2 min; the temperature was heated to 280°C at 5°C/min and kept for 20 min; the temperature was heated to 300°C at 5°C/min and kept for 2 min; a temperature of an injection port was 250°C and a split ratio was 1:10; a temperature of an injection port was 300°C, an injection amount was 1 uL and a flow rate of carrier was 1 ml/min; each group of the sample was repeatedly tested for 3 times; results were shown in FIG. 5;

[83] the result of the gas chromatographic analysis of the squalene standard substance was shown in the left of FIG. 5, and since no derivatization was conducted in determination of the squalene standard substance, peak appearance time of the squalene was about 15.017 min, which was about 2.7 min earlier than the peak appearance time in FIG. 4; and a standard curve of the squalene was shown in right of FIG. 5, an x-coordinate was concentration of squalene (ng/mL), a y-coordinate was a peak area and a linear regression equation was: y=1.9214x+6.089, R2 =0.9999; and

[84] 7) GC analysis of squalene:

[85] when the eluent of the sample obtained in step 3) consisted of the dichloromethane and methanol at the ratio of 9:1 (v/v), the obtained product of the 7th collection unit was analyzed through the GC analysis to determine the content of the squalene; a certain amount of the sample was weighed and dissolved in n-hexane, and the dissolved sample was filtered (through a filter membrane with a filter diameter of

0.22 um) for testing;

[86] the gas chromatographic conditions were the same as those in step 6);

[87] the unsaponifiable matter was detected according to the above method;

[88] according to calculation of the standard curve, the unsaponifiable matter squalene before the enrichment had a purity of 54.21% and the squalene after the enrichment had a purity of 49.18%;

[89] Therefore, it can be known that the enrichment method of the present disclosure would not lead to a large reduction in the content of the squalene; and the neutral alumina chromatographic column had an obvious effect on separating and purifying the batyl alcohol and could effectively separate the batyl alcohol and squalene in the shark liver oil.

[90] Example 2 A method for improving purity of batyl alcohol in shark liver oil:

-12-

[91] in step 3), 5 groups of the unsaponifiable matter samples were weighed, dissolved in dichloromethane and passed through the column, the loading amount of the unsaponifiable matter was 1, 2, 3, 4 and 5 g; the eluent was a mixed solution of dichloromethane and absolute methanol at 9:1 (v/v); the elution flow rate was 2 mL/min; the remaining operations were the same as those of Example 1;

[92] each group of the sample was repeatedly tested for 3 times; results were shown in FIG. 6;

[93] It can be seen from FIG. 6, the loading amount was within a range of 1 gto 3 g, the purity of the batyl alcohol increased with the increase of the loading amount, but as the loading amount continued to increase, the purity of the batyl alcohol showed a gradual decrease trend; a reason may be that when the loading amount was too low, the alumina chromatographic column cannot be fully utilized, resulting in waste; at the same time, large consumption of the eluent cannot effectively separate the batyl alcohol and impurities in the unsaponifiable matter; while when the loading amount was too large, the alumina chromatographic column was easily overloaded, resulting in a decrease in adsorption efficiency; therefore, the loading amount of 3 g was selected for subsequent optimization; and

[94] when the loading amount was 3 g, the batyl alcohol had the highest purity in the 7th tube.

[95] Example 3 A method for improving purity of batyl alcohol in shark liver oil:

[96] in step 3): 5 groups of the unsaponifiable matter samples were weighed and passed through the column; the eluent was a mixed solution of dichloromethane and anhydrous methanol (9:1, v/v); the loading amount was 3 g; the elution flow rate was 1,2, 3, 4 and 5 mL/min, respectively; the remaining operations were the same as those of Example 1;

[97] each group of the sample was repeatedly tested for 3 times; results were shown in FIG. 7;

[98] it can be seen from FIG. 7 that the purity of the batyl alcohol increased first and decreased as the elution flow rate increased; a reason may be that at a lower elution flow rate, retention time of the batyl alcohol in the alumina chromatographic column would be longer and neutral alumina had a strong adsorption effect, which causes part of the batyl alcohol to be adsorbed in the chromatographic column and cannot be eluted; when the elution flow rate was too high, the unsaponifiable matter would flow

-13- through the chromatographic column quickly and the batyl alcohol and impurities cannot be effectively separated; therefore, 2 mL/min was selected as the optimal elution flow rate; and

[99] when the elution flow rate was 2 mL/min, the batyl alcohol had the highest purity in the 7th tube.

[100] Example 4 A method for improving purity of batyl alcohol in shark liver oil:

[101] 1n step 3), the loading amount of the unsaponifiable matter was 3 g; the eluent was a mixed solution of dichloromethane and methanol at a volume ratio of 9:1; the elution flow rate was 2 mL/min; the remaining operations were the same as those of Example 1;

[102] every 40 mL was taken as one collection unit and 20 collection units were obtained; and preferably the 7th collection unit was selected; and

[103] obtained results were the batyl alcohol had a purity of 18.32% and the squalene had a purity of 49. 18%.

[104] Comparative example 1

[105] The eluent in Example 4 was changed that a ratio of the dichloromethane to the anhydrous methanol was 5:5, v/v; the remaining operations were the same as those of Example 4; and

[106] obtained results were the batyl alcohol had the highest purity of 15.76% and the squalene had the highest purity of 46.37%.

[107] Comparative example 2 The "neutral alumina chromatographic column" in Example 4 was changed to a "silica gel chromatography column", that is, in a preparation method of the chromatographic column, the neutral alumina was changed to silica gel; and the remaining operations were the same as those of Example 4.

[108] obtained results were the batyl alcohol had the highest purity of 15.23% and the squalene had the highest purity of 48.55%.

[109] Finally, it should be noted that the examples listed above are merely a few specific examples of the present disclosure. Apparently, the present disclosure would not be limited to the above examples, and many variations are possible. All modifications that can be directly derived or conceived by a person of ordinary skill in the art from the specification of the present disclosure should be regarded as falling into the protection scope of the present disclosure.

Claims

-14- Conclusions

A method for improving the purity of batyl alcohol in shark liver oil comprising the following steps: 1) extraction of unsaponifiables adding 1 mol/L of a potassium hydroxide ethanol solution to shark liver oil and reacting at (70 + 10)°C for (50 + 10) min, wherein a ratio of the shark liver oil to the potassium hydroxide-ethanol solution is 1g:(2.5-3.5 ml; and post-treating the resulting reaction liquid to obtain the unsaponifiables; and 2) column chromatography the selecting a neutral alumina chromatography column; and dissolving the unsaponifiables in dichloromethane, loading a obtained sample and passing it through the column, charging 2.5 - 3.5 g of the unsaponifiables, wherein an eluent is a mixed solution of dichloromethane and methanol is at a volume ratio of 9.5:0.5 - 8.5:1.5, wherein an eluent flow rate is 1.5 - 2.5 mL/min, and wherein a resulting eluent comprises enriched batyl alcohol.

The method for improving the purity of batyl alcohol in shark liver oil according to claim 1, wherein the post-treatment in step 1) specifically comprises: adding water to the obtained reaction liquid, performing an extraction with n-hexane and washing and rotary evaporation of a obtained extract to obtain the unsaponifiables.

The method for improving the purity of batyl alcohol in shark liver oil according to claim 1 or 2, wherein a preparation method of the chromatography column in step 2) specifically comprises: mixing neutral alumina having a particle size of 100-200 mesh and distilled water in a ratio of 1 g : (5 + 1) ml, adjusting a pH to 1.5 - 2.5 with hydrochloric acid, leaving for 8 - 12 min and washing

_15- of the treated alumina; and placing the washed alumina at 105°C for activation for (24 + Dh, packing the column by a drying method, eluting with dichloromethane to remove impurities in the column and performing equilibration for 2 hours.

The method for improving the purity of batyl alcohol in shark liver oil according to any one of claims 1 to 3, wherein in step 2) 3 g of the unsaponifiables are charged, the eluent is a mixed solution of the dichloromethane and the methanol at a volume ratio of 9:1 and the eluent flow rate is 2 mL/min.