Composition helpful for maintaining healthy level of blood fat and application thereof

Abstract

The invention provides a composition beneficial to maintaining the healthy level of blood fat and application of the composition. Specifically, the composition provided by the invention comprises phytosterol ester, fish oil and beta-glucan, and the weight ratio of the phytosterol ester to the fish oil to the beta-glucan is 1: (1-5): (10-100). The composition disclosed by the invention can be beneficial to maintaining the healthy level of blood fat, reducing / preventing low-density cholesterol accumulation and reducing the cholesterol level.

Description

Technical Field

[0001] The present invention relates to a composition that helps maintain healthy blood lipid levels and its application. Specifically, it relates to a composition comprising plant sterol esters, fish oil and β-glucan and its application in preparing a product that helps maintain healthy blood lipid levels. Background Art

[0002] Dyslipidemia is a major risk factor for cardiovascular disease. Hypercholesterolemia is a metabolic state characterized by elevated cholesterol concentrations in the blood. Elevated cholesterol concentrations in the blood can be caused by various factors, including an unbalanced diet, obesity, inherited genetic diseases, or other underlying health problems. Hypercholesterolemia has become a key target for primary and secondary prevention strategies for cardiovascular disease. Over the past few decades, various cholesterol-lowering drugs, including statins, have been developed to address the problem of hypercholesterolemia. However, clinical studies have shown that many drugs are associated with a range of adverse side effects.

[0003] Therefore, developing safe and effective management strategies to maintain healthy lipid levels, especially cholesterol levels, is crucial to minimize the occurrence of cardiovascular events and improve overall cardiovascular health outcomes. Summary of the Invention

[0004] An object of the present invention is to provide a product that helps maintain healthy blood lipid levels, particularly healthy cholesterol levels.

[0005] Another object of the present invention is to provide a use of a composition comprising plant sterol esters, fish oil and β-glucan in the preparation of a product that helps maintain healthy blood lipid levels, especially helps maintain healthy cholesterol levels, in an individual.

[0006] The inventors of this case discovered in their research that a composition of plant sterol esters, fish oil, and β-glucan in a certain ratio can help maintain healthy blood lipid levels, especially healthy cholesterol levels, by reducing / preventing the accumulation of low-density lipoprotein cholesterol and lowering cholesterol levels.

[0007] Specifically, on the one hand, the present invention provides a composition comprising plant sterol esters, fish oil and β-glucan, wherein the weight ratio of plant sterol esters, fish oil and β-glucan is 1:1~5:10~100.

[0008] In the present invention, the plant sterol esters, fish oil and β-glucan are all new food raw materials (new resource food), and each raw material should meet relevant quality requirements.

[0009] According to some specific embodiments of the present invention, in the composition of the present invention, the β-glucan is oat β-glucan.

[0010] According to some specific embodiments of the present invention, in the composition of the present invention, the total mass percentage of DHA and EPA in the fish oil is ≥10.0%, and the mass percentage of ω-3 polyunsaturated fatty acids is ≥12.0%.

[0011] According to some specific embodiments of the present invention, in the composition of the present invention, the total phytosterol content in the phytosterol ester is greater than 60%.

[0012] According to some specific embodiments of the present invention, in the composition of the present invention, the weight ratio of the phytosterol ester, fish oil and β-glucan is 1:1-3:20-80.

[0013] According to some specific embodiments of the present invention, in the composition of the present invention, the weight ratio of the phytosterol ester, fish oil and β-glucan is 1:1-3:20-50.

[0014] According to some specific embodiments of the present invention, in the composition of the present invention, the weight ratio of the phytosterol ester, fish oil and β-glucan is 1:1-3:20-40.

[0015] According to some specific embodiments of the present invention, in the composition of the present invention, the weight ratio of the phytosterol ester, fish oil and β-glucan is 1:1.5-2.5:20-50.

[0016] According to some specific embodiments of the present invention, in the composition of the present invention, the weight ratio of the phytosterol ester, fish oil and β-glucan is 1:1.5-2.5:20-40.

[0017] According to some specific embodiments of the present invention, in the composition of the present invention, the weight ratio of the phytosterol ester, fish oil and β-glucan is 1:1.5-2.5:25-35.

[0018] According to some specific embodiments of the present invention, in the composition of the present invention, the weight ratio of the phytosterol ester, fish oil and β-glucan is 1:1.5-2.5:30-35.

[0019] On the other hand, the present invention also provides a food comprising the composition of the present invention, that is, comprising the phytosterol esters, fish oil and β-glucan.

[0020] According to some specific embodiments of the present invention, in the food of the present invention, the content of phytosterol esters, fish oil and β-glucan in the food is, based on the content of dry matter:

[0021] Fish oil ≤ 6g / 100g food dry matter;

[0022] Phytosterol esters ≤ 7.8g / 100g food dry matter; and / or

[0023] β-glucan ≤10g / 100g food dry matter.

[0024] Specifically, the content of plant sterol esters, fish oil and β-glucan in food can be adjusted according to the specific category of food within the range of relevant quality requirements.

[0025] According to some specific embodiments of the present invention, the food of the present invention is a dairy product, for example, it can be a fermented dairy product (such as fermented milk, flavored fermented milk, fermented milk beverage, etc.), cheese, milk-containing beverage, solid beverage or milk powder, etc.

[0026] On the other hand, the present invention also provides the use of the composition or the food in preparing a product that helps maintain healthy blood lipid levels.

[0027] According to some specific embodiments of the present invention, in the application of the present invention, the method of helping to maintain healthy blood lipid levels includes one or more of the following applications:

[0028] Helps maintain healthy triglyceride levels in individuals;

[0029] Helps maintain healthy cholesterol levels in individuals;

[0030] Assists in lowering an individual's cholesterol levels;

[0031] Down-regulating the relative expression of one or more of the triglyceride and cholesterol metabolism-related genes APOA1A, APOA2, APOBA, APOA4B.3, CD36, HMGCRA, and FADS2 in an individual; and / or

[0032] Prevent individuals from developing high cholesterol conditions.

[0033] According to some specific embodiments of the present invention, in the application of the present invention, the individual is an individual on a high cholesterol diet.

[0034] According to some specific embodiments of the present invention, in the application of the present invention, the prevention of a high cholesterol condition in an individual includes reducing the probability and / or risk of a high cholesterol condition in an individual on a high cholesterol diet, or reducing or inhibiting further development of the condition in an individual who is not ill but is close to developing a high cholesterol condition or close to becoming a patient with hypercholesterolemia.

[0035] According to some specific embodiments of the present invention, in the application of the present invention, the product is food or health food.

[0036] According to some specific embodiments of the present invention, in the application of the present invention, the phytosterol esters, fish oil and β-glucan are used to prepare the product in the following amounts, calculated on a dry matter basis:

[0037] Fish oil ≤3g / day;

[0038] Phytosterol esters ≤ 3.9 g / day; and / or

[0039] β-glucan ≤5g / day.

[0040] According to some specific embodiments of the present invention, the specific formula and production method of the product, such as food, can be made with reference to similar products in the prior art, and the amounts of phytosterol esters, fish oil and β-glucan therein can be adjusted within the scope of the present invention.

[0041] In summary, the present invention provides a composition comprising probiotics and dietary fiber and its application. The composition helps maintain healthy blood lipid levels, especially helps maintain healthy cholesterol levels, which can be specifically manifested in reducing / preventing the accumulation of low-density lipoprotein cholesterol and lowering triglyceride levels. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 A typical graph of cholesterol fluorescence intensity in zebrafish tail vessels after treatment with the composition. The yellow dotted box in the figure indicates the analyzed area. DETAILED DESCRIPTION

[0043] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.

[0044] When numerical ranges are given in the examples, it should be understood that unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected.

[0045] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0046] Unless otherwise defined, the term "prevention" means effectively reducing the probability of an increase in blood cholesterol concentration and / or the occurrence of hypercholesterolemia caused by a high-cholesterol diet, that is, reducing the risk of an individual on a high-cholesterol diet developing or suffering from hypercholesterolemia, or reducing or inhibiting the further development of the condition in an individual who is not ill but is close to becoming ill with hypercholesterolemia. The purpose of "prevention" use is primarily to prevent rather than treat or cure the disease.

[0047] In addition to the specific methods, equipment, and materials used in the embodiments, based on the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials in the prior art that are similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0048] Unless otherwise noted, the experimental methods, detection methods, and preparation methods disclosed herein all employ conventional techniques in the art. In animal experiments, all reagents, materials, and test substances used were commercially available. The plant sterol esters, fish oil, and β-glucan used were all commercially available products that met the quality requirements for new food ingredients (new resource foods).

[0049] Example 1: Experimental Study on the Efficacy of Phytosterol Esters, Fish Oil, and Oat β-Glucan in Maintaining Healthy Blood Lipid Levels

[0050] 1. Experimental Materials

[0051] 1.1 Sample preparation information

[0052] Fish oil powder, Shanghai Laique Biotechnology Co., Ltd. Prepare a 50.0 mg / mL stock solution with 75% ethanol (only used to fully dissolve the fish oil powder and evenly disperse it in the zebrafish feeding system) and use it immediately.

[0053] Phytosterol esters, Shanghai Leabird Biotechnology Co., Ltd. Prepare a 50.0 mg / mL stock solution with 75% ethanol (only for fully dissolving and evenly dispersing the phytosterol esters in the zebrafish feeding system) and use immediately.

[0054] Oat β-glucan, Guangzhou Zhongkang Food Co., Ltd. Prepare a 2000 μg / mL stock solution with standard dilution water for immediate use.

[0055] Positive control: Pioglitazone hydrochloride tablets (hereinafter referred to as pioglitazone), white tablets, batch number 20220503, Jiangsu Deyuan Pharmaceutical Co., Ltd., store in a cool, dark place. Prepare a 10.0 mg / mL stock solution with ultrapure water for immediate use.

[0056] 1.2 Experimental animals

[0057] Zebrafish were maintained at 28°C in aquaculture water (water quality: 200 mg of instant sea salt per liter of reverse osmosis water, conductivity 450–550 μS / cm, pH 6.5–8.5, and hardness 50–100 mg / L CaCO₃). Zebrafish were bred and provided by the Hangzhou Huante Biotechnology Co., Ltd. Fish Breeding Center, Laboratory Animal Use License No. SYXK(Zhejiang)2022-0004. Animal husbandry and management were in compliance with AAALAC accreditation (certification number: 001458).

[0058] Wild-type AB zebrafish, bred by natural pair mating, were used to evaluate the cholesterol-lowering efficacy of the drug at 5 days post-fertilization (dpf).

[0059] 1.3 Instruments, consumables and reagents

[0060] A dissecting microscope (SZX7, OLYMPUS, Japan); a microinjector (IM300, Narishige, Japan); a needle puller (PC-10, Narishige, Japan); a CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); a motorized focus continuous zoom fluorescence microscope (AZ100, Nikon, Japan); a precision electronic balance (CP214, OHAUS, USA); a 6-well plate (Nest Biotech, China); an automatic sample rapid grinding device (JXFSTPRP-24L, Shanghai Jingxin Laboratory Equipment Technology Department, China); and a high-speed refrigerated centrifuge (Heraeus Fresco17, Thermo Fisher Scientific, China). Fisher, Germany); homogenizer (T10, IKA, Germany); ultrasonic cleaner (JP-010T, Shenzhen Jiemeng Cleaning Equipment Co., Ltd., China); fully automatic nucleic acid extractor (Auto-Pure32A, Hangzhou Aosheng Instrument Co., Ltd., China); conventional PCR amplifier (T100, BIO-RAD, Singapore); fluorescence quantitative PCR instrument (CFX Connect, BIO-RAD, Singapore); UV-visible spectrophotometer (Nanodrop2000, Thermo, Austria); microplate mini centrifuge (BE-6100, Haimen Qilin Bell Instrument Manufacturing Co., Ltd., China); optical adhesive sealing film B (MSB1001, Bio-rad, USA); low-skirt 96-well plate (transparent) (HSP9601, Bio-rad, USA).

[0061] cholesterlylBODIPY TM542 / 563C11 (cholesterol probe, batch number 2291600, Invitrogen, USA); pure egg yolk powder (batch number 20200809, Zhejiang Aiger Biotechnology Co., Ltd., China); D-(+)-glucose (batch number F2209126, Shanghai Aladdin Biochemical Technology Co., Ltd., China); anhydrous ethanol (batch number 20210107, Sinopharm Chemical Reagent Co., Ltd., China); dimethyl sulfoxide (DMSO, batch number BCCD8942, Sigma, Switzerland); methylcellulose (batch number C2004046, Shanghai Aladdin Biochemical Technology Co., Ltd., China); ChamQ Universal SYBR qPCR Master Mix (batch number 027E2201CD, Vazyme, China); FastKing cDNA First-Strand Synthesis Kit (Genomic Depleted) (Lot No. X0320, Tiangen Biochemical Technology (Beijing) Co., Ltd., China); Pre-packed Magnetic Beads Universal RNA Extraction Kit C (Lot No. TL2210001643C, Foshan Aowei Biotechnology Co., Ltd. (ONREW), China).

[0062] 2. Experimental Methods

[0063] 2.1 Evaluation of the efficacy of auxiliary cholesterol-lowering

[0064] Wild-type AB zebrafish (5 dpf) were randomly selected and placed in beakers, with 30 per beaker. Fish oil powder, phytosterol esters, oat β-glucan, and their combination were administered in water, respectively. A normal control group and a model control group were also established. Each beaker held 25 mL of water. All other experimental groups, except the normal control group, were administered a 0.15% egg yolk powder solution in water during the day and a 3% glucose solution in the evening to establish a hyperlipidemia model in zebrafish. After 32 hours of treatment at 28°C, a cholesterol probe was injected. Treatment continued for 16 hours. Ten zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope. Data were collected and analyzed using NIS-Elements D 3.20 advanced image processing software. Cholesterol fluorescence intensity in zebrafish tail vessels was analyzed and statistically analyzed to evaluate the cholesterol-lowering efficacy of fish oil powder, phytosterol esters, oat β-glucan, and their combination. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software. P < 0.05 indicated statistical significance.

[0065] 2.2 Study on the mechanism of auxiliary lipid-lowering effect

[0066] 5dpf wild-type AB zebrafish were randomly selected and placed in beakers, with 30 per beaker. A combination of fish oil powder, plant sterol esters, and oat β-glucan was administered in water, along with a positive control of pioglitazone at a concentration of 20.0μg / mL. A normal control group and a model control group were also established, each containing 25mL. Three parallel experiments were performed for each experimental group. Except for the normal control group, the remaining experimental groups were administered a 0.15% egg yolk powder solution in water during the day and a 3% glucose solution at night to establish a hyperlipidemia model in zebrafish. After treatment at 28°C for 48 hours, total RNA from each group was extracted using an automated nucleic acid extractor, and total RNA concentration and purity were determined using a UV-visible spectrophotometer. 2.00 μg of total RNA from zebrafish samples was synthesized into 20.0 μL of cDNA according to the instructions of the first-strand cDNA synthesis kit. The expression of β-actin, apoa1a, apoa2, apoba, apoa4b.3, cd36, hmgcra, and fads2 genes was detected by q-PCR. β-actin was used as an internal control for gene expression, and the relative RNA expression levels of apoa1a, apoa2, apoba, apoa4b.3, cd36, hmgcra, and fads2 genes were calculated. Statistical analysis was performed using SPSS 26.0 software. P < 0.05 indicated statistical significance.

[0067] 3. Experimental Results

[0068] 3.1 Evaluation of the efficacy of auxiliary cholesterol-lowering

[0069] A typical graph of cholesterol fluorescence intensity in zebrafish tail blood vessels after treatment with the composition is shown in Figure 1 The test data are shown in Tables 1 and 2.

[0070] Under these experimental conditions, compared with the model control group, the use of 31.2 μg / mL phytosterol esters, 62.5 μg / mL fish oil powder, and 1000 μg / mL oat β-glucan alone had no auxiliary cholesterol-lowering effect. However, the combination of these three samples (phytosterol esters + fish oil powder + oat β-glucan) showed auxiliary cholesterol-lowering effects at concentrations of 31.2, 62.5, and 1000 μg / mL. The experimental results showed that 31.2 μg / mL phytosterol esters, 62.5 μg / mL fish oil powder, and 1000 μg / mL oat β-glucan had a synergistic effect.

[0071] Table 1. Results of single sample auxiliary cholesterol-lowering efficacy evaluation experiment (n=10)

[0072]

[0073] Compared with the model control group, ***p<0.001.

[0074] Table 2. Results of experimental evaluation of the cholesterol-lowering efficacy of the composition (n=10)

[0075]

[0076] Compared with the model control group, ***p<0.001.

[0077] 3.2 Study on the mechanism of auxiliary lipid-lowering effect

[0078] 3.2.1 RNA extraction results and primer sequence information

[0079] At the end of the experiment, total RNA was extracted from zebrafish. RNA concentration and A260 / A280 ratio were measured using a UV-visible spectrophotometer. The A260 / A280 ratios were both between 1.8 and 2.2, indicating that the extracted zebrafish total RNA was of good quality and suitable for subsequent q-PCR experiments. Primer sequences are shown in Table 3.

[0080] Table 3. Primer sequence information

[0081]

[0082] 3.2.2 Mechanism research results

[0083] Apolipoproteins (Apo) are the protein components of plasma lipoproteins that bind and transport lipids to various tissues for metabolism and utilization. Different apolipoprotein phenotypes have varying degrees of lipid metabolism and utilization. The apoa1a gene, a subtype of apoa1, encodes apolipoprotein A-I, responsible for transporting cholesterol from the arterial wall to the liver for catabolism. The apoa2 gene encodes apolipoprotein A-II, which is involved in visceral fat accumulation and the metabolism of triglyceride-rich lipoproteins. These two genes are primarily present in high-density lipoprotein particles and chylomicrons, but not in low-density lipoproteins or very-low-density lipoproteins. The apoba gene encodes apolipoprotein B, a component of chylomicrons, low-density lipoproteins, and very-low-density lipoproteins, participating in the assembly of very-low-density lipoproteins with cholesterol and triglycerides. The apoa4b.3 gene, a subtype of apoa4, encodes apolipoprotein A-IV, primarily present in chylomicrons, low-density lipoproteins, and very-low-density lipoproteins, playing a crucial role in lipid transport and metabolism, particularly reverse cholesterol transport. CD36 expression is involved in fatty acid transport. Fatty acids are one of the body's main energy sources and the primary raw material for triglyceride synthesis. Both in vivo and in vitro, upregulated CD36 expression promotes adipogenesis and participates in the feedback regulation of lipolysis. Its loss can also mitigate the inflammatory response caused by obesity. Hydroxymethylglutaryl-CoA reductase A (HMGCRA) is the rate-limiting enzyme in the cholesterol synthesis pathway. The fads2 gene encodes D6D (delta-6 desaturase), which catalyzes the conversion of the essential fatty acids linoleic acid and α-linolenic acid into long-chain polyunsaturated fatty acids (LC-PUFA). LC-PUFA inhibits the activity of lipases and promotes the oxidative decomposition of fatty acids, thereby reducing serum triglyceride levels.

[0084] Under the experimental conditions, the combination of phytosterol esters, fish oil powder, and oat β-glucan (31.2, 62.5, and 1000 μg / mL) downregulated the relative expression of apoa1a, apoa2, apoba, apoa4b.3, cd36, hmgcra, and fads2 genes. See Table 4 for details.

[0085] Table 4. Experimental results of the mechanism of the sample's auxiliary blood lipid lowering efficacy (n=3)

[0086]

[0087] Compared with the model control group, *p<0.05, **p<0.01, ***p<0.001.

[0088] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

Claims

1. A composition comprising phytosterol esters, fish oil and β-glucan, wherein: The weight ratio of phytosterol ester, fish oil and beta-glucan is 1:1-5:10-100.

2. The composition according to claim 1, wherein The β-glucan is oat β-glucan.

3. The composition according to claim 1, wherein The total mass percentage of DHA and EPA in the fish oil is ≥10.0%, and the mass percentage of ω-3 polyunsaturated fatty acids is ≥12.0%.

4. The composition according to any one of claims 1 to 3, wherein The weight ratio of the phytosterol ester, fish oil and β-glucan is 1:1-3:20-80, preferably 1:1-3:20-50, and more preferably 1:1-3:20-40. A food comprising the composition according to any one of claims 1 to 4.

6. The food according to claim 5, wherein Calculated on dry matter, the content of phytosterol esters, fish oil and β-glucan in food is: Fish oil ≤ 6g / 100g food dry matter; Phytosterol esters ≤ 7.8g / 100g food dry matter; and / or β-glucan ≤10g / 100g food dry matter.

7. Use of the composition according to any one of claims 1 to 4 or the food according to claims 5 to 6 in preparing a product that helps maintain healthy blood lipid levels.

8. The use according to claim 7, wherein: The method of helping to maintain healthy blood lipid levels includes one or more of the following applications: Helps maintain healthy triglyceride levels in individuals; Helps maintain healthy cholesterol levels in individuals; Assists in lowering an individual's cholesterol levels; Down-regulating the relative expression of one or more of the triglyceride and cholesterol metabolism-related genes APOA1A, APOA2, APOBA, APOA4B.3, CD36, HMGCRA, and FADS2 in an individual; and / or Prevent individuals from developing high cholesterol conditions.

9. The use according to claim 8, wherein: The individual is on a high cholesterol diet; Preferably, said preventing a subject from developing a hypercholesterolemia condition comprises reducing the probability and / or risk of a subject on a high cholesterol diet developing a hypercholesterolemia condition, or reducing or inhibiting further progression of the condition in a subject who is not ill but is on the verge of developing a hypercholesterolemia condition or on the verge of becoming ill with hypercholesterolemia.

10. The use according to any one of claims 7 to 9, wherein: The product is a food or a health food; Preferably, the phytosterol esters, fish oil and β-glucan are used to prepare the product in the following amounts, calculated on a dry matter basis: Fish oil ≤3g / day; Phytosterol esters ≤ 3.9 g / day; and / or β-glucan ≤5g / day.

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