Application of cis-astaxanthin in preparation of lipid-lowering product

Abstract

The invention relates to an application of cis-astaxanthin in preparation of a lipid-lowering product. The cis-astaxanthin is astaxanthin as shown in a formula (I) and / or astaxanthin as shown in a formula (II). Compared with all-trans-astaxanthin, the specific cis-astaxanthin disclosed by the invention has the advantage that the lipid-lowering activity of the cis-astaxanthin is obviously better. The specific cis-astaxanthin not only can reduce lipid accumulation, the content of triglyceride and the ratio of oleic acid to stearic acid, but also can reduce the content of large lipid droplets and total cholesterol. The specific cis-astaxanthin disclosed by the invention not only can inhibit weight gain, but also can prevent and treat fatty liver, inhibit liver injury induced by obesity and reduce adipocyte size.

Description

Technical Field

[0001] The present invention relates to the technical field of astaxanthin, and more particularly to application of cis-astaxanthin in the preparation of lipid-lowering products. Background Art

[0002] Obesity may lead to pathological changes such as elevated total cholesterol (TC) and triglycerides (TG), which may further lead to various diseases or complications such as hyperlipidemia. The development of active substances with lipid-lowering activity is currently a hot topic in research.

[0003] Astaxanthin (3,3'-dihydroxy-4,4'-diketo-β,β'-carotene) is widely found in crustaceans, as well as fish, birds, algae, and fungi. Its molecular structure is divided into cis and trans forms, depending on the arrangement of the groups attached to the carbon-carbon double bond. Most astaxanthin in nature exists in the all-trans form, but it isomerizes to the cis form under the influence of heat, light, or other environmental factors.

[0004] Existing studies have reported that all-trans astaxanthin has lipid-lowering activity, but this activity still needs to be further improved. Summary of the Invention

[0005] The primary purpose of the present invention is to overcome the problem that the lipid-lowering activity of existing all-trans astaxanthin still needs to be further improved, and to provide the use of cis-astaxanthin in the preparation of lipid-lowering products. Compared with all-trans astaxanthin, the lipid-lowering activity of the specific cis-astaxanthin of the present invention is significantly superior.

[0006] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0007] The use of cis-astaxanthin in the preparation of lipid-lowering products, wherein the cis-astaxanthin is astaxanthin represented by formula (I) and / or astaxanthin represented by formula (II):

[0008]

[0009] The astaxanthin represented by formula (I) of the present invention is 9-cis astaxanthin; the astaxanthin represented by formula (II) is 13-cis astaxanthin.

[0010] Existing studies have shown that all-trans astaxanthin has certain lipid-lowering activity, such as the document Doi: 10.1039 / d3fo01403g.

[0011] Other studies have used all-trans astaxanthin as a lead compound to develop all-trans astaxanthin derivatives with improved lipid-lowering activity, but the extent of this improvement remains limited. For example, a study (DOI: 10.2174 / 1389201021666200626162301) compared the triglyceride-lowering activity of astaxanthin diester and all-trans astaxanthin. Compared to the control group, astaxanthin diester lowered triglycerides by approximately 1.27 times the level of all-trans astaxanthin.

[0012] The inventors of the present invention have found that compared with all-trans astaxanthin, the lipid-lowering activity of specific cis astaxanthin is significantly better. Specific cis astaxanthin can not only reduce lipid accumulation, triglyceride content and the ratio of oleic acid / stearic acid, but also reduce the content of large fat droplets and total cholesterol. The specific cis astaxanthin of the present invention can not only inhibit weight gain, but also prevent and treat fatty liver, inhibit obesity-induced liver damage and reduce fat cell size. Among them, the degree to which specific cis astaxanthin reduces triglycerides is at least 1.9 times that of all-trans astaxanthin, and the degree of improvement in effect is significantly better than existing technical means (such as group modification of all-trans astaxanthin to obtain derivatives).

[0013] The inventors of the present invention also found through transcriptomics, metabolomics, 16s microbiome and qPCR technical analysis that a specific cis-astaxanthin can effectively lower blood lipids by regulating the expression of lipid-lowering signaling pathway genes, lipid metabolism-related metabolites, metabolic pathways, lipid metabolism-related intestinal flora, and the regulation of obesity-related genes.

[0014] In addition, compared with all-trans astaxanthin, the cis-astaxanthin of the present invention can not only independently regulate the three metabolites of 3-ureidopropionic acid, cuminaldehyde and glycodeoxycholic acid, and independently regulate the two metabolic pathways of steroid synthesis and β-alanine, but also independently participate in the regulation of more obesity-related genes (including Fitm1, Acot2, Manf, TSPO, Cisd1, Fdps, Id1, Abca1, Foxa2 and Fgf2) and enrich the beneficial bacteria Enterorhabdus that are closely related to lipid metabolism.

[0015] Preferably, the cis-astaxanthin is a mixture of astaxanthin represented by formula (I) and astaxanthin represented by formula (II), and the mass ratio of astaxanthin represented by formula (I) to astaxanthin represented by formula (II) is 1:(1.5-2).

[0016] Preferably, the cis-astaxanthin is astaxanthin represented by formula (I):

[0017] When cis-astaxanthin is 9-cis-astaxanthin, cis-astaxanthin not only has a better effect on reducing lipid accumulation, triglyceride content, the number of large lipid droplets, and the ratio of oleic acid / stearic acid, but also has a better effect on improving athletic ability and has a better ability to downregulate the expression of sbp-1, mdt-15, fat-7 and daf-16 genes.

[0018] Preferably, the cis-astaxanthin is astaxanthin represented by formula (II):

[0019] When the cis-astaxanthin is 13-cis-astaxanthin, the ability of cis-astaxanthin to upregulate the expression of the daf-2 gene is better.

[0020] Preferably, the lipid-lowering product is a blood lipid-lowering product.

[0021] More preferably, the lipid-lowering product is a health product that assists in lowering blood lipids.

[0022] More preferably, the lipid-lowering product is a drug for preventing and / or treating diseases related to hyperlipidemia.

[0023] Further preferably, the hyperlipidemia-related disease is obesity and / or hyperlipidemia.

[0024] More preferably, the drug is a drug for lowering triglycerides and / or total cholesterol.

[0025] Further preferably, the drug is a drug that reduces lipid droplet content.

[0026] Specifically, the particle size of the lipid droplets is 2.5 to 3.3 μm.

[0027] More preferably, the drug is a drug that inhibits weight gain.

[0028] More preferably, the drug is a drug that inhibits the weight gain of the kidney and heart.

[0029] More preferably, the drug is a drug for preventing and treating fatty liver.

[0030] More preferably, the drug is a drug for inhibiting obesity-induced liver damage.

[0031] More preferably, the drug is a drug for reducing the size of fat cells.

[0032] Specifically, the fat cells are one or both of perirenal white fat cells and posterior spinal brown fat cells.

[0033] Further preferably, the drug is a drug that regulates the expression of genes in the lipid-lowering signaling pathway.

[0034] Specifically, the lipid-lowering signaling pathway gene is at least one of sbp-1, Mdt-15, daf-2, fat-7, daf-2 or daf-16.

[0035] Further preferably, the drug is a drug that regulates metabolites and / or metabolic pathways related to lipid metabolism.

[0036] Specifically, the lipid metabolism-related metabolite is at least one of 3-methyladenine, 3-ureidopropionic acid, glycodeoxycholic acid, estrone glucuronide, pyruvic acid, d-inositol-3,4,5,6-tetraphosphate and pyridoxal phosphate, 3-ureidopropionic acid or cuminaldehyde and glycodeoxycholic acid.

[0037] Specifically, the lipid metabolism-related metabolic pathway is at least one of the TCA cycle, vitamin B6 metabolism, thiamine metabolism, pyruvate metabolism, insulin metabolism, phosphate metabolism, amino acid synthesis and phosphoinositide metabolism, steroid synthesis or β-alanine metabolic pathway.

[0038] Further preferably, the drug is a drug that regulates intestinal flora related to lipid metabolism.

[0039] Specifically, the lipid metabolism-related intestinal flora is at least one of Colidextribacter, Lachnospiraceae_UCG-001 or Enterorhabdus.

[0040] The cis-astaxanthin of the present invention can reduce the content of Colidextribacter, increase the content of Lachnospiraceae_UCG-001, and enrich Enterorhabdus.

[0041] Preferably, the cis-astaxanthin is obtained by photo-iodine isomerization of all-trans-astaxanthin.

[0042] More preferably, the specific process of the photo-iodine isomerization is as follows: mixing the all-trans astaxanthin solution and the iodine solution, irradiating with light, removing the iodine, drying, and purifying to obtain the cis astaxanthin.

[0043] Further preferably, the mass ratio of all-trans astaxanthin in the all-trans astaxanthin solution to iodine in the iodine solution is 1:(0.5-0.7), and the illumination time is 8-12 min.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] Compared with all-trans astaxanthin, the lipid-lowering activity of specific cis astaxanthin is significantly better. Specific cis astaxanthin can not only reduce lipid accumulation, triglyceride content and the ratio of oleic acid to stearic acid, but also reduce the content of large fat droplets and total cholesterol. The specific cis astaxanthin of the present invention can not only inhibit weight gain, but also prevent and treat fatty liver, inhibit obesity-induced liver damage and reduce fat cell size. Among them, the degree to which specific cis astaxanthin reduces triglycerides is at least 1.9 times that of all-trans astaxanthin, and the degree of improvement in effect is significantly better than existing technical means (such as group modification of all-trans astaxanthin to obtain derivatives).

[0046] The specific cis-astaxanthin of the present invention can effectively lower blood lipids by regulating the expression of lipid-lowering signaling pathway genes, metabolites related to lipid metabolism, metabolic pathways, intestinal flora related to lipid metabolism, and the regulation of genes involved in obesity.

[0047] In addition, compared with all-trans astaxanthin, the cis-astaxanthin of the present invention can not only independently regulate the three metabolites of 3-ureidopropionic acid, cuminaldehyde and glycodeoxycholic acid, and independently regulate the two metabolic pathways of steroid synthesis and β-alanine, but also independently participate in the regulation of more obesity-related genes (including Fitm1, Acot2, Manf, TSPO, Cisd1, Fdps, Id1, Abca1, Foxa2 and Fgf2) and enrich the beneficial bacteria Enterorhabdus that are closely related to lipid metabolism. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Figure 1 is a diagram of the separation and purification of cis-astaxanthin; Figure A is a diagram of the separation of cis- and trans-astaxanthin mixture by silica gel column chromatography; Figure B is a diagram of the thin layer chromatography detection of color band samples by silica gel column chromatography; Figures C to E are HPLC spectra of color band samples No. 1 to 3 obtained by silica gel column chromatography.

[0049] Figure 2 These are Oil Red O staining experimental images; Figure A is the ORO-stained nematode imaging image; Figure B is the nematode fat content image.

[0050] Figure 3 This is a diagram showing the effects of AST geometric isomers on TG content in nematodes.

[0051] Figure 4 This is a diagram showing the effect of AST geometric isomers on the ratio of nematodes (C18:1Δ9 / C18:0).

[0052] Figure 5 This is a diagram showing the effect of AST geometric isomers on the movement speed of nematodes.

[0053] Figure 6Figure 2 is a graph showing the effects of AST geometric isomers on nematode gene expression; Figure A shows the expression results of sbp-1 and mdt-15 genes; Figure B shows the expression results of fat-7 genes; and Figure C shows the expression results of daf-2 and daf-16 genes.

[0054] Figure 7 Figure 3 is a diagram showing the effects of AST geometric isomers on lipid droplets in mutant nematodes; Figure A is a diagram of lipid droplets under fluorescence imaging; Figure B is a diagram showing the number of lipid droplets of different sizes.

[0055] Figure 8 These are graphs showing the growth indicators of mice; Figure A is a comprehensive graph of mice; Figure B is a graph showing the food intake of mice; Figure C is a graph showing the weight of mice at week 7; and Figure D is a graph showing the weight of mice at different times.

[0056] Figure 9 This is a diagram showing the effects of AST geometric isomers on the weight of various organs in mice.

[0057] Figure 10 These are graphs showing the measurement of physiological indicators of mice; among them, Graph A shows the relative content of serum TG; Graph B shows the relative content of liver TC; Graph C shows the relative content of liver TG; and Graph D shows the relative content of serum TC.

[0058] Figure 11 Figure 1 is a diagram showing the effects of AST geometric isomers on fat cells; Figure A shows white fat cells around the kidneys, brown fat cells on the back, and liver sections; Figure 1 is a diagram showing the effects of AST geometric isomers on fat cell diameter.

[0059] Figure 12 Figure 1 is a graph showing the effects of AST geometric isomers on intestinal flora; Figure A is a graph showing the relative content of the harmful bacterium Colidextribacter; Figure B is a graph showing the relative content of the beneficial bacteria Lachnospiraceae_UCG-001; Figure C is a Lefse analysis graph of all-E AST; and Figure D is a Lefse analysis graph of Z AST.

[0060] Figure 13 This is a heat map showing the correlation between differential metabolites, differential bacterial genera, and key obesity indicators.

[0061] Figure 14 Figure 3 is a map of genes significantly regulated by AST geometric isomers in obese mice; Figure A is a Venn diagram of differentially expressed genes; Figure B is a volcano diagram of differentially expressed genes between CK and MOD; Figure C is a volcano diagram of differentially expressed genes between MOD and all-EAST; and Figure D is a volcano diagram of differentially expressed genes between MOD and Z AST. DETAILED DESCRIPTION

[0062] In order to more clearly and completely describe the technical solution of the present invention, the present invention is further described in detail through specific embodiments below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Various changes can be made within the scope of the rights of the present invention.

[0063] The test data processing methods of various embodiments of the present invention are as follows:

[0064] Statistical analysis was performed using at least three independent biological replicates in three groups. Data are reported as the mean ± standard deviation (SD) of at least three replicates. One-way analysis of variance (Anova), LSD, and Student's test in SPSS 17.0 statistical software package (SPSS Inc., Chicago, IL) were used for significance analysis and post hoc comparisons. Differences were considered statistically significant when p value < 0.05.

[0065] The present invention uses nematodes and mice as models for research because nematodes are suitable for the verification of molecular mechanisms, while mice can provide long-term effects and complex physiological feedback that are closer to those of the human body. Combining the two studies will help to conduct a multi-dimensional analysis of the lipid-lowering activity of the specific cis-astaxanthin of the present invention.

[0066] Example 1 Preparation, Detection and Isolation of Cis-Astaxanthin

[0067] Preparation of cis-astaxanthin: The cis-conversion of astaxanthin (AST) was achieved by photo-iodine isomerization. 200 mg of all-trans astaxanthin (AST, structural formula: Dissolve AST in 100 mL of dichloromethane to obtain an iodine solution, dissolve 120 mg of iodine in 200 mL of dichloromethane to obtain an iodine solution, and dissolve 148.9 mg of sodium thiosulfate in 600 mL of deionized water to obtain a sodium thiosulfate solution. Mix the AST solution and iodine solution in a 1:2 volume ratio and irradiate under a fluorescent lamp for 10 minutes. Then, add the sodium thiosulfate solution to remove the iodine. The mixture is then vacuum-dried at 25°C in the dark. Next, perform solvent purification by adding 13.38 mL of ethanol and 2.00 mL of ethyl acetate (1:13, v / m). Ultrasonication is used to suspend the solids, and the mixture is allowed to stand at 4°C in the dark for 1 hour. The supernatant is then centrifuged (room temperature, 4000 rpm, 10 minutes) to obtain the supernatant, which is then filtered through 0.22 μm and the filtrate is spin-dried. Column chromatography was then performed using 200-300 mesh silica gel powder. 160 mL of silica gel powder was mixed with an eluent (n-hexane / methanol / dichloromethane, 1:0.0325:2, v / v). After removing bubbles, the column was wet-packed to obtain a pre-purified mixture of cis-astaxanthin (9-cis-astaxanthin accounting for 24.49 wt% and 13-cis-astaxanthin accounting for 44.02 wt%). The effluent from each tube of column chromatography was analyzed by thin layer chromatography (TLC) using n-hexane / methanol / dichloromethane (1:0.065:2, v / v) as the developing solvent. The high-purity all-trans-astaxanthin sample and the pre-purified cis-astaxanthin mixture were dissolved in dichloromethane, respectively. The silica gel plate was then tilted and placed in a chromatography tank containing the developing solvent after spotting with a 2.5 μL pipette. The sample run-through was observed.

[0068] During the column separation process, three color bands were generated, such as Figure 1 As shown in (A), they were collected separately and preliminarily identified by TLC. The thin layer plate showed that there were three main substances, such as Figure 1 As shown in (B), these spots correspond well to those of the initially purified cis-astaxanthin mixture (reference), and the three substances are gradually eluted, demonstrating good separation. Based on the spot plate results, spots 1-3 (band 1), 5-7 (band 2), and 11-18 (band 3) were combined and then used to identify the astaxanthin configuration and purity using HPLC.

[0069] Take 60mg of the initially purified mixture of cis-astaxanthin, dissolve it in 5mL of dichloromethane and apply the sample. Collect the colored effluent, combine the same components, evaporate to dryness, re-dissolve it, pass it through a 0.22μm organic filter membrane, blow it through with nitrogen to dryness, and freeze-dry to obtain the cis-astaxanthin mixture. Store it at -20°C in the dark and away from oxygen. The cis-astaxanthin mixture was re-dissolved in a methanol-dichloromethane solution, passed through a 0.22μm organic filter membrane, and analyzed by high-performance liquid chromatography (HPLC). The detection conditions were as follows: the mobile phase was pump A with first-grade water and pump B with a mixed solution of methanol / acetonitrile / dichloromethane (85 / 5 / 5, v / v). An isocratic elution was used, with a ratio of 5% for pump A and 95% for pump B, and a flow rate of 1mL / min. The injection volume was 20μL, the detection wavelength was 470nm, the column temperature was 32°C, and the run time was 25min. The classic Q value (Quenching Value) calculation method was used to determine the cis isomer. The Q value is currently recognized as a quick and simple method for determining the cis configuration. Due to the bending of the double bond in its molecular structure, the cis isomer produces an additional absorption peak in the range of 300-400nm, this peak is called the cis peak, while the all-trans isomer has only one main absorption peak. The ratio of the absorbance of the cis peak to the absorbance of the main peak is defined as the Q value, and the size of the Q value is used to determine the site of astaxanthin isomerization. At the same time, the Q value is obtained by comparing the HPLC retention time, spectral data, and the relative intensity of the cis peak (approximately 360nm) with the main absorption peak of the isomer to identify all-trans and cis astaxanthin. The astaxanthin content is calculated based on the peak area.

[0070] like Figure 1 As shown in (C to E), color bands 1, 2, and 3 correspond to all-trans (all-E), 9-cis (9-Z), and 13-cis (13-Z) astaxanthin, respectively, and the purity of 9-cis and 13-cis astaxanthin samples reached 95.07% and 90.87%, respectively.

[0071] Example 2: Lipid-lowering activity experiment using obese Caenorhabditis elegans as a model

[0072] (1) Cultivation of Caenorhabditis elegans

[0073] Buffer solution preparation: 1M potassium phosphate buffer: take 54.20gKH2PO4 and 17.85gK2HPO4, and make up to 500mL, and adjust the pH to 6.0; 1M CaCl2 buffer: take 11.1gCaCl2 and dissolve it in 100mL deionized water; 1MM MgSO4 buffer: accurately weigh 24.6gMgSO4·7H2O and dissolve it in 100mL deionized water; M9 buffer: take 7.56gNa2HPO4·12H2O, 1.5gKH2PO4, 2.5gNaCl, and 0.125gMgSO4·7H2O and make up to 500mL; after all buffer solutions are prepared, place them in a high-pressure steam sterilizer at 121℃ for 20min; lysis solution (protect from light and prepare immediately before use): mix 2mol / LNaOH, NaClO, and H2O in a volume ratio of 1:1:1 and shake well.

[0074] LB liquid medium: Accurately weigh 2.1 g of prepared LB broth medium powder into a conical flask, dissolve it in 100 mL of deionized water, shake until dissolved, seal the flask, and sterilize it by high-pressure steam at 121°C for 20 min. Store at room temperature until used.

[0075] Nematode Growth Medium (NGM): Add 1.2g NaCl, 1.0g tryptone, 6.8g technical agar powder, and 0.08g to a 500mL Erlenmeyer flask, then add 390mL deionized water and shake well. Seal with foil or newspaper, and autoclave at 121°C for 20min. Cool to approximately 70°C and place in a laminar flow hood. Add 400μL of sterilized 1M CaCl2 buffer, 1M MgSO4 buffer, 10mL of 1M potassium phosphate buffer, and 400μL of a 5mg / mL cholesterol-ethanol solution filtered through a 0.22μm microporous filter. Gently shake after each addition. Aliquot into 6cm Petri dishes and allow to dry in the laminar flow hood.

[0076] Solid medium for growing obese nematodes: 1.2g NaCl, 1.0g tryptone, 6.8g technical agar powder, and 0.08g of it are added to a 500mL Erlenmeyer flask, followed by 390mL of deionized water and shaken. Seal with foil or newspaper and autoclave at 121°C for 20min. Cool to approximately 70°C and place in a laminar flow hood. Add 400μL of sterilized 1M CaCl2 buffer, 1M MgSO4 buffer, 10mL of 1M potassium phosphate buffer, and 400μL of a 5mg / mL cholesterol-ethanol solution filtered through a 0.22μm microporous filter. Additionally, add 4mL of a pre-prepared 1mol / L anhydrous glucose solution (also filtered through a 0.22μm microporous filter) and mix with normal nematode growth medium at a ratio of 1:100 to induce obesity in nematodes. After adding each solution to the culture medium, shake it gently and then dispense it into 6 cm culture dishes and leave it in the clean bench to dry.

[0077] Culture of E. coli OP50: In a clean environment, take 100 μL of bacteria-free OP50 with OD = 0.4-0.7 into sterile LB medium, seal the medium, and place it in a constant temperature shaker at 37°C and 170 rpm for 12 hours until the OD value is in the range of 0.4-0.7.

[0078] Drug-treated NGM: A certain amount of astaxanthin powder was placed in a centrifuge tube and dissolved in an appropriate amount of dimethyl sulfoxide (DMSO) to a final concentration of 3 mM astaxanthin stock solution. After sonication at low temperature for 1 hour, the solution was heated at 80°C for 1 minute and then sonicated for 0.5-1 hour until the astaxanthin was completely dissolved. E. coli OP50 culture and astaxanthin stock solution were mixed at a ratio of 98:2 to achieve a final drug concentration of 60 μM. The experiment was divided into five groups: a normal control group (CK group), a high-fat group (MOD group), an all-E AST-treated group, a 9-Z AST-treated group, and a 13-Z AST-treated group. In a clean environment, a 98:2 ratio of OP50 and DMSO was gently dropped onto the center of the NGM for the normal and high-fat groups, while a 98:2 ratio of OP50 and DMSO / AST was dropped onto the center of the NGM containing 10 mM glucose for the drug-treated groups. The entire process was protected from light.

[0079] Cultivation of Nematodes: From hatching to larvae, nematodes undergo four growth stages: L1 (larval stage), L2, L3 (growing and developing stages), and L4 (adult stage). Adult worms then enter the reproductive stage (egg-laying stage) and the post-reproductive stage. All experiments require synchronization. Typically, we use L4 worms for synchronization. Synchronization is performed by repeatedly washing the plate with 1 mL of M9 buffer several times to elute the adult worms and eggs into a 2 mL sterile centrifuge tube and pelleting by centrifugation. Aspirate the supernatant and add an equal volume of lysis buffer. Manually shake the plate for 4-6 minutes until the edges of the worms become transparent or the worms are partially broken apart. Centrifuge to pellet the worms. Discard the supernatant and wash the plate 3-4 times with M9 buffer until the lysate becomes less odorous. Finally, discard the supernatant, retaining the egg solution. Gently pipette to mix thoroughly, pipette approximately 80 μL of eggs and evenly distribute them around the NGM culture medium (around the edge of the culture medium but not touching it). Place the plate containing the eggs in a 20°C incubator. Larvae will hatch after 9 hours.

[0080] (2) Oil Red O staining and quantification

[0081] Before staining, prepare a 5 mg / mL solution of Oil Red O in isopropanol. Dissolve the worms in a 37°C waterbath in a shaker to promote dissolution. Anesthetic agent (1% NaN3) was then prepared. Wash the worms with M9. After the final wash, place the tube in a 2 mL centrifuge tube and remove as much supernatant as possible. Add 50 μL of anesthetic, invert, and allow to stand for 20 minutes to anesthetize. Centrifuge and discard the supernatant to remove the anesthetic. Add 1 mL of 4% paraformaldehyde fixative, gently invert, pre-chill at -4°C for 15-30 minutes, then quickly freeze at -80°C for 15-30 minutes. Repeat three times to stiffen the worms. After the final thaw, centrifuge at 1500 rpm at 4°C for 1 minute, aspirate the supernatant, and wash twice with M9 to remove excess paraformaldehyde. Add 1 mL of 60% isopropanol and dehydrate at 1500 rpm at 4°C for 5 minutes. Repeat twice. After dehydration, centrifuge at 1500 rpm at 4°C for 1 minute and wash once with M9. Mix equal volumes of 2% Triton X-100 and ORO stain, incubate in a shaker at 25°C, 400 rpm, and stain in the dark for 12 hours. Remove the tube and wash twice with 60% isopropanol for 40 seconds to remove any loose color. Wash several more times with M9 until the solution turns light red.

[0082] The nematode has a simple and transparent structure. Its fat accumulation is mainly concentrated in the subcutaneous tissue and intestines behind the pharynx. Fat can bind to the fat-soluble dye ORO and appear red. The depth of the red can reflect the overall fat accumulation of the nematode. The darker the color, the more fat is bound to ORO. The results are as follows Figure 2As shown in (A-B), compared with the CK group, the overall fat content of the MOD group increased significantly by 88.72% (p < 0.01), indicating the successful establishment of a high-fat nematode model. Compared with MOD, the fat content of the all-E AST treatment group, the 9-Z AST treatment group, and the 13-Z AST treatment group decreased by 12.58%, 28.65%, and 20.95% (p < 0.05), respectively. The degree of decrease in the 9-Z AST treatment group and the 13-Z AST treatment group was 2.3 times and 1.7 times that of all-E AST, respectively. The effect of the two ZASTs in reducing fat content was significantly better than that of al1-E AST (p < 0.05). It can be seen that the effect of the cis-astaxanthin of the present invention in reducing lipid accumulation is significantly better than that of all-trans astaxanthin.

[0083] (3) Determination of triglyceride content

[0084] After washing with M9, collect over 500 nematodes into a new tube and homogenize using a 60 Hz bead mill with 30-second intervals of 30 seconds and 10-second intervals to obtain a homogenate. After centrifugation at 5000 rpm for 10 minutes, transfer the supernatant to a new tube and store at -20°C. TG and BCA quantification were performed according to the manufacturer's kit instructions.

[0085] Like humans, triglycerides (TG) are an important component of body fat in C. elegans and a key indicator of obesity (Bartels et al., 2009). However, the lipid composition of C. elegans is complex. Considering that ORO is a lipophilic dye, its staining range also involves lipid bodies in addition to TG. Therefore, this study further explored the regulation of AST on obesity by quantifying TG. The results are as follows: Figure 3 As shown. Compared with the CK group, the TG content of the MOD group increased significantly by 73.08% (p<0.05). This once again confirmed the successful establishment of a high-fat nematode model. The TG content of the all-E AST treatment group, the 9-Z AST treatment group, and the 13-Z AST treatment group decreased by 18.84%, 41.18%, and 35.94% (p<0.05), respectively. The degree of decrease in the 9-Z AST treatment group and the 13-Z AST treatment group was 2.2 times and 1.9 times that of the all-E AST treatment group, respectively; the TG-lowering effect of Z AST was significantly better than that of al1-E AST (p<0.05). It can be seen that the effect of the cis-astaxanthin of the present invention in reducing the content of triglycerides is significantly better than that of all-trans astaxanthin.

[0086] (4) Determination of fatty acid composition

[0087] Nearly 3000 nematodes were rinsed with M9 to completely remove residual OP50, and the adult worms were ground into a homogenate using the method (3) in Example 1, and the supernatant was obtained by centrifugation using the above method. Fatty acids were extracted from 3000 nematodes and incubated in a 2% sulfuric acid 98% methanol solution at 80°C for 2 hours with occasional shaking. Then, 1 mL of n-hexane was added to each sample, vortexed for 5 minutes, and allowed to stand for 15 minutes. Then, the organic solvent was blown dry with nitrogen and stored at -80°C. Before gas chromatography-mass spectrometry analysis, the fatty acids needed to be redissolved in chromatography-grade hexane. GC-MS chromatography used a DB-WAX column (60m×0.25m, 0.25m). The sampling temperature was set to 250°C. The oven temperature was programmed as follows: initial temperature was 50°C for 1 minute, and the programming temperature was increased to 175°C at a rate of 22.5°C / min. The temperature was increased to 240°C at a rate of 4°C / min and maintained for 15 minutes. The gas flow rate was 1.0 mL / min, and the split ratio was 20:1.

[0088] Fat is mainly stored in adipocytes in the form of lipid droplets. The size of lipid droplets is closely related to the fatty acid composition. The size of lipid droplets increases rapidly under the change of fatty acids. Oleic acid (C18:1Δ9) is an important substrate for the synthesis of TG. In the human body, a high oleic acid / stearic acid (C18:1Δ9 / C18:0) ratio will lead to an increase in TG and low-density lipoprotein (LDL). The results are as follows Figure 4 As shown in the results, compared with the CK group, the oleic acid / stearic acid ratio of the MOD group was significantly increased by 31.96% (p<0.05), indicating that the high-fat diet leads to abnormal fatty acid ratios in nematodes. Compared with the MOD group, the all-E AST treatment group, the 9-Z AST treatment group, and the 13-Z AST treatment group reduced the oleic acid / stearic acid ratio by 11.47%, 40.30%, and 26.02%, respectively (p<0.05); the degree of decrease in the 9-Z AST treatment group and the 13-Z AST treatment group was 3.5 times and 2.3 times that of the all-E AST treatment group, respectively. The reduction effect of Z AST was significantly better than that of al1-E AST (p<0.05). It can be seen that the effect of cis-astaxanthin of the present invention in reducing the oleic acid / stearic acid ratio is significantly better than that of all-trans astaxanthin.

[0089] (5) Determination of energy homeostasis

[0090] Energy imbalance, characterized by excess energy intake and insufficient energy expenditure, is known as positive energy balance and is currently considered a direct cause of weight gain. The effects of AST isomers on energy expenditure in nematodes were analyzed by measuring body sway. Locomotor behavior: Nematodes were transferred to new NGM cells, and their movements were recorded for 1 minute using the WormLab video acquisition system (MBF Biosciences, USA).

[0091] The results are as follows Figure 5 As shown in the results, in terms of energy consumption, the nematode movement frequency of the MOD group was reduced by 69.60% (p<0.05) compared with the CK group. Compared with the MOD group, the nematode movement frequency of the all-E AST treatment group, the 9-Z AST treatment group and the 13-Z AST treatment group increased by 49.72%, 65.10% and 58.42% (p<0.05), respectively; the improvement degree of the 9-Z AST treatment group and the 13-Z AST treatment group was 1.3 times and 1.2 times that of the all-E AST treatment group, respectively, and the improvement degree of Z AST was significantly better than that of all-EAST (p<0.05). It can be seen that the cis-astaxanthin of the present invention can maintain the energy homeostasis of nematodes by improving the motility, which is significantly better than that of all-trans astaxanthin.

[0092] (6) Determination of key gene expression in lipid-lowering signaling pathways

[0093] RNA extraction and cDNA reverse transcription: About 20 to 40 nematodes per group were placed in a sterile enzyme-free centrifuge tube and washed three times with DEPC water before mRNA extraction. After the last wash, the supernatant was removed and Trizol solution was added. The nematodes were lysed using the method (3) in Example 1. 200 μL of pre-cooled chloroform was added to the above lysed worm solution and shaken vigorously to form an emulsion. The solution was allowed to stand on ice for 5 minutes. Then, centrifuged at 4°C and 12000g for 15 minutes. The liquid was separated into layers. The supernatant was aspirated into a new enzyme-free centrifuge tube and an equal volume of pre-cooled isopropanol was added and mixed slightly. The tube was allowed to stand at -20°C for 10 minutes. The tube was removed and centrifuged at 4°C and 12000g for 10 minutes. The supernatant was discarded carefully. An appropriate amount of pre-cooled 75% ethanol (prepared with DEPC water) was added to thoroughly wash the tube cap and tube wall and shaken to prevent the precipitate from adhering to the wall. After standing on ice for 3 to 5 minutes, centrifuge at 12000g for 5 minutes at 4°C, discard the supernatant, open the bottle cap up and down in a clean environment, dry for 2 to 5 minutes, then add an appropriate amount of DEPC to dissolve the precipitate, and take a small amount for detection. Take a small amount of solution (1 μL) for nanodrop determination of RNA purity and concentration. Subsequently, the volume of RNA required for 1 μg reverse transcription is calculated based on 1 μL = 1000 ng. Finally, use the HiScript IIQ RT Super Mix qRT-PCR (+gDNA Removal) Kit (Nanjing Novozymes) to make a 20 μL system according to the instructions, and perform reverse transcription according to the instructions.

[0094] qPCR detection of gene expression: Forward and reverse primer sequences were designed based on the gene sequences provided by NCBI (completed by Sangon Biotech (Shanghai) Co., Ltd.), see Table 1.

[0095] Table 1 Primer sequences

[0096]

[0097]

[0098] Sbp-1 is homologous to the human lipogenesis gene, sterol regulatory element binding protein-1c (SREBP-1c), and is a key gene controlling the biosynthesis of several mammalian fatty acids, triglycerides, cholesterol, and phospholipids. Upregulation of Sbp-1 indicates increased lipogenesis. Furthermore, as a key factor encoding lipogenesis, sbp-1 is a key gene that activates the downstream target genes mdt-15 and pod-2 (the intermediate subunit responsible for final fatty acid synthesis) of this pathway, thereby downregulating the gene fat-7 responsible for regulating C18:1Δ9 synthesis, thereby inhibiting fat accumulation. Furthermore, SCDs are the rate-limiting enzymes for the conversion of saturated fatty acids to monounsaturated fatty acids. Fat-7, also a gene under SCDs, is responsible for regulating the conversion of C18:0 to C18:1Δ9, thereby promoting triglyceride synthesis.

[0099] The results of sbp-1 gene expression were as follows Figure 6 As shown in (A), compared with the CK group, the expression of sbp-1 in the MOD group increased significantly by 98.32% (p < 0.01). Compared with MOD, the all-E AST treatment group, the 9-Z AST treatment group, and the 13-Z AST treatment group significantly downregulated the expression of sbp-1 by 52.51%, 79.51%, and 78.51%, respectively (p < 0.05); the degree of downregulation in the 9-Z AST treatment group and the 13-Z AST treatment group was 1.5 times that of the all-E AST treatment group.

[0100] The results of mdt-15 gene expression were as follows Figure 6 As shown in (A), compared with the CK group, the expression of mdt-15 in the MOD group was upregulated by 42.86%. Compared with MOD, the all-E AST treatment group, the 9-Z AST treatment group, and the 13-Z AST treatment group significantly downregulated the expression of mdt-15 by 32.23%, 54.33%, and 34.19%, respectively (p < 0.05); the downregulation degree of the 9-Z AST treatment group and the 13-Z AST treatment group was 1.7 times and 1.1 times that of the all-E AST treatment group, respectively.

[0101] The results of fat-7 gene expression were as follows Figure 6As shown in (B), compared with the CK group, the fat-7 expression in the MOD group was significantly upregulated by 67.57% (p<0.05). Compared with the MOD group, the all-E AST treatment group, the 9-Z AST treatment group and the 13-Z AST treatment group significantly downregulated the expression of fat-7 by 57.65%, 84.04% and 70.80%, respectively (p<0.05); the downregulation degrees of the 9-Z AST treatment group and the 13-Z AST treatment group were 1.5 times and 1.2 times that of the all-E AST treatment group, respectively.

[0102] Insulin is the main signal in the central nervous system that regulates energy balance and has a long-term inhibitory effect on energy expenditure. Elevated insulin levels often occur in obesity. Important genes in the insulin signaling pathway (IIS signaling pathway) include: daf-2 and daf-16. Figure 6 As shown in (C), compared with CK, daf-2 in the MOD group was significantly upregulated by 120.60% (p<0.01). Compared with the MOD group, daf-2 was significantly downregulated by 32.84%, 48.56% and 59.93% after treatment with all-E AST, 9-Z AST and 13-Z AST, respectively (p<0.05); the degree of downregulation in the 9-Z AST treatment group and the 13-Z AST treatment group was 1.5 times and 1.8 times that of the all-E AST treatment group, respectively. Figure 6 As shown in (C), compared with the CK group, the MOD group significantly upregulated daf-16 by 404.19% (p < 0.01). Compared with the MOD group, the all-E AST treatment group, the 9-Z AST treatment group, and the 13-Z AST treatment group significantly downregulated daf-16 by 48.30%, 84.05%, and 73.70% (p < 0.01), respectively. The degree of downregulation in the 9-Z AST treatment group and the 13-Z AST treatment group was 1.7 times and 1.5 times that of the all-E AST treatment group, respectively.

[0103] From the above, it can be seen that the cis-astaxanthin of the present invention has a stronger regulatory effect on the key genes of the lipid-lowering signaling pathway, sbp-1, mdt-15, fat-7, daf-2 and daf-16, than all-E AST.

[0104] Example 3 Lipid droplet measurement experiment using mutant nematodes as a model

[0105] The ZXW618 [hkdIs618(dhs-3p::dhs-3::GFP)] mutant nematode is a transgenic mutant of Caenorhabditis elegans. This mutant, fused to the lipid droplet marker protein DHS-3, is useful for studying lipid droplet distribution. Over 20 mutant nematodes were anesthetized with 1% sodium azide and held rigid for 24 hours. The images were then taken using a 40-degree objective laser scanning confocal microscope with a 488 nm excitation filter and a 500 / 525 nm emission filter. All measurements were performed in triplicate and analyzed using Image J software to visualize lipid droplet size and distribution.

[0106] Fat is primarily stored in adipocytes in the form of lipid droplets, with over 50% of triglycerides stored primarily in large lipid droplets. Lipids stored in lipid droplets are generally considered to be used for metabolism and membrane synthesis. Their abundance, size, and distribution are related to the fat composition of an animal. Based on previous studies, the sizes of small lipid droplets (0–0.8 μm), medium lipid droplets (0.9–1.6 μm), medium-large lipid droplets (1.7–2.5 μm), and large lipid droplets (2.5–3.3 μm) have been defined.

[0107] The results are as follows Figure 7 As shown in (A-B), in terms of large lipid droplets, the MOD group significantly increased its content of large lipid droplets compared to the CK group, reaching 83.44% (p < 0.01), indicating that large lipid droplets are highly enriched in high-fat nematodes. Compared with the MOD group, the content of large lipid droplets was significantly reduced by 63.12%, 85.45%, and 70.71% after intervention with all-E AST, 9-Z AST, and 13-Z AST, respectively (p < 0.01). The degree of reduction in the 9-Z AST-treated group and the 13-Z AST-treated group was 1.4 times and 1.1 times that of the all-E AST-treated group, respectively. The reduction effect of Z AST was significantly greater than that of all-E AST (p < 0.01). This shows that the cis-astaxanthin of the present invention is significantly more effective in reducing the content of large lipid droplets than all-trans astaxanthin.

[0108] Example 4: Experiment on hypolipidemic activity using obese mice as a model

[0109] (1) Animal grouping and drug administration

[0110] HFD (High-Fat Diet) is a high-fat diet that is often used to induce obese mouse models in the laboratory. By feeding mice a high-fat diet, the pathological process of human obesity can be simulated. After the mice adapted to the environment for one week, they were randomly divided into a normal group (CK group), an obese group (MOD group), an all-E AST group, and a cis-astaxanthin (Z AST) group (25 mg / kg). The cis-astaxanthin in the cis-astaxanthin treatment group was the cis-astaxanthin mixture in Example 1. The normal group was given ordinary feed, and the obese group and the drug group were given a high-fat feed to induce obesity. The modeling was successful when the body weight of the mice in the high-fat diet group was greater than 20% of that in the normal group. After the modeling was successful, the normal group and the obese group were gavaged with a 0.25% sodium carboxymethyl cellulose-normal saline suspension. The drug group was gavaged with an astaxanthin-sodium carboxymethyl cellulose suspension, with free access to water during the period. Gavage was continued for 6 weeks. Use clean tweezers to pick up fresh feces from mice, divide them into sterilized centrifuge tubes, and store them in a -80°C ultra-low temperature freezer in time for later measurement of indicators.

[0111] (2) Determination of growth-related indicators

[0112] Food intake was measured daily by recording the amount of food given and remaining. Body weight was measured weekly using an electronic scale. After six weeks of continuous gavage, mice were anesthetized with carbon dioxide and sacrificed by cervical dislocation. Organs were dissected and carefully separated for immediate weighing to determine organ indices. Immediately after weighing, the specimens were placed in specimen bags and stored on dry ice.

[0113] The results are as follows Figure 8 As shown in (A~D), Figure 8 (B) It can be seen that AST intervention did not affect the food intake of mice, and there was no significant difference in the body weight of mice at the beginning of the experiment. Figure 8 (C) As shown, starting from week 7, the body weight of the MOD group was significantly higher than that of the CK group by 20%, indicating that the high-fat diet model was successfully established. Compared with the MOD group, the average body weight gain rate of mice in the all-E AST group was reduced by 21.46%, and that of mice in the Z AST group was reduced by 44.22% (p < 0.05). Figure 8 (D) It can be seen that at week 16, the body weight of mice in the all-E AST group and the Z AST group was significantly lower than that in the MOD group by 14.74% and 22.32%, respectively (p<0.05). The degree of decrease in the Z AST group was 1.5 times that of the all-E AST group. Z AST was significantly better than all-E AST in inhibiting body weight gain (p<0.05).

[0114] Figure 9 The figure shows the effect of AST geometric isomers on the weight of various organs in mice. Figure 9Compared with the CK group, the organ weights of the MOD group, except for the brain, were significantly increased (P<0.05). Compared with the MOD group, the organ weights of the all-E AST and Z AST groups were significantly reduced after intervention (P<0.05). Among them, the reduction in kidney and heart weights in the Z AST group was significantly greater than that in the all-E AST group.

[0115] It can be seen from this that the cis-astaxanthin of the present invention can inhibit body weight gain and inhibit the weight gain of the kidney and heart, and the effects are better than those of all-trans-astaxanthin.

[0116] (3) Physiological index measurement

[0117] After blood was collected from the eyeball, the mixture was centrifuged at 14,000 rpm and the supernatant was removed. After the liver was cut, 2 cm was taken and ground into a homogenate according to the method (3) in Example 1. The levels of TG, TC, aspartate aminotransferase, and alanine aminotransferase were determined according to the kit instructions.

[0118] Obesity causes excessive accumulation of TG and TC lipids in various organs of the body, with the most obvious accumulation in the blood and liver. Figure 10 (A~D) Figure 10 As shown in (A and D), compared with the CK group, the MOD group had extremely significant increases in serum TG and TC levels by 70.31% and 87.24%, respectively (p < 0.01), indicating that HFD-induced dyslipidemia in mice. Compared with the MOD group, the all-E AST and Z AST groups significantly reduced serum TG levels by 28.66% and 44.53%, respectively (p < 0.05), with the Z AST group experiencing a 1.6-fold decrease compared to the all-E AST group. The serum TC levels were also significantly reduced by 44.24% and 60.70%, respectively (p < 0.05), with the Z AST group experiencing a 1.4-fold decrease compared to the all-E AST group. Significant differences were observed between the all-E AST and Z AST groups.

[0119] from Figure 10 (B and C) As shown in Figure 3, compared with the CK group, the MOD group showed a highly significant increase in liver TG and TC levels by 85.22% and 92.71%, respectively, indicating that HFD successfully induced liver lipid accumulation in mice (p < 0.01). Compared with the MOD group, the all-E AST and Z AST groups significantly reduced liver TG levels by 25.95% and 85.23%, respectively (p < 0.05), with the Z AST group showing a 3.3-fold decrease compared to the all-E AST group. The liver TC levels were also reduced by 44.24% and 49.33% (p < 0.05), with the Z AST group showing a 1.1-fold decrease compared to the all-E AST group. Significant differences were observed between the all-E AST and Z AST groups.

[0120] Liver damage is one of the diseases induced by obesity. Aspartate aminotransferase (AST) is an enzyme related to liver damage, and its increase is the direct cause of liver damage. Figure 10 (E) Compared with the CK group, the MOD group showed a significant increase in AST levels by 89.77% (p < 0.01), indicating that obesity can cause liver damage. Compared with the MOD group, the all-E AST group and the ZAST group showed significant decreases of 28.76% and 40.72%, respectively (p < 0.05). The decrease in the Z AST group was 1.4 times greater than that in the all-E AST group.

[0121] The above results show that the cis-astaxanthin of the present invention not only has the ability to lower triglycerides and total cholesterol, but also can prevent and treat fatty liver and inhibit liver damage induced by obesity, and the effects are better than those of all-trans astaxanthin.

[0122] (4) Histological morphology observation

[0123] Perinephric white adipocytes and dorsal brown adipocytes were fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 5 μm thick paraffin sections. After hematoxylin-eosin (HE) staining, the morphology was examined under a light microscope to assess pathological changes.

[0124] Image-J analysis showed that white fat cells were round or oval, and some were squeezed together to form polygons. Brown fat tissue is usually darker than white fat and has a smaller overall diameter than white fat. Figure 11 As shown in (A-B). Compared with the CK group, the diameters of perirenal white adipocytes and dorsal brown adipocytes in the MOD group were significantly increased, by 54.01% and 74.90%, respectively, indicating that obesity significantly induces adipocyte hypertrophy (p < 0.05). Compared with the MOD group, intervention with both the all-E AST and Z AST groups reduced the size of perirenal white adipocytes and dorsal brown adipocytes. Regarding perirenal white adipocytes, intervention with the all-E AST and Z AST groups significantly reduced white adipocyte size by 34.71% and 47.08%, respectively (p < 0.05), with the reduction in the Z AST group being 1.4 times greater than that in the all-E AST group. Regarding dorsal brown adipocytes, intervention with the all-E AST and Z AST groups reduced brown adipocyte size by 59.14% and 70.25%, respectively (p < 0.05), with the reduction in the Z AST group being 1.2 times greater than that in the all-E AST group.

[0125] The above results show that the cis-astaxanthin of the present invention can reduce fat size, and the effect is better than that of all-trans astaxanthin.

[0126] (5) Influence of intestinal flora

[0127] The V3-V4 region of the bacterial 16S rRNA gene was amplified using universal primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). An 8-bp barcode sequence was added to the 5' end of each upstream and downstream primer to distinguish different samples. Finally, universal primers with barcode sequences were synthesized and amplified on an ABI 9700 PCR instrument (Applied Biosystems, Inc., USA). The DNA was then automatically purified using the Agencourt AMPure XP (Beckman Coulter Inc., USA) nucleic acid purification kit. The DNA was then sequenced.

[0128] At the phylum level, the differential bacterial genus Colidextribacter, a harmful bacterium, and Lachnospiraceae_UCG-001, a beneficial bacterium, were associated with AST intervention in obesity and obesity-related diseases. The results of the effects of AST geometric isomers on intestinal flora were as follows: Figure 12 (A~D) Figure 12 (A-B) It can be seen that the ZAST group significantly reduced the relative content of the harmful bacteria Colidextribacter (p<0.05) and significantly increased the relative content of the beneficial bacteria Lachnospiraceae_UCG-001 (p<0.05). Next, the linear discriminant analysis effect size (LEfSe) was further used to identify the differential bacteria in each group, with the threshold set at ≥3.5. Figure 12 (C-D) As shown, at the genus level, the all-E AST group had the most enriched genus, Mucispirillum; the Z AST group had the most enriched genus, Eubacterium fissicatena, Romboutsia, Enterorhabdus, Clostridium sensu stricto, and Coprococcus. Among them, Enterorhabdus is a beneficial bacterium closely related to lipid metabolism.

[0129] (6) Regulation of serum metabolites and metabolic pathways

[0130] Sample pretreatment and analysis: Serum was removed from a -80°C freezer and slowly thawed at 4°C. An appropriate amount of sample was added to a pre-chilled ACN:MeOH solution (v:v = 1:1) containing an internal standard. The sample was vortexed for 30 seconds and sonicated for 10 minutes. The sample was then allowed to stand at -20°C for 1 hour and centrifuged at 13,000 rpm for 15 minutes at 4°C. The supernatant was then freeze-dried. For mass spectrometry analysis, the sample was reconstituted with an appropriate amount of ACN:H₂O (v:v = 1:1), vortexed for 30 seconds, sonicated for 10 minutes, and centrifuged at 13,000 rpm for 15 minutes at 4°C. The supernatant was then aspirated into a vial for LC-MS / MS analysis. Additionally, 10 μl of each sample was mixed to create a QC sample, which was also vialed. Analysis was performed on a SciexExion LC ultra-high performance liquid chromatography system using the mobile phase parameters listed in the table below. The chromatographic column used was an ACQUITY UPLC HSS T3 (1.8 μm, 2.1 mm x 100 mm) purchased from Waters, with an injection volume of 2 μL, a flow rate of 0.3 mL / min, and a column temperature of 50°C.

[0131] Data processing: Filter individual peaks. Only peaks with missing values ​​in less than 50% of the actual samples are retained. Missing values ​​in the original data are simulated (missing value recoding). Data normalization is performed. After normalization using the sum of each sample's features, a series of multivariate pattern recognition analyses are performed, starting with principal component analysis (PCA). Principal component analysis (PCA) is a statistical method that converts data into linearly uncorrelated variables (i.e., principal components) through an orthogonal transformation. PCA can reveal the internal structure of the data, thereby better interpreting data variables. Metabolomics data can be considered a multivariate dataset that can be visualized in a high-dimensional data space coordinate system. PCA can then provide a relatively low-dimensional image (two-dimensional or three-dimensional), representing a "projection" of the original object onto the points containing the most information, effectively reducing the dimensionality of the data using a small number of principal components. Orthogonal partial least squares discrimination analysis (OPLS-DA) is a multivariate statistical analysis method used for discriminant analysis. Discriminant analysis is a common statistical analysis method that determines how the research object is classified based on the values ​​of several observed or measured variables. Its principle is to train the characteristics of different treated samples (such as observation samples and control samples) separately, generate training sets, and test the credibility of the training sets. PLSDA can reflect the differences between classification groups to the greatest extent. This method uses partial least squares regression to establish a relationship model between metabolite expression and sample category to achieve modeling prediction of sample category. The R2Y (model's interpretability of classification variable Y) and Q2 (model's predictability) obtained after cross-validation are used to judge the effectiveness of the model; finally, through permutation test, the order of classification variable Y is randomly changed multiple times to obtain different random Q2 values ​​to further test the effectiveness of the model.

[0132] Screening and enrichment analysis of differential metabolites: The inherent characteristics of LC-QTOF-MS-based metabolomics data require the use of multivariate statistical analysis methods to analyze the data. The chi-square criterion used is a Student's t-test P-value (P-value) less than 0.05, and the variable importance in the projection (VIP) of the first principal component of the OPLS-DA model greater than 1. Users can also select other chi-square criterion for differential metabolites based on their needs, such as a FOLD CHANGE value less than 0.5 or greater than 2, and a P-value less than 0.05. The differential metabolites are mapped to authoritative metabolite databases such as KEGG and PubChem to identify pathways in which all differential metabolites participate. Further metabolic pathway analysis is then performed on the differential metabolites to determine their relevance to the biological question. Comprehensive analysis of the pathways in which the differential metabolites reside (including enrichment analysis and topological analysis) allows for further pathway screening and identification of key pathways with the highest correlation with metabolite differences.

[0133] Endogenous metabolites play a crucial role in health and disease. Under normal conditions, they maintain a dynamic equilibrium within a specific range. However, obesity can lead to abnormal endogenous metabolites. To further understand the lipid-lowering effects of AST, serum metabolites were analyzed by LC-QTOF-MS metabolomics. Significantly differential metabolites were screened using VIP values ​​>1.0, FC values ​​>1.0 or <1.0, and p-values ​​<0.05. Ten metabolites closely related to lipid metabolism were identified, nine of which were enriched in KEGG pathways. Seven metabolites were co-regulated by all-E AST and Z AST, including 3-methyladenine, 3-ureidopropionic acid, glycodeoxycholic acid, estrone glucuronide, pyruvate, d-inositol-3,4,5,6-tetraphosphate, and pyridoxal phosphate. Three metabolites, 3-ureidopropionic acid, cuminaldehyde, and glycodeoxycholic acid, were individually regulated by ZAST.

[0134] KEGG pathway analysis was used to explore lipid metabolism pathways involved in AST-regulated metabolites. All-EAST and ZAST were jointly involved in the TCA cycle, vitamin B6 metabolism, thiamine metabolism, pyruvate metabolism, insulin metabolism, phosphate metabolism, amino acid synthesis, and phosphoinositide metabolism, primarily involving four metabolites: pyruvate, phosphoenolpyruvate, pyridoxal phosphate, and d-inositol-3,4,5,6-tetraphosphate. Z-AST was independently involved in the steroidogenesis and β-alanine metabolism pathways, primarily involving two metabolites: estrone glucuronide and 3-ureidopropionic acid.

[0135] Correlation heatmap of differential metabolites, differential bacterial genera and key obesity indicators Figure 13 As shown, the metabolites pyruvate and glycodeoxycholic acid were correlated with the gut microbiota Prevotella, Marvinbryantia, Coriobacteriaceae_UCG-002, and Colidextribacter, as well as key lipid-lowering indicators. The metabolite cuminaldehyde was correlated with the gut microbiota Lachnospiraceae_UCG-001 and Colidextribacter, as well as key lipid-lowering indicators. Cis-4-hydroxy-D-proline was correlated with the gut microbiota Lachnospiraceae_UCG-001 and Coriobacteriaceae_UCG-0023. Furthermore, 3-methyladenine was correlated with the beneficial bacteria Lachnospiraceae_UCG-0012. These metabolites showed significant negative correlations with harmful bacteria and key obesity indicators (R < -0.5, P < 0.05) and significant positive correlations with beneficial bacteria (R > 0.5, P < 0.05). Therefore, these results suggest that these bacterial genera and metabolites can be considered as biomarkers for obesity alleviation.

[0136] (7) Regulation of potential key genes and signaling pathways

[0137] RNA extraction and detection: Total RNA was extracted using the TRIzol method. The steps are as follows: Grind the tissue in a pre-chilled mortar and pestle. Once the tissue sample is slurried, add Trizol and store at room temperature for 5 minutes. Add 0.2 mL of chloroform and vigorously vortex the centrifuge tube to mix thoroughly. The tube should be incubated at room temperature for 5-10 minutes. Centrifuge at 12,000 rpm for 15 minutes, then aspirate 70% of the upper aqueous phase into a fresh tube, taking care not to aspirate protein between the two aqueous phases. Transfer the tube to the new tube, add an equal volume of -20°C pre-chilled isopropanol, mix thoroughly by inversion, and place on ice for 10 minutes. Centrifuge at 12,000 rpm for 15 minutes, then carefully discard the supernatant and wash the pellet with 75% DEPC-treated ethanol (stored at 4°C) at a ratio of 1 mL per 1 mL of Trizol. Wash the pellet, vortex to mix, and centrifuge at 12,000 rpm for 5 minutes at 4°C. Discard the ethanol, let the pellet dry at room temperature for 5 minutes, and dissolve it in DEPC-treated water. RNA quality was then tested using NanoDrop.

[0138] Library Construction and Sequencing: After all samples passed the test, 1.5 μg of RNA was collected from each sample and sent to Beijing Aoweisen Gene Technology Co., Ltd. for library construction and sequencing. The raw data obtained from sequencing were first filtered using Trimmomatic software (v0.33) to remove reads with sequencing adapters*, reads with an N (uncertain base) content greater than 10%, and reads with a low-quality base content (Q≤20) greater than 50%. Clean data were obtained. Sequencing data statistics showed that Q20 > 95% and Q30 > 89% (Q20, Q30: the ratio of bases with Phred quality values ​​greater than 20 and 30 to the total base count), indicating good sequencing quality and suitable for subsequent alignment and data analysis. Alignment to the reference genome (GADPH) was performed using STAR software (v2.5.2b).

[0139] Gene expression analysis: HTSeq (v0.5.4p3) software was used to analyze gene expression for each sample using the union model. FPKM (fragments per kilobase of exon model per million mapped reads) is the number of fragments per kilobase of length from a gene per million reads. FPKM accounts for the effects of sequencing depth and gene length on read counts and is used to measure gene expression. Generally, an FPKM value of 0.1 or 1 is used as the threshold for determining gene expression. In subsequent analyses, only genes with an FPKM > 1 were analyzed. Gene differential expression analysis was performed based on the read count data obtained from gene expression analysis. For samples with biological replicates, differential expression analysis was performed using DESeq. The P values ​​of the differential expression analysis results were adjusted for the false discovery rate (FDR) using the Benjamini and Hochberg method. The standard for screening differentially expressed genes was generally a P-value < 0.05. For samples without biological replicates, differential expression analysis between two samples was performed using the DEGseq (2010) R software package. P-values ​​were corrected using P-values, and the thresholds for differential expression were qvalue < 0.005 and |log2(foldchange)| > 1.

[0140] In addition to gut microbiota and metabolites, research has found that one of the reasons for the isoform-specific effects of dietary ingredients is the selective specificity between isoforms and gene targets. Transcriptomics provides a global view of gene expression and regulatory patterns.

[0141] Global Genetic Changes: RNA-Seq was used to investigate the lipid-lowering mechanisms of all-E AST and Z AST. Generally, a small difference between the total base count of raw sequencing data (Raw reads) and the base count obtained after quality control (Clean reads) and a Q30 value >80% indicate high base quality, indicating minimal sample loss and complete sequencing results. As shown in Table 2, the difference in base count between Raw and Clean reads is small, and the base quality value (Q30) is >90%. Overall, these data indicate relatively high sequencing data quality and are suitable for bioinformatics analysis.

[0142] Table 2 Transcriptomic GC content

[0143] Sample name Raw reads Raw bases Q30 CK1 40844450 6.12G 94.23％ CK2 43796502 6.56G 94.34％ CK3 39561694 5.93G 94.09％ CK4 42280284 6.34G 94.34％ MOD1 41411346 6.21G 94.41％ MOD2 42608752 6.39G 94.07％ MOD3 40530674 6.07G 94.03％ MOD4 43523346 6.52G 93.88％ all-E AST 1 42698788 6.4G 94.17％ all-E AST 2 39605300 5.94G 94.05％ all-E AST 3 42945182 6.44G 94.36％ all-E AST 4 42330986 6.34G 94.45％ Z AST 1 42066542 6.3G 94.12％ Z AST 1 43926380 6.58G 94.61％ Z AST 1 41972210 6.29G 95.08％ Z AST 1 39667474 5.95G 94.17％

[0144] according to Figure 14 As shown in Figures A to D, compared with the CK group, the MOD group showed significant changes in 482 genes (126 upregulated and 356 downregulated), indicating that obesity triggers genetic changes. AST intervention significantly alters the genes of obese mice. Overall, compared with the MOD group, intervention with the all-EAST group resulted in changes in 225 genes (114 upregulated and 112 downregulated), and with the ZAST group, in 626 genes (301 upregulated and 326 downregulated). Of these, 14 genes were co-regulated by both all-EAST and ZAST. Furthermore, 22 genes were significantly altered in the (CK vs MOD) & (MOD vs all-EAST) comparison, 54 genes were altered in the (CK vs MOD) & (MOD vs ZAST) comparison, and 44 genes were significantly altered in the (MOD vs all-EAST) & (MOD vs ZAST) comparison. This shows that the cis-astaxanthin of the present invention can significantly change the gene expression level of obese mice, and cis-astaxanthin regulates more gene numbers.

[0145] Among them, among the 54 changed genes in (CK vs MOD) & (MOD vs Z AST), 10 genes are differentially expressed genes unique to Z AST lipid-lowering. Specifically, Z AST significantly up-regulated Fitm1, Acot2 (regulating lipid droplet formation and decomposition), Manf (regulating white fat browning), TSPO, and Cisd1 (mediating fatty acid metabolism). Z AST significantly down-regulated Fdps (promoting cholesterol synthesis), Id1 (controlling adipocyte thermogenesis), Abca1, Foxa2, and Fgf2 (inducing adipocyte lipogenesis and lipid proliferation). However, only 5 genes are differentially expressed genes unique to all-E AST lipid-lowering. The above shows that the cis-astaxanthin of the present invention is involved in the regulation of more obesity-related genes.

[0146] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. The application of cis-astaxanthin in the preparation of lipid-lowering products, characterized in that: The cis-astaxanthin is astaxanthin represented by formula (I) and / or astaxanthin represented by formula (II):

2. The use according to claim 1, characterized in that The lipid-lowering product is a blood lipid-lowering product.

3. The use according to claim 2, characterized in that The blood lipid lowering product is a health product that assists in lowering blood lipids.

4. The use according to claim 2, characterized in that The lipid-lowering product is a drug for preventing and / or treating diseases related to hyperlipidemia.

5. The use according to claim 4, characterized in that The drug is a drug for lowering triglycerides and / or total cholesterol.

6. The use according to claim 4, characterized in that The drug is a drug for reducing fat droplet content.

7. The use according to claim 4, characterized in that The drug is a drug that regulates the expression of lipid-lowering signaling pathway genes.

8. The use according to claim 4, characterized in that The drug is a drug that regulates metabolites and / or metabolic pathways related to lipid metabolism.

9. The use according to claim 4, characterized in that The drug is a drug for regulating intestinal flora related to lipid metabolism.

10. The use according to claim 1, characterized in that The cis-astaxanthin is obtained by photo-iodine isomerization of all-trans-astaxanthin.

Images