TWI397522B - Method for preparing δ7,11,14-eicosatrienoic acid (δ7-etra), composition containing the same and use thereof - Google Patents

Description

Method for preparing Δ7,11,14-eicosatrienoic acid (Δ7,11,14-eicosatrienoic acid), group thereof Compound and its application

The present invention relates to a process for producing Δ7,11,14-eicosatrienoic acid (Δ7,11,14-eicosatrienoic acid) and its use.

△7-ETrA (△7,11,14-eicosatrienoic acid, also known as bishomopinolenic acid or dihomopinolenic acid) is a rare polyunsaturated fatty acid (PUFA) found in nature. It is only found in the gymnosperm The seeds of Pinus are only about 0.7% of the total fatty acids of pine seed oil (Wolff et al., JAOCS 74, 1583-1586, 1997). Since the content of this fatty acid is scarce, separation and purification are not easy, so so far, there is no literature on the biological activity and function of the industry.

In the past, certain specialty fatty acids were limited, lacked commercial sources, or were expensive, limiting the design and operation of the experiment. Since the 1970s, C2-extended synthesis of fatty acids has been used in various fatty acids (Meyers et al., J. Org. Chem. 39, 2778-2783, 1974; Herslof and Gronowitz, Chem. Scr. 22, 230-235, 1983). However, the synthesis of polyunsaturated fatty acids has only recently been successfully completed. In 2006, Kuklev and Smith published a series of organic synthesis methods to successfully convert some fatty acids of the n-3 PUFA family, such as alpha-linolenic acid (ALA) and stearidonic acid (SDA). Eicosapentaenoic acid (EPA) is synthesized as n-3 eicosatrienoic acid (n-3 ETrA), n-3 arachidonic acid (n-3 arachidonic acid) (n-3) AA) with n-3 docosapentaenoic acid (n-3 DPA) (Kuklev and Smith, Chem. Phys. Lipids 144, 172-177, 2006). However, there is no literature mentioning the method of artificial synthesis of Δ7-ETrA.

Previous studies have indicated that rosin oleic acid (PNA), the precursor of △7-ETrA, has an anti-inflammatory effect (Chuang et al., Lipids, 44: 217-224, 2009), and is also formulated with other fatty acids. Has similar efficacy (Cain et al., U.S. Patent No. 6,479,070, 2002). However, according to the results of animal and cell culture experiments, all of PNA can be biologically effective in cell culture, probably because PNA can be metabolized to Δ7-ETrA in cells, so △7-ETrA may be One of the main anti-inflammatory factors.

In addition, pine nut oil rich in PNA in recent years is also considered to be an important dietary factor for regulating appetite and weight loss (Hughes et al., Lipids Health Dis. 7, 6, 2008; Pasman et al., Lipids Health Dis. 7,10, 2008). In view of the correlation between PNA and Δ7-ETrA in anti-inflammatory effects, it is possible that the appetite and weight loss effects caused by PNA are also related to its metabolite Δ7-ETrA.

In view of the possible medical effects of Δ7-ETrA and the lack of its preparation methods, the inventors of the present invention are committed to research and innovation, and after years of painstaking research, developed the Δ7,11,14-eicosatrienoic acid of the present invention. (Δ7,11,14-eicosatrienoic acid) preparation method and application thereof.

One of the objects of the present invention is to propose a Δ7,11,14-eicosatrienoic acid (△7,11,14-eicosatrienoic acid, hereinafter referred to as Δ7-ETrA), including conversion from pine nut oleic acid (PNA) to Δ7,11,14-eicosatriene via a series of chemical synthesis acid.

Another object of the invention is to propose a composition comprising Δ7-ETrA and a physiologically or pharmaceutically acceptable carrier.

Another object of the present invention is to provide an anti-inflammatory use of Δ7-ETrA and the above composition.

A method for producing Δ7,11,14-eicosatrienoic acid (Δ7-ETrA) which satisfies the above purpose, comprising the following steps: 1. From pine nut oil Saponification, urea saturation and extraction concentrates 18% carbon pine nut oleic acid (PNA; △5,9,12-octadecatrienoic acid) in the form of free fatty acids; 2. Free octadecyl fatty acid ester 3. Alcoholization of the octadecyl fatty acid ester; 4. Bromination of the octadecyl fatty acid ester to form the octadecyl fatty acid ester bromide; 5. The octadecyl fatty acid ester bromide and 2, 4,4-Trimethyl-2-oxazoline (2,4,4-trimethl-2-oxazoline) to form a dimethyloxazoline reactant having twenty carbons; 6. Converting a 20-carbon dimethyl-2-oxazoline reactant into a twenty-carbon long-chain unsaturated fatty acid; and 7. using liquid chromatography for Δ7-ETrA separation and purification.

In the above method, the proportion of PNA in pine nut oil is basically increased first, and then the linoleic acid isomer used in the Kuklev and Smith method as a raw material (Fig. 1), so Kuklev and Smith can be used. The method comprises the organic synthesis of polyunsaturated fatty acids; then the separation and purification of Δ7-ETrA is carried out by liquid chromatography.

In the first step of the method of the present invention, linoleic acid (LA) and pine oleic acid (PNA), which are main components of pine nut oil, are obtained from pine nut oil by saponification, urea saturation and extraction. Concentrate in the form of free fatty acids.

In the first step of the process of the invention, the saponification step can be carried out with an alkali metal or alkaline earth metal hydroxide such as sodium hydroxide, potassium hydroxide, calcium hydroxide or the like. After saponification, the fatty acid is first dissociated with hydrochloric acid, and then the fatty acid is extracted with a non-polar organic solvent such as hexane. Subsequent to the urea saturation method, for example, in one embodiment, a urea-methanol solution is used to form crystals. Then, the obtained urea-methanol-oil solution was filtered. The filtrate is extracted with a non-polar organic solvent such as petroleum ether and concentrated to give a free fatty acid. This step can increase the concentration of pine nut oleic acid by several times, for example by about four times in one embodiment.

In the second step of the process of the invention, the resulting free fatty acid is esterified, for example, methyl ester, ethyl ester, propyl ester or the like. In one embodiment, methyl esterification is carried out using a boron trifluoride methanol-complex solution.

In the third step of the process of the invention, the resulting free fatty acid ester is subjected to alcoholization. In one embodiment, the system using LiAlH ₄ reduction of the esters to alcohols.

In the fourth step of the process of the invention, the resulting fatty alcohol is brominated. In one embodiment, the resulting alcohol is brominated using triphenylphosphine in bromine/dichloromethane. The bromide is purified by liquid chromatography such as thin layer chromatography (TLC).

In the fifth step of the process of the invention, the resulting bromide is reacted with 2,4,4-trimethl-2-oxazoline. In one embodiment, the reaction is carried out in tetrahydrofuran in the presence of n-butyllithium at -78 °C under nitrogen. After the reaction solution is acidified with hydrochloric acid, the desired reaction product is extracted with an organic solvent such as ethyl acetate.

In the sixth step of the process of the invention, the dimethyloxazoline reaction product is converted to a twenty carbon long chain unsaturated fatty acid ester. In one embodiment, the reaction is carried out by alcoholysis with methanol and acetonitrile, and hydrolysis with a base such as an alkali metal hydroxide such as sodium hydroxide, potassium hydroxide or the like followed by acidification with hydrochloric acid.

In the seventh step of the method of the invention, the resulting mixed product is purified by liquid chromatography such as thin layer chromatography (TLC). In one embodiment, silver ion thin film chromatography is used to separate the toluene/ethyl acetate/acetic acid (95/5/1, v/v/v). purification.

The twenty carbon fatty acid formed by the method of the present invention was analyzed by gas chromatography and gas chromatography to analyze the mass spectrometer, and it was confirmed that one of them was the C2-extended product Δ7-ETrA of PNA.

In another aspect, the invention provides a composition comprising Δ7-ETrA With a physiologically or pharmaceutically acceptable carrier.

The term "physiologically or pharmaceutically acceptable carrier" means that the substance or composition used must be compatible with the other ingredients of the formulation and not deleterious to the patient.

The composition of the present invention can be prepared into a dosage form suitable for use in the composition of the present invention by a technique known to those skilled in the art from the above-mentioned Δ7-ETrA and a physiologically or pharmaceutically acceptable carrier. Wherein the dosage form comprises, but is not limited to, a solution, an emulsion, a suspension, a powder, a lozenge, a pill, a lozenge, a troche, a chewing gum, a capsule, and the like or the like. The dosage form of the present invention.

Wherein the pharmaceutically acceptable carrier may comprise one or more agents selected from the group consisting of solvents, emulsifiers, suspending agents, decomposers, binders, excipients, stabilizers, chelating agents, diluents, gelling agents. Agents, preservatives, lubricants, surfactants, and other carriers similar or suitable for use in the present invention.

In the above composition, one or more dissolution aids, buffers, preservatives, coloring agents, perfumes, flavoring agents and the like which are usually used in the field of preparation may be appropriately added as needed.

On the other hand, the Δ7-ETrA synthesized or purified by the method of the present invention or a combination thereof is cultured in a macrophage, and tested by a lipopolysaccharide (LPS)-stimulated cell culture model of an inflammatory mode. It was found that a large amount of Δ7-ETrA was absorbed by cells, transferred into the cell phospholipids, and reduced the proportion of the eicosanoids precursor, arachidonic acid (AA), in the phospholipids. In addition, by regulating inducible nitric oxide synthase (iNOS) and type 2 cyclooxygenase (type II) Cyclooxygenase; COX-2) to reduce nitric oxide (NO), prostaglandin E2 (PGE2), cytokines (such as interleukin-6; IL-6) and other inflammatory biochemical mediators To slow down the inflammatory response; thereby inhibiting inflammation-related diseases such as cardiovascular disease, cancer, and arthritis. This has not been disclosed in the prior art.

Accordingly, the present invention provides anti-inflammatory use of Δ7-ETrA or a combination thereof.

The invention is illustrated by the following examples, but the invention is not limited by the following examples.

△7-ETrA chemical synthesis Embodiment 1

A. Preparation of free PNA

Saponification

15 g of commercially available pine nut oil was added to 40 ml of NaOH solution (6 g of NaOH, 0.06265 g of Na ₂ EDTA-2Na, 20 ml of H ₂ O, 20 ml of EtOH), and heated at 65 ° C for 40 minutes. After cooling to room temperature, deionized water (ddH ₂ O) was added and stirring was continued. 160 ml of n-hexane was added and stirred for 1 hour. The n-hexane layer was removed and the bottom layer was retained. Add 6 N HCl to the white precipitate and shake up and down to pH <1. The bottom layer was removed, an appropriate amount of n-hexane was added, and reverse osmosis (RO) water was added for extraction. The aqueous layer was removed, and the n-hexane layer was partitioned from Na ₂ SO ₄ and evaporated.

Commercially available pine nut oil was subjected to methylation treatment, and the composition and content of each fatty acid in pine nut oil were analyzed by gas chromatography (6890N, Agilent Technologies, USA), and the results are shown in Fig. 2. In the figure, C17:0 is the internal standard we added, and the analysis time has three higher peaks between 11 and 13 minutes, respectively. C18: 1n-9, C18: 2n-6 and PNA three unsaturated fatty acids, followed by C16:0 saturated fatty acids and C18:0 saturated fatty acids.

After the GC spectrum is analyzed by software, it can be converted into the following values. The results in Table 1 show that pine nuts oil contains a variety of fatty acids, of which C18:2n-6 accounted for the highest (46.4%), C18:1n-9 accounted for 26.6%, and PNA accounted for 16.8%.

2. Urea saturation

30 g of urea was dissolved in 90 ml of methanol and heated to 65 ° C until clear to clear to prepare a urea-methanol solution. About 15 g of the free PNA obtained above was added and stirred for 10-15 minutes. The mixture was allowed to stand at room temperature overnight, and crystals and precipitates were produced.

A free pine nut oil fatty acid solution of 15.5 ± 0.3 g was subjected to a urea saturation test, and the obtained product was analyzed by a gas chromatograph. The results from Figure 3 show that urea saturation significantly reduces the ratio of C18:1n-9 to C18:2n-6, but increases the proportion of PNA in the mixed solution.

After the map was analyzed by software, the results in Table 2 showed that the ratio of C18:2n-6 decreased from 43.4% to 34.6%, and the PNA from the original after the urea saturation experiment 16.75% rose to 61.7%.

The pine nut oil 15.5±0.3 g was purified by urea saturation to obtain 14.7±0.9 free pine nut oil fatty acid. The calculated recovery was 94.9±3.9%.

3. Extraction

The urea-methanol-oil solution was filtered using a filter paper. The first (1 ^st ) filtered solution was placed in a refrigerator at 4 ° C for 1 hour and then at -20 ° C for 2 hours. The urea-methanol-oil solution was further washed by adding 80 ml of methanol, and filtered again. The second (2 ^nd ) filtration solution was placed in a refrigerator at 4 ° C for 1 hour and then at -20 ° C for 2 hours. 1 ^st and 2 ^nd placing two 500 ml separatory funnel, respectively. Add 50 ml of water and 150 ml of petroleum ether at 1 ^st and shake up and down for 5 minutes. Allow to stand in two layers and drain the bottom layer to another 500 ml separatory funnel. Petroleum ether layer was collected and filtered again with Na ₂ SO ₄ removal of water using a filter paper. Add 50 ml of water and 150 ml of petroleum ether to the bottom layer and shake up and down for 5 minutes. Petroleum ether layer was collected and filtered again with Na ₂ SO ₄ removal of water using a filter paper. Concentrate using a reduced pressure vacuum concentrator. Repeat the above steps for 2 ^nd . Weigh the oil.

B. Methylation

12 tubes were weighed and weighed 0.25 g of the above oil, for a total of about 3 g. 2 ml of a boron trifluoride-methanol complex solution was separately added under heating. After heating for 20 minutes, it was cooled at room temperature. Add 2 ml of 0.9% NaCl and 4 ml of hexane. Cover the lid up and down and shake it about 30~40 times. Centrifuge at 1500 rpm for 1 minute. Take hexane The layer was dried with nitrogen.

C. Alcoholization

A 900 mg (24 mmol) LiAlH _{4 was} placed in 200 ml of diethyl ether and stirred at room temperature for 1 hour. 3.223 g (11 mmol) of the above methylated sample was added to the above solution and stirred for 2 hours. After the reaction, 20 ml of ethyl acetate and 20 ml of ethanol, 200 ml of water and 30 ml of 1N HCl were added to the mixture for continuous stirring. The ether layer was removed. The aqueous layer was extracted with 200 mL of ethyl acetate to leave an organic layer. Washed with water (2 × 250 mL) and saturated brine (200 mL) extracted wash, Na ₂ SO ₄ removal of water, stirred for 30 minutes. The extract was evaporated in vacuo to give a colourless oil.

3.1±0.1 g of methylated free pine nut oil fatty acid was taken, and the fatty acid methyl ester was reduced to alcoholate by the action of LiAlH ₄ , and then analyzed by thin layer chromatography. Figure 4 is the result of separation of the LiAlH ₄ reaction product via TLC. According to the literature, the Rf value of the newly synthesized product is about 0.4, and the result of this experiment is also about 0.4.

The alcoholate obtained by the conversion of LiAlH ₄ was 2.70 ± 0.02 g, and the recovery was 86.2 ± 3.4%.

D. Bromination

4.86 g of triphenylphosphine (Ph ₃ P) was placed in 160 ml of CH ₂ Cl ₂ . 1.16 g of Br _{2 was} added to 160 ml of CH ₂ Cl ₂ . The two solutions were mixed and stirred rapidly (addition of a little Ph ₃ P to completely react Br ₂ ) to form a pale yellow almost colorless solution. About 140 ml of Ph ₃ PBr _{2 was} added to about 2.62 g of the product of Step C (dissolved in CH ₂ Cl ₂ ) and stirred for 30 minutes. Then 50 ml of methanol was added. TLC was used to detect whether the reaction was complete. The mixture was extracted with hexane (3 x 20 mL). Purified by TLC (120 g EtOAc, 1% diethyl ether /hexane). Rf 0.4 (alcohol), Rf 0.95 (product).

The product of LiAlH ₄ was previously reacted with Ph ₃ PBr ₂ , and the new reactant was separated by column chromatography. Figure 5 shows the results of confirmation of the new reactants after purification and collection using TLC. Each point in the figure is a brominated synthesis product of pine nut oil fatty acid after separation.

The distance traveled by the new brominated product was calculated to be 0.95, similar to the literature. The yield of the brominated composition was 2.33 ± 0.14 g, and the recovery was 86.7 ± 4.5% (Table 3.5).

E. Bromide and 2,4,4-trimethyl-2-oxazoline reaction

3.0 g (26.4 mmol) of 2,4,4-trimethyl-2-oxazoline was placed in 70 ml of anhydrous tetrahydrofuran, kept under nitrogen and maintained at -78 ° C (dry ice plus acetone). Slowly inject (over 5 minutes) 12 ml of 2.44 M n-Butyllithium. 2.37 g (8.8 mmol) of the product was dissolved in 25 ml of tetrahydrofuran to a blue solution over ca. 20 min. The reaction was continued at -78 ° C for 2 hours. Inject 400 ml of ethyl acetate. Acidified by the addition of 200 ml of 1 N HCl. Washed with water (2 × 250 mL) and saturated brine (250 mL) the mixture was extracted wash, Na ₂ SO ₄ removal of water, stirred for 30 minutes. The extract was evaporated in vacuo to a yellow oil. Purified by TLC.

The brominated synthesis product was mixed with 2,4,4-trimethyl-2-oxazoline and reacted at -70 ° C to give the dimethyloxazoline product. The results in Figure 6 show that the Rf value of this product is about 0.5 as reported in the literature. The upper two points are unsynthesized complete products, while the larger intermediate material is the product of this time.

The product of pine nut oil free fatty acid dimethyloxazoline was 2.04 ± 0.17 g, and the recovery was about 87.3 ± 3.0% (Table 3.6).

F. alcoholysis

The above product was dissolved in 50 ml of methanol and 5 ml of acetonitrile was dissolved in 100 ml of methanol. The mixture was heated to boiling reflux for 12 hours, cooled and dissolved in EtOAc EtOAc. The mixture with water (2 × 250 mL) and saturated brine (250 ml) extracted wash, Na ₂ SO ₄ water removal, and evaporated in vacuo to give a yellow oil. The oil was purified through a column (60 g EtOAc, EtOAc EtOAc)

The fatty acid dimethyloxazoline product and the acetonitrile chloride/methanol solution can produce unsaturated fatty esters, which are separated by column chromatography and then analyzed by thin layer chromatography. The black spots in the left area of the seven (A) are unreacted reactants, and the right side is the new synthetic product of this reaction. After calculation, the reactant had an Rf value of 0.7.

The unsaturated fatty ester was collected, concentrated under reduced pressure in vacuo, and the product was identified by thin layer chromatography. In the figure, (B) shows that there is a large black dot in the middle of the TLC sheet, and its Rf value is also about 0.7. The recovery and recovery of unsaturated fatty esters were 1.71 ± 0.19 g and 83.9 ± 4.1%, respectively.

G. Hydrolysis of unsaturated fatty esters into unsaturated fatty acids

About 1 g (3.14 mmol) of the above product was dissolved in 50 ml of ethanol, and 10 ml of 1.5 N KOH, 30 ml of ethanol and 30 ml of water were added thereto, and the mixture was reacted at room temperature for 16 hours. 100 ml of water was added and the solution was acidified to pH = 4 with 1 N HCl. Extract with ethyl acetate (3 x 100 mL). The extract was washed with water (2 × 150 mL) and saturated brine (150 ml) and extracted wash, Na ₂ SO ₄ removal of water and evaporated in vacuo, the product could not be saturated fatty acids.

The resulting unsaturated fatty acid was analyzed by thin film layer chromatography. The results in Figure 8 show that the three-tube sample is consistent with the standard free fatty acid position on the TLC sheet, and the Rf values are all about 0.6. Further, the recovered amount of free fatty acid was 0.67 ± 0.06 g, and the recovery was 39.5 ± 3.4%.

The unsaturated fatty acid obtained by hydrolysis was subjected to fatty acid analysis by gas chromatography, and it was found that there were two higher peaks in about 16 minutes. After standard comparison, the two fatty acids were respectively C20:2n- 6 and may be fatty acids of Δ7-ETrA (Figure 9).

Among them, C20: 2n-6 accounted for 36.8%, and probably Δ7-ETrA accounted for about 59.0%.

H. Silver ion thin film chromatography purification

A 4% saturated silver nitrate/methanol/water (9/1, v/v) solution was prepared. Pour the silver nitrate solution into a glass tank. In a dim environment, the TLC sheet was quickly placed in the tank for about 1 minute. Remove and place in a ventilated box and dry in the dark for 40 minutes. The aluminum foil was placed in an oven at about 100 ° C for 20 minutes. The developing solution was toluene/ethyl acetate/acetic acid (95/5/1, v/v/v). The product to be separated is applied. The TLC sheet was placed in the expansion tank for 20 minutes. The separated silver nitrate gel was scraped off, and the obtained product was washed with a solvent and evaporated in vacuo to give a purified unsaturated fatty acid product.

In view of the fact that the synthesized fatty acids have Δ7-ETrA and C20:2n-6 and other trace fatty acids, silver ion thin layer chromatography is used to separate Δ7-ETrA.

After purification by silver ion thin film chromatography, the fatty acid is further subjected to a saponification reaction. Then, the mixed standard was compared with the unmethylated free Δ7-ETrA fatty acid and Δ7-ETrA methyl ester. The results of (Fig. 10) showed that most of the Δ7-ETrA methyl esters were converted to free. Δ7-ETrA, only a small portion of Δ7-ETrA did not dissociate via action. A slight faint spot on the TLC on-chip purified free Δ7-ETrA was also observed. In addition, the comparison of Δ7-ETrA methyl esters can be demonstrated as the unsaponifiable fatty acids (Fig. 3-10).

The Δ7-ETrA separated and purified by silver ion thin film chromatography was analyzed by a gas chromatograph, and it was found from Fig. 11 that there was a peak at about 16 to 17 minutes. Analysis by gas chromatography showed that the purity of Δ7-ETrA was about 96.5%.

The synthesized fatty acids were analyzed and identified by gas chromatography mass spectrometry to determine whether it is the Δ7-ETrA to be synthesized in our laboratory. We used DGLA as a standard to compare, because DGLA is an extension of GLA and can be used as a control group similar to the structure of Δ7-ETrA. It can be seen from Fig. 12 that the two graphs have a high degree of similarity: except for the shape and intensity of the mass spectrum, the molecular weights of the two fatty acid methyl esters are 320 MW. Although there is currently no commercially available standard for Δ7-ETrA, we can judge that the synthesized product is Δ7-ETrA.

Example 2 Analysis of the effect of △7-ETrA fatty acid on inflammatory index by liposome-stimulated macrophage model

Cell culture

(1) Experimental cells

Mouse macrophage RAW 264.7 (BCRC 60001) medium 10% FBS/DMEM

(2) Culture conditions and subculture

Subculture was performed when the cells were grown eight to nine minutes. The old medium in the dish was removed while a small amount of new medium was added, and the cells were scraped from the 75T flask using a spatula and placed in a centrifuge tube. After centrifugation, the culture medium was added, and the cells were cultured in an incubator containing 5% CO ₂ at a ratio of 1:3 to 1:6 at 37 °C.

Centrifuge the cells grown to nine minutes, centrifuge, remove the supernatant, store the cells in 7% DMSO, and store in a -80 ° C storage box. After an hour, it is stored in liquid nitrogen.

2. Measurement of NO production by LPS-stimulated macrophages

Reference is made to the method of Stuehr et., al et al. [J Exp Med. 169 , 1543-1555, 1989]. The nitric oxide produced in the cells is unstable and is quickly oxidized to nitrite (NO2 ^- ) and nitrate (NO3 ^- ). Therefore, the formation of NO can be indirectly determined by measuring the nitrite. The Griess reagent used in this experiment can react with nitrite to form a red solution with a maximum absorbance at a wavelength of 546 nm. It is easy to exhibit a dark color change due to the difference in the amount of nitrite accumulated in the cell culture medium to be tested.

Reagents:

Griess reagent: Griess A and Griess B reagent are mixed in a 1:1 ratio. Griess A: 0.1% N-(1-Naphthtl)ethylenediamine dissolved in dd H2O Griess B: 1% Sulfanilamide dissolved in 5% H3PO4 5% H3PO4: 5.9 ml H3PO4 (85%) plus dd H2O to 100 ml NaNO ₂ : 2 mM dissolved in dd H ₂ O

Next, the possible effects of different concentrations of Δ7-ETrA (0, 10, 25, 50, 100 and 200 μM) or PNA (50 and 100 μM) on NO secretion by LPS-stimulated RAW 264.7 cells were examined. The results in Figure 13 show that both Δ7-ETrA and PNA have the effect of inhibiting cell production or releasing NO, and the degree of inhibition exhibits a dose-dependent trend.

3. LPS-stimulated macrophage PGE ₂ Generated measurement

Determination of PGE2 in cell supernatants was performed using the principle of competitive immunoassay and using the commercial reagent set Prostaglandin E2 Enzyme Immunoassay Kit (Cayman Chemical) Co., USA), analytical determination. The PGE2 in the sample competes with a known fixed concentration of PGE2 molecule for a limited monoclonal antibody to PGE2. After the action, the PGE2 monoclonal antibody binds to the goat anti-mouse IgG secondary antibody linked to the bottom of the sample tank, washes away the unbound material, and adds the Ellman's reagent. The reaction result will be Produces a yellow substance.

After measuring the absorbance at 420 nm by ELISA reader, the stanard curve was drawn from the absorbance of the standard and the regression equation was obtained, and the concentration of PGE2 in the sample was converted by interpolation. In competitive immunoassays, the absorbance measured by the sample is inversely proportional to the concentration of PGE2.

Reagents:

Prostaglandin E2 kit using Cayman Chemical Company Prostaglandin E2 Enzyme Immunoassay Kit (514010, Cayman, USA) EIA buffer one vial of EIA buffer dissolved in 90 ml of pure water wash buffer 5 ml of wash buffer dissolved in 2 liters of pure Tracer in water in a bottle of Tracer dissolved in 6 ml of EIA buffer PGE2 Standard in EIA buffer for a series of dilutions Ellman's reagent Stop solution

Experimental steps:

1. Inject 200 ml of the wash solution (wash buffer) into each well of the 96-well plate coated with the secondary antibody, and pour it after wetting.

2. Then inject 50 microliters of EIA buffer (B ₀ , see below) or standard or diluted sample or quality control (QC) into the well.

3. Add 50 μl of PGE ₂ Ache Tracer and 50 μl of PGE ₂ EIA antibody in sequence, and incubate for 18 hours at room temperature, pour out the material that is not bound to the well with the wash solution (including PGE in the sample). ₂ and the reagent group of the tracer) wash away.

4. Add Ellman's reagent for 1.5 hours, and read the absorbance at 415 nm with an enzyme immunoassay. Calculate %B/B ₀ (Sample or standard bound/maximum bound):%B/B ₀ =(A _{standard or sample} -A _NSB )/AB ₀ -A _NSB where NSB (Non-specific Binding): indicates PGE ₂ tracer Non-specific binding to the amount of pores (blank test without primary antibody) B ₀ (Maixmum binding): indicates the maximum amount of PGE ₂ tracer non-specifically bound to the pore A _{standard or sample} indicates the absorbance of the standard and sample A _NSB indicates that the absorbance value A _{B0 of the NSB} indicates that the absorbance value of B ₀ constructs a standard curve with respect to the standard concentration at %B/B ₀ , and the PGE ₂ concentration in the sample is obtained by interpolation.

In order to detect the release amount of PGE ₂ , the number of RAW264.7 cells was fixed, cultured at different concentrations of Δ7-ETrA or PNA for 24 hours, and induced with 0.1 μg/ml LPS for 16 hours. The results in Figure 14 show that if macrophages are not stimulated by LPS, only a small amount of PGE _{2 is} released into the culture medium; conversely, the PGE ₂ released by the cells will be stimulated in large amounts by LPS. Under the treatment of different concentrations of Δ7-ETrA or PNA, there are cases of inhibition, and there is a dose-related tendency.

4. Measurement of iNOS and COX-2 protein expression in LPS-stimulated macrophages

When cells are subjected to external stimulation to cause a large secretion of NO or PGE ₂ , intracellular iNOS and COX-2 can be induced and expressed in large amounts. In the experiment, macrophages were co-cultured with LPS (0.1 μg/ml) for 16 hours, and the induced iNOS and COX-2 were inhibited by Δ7-ETrA.

(2) Reagents

Liquid A: 30% acrylamide-bisacrylamide (29:1) solution.

Solution B (separating colloidal buffer) formula:

Dissolve in 300 ml of water, adjust the pH to 8.8, and add water to 500 ml.

The C solution (aggregate colloidal buffer) is formulated as follows:

Dissolve in 300 ml of water, adjust the pH to 6.8, and add water to 500 ml. 10% SDS electrophoretic film was prepared in the following proportions.

The resolution gel solution formulation is as follows:

The formulation of the stacking gel solution is as follows:

The transfer buffer solution (10X concentration) is formulated as follows:

Add water to 1500 ml, adjust the pH to 8.3, quantify to 2 L, and dilute in ten times when used.

The NET solution recipe is as follows:

Dissolve in 1800 ml of water, adjust the pH to 8.0, and add water to 2 liters.

The wash buffer (PBST) solution is formulated as follows:

The alkaline phosphate buffer formulation is as follows:

Finally adjust the pH to 9.5 and quantify to 1 liter.

(3) Experimental steps

Cellular protein content determination

The treated cell pellet is added to the lysis buffer and frozen. Save. Before measuring the cellular protein content, it was homogenized in an ice bath with a sonicator, and then centrifuged at 10000 rpm for 10 minutes at 4 ° C. The upper layer was the cytoplasmic layer and the lower layer was the cell layer. . After appropriate dilution with the lysis buffer, the protein content was determined, 0.1 mg/ml of BSA (Bovine serum albumin) was placed, 10 μl of sample or standard was added to the well, and finally in each well. 200 μl of Dye reagent blue was added thereto, and allowed to stand at room temperature for 10 minutes, and then quantitatively analyzed by an ELISA reader at a wavelength of 595 nm.

SDS-PAGE electrophoresis

The quantified protein was mixed with an equal amount of sample buffer, heated at 100 ° C for 10 minutes, allowed to stand on ice for about 10 minutes, and then sequentially added to a 10% SDS-polyacrylamide gel hole to set the voltage. Electrophoresis was carried out at 150 volts, and the power was turned off after the dye in SDS-PAGE ran out of SDS-PAGE.

Western blotting

Transfer the protein on SDS-PAGE to the PVDF membrane. The PVDF membrane should be infiltrated in methanol for later use. After electrophoresis, the PVDF transfer membrane is used for transfer, and the sponge, filter paper, film, PVDF transfer film, filter paper, The sponge is fixed in a sandwich type plastic tray, the bubble is driven out and clamped, and then placed in a transfer tank, and the current is set at 400 microamperes for 1 hour. Then take it out. Add an appropriate amount of 5% skim milk powder solution to the PVDF membrane, perform blocking at 37 ° C for 30 minutes or 4 ° C overnight, then wash in buffer buffer (PBST) for 15 minutes (3 times 5 minutes), then add and dilute The primary antibody was applied to the PVDF membrane for 2 hours and then washed continuously with washing buffer for 15 minutes. The secondary antibody was diluted to the appropriate concentration and uniformly poured onto the PVDF membrane for 2 hours. Finally, wash with washing buffer for 15 minutes. The color of the BCIP ^® /NBT matrix is about 5~10 minutes (protected from light). Before the background is deepened, the coloring liquid is poured out, washed with distilled water or tap water several times, and dried to avoid fading. Scan and analyze the ribbon intensity.

Since inducible nitric oxide synthase (iNOS) is continuously expressed in externally stimulated macrophages, NO is produced in large quantities. The results of Fig. 15 show that different concentrations of △7-ETrA have the effect of inhibiting NO production. Therefore, we want to know whether the inhibition of NO production by △7-ETrA regulates the expression of iNOS. Western blotting protein analysis showed that △7-ETrA fatty acids can reduce the protein expression of iNOS (Fig. 15).

In addition, the above experimental results show that △7-ETrA also inhibits the production of PGE ₂ , so we further explore whether Δ7-ETrA inhibits the correlation between the expression of PGE ₂ and COX-2 by macrophages. As can be seen from the results of Fig. 16, there was no significant difference between the addition of 50 μM Δ7-ETrA and the positive control group LPS via stimulation alone. When the concentration of Δ7-ETrA reached 100 μM, △7-ETrA slightly inhibited the performance of COX-2. PNA has a slight inhibitory effect on COX-2.

5. Measurement of cytokine production by LPS-stimulated macrophages

IL-6 and TNF-α in the samples were measured using an eBioscience ELISA kit. Samples or standards are first added to the ELISA 96-well plate, sequentially added to the raw Biotin-conjugate monoclonal antibody (anti-mouse IL-6) and Streptavidin-HRP. By binding the immune antibody to IL-6 in the sample, the color will change from blue to yellow, and then the absorbance of the standard dilution of the standard can be converted to the concentration of IL-6 or TNF-α in the sample.

(2) Reagents

Immunoassay Kit IL-6 ELISA kit (88-7064-88, USA) Capture Antibody Biotin-Conjugate antibody Standard Streptavidin-Peroxidase (HRP) Wash Buffer Stabilized Chromogen Stop Stop solution

(3) Experimental steps

1. Coating: A disk (NUNC Maxisorp) was made using the capture antibody the day before.

2. First take 4 ml of coating buffer into the sample well; take 16 μl of 250x capture antibody into the sample well containing 4 ml of coating buffer.

3. Place 100 μl in a 96-well plate and place at 4 ° C for 12 hours.

4. Configure the wash buffer and assay diluent (Assay Diluent), rinse the 96-well plate 5 times with wash buffer, and take 200 μl of 1x assay dilution into a 96-well plate, then place at 4 ° C for 1 hour.

5. Dilute the sample and the standard product, wash the 96-well plate with the washing buffer 4 times, take the diluted sample and the standard product 100 μl to the 96-well plate, wrap the aluminum foil, and reverse the room temperature. It should be 2 hours.

6. Rinse the 96-well plate 5 times with wash buffer, place 16 μl of 250x detection antibody into the sample well containing 4 ml of 1x assay dilution, and add 100 μl to 96-well. The reaction was carried out for 1 hour in the pan.

7. Rinse the 96-well plate with wash buffer 5 times and place 16 μl of Avidin-HRP in a sample well containing 4 mL of 1x assay dilution for 30 minutes.

8. Rinse the 96-well plate 7 times with wash buffer and add 100 μl/well of 1x TMB substrate solution for 15 minutes at room temperature.

9. Add 50 μl of stop solution.

10. Measure the 450 nm absorbance by ELISA reader.

Figure 17 shows that the IL-6 produced by the cells due to the inflammatory reaction is reduced by the addition of Δ7-ETrA (0, 10, 25, 50 and 100 μM) and PNA (50 and 100 μM) in the culture solution. And the degree of inhibition of IL-6 is also a dose-related trend.

In addition, the effect of Δ7-ETrA fatty acid on TNF-α production by cells is shown in FIG. LPS stimulates cells to produce large amounts of TNF-α; while cultured at different concentrations of Δ7-ETrA (10-100 μM), it promotes the formation of TNF-α, and the degree of increase is related to the concentration of Δ7-ETrA added; PNA (50 and 100 μM) also promotes the formation of TNF-α.

6. Statistical analysis

Each experiment was repeated three times or more, and the results were expressed as mean ± SD. The data were analyzed by SPSS 12 (Statistical Package for the Social Sciences), and ANOVA was used for variance analysis and Deng multivariate domain test. (Duncan’s multiple range test) A comparison of significant differences was made. The results of the analysis are expressed in English letters.

The above results confirmed that Δ7-ETrA can be synthesized by chemically C2-lengthening from PNA. This is the first time in the art that PNA was synthesized from Δ7-ETrA.

Further, it was confirmed by the present invention for the first time that Δ7-ETrA and PNA can also inhibit the production of NO, IL-6 and PGE ₂ induced by LPS induction.

The detailed description of the preferred embodiments of the present invention is not intended to limit the scope of the present invention, and the equivalent implementations or modifications of the present invention should be included in the present invention. In the scope of patents.

Figure 1 shows the structure of PNA and GLA.

Figure 2 shows the GC analysis of the fatty acid composition of pine nut oil.

Figure 3 is a GC analysis of the fatty acid composition of pine nut oil solution after urea saturation.

Figure 4 shows the results of separation of the alcoholized product by TLC; the developing solution was hexane:ethyl acetate:acetic acid (1:1:0.01, v/v/v).

Figure 5 shows the results of separation of pine nut oil fatty acid bromination product by TLC by column chromatography; TLC developing solution is hexane:ethyl acetate:acetic acid (1:1:0.01, v/v/v).

Figure 6 shows the results of TLC separation of the brominated product of pine nut oil fatty acid and 2,4,4-trimethyl-2-oxazoline; the TLC developing solution is hexane:acetone (1:1, v/v).

Figure 7 (A) shows the product of pine nut oil fatty acid dimethyloxazoline via alcoholysis tube The results of column chromatography and TLC analysis; TLC developing solution was hexane: diethyl ether: acetic acid (4:1:0.1, v/v/v).

Figure 7 (B) shows the results of separation of the new product, concentration and TLC separation.

Figure 8 is a comparison of the fatty acid methyl ester with the free fatty acid standard by TLC analysis after hydrolysis; the TLC developing solution is hexane: diethyl ether: acetic acid (1:1: 0.01, v/v/v).

Figure 9 is a map of the synthesized fatty acid analyzed by gas chromatography.

Figure 10 shows the alignment of free Δ7-ETrA fatty acid with Δ7-ETrA methyl ester by TLC; the TLC developing solution is hexane:ethyl ether:acetic acid (80:20:1, v/v/v).

Figure 11 shows the GC spectrum measured for the saponified product.

Figure 12 is a gas chromatography/mass spectrogram; (A) DGLA-Δ8, 11, 14-20:3; (B) Δ7-ETrA-Δ7, 11, 14-20:3.

Figure 13 shows the effect of Δ7-ETrA and PNA on NO production in RAW264.7 cells; the values of the different superscript letters ^ABCDEF are significantly different (P<0.05).

Figure 14 shows the effect of Δ7-ETrA and PNA on the secretion of PGE ₂ by RAW264.7 cells; the values of the different superscript letters ^ABCDE were significantly different (P<0.05).

Figure 15 shows the effect of the addition of Δ7-ETrA and PNA on iNOS in RAW264.7 cells; the values of the different superscript letters ^ABCD were significantly different (P<0.05).

Figure 16 shows the effect of the addition of Δ7-ETrA and PNA on COX-2 in RAW264.7 cells; the values of the different superscript letters ^ABC were significantly different (P<0.05).

Figure 17 shows the effect of △7-ETrA and PNA on the secretion of IL-6 by RAW264.7 cells; the values of the different superscript letters ^ABCDEF were significantly different (P<0.05).

Figure 18 shows the effect of △7-EtrA and PNA on TNF-α secretion by RAW264.7 cells; the values of the different superscript letters ^ABCDE were significantly different (P<0.05).

Claims (9)

A method for preparing Δ7,11,14-eicosatrienoic acid (△7,11,14-eicosatrienoic acid, hereinafter referred to as Δ7-ETrA), comprising the following steps: 1. Saponification from pine nut oil, urea Saturating and extracting pine acid oleic acid (PNA; Δ5,9,12-octadecatrienoic acid) having eighteen carbons is concentrated as free fatty acid; 2. esterifying free octadecyl fatty acid; Alcoholizing the octadecyl fatty acid ester; 4. Brominating the octadecyl fatty acid ester to form the octadecyl fatty acid ester bromide; 5. The octadecyl fatty acid ester bromide with 2, 4, 4 - Trimethyl-2-oxazoline (2,4,4-trimethl-2-oxazoline) to form a dimethyloxazoline reactant having twenty carbons; The dimethyl-2-oxazoline reactant having twenty carbons is converted into a long-chain unsaturated fatty acid of twenty carbons; and 7. The Δ7-ETrA separation and purification is carried out by liquid chromatography. The method for preparing Δ7,11,14-eicosatrienoic acid according to claim 1, wherein the step 2 esterifying the boron trifluoride-methanol complex solution (boron trifluoride methanol- Complex solution). The method for producing Δ7,11,14-eicosatrienoic acid according to claim 1, wherein the step 3 alcoholizing uses the LiAlH ₄ to reduce the ester to an alcohol. Manufacture of Δ7,11,14-eicosatrienoic acid as described in item 1 of the patent application A process wherein the bromination of step 4 utilizes triphenylphosphine to bromine the resulting alcohol in bromine/dichloromethane. A method for producing Δ7,11,14-eicosatrienoic acid as described in claim 4, wherein the reaction of bromide with 2,4,4-trimethl-2-oxazoline in the step 5 This was carried out in tetrahydrofuran in the presence of n-butyllithium at -78 ° C under nitrogen. The method for preparing Δ7,11,14-eicosatrienoic acid according to the first aspect of the patent application, wherein the step 7 is carried out by liquid chromatography, and the Δ7-ETrA separation and purification system utilizes silver ions. The membrane was chromatographed and purified by separation of the developing solvent toluene/ethyl acetate/acetic acid (95/5/1, v/v/v). The use of a compound Δ7,11,14-eicosatrienoic acid for the preparation of an anti-inflammatory drug. A composition comprising Δ7,11,14-eicosatrienoic acid and a physiologically or pharmaceutically acceptable carrier. A use of the composition of claim 8 in the preparation of an anti-inflammatory drug.