Branched-chain fatty acid composition and its use in regulating brain neuroimmunity

Abstract

The present invention relates to a branched-chain fatty acid composition and its use in helping to regulate brain neuroimmunity, and belongs to the field of nutrient research. The branched-chain fatty acid composition provided by the present invention comprises C15:0 and C17:0, and more specifically comprises iso-C15:0, anteiso-C15:0, anteiso-C17:0 and iso-C17:0. The branched-chain fatty acid composition promotes the trend of LPS-induced microglia differentiation into M2 phenotype microglia, which is higher than the trend of promoting LPS-induced microglia differentiation into M1 phenotype microglia, and it also promotes the secretion of anti-inflammatory factors such as IL-4 and IL-10, and inhibits the secretion of pro-inflammatory factors such as NO, TNF-a, and IL-1β. Each branched-chain fatty acid in the branched-chain fatty acid composition has a synergistic effect, which can play a role in regulating brain neuroimmunity and maintaining the health of the central nervous system.

Description

Technical Field

[0001] The present invention relates to a branched fatty acid composition and its use in regulating brain neuroimmunity, and more specifically to a branched fatty acid composition containing C15:0 and C17:0 and its use in regulating brain neuroimmunity and nervous system health, belonging to the field of nutrient research. Background Art

[0002] Branched chain fatty acids (BCFA) refer to fatty acids with one or more branched alkyl groups or other functional groups on the fatty acid alkyl chain. Branched alkyl groups located on the second carbon atom at the alkyl end are called iso- or iso- fatty acids, and those located on the third carbon atom at the alkyl end are called anteiso- or anteiso- fatty acids. Due to the unique branched chain structure, BCFA has low freezing point and pour point, good thermal stability and oxidative stability, good solubility, low foaming, and good air and water permeability of the formed membrane. BCFA is widely present in nature, but the content is low. It is reported that a certain amount of BCFA with different structures exists in the milk and internal tissues of ruminants (such as cow and sheep milk and meat products), and in human milk. BCFA is more abundant in vernix and the intestines of newborns. The BCFA spectrum of meconium shows that BCFAs are the main components of the gastrointestinal tract of normal and healthy full-term newborns. The BCFA in the intestines of premature infants is much less than that of normal and healthy infants, and the probability of suffering from necrotizing enterocolitis is higher. For example, Reference 1 discloses that BCFA has the effect of inhibiting breast cancer, prostate cancer, bladder cancer and necrotizing enterocolitis, but its mechanism of action is still unclear.

[0003] The enteric nervous system is essential for the coordination of absorption, digestion, motility, secretion, regulation of intestinal microbiota, and maintenance of intact barrier function. It has a high degree of autonomy and forms an external connection with the central nervous system through the autonomic nervous system, forming the gut-brain axis. The communication mechanism of the gut-brain axis includes three main pathways: first, intestinal microorganisms stimulate the enteric nervous system and then transmit information to the central nervous system through the vagus nerve of the autonomic nervous system; second, intestinal microorganisms produce or induce intestinal epithelial secretory cells to produce metabolites or neurotransmitters, such as 5-HT, GABA, dopamine, short-chain fatty acids (SCFAs), etc. These substances enter the circulatory system and eventually cross the blood-brain barrier to achieve the purpose of regulating the central nervous system; third, intestinal microorganisms regulate the immune system by producing microbial-associated molecular patterns (MAMPs), promoting immune cells or cytokines, such as IL-6, IL-1a, IL1b, TNFa, etc., which in turn affect the central nervous system. Glial cells are immune cells of the central nervous system. Participating in the process of neuroinflammation and causing nerve damage repair is one of the key mechanisms for glial cells to exert their immune function. How to control neuroinflammation has long been the focus of treatment for degenerative diseases of the central nervous system and traumatic injury diseases. At present, there is no effective medical treatment for a large number of neurodegenerative diseases, such as Alzheimer's disease and stroke. The gut-brain axis has become a breakthrough point for medical treatment. The intestinal flora is regulated by drugs or fecal transplantation, the intestinal microenvironment is changed, and the intestinal flora is continuously restored from multiple dimensions of the microenvironment such as flora structure and short-chain fatty acids to improve the occurrence and development of neurodegenerative diseases. Reference 2 discloses the interaction between the intestinal microbiome and oligodendrocytes, as well as the role of metabolites derived from the intestinal microbiome in neurodegenerative diseases. It points out that the intestinal microbiome can regulate oligodendrocyte maturation and myelin production, and that changes in the intestinal microbiome are related to the pathogenesis of neurodegenerative diseases. But many mechanisms are still unclear.

[0004] Previous studies and patents have confirmed the efficacy of branched-chain fatty acids in inhibiting intestinal inflammation. The results showed that both iso-BCFAs and anteiso-BCFAs have a significant inhibitory effect on intestinal inflammation.

[0005] For example, Reference 3 discloses that branched-chain fatty acids are added to the CaCo2 cell culture system and gavage branched-chain fatty acids to mice with DSS-induced enteritis. The results show that BCFAs can induce high SUMO modification of intestinal proteins in CaCo2 cells, thereby reducing the expression of proinflammatory cytokines. In addition, BCFAs also inhibit intestinal inflammation caused by DSS, reduce the activity of intestinal cell deaminase, and promote intestinal epithelial integrity. Reference 4 discloses a composition comprising branched-chain fatty acids such as 14-methylpentadecanoic acid, 12-methyltridecanoic acid, and 13-methyltetradecanoic acid. The composition can inhibit the generation of ROS in the LPS-induced calf intestinal epithelial cell inflammation model; and can regulate the level of inflammatory factors in the intestine of DSS-induced colitis mice and improve the gastrointestinal health of mice. Reference 5 also discloses that branched-chain fatty acids have the function of inhibiting intestinal inflammation in DSS-induced enteritis mice and enhancing the resistance of the mouse intestine. Reference 6 discloses that branched-chain fatty acids can reduce the risk of necrotizing enterocolitis (NEC) and can also increase the expression of IL-10 in the intestine.

[0006] Citations

[0007] Reference 1: Wang Xiuwen, Wei Wei, Wang Xingguo, et al. Research progress on the sources and functions of branched-chain fatty acids[J]. China Oils and Fats, 2018, 43(12):5.

[0008] Cited literature 2: Loh, JS, Mak, WQ, Tan, LKS et al. Microbiota-gut-brain axis and its therapeutic applications in neurodegenerative diseases. Sig TransductTarget Ther 9, 37 (2024).

[0009] Reference 3: WO2023031226A1;

[0010] Reference 4: CN118416112B;

[0011] Reference 5: CN115300530A;

[0012] Reference 6: Ran-Ressler RR, et al. Branched chain fatty acids reduce theincidence of necrotizing enterocolitis and alter gastrointestinal microbialecology in a neonatal rat model.PLoS One.2011;6(12):e29032. Summary of the invention

[0013] Problem that the invention aims to solve

[0014] Currently, most of the treatments for neurodegenerative diseases target the intestinal flora and improve the occurrence and development of neurological diseases by regulating the intestinal microecological environment.

[0015] In the process of studying branched-chain fatty acids (BCFAs), the present invention unexpectedly discovered that BCFAs also have certain effects in regulating brain neuroimmunity, relieving and improving neuroinflammation. Therefore, on this basis, the present invention screened a branched-chain fatty acid composition that can maintain the health of the nervous system by regulating the phenotypic polarization of microglia.

[0016] It should be noted that the neuroinflammation mentioned in the present invention refers to a non-disease state in which the nerves are not healthy and full due to the presence of inflammatory factors.

[0017] Solutions for solving problems

[0018] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0019] [1]. A branched fatty acid composition, wherein the branched fatty acid comprises C15:0 and C17:0;

[0020] The C15:0 includes an isomeric form (iso-C15:0) and an anti-isomer form (anteiso-C15:0);

[0021] The C17:0 includes an isomeric form (iso-C17:0) and an anteisomeric form (anteiso-C17:0);

[0022] Based on the total mass of the C15:0 and C17:0 branched-chain fatty acids,

[0023] The content of iso-C15:0 is not less than 10%, the content of anteiso-C15:0 is 15% to 34%, the content of anteiso-C17:0 is 20% to 35%, and the content of iso-C17:0 is not less than 25%.

[0024] [2] The composition according to [1], wherein, based on the total weight of C15:0 and C17:0,

[0025] The content of iso-C15:0 is 10% to 20%, the content of anteiso-C15:0 is 19% to 34%, the content of anteiso-C17:0 is 22% to 35%, and the content of iso-C17:0 is 25% to 35%.

[0026] [3] The composition according to [1] or [2], wherein, based on the total mass of C15:0 and C17:0,

[0027] The content of iso-C15:0 is 13% to 18%, the content of anteiso-C15:0 is 19% to 32%, the content of anteiso-C17:0 is 23.5% to 35%, and the content of iso-C17:0 is 25% to 32%.

[0028] [4]. Use of the branched fatty acid composition described in any one of [1] to [3] in the preparation of food that helps regulate brain neuroimmunity.

[0029] [5]. The use according to [4], wherein the helping to regulate brain neuroimmunity includes regulating microglial polarization.

[0030] [6]. The use according to [5], wherein the regulation of microglia polarization comprises at least one of inhibiting microglia polarization to M1 phenotype, promoting microglia polarization to M2 phenotype, increasing the amount of IL-4 and / or IL-10 released by microglia, and reducing the amount of TNF-α, IL-1β and / or NO released by microglia.

[0031] [7]. Use of the branched fatty acid composition described in any one of [1] to [3] in the preparation of food for assisting in maintaining the health of the central nervous system.

[0032] [8] The use according to [7], wherein the assistance in maintaining the health of the central nervous system includes alleviating neuroinflammation for non-therapeutic purposes.

[0033] [9]. The use according to any one of [4] to [8], wherein the food is candy, a beverage, a dairy product or a baked product.

[0034]

[10] . The use according to [9], wherein the food is an oral preparation, and the oral preparation includes any one or more of tablets, pills, granules, powders, capsules and oral liquids.

[0035] Effects of the Invention

[0036] The branched fatty acid composition provided by the present invention comprises C15:0 and C17:0, and more specifically comprises iso-C15:0, anteiso-C15:0 and anteiso-C17:0 and iso-C17:0. The results after the branched fatty acid composition is applied to the brain microglia induced by LPS show that the trend of promoting the differentiation of microglia into M2 phenotype microglia is higher than the trend of promoting its differentiation into M1 phenotype microglia, thereby promoting the secretion of anti-inflammatory cytokines such as IL-4 and IL-10, and inhibiting the secretion of pro-inflammatory cytokines such as NO, TNF-a, and IL-1β. The iso-C15:0, anteiso-C15:0, anteiso-C17:0 and iso-C17:0 in the branched fatty acid composition have a synergistic effect, which can play a role in regulating the neuroimmune state of the brain and maintaining the health of the central nervous system. DETAILED DESCRIPTION

[0037] Various exemplary embodiments, features and aspects of the present invention will be described in detail below. The word "exemplary" used here means "used as an example, embodiment or illustrative". Any embodiment described here as "exemplary" is not necessarily interpreted as being superior or better than other embodiments.

[0038] In addition, in order to better illustrate the present invention, numerous specific details are provided in the following specific embodiments. It should be understood by those skilled in the art that the present invention can be implemented without certain specific details. In other examples, methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the subject matter of the present invention.

[0039] Unless otherwise stated, the units used in this specification are all international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0040] In this specification, the word "may" means both performing a certain process and not performing a certain process.

[0041] In this specification, the references to "some specific / preferred embodiments", "other specific / preferred embodiments", "embodiments", etc., mean that the specific elements (e.g., features, structures, properties and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not exist in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0042] Throughout the specification, “optional” and “optionally” mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0043] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints numerical values ​​A and B.

[0044] In the present invention, the word "may" includes both performing a certain process and not performing a certain process.

[0045] In the present invention, the term "a" or "an" or "the" may mean "one", and may also mean "one or more", "at least one" and "one or more than one".

[0046] In the present invention, the terms "comprising", "having", "including" or "containing" may mean inclusive or open-ended, and do not exclude additional, uncited elements or method steps. At the same time, "comprising", "having", "including" or "containing" may also mean closed, excluding additional, uncited elements or method steps.

[0047] In the present invention, the term "about" is used to define the numerical range and parameters of the present invention are approximate values, and the specific relevant values ​​are presented as accurately as possible. Unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in the present invention are modified by "about". Here, "about" usually means that the actual value is within ±5%, ±3%, ±1% or ±0.5% of a particular value or range.

[0048] Unless otherwise defined, other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0049] The present invention is mainly completed based on the following insights:

[0050] Branched-chain fatty acids have been widely reported to be used to improve intestinal health, and their other functions have yet to be explored and studied. The present invention uses LPS-induced brain neuroimmune cell microglia as an in vitro neuroinflammation model, first selects branched-chain fatty acids with less cytotoxicity and intervention concentrations, and then mixes branched-chain fatty acids of different concentrations to obtain a branched-chain fatty acid composition, and explores a branched-chain fatty acid composition that can improve neuroinflammation and thus regulate the brain's neuroimmune state.

[0051] I. Branched-chain fatty acid composition

[0052] The branched fatty acid composition of the present invention comprises C15:0 and C17:0, specifically comprises iso-C15:0, anteiso-C15:0, anteiso-C17:0 and iso-C17:0.

[0053] In the present invention, the "isomeric" or "isomeric branched-chain fatty acid (iso-BCFA)" refers to a fatty acid with a methyl branch on the second to last carbon atom of the carbon chain backbone of the fatty acid molecule. The "anteisomeric" or "anteisomeric branched-chain fatty acid (anteiso-BCFA)" refers to a fatty acid with a methyl branch on the third to last carbon atom of the carbon chain backbone of the fatty acid molecule.

[0054] The composition of the present invention may contain, in addition to the above-mentioned four branched fatty acids of iso-C15:0, anteiso-C15:0, anteiso-C17:0 and iso-C17:0, other types of branched or straight-chain fatty acids may be contained. The content of these other fatty acids in the composition may be 50% by mass or less, 30% by mass or less, or 10% by mass or less.

[0055] Further, in the composition, based on the total mass of the four branched-chain fatty acids of iso-C15:0, anteiso-C15:0, anteiso-C17:0 and iso-C17:0, the content of iso-C15:0 is not less than 10%, preferably 10% to 20%, and more preferably 13% to 18%, for example, 10%, 11%, 12%, 13%, 13.1%, 13.2%, 13.3%, 13.4%, 13.5%, 13.6%, 13.7%, 13.71%, 13.72%, 13.73%, 13.74%, 13.75%, 13.76%, 13.77%, 13.78%, 13.79%, 13.80%, 13.81%, 13.82%, 13.83%, 13.84%, 13.85%, 13.86%, 13.87%, 13.88%, 13.89%, 13.90%, 13.91%, 13.92%, 13.93%, 13.94%, 13.95%, 13.96%, 13.97%, 13.98%, 13.99%, 13.91 ...1%, 13.92%, 13.93%, 13.94%, %, 13.9%, 14%, 15%, 16%, 16.1%, 16.11%, 16.12%, 16.13%, 16.14%, 16.15%, 16.16%, 16.17%, 16.18%, 16.19%, 16.2%, 16.3%, 16.4%, 16.5%, 16.6%, 16.7%, 16.8%, 16.9%, 17%, 17.1%, 17.2%, 17.3%, 17.4%, 17.5%, 17.6%, 17.7%, 17.71%, 17.72%, 17.73%, 17.74%, 17.75%, 17.76%, 17.77%, 17.78%, 17.79%, 17.8%, 17 .9%, 18%, 19%, 20%; the content of anteiso-C15:0 is 15% to 34%, preferably 19% to 34%, more preferably 19% to 32%, for example 10%, 15%, 16%, 17%, 18%, 19%, 19.1%, 19.2%, 19.3%, 19.4%, 19.41%, 19.42%, 19.43%, 19.44%, 19.45%, 19.46%, 19.47%, 19.48%, 19.49%, 19.5%, 19.6%, 19.7%, 19.8%, 19.9%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28 %, 29%, 29.1%, 29.2%, 29.3%, 29.4%, 29.5%, 29.6%, 29.61%, 29.62%, 29.63%, 29.64%, 29.65%, 29.66%, 29.67%, 29.68%, 29.69%, 29.7%, 29.8%, 29.9% , 30%, 31%, 31.01%, 31.02%, 31.03%, 31.04%, 31.05%, 31.06%, 31.07%, 31.08%, 31.09%, 31.1%, 31.2%, 31.3%, 31.4%, 31.5%, 31.6%, 31.7%, 31.8%, 31.9%, 32%, 33%, 34%; the content of anteiso-C17:0 is 20% to 35%, preferably 25% to 35%, more preferably 25% to 32%, for example 20%, 20.5%, 21%, 21.5%, 22%, 22.5%, 23%, 23.1%, 23.2%, 23.3%, 23.4%, 23.5%, 23.6%, 23.61%, 23.62%, 23.63%, 23.64%, 23.65%, 23.66%, 23.67%, 23.68%, 23.69%, 23.70%, 23.71%, 23.72%, 23.73%, 23.74%, 23.75%, 23.76%, 23.77%, 23.78%, 23.79%, 23.80%, 23.81%, 23.82%, 23.83%, 23.84%, 23.85%, 23.86%, 23.87%, 23.88%, 23.89%, 23.90%, 23.91%, 23.92%, 23.93%, 23.94%, 23.95%, 23.96%, 23.97%, 23.98%, 23.99%, 23. 3.7, 23.8, 23.9, 24%, 24.5, 25%, 25.5%, 25.51%, 25.52%, 25.53%, 25.54%, 25.55%, 25.56%, 25.57%, 25.58%, 25.59%, 26%, 26.5, 27%, 27.5, 28%, 28.5, 29%, 29.5, 30%, 31%, 32%, 33%, 34%, 35%; the content of iso-C17:0 is not less than 25%, preferably 25% to 35%, more preferably 25 % to 32%, for example 25%, 25.5%, 26%, 26.5%, 27%, 27.5%, 27.6%, 27.7%, 27.71%, 27.72%, 27.73%, 27.74%, 27.75%, 27.76%, 27.77%, 27.78%, 27.79%, 27.8%, 28%, 28.1%, 28.2%, 28.3%, 28.4%, 28.5%, 28.6%, 28.61%, 28.62%, 28.63%, 28.64%, 28.65%, 28. 66%, 28.67%, 28.68%, 28.69%, 28.7%, 28.8%, 28.9%, 29%, 29.5%, 30%, 31%, 31.1%, 31.2%, 31.3%, 31.4%, 31.5%, 31.51%, 31.52%, 31.53%, 31.54%, 31.55%, 31.56%, 31.57%, 31.58%, 31.59%, 31.6%, 31.7%, 31.8%, 31.9%, 32%, 33%, 34%, 35%. .

[0056] The present invention does not specifically limit the source of branched fatty acids. For example, they can be separated and extracted from natural substances containing branched fatty acids, such as bacterial biofilms, animal sebum, mammary tissue, and plants; they can also be prepared by biological or chemical synthesis, such as synthesis using microorganisms or Caenorhabditis elegans. For example, in microorganisms or Caenorhabditis elegans, branched fatty acids are synthesized by enzymatic reactions using branched-chain amino acids as substrates. At present, the common method in the art is to separate and extract branched fatty acids from lanolin. Free lanolin alcohol and free lanolin acid soap can be prepared by saponification of lanolin, and then the branched fatty acids in lanolin are obtained through multiple steps such as alcohol soap separation process and preparation and extraction of fatty acids.

[0057] II. Food

[0058] The food of the present invention contains or uses the above-mentioned branched fatty acid complex, thereby containing C15:0 and C17:0 in the food of the present invention, specifically containing iso-C15:0, anteiso-C15:0, anteiso-C17:0 and iso-C17:0.

[0059] The present invention does not particularly limit the absolute content of C15:0 and C17:0 in food, as long as it complies with the requirements of relevant laws and regulations, for example, it complies with the quantitative limit of fatty acids in food in GB 5009.168-2016 "National Food Safety Standard Determination of Fatty Acids in Food". In some embodiments, the content of C15:0 is not higher than 0.0033g / 100g in solid food, for example, 0.0001 to 0.0033g / 100g; and is not higher than 0.0013g / 100g in liquid food, for example, 0.0001 to 0.0013g / 100g. In some embodiments, the C17:0 content in solid foods is not higher than 0.0066 g / 100 g, for example, 0.0001 to 0.0066 g / 100 g; and in liquid foods, the content is not higher than 0.0026 g / 100 g, for example, 0.0001 to 0.0026 g / 100 g.

[0060] The present invention does not particularly limit the types of food, and examples thereof include candies, beverages, dairy products or baked goods. For candies, examples include hard candies, crisp candies, gel candies, compressed candies and aerated candies. For beverages, examples include carbonated beverages, tea beverages, coffee beverages, fruit and vegetable juice beverages and lactic acid bacteria beverages. For dairy products, examples include fermented milk, cheese and milk powder. For baked goods, examples include bread, cakes and biscuits. In addition, the food of the present invention can also be a health food, such as various types of oral preparations, including tablets, pills, granules, powders, capsules and oral liquids.

[0061] In addition to C15:0 and C17:0 in the branched-chain fatty acid composition, depending on the type of food and the final needs of the applicable objects, in some embodiments, the food described in the present invention also contains any one or more of the following ingredients: plant product ingredients, animal dairy product ingredients, animal meat product ingredients, functional added ingredients and any acceptable excipients.

[0062] As for the plant product ingredients, examples include fruits such as figs, pomegranates, kiwis, tangerines, oranges, pineapples, strawberries, apples, bananas, grapes, pears, cherries, blueberries, blackberries, blackcurrants, cranberries, raspberries, melons, emblica chinensis and bilberry or their extracts; vegetables such as onions, cucumbers, tomatoes, cauliflower, carrots, spinach, kale, Brussels sprouts, garlic, basil, oregano or their extracts; rice (indica rice , japonica rice, glutinous rice), wheat (wheat, barley, oats, rye), corn, sorghum, millet, sorghum, yellow rice, buckwheat, soybean, broad bean, pea, mung bean, red bean, kidney bean and other cereals or their extracts; walnut, pistachio, cashew, hazelnut, almond, apricot, pine nut, peanut, melon seed, chestnut, macadamia, ginkgo and other nuts or their extracts; coffee or its extract; and some edible and medicinal plant-based Chinese medicinal materials or their extracts.

[0063] As for animal dairy ingredients, examples include fresh milk from mammals such as cows, sheep, and camels, as well as reprocessed dairy products such as whole milk powder, skim milk powder, concentrated whey protein powder, desalted whey powder, whey protein powder, and hydrolyzed whey protein powder.

[0064] Examples of animal meat ingredients include meat ingredients from pigs, cattle, sheep, aquatic animals or poultry.

[0065] Functional added ingredients include vitamin supplements, mineral supplements, nucleotide supplements, dietary fiber, functional polyunsaturated fatty acid supplements, and the like.

[0066] As for any acceptable excipients, examples include solvents, antioxidants, antibacterial agents, thickeners, diluents, cosolvents, stabilizers, emulsifiers, fillers, disintegrants, lubricants, coating materials, anti-caking agents, flavoring agents, sweeteners, edible flavors, edible colorings, etc.

[0067] III. Uses that help regulate brain neuroimmunity

[0068] The present invention unexpectedly discovered that a branched fatty acid composition comprising specific C15:0 and C17:0, specifically comprising iso-C15:0, anteiso-C15:0, anteiso-C17:0, and iso-C17:0 branched fatty acid composition has the effect of regulating the brain neuroimmune state. Moreover, when iso-C15:0, anteiso-C15:0, anteiso-C17:0 and iso-C17:0 are combined, there is a synergistic effect between the four, which can further enhance the efficacy of regulating the brain neuroimmune state. Therefore, the foods containing iso-C15:0, anteiso-C15:0, anteiso-C17:0, and iso-C17:0 branched fatty acid compositions of the present invention and the addition or use of the branched fatty acid compositions all have the effect of regulating brain neuroimmunity. The regulation of brain neuroimmunity described in the present invention is not for the purpose of preventing and treating diseases.

[0069] Furthermore, the present invention provides the use of a branched-chain fatty acid composition containing iso-C15:0, anteiso-C15:0, anteiso-C17:0, and iso-C17:0 in preparing foods that help regulate brain neuroimmunity.

[0070] In some embodiments, the effect of helping to regulate brain neuroimmunity includes regulating the polarization of microglia in brain neuroimmune cells. In some specific embodiments, the regulation of microglia polarization includes inhibiting microglia polarization to M1 phenotype, promoting microglia polarization to M2 phenotype, increasing the amount of IL-4, IL-10 released by microglia, and reducing the amount of TNF-α, IL-1β and / or NO released by microglia. In some specific embodiments, the regulation of microglia polarization includes inhibiting the trend of microglia polarization to M1 phenotype, promoting the trend of microglia polarization to M2 phenotype, increasing the amount of IL-4, IL-10 released by microglia, and reducing the amount of TNF-α, IL-1β and / or NO released by microglia. In some specific embodiments, the regulation of microglial polarization includes inhibiting the tendency of microglial cells to polarize toward the M1 phenotype, promoting the tendency of microglial cells to polarize toward the M2 phenotype, increasing the amount of IL-4 and IL-10 released by microglial cells, and reducing the amount of TNF-α, IL-1β and NO released by microglial cells.

[0071] Furthermore, the effect of helping to regulate brain neuroimmunity includes promoting the development of brain nerves and the nervous system.

[0072] In some embodiments, the nervous system is the central nervous system.

[0073] IV. Uses to assist in improving or maintaining the health of the central nervous system

[0074] The present invention unexpectedly discovered that the combination of iso-C15:0, anteiso-C15:0, anteiso-C17:0, and iso-C17:0 can play a role in assisting the improvement or maintenance of the health of the central nervous system, especially improving the discomfort or poor health caused by non-disease neuroinflammation. Moreover, when the above combination is performed, there is a synergistic effect between the four, which can further enhance the effect of improving neuroinflammation. Therefore, the branched-chain fatty acid composition containing iso-C15:0, anteiso-C15:0, anteiso-C17:0, and iso-C17:0 described in the present invention also has the effect of assisting the improvement or maintenance of the health of the central nervous system and improving the discomfort or poor health caused by non-disease neuroinflammation.

[0075] Furthermore, the present invention provides use of a branched-chain fatty acid composition containing iso-C15:0, anteiso-C15:0, anteiso-C17:0, and iso-C17:0 in preparing food for assisting in maintaining the health of the central nervous system.

[0076] In some embodiments, the assistance in maintaining the health of the central nervous system includes alleviating and improving neuroinflammation for non-therapeutic purposes, such neuroinflammation does not reach a medically pathological level.

[0077] Example

[0078] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0079] 1. Instruments and Reagents

[0080] Mouse microglial cell line BV-2 cells (Shanghai Enzyme Research Biotechnology, catalog number CC-Y2022), fetal bovine serum (Vicente, catalog number 085-150), high-glucose DMEM medium (Scott, catalog number CA0004-500ML), double antibody, culture bottles, well plates, centrifuge tubes, trypsin, dimethyl sulfoxide, lipopolysaccharide, kits, etc.

[0081] 2. Experimental Methods

[0082] 2.1 Cell culture

[0083] Mouse microglial cell line (BV2) was cultured in high-glucose DMEM medium (containing 10% fetal bovine serum) in a 5% CO 2 incubator (37° C., saturated humidity).

[0084] 2.2 Cytotoxicity assay

[0085] The CCK8 method (CCK8 kit was purchased from White Shark, catalog number BS350A) was used to detect the activity of BV2 microglia cells.

[0086] Evaluation method of cytotoxicity of nutrients: BV2 cells growing in logarithmic phase were selected and 5×10 4 Cells were seeded in 96-well plates at a density of 100 cells / mL. After overnight attachment, branched-chain fatty acids of different concentrations were added for treatment for 12 hours. After treatment, 10 μL of CCK8 solution was added to each well and incubated at 37°C for 4 hours. The absorbance at 450 nm was measured using an ELISA reader.

[0087] According to the formula: cell survival rate = [(absorbance of experimental well - absorbance of blank well) / (absorbance of control well - absorbance of blank well)] × 100% (wherein the experimental wells contain cells, culture medium, nutrients of different concentrations and CCK-8 solution; the blank wells contain culture medium and CCK-8 solution, but no cells and nutrients; the control wells contain cells, culture medium and CCK-8 solution, but no nutrients), the cell survival rate is finally calculated.

[0088] 2.3 Evaluation of anti-inflammatory efficacy

[0089] 1) Establishment of microglial inflammation model and nutrient intervention method

[0090] BV2 cells in logarithmic phase were selected and 5×10 4 The cells were seeded in 96-well plates at a number of 1 μg / mL and then adhered to the plate overnight before being divided into different groups for intervention. (1) Control group: conventional cell culture was used; (2) LPS group (model group): LPS (1 μg / mL) was used to intervene BV2 cells for 24 hours to establish a cellular neuroinflammation model; (3) Experimental group (example and comparative example group): After LPS stimulated BV2 cells for 24 hours, the six compositions were added for intervention for 12 hours.

[0091] 2) NO level measurement

[0092] The NO (nitric oxide) level in the culture medium of BV2 microglia was detected by nitrate reductase method. The experimental grouping and nutrient intervention method were as shown in 1) above, and the cell culture medium of each group of cells was collected. According to the instructions of the NO detection kit, the absorbance value of each tube was measured at a wavelength of 550nm and a light path of 0.5cm, and the NO content was calculated.

[0093] 3) Determination of inflammatory factor levels

[0094] ELISA was used to detect the content of IL-1β, TNF-α, IL-10, and IL-4 in the supernatant of BV2 cells. The experimental grouping and nutrient intervention methods were the same as those in 1) above. The cell supernatant of each group of cells was collected, and the ELISA kit instructions were followed. The absorbance values ​​of each group were measured in turn at a wavelength of 405 nm using an ELISA reader to detect the amount of IL-1β, TNF-α, IL-4, and IL-10.

[0095] 2.4 Detection of iNOS and CD206, markers of microglial M1 and M2 phenotypes

[0096] qRT-PCR was used to detect the mRNA expression levels of iNOS and CD206-related genes and then determine the changes in the content of M1 cell population and M2 cell population. The third-generation BV2 microglial cell suspension was inoculated in a 24-well culture plate with a cell density of 4×10 5 Each well was divided into groups according to the experimental design. After treatment, the cells were washed twice with PBS, using 200 μL per well. Digested with trypsin for 2.5 min, and digested with 400 μL of culture medium. The cells were blown up with a pipette and collected in enzyme-free centrifuge tubes. Total RNA of each group of cells was extracted. Reverse transcription was performed using a reverse transcription kit according to the operating procedures to obtain cDNA, and the amplified cDNA was mixed with primers, fluorescent probes and qPCR Master Mix. The corresponding PCR cycle program and parameters were set according to the requirements of qPCR, and the corresponding mRNA expression level was analyzed in the real-time PCR detection system. The specific primer information is as follows:

[0097] iNOS:

[0098] Upstream: 5'-GGCTTGCCCCTGGAAGTTT-3' (SEQ ID NO. 1),

[0099] Downstream: 5′-TGCAAGTGAAATCCGATGTGG-3′ (SEQ ID NO. 2);

[0100] CD206:

[0101] Upstream: 5'-TTCAGCTATTGGACGCGAGG-3' (SEQ ID NO. 3),

[0102] Downstream: 5'-GAATCTGACACCCAGCGGAA-3' (SEQ ID NO. 4);

[0103] GAPDH:

[0104] Upstream: 5'-GGTTGTCTCCTGCGACTTCA-3' (SEQ ID NO. 5),

[0105] Downstream: 5'-TGGTCCAGGGTTTCTTACTCC-3' (SEQ ID NO. 6).

[0106] 3. Cytotoxicity test of branched-chain fatty acids

[0107] In this study, six branched-chain fatty acids, iso-C14:0, iso-C15:0, anteiso-C15:0, iso-C16:0, iso-C17:0, and anteiso-C17:0, were selected to investigate the regulatory effects of different compositions on neuroinflammation. First, the toxic effects of the six branched-chain fatty acids on microglial cells were investigated. The results are shown in Table 1. It can be seen from the table that the cytotoxic effect of iso-C17:0 dropped to 90.79% when the concentration reached 25 micromoles / liter, and the cell viability of the other four branched-chain fatty acids was still above 100% at a concentration of 25%, so the concentration of branched-chain fatty acids in the present invention was uniformly determined to be 25 micromoles / liter.

[0108] Table 1 Effects of different concentrations of branched-chain fatty acids on microglial cell viability (%)

[0109]

[0110] 4. Inhibitory effects of branched-chain fatty acids on neuroinflammation

[0111] Microglia, as resident immune cells in the central nervous system, are key players in neuroinflammation. LPS-induced microglia activate the Toll-like receptor (TLR4) NF-kB signaling pathway, accelerate the transcription of inflammatory genes, and further cause the differentiation of microglia M1 phenotype and M2 phenotype.

[0112] M1 phenotype microglia secrete proinflammatory cytokines such as NO, IL-1β, and TNF-a, exacerbating the inflammatory response. NO free radicals are synthesized by enzymes such as NOS (eNOS) and iNOS through the l-arginine pathway. Under normal physiological conditions, iNOS is dormant in dormant cells. However, under pathological conditions, it produces a large amount of NO and plays a dual role in chronic infection and inflammation. Reducing the production of NO may be an effective strategy to improve inflammation; IL-1β has strong proinflammatory activity and can induce a variety of proinflammatory mediators, such as cytokines and chemokines; tumor necrosis factor-a (TNF-α) is a pleiotropic cytokine produced by a variety of cells during inflammatory responses and immune regulatory stimulation, which can induce cell apoptosis.

[0113] The M2 type can secrete anti-inflammatory cytokines such as IL-4 and IL-10 to reduce neuroinflammatory reactions. Among them, IL-4 is a typical type II immune cytokine, which plays an important immunomodulatory role in type II immune diseases such as asthma, allergic dermatitis and urticaria. Studies have shown that IL-4 can promote the differentiation of macrophages into M2 macrophages, thereby exerting an anti-inflammatory effect; interleukin 10 is a multi-cell-derived, multifunctional cytokine that regulates cell growth and differentiation, participates in inflammatory and immune responses, and is a recognized inflammatory and immunosuppressive factor.

[0114] Therefore, regulating the transformation of microglia to M2 is a potential strategy to inhibit neuroinflammation. The present invention selects the effects of different branched-chain fatty acids mixed in different proportions on LPS-induced BV2 microglial inflammation.

[0115] Examples 1 to 3

[0116] According to the method in step 1) of 2.3 above, a microglial inflammation model was established and nutrient intervention was performed. The composition of branched-chain fatty acids is shown in Table 2. The concentration of branched-chain fatty acids in the microglial inflammation model was 25 μmol / L. After the intervention, the NO level, inflammatory factor level, and iNOS and CD206 content were detected.

[0117] Comparative Examples 1 to 6

[0118] According to the method in step 1) of 2.3 above, a microglial inflammation model was established and nutrient intervention was performed. The composition of branched-chain fatty acids is shown in Table 2. The concentration of branched-chain fatty acids in the microglial inflammation model was 25 μmol / L. After the intervention, the NO level, inflammatory factor level, and iNOS and CD206 content were detected.

[0119] Table 2 Combination design of different ratios of different branched-chain fatty acids (%)

[0120] iso-C15:0 anteiso-C15:0 anteiso-C17:0 iso-C17:0 iso-C14:0 iso-C16:0 Example 1 17.79 19.41 35.00 27.75 - - Example 2 13.77 31.06 23.65 31.52 - - Example 3 16.18 29.62 25.51 28.69 - - Comparative Example 1 16.18 29.62 25.51 - 28.69 Comparative Example 2 16.18 29.62 - - 25.51 28.69 Comparative Example 3 - - 25.51 28.69 16.18 29.62 Comparative Example 4 15.00 35.00 25.00 25.00 - - Comparative Example 5 15.00 25.00 40.00 20.00 - - Comparative Example 6 5.00 25.00 45.00 25.00 - -

[0121] By applying different branched-chain fatty acid compositions to a microglial inflammation model, the inventors unexpectedly discovered that a composition comprising four branched-chain fatty acids composed of two different carbon chains and different structures, type a and type i, has a regulatory effect on neuroinflammation. The specific results are as follows:

[0122] Table 3 Effects of different branched-chain fatty acid compositions on LPS-induced pro-inflammatory cytokines in BV2 microglia

[0123] NO(μM / L) TNF-a (ng / L) IL-1β (ng / L) Control group 16.62±1.27 191.69±12.51 12.36±0.53 Model Group 29.35±0.36 254.96±2.71 20.32±0.53 Example 1 22.19±1.21 168.92±23.82 7.54±0.47 Example 2 20.86±1.32 149.54±5.73 7.62±0.68 Example 3 23.65±1.93 173.29±5.36 7.84±0.18 Comparative Example 1 24.59±1.22 202.89±4.95 8.79±1.12 Comparative Example 2 23.98±1.67 215.76±2.92 8.23±1.25 Comparative Example 3 24.69±1.09 237.55±1.89 9.12±0.98 Comparative Example 4 25.32±0.47 256.28±23.51 8.43±1.03 Comparative Example 5 25.01±1.54 216.49±12.23 9.45±2.29 Comparative Example 6 27.25±1.14 223.41±11.52 9.07±0.46

[0124] Table 3 shows the effect of branched fatty acid compositions mixed in different proportions on the production of pro-inflammatory factors by LPS-induced BV2 microglia. It can be seen from the table that the content of pro-inflammatory factors NO, TNF-a and IL-1β produced by microglia increased significantly after LPS induction, indicating that LPS induction causes microglia to be severely damaged and produce a large number of pro-inflammatory factors.

[0125] In the examples and comparative examples after co-treatment with different branched-chain fatty acid compositions and LPS, it can be seen that pro-inflammatory factors are reduced to varying degrees.

[0126] Among them, for the proinflammatory factor IL-1β, all branched fatty acid compositions including comparative examples and embodiments showed good reduction effects, and IL-1β was significantly lower than that of the control group, and after one-way ANOVA difference significance analysis, it was found that they all had extremely significant effects (p < 0.0001). Further comparison found that the IL-1β production of Examples 1-3 was 7.54-7.84ng / L, while the production of the comparative example was between 8.23-9.45ng / L. Obviously, the production of the proinflammatory factor IL-1β in the embodiment was lower than that in the comparative example; for the proinflammatory factor NO, it can be seen from the table that after treatment with the embodiments and comparative examples, they were all reduced compared with the model group. Except for comparative example 6, which had no significant difference with the model group (p > 0.05), the other groups all produced a significant effect of reducing NO production, but further Comparison found that the production of NO in Example 2 was 20.86 μM / L, the lowest among all groups, and the other two examples were also lower than the comparative example; for the proinflammatory factor TNF-a, the inventors found that all the compositions had a certain inhibitory effect on the production of TNF-a after treatment, but after difference analysis, it was found that only Examples 1-3 and Comparative Example 1 (p < 0.0001) had significant differences from the model group (p = 0.041), and Comparative Examples 2-6 had no significant difference from the model group (p > 0.05). In particular, Example 2 had the lowest production of TNF-a, and was significantly higher than Comparative Examples 1-6 (p < 0.01).

[0127] The above results indicate that the four branched fatty acids iso-C15:0, anteiso-C15:0, anteiso-C17:0 and iso-C17:0 in the fatty acid composition have a good effect in reducing the production of proinflammatory factors induced by LPS treatment at 13.77%-17.79%, 19.41%-31.06%, 23.65%-35% and 27.75%-31.52%, respectively.

[0128] Table 4 Effects of different branched-chain fatty acid compositions on LPS-induced pro-inflammatory cytokines in BV2 microglia

[0129] IL-10 (ng / L) IL-4 (ng / L) Control group 243.66±6.61 58.23±2.56 Model Group 202.28±3.16 35.59±9.73 Example 1 221.76±20.62 39.24±1.24 Example 2 244.75±23.42 41.03±1.42 Example 3 217.16±28.77 39.45±0.78 Comparative Example 1 183.62±16.59 32.58±5.69 Comparative Example 2 172.69±8.59 28.86±7.41 Comparative Example 3 163.98±12.25 29.58±1.32 Comparative Example 4 157.45±20.81 27.25±1.22 Comparative Example 5 173.94±18.34 27.58±1.41 Comparative Example 6 168.14±9.27 27.70±1.49

[0130] Table 4 shows the effects of different combinations of branched-chain fatty acid treatment on LPS-induced anti-inflammatory factors in BV2 microglia. It can be seen from the table that the production of IL-4 in Examples 1-3 is 35.59 ng / L higher than that in the model group, while the production of IL-4 in Comparative Examples 1-6 is equivalent to or lower than that in the model group. For the anti-inflammatory factor IL-10, Examples 1-3 have the effect of promoting the production of the anti-inflammatory factor IL-10, and after analysis of the significance of the difference, it was found that both Examples 1 and 2 could reach the level of the control group, with no significant difference from the control group (p>0.05), while the production of IL-4 in Comparative Examples 1-6 was at the level of the model group or lower than that of the model group, especially Example 2, whose production of IL-4 was significantly higher than that in Comparative Examples 1-6 (p<0.05). Therefore, the above conclusions show that Examples 2 and 3 have a regulatory effect on the production of anti-inflammatory factors.

[0131] In order to determine whether the inhibition of BV2 cell inflammatory response by the branched-chain fatty acid composition is related to phenotypic polarization, the present invention uses RT-qPCR to detect the mRNA expression of BV2 cell M1 phenotype marker iNOS and M2 phenotype marker CD206. The results are shown in Table 5.

[0132] Table 5 RT-PCR detection of iNOS and CD206 mRNA expression levels in BV2 cells of each group

[0133] iNOS CD206 Control group 1.03±0.14 1.05±0.22 Model Group 1.94±0.035 0.43±0.046 Example 1 0.70±0.052 4.80±0.16 Example 2 0.35±0.099 6.50±0.063 Example 3 0.53±0.037 3.46±0.076 Comparative Example 1 1.35±0.079 1.09±0.025 Comparative Example 2 1.62±0.079 0.89±0.023 Comparative Example 3 1.48±0.065 0.79±0.046 Comparative Example 4 1.72±0.083 0.63±0.056 Comparative Example 5 1.68±0.051 0.81±0.021 Comparative Example 6 1.43±0.078 0.59±0.036

[0134] As can be seen from the table, the mRNA expression of iNOS, a marker of M1 cell polarization, in the three examples was significantly lower than that in the model group (p < 0.0001), especially in Example 2, the mRNA expression of iNOS was significantly lower than that in the control group (p < 0.0001). The mRNA expression of CD206 in the three examples was significantly higher than that in the model group (p < 0.0001), and Example 2 was significantly higher than that in the control group (p < 0.001). The iNOS content in the six comparative examples was significantly higher than that in the three examples, and CD206 was significantly lower than that in the examples (p < 0.05). The above results further prove that the four branched fatty acids iso-C15:0, anteiso-C15:0, anteiso-C17:0 and iso-C17:0 have a good effect on reducing the production of pro-inflammatory factors induced by LPS treatment at 13.77%-17.79%, 19.41%-31.06%, 23.65%-35% and 27.75%-31.52%, respectively, among which Example 2 in particular has the best effect of promoting the production of anti-inflammatory factors.

Claims

1. A branched-chain fatty acid composition, characterized in that: The branched-chain fatty acids include C15:0 and C17:0; The C15:0 includes an isomeric form (iso-C15:0) and an anti-isomer form (anteiso-C15:0); The C17:0 includes an isomeric form (iso-C17:0) and an anteisomeric form (anteiso-C17:0); Based on the total mass of the C15:0 and C17:0 branched-chain fatty acids, The content of iso-C15:0 is not less than 10%, the content of anteiso-C15:0 is 15% to 34%, the content of anteiso-C17:0 is 20% to 35%, and the content of iso-C17:0 is not less than 25%.

2. The composition according to claim 1, characterized in that Based on the total mass of C15:0 and C17:0, The content of iso-C15:0 is 10% to 20%, the content of anteiso-C15:0 is 19% to 34%, the content of anteiso-C17:0 is 22% to 35%, and the content of iso-C17:0 is 25% to 35%.

3. The composition according to claim 1 or 2, characterized in that Based on the total mass of C15:0 and C17:0, The content of iso-C15:0 is 13% to 18%, the content of anteiso-C15:0 is 19% to 32%, the content of anteiso-C17:0 is 23.5% to 35%, and the content of iso-C17:0 is 25% to 32%.

4. Use of the branched-chain fatty acid composition according to any one of claims 1 to 3 in the preparation of food that helps regulate brain neuroimmunity.

5. The use according to claim 4, characterized in that The described contribution to regulating brain neuroimmunity includes regulating microglial polarization.

6. The use according to claim 5, characterized in that The regulation of microglial polarization includes at least one of inhibiting microglial polarization to M1 phenotype, promoting microglial polarization to M2 phenotype, increasing the amount of IL-4 and / or IL-10 released by microglial cells, and reducing the amount of TNF-α, IL-1β and / or NO released by microglial cells.

7. Use of the branched-chain fatty acid composition according to any one of claims 1 to 3 in the preparation of food for assisting in maintaining the health of the central nervous system.

8. The use according to claim 7, characterized in that The assistance in maintaining the health of the central nervous system includes alleviating neuroinflammation for non-therapeutic purposes.

9. The use according to any one of claims 4 to 8, characterized in that: The food is candy, beverage, dairy product or baked food.

10. The use according to claim 9, characterized in that The food is an oral preparation, which includes any one or more of tablets, pills, granules, powders, capsules and oral liquids.

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