TWI713876B - Use of radish seed fermentation product for manufacture of composition for inhibiting foam cell formation - Google Patents

Abstract

The present invention provides use of a radish seed fermentation product for manufacture of composition for inhibiting foam cell formation. The radish seed fermentation product is prepared by three-stage fermentation of a radish seed water extract with Saccharomyces cerevisiae, Lactobacillus plantarum, and Acetobacter aceti. Said fermentation product suppresses the gene expression of oxidized low-density lipoprotein receptors and reduces cellular fat accumulation, and thereby inhibits foam cell formation, leading to decreased risk of atherosclerosis and prevention of cardiovascular diseases in individuals.

Description

Use of fermented raspberry seeds for preparing a composition for inhibiting foam cell formation

The present invention relates to the health care use of a plant fermented product, in particular to the use of a raspberry seed fermented product for preparing a composition for inhibiting the formation of foam cells.

Heart and cerebrovascular diseases are the second of the top ten causes of death among Chinese in recent years. The main cause of the disease is atherosclerosis. In short, atherosclerosis is a condition in which the accumulation of cholesterol in the arteries causes the wall of the blood vessel to thicken, lose its elasticity, and the diameter of the vessel becomes smaller, so blood flow is not smooth. Previous studies have shown that during the development of arteriosclerosis, excessive low-density lipoprotein (LDL) in the blood carries cholesterol that accumulates on the inner wall of the artery and is oxidized into cytotoxic oxidized low-density lipoprotein (oxidized low-density lipoprotein). -density lipoprotein, ox-LDL), which causes inflammation of blood vessels, attracts monocytes into the vascular endothelium and differentiates into macrophages. Afterwards, macrophages will bind to oxidized low-density lipoproteins through protein receptors on the cell surface and swallow them, forming foam cells containing a large number of oil droplets. The accumulation of foam cells may cause the formation of fatty plaques. Together with the large amount of collagen fibers secreted by vascular smooth muscle cells, atherosclerotic plaques may eventually form, which may aggravate inflammation and increase the chance of thrombosis, and even cause angina, Acute myocardial infarction, and stroke.

The first strategy to avoid atherosclerosis is to reduce blood fat content through balanced diet or exercise, especially to reduce triglycerides and low-density lipoprotein cholesterol. Reducing the risk factors of atherosclerosis, such as high blood pressure, diabetes, smoking, etc., is also an important way to prevent atherosclerosis. Other strategies include reducing blood vessel inflammation and inhibiting foam cell formation. In view of the complex causes of atherosclerosis, in order to prevent its occurrence and deterioration from various aspects, it is really necessary to develop a novel composition with the goal of inhibiting the formation of foam cells to enhance the prevention and treatment effect of atherosclerosis.

For this reason, one object of the present invention is to provide a use of fermented scutellaria for preparing a composition for inhibiting the formation of foam cells, wherein the fermented scutellaria is prepared by the following steps: (a) extracting one with water Raphanus raphanistrum subsp. sativus) to obtain a raphanus raphanistrum subsp. sativus, (b) adding a Saccharomyces cerevisiae to the raphanus raphanistrum subsp. sativus and fermenting to obtain a first intermediate ferment, (c ) Adding a Lactobacillus plantarum to the first intermediate fermentation product and performing fermentation to obtain a second intermediate fermentation product, and (d) adding an Acetobacter aceti to the second intermediate fermentation product And fermented to obtain the fermented raspberry seed.

In an embodiment of the present invention, the fermentation time of the Saccharomyces cerevisiae is 1 to 2 days, the fermentation time of the Lactobacillus embryos is 1 to 3 days, and the fermentation time of the Acetobacter acetobacter is 14 to 21 days.

In an embodiment of the present invention, the addition amount of the saccharomyces cerevisiae is 0.01% to 0.5% by weight of the raspberry extract, and the addition amount of the Lactobacillus embryonicum is 0.01% to 0.25% by weight of the raspberry extract, And the addition amount of the Acetobacter acetobacter is 1% to 20% of the weight of the raspberry extract.

In an embodiment of the present invention, the weight ratio of the raspberry seed to water is 1:5 to 1:10, and the extraction is performed at 50°C to 100°C.

In one embodiment of the present invention, the concentration of the fermented raspberry seed is at least 2% w/v.

In one embodiment of the present invention, the fermented raspberry seed reduces the fat accumulation of a macrophage, because it inhibits the gene expression of the binding receptor of low-density lipoprotein monoxide in the macrophage, thereby reducing the Uptake of oxidized low-density lipoprotein by macrophages.

In an embodiment of the present invention, the binding receptor for the oxidized low-density lipoprotein is a leukocyte differentiation antigen 36 (cluster of differentiation 36, CD36), a macrophage scavenger receptor (MSR) ), or a combination thereof.

The present invention is based on cell experiments revealing that the fermented scutellaria seed after a specific fermentation process can inhibit the gene expression of oxidized low-density lipoprotein binding receptors in macrophages, reduce fat accumulation in macrophages, and ultimately inhibit foam cell formation. Therefore, the present invention provides the use of the fermented raspberry seeds for preparing a composition that inhibits the formation of foam cells. The composition can have a powder, granule, solution, gel, or paste dosage form, and can be made into food, drink, and medicine. , Reagents or nutritional supplements, administered to a body by oral means.

Given that previous studies have shown that foam cell formation is a consistent pathogenic factor in the development of atherosclerosis, administering a scutellaria fermented substance that can inhibit foam cell formation to an individual can prevent foam cells from accumulating on the inner wall of the individual’s blood vessels, thereby reducing the occurrence The risk of atherosclerosis and prevention of cardiovascular disease.

The following will further illustrate the implementation of the present invention in conjunction with the drawings. The following examples are used to illustrate the inventive features and applications of the present invention, rather than limiting the scope of the present invention. Anyone familiar with the art will not depart from Within the spirit and scope of the present invention, some changes and modifications can be made. Therefore, the protection scope of the present invention shall be subject to those defined by the appended patent scope.

Figure 1 shows that macrophages are treated with resveratrol, water extract of scutellaria, or fermented scutellaria, and then co-cultured with oxidized low-density lipoprotein (ox-LDL) and lipopolysaccharide (LPS). The relative content of Oil Red O.

Figure 2 shows that after macrophages are treated with water extract or fermented scutellaria seed and co-cultured with oxidized low-density lipoprotein (ox-LDL) and lipopolysaccharide (LPS), their blood cell differentiation antigen 36 (CD36) and The relative expression of the macrophage removal receptor (MSR) gene.

The present invention provides a use of fermented raspberry seeds for preparing a composition for inhibiting foam cell formation. The fermented scutellaria of the present invention is prepared by water extraction and three-stage fermentation. Specifically, the preparation of the fermented scutellaria scutellariae includes the following steps: (a) extracting a scutellaria scutellaria with water to obtain a scutellaria scutellaria extract , (B) adding a Saccharomyces cerevisiae to the raspberry seed extract and fermenting to obtain a first intermediate fermented product, (c) adding a Lactobacillus embryo to the first intermediate fermented product and fermenting to obtain a second intermediate fermented product Fermented product, and (d) adding an Acetobacter acetobacter to the second intermediate fermented product and fermenting to obtain the scutellaria fermented product. The fermented scutellaria seed has been experimentally confirmed to inhibit the gene expression of oxidized low-density lipoprotein binding receptors in macrophages, such as the gene expression of CD36 and MSR, and reduce the accumulation of fat in macrophages, so it can inhibit foam Cell formation.

definition

The numerical values used herein are approximate values, and all experimental data are expressed in the range of 20%, preferably in the range of 10%, and most preferably in the range of 5%.

Materials and Methods microorganism

Purchased Saccharomyces cerevisiae (BCRC 20271), Lactobacillus embryo (BCRC 910805), and Acetobacter acetobacter (BCRC 11688) from the Bioresource Collection and Research Center (BCRC) of the Food Industry Development Institute.

Cell culture

The following examples use the human monocytic cell line THP-1 (ATCC TIB-202) purchased from the American Type Culture Collection (ATCC). The THP-1 cells were cultured at 37° C. and 5% carbon dioxide with the addition of 10% fetal bovine serum (FBS), 0.05 mM 2-mercaptoethanol (2-mercaptoethanol), 100 units/mL penicillin, and 100μg/mL streptomycin RPMI 1640 medium (Gibco RPMI medium 1640; Thermo Fisher Scientific), hereinafter referred to as RPMI cell culture medium.

Preparation of oil red O dye solution

The dye Oil red O (purchased from Sigma) was thoroughly dissolved in 100% isopropanol (isopropanol; purchased from Echo chemical) to prepare a 30 mg/mL oil red O stock solution. In order to obtain a usable Oil Red O dye solution, the stock solution was diluted with secondary water to a concentration of 18mg/mL immediately before use and filtered with a 0.22m filter membrane.

Oil red O stain

The fat content in the cells was determined by oil red O staining. Before staining, the cells were washed with phosphate buffered saline solution (abbreviated as PBS solution; purchased from Thermo Fisher Scientific), and then fixed with 10% formaldehyde (formaldehyde; purchased from Echo chemical) for 10 minutes. After the fixed cells were washed once with PBS solution, they were rinsed with 60% isopropanol for 15 seconds. Thereafter, stain the cells with Oil Red O staining solution for 1 minute, and then de-stain with 60% isopropanol for 15 seconds. After staining, the cells were washed with PBS solution and then observed under a microscope (ZEISS Axio Vert.A1), or the intracellular stain was dissolved with 100% isopropanol for quantification. The quantitative results are statistically analyzed by the student t test of Excel software.

Gene expression analysis

Quantitative polymerase chain reaction (qPCR) is used to determine the gene expression of protein receptors for uptake of oxidized low-density lipoprotein in macrophages. The steps are briefly described as follows. According to the manufacturer’s instructions, use RNA Extraction Kit (Geneaid) to isolate ribonucleic acid (RNA) from cells, and reverse transcribe 2000ng RNA into cDNA at 37°C with SuperScript® III Reverse Transcriptase (Invitrogen) . Afterwards, use the qPCR kit (KAPA CYBR FAST qPCR Kit (2X); KAPA Biosystems), CD36, MSR and other target genes and the β-actin (β-actin) gene ACTB primer pair as an internal control (Table 1 ) Perform qPCR on the aforementioned cDNA in a PCR reaction machine (Step One Plus Real-Time PCR system; Applied Biosystems) to obtain a melting curve.

Finally, the 2- ^△△CT method was used to determine the relative expression of the target gene. The method is the threshold cycle (C _T) GAPDH gene as an internal control of the reference cycle threshold gene, calculated by the following formula relative fold change: C gene of △ C _T = the experimental group or the control group _T - internal control C _T

△△ C _T = test group △ C _T - △ C _T of the control group

Multiple change=2 ^-△△Ct average

The statistical analysis department uses the STDEV function in Excel software to calculate the standard deviation of the relative expression of each gene, and calculates the statistical difference with the one-tailed student t test (TTEST).

Example 1 Preparation of fermented raspberry seeds

First, wash the mature dried seeds of the radish (i.e. scutellaria), and then mix the water with the scutellaria to extract and obtain a scutellaria extract. The weight ratio of the raspberry seed to water is 1:5 to 1:10. The extraction temperature is between 50°C and 100°C, preferably between 80°C and 95°C. The extraction time in this embodiment is 0.5 to 3 hours. Before the start of the extraction, 5% to 15% (w/v) glucose is added to the mixture of scutellaria and water.

The raspberry extract obtained through the above steps is cooled to room temperature and used for three-stage fermentation. The optimal fermentation conditions for each stage are as follows:

Stage 1: adding Saccharomyces cerevisiae (BCRC 20271) to the raspberry seed extract and fermenting at 25°C to 35°C for 1 to 2 days to obtain a first fermented product. The addition amount of the saccharomyces cerevisiae is 0.01% to 0.5% by weight of the raspberry seed extract.

Stage 2: Add Lactobacillus embryonicum (BCRC 910805) to the first fermented product and ferment at 25°C to 35°C for 1 to 3 days to obtain a second fermented product. The addition amount of the Lactobacillus embryonicum is 0.01% to 0.25% of the weight of the raspberry extract.

Stage 3: Adding Acetobacter aceti (BCRC 11688) to the second fermented product and fermenting at 25°C to 35°C for 14 to 21 days to obtain a raspberry fermented product. The addition amount of the Acetobacter acetobacter is 1% to 20% of the weight of the raspberry extract.

The fermented raspberry seed can be filtered through a 200-400 mesh (mesh) filter to remove residual solids. The filtered fermented raspberry seed can be further concentrated under reduced pressure at 45°C to 70°C to obtain a concentrated product. The concentrated fermented scutellaria can be optionally added with 1-3% (w/w) citric acid and 40-70% (w/w) isomalto-oligosaccharides. Finally, the processed scutellaria seed fermented material is sterilized at 95°C to 120°C for 70 to 90 minutes before subsequent use.

Example 2 The fermented raspberry seed reduces the fat accumulation of macrophages and the formation of foam cells

In order to explore the effect of the fermented scutellaria of the present invention on the formation of foam cells by macrophages, this example uses oil red O staining to analyze the macrophages differentiated from the human monocyte line THP-1 after being treated with the fermented scutellaria. , Its fat content changes. First, THP-1 cells were seeded at 2×10 ⁵ cells/well in each well of a 6-well plate containing 2 mL of RPMI cell culture medium, and then 500 nM phorbol-12-myristate-13-ethyl ester (phorbol 12-myristate 13-acetate, PMA; Sigma-Aldrich) treated the cells for 48 hours to induce differentiation into macrophages. Thereafter, the cell culture medium was updated and the macrophages were cultured at 37°C for 48 hours, and then the cells in each well were treated in the following manner: (a) 2mL RPMI medium was applied for 1 hour (control group); (b) 2mL RPMI medium was applied For 1 hour, apply 50μg/mL human oxidized low-density lipoprotein (purchased from Athens Research & Technology) and 100ng/mL lipopolysaccharide (LPS; Sigma) (ox-LDL/LPS group); (c) administration Take 2mL of RPMI medium containing 2.5μM resveratrol for 1 hour, then apply 50μg/mL oxidized low-density lipoprotein and 100ng/mL lipopolysaccharide (ox-LDL/LPS+resveratrol group); (d) apply Take 2 mL of RPMI cell culture medium containing 2% (w/v) Scutellaria serrata water extract for 1 hour, then apply 50μg/mL oxidized low-density lipoprotein and 100ng/mL lipopolysaccharide (ox-LDL/LPS+ Scutellaria scutellaria water extraction Group); and (e) 2mL of RPMI cell culture medium containing 2% (w/v) scutellaria seed fermented product was applied for 1 hour, and then 50μg/mL oxidized low-density lipoprotein and 100ng/mL lipopolysaccharide (ox -LDL/LPS+ Scutellaria seed fermentation group). After the aforementioned groups of cells were cultured at 37°C for 24 hours, the medium was removed, the cells were washed with PBS solution, and then oil red O staining and quantitative analysis were performed.

Figure 1 shows the relative content of Oil Red O in the cells of the aforementioned groups, which is the percentage relative to the Oil Red O content of cells in the control group; ^## indicates p<0.01 compared to the control group, ** and *** indicate respectively Compared with ox-LDL group, p<0.01 and p<0.001, ^▲▲ means p<0.01 compared with ox-LDL+Scutellaria serrata water extract group. According to Figure 1, the administration of oxidized low-density lipoprotein and lipopolysaccharides that stimulate inflammation can significantly increase the fat content in macrophages, but the extra treatment of the fermented scutellariae significantly inhibits the increase in the fat content and makes the oil in the cells The relative content of red O dropped to about 80%. In addition, the water extract of Scutellaria serrata could not significantly reduce the increased fat stimulated by ox-LDL/LPS. This result shows that the fermented scutellaria of the present invention can effectively reduce the accumulation of fat in macrophages, thereby inhibiting the formation of foam cells.

Example 3 The gene expression of scutellaria fermented product inhibiting oxidized low-density lipoprotein binding receptor

In order to explore the effect of the scutellaria scutellariae fermented product on the expression of macrophages' fat uptake-related genes, qPCR was used to determine the oxidized low-density lipoproteins of the macrophages derived from THP-1 cell differentiation after being treated with the scutellaria scutellaria fermentation product. Combined with changes in gene expression of receptor white. First, THP-1 cells were seeded into each well of a 6-well plate containing 2 mL of RPMI cell culture medium at 2×10 ⁵ cells/well, and then treated with 500 nM PMA for 48 hours to induce differentiation into macrophages. Thereafter, the cell culture medium was updated and the macrophages were cultured at 37°C for 48 hours, and then the cells in each well were treated in the following manner: (a) 2mL RPMI medium was applied for 1 hour (control group); (b) 2mL RPMI medium was applied For 1 hour, apply 50μg/mL oxidized low-density lipoprotein and 100ng/mL lipopolysaccharide (ox-LDL/LPS group); (c) apply 2mL of water extract containing 2% (w/v) scutellaria RPMI cell culture medium was applied for 1 hour, and then 50μg/mL oxidized low-density lipoprotein and 100ng/mL lipopolysaccharide (ox-LDL/LPS+Scutellaria serrata water extract group) were applied; and (d) 2mL containing 2% (w /v) The RPMI cell culture medium of the scutellaria fermented product is applied for 1 hour, and then 50μg/mL oxidized low-density lipoprotein and 100ng/mL lipopolysaccharide (ox-LDL/LPS+ scutellaria fermented group) are applied. The aforementioned groups of cells were cultured at 37°C for 24 hours and then used for qPCR analysis.

Figure 2 shows the relative expression levels of blood cell differentiation antigen 36 (CD36) and macrophage clearance receptor (MSR) genes of the aforementioned cells in each group, which is the ratio of the RNA content of the same gene in the control group of cells; * and * *Respectively indicate p<0.05 and p<0.01 compared to the ox-LDL/LPS group. According to Figure 2, the administration of oxidized low-density lipoproteins and lipopolysaccharides that stimulate inflammation can slightly increase the gene expression of MSR in macrophages, but the additional treatment of the scutellariae fermented product significantly inhibited the gene expression of MSR. In addition, the fermented Scutellaria spp. will make the expression of CD36 gene reduced under ox-LDL/LPS stimulation more significantly decreased. In contrast, compared to the ox-LDL/LPS group, the additional treatment of the water extract of Scutellaria serrata only significantly reduced the gene expression of MSR, but did not significantly affect the gene expression of CD36. This result indicates that the fermented scutellaria can more comprehensively inhibit the gene expression of the oxidized low-density lipoprotein binding receptor in macrophages, thus effectively reducing the uptake of oxidized low-density lipoprotein by macrophages and the accumulation of intracellular fat .

In summary, the above experiments show that the fermented raspberry seed after a specific fermentation process can inhibit the gene expression of the oxidized low-density lipoprotein binding receptor in macrophages, reduce the accumulation of fat in the macrophages, and ultimately inhibit the formation of foam cells . Therefore, the present invention provides the use of the fermented raspberry seeds for preparing a composition that inhibits the formation of foam cells. The composition can have a powder, granule, solution, gel, or paste dosage form, and can be made into food, drink, and medicine. , Reagents or nutritional supplements, administered to a body by oral means.

<110> Dajiang Biomedical Co., Ltd.

<120> Use of fermented scutellaria for preparing a composition for inhibiting foam cell formation

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Claims (10)

The use of a scutellaria fermented product for the preparation of a composition that directly inhibits the uptake of fat by macrophages, wherein the scutellaria fermented product is prepared by the following steps: (a) extracting a scutellaria scutellarum with water to obtain a scutellaria extract (B) adding a Saccharomyces cerevisiae to the raspberry seed extract and fermenting to obtain a first intermediate ferment; (c) adding a Lactobacillus embryo to the first intermediate ferment and fermenting to obtain a second Intermediate fermented product; and (d) adding an Acetobacter acetobacter to the second intermediate fermented product and fermenting to obtain the scutellaria seed fermented product. The use described in item 1 of the scope of patent application, wherein the fermentation time of the Saccharomyces cerevisiae is 1 to 2 days, the fermentation time of the Lactobacillus embryonicum is 1 to 3 days, and the fermentation time of the Acetobacter acetobacter is 14 to 21 days. The use described in item 1 of the scope of the patent application, wherein the addition amount of the Saccharomyces cerevisiae is 0.01 to 0.5% by weight of the raspberry extract, and the addition amount of the Lactobacillus spp. is 0.01 to 0.25 by the weight of the raspberry extract %, and the addition amount of the Acetobacter acetobacter is 1 to 20% of the weight of the raspberry extract. The use as described in item 1 of the scope of patent application, wherein the weight ratio of raspberry seed to water is 1:5 to 1:10. The use as described in item 1 of the scope of patent application, wherein the extraction is carried out at 50°C to 100°C. The use as described in item 1 of the scope of patent application, wherein the concentration of the fermented raisin seed is at least 2% w/v. The use as described in item 1 of the scope of patent application, wherein the fermented raspberry seed reduces the uptake of oxidized low-density lipoprotein by the macrophages. The use described in item 7 of the scope of the patent application, wherein the fermented raspberry seed inhibits the gene expression of the low-density lipoprotein monoxide binding receptor in the macrophage. The use described in item 8 of the scope of application, wherein the oxidized low-density lipoprotein binding receptor is a leukocyte differentiation antigen 36 (cluster of differentiation 36), a macrophage scavenger receptor, Or a combination. The use described in item 1 of the scope of the patent application, wherein the composition has a dosage form of powder, granule, solution, colloid, or paste.

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