Preparation for reducing blood fat and application of algae extract in preparation of preparation for reducing blood fat

Abstract

The invention provides a preparation for reducing blood fat and application of an algae extract in preparation of the preparation for reducing blood fat. The preparation for reducing blood fat comprises an algae extract, the algae extract is crude algae oil or refined algae oil, the crude algae oil comprises EPA and polar fat, the polar fat accounts for 30%-100% of the crude algae oil, the refined algae oil comprises rTG type EPA, and the algae extract can reduce blood fat at least by reducing LDL-C. According to the invention, the preparation for reducing blood fat and the application of the algae extract in preparation of the preparation for reducing blood fat can be provided.

Description

Technical Field

[0001] The present disclosure relates to the field of biopharmaceutical industry, and specifically to a lipid-lowering preparation and the use of an algae extract in the preparation of the lipid-lowering preparation. Background Art

[0002] EPA (eicosapentaenoic acid), as an ω-3 polyunsaturated fatty acid, has long been studied and applied in the management of blood lipid health. EPA is mainly ingested through food, such as fish oil rich in ω-3. Scientific research shows that EPA has the function of regulating blood lipids and improving cardiovascular health, which is mainly achieved through several mechanisms: 1) Lowering triglyceride (TG) levels: EPA can effectively lower triglyceride levels in the blood, which is especially important for the prevention and treatment of hypertriglyceridemia. By lowering triglyceride levels, EPA helps reduce the risk of cardiovascular disease. 2) Improve vascular health: EPA can also improve the elasticity of blood vessels, promote the function of vascular endothelial cells, and help reduce the risk of arteriosclerosis. 3) Anti-inflammatory effect: EPA has anti-inflammatory effects and can reduce inflammatory responses in the body. Inflammation is an important contributing factor to cardiovascular disease.

[0003] In recent years, large clinical trials such as REDUCE-IT have shown that supplementing with high-purity EPA can significantly reduce the risk of CVD and reduce the volume of coronary plaques, while studies such as VITAL and STRENGTH using a combination of EPA+DHA have failed to obtain significant cardiovascular clinical benefits. The "Guidelines for Blood Lipid Management in China (2023)" recommends the use of high-purity EPA to assist in the treatment of cardiovascular disease. In the MARINE and ANCHOR studies, subjects took 2g or 4g of 97% ethyl ester EPA (EE-type EPA) per day, which significantly reduced the level of triglycerides in the blood and red blood cells after 12 weeks. Moreover, the amount of triglyceride reduction is dose-dependent with the amount of EPA taken. In the MARINE study, 4g and 2g of EE-type EPA per day reduced TG levels by -27% and -7%, respectively. In the ANCHOR study, 4g and 2g of EE-type EPA per day reduced TG levels by -18% and -6%, respectively.

[0004] However, studies such as REDUCE-IT have found that omega-3 fatty acid intake can increase the risk of atrial fibrillation in a dose-dependent manner. The higher the intake, the higher the risk. The European Medicines Agency has issued a warning: EPA or DHA intake of more than 2g will increase the risk of atrial fibrillation. Meta-analysis shows that the risk increases by 11% for every 1g / d increase.

[0005] Currently, EPA is mainly derived from marine fish, but it is difficult to completely separate EPA and DHA in fish oil, and the EPA resources in fish oil are gradually depleted due to marine pollution and overfishing. Extracting EPA from microalgae, the primary producer of EPA, has become a new path. Microalgae, such as Nannochloropsis spp., only synthesize EPA but not DHA, providing excellent raw materials for the preparation of pure EPA products.

[0006] Microalgae-derived EPA raw materials can avoid the risk of atrial fibrillation caused by excessive intake of Omega-3 by taking effect at low doses due to their special lipid composition. Currently, the results of two human efficacy trials of Pseudochlorophyll extract have been published. One of them is a randomized double-blind controlled trial of 120 people. The subjects were randomly divided into two groups. One group took 1g of Pseudochlorophyll extract (containing 250mg EPA, 150mg polar lipids, 40mg ARA, 90mg palmitoleic acid) every day, and the other group took a placebo every day for 12 weeks. Compared with the placebo group, the EPA supplement of Pseudochlorophyll oil significantly increased the Omega-3 index and plasma EPA concentration, from 4.96±0.90 and 0.82±0.37% at baseline to 5.75±0.90 and 1.27±0.36 at week 12, respectively. Very low-density lipoprotein cholesterol (VLDL) was reduced by 25%, resulting in a significant reduction in total cholesterol.

[0007] The second study was a real-world post-market cohort study in which 480 normolipidemic consumers aged 18 years and older took 1000-1100 mg of Nannochloropsis extract (containing 250 mg EPA, 150 mg polar lipids, 40 mg ARA, 90 mg palmitoleic acid) daily. Changes in TG, RC, LDL, HDL, TC, hs-CRP, glucose, and HbA1c were monitored at the start of supplementation, 3 months, and 6 months. Participants had normal mean TG levels at baseline (1.62 ± 0.60 mmol / L) and experienced significant and sustained reductions in triglycerides at 3 months (-8.0%; p < 0.001) and 6 months (-14.2%; p < 0.001). Furthermore, after 6 months of supplementation, TC and Non-HDL-C decreased by 5.0% (p<0.001) and 5.5% (p<0.001), respectively, mainly due to a 14.9% reduction in cholesterol remnant (RC) (p<0.001).

[0008] In the above two human studies of Microchloropsis algae extracts, some blood lipid indicators were reduced when the intake was 250mg EPA, which is much lower than the intake of 2g or 4g of ethyl ester EPA in large clinical studies such as REDUCE-IT mentioned above, and can reduce the risk of atrial fibrillation due to taking EPA to lower blood lipids. However, in these two human clinical studies of Microchloropsis algae extracts, there was no significant decrease in LDL (low-density lipoprotein) levels. The "Guidelines for Blood Lipid Management in China (2023)" recommends LDL-C (low-density cholesterol, which is the cholesterol in low-density lipoprotein (LDL)) as the primary target for blood lipid intervention. At present, the level of LDL is mainly controlled by statins, but statins have side effects such as promoting rhabdomyolysis, and better intervention methods without toxic side effects are still needed. Summary of the invention

[0009] The present disclosure is made in view of the above-mentioned prior art conditions, and its purpose is to provide a lipid-lowering preparation and the use of algae extract in the preparation of lipid-lowering preparation.

[0010] To this end, the first aspect of the present disclosure provides a lipid-lowering preparation, including an algae extract, wherein the algae extract is crude algae oil or refined algae oil, wherein the crude algae oil includes EPA and polar lipids, wherein the polar lipids account for 30% to 100% of the crude algae oil, and wherein the refined algae oil includes rTG-type EPA, wherein the algae extract at least lowers blood lipids by lowering LDL-C. In the present disclosure, it is verified that crude algae oil and refined algae oil can be used as efficient lipid-lowering raw materials, and can mainly lower blood lipids by lowering LDL-C, and therefore, a lipid-lowering preparation comprising an algae extract can be provided.

[0011] In the preparation involved in the first aspect of the present disclosure, optionally, the EPA in the refined algae oil accounts for 1% to 100%, and the EPA in the crude algae oil accounts for 1% to 70%.

[0012] In the preparation involved in the first aspect of the present disclosure, optionally, when the preparation is administered to a subject, the dosage is no more than 2000 mg / d in terms of EPA content.

[0013] In the preparation involved in the first aspect of the present disclosure, optionally, the refined algae oil also includes unsaturated fatty acid lipids other than EPA, and the unsaturated fatty acid lipids are selected from any one or more combinations of palmitoleic acid (C16:1), oleic acid (C18:1n9c), linoleic acid (C18:2n6c) and arachidonic acid (C20:4n6).

[0014] In the preparation involved in the first aspect of the present disclosure, optionally, the algae extract is derived from Pseudomonas aeruginosa.

[0015] In the preparation involved in the first aspect of the present disclosure, optionally, the polar lipid includes phospholipids, glycolipids and / or betaine esters.

[0016] In the preparation involved in the first aspect of the present disclosure, optionally, the refined algae oil is obtained by further refining the crude algae oil, and the refining includes a re-esterification step.

[0017] A second aspect of the present disclosure provides a use of an algae extract in preparing a lipid-lowering preparation, wherein the algae extract is the algae extract described in any one of the first aspect of the present disclosure.

[0018] The third aspect of the present disclosure provides an application of an algae extract in the preparation of a preparation for lowering LDL-C, wherein the algae extract is crude algae oil or refined algae oil, the crude algae oil comprises EPA and polar lipids, the polar lipids account for 30% to 100% of the crude algae oil, and the refined algae oil comprises rTG-type EPA.

[0019] The fourth aspect of the present disclosure provides a preparation for lowering LDL-C, comprising an algae extract, wherein the algae extract is crude algae oil or refined algae oil, the crude algae oil comprises EPA and polar lipids, the polar lipids account for 30% to 100% of the crude algae oil, and the refined algae oil comprises rTG-type EPA.

[0020] In the present invention, microalgae are used as raw materials to extract EPA algae oil, which has multiple advantages:

[0021] First, the disclosure provides two efficient lipid-lowering raw materials (algae extracts). Among them, rTG type EPA algae oil (refined algae oil) has a special fatty acid composition, and has a more significant effect than 97% EE type EPA fish oil in terms of lipid-lowering (specifically, see subsequent experimental data). And natural EPA algae oil (crude algae oil) has a more significant lipid-lowering effect compared with refined algae oil because it is rich in polar lipids. In the present invention, it is found that the refined algae oil with an EPA content of 60% is better than the EE type EPA fish oil with an EPA content of 97%, which shows that the higher the EPA purity is, the better. And the crude algae oil containing only 25% EPA is better than the refined algae oil containing 60% EPA, indicating that polar lipids can significantly improve the lipid-lowering effect of algae oil. This low-dose, low-purity natural EPA algae oil that takes effect is of great significance for the treatment and prevention of cardiovascular-related diseases (such as atherosclerosis, myocardial infarction, coronary heart disease, etc.). Since only 500 mg of EPA from natural EPA algae oil can lower the three blood lipid indicators of TC, TG and LDL-C, which is much lower than the intake of 2g or 4g of fish oil, it can also reduce the risk of atrial fibrillation due to taking EPA to lower blood lipids.

[0022] Secondly, because it is extracted from natural microalgae, the preparation has higher biosafety and lower risk of side effects, and provides vegetarians and those who are allergic to fish oil with plant-based EPA other than fish oil.

[0023] Furthermore, the sustainability and environmental friendliness of the algae extract used in the present invention are also important advantages. The powerful carbon fixation ability of microalgae can help save energy and reduce emissions.

[0024] According to the present disclosure, a lipid-lowering preparation and the use of an algae extract in the preparation of a lipid-lowering preparation can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a schematic diagram of part of the experimental results of Experiment 1 involved in the example of the present disclosure.

[0026] Figure 2 It is a schematic diagram of part of the experimental results of Experiment 2 involved in the example of the present disclosure. DETAILED DESCRIPTION

[0027] Hereinafter, preferred embodiments of the present disclosure will be described in detail with reference to the accompanying drawings. In the following description, the same reference numerals are given to the same components, and repeated descriptions are omitted.

[0028] It should be noted that the terms "including" and "having" and any variations thereof in the present disclosure, such as a process, method, system, product or device that includes or has a series of steps or units, are not necessarily limited to those steps or units clearly listed, but may include or have other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0029] The first aspect of the present disclosure provides a lipid-lowering preparation, including an algae extract. In the present disclosure, it is verified that the algae extract has a highly effective lipid-lowering effect and can be applied in a lipid-lowering preparation, so a lipid-lowering preparation containing an algae extract can be provided. In the present disclosure, the algae extract is derived from microalgae, so the algae extract can also be referred to as a microalgae extract, or simply referred to as an "extract".

[0030] A second aspect of the present disclosure provides a use of an algae extract in preparing a lipid-lowering preparation.

[0031] The third aspect of the present disclosure provides an application of an algae extract in the preparation of a preparation for lowering LDL-C. In the present disclosure, it is verified that the algae extract has a highly effective effect of lowering LDL-C, and can be used in the preparation of a preparation for lowering LDL-C. It should be noted that the preparation for lowering LDL-C can be used as a drug in the medical field and can also be used as an experimental agent for related scientific research.

[0032] A fourth aspect of the present disclosure provides a formulation for lowering LDL-C, comprising an algae extract.

[0033] Hereinafter, the algae extract and its application according to the present disclosure will be described in detail.

[0034] In some examples, the lipid-lowering formulation can include an algae extract.

[0035] In some examples, the algae extract can be crude algae oil. In some examples, the crude algae oil can include polar lipids and EPA. In the present disclosure, it is verified that the crude algae oil containing a predetermined content of polar lipids can reduce blood lipids, particularly reduce LDL-C. Thus, a preparation for reducing blood lipids or reducing LDL-C and the application of crude algae oil in the preparation of a preparation for reducing blood lipids or reducing LDL-C can be provided. In the present disclosure, crude algae oil can also be referred to as natural algae oil or natural EPA algae oil.

[0036] In the present disclosure, polar lipids refer to a class of lipid molecules containing polar groups. These polar groups make polar lipids have a certain solubility or hydrophilicity in water, while their hydrophobic fatty acid chains make them lipophilic in non-polar environments. Polar lipids are usually composed of two parts: one or more long-chain fatty acids (hydrophobic part) and a head containing polar groups (hydrophilic part). When a fatty acid at the SN1 or SN2 position of a glyceride-type polar lipid is removed, a hemolytic polar lipid can be obtained.

[0037] In some examples, the polar lipids in the crude algae oil can account for 1% to 100%. For example, the polar lipids in the crude algae oil can account for 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111 0%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0038] In some examples, preferably, the polar lipids in crude algae oil can account for 30% to 100%. In existing studies, crude algae oil with low content of polar lipids has no significant decrease in the level of LDL-C, and in the present disclosure, it is verified that crude algae oil with high content of polar lipids has a good lipid-lowering effect, especially the effect of reducing LDL-C. Therefore, providing crude algae oil with suitable polar lipids can help improve the lipid-lowering effect of crude algae oil. In some examples, preferably, the polar lipids in crude algae oil can account for 70% to 90%. In some examples, preferably, the polar lipids in crude algae oil can account for no less than 70%.

[0039] Specifically, in addition to the lipid-lowering effect of EPA, the polar lipids of crude algae oil have unique lipid-lowering effects. In the EPA fish oil lipid-lowering clinical experiment, in general, the effect of high-purity EPA on reducing TG is relatively significant, but there is no significant reduction effect on LDL-C, so in these clinical experiments, many subjects need to take statins at the same time to control LDL-C levels. Therefore, the applicant believes that in the subsequent experiments of the present disclosure, the significant reduction of LDL-C is likely to come from the effect of its polar lipids. On the other hand, in the real-world clinical experiment of Microcystis pseudocystis algae oil (containing 250mgEPA, 150mg polar lipids, 40mgARA, 90mg palmitoleic acid), the polar lipid content is 15%, and its effect of reducing LDL-C is not significant. The polar lipid content of natural EPA algae oil in the present invention is about 70%, which has a significant effect of reducing LDL-C. Therefore, when the EPA content is the same, the content of polar lipids has a certain influence on the reduction effect of LDL-C, and the high content of polar lipids is more helpful to reduce LDL-C.

[0040] In some examples, polar lipids may include phospholipids, glycolipids and / or betaine esters. In other words, in some examples, crude algae oil may contain phospholipids, glycolipids and / or betaine esters. In some examples, preferably, crude algae oil may contain lysophospholipids and lysobetaine esters. In some examples, preferably, crude algae oil may contain lysophospholipids and lysobetaine esters. In some examples, preferably, crude algae oil may contain lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE) and lysobetaine esters (LDGTS). In this case, LDGTS has the effect of reducing the risk of plaque formation by increasing the activity of paraoxonase (PON1) and preventing LDL-C oxidation, and can also improve the efficiency of HDL in binding to the outflow of cholesterol in macrophages, and improve the efficiency of HDL in transporting cholesterol to the liver.

[0041] In some examples, crude algae oil may contain any one or more of DGTS: diacylglycerol trimethyl homoserine, LDGTS: lysodiacylglycerol trimethyl homoserine, Cer: ceramide, MGDG: monogalactosyl diacylglycerol, DGDG: digalactosyl diacylglycerol, SQDG: thioisosorhamnosyl diacylglycerol, DGGA: diacylglycerol glucuronic acid, CE: cholesterol ester, CL: cardiolipin, LPA: lysophosphatidic acid, LPE: lysophosphatidylethanolamine, LPG: lysophosphatidylglycerol, LPC: lysophosphatidylcholine, LPI: lysophosphatidylinositol, PE: phosphatidylethanolamine, PG: phosphatidylglycerol, PI: phosphatidylinositol, PC: phosphatidylcholine, and PI-Cer: inositol phosphoceramide. In this case, these components are all polar lipids, which can help improve the lipid-lowering effect of crude algae oil.

[0042] In some examples, the crude algal oil may contain any one or more of FFA: free fatty acids, MG: monoglycerides, DG: diglycerides, and TG: triglycerides.

[0043] In some examples, the EPA in the crude algae oil can account for 1% to 100%. For example, the EPA in the crude algae oil can account for 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111 %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In some examples, the EPA in the crude algae oil can account for 1% to 70%.

[0044] In some examples, polar lipids in crude algae oil can be linked to fatty acids. In some examples, a portion of EPA in crude algae oil can be linked to polar lipids.

[0045] In some examples, crude algae oil can be used after being mixed with fatty acids or fatty acid-containing substances other than the fatty acids contained in the crude algae oil. The applicant has found that crude algae oil can promote the absorption of fatty acids or fatty acid-containing substances by users, thereby promoting the absorption of fatty acids or fatty acid-containing substances by users by utilizing the characteristics of crude algae oil itself, thereby helping to further improve the lipid-lowering effect.

[0046] In the present disclosure, the algae extract can be extracted from algae. The algae can be selected from Cryptophyceae, Chlorophyceae, Diatomaceous Algae, Chrysophyceae, Macrophyceae and Euchlorophyceae, etc.

[0047] In some examples, the algae extract can come from unicellular microalgae. In some examples, the unicellular microalgae can be cyanobacteria, green algae or red algae, etc. In some examples, preferably, the unicellular microalgae is pseudo-microalgae. That is to say, in some examples, preferably, the algae extract can be derived from pseudo-microalgae (Nannochloropsis sp.). Pseudo-microalgae, also known as micro-microalgae or micro-green algae, is a widely distributed marine algae, and the cells accumulate EPA and other Omega-3 polyunsaturated fatty acids in large quantities, and the pseudo-microalgae contains abundant natural polar lipids, which have special health effects and extremely high bioavailability. In this case, the microalgae extract derived from pseudo-microalgae contains abundant natural polar lipids. In addition, pseudo-microalgae is a new food raw material, and the microalgae extract is derived from a new food raw material, has high nutritional and medicinal value, and has high biosafety, and has no obvious toxic side effects when eaten for a long time. In the present disclosure, the crude algae oil derived from pseudo-microalgae can also be referred to as polar lipid pseudo-microalgae oil.

[0048] In some examples, the algae extract may include fatty acids. Omega-3 polyunsaturated fatty acids such as EPA are accumulated in large quantities in the cells of Nannochloropsis algae, and therefore, the algae extract extracted therefrom also contains certain fatty acids.

[0049] In some examples, the algae can be extracted using an organic solvent. In some examples, defatting the algae can include extracting the algae using an organic solvent. Specifically, the algae can be mixed with an organic solvent, and the fat in the algae will dissolve in the organic solvent, and then filtered to obtain defatted algae tissue (referred to as defatted algae tissue). The filtrate obtained by filtration contains polyunsaturated fatty acids, which can be further processed to extract the polyunsaturated fatty acids for other applications. This can help improve the utilization rate of algae raw materials.

[0050] In some examples, the method of preparing crude algae oil may include the following steps: contacting raw algae with an organic solvent; and removing the organic solvent to obtain crude algae oil.

[0051] In some examples, the organic solvent may include any one or more combinations of ethanol, n-hexane, petroleum ether and n-heptane. In some examples, preferably, the organic solvent may be ethanol. Thus, it is possible to help extract the algae efficiently. That is, in some examples, preferably, the method for preparing crude algae oil may include the following steps: contacting the original algae with ethanol; and removing the ethanol in the second step to obtain crude algae oil.

[0052] In some examples, the ratio of organic solvent to algae can be 2:1 to 10:1. This can help to efficiently extract algae. In some examples, the mass ratio of organic solvent to algae can be 10:1, 9.5:1, 9:1, 8.5:1, 8:1, 7.5:1, 7:1, 6.5:1, 6:1, 5.5:1, 5:1, 4.5:1, 4:1, 3.5:1, 3:1, 2.5:1 or 2:1.

[0053] In some examples, the algae may be algae with intact cell walls. The applicant has found that breaking the cell walls before defatting is not only unnecessary but may also result in poor defatting effect. Therefore, in this embodiment, using algae with intact cell walls for defatting can help simplify the process and improve the extraction efficiency on the one hand, and also help to extract the fat in the algae as much as possible on the other hand.

[0054] In some examples, the algae may be dry algae. Dry algae refers to algae with a moisture content of less than 5wt%. In this case, compared to wet algae, dry algae will not introduce too much water during the defatting process, which can help improve the extraction efficiency, and can help simplify the subsequent process of reusing the organic solvent, thereby helping to improve efficiency and reduce costs in the large-scale preparation process. Regarding the reuse of organic solvents, taking ethanol as an organic solvent as an example, ethanol can usually be recycled by distillation, but ethanol and water easily form azeotropes, and it is difficult to separate the water from ethanol by distillation alone. At this time, other operations need to be introduced to separate the two, which is relatively complicated. Therefore, in this embodiment, the use of dry algae for extraction will not introduce too much water, which can help simplify the subsequent process of reusing the organic solvent.

[0055] In some examples, the algae may be dried to obtain dry algae, and then the dry algae may be extracted. In other words, in some examples, the dry algae may be defatted. In some examples, the original algae may be flocculated, deflocculated, and then dried to obtain dry algae.

[0056] In some examples, the algae extract may be refined algae oil. In some examples, the algae extract may be crude algae oil or refined algae oil. In some examples, the algae extract may also be a mixture of crude algae oil and refined algae oil.

[0057] In some examples, refined algae oil can include rTG type EPA. Wherein, "rTG" refers to "re-esterified triglyceride". Both rTG type EPA and EE type EPA (ethyl ester type EPA) need to be hydrolyzed by pancreatic lipase to release EPA in the form of free fatty acids to be absorbed by the small intestine. Compared with the EE type, the rTG form is closer to the form in the natural state, and may be more easily recognized and hydrolyzed by the human enzyme system. Therefore, the EPA of the rTG type has better absorption and utilization than the EE type EPA, and has a more significant lipid-lowering effect. In the present disclosure, refined algae oil may also be referred to as rTG refined algae oil, rTG type EPA algae oil, rTG type pseudo-microsphere algae oil or refined algae oil.

[0058] In some examples, refined algae oil can also include unsaturated fatty acid lipids other than EPA. In some examples, unsaturated fatty acid lipids can include one or more of palmitoleic acid (C16:1), oleic acid (C18:1n9c), linoleic acid (C18:2n6c) and arachidonic acid (C20:4n6). In this case, unsaturated fatty acid lipids have a certain contribution to the reduction of TC and LDL-C in refined algae oil, which helps to improve the effect of refined algae oil in reducing blood lipids, such as palmitoleic acid has a significant anti-inflammatory effect and has a certain reduction effect on LDL-C, and linoleic acid can esterify cholesterol to reduce the cholesterol level in serum and liver.

[0059] In some examples, the EPA in refined algae oil can account for 1% to 100%. For example, the EPA in refined algae oil can account for 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111 EPA in crude algae oil can be 1% to 70%.

[0060] In some examples, the refined algae oil can be obtained by further refining the crude algae oil. In some examples, the refining includes a re-esterification step. In some examples, the method for preparing the refined algae oil may include the following steps: preparing the crude algae oil; hydrolyzing and separating the crude algae oil to obtain a free fatty acid layer; molecularly distilling the free fatty acid layer; mixing the light phase obtained by molecular distillation with glycerol, and stopping the reaction to obtain the refined algae oil.

[0061] In some examples, the algae extract can be prepared by the preparation method of the present disclosure, or can be prepared by other methods.

[0062] In the present disclosure, the lipid-lowering / LDL-C-lowering preparation can be food, cosmetics or medicine. The algae extract disclosed in the present disclosure is extracted from natural microalgae, which is a new type of food raw material with high biosafety and low risk of side effects, and can be applied to food or medicine.

[0063] In some examples, the algae extract can be used externally. In some examples, the algae extract can be used internally. In some examples, the lipid-lowering / LDL-C-lowering formulation can be administered orally or by injection into the human body.

[0064] In some examples, preferably, the food may be a functional food. In some examples, the food may include biscuits, candies, cakes and beverages. In some examples, preferably, the dosage form of the medicine may be an oral liquid, granules, tablets, capsules, powders, powders, pills, solutions, syrups, decoctions, patches, gels, creams or sprays. Thus, it is easy to store and use.

[0065] In some examples, the lipid-lowering / LDL-C-lowering formulation can be composed of an algae extract.

[0066] In some examples, the lipid-lowering / LDL-C lowering preparation may also include other functional adjuvants. In some examples, the lipid-lowering / LDL-C lowering preparation may also include auxiliary materials permitted by pharmacy, ordinary food, health food, special medical food, and cosmetics regulations. For example, the lipid-lowering / LDL-C lowering preparation may include auxiliary materials such as diluents, thickeners, sweeteners, antioxidants, vitamins, plasticizers and / or diluents.

[0067] In some examples, when a lipid-lowering / LDL-C-lowering preparation is administered to a subject, the dosage may not exceed 2000 mg / d based on the content of EPA. In this case, while achieving the lipid-lowering / LDL-C-lowering effect, the risk of atrial fibrillation caused by taking EPA to lower blood lipids can be reduced as much as possible. In some examples, when a lipid-lowering / LDL-C-lowering preparation is administered to a subject, the dosage may be 1 mg / d-2000 mg / d based on the content of EPA. For example, the dosage can be 1 mg / d, 100 mg / d, 150 mg / d, 200 mg / d, 250 mg / d, 300 mg / d, 350 mg / d, 400 mg / d, 450 mg / d, 500 mg / d, 550 mg / d, 600 mg / d, 650 mg / d, 700 mg / d, 800 mg / d, 900 mg / d, 1000 mg / d, 1100 mg / d, 1200 mg / d, 1300 mg / d, 1400 mg / d, 1500 mg / d, 1600 mg / d, 1700 mg / d, 1800 mg / d, 1900 mg / d or 2000 mg / d. In some examples, preferably, when administering a lipid-lowering / LDL-C-lowering preparation to a subject, the dosage may be 200 mg / d-1200 mg / d based on the EPA content.

[0068] In some examples, the algae extract can be diluted / concentrated before use according to actual needs.

[0069] The present disclosure also provides an application of an algae extract in the preparation of a medicament for treating hyperlipidemia. The hyperlipidemia is caused by at least high LDL-C. The algae extract can treat hyperlipidemia by at least lowering LDL-C. For example, the hyperlipidemia can be hypercholesterolemia or a mixed hyperlipidemia in which both serum total cholesterol and triglyceride levels are elevated.

[0070] In order to further illustrate the present disclosure, the preparation method and application of the algae extract provided by the present disclosure are described in detail below in conjunction with the examples, and the beneficial effects achieved by the present disclosure are fully illustrated in conjunction with the comparative examples.

[0071] It should be noted that, in the examples of the present disclosure, unless otherwise specified, the reagents and instruments used are all commercially available products.

[0072] The natural EPA algae oil and rTG EPA algae oil used in the examples of the present invention are prepared and provided by Xiaozao Technology (Anji) Co., Ltd., and the remaining reagents are conventionally purchased raw material reagents.

[0073] The preparation process of natural algae oil (crude algae oil) is as follows:

[0074] The first step is to contact the dry algae with ethanol, wherein the dry algae is obtained by flocculating, deflocculating, desalting and drying algae biomass, and the water content of the dry algae is less than 5wt%, and the dry algae is algae with intact cell walls of the genus Nannochloropsis; the second step is to remove the ethanol to obtain natural algae oil, wherein the content of polar lipids in the natural algae oil is about 70%-75%.

[0075] The preparation process of rTG type EPA algae oil (refined algae oil) is as follows:

[0076] After obtaining the crude extract by ethanol extraction of microalgae, free fatty acids are obtained by hydrolysis. The free fatty acids are heated to 60-80 ° C, and purified water at 60-80 ° C is added in a ratio of free fatty acids: purified water = 4: 1 to 5: 1, stirred evenly and allowed to stand for stratification, and the water layer is discarded. Stirring is continued until the water in the free fatty acids is completely evaporated. Four-stage molecular distillation is performed, the molecular distillation four-stage light phase is mixed with glycerol, vacuumed, heated to 45 to 55 ° C, stirred and mixed for 20 to 45 minutes, enzyme powder is added, vacuumed to -0.095MPa, the acid value is monitored, and the reaction is stopped when the set value is reached, and rTG type EPA algae oil (refined algae oil) is obtained. The content of unsaturated fatty acids in the obtained refined algae oil is shown in Table 1.

[0077] Table 1

[0078]

[0079] As shown in Table 1, the content of EPA in the refined algae oil is about 60%, so it is hereinafter referred to as 60% EPA rTG type Nannocystis oil.

[0080] Experiment implementation:

[0081] Experimental principle: Feeding animals with feed containing cholesterol, sucrose, lard, and sodium cholate can form an animal model of lipid metabolism disorder. Then, giving the animals test samples can detect the effects of the test samples on hyperlipidemia, and determine the effects of the test samples on lipid absorption, lipoprotein formation, and lipid degradation or excretion.

[0082] Experimental animals: healthy adult male rats, weighing 200±20g, preferably SD rats, 10 rats per group for high-dose EPA comparison experiments, and 15 rats per group for low-dose EPA comparison experiments.

[0083] Model feed: add 20.0% sucrose, 15% lard, 1.2% cholesterol, 0.2% sodium cholate, appropriate amount of casein, calcium hydrogen phosphate, stone powder, etc. to the maintenance feed. In addition to crude fat, the moisture, crude protein, crude fat, crude fiber, crude ash, calcium, phosphorus, calcium: phosphorus of the model feed must meet the national standards for maintenance feed.

[0084] Adaptation period: Rats were fed maintenance diet under the barrier system and observed for 5-7 days.

[0085] Modeling period: The rats were randomly divided into groups according to body weight, of which 10 rats were given maintenance feed as the blank control group, and the remaining experimental group rats were given model feed as the model control group. The body weight was weighed once a week. After the model control group was given the model feed for 1-2 weeks, the blank control group and the model control group rats were blooded without fasting (inner canthus or tail of the eye), and the serum was separated as soon as possible after blood collection to measure the serum TC, TG, LDL-C, and HDL-C levels. The model control group was randomly divided into the required number of groups according to the TC level. After grouping, there was no significant difference in TC, TG, LDL-C, and HDL-C between the groups; compared with the blank control group, the differences in TC, TG, and LDL-C were significant, and the model was determined to be established.

[0086] Administration of test samples: After grouping, the experimental group was orally administered the test samples every day, while the blank control group and the model control group were simultaneously administered the same volume of the corresponding solvents. The blank control group continued to be administered maintenance feed, while the model control group and the experimental group continued to be administered model feed. Body weights were measured regularly, and blood was collected without fasting at the end of the experiment. Serum was separated as soon as possible after blood collection to measure serum TC, TG, LDL-C, and HDL-C levels.

[0087] Administration time of test samples: The administration time of test samples is 30 days to 45 days.

[0088] Experiment 1: High-dose EPA comparison experiment

[0089] Each group of 10 rats was gavaged for 30 days according to the following experimental settings.

[0090] The embodiments and comparative examples are set up as shown in Table 2.

[0091] Table 2

[0092]

[0093] The results of Experiment 1 are as follows Figure 1 , as shown in Table 3-1 and Table 3-2. Figure 1 It is a schematic diagram of part of the experimental results of Experiment 1 involved in the example of the present disclosure.

[0094] Table 3-1

[0095]

[0096] Table 3-2

[0097]

[0098]

[0099] Note: In Table 3-1 and Table 3-2, the blank control group was compared with the model control group using the independent sample T test. Each drug-treated group was compared with the model control group using one-way analysis of variance; compared with the negative control group, **: P < 001; compared with the model control group, ▲: P < 0.05, ▲▲: P < 0.01.

[0100] rTG-type Pseudo-Nanochloa oil has a special fatty acid composition and is more effective in lowering blood lipids than 97% EPA EE-type fish oil: when the EPA dosage is 4g / d (animal experiment converted to human dosage), the rTG-type Pseudo-Nanochloa oil can reduce the total cholesterol (TC) content in the blood of hyperlipidemia model rats by 24.8% and the low-density lipoprotein cholesterol (LCL-C) content by 46.8%, while 97% EPA EE-type fish oil has no significant effect on reducing TC, TG and LCL-C.

[0101] The possible reasons why rTG type Pseudo-Nannoglossum oil has a better lipid-lowering effect than 97% EPA EE type fish oil are: 1) In addition to EPA (C20:5n3), rTG type EPA algae oil also contains unsaturated fatty acid lipids such as palmitoleic acid (C16:1), oleic acid (C18:1n9c), linoleic acid (C18:2n6c), and arachidonic acid (C20:4n6), which also contribute to the rTG type EPA algae oil in reducing TC and LDL-C. For example, palmitoleic acid has a significant anti-inflammatory effect and has a certain effect on reducing LDL-C, and linoleic acid can esterify cholesterol and thus reduce cholesterol levels in serum and liver.

[0102] 2) Both rTG EPA and EE EPA need to be hydrolyzed by pancreatic lipase to release EPA in the form of free fatty acids before they can be absorbed by the small intestine. Compared with EE, rTG is closer to its natural form and may be easier to be recognized and hydrolyzed by the human enzyme system. Therefore, rTG EPA has better absorption and utilization than EE EPA and has a more significant lipid-lowering effect.

[0103] Experiment 2: Low-dose EPA comparison experiment

[0104] Each group of 15 rats was gavaged for 45 days according to the following experimental settings.

[0105] The embodiments and comparative examples are set up as shown in Table 4.

[0106] Table 4

[0107]

[0108] The results of Experiment 2 are as follows Figure 2 , as shown in Tables 5-1, 5-2 and 5-3. Figure 2 It is a schematic diagram of part of the experimental results of Experiment 2 involved in the example of the present disclosure.

[0109] Table 5-1

[0110] Group TC(mmol / L) △TC TG (mmol / L) △TG Blank control group 1.74±0.34 - 1.72±0.33 - Model control group <![CDATA[4.87±0.33 # ]]> - <![CDATA[3.62±0.16 # ]]> - Positive control group (simvastatin) <![CDATA[3.59±0.35 * ]]> -26.3％ <![CDATA[2.71±0.34 * ]]> -25.1％ Refined algae oil1 4.95±0.42 1.6％ 3.53±0.24 -2.5％ Refined algae oil 2 <![CDATA[4.59±0.40 * ]]> -5.7％ 3.64±0.23 0.6％ Natural algae oil 1 5.02±0.40 3.1％ 3.48±0.22 -3.9％ Natural algae oil 2 <![CDATA[4.53±0.39 * ]]> -7.0％ <![CDATA[3.13±0.41 * ]]> -13.5％ Fish oil 4.95±0.28 1.6％ 3.82±0.49 5.5％ 50 / 20 Fish Oil 4.92±0.49 1.0％ <![CDATA[3.37±0.18 * ]]> -6.9％

[0111] Table 5-2

[0112]

[0113]

[0114] Table 5-3

[0115] Group apoA-1 (μg / mL) △apoA-1 apoB100 (pg / mL) △apoB100 Blank control group 0.22±0.06 - 656.8±83.6 - Model control group <![CDATA[0.18±0.03 # ]]> - <![CDATA[744.1±57.7 # ]]> - Positive control group (simvastatin) <![CDATA[0.25±0.10 * ]]> 38.9％ <![CDATA[683.2±61.9 * ]]> -8.2％ Refined algae oil1 0.22±0.09 22.2％ 731.9±80.3 -1.6％ Refined algae oil 2 <![CDATA[0.23±0.08 * ]]> 27.8％ <![CDATA[660.1±61.9 * ]]> -11.3％ Natural algae oil 1 <![CDATA[0.21±0.04 * ]]> 16.7％ 713.0±71.9 -4.2％ Natural algae oil 2 <![CDATA[0.22±0.05 * ]]> 22.2％ <![CDATA[696.2±58.6 * ]]> -6.4％ Fish oil <![CDATA[0.23±0.07 * ]]> 27.8％ <![CDATA[679.2±76.5 * ]]> -8.7％ 50 / 20 Fish Oil 0.19±0.04 5.6％ <![CDATA[663.3±71.2 * ]]> -10.9％

[0116] Note: #: There is a significant difference compared with the blank control group (p<0.05); *: There is a significant difference compared with the model control group (p<0.05).

[0117] Natural algae oil is rich in polar lipids such as phospholipids, glycolipids and betaine esters, so it has a more significant lipid-lowering effect than rTG-type EPA algae oil: see Table 5-1 to Table 5-3, the high-dose group (334 mg / kgBW) natural algae oil 2 can reduce the triglyceride (TG) content in the blood of hyperlipidemia model rats by 13.5%, the total cholesterol (TC) content by 7.0%, the LDL-C content by 17.1%, the apoA-1 content by 22.2%, and the apoB100 content by 6.4%; the high-dose group (334 mg / kgBW) refined algae oil 2 can reduce the total cholesterol content in the blood of hyperlipidemia model rats by 5.7%, the LDL-C content by 12.8%, the apoA-1 content by 27.8%, and the apoB100 content by 11.29%, but there is no significant change in triglyceride content. Therefore, under equal dosage conditions, although the EPA content in natural algae oil is only 25%, its lipid-lowering effect is better than that of refined algae oil containing 60% EPA.

[0118] The possible reasons why natural algae oil has a better lipid-lowering effect than refined algae oil are: 1) Since polar lipid EPA such as phospholipids, glycolipids and betaine esters in natural algae oil does not need to be hydrolyzed by pancreatic lipase to become EPA in the form of free fatty acids and then absorbed by the small intestine, the bioavailability of EPA can be greatly improved. Even if the EPA content in natural algae oil is lower than that in refined algae oil, the EPA content entering the blood may be higher than rTG EPA.

[0119] 2) There is a special polar lipid in natural algae oil called lyso-betaine ester (Lyso-DGTS), which can reduce the risk of plaque formation by increasing the activity of paraoxonase (PON1) and preventing LDL-C oxidation. It can also increase the efficiency of HDL binding to cholesterol outflow in macrophages and increase the efficiency of HDL transporting cholesterol to the liver.

[0120] 3) When the liver is in a state of overnutrition, the expression of Mfsd2a, an active transporter for transporting lysopolar lipids, will be upregulated; the upregulated Mfsd2a absorbs more lysopolar lipids from the blood into the liver, inducing the formation of small lipid droplets that help maintain lipid homeostasis in the liver, and inhibiting the formation of large lipid droplets that store a large amount of TG that are harmful to liver health; the lysopolar lipids absorbed by Mfsd2a from the blood can be converted into phospholipids, betaine esters, etc. through reacylation by LPCAT3, replenishing the PC pool in the liver, and consuming free fatty acids during the reacylation process, reducing the formation of TG; the lysopolar lipids absorbed by Mfsd2a from the blood can inhibit the process of liver synthesis of fat from non-lipid precursors (such as glucose), which may further reduce LDL in the blood. Therefore, in addition to EPA, the polar lipids in natural algae oil also play a role in promoting blood lipid health.

[0121] In addition, referring to Table 5-1 to Table 5-3, compared with 33 / 22 fish oil and 50 / 20 fish oil containing both EPA and DHA, the high-dose group (334 mg / kg BW) natural algae oil 2 has better effects on lowering LDL-C, TG and TC than both 33 / 22 fish oil and 50 / 20 fish oil, when the EPA dosage is lower than that of 33 / 22 fish oil and 50 / 20 fish oil. In other words, compared with fish oil containing both EPA and DHA, natural algae oil can achieve better blood lipid-lowering effects with a smaller EPA intake.

[0122] In summary, the present disclosure can provide two highly effective lipid-lowering raw materials (algae extracts), which can achieve better lipid-lowering effects, especially the effect of reducing LDL-C, when low-dose EPA is taken compared to fish oil. In addition, due to the low intake of EPA, the risk of atrial fibrillation caused by taking EPA to lower blood lipids can also be reduced.

[0123] In summary, the present disclosure can provide a lipid-lowering preparation and the use of an algae extract in the preparation of a lipid-lowering preparation, which can at least achieve lipid-lowering by lowering LDL-C.

[0124] Although the present disclosure is specifically described above in conjunction with the accompanying drawings and examples, it is to be understood that the above description does not limit the present disclosure in any form. Those skilled in the art may modify and change the present disclosure as needed without departing from the essential spirit and scope of the present disclosure, and these modifications and changes all fall within the scope of the present disclosure.

Claims

1. A preparation for lowering blood lipids, characterized in that: It comprises an algae extract, wherein the algae extract is crude algae oil or refined algae oil, the crude algae oil comprises EPA and polar lipids, the polar lipids account for 30% to 100% of the crude algae oil, the refined algae oil comprises rTG type EPA, and the algae extract achieves blood lipid lowering by at least lowering LDL-C.

2. The preparation according to claim 1, characterized in that The EPA content in the refined algae oil is 1% to 100%, and the EPA content in the crude algae oil is 1% to 70%.

3. The preparation according to claim 1, characterized in that When the preparation is administered to a subject, the dosage is no more than 2000 mg / d based on the EPA content.

4. The preparation according to claim 1, characterized in that The refined algae oil also includes unsaturated fatty acid lipids other than EPA, and the unsaturated fatty acid lipids are selected from any one or more combinations of palmitoleic acid (C16:1), oleic acid (C18:1n9c), linoleic acid (C18:2n6c) and arachidonic acid (C20:4n6).

5. The preparation according to claim 1, characterized in that The algae extract is derived from Pseudomonas aeruginosa.

6. The preparation according to claim 1, characterized in that The polar lipids include phospholipids, glycolipids and / or betaine esters.

7. The preparation according to claim 1, characterized in that The refined algae oil is obtained by further refining the crude algae oil, wherein the refining comprises a re-esterification step.

8. Use of an algae extract in preparing a lipid-lowering preparation, characterized in that: The algae extract is the algae extract according to any one of claims 1 to 7.

9. Use of an algae extract in preparing a preparation for lowering LDL-C, characterized in that: The algae extract is crude algae oil or refined algae oil, the crude algae oil comprises EPA and polar lipids, the polar lipids account for 30% to 100% of the crude algae oil, and the refined algae oil comprises rTG type EPA.

10. A preparation for lowering LDL-C, characterized in that: It comprises an algae extract, wherein the algae extract is crude algae oil or refined algae oil, the crude algae oil comprises EPA and polar lipids, the polar lipids account for 30% to 100% of the crude algae oil, and the refined algae oil comprises rTG type EPA.

Images