Application of plasmalogen in preparation of product for improving activity of lithocholic acid synthetase

Abstract

The invention discloses application of mussel plasmalogen in preparation of a product for improving activity of lithocholic acid synthase. The mussel plasmalogen comprises mussel plasmalogen phosphatidylethanolamine and / or mussel plasmalogen phosphatidylserine. The mussel plasmalogen comprises mussel plasmalogen phosphatidylethanolamine and The mussel plasmalogen promotes the biosynthesis of lithocholic acid by enhancing the activity of lithocholic acid synthetase, significantly reduces the content of a lithocholic acid synthesis precursor and significantly improves the lithocholic acid level.

Description

Technical Field

[0001] This invention relates to the field of bioactive substances technology, specifically to the application of mussel phospholipids. Background Technology

[0002] Bile acids are a collective term for a class of cholanonic acids present in bile, existing in the form of sodium or potassium salts, and are the main organic components of bile. Bile acids are converted from cholesterol in liver cells and are essential for the digestion and absorption of lipids (including fat-soluble vitamins).

[0003] Bile acids in normal human bile can be structurally classified into two main categories: free bile acids, including cholic acid, deoxycholic acid, chenodeoxycholic acid, and a small amount of lithocholic acid (LCA); and conjugated bile acids, which are products of the combination of the above free bile acids with glycine or taurine. Based on their origin, bile acids can be divided into primary and secondary bile acids. Primary bile acids, including cholic acid and chenodeoxycholic acid, are bile acids directly synthesized from cholesterol in hepatocytes. Secondary bile acids, including deoxycholic acid and lithocholic acid, are bile acids produced in the intestines through bacterial action and the removal of the 7-hydroxyl group.

[0004] Lithocholic acid is an important secondary bile acid produced by the metabolism of primary bile acids by gut microbiota. It has significant probiotic effects in fat digestion and absorption, anti-inflammation, maintenance of intestinal barrier function, regulation of lipid and glucose metabolism, muscle regeneration, and anti-aging. Researchers found that 70% calorie restriction significantly increased the level of the bile metabolite lithocholic acid in the blood of mice. Supplementing mice with lithocholic acid alone could reproduce the effects of calorie restriction on model organisms, such as activating AMPK, promoting muscle regeneration, recovery of grip strength and running ability in mice, and prolonging the lifespan of fruit flies and nematodes (Qi Qu et al., Lithocholic acid phenocopies anti-aging effects of calorie restriction, Nature, 2025, 643:192–200).

[0005] Patent application CN 202210481200.8 provides an effective lipid composition for improving metabolism, used to increase the level of one or more of the following free bile acids in the serum of a subject: chenodeoxycholic acid, deoxycholic acid, lithocholic acid, ursodeoxycholic acid, α-mouse cholic acid, β-mouse cholic acid, and ω-mouse cholic acid. This is mainly achieved by influencing the intestinal microbiota to increase the content of total bile acids and taurine-conjugated bile acids in the subject's serum.

[0006] In daily life, lithocholic acid supplementation is mainly achieved through calorie restriction, which carries the risk of metabolic and nutritional imbalances. Therefore, there is a need to develop safer and more efficient methods for lithocholic acid supplementation. Finding substances that can regulate the activity of specific enzymes to increase the production of lithocholic acid in the body is a new approach to increasing its levels.

[0007] Figure 1 This indicates a key enzyme in the lithocholic acid synthesis pathway in the body. For example... Figure 1 As shown, conjugated chenodeoxycholic acid (TCDCA) that does not enter the enterohepatic circulation enters the intestine via bile acid transport proteins such as ATP-binding cassette transporters and sodium / bile acid cotransporters. Under the action of bile saline hydrolase (BSH), it is converted into chenodeoxycholic acid (CDCA) or cholic acid (CA). Finally, under the action of 7α-dehydroxylase, the hydroxyl group is removed to generate lithocholic acid (LCA). The key step in this process begins with the hydrolysis of conjugated chenodeoxycholic acid by bile saline hydrolase.

[0008] Figure 2 This indicates the structure of acetal phospholipids, a class of phospholipids in which a long-chain hydrocarbon group is linked to the sn-1 position of glycerol via an ether bond. Acetal phospholipids mainly include acetal phosphatidylethanolamine and / or acetal phosphatidylserine. Figure 2 A represents the structure of acetal phosphatidylethanolamine. Figure 2 B represents the structure of acetal phosphatidylserine.

[0009] Phospholipids are widely found in aquatic foods and possess significant nutritional functions. Due to the presence of an ether bond at the sn-1 position, phospholipids can enter the large intestine, rich in gut microbiota, in their intact form, thereby regulating microbial activity. However, there are no research reports on the application of phospholipids in enhancing the activity of lithocholic acid synthase. Summary of the Invention

[0010] This invention discloses the application of mussel acetal phospholipids in the preparation of products that enhance the activity of lithocholic acid synthase in vivo.

[0011] This invention also discloses the application of mussel acetal phospholipids in the preparation of products that increase the level of lithocholic acid in vivo.

[0012] This invention also discloses the application of mussel acetal phospholipids in the preparation of intestinal flora products that regulate the conversion of bound chenodeoxycholic acid to lithocholic acid.

[0013] This invention confirms that mussel acetal phospholipids promote the biosynthesis of lithocholic acid by enhancing the activity of lithocholic acid synthase.

[0014] Furthermore, the aforementioned lithocholic acid synthase can be a bile salt hydrolase and / or a 7α-dehydroxylase.

[0015] Furthermore, the mussel acetal phospholipid provided by this invention can significantly reduce the content of lithocholic acid synthesis precursors and increase lithocholic acid levels.

[0016] Furthermore, the amount of mussel phospholipid provided by the present invention added to food can be 1 to 2%.

[0017] Specifically, the present invention is as follows.

[0018] 1. Application of mussel acetal phospholipids in the preparation of products that enhance the activity of lithocholic acid synthase in vivo.

[0019] 2. The application as described in item 1, characterized in that: the lithocholic acid synthase is a bile salt hydrolase and / or a 7α-dehydroxylase.

[0020] 3. The application as described in item 1, characterized in that: the mussel acetal phospholipid comprises mussel acetal phosphatidylethanolamine and / or mussel acetal phosphatidylserine.

[0021] 4. The application as described in item 2, characterized in that: the mussel acetal phospholipid comprises mussel acetal phosphatidylethanolamine and / or mussel acetal phosphatidylserine.

[0022] 5. Application of mussel phospholipids in the preparation of products that increase lithocholic acid levels in vivo.

[0023] 6. The application as described in item 5, characterized in that: the mussel acetal phospholipid comprises mussel acetal phosphatidylethanolamine and / or mussel acetal phosphatidylserine.

[0024] 7. The application as described in any one of items 1 to 6, characterized in that: the article is an article for regulating intestinal flora.

[0025] 8. The application as described in any one of items 1 to 6, characterized in that: the amount of mussel phospholipid added to the food is 1 to 2%.

[0026] 9. The application as described in item 7, characterized in that: the amount of mussel phospholipid added to the food is 1-2%.

[0027] The beneficial effects of this invention are as follows:

[0028] (1) This invention is the first to discover and demonstrate through experiments that mussel acetal phospholipids can significantly increase the level of key enzymes for the generation of lithocholic acid in vivo.

[0029] (2) This invention can increase the level of key enzymes for lithocholic acid production in vivo by supplementing mussel phospholipids in vivo. Using mussel phospholipids to prepare products that increase lithocholic acid in vivo provides a new approach to increasing the level of lithocholic acid in vivo. Attached Figure Description

[0030] Figure 1 This indicates a key enzyme in the lithocholic acid synthesis pathway.

[0031] Figure 2 This indicates the structure of acetal phospholipids.

[0032] Figure 3 This is a thin-layer chromatography chromatogram of acetal phospholipids according to an embodiment of the present invention.

[0033] Figure 4 This is a total ion current chromatogram of an embodiment of the present invention.

[0034] Figure 5 This is the purity identification result of acetal phosphatidylethanolamine and acetal phosphatidylserine in one embodiment of the present invention.

[0035] Figure 6 This is the result of measuring the change in the content of key bile acids during the synthesis of lithocholic acid in one embodiment of the present invention.

[0036] Figure 7 This indicates the bile acid content of the intestinal flora enzymatic hydrolysate after 24 hours in one embodiment of the present invention. Detailed Implementation

[0037] To better understand this invention, the following embodiments are provided in conjunction with the accompanying drawings. It should be understood that the embodiments of this invention are for illustrative purposes only and not for limiting the invention; the scope of protection of this invention is defined solely by the claims. The embodiments provided are merely preferred embodiments and are not intended to limit the invention in any way. Those skilled in the art can make changes, equivalent substitutions, or modifications based on the content of this invention to form different implementations. However, any changes and modifications, and any equivalent substitutions made to the method of this invention without departing from the inventive concept are within the scope of protection of this invention.

[0038] Unless otherwise specified, all materials used in the embodiments of this invention were purchased from the market.

[0039] Example 1: Extraction of total lipids from mussels

[0040] Fresh mussels are freeze-dried in a freeze dryer to remove moisture, producing dried mussels. The freeze-drying conditions are: temperature -50℃, vacuum degree <10kPa, and time 48h.

[0041] Dried mussels were pulverized and extracted with a chloroform / methanol (2:1, v / v) solution at a material-to-liquid ratio of 1:8 (m / v) for 24 h with stirring. The extract was filtered through medium-speed filter paper with a pore size of 30–50 μm. A 0.58% NaCl solution was added to the extract, and after thorough mixing, the mixture was allowed to stand and separate into layers. The lower chloroform layer was collected and dried by rotary evaporation to obtain total mussel lipids. The rotary evaporation conditions were: vacuum degree between 0.06 and 0.08 MPa, rotation speed between 30 and 60 rpm, cooling temperature between -10°C, and water bath temperature between 40 and 50°C.

[0042] To improve the lipid extraction rate, the filter residue was subjected to a second extraction following the steps described above.

[0043] Example 2: Extraction of mussel acetal phosphatidylethanolamine and mussel acetal phosphatidylserine

[0044] The prepared total lipids from mussels were dissolved in a certain volume of chloroform solution and then mixed with activated column chromatography silica gel (reagent grade 200-300 mesh, Shanghai Maclean Biochemical Technology Co., Ltd., item number C820913). The chloroform was removed by rotary evaporation, and the lipids were fully adsorbed onto the column chromatography silica gel.

[0045] The silica gel for column chromatography was loaded onto the column using a dry method, with chloroform as the packing solvent. Neutral lipids were removed sequentially with chloroform and chloroform:methanol (9:1, v / v) solution, followed by elution with chloroform:methanol 2:1 (v / v) solution and chloroform:methanol 1:1 (v / v) solution, yielding mussel acetal phosphatidylethanolamine and mussel acetal phosphatidylserine, respectively.

[0046] Thin-layer chromatography (TLC) was used to determine the composition of the eluent throughout the elution process. After stepwise collection, the eluent was developed using a Yinlong TLC silica gel pre-prepared plate (HSG type, Yantai Chemical Industry Research Institute) in a chloroform:methanol:water ratio of 65:25:4, with iodine vapor for color development to confirm the lipid components.

[0047] The thin-layer chromatography chromatogram of mussel acetal phospholipids eluted is shown below. Figure 3 . Figure 3 A is a thin-layer chromatography chromatogram of mussel acetal phosphatidylethanolamine, with phosphatidylethanolamine (Shanghai Yuanye Biotechnology Co., Ltd., product number B74432) as the standard. 1-4 are the mussel acetal phosphatidylethanolamine fractions collected by elution with chloroform:methanol 2:1 solution. Figure 3 B is a thin-layer chromatography chromatogram of mussel acetal phosphatidylserine, with phosphatidylserine (Shanghai Yuanye Biotechnology Co., Ltd., product number S27340) as the standard. 1-4 are the mussel acetal phosphatidylserine fractions collected by elution with chloroform:methanol 1:1 solution.

[0048] Example 3: Purity Identification of Pyralphospholipids

[0049] Purity was determined using an electrostatic field orbital trap high-resolution mass spectrometer (Q-exactive quadrupole Orbitrap mass spectrometer, Thermo Fisher Scientific China Co., Ltd.) equipped with an ACQUITY UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm) (Waters Corporation, USA) and an electrospray ionization (ESI) source. Mobile phase A: acetonitrile / water (60:40, v / v) + 10 mmol ammonium formate; mobile phase B: isopropanol / acetonitrile (90:10, v / v).

[0050] The total ion chromatogram (TCI) was quantified and analyzed using LipidSearch 4.2.21 software (Thermo Fisher Scientific (China) Co., Ltd.). Based on mass spectrometry database matching of lipid species, characteristic peaks were automatically extracted and compared with known lipid libraries to identify the target components acetal phosphatidylethanolamine and acetal phosphatidylserine. The peak areas on the TCI were quantitatively analyzed, and the relative abundance of the target components was calculated.

[0051] The total ion current chromatogram is shown in the image. Figure 4 . Figure 4 A is the total ion chromatogram of mussel acetal phosphatidylethanolamine. Figure 4 B is the total ion chromatogram of mussel acetal phosphatidylserine.

[0052] Analysis results as follows Figure 5 As shown. Figure 5 A represents the mussel acetal phosphatidylethanolamine sample, with a purity of 80.68 ± 1.98%. Figure 5 B represents the mussel acetal phosphatidylserine sample, with a purity of 91.01 ± 0.88%.

[0053] Example 4: Determination of fatty acid composition of mussel phospholipids

[0054] Take 2 mg of mussel acetal phosphatidylethanolamine and mussel acetal phosphatidylserine into methyl esterification tubes, add 200 μg of triglyceride type pentadecanoic acid standard, and blow nitrogen gas through the tubes to dry the solvent.

[0055] Add 2 mL of hydrochloric acid:methanol 1:5 (v:v) solution, purge with nitrogen to prevent fatty acid oxidation, tighten the cap, and methylate in a metal bath at 90°C for 3 h. During methylation, vortex every 30 min to mix the liquid and ensure complete lipid methylation.

[0056] After methylation, cool the methylation tube to room temperature, add 1.5 mL of n-hexane, vortex to mix, and allow to stand for separation. Take the upper n-hexane layer into a 1.5 mL centrifuge tube, concentrate and dry the liquid in the tube using nitrogen blowing, and redissolve it with an appropriate amount of chromatographic grade n-hexane.

[0057] Fatty acid composition analysis was performed on acetal phosphatidylethanolamine and acetal phosphatidylserine derived from mussels.

[0058] A 1 μL sample solution of the reconstituted sample was analyzed by gas chromatography (GC). Specific conditions were: injection volume 2 μL; split mode, split ratio 10:1; constant flow mode. The detector and injector temperatures were maintained at 250 °C and 240 °C, respectively. The temperature was increased from 170 °C to 240 °C at a rate of 3 °C / min and held at 240 °C for 35 min. Nitrogen was used as the carrier gas at a flow rate of 1.0 mL / min. The entire analysis process took 48.5 min.

[0059] The analysis results are shown in Table 1.

[0060] Table 1. Fatty acid composition of phosphatidylcholine from mussels

[0061] For the two phosphatal samples, mussel phosphatidylethanolamine and mussel phosphatidylserine, the saturated fatty acids were mainly C16:0 and C18:0, while the unsaturated fatty acids were mainly C20:5n-3 (relative content 24.45% and 6.58%), C22:6n-3 (relative content 14.17% and 11.23%), and C20:4n-6 (relative content 1.42% and 3.90%). They had a high content of n-3 polyunsaturated fatty acids, especially DHA and EPA, showing high nutritional value.

[0062] Example 5: Mouse feeding experiment

[0063] Phosphatidylserine was purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number S20166. Phosphatidylethanolamine was purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number S30050.

[0064] Eight-week-old SPF-grade male C57b1 / 6 mice were acclimatized for one week and then randomly divided into five groups: control (N), mussel acetal phosphatidylserine (pPS), mussel acetal phosphatidylethanolamine (pPE), phosphatidylserine (PS), and phosphatidylethanolamine (PE), with eight mice in each group. The normal control group was fed ordinary bar-shaped feed (containing 5% corn oil), while the feeds of each phospholipid group were supplemented with the corresponding phospholipid at a final concentration of 2% in addition to the feed of the control group.

[0065] Fresh feces were collected from each group of mice on the sixth day.

[0066] Mice were sacrificed on day 7, and serum and intestinal contents were collected. An appropriate amount of serum or intestinal contents was centrifuged, and the supernatant was collected. Acetonitrile at -20℃ was added to remove protein, and the mixture was vortexed. The supernatant was then centrifuged at 12000g and concentrated under reduced pressure at 45℃ and 1500rpm. The supernatant was then reconstituted with 100μL of methanol-water (85:15, v:v) solution and centrifuged at 14800rpm and 4℃ for 10min.

[0067] Example 6: Effect of mussel phospholipids on in vivo lithocholic acid synthesis

[0068] The composition of bile acids in serum and intestinal contents extracts from mice in each group in Example 5 was determined, with a focus on analyzing the content and proportion of lithocholic acid and its synthetic intermediates. 70 μL of the collected supernatant was injected into an LC-MS system for analysis to determine the composition of key bile acids involved in the lithocholic acid synthesis process in serum and intestinal contents extracts.

[0069] A Gilent G6410 Triple Quad (QQQ) mass spectrometer (Agilent Technologies (China) Co., Ltd.) equipped with an electrospray ionization (ESI) source was coupled with an Agilent 1260 Infinity liquid chromatography system (Agilent Technologies (China) Co., Ltd.) for liquid chromatography separation and mass spectrometry detection.

[0070] A ZORBAX SB-C18 reversed-phase column (250 × 4.6 mm, 5 μm inner diameter, Agilent Technologies (China) Co., Ltd.) was used. The injection volume was 10 μL. The mobile phase consisted of methanol (A) and 5 mmol / L ammonium acetate aqueous solution (B, containing 0.1% formic acid), with a total flow rate of 0.5 mL / min. The gradient curves of the LC pump under the final chromatographic conditions are as follows: 0 min, 10:90; 1.4 min, 50:50; 2.8 min, 65:35; 4.2 min, 70:30; 8.4 min, 75:25; 15.4 min, 80:20; 28 min, 92:8; 38 min, 93:7; 56 min, 93:7; 60 min, 10:90.

[0071] ESI in the mass spectrometer was performed using the following parameters: ionization, negative ion mode; capillary voltage, 3.5 kV; nebulizer gas pressure (N2), 40 psi; ion source temperature, 300 °C; dry gas (N2) flow rate, 10.0 L / min; dry gas temperature, 350 °C. The mass range in FS mode was recorded as m / z 300 to 600. Data analysis was performed using Agilent MassHunter Workstation Software Qualitative Analysis.

[0072] Figure 6 The results represent the changes in the content of key bile acids during lithocholic acid synthesis. All results are expressed as Mean ± SEM. Significance was analyzed using one-way ANOVA and then tested using Tukey's test. Different letters above the data bars indicate significant differences between groups (p < 0.05).

[0073] The results are as follows Figure 6 As shown, in the intestinal contents of mice, compared with the control group (N), dietary intervention with mussel phosphatidylserine (pPS), mussel phosphatidylethanolamine (pPE), phosphatidylserine (PS), and phosphatidylethanolamine (PE) significantly increased the production of lithocholic acid (LCA) by 54.90%, 45.94%, 26.79%, and 24.67%, respectively. Furthermore, the intervention with mussel phosphatidylserine significantly increased the consumption of lithocholic acid precursors, bound chenodeoxycholic acid (TCDCA) and chenodeoxycholic acid (CDCA).

[0074] In mouse serum, compared with the control group (N), dietary intervention with mussel phosphatidylserine (pPS), mussel phosphatidylethanolamine (pPE), phosphatidylserine (PS), and phosphatidylethanolamine (PE) increased lithocholic acid (LCA) production by 26.67%, 17.22%, 8.20%, and 2.73%, respectively, with pPS and pPE showing significant increases. Furthermore, regarding the consumption of lithocholic acid precursors conjugated chenodeoxycholic acid (TCDCA) and chenodeoxycholic acid (CDCA), both pPS and pPE showed significant promoting effects, indicating that mussel phosphatidylserine promotes lithocholic acid production in vivo. In summary, mussel phosphatidylserine (pPS) exhibits the best effect in promoting lithocholic acid production.

[0075] Example 7: Effect of mussel acetal phospholipids on in vitro lithocholic acid synthase activity

[0076] Fresh feces from mice in each group in Example 5 were collected, dissolved in sterile PBS solution, and prepared into a 10% suspension. The suspension was centrifuged at 2000g and 4℃ for 10 min, and the supernatant was collected. The suspension was then centrifuged at 10000g and 4℃ for 10 min, and the supernatant was removed to obtain intestinal microbial precipitate.

[0077] The intestinal microbial pellet was resuspended in Tris-HCl buffer containing lysozyme and EDTA and incubated at 37°C for 30 min. The bacterial cells were lysed under ice bath conditions, and then the supernatant was collected by centrifugation at 12000g for 30 min at 4°C to obtain the bacterial enzyme lysate.

[0078] Prepare enzyme activity reaction solutions containing 5 mM ATP, 0.1 mM Coenzyme A, 1.25 mM NADH, 0.5 mM nicotinamide, 2.25 mM glutathione, and 10 mM MgCl2. Add 1 mL of each group's bacterial enzyme lysate (containing 3 mg of protein) to the enzyme activity reaction solution. Add 200 μmol / L of either lithocholic acid precursor-bound chenodeoxycholic acid or free chenodeoxycholic acid to each reaction solution. Incubate at 37°C for 30 min, then add an equal volume of pre-cooled methanol solution at -20°C to terminate the reaction.

[0079] The amount of substrate consumed and the amount of product generated were determined by LC-MS.

[0080] Figure 7 This indicates the concentration of conjugated chenodeoxycholic acid (TCDCA) and lithocholic acid (LCA) in bile acids after 24 hours of in vitro enzymatic hydrolysis of mouse intestinal flora. The consumption of TCDCA can represent the activity of bile salt hydrolase (BSH), and the production of lithocholic acid can represent the activity of 7α-dehydroxylase. Figure 7 It can be seen that, among the experimental groups, the mussel acetal phosphatidylserine (pPS) group had the lowest content of bound chenodeoxycholic acid and the highest content of lithocholic acid, which was significant, and showed the strongest bile salt hydrolase and 7α-dehydroxylase activity.

Claims

1. Use of a mytilus edulis aldehyde plasmalogen in the preparation of a product for increasing the activity of a lithocholic acid synthesis enzyme in vivo.

2. Use according to claim 1, characterized in that: The lithocholic acid synthesis enzyme is a bile salt hydrolase and / or a 7a- dehydroxylase.

3. Use according to claim 1, characterized in that: The mytilus edulis aldehyde plasmalogen is a mytilus edulis aldehyde plasmalogenethanolamine and / or a mytilus edulis aldehyde plasmalogenserine.

4. The use according to claim 2, characterized in that: The mytilus edulis aldehyde plasmalogen comprises a mytilus edulis aldehyde plasmalogenethanolamine and / or a mytilus edulis aldehyde plasmalogenserine.

5. Use of a mytilus edulis aldehyde plasmalogen in the preparation of a product for increasing the level of lithocholic acid in vivo.

6. Use according to claim 5, wherein: The mytilus edulis aldehyde plasmalogen comprises a mytilus edulis aldehyde plasmalogenethanolamine and / or a mytilus edulis aldehyde plasmalogenserine.

7. Use according to any one of claims 1 to 6, wherein: The product is a product for modulating the intestinal flora.

8. Use according to any one of claims 1 to 6, wherein: The mytilus edulis aldehyde plasmalogen is added to the food in an amount of 1-2%.

9. The use according to claim 7, characterized in that: The mytilus edulis aldehyde plasmalogen is added to the food in an amount of 1-2%.

Images