Method for efficiently preparing high-purity chrysolic acid based on torreya grandis oil

Abstract

The invention discloses a method for efficiently preparing high-purity chrysolic acid based on torreya grandis oil, which comprises the following steps: dissolving urea in ethanol, adding free fatty acid, and carrying out inclusion reaction to obtain preliminarily purified torreya grandis oil fatty acid; dissolving the primarily purified torreya grandis oil fatty acid in ethanol, adding Lipozyme RM C lipase, and carrying out enzymatic reaction to obtain secondarily purified torreya grandis oil fatty acid; and dissolving the secondarily purified torreya grandis oil fatty acid in a mobile phase, injecting the obtained sample solution into a reversed-phase chromatographic column of octadecylsilane chemically bonded silica, collecting fractions, merging, and carrying out rotary evaporation until the fractions are dry, thereby obtaining the high-purity aureinic acid. According to the preparation method, urea inclusion preliminary enrichment, enzymatic selective esterification secondary enrichment and preparative chromatography fine purification are combined, the preparation method of the high-purity aureinic acid is reasonable in process route, remarkable in synergistic effect and extremely high in product purity, and the defects that in the prior art, the aureinic acid purification efficiency is low, and the purity is insufficient are overcome.

Description

Technical Field

[0001] This invention belongs to the field of pine acid separation and purification technology, specifically involving a method for efficiently preparing high-purity pine acid based on torreya oil. Background Technology

[0002] Torreya grandis is an economic tree species unique to China. Its fruit, Torreya grandis seed oil, contains a characteristic fatty acid called pinocembrinic acid. Pinocembrinic acid is a unique Δ5-polyunsaturated fatty acid, characterized by a double bond on the fifth carbon atom near the carboxyl terminus. Studies have shown that this special structure gives it several significant health benefits, including lowering lipids and providing antioxidant effects. Pinocembrinic acid accounts for approximately 9% of the total fatty acids in Torreya grandis oil.

[0003] Currently, numerous studies have been conducted on the separation and purification techniques for polyunsaturated fatty acids. The main methods include urea inclusion, low-temperature crystallization, molecular distillation, enzyme-catalyzed reactions, chromatography (including silver nitrate complexation chromatography), and supercritical CO2 extraction. However, single separation and purification methods often have limitations. For example, urea inclusion is effective at removing saturated and monounsaturated fatty acids, but its separation effect on structurally similar polyunsaturated fatty acids (such as linoleic acid) is limited. Enzymatic selective esterification can utilize the differences in catalytic efficiency of lipases on different substrates for separation, but it is difficult to achieve extremely high purity in one step. Chromatographic methods (such as preparative liquid chromatography) offer high separation precision, but if the purity of the raw material is too low, it will lead to low loading capacity, high cost, and high solvent consumption.

[0004] Therefore, there is an urgent need to develop an efficient, economical, and high-purity method for preparing pine acid. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of the invention, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0006] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0007] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a method for efficiently preparing high-purity pinocembryonic acid based on torreya oil.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution: Urea was dissolved in ethanol and free fatty acids were added. The mixture was stirred and refluxed until homogeneous to carry out an inclusion reaction. The solid and liquid phases were separated, and the liquid phase was evaporated to dryness. Water was added and the pH of the solution was adjusted to acidity. After extraction with an organic solvent, the mixture was washed with water, and the water and solvent were removed in sequence to obtain preliminarily purified Torreya grandis oil fatty acids. The preliminarily purified Torreya grandis oil fatty acids were dissolved in ethanol and Lipozyme RM C lipase was added to carry out the enzymatic reaction. The product was centrifuged to remove the enzyme and then extracted and separated. The lower aqueous phase was collected and washed, acidified, extracted, washed with water and evaporated to dryness in sequence to obtain the second-purified Torreya grandis oil fatty acids. The fatty acids from the second-purified Torreya grandis oil were dissolved in the mobile phase, and the resulting sample solution was injected into a reversed-phase chromatography column of octadecylsilane-bonded silica gel. The fractions were collected and combined, and then evaporated to dryness to obtain high-purity pine acid.

[0009] As a preferred embodiment of the method for efficiently preparing high-purity pine acid based on torreya oil according to the present invention, wherein: urea is dissolved in ethanol and free fatty acids are added, wherein the volume-to-mass ratio of ethanol to urea is 2~5:1.

[0010] As a preferred embodiment of the method for efficiently preparing high-purity pinocembryonic acid based on torreya oil according to the present invention, the mass ratio of the free fatty acid to urea is 1:3~5.

[0011] As a preferred embodiment of the method for efficiently preparing high-purity pinocembryonic acid based on torreya oil according to the present invention, the inclusion reaction time is 12~24h and the inclusion temperature is -20~-10℃.

[0012] As a preferred embodiment of the method for efficiently preparing high-purity pine acid based on torreya oil according to the present invention, wherein: the preliminarily purified torreya oil fatty acids are dissolved in ethanol and Lipozyme RM C lipase is added, wherein the molar ratio of the preliminarily purified torreya oil fatty acids to ethanol is 1:1~5.

[0013] As a preferred embodiment of the method for efficiently preparing high-purity pine acid based on Torreya grandis oil according to the present invention, the amount of Lipozyme RM C lipase added is 1~5wt% compared to the amount of fatty acids in the preliminarily purified Torreya grandis oil.

[0014] As a preferred embodiment of the method for efficiently preparing high-purity pinocembryonic acid based on torreya oil according to the present invention, the enzymatic reaction is carried out at a temperature of 30~40℃ for a time of 4~8h.

[0015] As a preferred embodiment of the method for efficiently preparing high-purity pinocembryonic acid based on Torreya grandis oil according to the present invention, wherein: the fatty acids of Torreya grandis oil that have undergone secondary purification are dissolved in a mobile phase, wherein the mobile phase is composed of acetonitrile and water, and the volume ratio of acetonitrile to water is 70~85:30~15.

[0016] As a preferred embodiment of the method for efficiently preparing high-purity pinocembryonic acid based on torreya oil according to the present invention, wherein: the obtained sample solution is injected into a reversed-phase chromatography column of octadecylsilane-bonded silica gel, wherein the injection flow rate is 10~20mL / min.

[0017] As a preferred embodiment of the method for efficiently preparing high-purity pinocembryonic acid based on Torreya grandis oil according to the present invention, the method for preparing the free fatty acid includes, Torreya oil is mixed with potassium hydroxide, distilled water, and anhydrous ethanol for saponification. The resulting product is converted into water-insoluble free fatty acids using a strong acid. Organic solvents are added to extract the fatty acids, and the organic phase is recovered and washed to remove water and solvent, thus obtaining the free fatty acids.

[0018] Beneficial effects of this invention: (1) The present invention combines urea inclusion pre-enrichment, enzyme-catalyzed selective esterification secondary enrichment and preparative chromatography fine purification to provide a reasonable process route, significant synergistic effect and extremely high product purity preparation method for pine acid, overcoming the defects of low purification efficiency and insufficient purity of pine acid in the prior art.

[0019] (2) Synergistic Processing and Step-by-Step Purification: This invention organically combines three methods, giving full play to their respective advantages. Urea inclusion efficiently removes saturated fatty acids (C16:0, C18:0) and monounsaturated fatty acids (C18:1) with extremely high content in the raw materials; enzymatic esterification precisely removes polyunsaturated fatty acid impurities (C18:2, linoleic acid) with more similar structures; preparative chromatography, as the final purification method, ensures the purity of the product.

[0020] (3) High purity and significant effect: By comprehensively optimizing the material ratio, temperature and time of each step, this invention has determined an efficient process range, ensuring the stability and efficiency of the method. This method can improve the purity of pine acid, which is only 9.89 ± 0.47% in Torreya grandis oil, to more than 99% after three steps, laying a material foundation for subsequent nutritional research and high value-added applications. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The chromatogram is obtained by reversed-phase chromatography analysis of the fatty acids of Torreya grandis oil purified in Example 1 of this invention.

[0023] Figure 2 This is a comparison chart showing the purity and yield of pinocembryonic acid in the preliminarily purified torreya oil fatty acids prepared by different proportions of free fatty acids and urea in Examples 1-2 and Comparative Example 1 of the present invention.

[0024] Figure 3 This is a comparison chart showing the purity and yield of pinocembryonic acid in the preliminarily purified torreya oil fatty acids prepared by different proportions of ethanol and urea in Examples 1, 3 and Comparative Example 2 of the present invention.

[0025] Figure 4 This is a comparison chart showing the purity and yield of pinocembryonic acid in the preliminarily purified torreya oil fatty acids obtained at different inclusion times in Examples 1, 4 and Comparative Example 3 of this invention.

[0026] Figure 5 This is a comparison chart showing the purity and yield of pinocembryonic acid in the preliminarily purified torreya oil fatty acids obtained at different inclusion temperatures in Examples 1, 5 and Comparative Example 4 of this invention.

[0027] Figure 6 This is a comparison chart showing the purity and yield of pinocembryonic acid in the secondary purified torreya oil fatty acids obtained from different amounts of ethanol added in Examples 1, 6 and Comparative Example 5 of this invention.

[0028] Figure 7 This is a comparison chart showing the purity and yield of pinocembryonic acid in the secondary purified Torreya grandis oil fatty acids obtained from different amounts of Lipozyme RM C lipase added in Examples 1, 7 and Comparative Example 6 of this invention.

[0029] Figure 8 This is a comparison chart showing the purity and yield of pinocembryonic acid in the secondary purified Torreya grandis oil fatty acids obtained at different enzymatic reaction temperatures in Examples 1, 8 and Comparative Example 7 of this invention.

[0030] Figure 9 This is a comparison chart showing the purity and yield of pinocembryonic acid in the secondary purified Torreya grandis oil fatty acids obtained at different enzymatic reaction times in Examples 1, 9 and Comparative Example 8 of this invention.

[0031] Figure 10The diagram shows the separation effect of different mobile phases in Examples 1 and 10 of the present invention.

[0032] Figure 11 The diagram shows the separation effect at different flow rates in Embodiments 1, 11 and Comparative Example 9 of the present invention. Detailed Implementation

[0033] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0034] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0035] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0036] Unless otherwise specified, all raw materials used in the embodiments of this invention are commercially available.

[0037] In this invention, the ethanol concentration is 95%.

[0038] In this invention, the yield = mass of pine acid in the product of this step / mass of pine acid in the raw materials of this step * 100%.

[0039] Example 1 This embodiment provides a method for efficiently preparing high-purity pinocembryonic acid based on Torreya grandis oil, specifically: (1) Preparation of fatty acids: Torreya oil, potassium hydroxide, distilled water and anhydrous ethanol are mixed in a mass ratio of 1:0.25:1:1. Nitrogen gas is introduced at 80°C, and the mixture is stirred and refluxed for 2 h of saponification reaction. After cooling to room temperature, the pH is adjusted to 1~2 with concentrated hydrochloric acid to separate the solution into layers. Hexane is added to extract fatty acids twice. The upper organic phase is recovered and washed with distilled water until neutral. Anhydrous sodium sulfate is added to remove water. The solvent is removed by rotary evaporation under reduced pressure at 70°C to obtain free fatty acids, which are stored at -20°C for later use. (2) Urea inclusion: Take urea and ethanol, stir and reflux at 80°C to completely dissolve urea, wherein the volume mass ratio of ethanol to urea is 3:1, add it to the free fatty acid obtained in step (1), wherein the mass ratio of free fatty acid to urea is 1:4, introduce nitrogen, stir and reflux for 1 hour, cool to room temperature, place at -10°C for low temperature inclusion reaction for 12 hours, separate solid and liquid, take liquid phase and evaporate to dryness, add distilled water, adjust pH to 2~3 with concentrated hydrochloric acid, extract with n-hexane 3 times, wash away residual urea with distilled water, add anhydrous sodium sulfate to remove water, and evaporate under reduced pressure at 60°C to remove solvent, thus obtaining preliminarily purified Torreya oil fatty acid; (3) Enzymatic selective esterification: The preliminarily purified Torreya oil fatty acids obtained in step (2) were mixed with ethanol at a molar ratio of 1:3 and Lipozyme RM C lipase was added. The amount of Lipozyme RM C lipase added was 3wt% compared with the preliminarily purified Torreya oil fatty acids. The enzymatic reaction was carried out for 6 h under a water bath at 30℃. The obtained product was centrifuged to remove the enzyme, and hexane, ethanol and 1.9% (w / v) sodium hydroxide solution were added for extraction and separation. The lower aqueous phase was collected, washed with hexane, acidified with hydrochloric acid, and extracted with hexane. The hexane phase was washed with water and the solvent was evaporated to obtain the second-purified Torreya oil fatty acids. (4) Preparative chromatography: Acetonitrile and water were mixed evenly at a volume ratio of 75:25 as the mobile phase. 200 mg of the secondary purified Torreya grandis oil fatty acid obtained in step (3) was dissolved in the mobile phase. The resulting sample solution was injected into an octadecylsilane-bonded silica gel (C18) reversed-phase column at a flow rate of 15 mL / min. The chromatogram is shown below. Figure 1 As shown, according to Figure 1 The characteristic peak of pine acid was observed between 41.6 and 49.6 min. Starting from 41.6 min, one fraction was collected every minute for a total of 8 fractions. The fractions were combined and rotary evaporated to dryness to obtain the final high-purity pine acid.

[0040] The initial purity of pine acid in the free fatty acids obtained in step (1) of Example 1 was 9.89±0.47% by gas chromatography analysis. The purity of pine acid in the preliminarily purified torreya oil fatty acids obtained in step (2) was 47.97±0.99%, and the yield was 29.5±1.17%. At this time, urea molecules form tubular lattices during crystallization, which tend to encapsulate straight-chain, saturated or low-unsaturation fatty acid molecules, while excluding branched or high-unsaturation fatty acids (such as pine acid) from the lattice, thus keeping them in the liquid phase.

[0041] Further analysis of the secondary purified Torreya grandis oil fatty acids obtained in step (3) showed that the purity of pine acid was 70.71±1.01%, with a yield of 25.42±1.08%. This step utilized the fact that the esterification catalytic activity of Lipozyme RM C lipase on the main impurity fatty acids (linoleic acid and oleic acid) in Torreya grandis oil was higher than its activity on the target product pine acid. Under these reaction conditions, the impurity fatty acids were preferentially esterified to ethyl esters, while pine acid was mainly retained in the form of free fatty acids. Subsequently, through acid-base extraction, the esterified impurities (ethyl esters) remained in the organic phase, while the target product pine acid was separated out as a sodium salt of fatty acids entering the aqueous phase. At this point, the purity of pine acid could reach over 60%.

[0042] Finally, the purity of pine acid in the final product obtained by analysis step (4) is 99.69±0.04%, and the yield of this step is 67.08±0.07%. This step utilizes the difference in the partition coefficient of pine acid and other residual impurity fatty acids (mainly linoleic acid) on the C18 reversed-phase chromatographic column, and achieves the precise separation and collection of pine acid by optimizing the mobile phase and flow rate.

[0043] Example 2 The difference between this embodiment and embodiment 1 is that the mass ratio of free fatty acids to urea in step (2) is adjusted to 1:3 and 1:5 respectively, while the rest of the preparation methods are the same as in embodiment 1.

[0044] Comparative Example 1 The difference between this comparative example and Example 1 is that only the mass ratio of free fatty acids to urea in step (2) is adjusted to 1:1 and 1:2 respectively, while the rest of the preparation methods are the same as in Example 1.

[0045] The purity and yield of pinocembryonic acid in the preliminarily purified Torreya grandis oil fatty acids prepared by reacting different proportions of free fatty acids with urea in Examples 1-2 and Comparative Example 1 are shown in Table 1 and 2. Figure 2 As shown.

[0046] Table 1. Effect of the ratio of free fatty acids to urea on the purity of pinocembryonic acid in preliminarily purified Torreya grandis oil fatty acids.

[0047] From Table 1 and Figure 2 As can be seen, when the mass ratio of free fatty acids to urea is in the range of 1:3 to 1:5, a purity of over 40% can be obtained, which is significantly better than the ratios of 1:1 and 1:2. However, the purity of pinocembryonic acid in the preliminarily purified torreya oil fatty acids corresponding to the mass ratio of 1:4 in Example 1 is the highest.

[0048] Example 3 The difference between this embodiment and embodiment 1 is that only the volume-to-mass ratio of ethanol to urea in step (2) is adjusted to 2:1, 4:1, and 5:1 respectively, while the rest of the preparation methods are the same as in embodiment 1.

[0049] Comparative Example 2 The difference between this comparative example and Example 1 is that only the volume-to-mass ratio of ethanol to urea in step (2) is adjusted to 6:1, while the rest of the preparation methods are the same as in Example 1.

[0050] The purity and yield of pinocembryonic acid in the preliminarily purified Torreya grandis oil fatty acids prepared by reacting different ratios of ethanol and urea in Examples 1, 3, and Comparative Example 2 are shown in Table 2. Figure 3 As shown.

[0051] Table 2. Effect of urea concentration on the purity of pinocembryonic acid in preliminarily purified Torreya grandis oil fatty acids

[0052] Comparison Table 2 or Figure 3 When the ratio of 95% ethanol to urea is in the range of 2 to 5:1, a high purity of over 42% can be obtained in urea-ethanol solutions of different concentrations.

[0053] Example 4 The difference between this embodiment and embodiment 1 is that only the inclusion time in step (2) is adjusted to 16h, 20h and 24h respectively, while the rest of the preparation methods are the same as in embodiment 1.

[0054] Comparative Example 3 The difference between this comparative example and Example 1 is that only the inclusion time in step (2) is adjusted to 8 hours, while the rest of the preparation methods are the same as in Example 1.

[0055] The purity and yield of pinocembryonic acid in the preliminarily purified Torreya grandis oil fatty acids obtained in step (2) of Examples 1, 4, and Comparative Example 3 were tested, and the results are shown in Table 3. Figure 4 As shown.

[0056] Table 3. Effect of inclusion time on the purity of pinocembryonic acid in pre-purified Torreya grandis oil fatty acids

[0057] According to Table 3 and Figure 4 At an inclusion temperature of -10℃, the purity of pine acid is highest when the inclusion time is 12h, while the purity is lower when the inclusion time is as low as 8h because an effective tubular lattice cannot be formed.

[0058] Example 5 The difference between this embodiment and embodiment 1 is that only the inclusion temperature in step (2) is adjusted to -20℃, while the rest of the preparation methods are the same as in embodiment 1.

[0059] Comparative Example 4 The difference between this embodiment and embodiment 1 is that only the inclusion temperature in step (2) is adjusted to 4°C and 20°C, while the rest of the preparation methods are the same as in embodiment 1.

[0060] The purity and yield of pinocembryonic acid in the preliminarily purified Torreya grandis oil fatty acids obtained in step (2) of Examples 1, 5, and Comparative Example 4 are shown in Table 4. Figure 5 As shown.

[0061] Table 4. Effect of inclusion temperature on the purity of pinocetic acid in pre-purified Torreya grandis oil fatty acids

[0062] From Table 4 and Figure 5 It can be seen that the inclusion temperature has a significant effect on the purity of pinocortic acid in the preliminarily purified Torreya grandis oil fatty acids. When the temperature is as high as 4℃, the purity of pinocortic acid begins to decrease. Torreya grandis oil fatty acids with higher purity of pinocortic acid can only be obtained when the temperature is between -20~-10℃.

[0063] Example 6 The difference between this embodiment and Example 1 is that the molar ratio of the preliminarily purified torreya oil fatty acids to ethanol in step (3) is adjusted to 1:1 and 1:5 respectively, while the rest of the preparation methods are the same as in Example 1.

[0064] Comparative Example 5 The difference between this comparative example and Example 1 is that only the molar ratio of the preliminarily purified torreya oil fatty acids to ethanol in step (3) is adjusted to 1:7 and 1:9 respectively, while the rest of the preparation methods are the same as in Example 1.

[0065] The purity and yield of pinocembryonic acid in the secondary purified Torreya grandis oil fatty acids obtained in step (3) of Examples 1, 6, and Comparative Example 5 are shown in Table 5. Figure 6 As shown.

[0066] Table 5. Effect of ethanol addition on the purity of pinocembryonic acid in the fatty acids of secondary purified Torreya grandis oil.

[0067] Compare Table 5 or Figure 6 The purity of the product after secondary purification with different amounts of ethanol added was approximately 64% in the range of 1:1 to 5. When the ratio was 1:3, the purity of the product reached more than 70%, which was significantly better than the product purified with other amounts of ethanol.

[0068] Example 7 The difference between this embodiment and embodiment 1 is that only the amount of Lipozyme RM C lipase added in step (3) is adjusted to 1wt% and 5wt% respectively, while the rest of the preparation methods are the same as in embodiment 1.

[0069] Comparative Example 6 The difference between this comparative example and Example 1 is that the amount of Lipozyme RM C lipase added in step (3) is adjusted to 7wt% and 9wt% respectively, while the rest of the preparation methods are the same as in Example 1.

[0070] The purity and yield of pinocembryonic acid in the secondary purified Torreya grandis oil fatty acids obtained in step (3) of Examples 1, 7, and Comparative Example 6 are shown in Table 6. Figure 7 As shown.

[0071] Table 6. Effect of Lipozyme RM C lipase addition on the purity of pinocembryonic acid in the fatty acids of secondary purified Torreya grandis oil.

[0072] From Table 6 or Figure 7 It can be seen that the amount of Lipozyme RM C lipase added has a significant impact on the purity of the product pine acid. Specifically, only when the amount of lipase added is 1~5wt% can a product with a purity greater than 63% be obtained. However, further increasing the amount added to 7wt% will lead to a significant decrease in purity.

[0073] Example 8 The difference between this embodiment and Example 1 is that only the temperature of the enzymatic reaction in step (3) is adjusted to 40°C, while the rest of the preparation methods are the same as in Example 1.

[0074] Comparative Example 7 The difference between this comparative example and Example 1 is that only the temperature of the enzymatic reaction in step (3) is adjusted to 20°C and 50°C respectively, while the rest of the preparation methods are the same as in Example 1.

[0075] The purity and yield of pinocembryonic acid in the secondary purified Torreya grandis oil fatty acids obtained in step (3) of Examples 1, 8, and Comparative Example 7 are shown in Table 7. Figure 8 As shown.

[0076] Table 7. Effect of enzymatic reaction temperature on the purity of pinocembryonic acid in the fatty acids of Torreya grandis oil after secondary purification.

[0077] According to Table 7 or Figure 8When the enzymatic reaction temperature is 30~40℃, the purity of the obtained product is high, with 30℃ being the best, at 70.71±1.01%.

[0078] Example 9 The difference between this embodiment and Example 1 is that the time of the enzymatic reaction in step (3) is adjusted to 4h and 8h respectively, while the rest of the preparation methods are the same as in Example 1.

[0079] Comparative Example 8 The difference between this comparative example and Example 1 is that only the time of the enzymatic reaction in step (3) is adjusted to 10h and 12h respectively, while the rest of the preparation methods are the same as in Example 1.

[0080] The purity and yield of pinocembryonic acid in the secondary purified Torreya grandis oil fatty acids obtained in step (3) of Examples 1, 9, and Comparative Example 8 are shown in Table 8. Figure 9 As shown.

[0081] Table 8. Effect of enzyme reaction time on the purity of pinocembryonic acid in the fatty acids of Torreya grandis oil after secondary purification.

[0082] According to Table 8 or Figure 9 It can be seen that under the conditions of the amount of ethanol added, the amount of Lipozyme RM C lipase added, and the corresponding reaction temperature in Example 1, if the reaction time exceeds 8 hours, the purity of the pine acid in the product will decrease, and the preparation of the high-purity pine acid of the present invention cannot be achieved.

[0083] Example 10 The difference between this embodiment and Example 1 is that the volume ratio of the mobile phase acetonitrile to water in step (4) is adjusted to 70:30, 80:20, and 85:15, respectively. The rest of the preparation methods are the same as in Example 1.

[0084] Comparison of separation effects of different mobile phases in Examples 1 and 10 Figure 10 As shown, when the volume percentage of acetonitrile is in the range of 70%-85%, effective separation of pine acid from other impurity fatty acids can be achieved.

[0085] Example 11 The difference between this embodiment and Example 1 is that only the flow rate in step (4) is adjusted to 10 mL / min and 20 mL / min respectively, while the rest of the preparation methods are the same as in Example 1.

[0086] Comparative Example 9 The difference between this comparative example and Example 1 is that only the flow rate in step (4) is adjusted to 5 mL / min and 25 mL / min, while the rest of the preparation methods are the same as in Example 1.

[0087] A comparison of the separation effects of different flow rates in Examples 1, 11, and 9: Figure 11 As shown in the figure, when the flow rate is only 5 mL / min, many impurities cannot be well separated, while when the flow rate is higher than 25 mL / min, reagents are wasted and the column is subjected to higher column pressure. Therefore, a flow rate in the range of 10~20 mL / min can achieve good chromatographic peak resolution within a reasonable time.

[0088] Comparative Example 10 The difference between this comparative example and Example 1 is that only the mobile phase in step (4) was changed to methanol and water; the rest of the preparation methods were the same as in Example 1. The results showed that, compared to using acetonitrile and water as the mobile phase, using methanol and water as the mobile phase resulted in a longer separation time and overlap between the target peak and other peaks, making effective separation impossible.

[0089] In summary, this invention achieves high-purity pine acid preparation through precise control of parameters in three steps: initial enrichment via urea inclusion complexation, secondary enrichment via enzymatic selective esterification, and fine purification via preparative chromatography. This overcomes the shortcomings of existing technologies, such as low efficiency and insufficient purity of pine acid. This method can improve the purity of pine acid in Torreya grandis oil from only 9.89 ± 0.47% to over 99% through three steps.

[0090] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for efficiently preparing high-purity abietic acid based on torreya grandis oil, characterized by comprising the steps of: The method comprises the following steps: ​ The urea is dissolved in ethanol and free fatty acid is added, and the mixture is stirred and refluxed to uniformly carry out the inclusion reaction, and then solid-liquid separation is performed, and the liquid phase is rotary evaporated to dryness, and then water is added and the pH of the solution is adjusted to be acidic, and then the solution is extracted with an organic solvent, and then water washing, water removal and solvent removal are sequentially performed, so that the initially purified Torreya grandis oil fatty acid is obtained; The initially purified Torreya grandis oil fatty acid is dissolved in ethanol and Lipozyme RM C lipase is added, and then an enzymatic reaction is performed, and then the obtained product is centrifuged to remove the enzyme, and then extraction separation is performed, and then the lower aqueous phase is collected, and then washing, acidification, extraction, water washing and rotary drying are sequentially performed, so that the secondly purified Torreya grandis oil fatty acid is obtained. The secondly purified Torreya grandis oil fatty acid is dissolved in a mobile phase, and then the obtained sample solution is injected into a reversed-phase chromatographic column of octadecylsilane-bonded silica gel, and then the fractions are collected and combined, and then rotary evaporation is performed to dryness, so that the high-purity abietic acid is obtained.

2. The method for efficiently preparing high-purity abietic acid based on torreya grandis oil according to claim 1, characterized by: In the step of dissolving the urea in ethanol and adding the free fatty acid, the volume-to-mass ratio of ethanol to urea is 2-5:

1.

3. The method for efficiently preparing high-purity abietic acid based on torreya grandis oil according to claim 2, characterized by: The mass ratio of the free fatty acid to urea is 1:3-5.

4. The method for efficiently preparing high-purity abietic acid based on torreya grandis oil according to claim 1, characterized by: The inclusion reaction is performed for 12-24 hours at a temperature of-20--10℃.

5. The method for efficiently preparing high-purity abietic acid based on torreya grandis oil according to claim 1, characterized by: In the step of dissolving the initially purified Torreya grandis oil fatty acid in ethanol and adding Lipozyme RM C lipase, the molar ratio of the initially purified Torreya grandis oil fatty acid to ethanol is 1:1-5.

6. The method for efficiently preparing high-purity abietic acid based on torreya grandis oil according to claim 5, characterized by: The Lipozyme RM C lipase is added in an amount of 1-5 wt% relative to the initially purified Torreya grandis oil fatty acid.

7. The method for efficiently preparing high-purity abietic acid based on Japanese nut oil according to claim 1, characterized by: The enzymatic reaction is performed at a temperature of 30-40℃ for 4-8 hours.

8. The method for efficiently preparing high-purity abietic acid based on Japanese nut oil according to claim 1, characterized by: In the step of dissolving the secondly purified Torreya grandis oil fatty acid in a mobile phase, the mobile phase is composed of acetonitrile and water, and the volume ratio of the acetonitrile to water is 70-85:30-15.

9. The method for efficiently preparing high-purity abietic acid based on Japanese nut oil according to claim 1, characterized by: The obtained sample solution is injected into a reversed-phase chromatographic column of octadecylsilane-bonded silica gel, and the flow rate during the injection is 10-20 mL / min.

10. The method for efficiently preparing high-purity abietic acid based on Japanese nut oil according to claim 1, characterized by: The method for preparing the free fatty acid comprises the following steps: The Torreya grandis oil is uniformly mixed with potassium hydroxide, distilled water and anhydrous ethanol to perform a saponification reaction, and then the obtained product is converted into free fatty acid that is insoluble in water by using a strong acid, and then an organic solvent is added to extract the fatty acid, and then the organic phase is recovered and subjected to washing, water removal and solvent removal, so that the free fatty acid is obtained.

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