KR20210053165A - Method For Astaxanthin Monoester Through Transesterification - Google Patents

Abstract

The present invention relates to a method for producing astaxanthin monoester through transesterification and, more particularly, to a method for producing astaxanthin monoester, which comprises: a step 1-1 to prepare a basic alcohol solution; a step 1-2 to transesterify astaxanthin; a step 1-3 to extract astaxanthin; and a step 2 to synthesize the astaxanthin monoester.

Description

Method for producing astaxanthin monoester through transesterification {Method For Astaxanthin Monoester Through Transesterification}

The present invention relates to a method for producing astaxanthin monoester, and more specifically, the 1-1 step of preparing a basic alcohol solution, the 1-2 step of transesterification of astaxanthin, and the extraction of astaxanthin It relates to a method for producing an astaxanthin monoester comprising a step 1-3 and a second step of synthesizing an astaxanthin monoester.

Astaxanthin is a type of carotenoid pigment that can be separated from lobster, and is known as a pigment that gives red color to fish flesh such as salmon and trout, or gives red color when crustaceans such as shrimp are heated. Astaxanthin has an antioxidant function, so it is increasingly used as a material for promoting human health functions as it has preventive and therapeutic activities against various diseases caused by oxidative phosphorus in the body.

Astaxanthin is more efficient than the free form without esters, since the ester form with one or two esters is better absorbed by fish and easily migrates to the muscles when used as feed. Since it is stable to oxygen, a method of producing a diester type of free astaxanthin as a raw material is known. Further, according to Prior Document 1 (Japanese Patent No. 4410675), it is known that monoester-type astaxanthin is superior to diester-type astaxanthin in oral absorption. However, there is a problem in that the efficiency is lowered because monoester-type astaxanthin is not produced.

An object of the present invention is to solve the problems of the prior art and technical problems that have been requested from the past.

Specifically, the present invention provides a 1-1 step of preparing a basic alcohol solution, a 1-2 step of transesterification of astaxanthin, a 1-3 step of extracting astaxanthin, and an astaxanthin monoester. An object of the present invention is to provide a method for producing astaxanthin monoester comprising a second step of synthesizing.

One embodiment of the present invention is a step 1-1 of preparing a basic alcohol solution; A 1-2 step of transesterification reaction of astaxanthin; It relates to a method for producing an astaxanthin monoester comprising: a 1-3 step of extracting astaxanthin; a second step of synthesizing an astaxanthin monoester.

Here, the 1-1st step of preparing the basic alcohol solution includes the 1-1-1st step of mixing sodium hydroxide with distilled water; Step 1-1-2 of mixing the mixture of step 1-1-1 with one or more mixtures selected from methanol, ethanol, and propanol; Step 1-1-3 of dehydrating the mixture of step 1-1-2; Step 1-1-4 of repeating the step 1-1-3 2 to 4 times while removing the precipitate; And a step 1-1-5 of filtering the mixture of steps 1-1-4 using a fine filter.

In addition, step 1-2 of the transesterification reaction of astaxanthin may include: a 1-2-1 step of mixing the supercritically extracted astaxanthin into the mixture of steps 1-1-5; Step 1-2-2 of reflux stirring the mixture of step 1-2-1 after vortexing; And a step 1-2-3 of cooling and neutralizing the mixture of step 1-2-2 to room temperature.

In addition, in the 1-3 step of extracting the astaxanthin, the mixture of steps 1-2-3 in which the astaxanthin transesterification reaction is completed is one or two selected from hexane and petroleum ether. 1-3-1 step of vortexing by mixing with a species of compound; A 1-32 step of separating the upper layer by centrifuging the mixture by rotating the mixture at high speed; A 1-33 step of drying the separated first 1-32 step mixture under reduced pressure; A 1-3-4 step of mixing and dissolving the mixture of step 1-3-3 in a compound selected from dichloromethane, dichloroethane, and dichlorobutane; Includes.

In addition, the second step of synthesizing astaxanthin monoester may include a 2-1-1 step of mixing an activator, a catalyst, and a saturated fatty acid with the compound of step 1-3-4; Step 2-1-2 of reacting the mixture of step 2-1-1 at room temperature for 1 hour to 3 hours; A 2-1-3 step of repeating the 2-1-2 step 2 to 4 times; And a step 2-1-4 of evaporating the mixture of step 2-1-3 under reduced pressure.

According to the method for producing an astaxanthin monoester of the present invention, it is possible to produce an astaxanthin monoester having excellent production efficiency.

Figure 1. Chemical conversion of natural astaxanthin (Ast-N) to ester-type astaxanthin with free astaxanthin (Ast-F) and decanoic acid (Ast-E). R1 is a carbon chain of a different length, known to range from 12 to 24, and R2 is derived from decanoic acid (C10).
Figure 2. HPLC chromatograms of (A) Ast-F, (B) Sample 1, (C) Sample 2, and (D) Sample 3 are shown.
Figure 3. DPPH radical scavenging activity in astaxanthin in (A) Sample 1, (B) Sample 2, and (C) Sample 3 is shown. Results are expressed as mean±SD of 3 independent experiments.
Figure 4. (A) B16F10, (B) HaCaT, (C) shows the cytotoxicity of Raw 264.7 cells exposed to different forms of astaxanthin. Out of 3 independent experiments, results are expressed as mean±SD.
Figure 5. Cellular antioxidant activity of each sample (1 to 3) containing 2.5 and 5 μM of different forms of astaxanthin in (A) B16F10 cells and (B) HaCaT cells. Results are expressed as mean±SD of 3 independent experiments.
Figure 6. Anti-inflammatory activity of astaxanthin samples (1 to 3) against LPS-stimulated Raw 274.7 macrophages. Results are expressed as mean±SD of 3 independent experiments.
Figure 7. Gene expression associated with anti-inflammatory and antioxidant activity is shown. (A) IL-1β, (B) IL-6, (C) TNF-α, (D) NOX-2, (E) Nrf2, (F) MnSOD. Data represent the mean ± SD of three individual experiments. *p <0.05, **p <0.01 vs. control group; #p <0.05, #p <0.01 vs. LPS group
Fig. 8. A schematic diagram of samples 1 to 3 of astaxanthin is shown.
Fig. 9 shows a calibration curve obtained by measuring the absorbance of 474 nm astaxanthin as an analysis standard at different concentrations. Results are expressed as the mean ± SD of three independent experiments.

Hereinafter, in order to describe in detail enough that a person having ordinary knowledge in the technical field of the present invention can easily implement the technical idea of the present invention, a most preferred embodiment of the present invention will be described with reference to the accompanying drawings. . In addition, in describing the present invention, when it is determined that a detailed description of a related known configuration or function may obscure the subject matter of the present invention, a detailed description thereof will be omitted.

In the following, specific steps of the manufacturing method of the astaxanthin monoester of the present invention will be described.

Step 1-1 of preparing a basic alcohol solution of the method for producing astaxanthin monoester of the present invention may include the following steps. For example, step 1-1-1 of mixing sodium hydroxide with distilled water; Step 1-1-2 of mixing the mixture of step 1-1-1 with one or more mixtures selected from methanol, ethanol, and propanol; Step 1-1-3 of dehydrating the mixture of step 1-1-2; Step 1-1-4 of repeating the step 1-1-3 2 to 4 times while removing the precipitate; It may include; steps 1-1-5 of filtering the mixture of steps 1-1-4 using a fine filter.

Here, in step 1-1-1 of mixing sodium hydroxide with distilled water, 0.01g to 0.1g of sodium hydroxide may be mixed with 0.01ml to 1ml of distilled water. That is, the mixing ratio of distilled water and sodium hydroxide (sodium hydroxide/distilled water ml/g) 0.01 ml/g to 10 ml/g, more preferably 0.1 ml/g to 1 ml/g may be used.

In addition, in step 1-1-2, the mixture of step 1-1-1 may be mixed with 50 ml of a mixture of one or two or more selected from methanol, ethanol, and propanol. That is, the mixture of step 1-1-1 with respect to one or two or more mixtures selected from methanol, ethanol, and propanol may use 50:0.1 to 1 in a volume ratio.

In addition, step 1-1-3 may proceed with a dehydration reaction by preferably using a compound represented by the following Formula 1-1 or Formula 1-2.

Step 1-1-4 may repeat the step 1-1-3 2 to 4 times while removing the precipitate. More preferably, steps 1-1-3 may be repeated 3 times. In addition, in steps 1-1-5, the mixture of steps 1-1-4 may be filtered using a filter of 0.1 μm to 0.5 μm. More preferably, a 0.2 μm filter may be used.

Step 1-2 of transesterification reaction of astaxanthin in the method for producing an astaxanthin monoester of the present invention is a first step of mixing the supercritically extracted astaxanthin into the mixture of steps 1-1-5. Step 2-1; Step 1-2-2 of reflux stirring the mixture of step 1-2-1 after vortexing; It may include a step 1-2-3 of cooling and neutralizing the mixture of the first step 1-22 to room temperature.

In step 1-2-2, the mixture in step 1-2-1 is vortexed and stirred under reflux at 40°C to 60°C, and the mixture is cooled and neutralized at room temperature in step 1-2-3. Taxanthine methanol can be effectively synthesized.

In the 1-3 steps of extracting astaxanthin in the method for producing astaxanthin monoester of the present invention, the mixture of steps 1-2-3 in which the astaxanthin transesterification reaction is completed is mixed with hexane and petroleum ether. 1-3-1 step of vortexing by mixing with one or two compounds selected from); A 1-32 step of separating the upper layer by centrifuging the mixture by rotating the mixture at high speed; A 1-33 step of drying the separated first 1-32 step mixture under reduced pressure; And a step 1-3-4 of mixing and dissolving the mixture of step 1-3-3 in one type of compound selected from dichloromethane, dichloroethane, and dichlorobutane.

Here, in the first step 1-3-1, the mixing ratio of the astaxanthin methanol and hexane or petroleum ether, which is the mixture of the first step 1-2-3, in which the astaxanthin transesterification reaction is completed, is 1:5 to 10 It can be mixed in a volume ratio of. More preferably, the mixing ratio of astaxanthin methanol and hexane or petroleum ether may be mixed in a volume ratio of 1:7.

In addition, in step 1-3-2, the upper layer portion may be separated by centrifugation by rotating the mixture in step 1-3-1 at 3000 rpm to 4000 rpm. In addition, step 1-3-3 can be obtained by lowering the pressure of the separated step 1-3-2 mixture and drying the temperature at 40°C to 60°C under reduced pressure for 1 to 5 hours.

If the ester reaction of the present invention is summarized, it can be expressed as follows.

The step of esterifying using a saturated fatty acid in the method for producing astaxanthin monoester of the present invention is a 2-1-1 of mixing an activator, a catalyst, and a saturated fatty acid with the compound of step 1-3-4. step; Step 2-1-2 of reacting the mixture of step 2-1-1 at room temperature for 1 hour to 3 hours; A 2-1-3 step of repeating the 2-1-2 step 2 to 4 times; It may include; a 2-1-4 step of evaporating by depressurizing the mixture of the step 2-1-3.

As the activator to be mixed in step 2-1-1, a water-soluble R1N=C=NR2 (here, R1 and R2 are hydrogen atoms, or alkyl having 1 to 10 carbon atoms in which unsubstituted or carbon atoms are substituted with nitrogen atoms) structure. A compound having may be used, and preferably, pH4.0 to pH6.0 may be used. For example, the use of the compound represented by the following formula (1) is useful for activating the carboxyl group of a saturated fatty acid.

[Formula 2]

The catalyst to be mixed in step 2-1-1 may be a nucleophilic catalyst, and preferably, the following formula (3) (where R is a hydrogen atom or an alkyl having 1 to 10 carbon atoms unsubstituted or substituted with a nitrogen atom) ) Can be used.

[Formula 3]

Preferably, R may use an NMe2 group, and when the compound of Formula 4 is used, there is an advantage of stabilizing resonance of the pyridine structure.

[Formula 4]

As the saturated fatty acid to be mixed in step 2-1-1, a compound represented by the following formula (5) (where n is 2 to 5) may be used.

[Formula 5]

Preferably, a compound represented by the following formula (6) may be used.

[Formula 6]

When the activator, catalyst, and saturated fatty acid of the present invention use a mass ratio of 1:0.5 to 1.5:0.5 to 1.5, the efficiency of synthesizing astaxanthin monoether due to activation of the carboxyl group increases.

Meanwhile, in step 2-1-2, the mixture of step 2-1-1 may be reacted at 10° C. to 30° C. for 1 to 3 hours. More preferably, the mixture of the step 2-1-1 may be reacted at 20° C. for 1 hour to 3 hours. In addition, step 2-1-4 can be obtained by lowering the pressure of the mixture and drying the mixture under reduced pressure at 40°C to 60°C for 1 to 5 hours.

The synthesis reaction of astaxanthin monoether of the present invention can be summarized as follows.

(Experiment)

2.1. Chemical changes in natural astaxanthin

Natural astaxanthin oleoresin derived from Hematococcus prualis, isolated by supercritical liquid extraction using carbon dioxide, was purchased from Yunnan Alphy Becheth (Chuxiong, China) and stored at -20℃ to avoid thermal degradation. This product was found to contain 10% astaxanthin. Ast-N extracted from Hematococcus prualis (Sample 1 dissolved in acetone) was first converted to Ast-F by trans-esterification in order to remove fatty acid chains of different lengths. Specifically, sodium hydroxide (0.0072 g, Samchun Chemical) was added to 50 mL of methanol (Samchun Chemical) and anhydrous sodium sulfate (Samchun Chemical) was mixed to remove residual moisture. The prepared basic alcohol solution is filtered with a 0.2 μm syringe filter, and 1 g of Ast-N is added to a shaking incubator at 50°C and incubated for 4 hours, and then a molecule named Ast-F is extracted with petroleum ether (Samchun Chemical). did. The ether was evaporated, and the resulting Ast-F was dissolved in dichloromethane (Samchun Chemical) or acetone (HPLC grade, Samchun Chemical) for esterification reaction or HPLC analysis, and was named Sample 2. Finally, Ast-F in Sample 2 was transformed into Ast-E by the Steglych esterification method using decanoic acid (Tokyo Chemical Industries, Japan), a C10 straight chain saturated fatty acid. Specifically, Ast-F of dichloromethane was sequentially mixed with a compound of formula 6 (0.12 mM), a compound of formula 2 (0.14 mM), and a compound of formula 4 (0.11 mM). Incubated for 2 hours at 20 ℃. This process was repeated 3 times and dichloromethane was evaporated. The resulting Ast-E was dissolved in acetone (Sample 3) for further analysis.

2.2. HPLC measurement

As the measurement conditions, Waters symmetry C ₁₈ column (4.6 x 250mm, 5μm, waters, USA), UV/visible detector (474nm), the column temperature was maintained at 25°C, and the flow rate was set to 1mL/min. The mobile phase is solvent A (methanol: tert- butyl methyl ether: 1% (w/v) phosphoric acid, 81:15: 4.v/v), solvent B (methanol: tert- butyl methyl ether: 1% (w/ v) phosphoric acid, 16:80:4, v/v) was used, and the solvent A was 100% for each mobile phase solvent from 0 to 15 minutes, and the solvent B was added to 0 to 100 depending on time until 23 minutes. The% linear concentration gradient was gradually increased, and after that, the ratio of solvent B was maintained at 100% for 4 minutes, and then the ratio of solvent A was maintained at 100% for 8 minutes. All samples including the standard material were diluted in acetone and used. Reference recovered energy-efficient astaxanthin from microalgae.

2.3. In vitro testing for antioxidant activity

A DPPH free radical removal assay was performed to measure the antioxidant activity of astaxanthin in vitro. Specifically, 20 μL of each sample (Samples 1-3) was added to 180 μL of DPPH (1,1-diphenyl-2-picryl hydrazyl) (DPPH; 10 mM; Sigma-Aldrich) dissolved in methanol. After incubation at 37° C. for 30 minutes, the optical density (OD) was evaluated at 517 nm in a microplate reader. The results representing the DPPH free radical removal analysis (%) were expressed using the following equation (%) = [1-(OD _Sample /OD _Control )] × 100 (%). Here, OD _sample is the absorbance when there is an astaxanthin sample, and OD _control is the absorbance when there is no astaxanthin sample.

2.4. Cytotoxicity test

RAW 264.7 and B16F10 cells were purchased from Cellline Bank (Seoul, Korea), and HaCaT cells were purchased from ATCC (Manassas, VA, USA). RAW 264.7, B16F10, HaCaT cells were first seeded with an uncoated 96-well cell culture plate at a density of ^{5 × 10 4} , 1 × 10 ⁴ , and 0.8 × 10 ^{4 cells/well, respectively, and 10% (v/v)} High glucose (4.5 g/L) DMEM (Dulbecco's modified Eagle's medium) medium supplemented with Fetal bovine serum (FBS) and 1% penicillin/streptomycin (Gibco, Grand Island, NY, USA) at 37°C. Incubated for 24 hours. Next, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was added to each well at a concentration of 0.5mg/mg. Incubated in the dark for 24 hours at 37 ℃. After adding 50 μL of a 20% SDS solution to each well, the plate was incubated in the dark overnight. Finally, the OD was evaluated at 595 nm in a microplate reader. In order to exclude the effect of acetone on cell growth, the final acetone concentration of the astaxanthin sample was less than 5%, and was used as a blank for calculating cell viability. The compound was considered to have cytotoxic potential if the cell viability decreased to less than 70% of the blank.

2.5. Cellular antioxidant activity test (CAA)

To measure the ROS production of B16F10 and HaCaT cells, 2',7'-DCFH-DA (2',7'-dichlorofluorescein 3'6'-diacetate; Sigma-Aldrich), an oxidation sensitive indicator, was used. In the presence of ROS, DCFH is oxidized to fluorescent DCF (2',7'-dichlorofluorescein), which can be measured in a microplate reader. All cell line cells were seeded in a 96-well cell-alignment plate at ^{a density of 0.8 to 1 × 10 4} cells/well. Cells in DMEM supplemented with 10% (v/v) FBS and 1% penicillin/streptomycin were incubated in 5% CO2 for 24 hours, and then the cells were washed with 1x PBS. After adding 10 μM DCFH-DA to each well, it was incubated at 37° C. for 1 hour. In order to measure the oxidation reaction occurring only inside the cells, the excess indicator in the medium was removed by washing with 1x PBS. Next, astaxanthin samples (samples 1 to 3) were _{added to each well with hydrogen peroxide (H 2} O ₂ , 1 mM, Sigma-Aldrich) to induce oxidative stress for 30 minutes, and finally, fluorescence intensity was measured in a microplate reader. Measurements were made with excitation and emission wavelengths of 485 and 530 nm, respectively. Dose response curves were established using blanks not treated with _{H 2} O _2. The ROS removal activity was expressed as CAA (%) using the following equation (%) = [(Ic-Is)/Ic] × 100. Here, Is is the intensity of the sample exposed to _{H 2} O ₂ in the presence of astaxanthin at different concentrations, Ic is the intensity of the control sample exposed to _{H 2} O _{2 in the absence of astaxanthin.}

2.6. Anti-inflammatory test

To measure the production of nitrogen oxides (NO) in RAW 264.7 cells, the NO concentration was measured through the Griess reaction. RAW 264.7 macrophage cells were first seeded in 96-well cell-culture plates at ^{a density of 5 × 10 4} cells/well. Cells in DMEM supplemented with 10% (v/v) FBS and 1% penicillin/streptomycin were incubated at 37° C. for 24 hours in 5% CO2, and 1 μg/mL LPS (lipopolysaccharides; Sigma-Aldrich) for control Was added to each well except that. Astaxanthin samples (Samples 1-3) were added to each well at various concentrations. After incubation for two days, 100 μL of the supernatant was collected from each well and 100 μL of Griess reagent (1% sulfanylamide in 5% phosphoric acid and 0.1% naphthylethylenediamine dihydrochloride in water) was added to a separate 96-well plate. Mixed in. After incubation at 25° C. for 15 minutes, OD at 540 nm was measured in a microplate reader. Production (%) was calculated using the following equation (%) = (As/Ac) × 100. Here, Ac is the absorbance of a sample exposed to LPS in the presence of astaxanthin at different concentrations, and Ac is the absorbance of a control sample exposed to LPS in the absence of astaxanthin.

2.7. mRNA analysis

RAW 264.7 cells were initially treated with astaxanthin samples (samples 1-3) as described above, and then total RNA was extracted using NucleoSpin RNA Plus (Macherey-Nagel, Duren, Germany). Next, 150ng of total RNA was converted into cDNA using an M-MLV cDNA synthesis kit (Enzynomics, Daejeon, Korea), and gene expression was analyzed by qPCR (quantitative polymerase chain reaction). Specifically, qPCR was performed at 95°C for 10 minutes using a specific gene primer (500nM; Table 1), and then 95°C for 10 seconds, 60°C for 15 seconds, and 72°C for 30 seconds at 35 cycles 1X PreMix (Enzynomics). Each experiment was repeated at least 3 times, and qPCR data were expressed as mean ± standard deviation (S.D.). Statistically significant differences were calculated by one-way batch analysis according to Dunnett's test (Minitab, version 18). Double-sided p-values of less than 0.05 were considered statistically significant.

[Table 1] Gene-specific primers for qPCR experiments

3. Results and discussion

3.1. Chemical changes in astaxanthin

Astaxanthin exists in various structural forms in nature. In particular, Hematococcus pruviais, known as the most abundant source of Ast-N, has three forms: Ast-F, Ast-mE, and Ast-dE. In the case of ester-formed astaxanthin (Ast-E) including Ast-mE and Ast-dE, fatty acids with carbon chains of different lengths are bonded to Ast-F through an ester bond. To prepare other forms of astaxanthin, Ast-N (sample 1) consisting of Ast-mE and Ast-dE was first converted to Ast-F by removing fatty acids using dehydrated basic methanol (step 1 in Fig. 1). I did. In particular, saponification can be used to prepare Ast-F, but the fatty acid after the saponification reaction contains an active carboxyl group capable of binding to Ast-F in the subsequent esterification reaction, so Ast- It is difficult to prepare E (decanoic acid, step 2 in Fig. 1). In contrast, since the transesterification used in this example produces Ast-F in addition to the fatty acid methyl ester (Sample 2), the added decanoic acid is Ast- It only reacted with the hydroxyl group of F. As a result, Ast-E (Sample 3) with a short-length fatty acid chain (C10) was prepared to analyze various types of antioxidant activity of astaxanthin (Ast-F, Ast-mE, Ast-dE) with a constant fatty acid chain length. Can be facilitated.

3.2. Characterization of astaxanthin

Prepared samples (Samples 1-3) containing different forms of astaxanthin (Ast-N, Ast-F, Ast-E) were characterized by HPLC. First, the interpretation standard of AST-F was analyzed by HPLC. As shown in Fig. 2A, the hold time was centered at 3.963 minutes. Similarly, chromatograms of other forms of astaxanthin were obtained. As shown in Fig. 2B, the main peaks of Ast-N (Sample 1) were 11.477, 13.627, 16.543, 20.450, and 21.373 minutes. Since Ast-N contains astaxanthin bound to fatty acids of different lengths, the first 3 peaks and the next 2 peaks were predicted to be Ast-mE and Ast-dE, respectively. In addition, we confirmed that the retention time of Ast-F in Sample 2 was centered at 3.943 min after the transesterification reaction (FIG. 2C), which was in good agreement with the results for analysis grade Ast-F (FIG. 2A). These results clearly show that fatty acids of different lengths were removed in Ast-E. Finally, a chromatogram was obtained after esterification with decanoic acid (sample 3). According to the results of FIG. 2D, the main peaks of Sample 3 were 3.787, 6.863, and 14.973 minutes. The first peak corresponds to Ast-F, the last two peaks correspond to Ast-mE, the last two peaks correspond to Ast-mE linked to decanoic acid, and the second peak corresponds to Ast-mE linked to long fatty acids. In particular, small fatty acids having an active carboxyl group can be produced as a by-product along with fatty acid methyl esters due to the generation of water during transesterification (step 1; Fig. 1) (step 2; Fig. 1), so that Ast-mE having long fatty acids is also It can be produced upon esterification (step 2; Fig. 1). The relative amounts of all forms of astaxanthin (Ast-F, Ast-mE, Ast-dE) in the three samples (1, 2, 3) were measured by measuring the area under the peak (FIG. 2). As shown in Table 2, the amount of Ast-mE in Sample 3 was higher than the amount of Ast-dE, suggesting that the composition of Sample 3 was different from the composition of Sample 1, which was more abundant than Ast-dE. Overall, other forms of astaxanthin were successfully synthesized by chemical transformation.

Table 2 below shows three samples (1) determined by measuring the area under the peak (Fig. 2) and calculating the relative ratio of all forms of astaxanthin (Ast-F, Ast-mE, Ast-dE) found in each sample. , 2, 3) represent the relative amounts of all forms of astaxanthin.

[Table 2]

3.3. In vitro antioxidant activity of astaxanthin

The biological activity of the prepared different forms of astaxanthin was evaluated. First, in vitro antioxidant activity was investigated by DPPH vitrification removal test. In principle, the hydrogen donating ability of the antioxidant reduces the free vitrified DPPH (purple) to a stable DPPH (yellow), resulting in a decrease in absorbance at 517 nm. Astaxanthin at various concentrations (Samples 1 to 3) was tested as shown in FIG. 3 to measure DPPH vitrification activity. As antioxidants, all other forms of astaxanthin showed concentration-dependent vitrification activity. The radical elimination activity of Sample 1 and Sample 3 was slightly higher than that of Sample 2. Importantly, Sample 3, which contained a higher amount of Ast-mE than Ast-dE, showed slightly higher antioxidant activity than that of Sample 1, which had a higher amount of Ast-dE than Ast-mE. However, although Sample 3 showed the best radical vitrification removal activity, the overall difference in antioxidant activity in vitro among the three samples was not significant.

3.4. Cell viability

The cytotoxicity of three astaxanthin samples (1~3) against B16F10 (murine melanoma), HaCaT (human keratinocyte), and Raw 264.7 (murine macrophage) cells was evaluated by MTT test. The results of FIG. 4 show that all three astaxanthin samples were cytotoxic to 15 μm. A significant decrease in cell viability of less than 70%, sensitive to environmental conditions, was observed at concentrations above 10 μM. Based on these results, the maximum concentration for evaluating the cellular activity of the astaxanthin sample was established. Specifically, 5 μM astaxanthin was used.

3.5. Measurement of cellular antioxidant activity

A well-established CAA test relying on an intracellular stress-based model was performed. Specifically, _{exposure to H 2} O ₂ induced oxidative stress in B16F10 and HaCaT cells. H ₂ O ₂ easily penetrates the cell membrane through the Fenton reaction to generate hydroxyl radicals, the most reactive form of oxygen. I can. Next, the ROS production of B16F10 was measured by adding DCFH-DA, an oxidation sensitive index. In principle, after entering the cells, DCFH-DA is converted to DCFH by cellular deacetylase enzyme, and if ROS is present, it is converted to fluorescent DCF. Is oxidized. Therefore, when the antioxidant activity is strong, the fluorescence signal generated by preventing DCFH oxidation decreases. CAA (%), which is inversely proportional to the fluorescence signal, was calculated (see experimental section). As shown in FIG. 5, among the three samples, Sample 2 composed of Ast-F showed the lowest antioxidant capacity in B16F10 cells and HaCaT cells (FIGS. 5 (A) and (B)). Importantly, Sample 3, which contained more Ast-mE than Ast-dE, had a higher antioxidant capacity than Ast-N (Sample 1), which had more Ast-mE. These results demonstrate that Sample 3, especially Ast-mE, is important for enhancing cellular antioxidant activity. Interestingly, unlike the in vitro test for antioxidant activity (Fig. 3), a distinct difference in antioxidant activity was observed among the three samples in the intracellular antioxidant test (Fig. 5), which is greater in the intracellular antioxidant activity than in the in vitro antioxidant activity. Show that it has an effect. Based on this, by removing radicals from the terminal ring cells of the polyene chain and astaxanthin, Ast-mE can be easily inserted into the double layer of phospholipids, and thus has excellent antioxidant activity.

3.6. Anti-inflammatory activity of astaxanthin

It is known that antioxidant activity and anti-inflammatory activity are closely related. In addition, astaxanthin is known as a powerful antioxidant that stops the induction of inflammation in the biological system. To evaluate the anti-inflammatory activity of astaxanthin, murine macrophage Raw 264.7 cells were stimulated by LPS to create and release various immunologically active mediators such as tumor necrosis factor (TNF)-α, interleukin-1β, and IL-6. This mediator induces transcription of nitrogen oxide synthase, which can generate large amounts of NO, and is thought to be involved in the cytotoxic effect of inflammation. The resulting level of NO was determined by the Griess reaction, a spectrophotometric measurement performed to measure the nitrate ion level. As the production of NO increases, the colorimetric signal increases, indicating that the anti-inflammatory activity is less effective. The results in Fig. 6 show the inhibition (%) of NO production by three astaxanthin samples (1 to 3) (see experimental section). The highest anti-inflammatory activity was observed in Sample 3 containing Ast-mE, supporting the important role of Ast-mE in the antioxidant and anti-inflammatory activity of astaxanthin depending on cellular antioxidant activity.

3.7. Gene expression related to anti-inflammatory and antioxidant activity

Based on the results on the chemical transformation, properties, and biological activity of astaxanthin, the mechanism of this improved activity for Sample 3 was investigated. To better understand the inhibitory effect of astaxanthin samples on inflammation, mRNA expression of inflammatory cytokines was first investigated by qPCR in LPS-stimulated RAW 264.7 macrophages (see experimental section). LPS-activated macrophages produce excessive amounts of inflammatory cytokines including IL-1β, IL-6, and TNF-α and other inflammatory mediators such as NO to promote the development and progression of inflammation. 7A-C, the level of gene expression of inflammatory cytokines was significantly increased in LPS-stimulated RAW 264.7 macrophages compared to the remaining RAW 264.7 macrophages.

In contrast, other astaxanthin samples (1-3) significantly reduced the mRNA levels of IL-1β, IL-6 and TNF-α. Most importantly, Sample 2 was the least effective sample in reducing the levels of IL-1β, IL-6, and TNF-α expression, and Sample 3 more effectively reduced inflammatory cytokine levels (p <0.01) compared to Sample 1. Made it. These results indicate that Sample 3 prepared by chemical transformation most effectively inhibited the production of inflammatory cytokines in active macrophages among the three forms of astaxanthin. These results are consistent with the results of FIG. 6 on the anti-inflammatory effect of astaxanthin.

Next, the expression of genes related to antioxidant activity was analyzed. In addition to the ROS removal function, astaxanthin is known to reduce ROS production in LPS-stimulated macrophages by inhibiting the expression of NOXs, an important enzyme that generates ROS in macrophages by donating electrons to oxygen molecules. As shown in Fig. 7D, significant induction of Nox2 expression by LPS was found in the presence of astaxanthin. In particular, Sample 3 caused the greatest reduction in the expression of Nox2 (p <0.01). Furthermore, the expression of nuclear factor E2-related factor 2 (Nrf2), which plays an important role in the antioxidant defense mechanism of resistance and prevents oxidative stress by stimulating the production of antioxidant enzymes, was increased by LPS stimulation. The expression was also increased (Figs. 7E and F). Importantly, sample 3 prepared by chemical transformation was most effective in reducing the expression of Nrf2 and MnSOD. These results are consistent with the results of FIG. 5 on the antioxidant effect of astaxanthin. Overall, the highest anti-inflammatory and antioxidant activity was observed in Sample 3, which contained more Ast-mE than Ast-dE.

In this example, various forms of astaxanthin, Ast-F, Ast-ME, and Ast-DE by chemical conversion were prepared to investigate the degree of esterification and their effects on biological activities such as antioxidant and anti-inflammatory activities. Here, through gene expression analysis, we found out how astaxanthin performs antioxidant and inflammatory activities. Chemically synthesized sample 3, which contained more Ast-mE than Ast-dE, showed the strongest antioxidant and anti-inflammatory activity. However, the conversion efficiency of astaxanthin was not 100%, especially in the esterification reaction (step 2; FIG. 1), as shown in the HPLC results. Therefore, Ast-F remained in sample 3, making it difficult to identify the most suitable structure for antioxidant activity. In addition, the effect of fatty acid chain length should be evaluated. However, this example is the first study to prove that Ast-mE is the most effective compared to other forms by investigating the effect of esterification of astaxanthin on biological activity. This example also suggests that structural transformation through chemical modification can be applied to astaxanthin freely formed from other sources to increase antioxidant capacity. Thus, astaxanthin can be applied as a food supplement and/or as a functional ingredient to reduce oxidative stress. These results provide the basis for an in-depth investigation of astaxanthin and other antioxidant molecules, enabling the development of new antioxidants and anti-inflammatory molecules.

(Abbreviations used)

Ast-N natural astaxanthin

Astaxanthin in Ast-F-free form

Ast-E ester-type astaxanthin

Diester form of Ast-dE astaxanthin

Monoester form of Ast-mE astaxanthin

CAA cell antioxidant test

DCFH-DA 2′,7′-Dichlorofluorescein 3′6′-diacetate

DCF 2',7'-dichlorofluorescein

DMEM Dulbecco's Modified Eagle's Medium

FBS fetal bovine serum

IL interleukin

LPS lipopolysaccharide

MT 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide

NO nitric oxide

OD optical density

qPCR Quanitative polymerase chain reaction

ROS reactive oxygen species

TNF tumor necrosis factor

Nrf nuclear factor E2 related factor

Claims (5)

1-1 step of preparing a basic alcohol solution;
A 1-2 step of transesterification reaction of astaxanthin;
Step 1-3 of extracting astaxanthin;
A second step of synthesizing astaxanthin monoester; method for producing astaxanthin monoester comprising.
The method according to claim 1,
The step 1-1 of preparing the basic alcohol solution,
Step 1-1-1 of mixing sodium hydroxide with distilled water;
Step 1-1-2 of mixing the mixture of step 1-1-1 with one or more mixtures selected from methanol, ethanol, and propanol;
Step 1-1-3 of dehydrating the mixture of step 1-1-2;
Step 1-1-4 of repeating the step 1-1-3 2 to 4 times while removing the precipitate;
A method for producing astaxanthin monoester comprising a; step 1-1-5 of filtering the mixture of steps 1-1-4 using a fine filter.
The method according to claim 2,
The 1-2 step of transesterification reaction of the astaxanthin,
1-2-1 step of mixing the supercritically extracted astaxanthin into the mixture of step 1-1-5;
Step 1-2-2 of reflux stirring the mixture of step 1-2-1 after vortexing;
A method for producing an astaxanthin monoester comprising: a 1-2-3 step of cooling and neutralizing the mixture of the step 1-2-2 to room temperature.
The method of claim 3,
Step 1-3 of extracting the astaxanthin,
Step 1-3-1 of vortexing by mixing the mixture of step 1-2-3, in which the astaxanthin transesterification reaction is completed, with one or two compounds selected from hexane and petroleum ether ;
A 1-32 step of separating the upper layer by centrifuging the mixture by rotating the mixture at high speed;
A 1-33 step of drying the separated first 1-32 step mixture under reduced pressure;
A 1-3-4 step of mixing and dissolving the mixture of step 1-3-3 in a compound selected from dichloromethane, dichloroethane, and dichlorobutane; Method for producing astaxanthin monoester comprising a.
The method of claim 4,
The second step of synthesizing astaxanthin monoester,
A 2-1-1 step of mixing the compound of step 1-3-4 with an activator, a catalyst, and a saturated fatty acid;
Step 2-1-2 of reacting the mixture of step 2-1-1 at room temperature for 1 hour to 3 hours;
A 2-1-3 step of repeating the 2-1-2 step 2 to 4 times;
A method for producing an astaxanthin monoester comprising a 2-1-4 step of evaporating the 2-1-3 step mixture under reduced pressure.

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