How is astaxanthin synthesized?

 Astaxanthin (containing 40 carbon atoms) is a non-vitamin A carotenoid and is widely used in the production of health products, pharmaceuticals, cosmetics, food and feed additives.  In food, it can not only color, but also effectively play a role in preserving freshness, preventing discoloration, tastelessness, and deterioration, and can also be used for coloring beverages, food, and seasonings.  Astaxanthin has brilliant color, and can be unintentionally bound to actin, and is widely used in aquatic feeds to improve the skin and muscle color of farmed fish and increase the disease resistance of fish and shrimp[1,2] .

 

The following methods for the total synthesis of astaxanthin have been reported in the literature. (1) Taking Canthaxanthin (containing 40 carbon atoms) as the starting material, treating the two carbonyl groups with lithium diisopropylammonium to form the dienophile anion, then protecting it with trimethylchlorosilane to form the dienophile silyl ether, and then oxidizing the dienophile silyl ether with peroxyacid to obtain astaxanthin selectively.  Although the reaction step is short, but keratine is very expensive, two-step yield 55% ~ 65%, so this route is not economical [3]; (2) C9 + C1 → C10, 2C10 + C20 → C40 route, 6-oxoisophorone (C9) as the starting material, after a multi-step reaction, and then in the strong alkali and chloromethyltrimethylsilane (C1) addition reaction to obtain C10 enol silicone ether, then with C20 bisacetal, and then with C20 bisacetal to obtain C10 enol silicone ether, and then with C20 bisacetal. C10 enol silyl ether was obtained by the addition reaction with chloromethyltrimethylsilane (C1) under the action of strong base, and then condensed with C20 acetal to form C40 skeleton under the catalytic reaction of Lewis acid, and then dealkoxylated to form a conjugated double bond under the action of strong base to obtain astaxanthin [4]; (3) C9 + C6 → C15, 2C15 + C10 → C40 Route, 6-oxoisophorone (C9) was used as starting material, and then hexacarbonyloxyalkynyl alcohol (C6) was used as starting material, and then reacted with 2,7-dimethylsilyl (2,7-DM) and hexacarboxylic alcohol (C6) in a multi-step reaction to form C15 triphenyl phosphonate, and then reacted with 2,7-DM to obtain C15 triphenyl phosphonate. C15 triphenylphosphine salt was formed by a multi-step reaction with hexadecynyl alcohol (C6), and then reacted with 2,7-dimethyl-2,4,6-octatriene-1,8-dialdehyde (C10) in the presence of alkali to form astaxanthin.  Although the process is long and complicated, this route is the only one that has been industrialized so far[5-9] .

 

In this paper, the synthesis route of 2C15+C10→ C40 was adopted for the preparation of astaxanthin, in which a new route was designed for the synthesis of the key intermediate C15, i.e., C13+C2→ C15 (Scheme 1).  A total of 9 steps were carried out to obtain astaxanthin using α-violet ketone (C13) as the starting material.  On the basis of the literature[10] , the conditions for the preparation of compound 6 were optimized, so that the yield of each reaction step was above 85%.  In particular, the purification of compound 4, which was not solved in the literature, was accomplished, so that the whole route could be completed successfully.  The preparation of compounds 7-9 is also the key success or failure point of this route.  It was found that the synthesis reactions of compounds 7-9 were very selective and high in yield, and almost all of them were quantitative reactions.  In the selective epoxidation reaction of compound 7, it is known from chemical theory that the rate of addition of enolated double bonds to peroxyacid is 100,000 times higher than that of normal double bonds, and this is confirmed in our experiments, i.e., peroxyacid preferentially epoxidizes with enolated double bonds, and there is no heterogeneous point of oxidation of normal double bonds.  The key intermediate 6-hydroxy-3-(3-hydroxy-3-methyl-1,4-pentadienyl)-2,4,4-trimethyl-2-cyclohexen-1-one (9) was synthesized in 68% yield.  Astaxanthin was further synthesized according to the methodology of [5], with a total yield of 38% in 9 steps.  The present paper provides a new route for the synthesis of astaxanthin, which mainly refers to the synthesis of the key C15 intermediate compound 9 using the new route. The starting materials used are easily available, the reaction is highly selective and the overall yield is high.

 

1 Experimental component

1.1 Instruments and reagents

Main Instruments: Agilent 4890D Gas Chromatograph, GC/MS Mass Spectrometer (Agilent 6890N Network GC System, Agilent 5973 Network Mass Selective Detector), INOVA-400 Nuclear Magnetic Resonance Analyzer (TMS internal standard, CDCl3 solvent), PE PE-HPLC- 900 High Performance Liquid Chromatograph. ), PE-HPLC-900 high performance liquid chromatograph (PE, USA).

Reagents: α-Violanone (96% GC, industrial product), m-chloroperoxybenzoic acid (industrial product, 85%), 2,7-dimethyl-2,4,6-octatriene-1,8-dialdehyde were self-manufactured, and the rest of the raw materials and solvents were reagent grade.

 

1.2 Synthesis

1.2.1 Preparation of 4-(2,6,6-trimethyl-2,3-epoxy-1-cyclohexyl)-3-buten-2- ketone (3)

Referring to the literature [10], α-violet ketone 202.0 g (1.0 mol, 95%) was dissolved in dichloromethane (500 mL), cooled to below 5 ℃, and m-chloroperoxybenzoic acid (207.0 g, 1.05 mol, 85%) was added dropwise into a dichloromethane solution (500 mL) at a controlled temperature below 5 ℃, and then the reaction was carried out at 10 ℃ for about 1 h. The endpoint of the reaction was reached when the raw material disappeared from the TLC and GC traces. The reaction was carried out at 10 ℃ for about 1 h, and the end point was reached when the TLC and GC traces disappeared.  The reaction was carried out at 10 ℃ for about 1 h. The end point was reached when the raw material disappeared by TLC and GC. The filter was filtered, the cake was washed with dichloromethane (500 mL), and the dichloromethane solution was combined and washed with aqueous sodium sulfite (10%), aqueous sodium hydroxide (5%), and water, and then concentrated to obtain the epoxide 3 (209.0 g), which was analyzed by GC with purity of 94% and yield of 95%.

 

1.2.2 Preparation of 4-(2,6,6-trimethyl-3-hydroxy-1-cyclohexen-1-yl)-3-buten-2-one (4)

Referring to the literature [10], epoxide 3 (209.0 g) was dissolved in methanol (800 mL), 30% of sodium methanol methanol solution (50 mL), reflux reaction for 3 h, cooled to room temperature, neutralized with ice acetic acid (5 mL), methanol solution was recovered under reduced pressure, water was added, and the solvent was evaporated after drying to obtain the crude (211.0 g), the purity of the GC analysis was 87%.

 

1.2.3 Purification of 4-(2,6,6-trimethyl-3-hydroxy-1-cyclohexen-1-yl)-3-buten-2-one (4)

The crude product of the above reaction (211.0 g) was added with succinic anhydride (110.0 g, 1.1 mol), refluxed for 5 h, cooled down and extracted with aqueous sodium carbonate, the organic layer was discarded, the aqueous layer was acidified with 10% hydrochloric acid to pH=1~2, then extracted with ethyl acetate, dried and evaporated to remove the solvent, then added with methanol (800 mL) and a 30% methanolic solution of sodium methanolate (170.0 g, 1.05 mol), refluxed for 3 h, cooled down to room temperature, recovered by methanol solution under reduced pressure, added with water, evaporated to remove the solvent, then isolated and reacted. 1.05 mol), refluxed for 3 h, cooled to room temperature, recovered under reduced pressure from methanol solution, added water, extracted with ethyl acetate, dried and evaporated to remove the solvent, to obtain the pure hydroxyketone compound 4 (177.4 g) after separation and removal of impurities, GC analysis of the purity of 98%, the yield of 85%.

 

1.2.4 Preparation of 5-(2,6,6-trimethyl-3-hydroxy-1-cyclohexen-1-yl)-3-methyl-3-hydroxy-1,4-pentadiene (5)

Referring to the literature [10], hydroxyketone compound 4 (192.3 g) was dissolved in tetrahydrofuran (1000 mL), cooled to below -30 ℃, and then dropwise added with tetrahydrofuran solution of vinyl chloride Grignard reagent (700 mL, 3.3 mol/L), and reacted for 1 h at 0 ℃. Ether (1000 mL) and saturated aqueous ammonium chloride (250 mL) were added. Add ether (1000 mL) and ammonium chloride saturated aqueous solution (250 mL), filter, dry the filtrate with anhydrous magnesium sulfate, evaporate the solvent to obtain the diol compound 5 (208.5 g), yield 96%.

 

1.2.5 Preparation of 5-(2,6,6-trimethyl-3-oxo-1-cyclohexen-1-yl)-3-methyl-3-hydroxy-1,4-pentadiene (6)

Referring to the literature [10], the diol compound 5 (208.5 g) was dissolved in a mixed solution of acetone and dichloromethane (2000 mL, V:V=1 ∶ 1), added aluminum isopropanol (450.0 g), refluxed for 5 h, cooled to room temperature, neutralized with sulfuric acid (w = 10%) and acidified to pH=3-4, the organic phase was dried with anhydrous magnesium sulfate, and evaporated the solvent, to obtain compound 6 (200.6 g), yield 96%. The organic phase was dried with anhydrous magnesium sulfate, and the solvent was evaporated to give compound 6 (200.6 g) in 96% yield.

 

1.2.6 Preparation of 5-(2,6,6-trimethyl-3-trimethylsilyloxy-1,3-cyclohexadien-1-yl)-3-methyl-3-trimethylsilyloxy-1,4-pentadiene (7)

Compound 6 (24.0 g) was dissolved in anhydrous tetrahydrofuran, 250 mL of 2 mol/L lithium diisopropylammonium (LDA) solution was added dropwise at -10 ℃, completed the addition, and stirred for 30 min at -10 ℃, 24.0 g of hexane solution of trimethylchlorosilane was added dropwise, completed the addition, and stirred for 1 h at 0 ℃, 200 mL of water was added, and 200 mL of hexane was added. After dropping and stirring for 1 h at -10 ℃, 200 mL of water was added, 200 mL of hexane was added, the organic layer was washed with 5% sodium bicarbonate solution, and the solvent was evaporated under pressure reduction to obtain 35.7 g of light yellow oily 7. Yield 97%.  1H NMR (CDCl3, 400 MHz) δ: 0.12-0.20 [m, 18H, Si(CH3)3], 0.98 (s, 6H, 2CH3), 1.45 (s, 3H, CH3), 1.76 (s, 3H, CH3), 2.03 (d, J=5.0 Hz, 2H, CH2), 4.85 (t, J=4.5 Hz, 1H, cyclohexene hydrogen). , 1H, cycloalkenyl hydrogen), 5.02 (dd, J=1.2, 11.0 Hz, 1H, side-chain-terminal cis-alkenyl hydrogen), 5.20 (dd, J=1.2, 17.0 Hz, 1H, side-chain-terminal trans-alkenyl hydrogen), 5.54 (d, J= 16.0 Hz, 1H, HC = CH), 5.99 (d, J= 16.0 Hz, 1H, HC = CH), 5.90 (dd, J= 5.0 Hz, 1H, CH 5.90 (dd, J=11.0, 17.0 Hz , 1H, = CH).

 

1.2.7 Preparation of 5-(2,6,6-trimethyl-3,4-epoxy-3-trimethylsilyloxy-1,3-cyclohexadien-1-yl)-3-methyl-3-trimethylsilyloxy-1,4-pentadiene (8)

Compound 7 (30.0 g) was dissolved in 200 mL of anhydrous dichloromethane, 18.0 g of m-chloroperoxybenzoic acid dissolved in 200 mL of dichloromethane was added dropwise at -20 ℃, the dropwise addition was completed at -20 ℃, then stirred for 30 min at -10 ℃, and mixed with 10% sodium dithionite solution 100 mL. Then mix with 10% sodium dithionite solution for 1 h. The organic layer was washed with 200 mL of water, dried with anhydrous sodium sulfate, and evaporated under pressure to remove the solvent to obtain 30.0 g of light yellow oil 8 in 95% yield.  1H NMR (CDCl3, 400 MHz) δ: 0.12-0.20 [m, 18H, Si(CH3)3], 0.98 (s, 6H, 2CH3), 1.45 (s, 3H, CH3), 1.73 (s, 3H, CH3), 1.92 (d, J=5.0 Hz, 2H, CH2), 2.85 (t, J=4.5 Hz, 1H, cyclopentamethylene), 1.85 (t, J=4.5 Hz, 1H, cyclopentamethylene). , 1H, last cyclic methyl group), 5.02 (dd, J= 1.2, 11.0 Hz, 1H, side-chain-terminal cis-alkenylhydride), 5.20 (dd, J=1.2, 17.0 Hz, 1H, side-chain-terminal trans-alkenylhydride), 5.54 (d, J=16.0 Hz, 1H, HC ＝ CH), 6.01 (d, J=16.0 Hz, 1H, HC ＝ CH) , 5.89 (dd, J = 11.0, 17.0 Hz, 1H, ＝CH).

 

1.2.8 Synthesis of 6-hydroxy-3-(3-hydroxy-3-methyl-1,4-pentadienyl)-2,4,4-trimethyl-2-cyclohexen-1-one (9)

 

30.0 g of oil 8 was mixed with 200 mL of methanol and 2.0 g of potassium carbonate, refluxed for 2 h. After cooling, the solid was filtered off, and the methanol was removed by evaporation under pressure to give compound 9 (20.0 g).  The yield was essentially quantitative.  1H NMR (CDCl3, 400 MHz) δ: 1.14 (s, 3H, CH3), 1.28 (s, 3H, CH3), 1.46 (s, 3H, CH3), 1.86 (s, 3H, CH3), 1.80 (t, J=15.0 Hz, 2H, CH2), 2.18 (s, 1H, OH), 3.68 (s, 1H, OH), 4.33 (q, 1H, OH), 2.18 (s, 1H, OH), 3.68 (s, 1H, OH). OH), 4.33 (q, J=6.0 Hz, 1H, cyclic methyl group), 5.13 (dd, J=1.2, 11.0 Hz, 1H, cis-alkenyl hydrogen at the end of the side chain), 5.27 (dd, J=1.2, 17.0 Hz, 1H, trans-alkenyl hydrogen at the end of the side chain), 5.75 (d, J=16.0 Hz, 1H, HC=CH), 6.24 (d, J=16.0 Hz, 1H, HC=CH), 6.24 (d, J=16.0 Hz, 1H, HC=CH), 6.20 (d, J=15.0 Hz, 1H, CH J= 16.0 Hz, 1H, HC = CH), 5.97 (dd, J=11.0, 17.0 Hz, 1H, CH =); ESI-MS m/z (%): 251 (M++1, 100), 249 (M+-1, 55), 233 (18). 1H NMR is in agreement with literature [5].

 

1.2.9 Synthesis of astaxanthin(1)

Referring to the synthesis method in the literature [5], 100.0 g of compound 9 was dissolved in 400 mL of dichloromethane, and added dropwise into 104.0 g of 48% HBr aqueous solution within 30 min at 0 ℃, the mixture was stirred at 0 ℃ for 30 min, 360 mL of water was added, the organic phase was separated, the aqueous phase was extracted with 50 mL of dichloromethane, the organic phase was combined and mixed with 360 mL of water, 47.0 g of solid NaHCO3 was added, and then each phase was stirred together briefly, the organic phase was separated, washed with 360 mL of water, and 3 mL of 1,2-solid NaHCO3 was added. The aqueous phase was extracted once with 50 mL of dichloromethane, the organic phase was combined and mixed with 360 mL of water, 47.0 g of solid NaHCO3 was added, the phases were briefly stirred together again, the organic phase was separated, washed with 360 mL of water, 3 mL of 1,2-butyl epoxide was added, and 105.0 g of triphenylphosphine was added while the mixture was cooled to ≤10 ℃. The mixture was stirred at room temperature for 18 h, and then 2,7-dimethyl-2,4,6-octatriene-1,8-dialdehyde was added.  The mixture was cooled to 0 °C and 58 g of 30% sodium methanol solution was added at 0 °C. The mixture was stirred at 0 °C for 3 h. 500 mL of water was added and the organic phase was separated. The aqueous phase was extracted twice with 100 mL of dichloromethane, the organic phases were combined, the organic phases were washed once with 300 mL of water, and the dichloromethane was evaporated, with the addition of methanol up to a boiling point of 65 °C. The suspension was refluxed for 15 h and then the mixture was allowed to reach its boiling point.  The suspension was refluxed for 15 h, then cooled to 0 ℃, and the resulting crystals were filtered out, dissolved in 500 mL of dichloromethane, replaced the solvent with methanol again as described above, and the filter cake was dried to yield 62.0 g (75% of the theoretical value) of astaxanthin. The purity was 98% by HPLC. ESI-MS and 1H NMR confirmed that it was consistent with the control.

 

2 Results and Discussion

When m-chloroperoxybenzoic acid (MCPB) was used in the first step to reduce the oxidation of α-violet ketone, the amount of MCPB was slightly overdosed by 5%~10% to ensure the complete epoxidation of α-violet ketone.  In the early stage of the reaction, it is easy to follow the reaction progress by TLC, but in the late stage of the reaction, it is necessary to use gas chromatography (GC) to follow the reaction progress due to the presence of impurities of β-violanone, which may interfere with the judgment of the end point of the reaction.  When the α-violet ketone material disappeared, the gas chromatographic analysis showed that the target product was in the range of 94% to 95%, and then the reaction should be terminated in time.  If the reaction continues, an impurity peak close to the main peak will increase significantly, from 1% to 10%.  Therefore, tracking the reaction with gas chromatography is one of the key factors in controlling the reaction.

When the epoxide is rearranged to the target product hydroxyketone compound 4 under alkaline catalytic reaction, an impurity is generated, which is reported to be a diketone compound in the literature, and is separated by a chromatographic column.  We analyzed two impurity peaks by gas chromatography, the impurity at about 8% was bis(ketone)11 as reported in the literature, and the impurity at 2%-3% was the product of hydroxyl dehydration to alkene 12.

 

The purity of hydroxyketone compound 4 will greatly affect the reaction yield and purity of the following fifteen-carbon synthesis unit, and it is also one of the key control points of this route. The chromatographic column separation method adopted in the literature is unsuitable for industrial production, so we creatively proposed the following purification method, i.e., using the hydroxyl group on the hydroxyketone compound 4 to form an ester with succinic anhydride, and then the ester-forming product was dissolved in alkaline aqueous solution, and the impurities insoluble in water were extracted with a non-water solvent, and then the ester-forming product was esterified with methanol under alkaline catalysis to obtain pure hydroxyketone compound 4 in 85% yield. The ester-forming product was dissolved in alkaline aqueous solution, and the water-insoluble impurities were extracted by a non-water-soluble solvent, and then the ester-forming product was esterified with methanol under alkaline catalyst to obtain the pure hydroxyketone compound 4 in 85% yield. The above post-processing method has not been reported in the literature.

 

3 Conclusion

The industrialized α-violet ketone product was used as the starting material, and the key intermediate 6-hydroxy-3-(3-methyl-3-hydroxy-1,4-pentadienyl)-2,4,4-trimethyl-2-cyclohexen-1-one was obtained by a multistep reaction using the industrially available conventional isolation method, and the C15 triphenylphosphine salt was obtained by the reaction of hydrobromic acid and triphenylphosphorane, and condensation of the C15 triphenylphosphorane salt with 2,7-dimethyl-2,4,6-octatriene-1,8-diels-Alder in the presence of alkali. C15 triphenylphosphine salt was then reacted with hydrobromic acid and triphenylphosphine to obtain C15 triphenylphosphine salt, which was condensed with 2,7-dimethyl-2,4,6-octatriene-1,8-dialdehyde in the presence of alkali, and then recrystallized to obtain astaxanthin with a purity of 98%.  HPLC, ESI-MS and 1H NMR confirm that it is consistent with the control. The total yield of the 9-step reaction was 38%.

 

References:

1 Li, H. M.; Gao, L. Fine Chem. 2003, 20(1), 32 (in Chinese). (Li, H. M.; Gao, L. Fine Chem. 2003, 20(1), 32.)

2 Meyers, S. Pure Appl. Chem. 1994, 66(5), 1069.

3 Bemhard, K.; Mayer, H. Pure Appl. Chem. 1991, 63(1), 35.

4 Ruttimann, A. Pure Appl. Chem. 1999, 71(12), 2285.

5 Zell, R.; Broger, E. A.; Crameri, Y.; Wagner, H. P.; Dinkel, J.; Schlageter, M.; Lukac, T. Helv. Chim. Acta 1981, 64, 2436.

6 Widmer, E. Pure Appl. Chem. 1985, 57(5), 741.

7 Mayer, H. Pure Appl. Chem. 1994, 66(5), 931.

8 Ernst, H.; Dobler, W.; Paust, J.; Rheude, U. US 5455362, 1995 [Chem. Abstr. 1995, 122, 265715f].

9 Ernst, H. Pure Appl. Chem. 2002, 74(8), 1369.

10 Rosenberger, M.; McDougal, P.; Bahr, J. J. Org. Chem. 1982, 47(11), 2130.

 

How does astaxanthin protect against reactive oxygen species-induced mitochondrial damage?

 Astaxanthin is widely found in nature, especially in shrimp, crab, fish, algae, yeast and bird feathers, and plays a role in color development. Astaxanthin is a kind of carotenoid, has reduced lipid peroxidation, scavenging free radicals, delay aging, inhibit tumorigenesis and improve immunity, prevention of cardiovascular disease and other physiological functions[1 ,2] . In recent years, astaxanthin has been widely used in medicine, and has received more and more attention [3].

 

1 Material

1.1 Instruments

Allegra 64R low-temperature high-speed centrifuge (Beckman); 722 visible spectrophotometer (Shanghai Spectrum Instrument Co., Ltd.); Wellscan MK 3 enzyme labeling instrument (Bio-Rad, USA); Adventurer electronic balance (Ohaus, USA).

 

1.2 Drugs and reagents

Astaxanthin (Sigma, purity 95%); 3% hydrogen peroxide (H2O2) and vitamin C (China Pharmaceutical Shanghai Chemical Company); calf serum albumin (BSA) (Roche); superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), nitric oxide (NO) and ATPase kit (Nanjing Jianjian Bioengineering Institute); dimethyl sulfoxide (DMSO) (Sigma, purity ≥999%); other reagents were domestic analytical purity. (Nanjing Jianjian Institute of Biological Engineering); dimethyl sulfoxide (DMSO) (Sigma, purity ≥99.9%); other reagents were domestic analytical purity.

 

1.3 Laboratory animals

Clean grade Wistar rats, weighing 180~210 g, provided by Harbin Medical University, Certificate of Conformity No. P00101014.

 

2 Methodology

2.1 Isolation and Preparation of Mitochondria [4]

Differential gradient centrifugation for the preparation of mitochondria: rats were killed by decapitation, the livers were dissected quickly and weighed accurately, and 10 % of the tissue homogenate was prepared by adding cold physiological saline in the ratio of 1:10 (w/v), and then centrifuged at 1,500 r/min for 10 min in a low-temperature and low-speed centrifuge to discard the precipitate, and then centrifuged the supernatant at 10,000 r/min for 15 min, and then the precipitate was mitochondria, which was suspended in 1 mL of the homogenate. The mitochondria were suspended in 1 mL of homogenate, and the protein was quantified by the Caulmers Brilliant Blue method, and the protein concentration of the mitochondrial suspension was adjusted to 2 g/L. The above processes were operated at 4 ℃.

 

2.2 H2O2 damage to mitochondria

The experiments are shown in Table 1 , mitochondrial protein suspension was added with sodium chloride, H2O2, vitamin C and different amounts of astaxanthin to form a mixture, and the mitochondria were incubated in a 37 ℃ water bath [5] for 1h.

 

2.3 Measurement of biochemical indicators

Mitochondrial MDA, NO and GSH contents as well as SOD and ATPase activities were determined according to the kit instructions.

 

2.4 Statistical treatment of data

The data were statistically analyzed by SPSS15.0 software and expressed as group

Differences were analyzed by one-way ANOVA, and P < 0.05 was considered ± statistically significant.

 

3 Results

3.1 Effect of astaxanthin on mitochondrial MDA content

See Figure 1 . As shown in Figure 1, the MDA content in the mitochondria of the H2O2 damage group was significantly higher than that of the control group, and the difference was statistically significant (P<0.01). The high dose group of astaxanthin could significantly inhibit the increase of MDA content caused by reactive oxygen species, and there was no significant difference with the control group (P>0.05).

 

3.2 Effect of astaxanthin on mitochondrial NO content

See Figure 2 . As shown in Figure 2, the NO content in the mitochondria of the H2 O2 damage group was significantly higher than that of the control group, and the difference was statistically significant (P<0.01). The high dose group of astaxanthin could significantly inhibit the increase of NO content caused by reactive oxygen species, and there was no significant difference with the control group (P>0.05).

 

3.3 Effect of astaxanthin on mitochondrial GSH content

See Figure 3. As shown in Figure 3, the GSH content in mitochondria of the H2O2 damage group was significantly lower than that of the control group, and the difference was statistically significant (P<0.01). The high dose group of astaxanthin could obviously inhibit the decrease of GSH content caused by reactive oxygen species, and there was no significant difference with the control group (P>0.05).

 

3.4 Effect of astaxanthin on mitochondrial SOD content

See Figure 4. As shown in Figure 4, the mitochondrial SOD activity in the H2O2 damage group was significantly lower than that in the control group, and the difference was statistically significant (P<0.01). Astaxanthin high-dose group can obviously inhibit the reduction of SOD activity caused by reactive oxygen species, and there is no significant difference with the control group (P>0.05).

 

3.5 Effects of astaxanthin on mitochondrial ATPase activity

See Figure 5. As shown in Figure 5, the activities of Na+,K+-ATPase, Mg2+-ATPase and Ca2+-ATPase in the H2O2 injury group were significantly lower than those in the control group (P<0.01). The activities of three kinds of ATPase in the low, medium and high doses of astaxanthin were stronger than those in the H2O2 injury group, and the ATPase activities increased with the increase of the dose of astaxanthin.

 

4 Discussion

Reactive oxygen species are the most important free radicals in the human body. Excessive reactive oxygen species can cause oxidative damage to biological macromolecules such as proteins and nucleic acids, resulting in aging, tumors, diabetes, and autoimmune diseases[6] . Hydrogen peroxide is the most common reactive oxygen species.

 

Mitochondria are membrane organelles of eukaryotic cells that perform important functions such as synthesizing ATP, generating ROS, regulating cellular oxidation/reduction signals, controlling apoptosis and gene expression. It has been found that mitochondria have a cytosol-independent nitric oxide synthase (NOS) system, which generates NO that directly damages the mitochondria. H2O2 damage to mitochondria generates large amounts of NO, which attacks the unsaturated fatty acids on the mitochondrial membrane, resulting in an increase in MDA and a decrease in mitochondrial activity[7,8] . The possible mechanism of astaxanthin to reduce NO production is the inhibition of NOS activity. H2O2 decreases SOD activity, depletes GSH, and decreases ATPase activity in mitochondria, resulting in abnormal mitochondrial energy metabolism and affecting the normal growth of the cells[9, 10] . Mitochondria are also the main calcium reservoir of the cell, and H2O2 reduces the activities of mitochondrial Ca2+-ATPase and Mg2+-ATPase, decreasing the Ca2+ uptake by mitochondria, and then cytoplasmic Ca2+ overload occurs, which affects the metabolism of the cell and even leads to the death of the cell.The activity of Na+-K+-ATPase of the mitochondrial membrane is decreased by H2O2, which leads to the retention of mitochondrial Na+ , swelling and even rupture of the mitochondria. H2O2 reduces Na+-K+-ATPase activity in the mitochondrial membrane, causing mitochondrial Na+ retention and mitochondrial swelling and rupture[11] . The results of this study showed that astaxanthin can increase mitochondrial SOD activity, reduce GSH consumption, and effectively counteract the reduction of Ca2+-ATPase, Mg2+-ATPase, and Na+-K+-ATPase activities, which is of positive significance in protecting mitochondria from oxidative damage by reactive oxygen species.

 

References:

[1] ZHU Mingjun, ZONG Hua, WU Zhenqiang, et al. Progress of astaxanthin research[J]. Food Industry Science, 2000, 21:79-81.

[2] Jyonouchi H, Sun S , Iijima K, et al. Antitumor activity of astaxanthin and its mode of action[J]. Nutr Cancer, 2000, 36:59- 65.

[3] Guerin M, Huntley M E, Olaizola M. Haematococcus astaxanthin application for hunman health and nutrition[J]. Trends Biotechnol, 2003, 21(5):210-6.

[4] PANG Hui, TANG Guifang, WANG Yanhong. Protective effects of spirulina polysaccharides on oxidative damage in rat liver mitochondria[J]. Guangdong Medicine, 2006, 27(12):1786-1787

[5] YU Tengfei, LIU Ping, WANG Donghai, et al. Protective effect and mechanism of piperine on myocardial mitochondria of rats with hydrogen peroxide injury[J]. Journal of Pharmacy of the People's Liberation Army, 2008, 24(1):7-9.

[6] Remirez D, Tafazoli S, Delgado R, et al. Preventing hepatocyte oxidative stress cytotoxicity with Mangifera indical extract (Vimang) [J]. Drug Metabol Drug Interact, 2005, 21( 1):19-35.

[7] Lin H I, Chou S J, Wang D, et al. Reperfusion liver injury induces down-regulation of eNOS and up-regulation of iNOS in lung tissues[J]. Transplant Proc, 2006, 38(7):2203-2206.

[8] SHANG Tao, ZHANG Jian-Xin, LI Lan-Fang, et al. Effects of NG-nitro-L-arginine on endotoxic lung mitochondrial injury in rats[J].  Chinese Journal of Pathophysiology, 2008 , 24

(4): 920-924.

[9] Gao Meijuan, Liu Ximangan, Gao Gui, et al. Antimitochondrial lipid peroxidation by glutathione[J]. Journal of Biological Chemistry, 1997 , 13(3):287-291.

[10] CHEN Rong, ZHENG Weiyun, YU Qun, et al. Effects of oil pollution on the glutathione content and related enzyme activities of the oyster Crassostrea virginica[J].   Journal of Oceanography, 2003 , 25:226-229.

[11] JIAO Shuping, NI Haijing, XUE Lijuan.  Protective effects of mountain grape polyphenols on mitochondrial oxygenation injury in rat myocardium[J]. Journal of Jilin University, 2008, 34(1):117- 119.

 

Can astaxanthin improve hyperlipidemia symptoms?

 Astaxanthin (astaxanthin) is known as 3, 3 ′ - dihydroxy - β-carotene - 4, 4 ′ - ketone, for the lutein-like carotenoids, naturally occurring in crustaceans (such as shrimp, crab shells), fish (such as salmon), bird feathers and some microalgae. Recent studies have shown that astaxanthin has a strong oxidative function, can effectively quench the body oxidized free radicals and reactive oxygen species, with the prevention and treatment of cardiovascular disease, prevention and treatment of atherosclerosis, inflammation, intervention in diabetes and other aspects of nutritional health care.

 

Astaxanthin has a variety of isomers, it is generally believed that algae-derived levulinic astaxanthin has the highest biological activity in vivo, the U.S. FDA approved natural algae-derived astaxanthin as a nutritional supplement in 1999, and China's Food and Drug Administration also approved in May 2012 the license of Astaxanthin as a health food. Astaxanthin has been widely recognized as an excellent antioxidant/anti-inflammatory agent by the global food and pharmaceutical industries, and is widely used as a functional food. International pharmaceutical giants are also developing astaxanthin derivatives to treat and prevent cardiovascular diseases.

Hyperlipidemia is defined as a condition in which plasma concentrations of lipid components such as TC, TG, and low-density lipoprotein (LDL) are above normal and HDL is below normal. The main hazard of hyperlipidemia is atherosclerosis, which leads to many related diseases. The main effects of astaxanthin in ameliorating hyperlipidemia are lowering of TG and elevation of high-density lipoprotein cholesterol (HDL-C). Fassett RG, in Mar Drugs [3], reviewed a number of reports on the lipid regulation of astaxanthin in animal experiments and clinical studies, and described the potential use of astaxanthin as a future preventive or therapeutic agent for cardiovascular disease (the following studies refer to natural astaxanthin of algal origin). The potential of astaxanthin as a future preventive or therapeutic drug for cardiovascular diseases is also discussed (all astaxanthin in the following studies are natural astaxanthin of algal origin).

 

1 Animal studies on lipid regulation by astaxanthin

Early animal studies have shown that astaxanthin can regulate blood lipids . Luo Renyong et al. reported the effect of astaxanthin on blood lipids in SD rats. The researchers randomly divided SD rats into 4 groups according to total cholesterol levels: low, medium, high, and control, corresponding to 3 increasing concentrations of astaxanthin solution by gavage for 30d. Total cholesterol TC, TG and HDL-C were measured, and the results showed that the TC and TG of the control group were higher than those before the test. In comparison, the TC of all astaxanthin groups was lower than that of the control group. The TG of the medium- and high-dose natural astaxanthin groups was lower than that of the control group, so astaxanthin has a lipid-lowering function. This conclusion was also confirmed by Hussein et al, who showed that astaxanthin could not only increase HDL-C but also improve the quality of blood lipid, and the blood sugar level was lower than that of the control group. Hussein et al. showed that in rat model animals suffering from metabolic syndrome, astaxanthin not only significantly increased the mean HDL-C value, but also significantly improved the level of lipocalin and lowered the plasma level of TG and free fatty acids. In mice, it was found that the addition of astaxanthin significantly reduced liver and plasma TG levels in mice fed high-fat food, and compared with the high-fat food group without astaxanthin, liver weight, liver TG, plasma TG and TC were significantly reduced in obese mice, and the increase in body mass and adipose tissue was also significantly inhibited, which suggests that astaxanthin has a high value for the improvement of hyperlipidemia. These results indicate that astaxanthin has a high value in improving hyperlipidemia.

 

In addition to the above, another study reported that astaxanthin significantly reduced oxidized low-density lipoprotein (Ox-LDL). In this study, normal rats were induced with streptozotocin (STZ), which induced a complex over-oxidative stress and vascular endothelial dysfunction, increasing serum Ox-LDL and aortic malondialdehyde (MDA) levels, while astaxanthin significantly reduced serum Ox-LDL and aortic MDA levels.Yang et al. also demonstrated that astaxanthin can significantly improve lipid metabolism and cholesterol metabolism. Yang et al. also reported that astaxanthin significantly improved lipid metabolism and cholesterol metabolism. Although β-carotene is a carotenoid, it does not cause changes in plasma cholesterol.

 

2 Clinical studies on the regulation of blood lipids by astaxanthin

Yoshida et al. 2010 reported that astaxanthin can significantly regulate blood lipids, the researchers adopted a randomized, double-blind model, selecting 61 volunteers aged 20-65 years without obesity (BMI<25kg/m2), and gave astaxanthin (0, 6, 12, 18mg/d) for 12 weeks, the study showed that after taking astaxanthin, TG was significantly reduced by 30%, HDL-C and lipocalin were significantly increased by 30%-40%, and lipocalin changes were correlated with HDL-C changes. The study showed that TG was significantly reduced by about 30%, HDL-C and lipocalin were significantly increased by 30%-40%, and the changes in lipocalin were positively correlated with the changes in HDL-C in the experimental group after the administration of astaxanthin. In this study, it was also found that body mass index (BMI) and LDL-C were not significantly affected before and after the trial (in all of the above dose groups), and that changes in TG, HDL-C, and lipocalin were independent of age and BMI.

 

Consistent with this finding, a study by Choi et al. in obese volunteers showed that astaxanthin also significantly reduced LDL and ApoB, further suggesting that astaxanthin is a good regulator of blood lipids regardless of whether one is obese or not. Earlier studies have also reported that astaxanthin significantly increased total antioxidant status, HDL levels, and TG concentrations in the blood. In their study, 15 healthy menopausal women were divided into three groups, one control group and two groups were given 2mg and 8mg/day of astaxanthin respectively. After eight weeks of administration, it was found that HDL concentration in the 2mg group increased from (50.6 ± 5 . 8) mg/dL to (50.6 ± 5 . 8) mg/dL. 8) mg/dL to (60 .4 ± 7 . 1) mg/dL , corresponding to an increase from (44 .4 ± 10 .7) mg/dL to (49 .4 ± 2 .7) mg/dL in the 8-mg group; TG concentrations in the 2-mg group declined from) (171 .6 ± 67 .4) mg/dL to (145 .8 ± 5 .1) mg/dL . (171.6±67.4) mg/dL to (145.8±5.1) mg/dL in the 2-mg group, with significant differences in all results. It was also reported that astaxanthin significantly reduced the levels of 12- and 15-hydroxy fatty acids in the body, indicating that astaxanthin can significantly reduce free fatty acids.

 

In addition to the above clinical studies, Iwamoto et al. reported that astaxanthin also significantly inhibited low-density lipoprotein (LDL) oxidation in a study of 24 volunteers who received astaxanthin (1.8, 3.6, 14.4, and 21.6 mg/d for 2 weeks), and the study showed a significant prolongation of low LDL oxidation time. The fourth edition of Internal Medicine has made it clear that the core cause of atherosclerosis is not LDL but oxidized low-density lipoprotein (Ox-LDL), and therefore astaxanthin's inhibition of the oxidation of LDL to Ox-LDL is even more significant in the inhibition of atherosclerosis.

 

3 Summary

Both animal studies and randomized, double-blind, placebo-controlled clinical studies in human beings have fully demonstrated that astaxanthin can significantly improve hyperlipidemia, specifically by raising HDL and lowering TG and Ox-LDL. Compared with existing lipid-lowering statins and fibrates, natural astaxanthin of algal origin does not have any toxic side effects, and has a higher degree of safety, so astaxanthin has the potential of becoming a drug (safe prophylactic drug) to improve hyperlipidemia. Therefore, astaxanthin is expected to be a potential drug (safe preventive drug) to improve hyperlipidemia.

 

References:

[1] WU Wanqiang,LIU Xuebo . Research progress of astaxanthin biofunctions[J] . Agricultural Products Processing, 2012(9):91-96.

[2] Riccioni G. Marine Carotenoids and Cardiovascular Risk Markers [J].Mar Drugs,2011,9(7):1166-1175.

[3] Fassett RG,Coombes JS.Astaxanthin:a potential therapeutic agent in cardiovascular disease[J]. Mar Drugs,2011,9(3):447-465.

[4] LUO Ren-Yong, ZENG Yong-Lan. Experimental study on lipid-lowering function of natural astaxanthin soft capsules[J]. Modern Preventive Medicine,2009,36(4):731-732.