CN111153827B - Preparation method and application of benzylamine omega-3 unsaturated fatty acid - Google Patents

Abstract

The invention discloses a preparation method of benzylamine omega-3 unsaturated fatty acid and application thereof, wherein raw materials rich in omega-3 unsaturated fatty acid and esters thereof are used as initial reactants, and free omega-3 unsaturated fatty acid mixture is prepared by ester hydrolysis reaction; then preparing a mixture of benzylamine fatty acid compounds by using a carbodiimide condensation method, wherein in the benzylamine synthesis process, benzylamine reaction can be completed preferentially by controlling synthesis reaction conditions; under the optimized chromatographic condition, the macamide can be effectively separated from other fatty acid (such as linoleic acid, linolenic acid and the like) derivatives and other impurities and byproducts (such as residual reagents and the like) in a complex synthesis system, and finally, two macamide monomers with high unsaturation degree, namely benzylamine total-cis-5, 8,11,14, 17-eicosapentaenoic acid and benzylamine total-cis-4, 7,10,13,16, 19-docosahexaenoic acid, can be simultaneously obtained, and can be applied to the treatment of ischemic brain injury with small toxic and side effects, safety and effectiveness.

Description

Preparation method and application of benzylamine omega-3 unsaturated fatty acid

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a preparation method and application of benzylamine omega-3 unsaturated fatty acid.

Background

Macamides are a class of amide fatty acid derivatives found in the natural plant maca and can be formed by the condensation of different kinds of fatty acids with benzylamine, wherein the form of the benzylamine group also includes m-methoxylation and p-hydroxylation. The maca has unique medical and edible values, and the characteristic active ingredients of the maca become a hotspot for extensive research and attention of students and markets at home and abroad. Forty or more maca amide compounds have been discovered to date.

Macaamide has two sources, namely natural separation and chemical synthesis. The natural source of the maca amide has the advantages of multiple types, low content and close physicochemical properties, and the problems of difficult separation and extraction of the maca amide monomer and high batch preparation cost exist.

The current chemical synthesis method of the macamide is to react a fatty acid monomer with benzylamine. In theory, higher unsaturation fatty acid compounds have potentially higher biological activities such as oxidation resistance. Benzylated trans-cis-5, 8,11,14, 17-eicosapentaenoic acid and benzylated trans-cis-4, 7,10,13,16, 19-docosahexaenoic acid are two macamide compounds which theoretically have the highest degree of unsaturation and have not been found in natural plants. In the existing method for preparing the benzylated trans-cis-5, 8,11,14, 17-eicosapentaenoic acid and the benzylated trans-cis-4, 7,10,13,16, 19-docosahexaenoic acid, firstly, monomer compounds of the eicosapentaenoic acid and the docosahexaenoic acid need to be obtained, the process not only needs to obtain high monomer raw material cost, but also has unstable chemical properties of the eicosapentaenoic acid and the docosahexaenoic acid, and oxidation and degradation easily occur in the storage and synthesis processes, a large number of homologs and analogues are generated, and the benzylated trans-cis-4, 8,11,14, 17-eicosapentaenoic acid and the benzylated trans-cis-4, 7,10,13,16, 19-docosahexaenoic acid with high purity are difficult to obtain. If the monomer compounds of eicosapentaenoic acid and docosahexaenoic acid are not used as the starting materials, but low-cost fatty acid mixtures, such as various animal and vegetable oils rich in unsaturated fatty acids, are used as the raw materials, the physicochemical properties of most fatty acid monomers are very similar, and the content of eicosapentaenoic acid and docosahexaenoic acid in most oils is low. Resulting in very low purity of benzylated trans-cis-5, 8,11,14, 17-eicosapentaenoic acid and benzylated trans-cis-4, 7,10,13,16, 19-docosahexaenoic acid, even being difficult to detect alone.

Disclosure of Invention

The invention aims at solving at least one of the technical problems in the prior art, and provides a method for simultaneously preparing two benzylated omega-3 unsaturated fatty acids, which can utilize substances containing various mixed fatty acids as starting materials, and simultaneously synthesize high-purity benzylated cis-5, 8,11,14, 17-eicosapentaenoic acid and benzylated cis-4, 7,10,13,16, 19-docosahexaenoic acid after benzylation.

The preparation method of benzylamine omega-3 unsaturated fatty acid provided by the invention comprises the following steps:

(1) Carrying out ester hydrolysis on a raw material rich in omega-3 unsaturated fatty acid and esters thereof to obtain a mixture of free omega-3 unsaturated fatty acid;

(2) Adding a condensing agent, a catalyst and an alkali additive into a mixture of free omega-3 unsaturated fatty acid, mixing for 10-24 hours, and then adding benzylamine or substituted benzylamine for acid-amine condensation reaction;

(3) Drying the reaction solution after the reaction in the step (2), extracting, separating by liquid chromatography, and drying to obtain benzylamine omega-3 unsaturated fatty acid;

the chemical structural general formula of the benzylamine omega-3 unsaturated fatty acid is as follows:

wherein R is ₁ =all-cis-5, 8,11,14, 17-eicosapentaenoic acid or all-cis-4, 7,10,13,16, 19-docosahexaenoic acid; r is R ₂ =h or methoxy; r is R ₃ =h or OH.

Further, the raw material rich in omega-3 unsaturated fatty acids and esters thereof is selected from microalgae oil and/or fish oil. Through analysis and screening of dozens of common animal and vegetable oil raw materials, the two raw materials are found to be rich in eicosapentaenoic acid and eicosahexaenoic acid.

In the step (1), the microalgae oil and/or fish oil is/are added into sodium hydroxide-methanol solution to react at 10-60 ℃.

Further, the concentration of the sodium hydroxide-methanol solution is 0.1-2 mol/L.

Further, the volume ratio of the microalgae and/or fish oil to the sodium hydroxide-methanol solution is 1: 2-1: 20, the hydrolysis time of the ester is 30-120 min.

Further, the condensing agent is at least one selected from EDC HCl (1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride), DIC (diisopropylcarbodiimide) and DCC (dicyclohexylcarbodiimide), preferably EDC HCl.

Further, the catalyst is at least one selected from DMAP (4-dimethylaminopyridine), HOBt (1-hydroxybenzotriazole) and HOAT (1-hydroxy-7-azabenzotriazole), preferably HOBt.

Further, the alkali additive is Et ₃ N (triethylamine) and/or DIPEA (N, N-diisopropylethylamine), preferably Et ₃ N。

Further, in step (2), the substituted benzylamine is selected from p-hydroxybenzylamine or m-methoxybenzylamine.

Further, in the step (2), the molar ratio of the benzylamine or the substituted benzylamine, the free omega-3 unsaturated fatty acid, the condensing agent, the catalyst and the base additive is 0.5 to 2:0.5 to 2:0.5 to 2:0.5 to 2:1 to 5.

Further, the temperature of the acid amine condensation reaction is 10-65 ℃ and the reaction time is 2-10 h.

Further, in step (3), the extraction method includes the steps of: adding an acid solution and an organic solvent, and taking an organic layer; fully oscillating the organic layer with alkali solution, and completely reacting; then adding acid solution for washing, and taking organic layer solution. The water-soluble, acidic material (e.g., unreacted fatty acid) in the reaction mixture will dissolve in the acid solution, while the macamide analog will dissolve in the organic solvent, allowing for efficient separation.

In some preferred embodiments, the acid solution is HCl solution and the organic solvent is selected from n-hexane.

Further, the liquid chromatography is carried out by using methanol/water solution as a mobile phase and a C18 chromatographic column, wherein the volume ratio of methanol to water is preferably 90:10, finally collecting the methanol solution.

The invention also provides application of the benzylamine omega-3 unsaturated fatty acid in preparing a medicament for treating ischemic brain injury.

Further, the medicine for treating ischemic brain injury is in the form of oral preparation, injection or sublingual buccal preparation.

The invention also provides a composition for treating ischemic brain injury, comprising benzylamine omega-3 unsaturated fatty acid.

Further, the composition further comprises any one or more of a solvent, a propellant, a solubilizer, a cosolvent, an emulsifier, a colorant, a binder, a disintegrant, a filler, a lubricant, a wetting agent, an osmotic pressure regulator, a stabilizer, a glidant, a flavoring agent, a preservative, a suspending agent, a coating material, a fragrance, an anti-adhesive agent, an integrating agent, a permeation enhancer, a pH regulator, a buffer, a plasticizer, a surfactant, a foaming agent, an antifoaming agent, a thickener, a inclusion agent, a humectant, an absorbent, a diluent, a flocculant and a deflocculant, a filter aid, a release retarder, and the like, which are used for preparing the composition into an oral preparation, an injection preparation or a sublingual buccal preparation.

Compared with the prior art, the invention adopts the raw materials rich in omega-3 unsaturated fatty acid and esters thereof as initial reactants, and prepares the free omega-3 unsaturated fatty acid mixture by utilizing the ester hydrolysis reaction; then preparing a mixture of benzylamine fatty acid compounds by using a carbodiimide condensation method, wherein in the benzylamine synthesis process, benzylamine reaction can be completed preferentially by controlling synthesis reaction conditions; under the optimized chromatographic condition, the method can effectively separate other fatty acid (such as linoleic acid, linolenic acid and the like) derivatives and other impurities and byproducts (such as residual reagents and the like) in a complex synthesis system, and finally, two macamide monomers with high unsaturation degree, namely benzylamine trans-cis-5, 8,11,14, 17-eicosapentaenoic acid and benzylamine trans-cis-4, 7,10,13,16, 19-docosahexaenoic acid, can be obtained simultaneously.

The invention has the following technical effects:

(1) The method can synthesize high-purity benzylated cis-5, 8,11,14, 17-eicosapentaenoic acid and benzylated cis-4, 7,10,13,16, 19-docosahexaenoic acid under the condition of taking microalgae oil or fish oil containing various fatty acids as raw materials without taking difficult and expensive fatty acid monomers as starting materials.

(2) The benzyl amination all-cis-5, 8,11,14, 17-eicosapentaenoic acid and benzyl amination all-cis-4, 7,10,13,16, 19-docosahexaenoic acid are applied to treat ischemic brain injury including neonatal ischemic anoxic encephalopathy, cerebral apoplexy, ischemia-reperfusion injury and transient cerebral ischemia attack, and a novel treatment way is provided for preventing ischemic brain injury repair medicines or treating ischemic brain injury diseases, and the preparation method is small in toxic and side effect, safe and effective.

Drawings

The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:

FIG. 1 is a chromatogram of the mixture of macamides of example 1 after separation by liquid chromatography;

FIG. 2 is a high performance liquid chromatogram of the P1 peak collected after purification;

FIG. 3 is a secondary mass spectrum of the P1 peak collected after purification;

FIG. 4 is a high performance liquid chromatogram of the P2 peak collected after purification;

FIG. 5 is a secondary mass spectrum of the P2 peak collected after purification;

FIG. 6 is a graph of TTC staining of the protective effect of benzylamine eicosapentaenoic acid on brain tissue of neonatal mice after hypoxia-ischemia;

FIG. 7 is a Nile's staining chart of benzylamine eicosapentaenoic acid on brain tissue pathological damage of neonatal mice after hypoxia-ischemia;

FIG. 8 is the results of a cliff avoidance test of benzylamine eicosapentaenoic acid on neonatal mice;

FIG. 9 is the results of a forearm suspension test of benzylamine eicosapentaenoic acid on neonatal mice;

FIG. 10 is the protective effect of benzylated docosahexaenoic acid on hypoxic-ischemic neurons.

Detailed Description

The technical scheme of the invention is further described below by combining specific embodiments.

Example 1

This example provides a method for simultaneously preparing two benzylated omega-3 unsaturated fatty acids comprising the steps of:

(1) 10mL of microalgae oil is measured, 100mL of 0.5M NaOH-CH is added ₃ The OH solution was reacted in a water bath at 50℃for 60min to effect ester hydrolysis, with shaking during the reaction. After ester hydrolysis is finished, placing the reaction liquid in a separating funnel, and standing the solution; 30mL of n-hexane and distilled water were added, and the unsaponifiable matter was removed, leaving a hydrated layer. Then 30mL of 3mol/L hydrochloric acid is added into the hydration layer

Standing the solution for 5min; adding 30mL of normal hexane into the hydrolyzed fatty acid for extraction, and repeating for 2-3 times; washing n-hexane layer with distilled water to neutrality, dewatering with anhydrous sodium sulfate, standing, and rotary evaporating to obtain mixture of free omega-3 unsaturated fatty acid.

(2) Into a 250mL Erlenmeyer flask, 100mL of methylene chloride was added as a reaction solvent, and 250 mu L, EDC. HCl 156mg and HOBT. H of the mixture of free omega-3 unsaturated fatty acids prepared in the step (1) was added ₂ O103 mg and 263. Mu.L triethylamine were mixed uniformly and reacted under stirring by a magnetic stirrer for 20 hours. After the completion of the reaction, 80. Mu.L of benzylamine was added thereto to carry out an acid-amine condensation reaction for 4 hours.

(3) After the acid amine condensation reaction is completed, placing the reaction solution in a rotary evaporator, and carrying out vacuum concentration at 35 ℃; after rotary evaporation and drying, 200mL of 0.3M HCl aqueous solution is added, and water-soluble and acidic substances are dissolved and are subjected to ultrasonic treatment for 2-3 min. Then 200mL of n-hexane is added, and ultrasonic treatment is carried out for 10min, so that the macamide analogue is dissolved into the n-hexane; after delamination, the lower hydration layer was discarded to obtain the upper n-hexane solution. Then adding 50mL of 0.29mol/L hydrochloric acid aqueous solution, shaking uniformly, shaking vigorously, discarding the lower hydration layer, and leaving an n-hexane organic layer; then 50mL of 0.125mol/L NaOH solution is added to the n-hexane organic layer, the mixture is shaken well, the mixture is vigorously shaken (ultrasound can be carried out when the solution is turbid), and the lower hydration layer is discarded. Repeating the above steps for 2-3 times until the n-hexane layer is clear and transparent, and has no obvious impurity. Finally, the n-hexane solution (i.e. the n-hexane organic layer remained after the acid-washing and alkali-washing) is subjected to rotary evaporation to obtain the maca amide mixture.

And (3) loading the macamide mixture through liquid chromatography, separating by using methanol/water (90:10) as a mobile phase, collecting a target peak through a C18 chromatographic column, carrying out vacuum rotary evaporation concentration on the collected methanol solution, blowing off an organic solvent by using a water bath nitrogen instrument, and carrying out freeze-drying treatment by using a freeze dryer when a small amount of water is remained to obtain the benzylamine omega-3 unsaturated fatty acid.

The chromatogram obtained by separating the maca amide mixture by liquid chromatography is shown in figure 1, and the chromatogram is shown in figure 1, wherein the chromatogram has complex components and comprises a large amount of various non-target fatty acids in microalgae oil, benzylamine products thereof, synthetic reagent residues, synthetic byproducts and the like; and the retention time is 23min and 32min, two main chromatographic peaks appear, a fraction collector is used for collecting a P1 peak and a P2 peak, the P1 peak and the P2 peak are respectively subjected to vacuum rotary evaporation concentration, then a nitrogen blower is used for removing methanol and water, and then a freeze drying method is used for completely removing the methanol and the water.

And analyzing the content and the components of the collected P1 and P2 components by adopting a high performance liquid chromatography and a mass spectrometry. The high performance liquid chromatography chromatogram of the P1 peak in the graph in FIG. 1 is shown in FIG. 2, and the purity of the substance corresponding to the P1 is calculated to be more than 95% according to the graph in FIG. 2; and the secondary mass spectrum of the substance is shown in figure 3, and the substance corresponding to the P1 peak can be identified as benzylamine eicosapentaenoic acid according to figure 3.

The high performance liquid chromatogram of the P2 peak in the graph in FIG. 1 is shown in FIG. 4, and the purity of the substance corresponding to the P2 is calculated to be more than 95% according to the graph in FIG. 4; and the secondary mass spectrum of the substance is shown in figure 5, and the substance corresponding to the P2 peak can be identified as benzylamine docosahexaenoic acid according to figure 5.

The total conversion of benzylated eicosapentaenoic acid and benzylated docosahexaenoic acid was 28.7% based on unsaturated fatty acid.

Example 2

This example provides a method for simultaneously preparing two benzylated omega-3 unsaturated fatty acids comprising the steps of:

(1) 10mL of fish oil is measured, 200mL of 0.2MNaOH-CH is added ₃ The OH solution reacts in a water bath at 25 ℃ for 120min to carry out ester hydrolysis,the oscillation is maintained during the reaction. After ester hydrolysis is finished, placing the reaction liquid in a separating funnel, and standing the solution; 30mL of n-hexane and distilled water were added, and the unsaponifiable matter was removed, leaving a hydrated layer. Then 30mL of 3mol/L hydrochloric acid solution is added into the hydration layer, and the mixture is kept stand for 5min; adding 30mL of normal hexane into the hydrolyzed fatty acid for extraction, and repeating for 2-3 times; washing n-hexane layer with distilled water to neutrality, dewatering with anhydrous sodium sulfate, standing, and rotary evaporating to obtain mixture of free omega-3 unsaturated fatty acid.

(2) Into a 250mL Erlenmeyer flask, 100mL of methylene chloride was added as a reaction solvent, 200. Mu. L, DIC 195mg of the mixture of the free omega-3 unsaturated fatty acids prepared in the step (1), 95mg of HOAT and 234. Mu.L of DIPEA were added, and the mixture was uniformly mixed and reacted for 12 hours under stirring by a magnetic stirrer. After the completion of the reaction, 80. Mu.L of m-methoxybenzylamine was added thereto to carry out an acid-amine condensation reaction for 8 hours.

(3) After the acid amine condensation reaction is completed, placing the reaction solution in a rotary evaporator, and carrying out vacuum concentration at 35 ℃; after rotary evaporation and drying, 200mL of 0.3M HCl aqueous solution is added, and water-soluble and acidic substances are dissolved and are subjected to ultrasonic treatment for 2-3 min. Then 200mL of n-hexane is added, and ultrasonic treatment is carried out for 10min, so that the macamide analogue is dissolved into the n-hexane; after delamination, the lower hydration layer was discarded to obtain the upper n-hexane solution. Then adding 50mL of 0.29mol/L hydrochloric acid aqueous solution, shaking uniformly, shaking vigorously, discarding the lower hydration layer, and leaving an n-hexane organic layer; then 50mL of 0.125mol/L NaOH solution is added to the n-hexane organic layer, the mixture is shaken well, the mixture is vigorously shaken (ultrasound can be carried out when the solution is turbid), and the lower hydration layer is discarded. Repeating the above steps for 2-3 times until the n-hexane layer is clear and transparent, and has no obvious impurity. Finally, the n-hexane solution (i.e. the n-hexane organic layer remained after the acid-washing and alkali-washing) is subjected to rotary evaporation to obtain the maca amide mixture.

And (3) loading the macamide mixture through liquid chromatography, separating by using methanol/water (90:10) as a mobile phase, collecting a target peak through a C18 chromatographic column, carrying out vacuum rotary evaporation concentration on the collected methanol solution, blowing off an organic solvent by using a water bath nitrogen instrument, and carrying out freeze-drying treatment by using a freeze dryer when a small amount of water is remained to obtain m-methoxybenzyl aminated omega-3 unsaturated fatty acid.

After the macamide mixture is separated by liquid chromatography, a fraction collector is used for collecting fractions corresponding to two main chromatographic peaks, vacuum rotary evaporation concentration is carried out on the fractions respectively, then a nitrogen blower is used for removing methanol and water, and a freeze drying method is used for completely removing the methanol and the water. And (3) carrying out content and ingredient analysis on the components by adopting a high performance liquid chromatography and mass spectrometry. Two main peaks are identified as m-methoxybenzylamine-eicosapentaenoic acid and m-methoxybenzylamine-docosahexaenoic acid respectively; the purity of the corresponding substances is more than 95 percent;

the total conversion of m-methoxybenzylated eicosapentaenoic acid and m-methoxybenzylated docosahexaenoic acid was 22.7% based on unsaturated fatty acid.

Comparative example 1

(1) 100mL of methylene chloride was added to a 250mL Erlenmeyer flask as a reaction solvent, and 250. Mu. L, EDC. HCl 156mg, HOBT. H of microalgae oil was added ₂ O103 mg and 263. Mu.L triethylamine were mixed uniformly and reacted under stirring by a magnetic stirrer for 20 hours. After the completion of the reaction, 80. Mu.L of benzylamine was added thereto to carry out an acid-amine condensation reaction for 4 hours.

(3) After the acid amine condensation reaction is completed, placing the reaction solution in a rotary evaporator, and carrying out vacuum concentration at 35 ℃; after rotary evaporation and drying, 200mL of 0.3M HCl aqueous solution is added, and water-soluble and acidic substances are dissolved and are subjected to ultrasonic treatment for 2-3 min. Then 200mL of n-hexane is added, and ultrasonic treatment is carried out for 10min, so that the macamide analogue is dissolved into the n-hexane; after delamination, the lower hydration layer was discarded to obtain the upper n-hexane solution. Then adding 50mL of 0.29mol/L hydrochloric acid aqueous solution, shaking uniformly, shaking vigorously, discarding the lower hydration layer, and leaving an n-hexane organic layer; then 50mL of 0.125mol/L NaOH solution is added to the n-hexane organic layer, the mixture is shaken well, the mixture is vigorously shaken (ultrasound can be carried out when the solution is turbid), and the lower hydration layer is discarded. Repeating the above steps for 2-3 times until the n-hexane layer is clear and transparent, and has no obvious impurity. Finally, the n-hexane solution (i.e. the n-hexane organic layer remained after the acid-washing and alkali-washing) is subjected to rotary evaporation to obtain the maca amide mixture.

Loading the macamide mixture through liquid chromatography, and separating by using methanol/water (90:10) as a mobile phase and a C18 preparation column; fractions at the retention times of 23min and 32min were collected using a fraction collector, and the fractions were concentrated by rotary evaporation under vacuum, followed by removal of methanol and water using a nitrogen blower, and then complete removal of methanol and water using a freeze-drying method. And (3) carrying out content analysis on the components by adopting a high performance liquid chromatography. The purity of the benzylamine-eicosapentaenoic acid is identified to be 14.3%, and the benzylamine-docosahexaenoic acid is identified; the purity of the corresponding material was 9.82%.

The total conversion of benzylated eicosapentaenoic acid and benzylated docosahexaenoic acid was 0.73% based on the unsaturated fatty acid.

Comparative example 2

0.02mol HOAT,0.02mol EDC.HCl and 0.02mol of DIEPA are respectively weighed into 30mL of DCM, 0.01mol of m-methoxybenzylamine and 0.01mol of eicosapentaenoic acid are added, and the mixture is stirred at room temperature overnight; 50ml of deionized water is added, stirring is carried out for 30min at room temperature, chloroform is added for extraction, and the volume of an extraction layer is not reduced any more until the product is obtained. The main component of the product is m-methoxybenzylamine-eicosapentaenoic acid with the purity of 45.5 percent.

Example 3

This example explores the protective effect of benzylated eicosapentaenoic acid and benzylated docosahexaenoic acid on hypoxic ischemic brain injury and hypoxic ischemic neurons in neonatal mice.

1. Protection of anoxic and ischemic neurons by benzylamine docosahexaenoic acid

Performing hypoxia treatment according to the following method, and establishing a hypoxia-ischemia brain injury model in vitro:

c57BL/6 mice on postnatal day 7 were anesthetized with isoflurane to be unresponsive to noxious stimuli (deep anesthesia). After cleansing the skin with alcohol, a midline ventral incision was made in the anterior cervical portion. Under an dissecting microscope, the left common carotid artery is peeled off, the electric coagulator is used for cauterization, and the skin incision is bonded by medical biological glue. The mice remained warm after surgery and returned to their home cage after 30min of recovery on a heating pad (33 ℃). After 2 to 3 hours, the animals are placed in a closed anoxic box with constant temperature water bath at 37 ℃ and 8%O is continuously introduced into the anoxic box ₂ +92％N ₂ The gas flow rate of the mixed gas is 1.5 to the upper part2L/min, for 90min. After hypoxia exposure, mice were recovered on a heated pad (33 ℃) for 30min and then returned to their home cage.

The dyeing method according to the present embodiment includes:

(1) TTC staining experiments.

The mice were anesthetized and then were either directly or perfused with normal saline. Freezing at-20deg.C in refrigerator for about 20min, cutting into 4-5 pieces, and cutting into pieces every 2 mm. Placing the slice in 1% -2% TTC, and dyeing for 15-30 min in dark, and turning over the brain slice from time to make the slice contact with the dyeing liquid uniformly.

(2) Nib staining experiment

Freezing slice is fixed with 4% paraformaldehyde for 30-40 s, washing with water for 2-3 s, dyeing with Nile's dyeing liquid for 40min, washing with distilled water, sequentially placing in 70% ethanol for 3-5 s,80% ethanol for 3-5 s,95% ethanol for 3-5 s, absolute ethanol (I) for 3-5 s, absolute ethanol (II) for 3-5 s, xylene (I) for 3min, xylene (II) for 3min, and finally sealing with neutral resin.

The neurobehavioral experiments involved in this example included:

(1) Cliff avoidance test: the mice were placed on the border of the plank platform and the time for the mice to recede or side-shift away from the border was recorded, the shorter the time, the better the neuroreflex development was demonstrated.

(2) Forelimb suspension experiments: an iron wire with the diameter of 1.5mm is placed on a frame with the height of 15cm, a soft elastic sponge cushion is placed below the iron wire, a mouse is placed on the iron wire lightly to ensure that timing is started after the forelimbs of the mouse hold the iron wire, the timing is ended when the mouse falls, the measurement of the experimental mouse is performed three times once, and the longest time is recorded as forelimb suspension time. The experiment can reflect the muscle strength and the exercise coordination ability of the mice, and the shorter the time is, the worse the muscle strength and the exercise coordination ability are represented.

Experimental group settings:

60C 57BL/6 mice of seven days of age were randomly divided into three groups of 20 mice each, the three groups being: a sham-operated control group, a hypoxic ischemia+pbs model group, and a hypoxic ischemia+benzylated eicosapentaenoic acid group. After the establishment of the hypoxia-ischemia model of the mice, the benzyl aminated eicosapentaenoic acid (250 mug/mouse) is injected into the abdominal cavity for 3 days; PBS groups were injected with equal amounts of PBS; the sham group was not specially treated. The three groups of mice were kept under the same conditions.

Results:

fig. 6 is a TTC staining pattern at day 7 post-operation for three groups of neonatal mice. In the figure, it can be seen that the mice in the sham surgery group had normal left brain morphology and the PBS model group had damaged left brain tissue structure, resulting in a significant increase in infarct size. After the treatment of the benzylamine eicosapentaenoic acid, the damage of brain tissues is reduced, and the infarct area of the cerebral ischemia area is reduced, which shows that the benzylamine eicosapentaenoic acid has the effect of relieving the anoxic ischemic brain damage.

FIG. 7 is a Nitype staining pattern of three groups of neonatal mice on day 7 post-surgery. In the figure, the neurons in the sham operation group are normal in morphological structure, the number of Nib bodies is large and are uniformly distributed, the neurons in the brain tissue of the PBS model group are seriously damaged, and the number of Nib bodies is obviously reduced. After the treatment of benzylamine eicosapentaenoic acid, the number of Nib's corpuscles is increased, and the damage degree of neurons in brain tissues is improved.

On days 1, 3 and 7 after the neonatal mice underwent the hypoxic-ischemic treatment, cliff avoidance test and forelimb suspension test were performed on three groups of mice, respectively, and the test results are shown in fig. 8 and 9, respectively.

Comparing the results of the PBS group and the benzylated eicosapentaenoic acid group in FIGS. 8-9, it is clear that the cliff avoidance test time of the mice in the benzylated eicosapentaenoic acid group is shortened, and the forelimb suspension time is prolonged, which indicates that the benzylated eicosapentaenoic acid improves the neurological deficit caused by hypoxia ischemia of the newborn mice.

2. Protection of anoxic and ischemic neurons by benzylamine docosahexaenoic acid

Taking brain tissue of a C57BL/6 mouse within 24 hours of new generation, shearing, adding papain, and digesting for 20 minutes at 37 ℃; after centrifugation, adding a neuron culture medium to perform cell culture; after 7-9 days, cell tests were performed.

The cell culture medium was changed to serum-free DMEM, 1mM benzylated docosahexaenoic acid was added, and subjected to 3 hours of hypoxia treatment (in vitro simulated hypoxia-ischemia damage), and after the cell culture medium was changed to neuronal culture medium and normal culture was performed for 24 hours, the survival of the cells was detected by CCK-8 assay.

The survival of the cells is shown in FIG. 10. FIG. 10 shows that neurons undergo hypoxia and ischemia with significantly reduced survival, and that benzylated docosahexaenoic acid treatment significantly improves cell survival, and protects neurons.

The experimental results reflect that the behenyl hexaenoic acid benzylamine and the behenyl hexaenoic acid benzylamine have a protective effect on ischemic brain injury, so that the behenyl hexaenoic acid benzylamine and the behenyl hexaenoic acid benzylamine can be used for preparing medicines for treating ischemic brain injury and can be used for preventing and treating ischemic brain injury diseases including neonatal ischemic hypoxic brain diseases, cerebral apoplexy, ischemia-reperfusion injury, transient cerebral ischemia attacks and the like. In actual production, the benzylated docosahexaenoic acid and/or the benzylated docosahexaenoic acid can be combined with any one or more of the pharmaceutical excipients such as solvent, propellant, solubilizer, cosolvent, emulsifier, colorant, binder, disintegrant, filler, lubricant, wetting agent, osmotic pressure regulator, stabilizer, glidant, flavoring agent, preservative, suspending agent, coating material, aromatic, anti-adhesive agent, integrating agent, permeation enhancer, pH regulator, buffer, plasticizer, surfactant, foaming agent, antifoaming agent, thickener, inclusion agent, humectant, absorbent, diluent, flocculant and deflocculant, filter aid, release retarder, etc., and the composition is prepared into oral preparation, injection preparation or sublingual buccal preparation.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (1)

1. A preparation method of benzylamine omega-3 unsaturated fatty acid is characterized by comprising the following steps: the method comprises the following steps:

(1) Carrying out ester hydrolysis on a raw material rich in omega-3 unsaturated fatty acid and esters thereof to obtain a mixture of free omega-3 unsaturated fatty acid, wherein the raw material rich in omega-3 unsaturated fatty acid and esters thereof is selected from microalgae oil; the method for hydrolyzing the ester comprises the following steps:

adding microalgae oil into sodium hydroxide-methanol solution, and reacting at 10-60 ℃; the concentration of the sodium hydroxide-methanol solution is 0.1-2 mol/L; the volume ratio of the microalgae oil to the sodium hydroxide-methanol solution is 1: 2-1: 20, the hydrolysis time of the ester is 30-120 min;

(2) Adding a condensing agent, a catalyst and an alkali additive into a mixture of free omega-3 unsaturated fatty acid, reacting for 10-24 h, and then adding benzylamine for acid-amine condensation reaction; the molar ratio of the benzylamine to the free omega-3 unsaturated fatty acid to the condensing agent to the catalyst to the alkali additive is 0.5-2:0.5-2:1-5; the condensing agent is EDC.HCl, the catalyst is HOBt, and the alkali additive is triethylamine; the temperature of the acid amine condensation reaction is 10-65 ℃ and the reaction time is 2-10 h;

(3) Drying the reaction solution after the reaction in the step (2), extracting, separating by liquid chromatography, and drying to obtain benzylamine omega-3 unsaturated fatty acid; the extraction method comprises the following steps: adding an acid solution and an organic solvent, and taking an organic layer; fully oscillating the organic layer with alkali solution, and completely reacting; then adding an acid solution for washing, and taking an organic layer solution; the acid solution adopts HCl solution, and the organic solvent is n-hexane; the liquid chromatographic separation is carried out by taking methanol/water solution as a mobile phase and a C18 chromatographic column, wherein the volume ratio of methanol to water is 90:10;

the chemical structural general formula of the benzylamine omega-3 unsaturated fatty acid is as follows:

wherein R is ₁ =all-cis-5, 8,11,14, 17-eicosapentaenoic acid or all-cis-4, 7,10,13,16, 19-docosahexaenoic acid; r is R ₂ =H；R ₃ =H。

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