TW202122075A - Metabolite of lactic acid bacteria and use of the metabolite for preparing vascular health care composition - Google Patents

Abstract

A metabolite of lactic acid bacteria, containing at least one biologically active substance selected from the group consisting of 2-hydroxy-4-methylpentanoic acid and 2-hydroxy-3-methylbutanoic acid.

Description

Metabolite of lactic acid bacteria and its use for preparing vascular health care composition

The invention relates to a metabolite of bacteria, in particular to the metabolite of lactic acid bacteria, and the use of the metabolite of lactic acid bacteria for preparing a vascular health care composition.

The vascular system shuttles around the body, allowing the blood to carry oxygen, nutrients and various chemical substances to various organs. It is an extremely important transport network. However, as the years grow older, blood vessels will gradually lose their elasticity, and the structure of the blood vessels will begin to change. Large blood vessel diseases look like swelling and deformation, while small blood vessel diseases will become blocked.

The main cause of common cardiovascular diseases is blood vessel obstruction. The process starts with normal blood vessels, possibly due to fat accumulation or other causes of coronary atherosclerosis, which gradually evolves into vascular stenosis, and finally evolves into vascular obstruction. . If atherosclerosis occurs in the coronary arteries, the coronary arteries are blocked, which can cause coronary heart disease and affect the normal function of the heart. If atherosclerosis in the blood vessels of the brain causes damage to brain cells and even rupture of cerebrovascular vessels, significant sequelae are often left even if cured. In addition, blood vessel embolism caused by atherosclerosis may also damage the valve and cause valvular heart disease.

In summary, based on the improvement of modern people's living standards and the improvement of the concept of health care, it is indeed necessary to develop a cardiovascular health care composition.

In one embodiment, a metabolite of lactic acid bacteria includes at least one biologically active substance selected from the group consisting of 2-hydroxy-4-methylpentanoic acid (2-hydroxy-4-methylpentanoic acid) and 2-hydroxy-3-methylbutanoic acid (2-hydroxy-3-methylbutanoic acid).

In some embodiments, the lactic acid bacteria is Lactobacillus plantarum TCI028, and the deposit number of Lactobacillus plantarum TCI028 is BCRC 910805.

In some embodiments, the metabolites of lactic acid bacteria further include: 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (1,2,3,4-tetrahydro-β-carboline-3 -carboxylic acid).

In one embodiment, a metabolite of lactic acid bacteria is used to prepare a cardiovascular health care composition, wherein the lactic acid bacteria is Lactobacillus plantarum TCI028, and the deposit number of Lactobacillus plantarum TCI028 is BCRC 910805.

In some embodiments, the aforementioned metabolite of Lactobacillus plantarum TCI028 contains at least one biologically active substance selected from the group consisting of: 1,2,3,4-tetrahydro-β-carboline-3-carboxy Acid, 2-hydroxy-4-methylpentanoic acid and 2-hydroxy-3-methylbutanoic acid.

In some embodiments, the metabolite of Lactobacillus plantarum TCI028 is prepared by a method including the following steps: Lactobacillus plantarum TCI028 is cultivated in a medium to obtain a culture of Lactobacillus plantarum TCI028, and then the Lactobacillus plantarum TCI028 culture is obtained. The culture of TCI028 was centrifuged and the supernatant was collected to obtain the metabolites of lactic acid bacteria.

In some embodiments, the aforementioned cardiovascular health care composition is used to inhibit MSR1 gene expression.

In some embodiments, the aforementioned cardiovascular health care composition is used to increase SCARB1 gene expression.

In one embodiment, a 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid is used to prepare a cardiovascular health care composition.

In some embodiments, the aforementioned vascular health care composition is used to inhibit MSR1 gene expression.

In some embodiments, the aforementioned vascular health care composition is used to increase SCARB1 gene expression.

In some embodiments, the aforementioned 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid is obtained by separation and purification from the methanol layer extract of the culture supernatant of Lactobacillus plantarum TCI028, and the plant The deposit number of Lactobacillus TCI028 is BCRC 910805.

In one embodiment, a 2-hydroxy-4-methylvaleric acid is used to prepare a cardiovascular health care composition. In some embodiments, the aforementioned cardiovascular health care composition is used to inhibit MSR1 gene expression.

In some embodiments, the aforementioned 2-hydroxy-4-methylvaleric acid is obtained by separating and purifying the methanol layer extract of the culture supernatant of Lactobacillus plantarum TCI028, and the deposit number of Lactobacillus plantarum TCI028 is BCRC 910805.

In one embodiment, a 2-hydroxy-3-methylbutanoic acid is used to prepare a cardiovascular health care composition. In some embodiments, the aforementioned cardiovascular health care composition is used to inhibit MSR1 gene expression.

In some embodiments, the aforementioned 2-hydroxy-3-methylbutyric acid is obtained by separation and purification from the methanol layer extract of the culture supernatant of Lactobacillus plantarum TCI028, and the deposit number of Lactobacillus plantarum TCI028 is BCRC 910805.

In summary, according to any embodiment, 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid, 2-hydroxy-4-methylpentanoic acid, 2-hydroxy-3-methyl Butyric acid or its combination can inhibit the expression of MSR1 gene, thereby providing cardiovascular health care. In some embodiments, 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid, 2-hydroxy-4-methylpentanoic acid, or 2-hydroxy-3-methylbutanoic acid is the source From Lactobacillus plantarum TCI028 with deposit number BCRC 910805. In some embodiments, 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid can also increase the expression of SCARB1 gene, thereby providing cardiovascular health care.

In one embodiment, at least one of the following compounds can be used to prepare a cardiovascular health care composition: 2-hydroxy-4-methylpentanoic acid, 2-hydroxy-3-methylbutyl Acid (2-hydroxy-3-methylbutanoic acid), and 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (1,2,3,4-tetrahydro-β-carboline-3- carboxylic acid).

Among them, the structural formulas of the above-mentioned various compounds are shown in Table 1 below:

Table I 2-hydroxy-4-methylvaleric acid 2-hydroxy-3-methylbutyric acid 1,2,3,4-Tetrahydro-β-carboline-3-carboxylic acid

In other words, when a recipient receives at least one of the aforementioned compounds or a cardiovascular health care composition prepared therefrom, it helps the recipient's cardiovascular health care. Therefore, the aforementioned compounds can also be referred to as biologically active substances. Among them, the recipient may be human.

In some embodiments, the aforementioned at least one compound or a cardiovascular health care composition prepared therefrom can inhibit the expression of MSR1 gene, thereby providing cardiovascular health care.

In some embodiments, 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid or a cardiovascular health care composition prepared therefrom can also increase the expression of SCARB1 gene, thereby providing cardiovascular health care.

In some embodiments, the above-mentioned various biologically active substances may be artificially synthesized or derived from Lactobacillus plantarum .

In some embodiments, the various biologically active substances mentioned above are metabolites of Lactobacillus plantarum.

In some embodiments, the Lactobacillus plantarum may be Lactobacillus plantarum TCI028. Among them, Lactobacillus plantarum TCI028 (Lactobacillus plantarum TCI028 ) is deposited at the Food Industry Development Institute under the deposit number BCRC 910805, and at the German National Culture Collection under the deposit number DSM33108.

In some embodiments, Lactobacillus plantarum TCI028 is a strain isolated from garlic.

Lactobacillus plantarum TCI028 is a gram-positive anaerobe. The colony on the surface is about 3mm in diameter, convex, round, smooth, dense, white, and occasionally light yellow or dark yellow. The growth temperature of Lactobacillus plantarum TCI028 is 15°C to 45°C, and it lives in an environment with a pH value of 5 to 7.

In other words, the metabolite of Lactobacillus plantarum TCI028 can be used to prepare a cardiovascular health care composition.

In some embodiments, the metabolite of Lactobacillus plantarum TCI028 is prepared by a method including the following steps: Lactobacillus plantarum TCI028 is cultivated in a medium to obtain a culture of Lactobacillus plantarum TCI028, and then the Lactobacillus plantarum TCI028 culture is obtained. The culture of TCI028 is centrifuged and the supernatant (also called culture supernatant) is collected to obtain the metabolites of lactic acid bacteria. In other words, the metabolites of Lactobacillus plantarum TCI028 include substances that are metabolized and secreted into the culture medium by Lactobacillus plantarum TCI028.

In some embodiments, the "metabolite" may include substances secreted into the culture solution after being metabolized by Lactobacillus plantarum TCI028 when Lactobacillus plantarum TCI028 is cultured, but does not include the substance of Lactobacillus plantarum TCI028.

In some embodiments, the metabolites of plant lactic acid bacteria include at least one biologically active substance selected from the group consisting of 2-hydroxy-4-methylpentanoic acid and 2-hydroxy-3-methylbutanoic acid .

In some embodiments, 2-hydroxy-4-methylvaleric acid is obtained by separation and purification from the methanol layer extract of the culture supernatant of Lactobacillus plantarum TCI028.

In some embodiments, 2-hydroxy-4-methylvaleric acid is obtained from the 40% methanol layer extract of the culture supernatant of Lactobacillus plantarum TCI028.

In some embodiments, 2-hydroxy-4-methylvaleric acid is a 40% methanol layer extract obtained by column chromatography on the culture supernatant of Lactobacillus plantarum TCI028 with a 40% methanol aqueous solution as the extraction solvent , And then use 20% methanol aqueous solution as the extraction solvent for fast column chromatography.

In some embodiments, 2-hydroxy-4-methylvaleric acid can be used to inhibit MSR1 gene expression.

In some embodiments, 2-hydroxy-3-methylbutyric acid is obtained by separation and purification from the methanol layer extract of the culture supernatant of Lactobacillus plantarum TCI028.

In some embodiments, 2-hydroxy-3-methylbutyric acid is obtained by separating and purifying the 40% methanol layer extract of the culture supernatant of Lactobacillus plantarum TCI028.

In some embodiments, 2-hydroxy-3-methylbutyric acid is a 40% methanol layer extract obtained by column chromatography on the culture supernatant of Lactobacillus plantarum TCI028 with a 40% methanol aqueous solution as the extraction solvent , And then use 20% methanol aqueous solution as the extraction solvent for fast column chromatography.

In some embodiments, 2-hydroxy-4-methylbutanoic acid can be used to inhibit MSR1 gene expression.

In some embodiments, the metabolites of plant lactic acid bacteria may further include: 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid.

In some embodiments, 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid is isolated and purified from the methanol layer extract of the culture supernatant of Lactobacillus plantarum TCI028.

In some embodiments, 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid is obtained by separation and purification from the 40% methanol layer extract of the culture supernatant of Lactobacillus plantarum TCI028.

In some embodiments, 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid uses 40% methanol aqueous solution as the extraction solvent to carry out the column on the culture supernatant of Lactobacillus plantarum TCI028 The 40% methanol layer extract obtained by chromatography is obtained by fast column chromatography with 80% methanol aqueous solution as the extraction solvent.

In some embodiments, 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid can be used to inhibit MSR1 gene expression.

In some embodiments, 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid can be used to increase SCARB1 gene expression.

In some embodiments, the aforementioned cardiovascular health care composition is used to inhibit MSR1 gene expression.

In some embodiments, the aforementioned cardiovascular health care composition is used to increase SCARB1 gene expression.

In some embodiments, the aforementioned cardiovascular health care composition may be a medicine. In other words, the medicine contains an effective content of the aforementioned at least one biologically active substance.

In some embodiments, the aforementioned cardiovascular health care composition may be an edible composition. In other words, the edible composition contains an effective content of the aforementioned at least one biologically active substance. In some embodiments, the edible composition can be made into a food product or can be a food additive, which means that a food product is made by adding it during food preparation by a conventional method, or it can be used in a food product. Added during the production process. Here, the food product may be a product formulated with edible materials for human or animal ingestion.

In some embodiments, the food product may be, but is not limited to: beverages, fermented foods, bakery products, health foods, and dietary supplements.

Example 1: Screening and identification of strains

Garlic juice obtained by grinding garlic, or homogenizing garlic by adding garlic to sterile water to obtain a homogenized garlic liquid. Coat the garlic juice or garlic homogenate on a solid medium (BD Difco™ Lactobacilli MRS Broth, DF0881-17-5) and culture it at 28°C until a single colony is formed. Pick multiple single colonies, and use this single colony for bacterial species identification. The 16S ribosomal gene (16SrDNA) sequence (ie SEQ ID NO: 1) of this isolated strain was obtained by polymerase chain reaction (PCR). Next, the SEQ ID NO:1 sequence was compared with the 16S ribosomal gene (16SrDNA) of other subspecies of Lactobacillus plantarum on the website of the National Center for Biotechnology Information (NCBI). The similarity between the 16S ribosomal gene sequence and other subspecies of Lactobacillus plantarum is shown in Table 2. It can be seen that these single colonies are more than 95.85 similar to other Lactobacillus plantarum, so they are named Lactobacillus plantarum TCI028. The name of the bacteria and the results of the identification of the bacteria are shown in Table 2 below:

Table II Strain number Lactobacillus The sequence of the strain Comparative subspecies (indicated by subspecies number) Similarity TCI028 Lactobacillus plantarum TCI028 SEQ NO:1 SC12 96.49% LY21 96.85% NWAFU1517 95.78% KLDS 1.0708 95.85% LCN 56 95.85%

In addition, Lactobacillus plantarum TCI028 is deposited at the Food Industry Development Institute under the deposit number BCRC 910805, and at the German National Culture Collection under the deposit number DSM33108.

Example 2: Preparation of metabolites

1. Inoculate Lactobacillus plantarum TCI028 with 1% (v/v) planting amount in the culture medium (MRS) and incubate at 37°C for 18 hours to obtain a bacterial solution. After centrifuging the bacterial solution at 5000 rpm for 20 minutes, the culture supernatant was collected. Here, the culture supernatant was taken as the TCI028 sample for subsequent analysis.

2. Take 2L of TCI028 sample into Luna® analytical column (5μm, C18(2), filled with Diaion HP-20 packing material (pore size 260 Å, particle size 0.5 mm, purchased from Mitsubishi Chemical Co., Japan) 100 Å, 70 x 7 mm, purchased from Phenomenex, USA), and a gradient of 100% water, 20% methanol aqueous solution, 40% methanol aqueous solution, 60% methanol aqueous solution, and 100% methanol extraction solvents were applied to the TCI028 sample. Perform Column Chromatography to separate five layers. The five strata are called stratification 1, stratification 2, stratification 3, stratification 4 and stratification 5 respectively.

Among them, the weight of the solids obtained after removing the extraction solvent in layer 1 to layer 5 is 20.9 grams (that is, the solids obtained in layer 1) and 6.3 grams (that is, the solids obtained in layer 2). ), 4.3 grams (that is, the solids obtained in layer 3), 2.9 grams (that is, the solids obtained in layer 4), and 2.7 grams (that is, the solids obtained in layer 5).

3. Use chromatographic sheets (TLC aluminium sheets, RP-18 F ₂₅₄ -S, 0.25 mm, Merck, Germany) for the layer 3 extracted with 40% methanol aqueous solution, and the gradient of 20% methanol aqueous solution, 40 % Methanol aqueous solution, 60% methanol aqueous solution, 80% methanol aqueous solution and 100% methanol extraction solvent were subjected to fast column chromatography, namely Thin-Layer Chromatography, to separate 5 sub-layers ( Hereinafter referred to as sub-layer 1, sub-layer 2, sub-layer 3, sub-layer 4 and sub-layer 5).

4. Perform reversed-phase chromatography (RPC) on sub-layer 1 with an extraction solvent of methanol and water with a volume ratio of 1:9 to obtain two compounds TCI-028-002 and TCI-028- 003. Among them, reverse chromatography is to inject Diaion HP-20 packing material (pore size 260 Å, particle size 0.5 mm, purchased from Mitsubishi Chemical Co., Japan) into the packed Luna® analytical column (5μm, C18(2), 100 Å, 250 x 10 mm, purchased from Phenomenex, USA).

The chemical structures of compound TCI-028-002 and compound TCI-028-003 were analyzed by hydrogen nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry. Here, the hydrogen nuclear magnetic resonance spectra of compound TCI-028-002 and compound TCI-028-03 are shown in Figure 1 to Figure 2, and the electrospray ionization of compound TCI-028-002 and compound TCI-028-03 are obtained. The mass spectra are shown in Figure 3 to Figure 4. In addition, the chemical structure and chemical name of each compound were determined according to the obtained hydrogen nuclear magnetic resonance spectrum and electrospray ionization mass spectrometry, as shown in Table 3 below.

圖1與圖3 TCI-028-003 2-羥基-3-甲基丁酸(2-hydroxy-3-methylbutanoic acid)

圖2與圖4 Table Three Compound Compound name Chemical structure Schema TCI-028-002 2-hydroxy-4-methylpantaoic acid

Figure 1 and Figure 3 TCI-028-003 2-hydroxy-3-methylbutanoic acid

Figure 2 and Figure 4

Among them, the 1D and 2D spectra of the NMR spectrometer use Ascend 400 MHz, Bruker Co. (Germany), and the chemical shift is represented by δ, and the unit is ppm. The electrospray ionization mass spectrometer is tandem mass spectrometry-two-dimensional ion trap tandem Fourier transform mass spectrometry and ESI-MS/MS: measured by Bruker amaZon SL system, and the unit is m/z.

5. Sublayer 4 is subjected to reverse chromatography with an extraction solvent of methanol and water with a volume ratio of 4:6 to obtain compound TCI-028-001 for purification. Among them, reverse chromatography is to inject Diaion HP-20 packing material (pore size 260 Å, particle size 0.5 mm, purchased from Mitsubishi Chemical Co., Japan) into the packed Luna® analytical column (5μm, C18(2), 100 Å, 250 x 10 mm, purchased from Phenomenex, USA.

The chemical structure of compound TCI-028-001 was analyzed by hydrogen nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry. Here, the hydrogen nuclear magnetic resonance spectrum of compound TCI-028-001 is shown in FIG. 5, and the electrospray ionization mass spectrum of compound TCI-028-001 is shown in FIG. 6. In addition, the chemical structure and chemical name of compound TCI-028-001 were determined according to the obtained hydrogen nuclear magnetic resonance spectrum and electrospray ionization mass spectrum, as shown in Table 4 below.

圖5與圖6 Table Four Compound Compound name Chemical structure Schema TCI-028-001 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (1,2,34-tetrahydro-β-carboline-3-carboxylic acid)

Figure 5 and Figure 6

Among them, reverse chromatography is performed with Agilent 1200 series high performance liquid chromatography (High Performance Liquid Chromatography, HPLC). In the Agilent 1200 series high performance liquid chromatograph, the degassing device is Agilent vacuum degassing device 1322A; the extraction solvent delivery system is Agilent quaternary pump G1311A; the variable wavelength detector (Multiple Wavelength Detector, MWD) G1314B; Diode Array Detector (DAD) is Agilent 1260 Infinity DAD VL G1315D, and its detection wavelengths are 210 nm, 280 nm, 320 nm and 365 nm (Agilent Germany).

Among them, fast column chromatography is performed with a medium pressure liquid chromatography (MPLC) (model Combi Flash ^® Rf+, purchased from Teledyne ISCO), and the filling material is Merck LiChroprep ^® RP-18 (pore size 40 -63 um, model Art. 0250).

Example 3: Detection of atherosclerosis-related genes

1. Human monocytic cell line (hereinafter referred to as THP-1 cells) (purchased from the American Type Culture Collection ATCC; Model: TIB-202), using 1.5×10 ⁵ cells per well , Inoculate in a 6-well culture dish containing 2 mL of cell culture medium per well.

Here, the cell culture medium is supplemented with 10% fetal bovine serum (FBS), 0.05mM 2-mercaptoethanol (2-mercaptoethanol), 100 units/mL penicillin and 1ug/1mL streptomycin RPMI 1640 medium (Gibco RPMI medium 1640; Thermo Fisher Scientific).

2. Replace 500nM phorbol-12-myristate 13-acetate (PMA) to the cell culture medium and incubate at 37°C, 5% CO ₂ for 48 hours, To stimulate THP-1 cells to differentiate into macrophages.

3. Replace the fresh cell culture medium and continue to culture for 48 hours under monitoring at 37°C and 5% CO _2.

4. Remove the cell culture medium from each well of the culture plate.

5. Divide the differentiated cells into 5 groups, namely the experimental group 1, the experimental group 2, the experimental group 3, the control group and the control group, and add the following experimental medium to each group: Experimental group 1: Add 2 mL of cell culture medium containing 20μg/ml compound TCI-028-001 and 100ng/ml lipopolysaccharides (LPS) to each well. Experimental group 2: Add 2 mL of cell culture medium containing 20 µg/ml compound TCI-028-002 and 100 ng/ml lipopolysaccharide to each well. Experimental group 3: Add 2 mL of cell culture medium containing 20 μg/ml compound TCI-028-003 and 100 ng/ml lipopolysaccharide to each well. Control group: Add 2 mL of cell culture medium containing 100 ng/ml lipopolysaccharide to each well. Control group: Add 2 mL of cell culture medium to each well (that is, no additional compounds and lipopolysaccharides are added).

6. After culturing each group for 48 hours, the cells of each group were destroyed by cell membrane with cell lysate to form a cell solution.

7. Use the RNA extraction reagent kit (purchased from Geneaid, Taiwan, Lot No.FC24015-G) to extract RNA in the cell solution of each group. Then, each group takes the RNA extracted from 2000 nanograms (ng) as the template, and uses SuperScript ^® III reverse transcriptase (purchased from Invitrogene, USA) to perform reverse transcription with primer bonding. Subsequent use of ABI StepOnePlusTM Real-Time PCR system (Thermo Fisher Scientific, USA) and KAPA SYBR FAST (2x) (purchased from KAPA Biosystems, USA) were used to combine the reverse transcription products with the MSR1 gene combinations shown in Table 5 The primers were used for quantitative real-time reverse transcription polymerase chain reaction to observe the expression level of MSR1 gene in each group of cells. In addition, using the ABI StepOnePlusTM Real-Time PCR system and KAPA SYBR FAST (2x) (purchased from KAPA Biosystems, USA), the reverse transcription products of the control group, the control group and the experimental group 1 are shown in Table 3. The combined primers of the SCARB1 gene perform quantitative real-time reverse transcription polymerase chain reaction to observe the expression level of the SCARB1 gene in each group of cells. Here, the instrument setting conditions for quantitative real-time reverse transcription polymerase chain reaction are 95°C for 1 second, 60°C for 20 seconds, a total of 40 cycles, and the 2- ^ΔCt method is used for gene quantification. Here, quantitative real-time reverse transcription polymerase chain reaction using cDNA can indirectly quantify the mRNA expression level of each gene, and then infer the expression level of the protein encoded by each gene.

Table 5 Primer name Serial number sequence length MSR1-F SEQ ID NO: 2 ACATGGGAATGCAATAGATGAAATC 25 MSR1-R SEQ ID NO: 3 GCACTTGTTCTTCTTTCATAGCCATA 26 SCARB1-F SEQ ID NO: 4 ACTCCGACTCTGGGCTCTTCA twenty one SCARB1-R SEQ ID NO: 5 GGCCTCCGGGCTGTAGAA 18 ACTB-F SEQ ID NO: 6 CATGTACGTTGCTATCCAGGC twenty one ACTB-R SEQ ID NO: 7 CTCCTTAATGTCACGCACGAT twenty one

It is important to note that the expression levels of each gene shown in the diagrams mentioned below are presented in relative magnifications, in which the STDEV formula of Excel software is used to calculate the standard deviation, and the one-tailed Student t-test (Student t-test) is used to calculate the standard deviation. Analyze whether there are statistically significant differences. In the diagram, "*" represents the p-value is less than 0.05, "**" represents the p-value is less than 0.01, and "***" represents the p-value is less than 0.001. The more "*", the more significant the statistical difference.

Refer to Figure 7. Compared with the control group, the expression level of the MSR1 gene in the control group is regarded as 1 (that is, 100%), and the expression level of the MSR1 gene in the experimental group 1 is 0.66 (that is, 66%). Compared with the control group, the expression level of MSR1 gene in experimental group 2 was 0.91 (ie 91%). Compared with the control group, the expression level of the MSR1 gene in the experimental group 3 was 0.92 (that is, 92%). Compared with the control group, the expression level of the MSR1 gene in the control group was 1.47 (that is, 147%).

Comparing the control group and the control group shows that when the cells are induced by lipopolysaccharide, the expression level of the MSR1 gene will increase. Further comparing each experimental group with the control group or the control group, it can be seen that the expression level of MSR1 gene of the cells treated with compound TCI-028-001, compound TCI-028-002, and compound TCI-028-003 are all reduced, and It is even lower than the expression level when no lipopolysaccharide is induced. Among them, after treatment with TCI-028-001 compound, the expression of MSR1 gene of the cells decreased the most. It can be seen that the compound TCI-028-001, the compound TCI-028-002 and the compound TCI-028-003 can reduce the expression of the MSR1 gene related to atherosclerosis, and the compound TCI-028-001 has the best effect. .

Refer to Figure 8. Compared with the control group, the expression level of the SCARB1 gene in the control group is regarded as 1 (that is, 100%), and the expression level of the SCARB1 gene in the experimental group 1 is 1.72 (that is, 172%). Compared with the control group, the expression level of the SCARB1 gene in the control group was 0.78 (ie 78%).

Comparing the control group and the control group shows that when the cells are induced by lipopolysaccharide, the expression level of SCARB1 gene will decrease. Further comparing the experimental group 1 with the control group or the control group, it can be seen that after treatment with compound TCI-028-001, the expression level of the SCARB1 gene of the cells will increase, and even higher than the expression level during induction without lipopolysaccharide .

It can be seen that the metabolites of Lactobacillus plantarum TCI028 can inhibit the expression of MSR1 gene of immune cells and increase the expression of SCARB1 gene of immune cells, thereby avoiding atherosclerotic cardiovascular disease.

Although the technical content of the present invention has been disclosed in the preferred embodiments as above, it is not intended to limit the present invention. Anyone who is familiar with this technique and makes some changes and modifications without departing from the spirit of the present invention should be covered by the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope of the attached patent application.

no.

[Figure 1] is the hydrogen nuclear magnetic resonance spectrum of compound TCI-028-002. [Figure 2] is the hydrogen nuclear magnetic resonance spectrum of compound TCI-028-003. [Figure 3] shows the electrospray ionization mass spectrum of compound TCI-028-002. [Figure 4] shows the electrospray ionization mass spectrum of compound TCI-028-003. [Figure 5] is the hydrogen nuclear magnetic resonance spectrum of compound TCI-028-001. [Figure 6] is the electrospray ionization mass spectrum of compound TCI-028-001. [Figure 7] is a data graph of the expression level of MSR1 gene. [Figure 8] is a data diagram of the expression level of the SCARB1 gene.

Food Industry Development Research Institute (Taiwan); December 13, 2006; Deposit number: BCRC 910805.

Claims (18)

A metabolite of lactic acid bacteria, containing at least one biologically active substance selected from the group consisting of 2-hydroxy-4-methylpentanoic acid and 2-hydroxy-3- 2-hydroxy-3-methylbutanoic acid. The metabolite of lactic acid bacteria according to claim 1, wherein the lactic acid bacteria is Lactobacillus plantarum TCI028, and the deposit number of Lactobacillus plantarum TCI028 is BCRC 910805. The metabolites of lactic acid bacteria as described in claim 1, further including: 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (1,2,3,4-tetrahydro-β-carboline- 3-carboxylic acid). A metabolite of lactic acid bacteria is used to prepare a cardiovascular health care composition, wherein the lactic acid bacteria is Lactobacillus plantarum TCI028, and the deposit number of Lactobacillus plantarum TCI028 is BCRC 910805. The use according to claim 4, wherein the metabolite of the Lactobacillus plantarum TCI028 contains at least one biologically active substance selected from the group consisting of: 1,2,3,4-tetrahydro-β-carba Phthaloline-3-carboxylic acid (1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid), 2-hydroxy-4-methylpentanoic acid (2-hydroxy-4-methylpentanoic acid) and 2- 2-hydroxy-3-methylbutanoic acid. The use according to claim 4, wherein the metabolite of the Lactobacillus plantarum TCI028 is prepared by a method comprising the following steps: culturing the Lactobacillus plantarum TCI028 in a medium to obtain the Lactobacillus plantarum TCI028 Then, the culture of the Lactobacillus plantarum TCI028 is centrifuged and the culture supernatant is collected to obtain the metabolite of the lactic acid bacteria. The use according to claim 4, wherein the cardiovascular health care composition is used to inhibit MSR1 gene expression. The use according to claim 4, wherein the cardiovascular health care composition is used to increase SCARB1 gene expression. A use of 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid for preparing a cardiovascular health care composition. The use according to claim 9, wherein the cardiovascular health care composition is used to inhibit MSR1 gene expression. The use according to claim 9, wherein the cardiovascular health care composition is used to increase SCARB1 gene expression. The use according to claim 9, wherein the 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid is separated and purified from the methanol layer extract of the culture supernatant of Lactobacillus plantarum TCI028 And the deposit number of the Lactobacillus plantarum TCI028 is BCRC 910805. A use of 2-hydroxy-4-methylvaleric acid for preparing a cardiovascular health care composition. The use according to claim 13, wherein the cardiovascular health care composition is used to inhibit MSR1 gene expression. The use according to claim 13, wherein the 2-hydroxy-4-methylvaleric acid is obtained by separation and purification of the methanol layer extract of the culture supernatant of Lactobacillus plantarum TCI028, and the deposit of the Lactobacillus plantarum TCI028 The serial number is BCRC 910805. A use of 2-hydroxy-3-methylbutanoic acid for preparing a cardiovascular health care composition. The use according to claim 16, wherein the cardiovascular health care composition is used to inhibit MSR1 gene expression. The use according to claim 16, wherein the 2-hydroxy-3-methylbutyric acid is obtained by separating and purifying the methanol layer extract of the culture supernatant of Lactobacillus plantarum TCI028, and the deposit of Lactobacillus plantarum TCI028 The serial number is BCRC 910805.

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