Branched chain fatty acid compositions and uses thereof to modulate brain neural immunity

Abstract

The invention relates to a branched chain fatty acid composition and application thereof to regulation of brain nerve immunity, and belongs to the field of nutrient substance research. The present invention provides a branched chain fatty acid composition comprising C15: 0 and C17: 0. After the branched chain fatty acid composition is used for promoting LPS-induced microglial cells, the trend of promoting the microglial cells to be differentiated into M2 phenotype microglial cells is higher than the trend of promoting the microglial cells to be differentiated into M1 phenotype microglial cells, meanwhile, secretion of anti-inflammatory factors such as IL-4 and IL-10 is promoted, and secretion of proinflammatory factors such as NO, TNF-a and IL-1beta is inhibited. C15: 0 and C17: 0 in the branched chain fatty acid composition have a better synergistic effect and play a role in regulating brain nerve immunity.

Description

Technical Field

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

[0002] The human intestine performs complex physiological functions, including absorption, digestion, motility, secretion, regulation of intestinal microbiota, and maintaining complete barrier function. The enteric nervous system is crucial to the coordination of these processes. It has a high degree of autonomy and forms an external connection with the central nervous system through the autonomic nervous system, forming a gut-brain axis. The intestine is also the largest immune organ in the human body, and neuroimmunity is a two-way regulation and interaction between the nervous system and the immune system, which is a complex and important physiological process. Glial cells are immune cells of the central nervous system. Among them, participating in the neuroinflammatory process 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 lesions 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 microbiota 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. For example, the study cited in Reference 1 found that an imbalance in the intestinal flora can produce bacterial metabolites that affect peripheral immune cells. Once peripheral immune cells are affected, they promote inflammation in the central nervous system, leading to Tau aggregation and neurodegeneration in the brain. Short-term antibiotic treatment or feeding under sterile conditions can reshape or eliminate the intestinal flora and reduce its metabolites, thereby exerting a neuroprotective effect and reducing Tau aggregation and neurodegeneration. However, many mechanisms are still unclear.

[0003] 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. BCFA has good physical and chemical properties due to its unique branched structure: 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. Many studies have shown that BCFA has unique physiological regulatory functions, including inhibiting inflammation, anti-cancer effects and improving the intestine, thereby reducing the incidence of necrotizing enterocolitis in premature infants. Many previous studies or 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 effect in inhibiting intestinal inflammation.

[0004] For example, in Reference 2, a composition containing branched-chain fatty acids such as 14-methylpentadecanoic acid, 12-methyltridecanoic acid, and 13-methyltetradecanoic acid is disclosed. The composition is added to a cell inflammation model induced by LPS to inhibit the generation of ROS in calf intestinal epithelial cells; the composition is fed to DSS-induced colitis mice to regulate the level of inflammatory factors in the mouse intestine and improve the gastrointestinal health of the mice.

[0005] Reference 3 discloses that branched-chain fatty acids were added to the CaCo2 cell culture system and administered orally to mice with DSS-induced enteritis. The results showed that BCFAs can induce hyper-SUMOylation of intestinal proteins in CaCo2 cells, thereby reducing the expression of pro-inflammatory cytokines. In addition, BCFAs also inhibit intestinal inflammation caused by DSS, reduce the activity of intestinal cell deaminase, and promote intestinal epithelial integrity.

[0006] Similarly, Reference 4 also discloses that branched-chain fatty acids can inhibit intestinal inflammation in mice with DSS-induced enteritis and enhance the resistance of the mouse intestine. Reference 5 also discloses that branched-chain fatty acids can reduce the risk of necrotizing enterocolitis (NEC) and increase the expression of IL-10 in the intestine.

[0007] Citations

[0008] Cited literature 1: Seo D, O'Donnell D, Jain N, et al. ApoE isoform-and microbiota-dependent progression of neurodegeneration in a mouse model of tauopathy[J].Science, 2023, 379(6628):eadd1236.

[0009] Reference 2: CN118416112B;

[0010] Reference 3: WO2023031226A1;

[0011] Reference 4: CN115300530A;

[0012] Reference 5: 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] The gut-brain axis is currently an important mechanism for the occurrence of neurodegenerative phenomena, and more research is focused on using the intestinal flora as a target to regulate the intestinal microecological environment through the flora to improve the occurrence and development of neurological diseases.

[0015] In the process of studying branched-chain fatty acids, the present invention unexpectedly discovered that branched-chain fatty acids also have certain effects in regulating brain neuroimmunity, relieving and improving neuroinflammation, and further screened a branched-chain fatty acid composition that can regulate inflammation caused by nerve damage by regulating microglial phenotype polarization.

[0016] It should be noted that the neuroinflammation mentioned in this specification refers to the 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 and C17:0 are in the anti-isomer form;

[0021] In terms of quality,

[0022] The content ratio of the C15:0 to the C17:0 is 3:7 to 6:4.

[0023] [2] The composition according to [1], wherein

[0024] The content ratio of the C15:0 to the C17:0 is 3.5:6.5 to 5.5:4.5.

[0025] [3] The composition according to [1] or [2], wherein, based on the total mass of the branched fatty acid composition,

[0026] The content of C15:0 is 30% to 60%, and the content of C17:0 is 40% to 70%.

[0027] [4] Use of the composition described in any one of [1] to [3] in the preparation of a food that helps regulate brain neuroimmunity.

[0028] [5]. The use according to [4], wherein the helping to regulate brain neuroimmunity includes regulating microglial polarization.

[0029] [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.

[0030] [7]. The use according to any one of [4] to [6], wherein the food is candy, a beverage, a dairy product or a baked product.

[0031] [8]. The use according to [7], wherein the candy comprises at least one of hard candy, jelly candy, crisp candy, compressed candy and aerated candy; the beverage comprises at least one of carbonated beverage, tea beverage, coffee beverage, fruit and vegetable juice beverage and lactic acid bacteria beverage; the dairy product comprises at least one of fermented milk, cheese and milk powder; and the baked food comprises at least one of bread, cake and biscuits.

[0032] [9]. 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.

[0033]

[10] . The use according to [9], wherein the assistance in maintaining the health of the central nervous system includes alleviating neuroinflammation for non-therapeutic purposes.

[0034] Effects of the Invention

[0035] After the branched fatty acid composition provided by the present invention is applied to brain microglia induced by LPS, the tendency of promoting the differentiation of microglia into M2 phenotype microglia is higher than the tendency of promoting the 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. C15:0 and C17:0 in the branched fatty acid composition have a good synergistic effect and play a role in regulating brain neuroimmunity. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The RT-PCR detection of the expression levels of iNOS and CD206 mRNA in BV2 cells of each group is shown; where **** represents p<0.0001. 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 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. In some preferred embodiments, both C15:0 and C17:0 are anteiso.

[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] Further, for the content ratio of the above C15:0 and C17:0 in the mass fatty acid composition, it has been found that the technical effect of the present invention can be obtained by setting the following mass ratio, the content ratio of the C15:0 to the C17:0 is 3:7 to 6:4. In some preferred embodiments, the content ratio of the C15:0 to the C17:0 is 3.5:6.5 to 5.5:4.5, more preferably 6:13 to 7:6, more preferably 7:13 to 13:12. Illustratively, the content ratio of the C15:0 to the C17:0 is 3:7, 3.5:7, 3.5:6.5, 4:6.5, 4:7, 4.5:7, 4.5:6.5, 5:7, 5.5:7, 5.5:6.5, 5.5:6, 6:7, 6.5:7, 7:7, 7.5:7, 8:7, 8.5:7, 9:7, 9.5:7, 10:7, 10.5:7, 3:6, 3.5:6, 4:6, 4.5:6, 5:6, 5.5:6, 6:6, 6.5:6, 7:6, 7.5:6, 8:6, 8.5:6, 9:6, 2.1:5, 2.2:5, 2.3:5, 2.4:5, 2.5:5, 3:5, 3.5:5, 4:5, 4.5:5, 5:5, 5.5:5, 5.5:4.5, 5.5:4, 6:5, 6.5:5, 7:5, 7.5:5, 1.68:4, 2:4, 3:4, 4:4, 5:4, 6:4, 6:13, 7:13, 8:13, 9:13, 10:13, 11:13, 12:13, 13:13, 14:13, 13:12, 12:12, 11:12, 10:12, 9:12, 8:12, 7:12.

[0055] In addition, for the above-mentioned branched fatty acid composition of the present invention, in addition to containing the above-mentioned C15:0 and C17:0 two kinds of fatty acids, it is optional to contain or not contain other kinds of fatty acids. These other kinds of fatty acids can be branched or linear. And, the amount of these other kinds of fatty acids is not particularly limited in principle. In some preferred embodiments, based on the gross mass of the branched fatty acid composition, the total content of the above-mentioned C15:0 and C17:0 can be 10% to 100%, such as 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%.

[0056] In some embodiments, based on the total mass of the branched fatty acid composition, the content of C15:0 is 30% to 60%, preferably 35% to 55%, more preferably 35% to 53%, and more preferably 38% to 52%, for example 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 37.5%, 38%, 38.5%, 39%, 39.5%, 40%, 40.5%, 41%, 41.5%, 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%, %, 57%, 58%, 59%, 60%; the C17:0 is 40% to 70, preferably 45% to 65%, more preferably 47% to 65%, more preferably 48% to 62%, for example 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 58.5%, 58.7%, 58.8%, 59%, 60%, 61%, 61.5%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%.

[0057] 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.

[0058] II. Food

[0059] The food of the present invention contains or uses the branched-chain fatty acid complex, thereby containing C15:0 and C17:0. In some preferred embodiments, both C15:0 and C17:0 are anteisomers.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] Examples of animal meat ingredients include meat ingredients from pigs, cattle, sheep, aquatic animals or poultry.

[0066] Functional added ingredients include vitamin supplements, mineral supplements, nucleotide supplements, dietary fiber, functional polyunsaturated fatty acid supplements, and the like.

[0067] 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.

[0068] III. Uses that help regulate brain neuroimmunity

[0069] The present invention unexpectedly discovered that a branched fatty acid composition comprising C15:0 and C17:0, especially a branched fatty acid composition comprising trans-isomer C15:0 and trans-isomer C17:0, has the effect of regulating brain neuroimmunity. Moreover, when C15:0 and C17:0 are combined, there is a synergistic effect between the two, which can further enhance the efficacy of regulating brain neuroimmunity. Therefore, the branched fatty acid composition containing C15:0 and C17:0 described in the present invention and the food added or used with the branched fatty acid composition 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.

[0070] Furthermore, the present invention provides the use of a branched-chain fatty acid composition containing C15:0 and C17:0 in preparing a food that helps to regulate brain neuroimmunity.

[0071] 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.

[0072] Furthermore, the effect of helping to regulate brain neuroimmunity includes promoting the development of brain nerves and the nervous system.

[0073] In some embodiments, the nervous system is the central nervous system.

[0074] IV. Uses to assist in improving or maintaining the health of the central nervous system

[0075] The present invention unexpectedly discovered that combining C15:0 and C17:0, for example, combining the trans-isomer C15:0 and the trans-isomer C17:0, can play a role in assisting the improvement or maintenance of the health of the central nervous system, especially can improve the discomfort or unhealthy fullness caused by non-disease neuroinflammation. Moreover, when C15:0 and C17:0 are combined, there is a synergistic effect between the two, which can further enhance the effect of improving neuroinflammation. Therefore, the branched-chain fatty acid composition containing C15:0 and 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 unhealthy fullness caused by non-disease neuroinflammation.

[0076] Furthermore, the present invention provides use of a branched-chain fatty acid composition containing C15:0 and C17:0 in preparing food for assisting in maintaining the health of the central nervous system.

[0077] 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.

[0078] Example

[0079] 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.

[0080] 1. Instruments and Reagents

[0081] 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.

[0082] 2. Experimental Methods

[0083] 2.1 Cell culture

[0084] 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).

[0085] 2.2 Cytotoxicity assay

[0086] The CCK8 method (CCK8 kit was purchased from White Shark, catalog number BS350A) was used to detect the activity of BV2 microglia cells.

[0087] 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.

[0088] 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.

[0089] 2.3 Evaluation of anti-inflammatory efficacy

[0090] 1) Establishment of microglial inflammation model and nutrient intervention method

[0091] 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, 10 compositions were added for intervention for 12 hours.

[0092] 2) NO level measurement

[0093] 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.

[0094] 3) Determination of inflammatory factor levels

[0095] ELISA was used to detect the contents 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 above. The cell supernatant of each group of cells was collected, and the operation was performed according to the instructions of the ELISA kit. The absorbance value of each group was measured in turn at a wavelength of 405nm using an enzyme marker to detect the amount of IL-1β, TNF-α, IL-4, and IL-10.

[0096] 2.4 Detection of iNOS and CD206, markers of microglial M1 and M2 phenotypes

[0097] qRT-PCR was used to detect the mRNA expression levels of iNOS and CD206-related genes, and then the content changes of M1 cell population and M2 cell population were determined. The third-generation BV2 microglial cell suspension was inoculated in a 24-well culture plate with a cell density of 4×105 cells / well, and the cells were treated in groups according to the experimental design. After the treatment, the cells were washed twice with PBS, using 200μL per well. Trypsin digestion was performed for 2.5min, and the digestion was terminated with 400μL of culture solution. The cells were blown with a pipette and collected in an enzyme-free centrifuge tube. Total RNA of each group of cells was extracted. Reverse transcription was performed using a reverse transcription kit according to the operating steps 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:

[0098] iNOS:

[0099] Upstream: 5'-GGCTTGCCCCTGGAAGTTT-3' (SEQ ID NO. 1),

[0100] Downstream: 5′-TGCAAGTGAAATCCGATGTGG-3′ (SEQ ID NO. 2);

[0101] CD206:

[0102] Upstream: 5'-TTCAGCTATTGGACGCGAGG-3' (SEQ ID NO. 3),

[0103] Downstream: 5'-GAATCTGACACCCAGCGGAA-3' (SEQ ID NO. 4);

[0104] GAPDH:

[0105] Upstream: 5'-GGTTGTCTCCTGCGACTTCA-3' (SEQ ID NO. 5),

[0106] Downstream: 5'-TGGTCCAGGGTTTCTTACTCC-3' (SEQ ID NO. 6).

[0107] 3. Cytotoxicity test of branched-chain fatty acids

[0108] In the present invention, four branched fatty acids, iso-C15:0, anteiso-C15:0, 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 four branched fatty acids on microglial cells were investigated, and 5, 10, 25, 50, 75, and 100 micromoles / liter of branched fatty acids were used for treatment, respectively. The results are shown in Table 1. It can be seen from the table that the cytotoxic effect of iso-C17:0 decreased to 90.79% when the concentration reached 25 micromoles / liter, and the cell viability of the other three branched fatty acids was still above 100% at a concentration of 25%, so the concentration of branched fatty acids in the present invention was uniformly determined to be 25 micromoles / liter.

[0109] Table 1 Effects of different concentrations of branched-chain fatty acids on microglial cell viability (%)

[0110]

[0111] 4. Inhibitory effects of branched-chain fatty acids on neuroinflammation

[0112] 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.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] Embodiments 1 to 5

[0117] 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.

[0118] Comparative Examples 1 to 5

[0119] 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.

[0120] Table 2 Combination design of different ratios of different branched-chain fatty acids (%)

[0121] anteiso-C15:0 anteiso-C17:0 iso-C15:0 iso-C17:0 Example 1 41.23 58.77 Example 2 38.49 61.51 Example 3 51.97 48.03 Example 4 35.00 65.00 Example 5 53.00 47.00 Comparative Example 1 65.00 35.00 Comparative Example 2 41.23 58.77 Comparative Example 3 58.77 41.23 Comparative Example 4 58.77 41.23 Comparative Example 5 41.23 58.77

[0122] By applying different branched-chain fatty acid compositions to a microglial inflammation model, the inventors unexpectedly discovered that two medium-chain branched-chain fatty acids of type a (anteiso) regulate neuroinflammation. The specific results are as follows:

[0123] Table 3 Effects of different branched-chain fatty acid compositions on LPS-induced pro-inflammatory cytokines in BV2 microglia

[0124] 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 15.71±1.59 165.79±23.43 5.35±0.47 Example 2 18.62±1.75 170.44±11.07 6.05±0.52 Example 3 18.86±2.22 171.49±20.15 7.26±0.56 Example 4 21.34±1.55 186.07±9.57 7.427±0.39 Example 5 21.01±0.84 193.64±12.73 7.37±0.54 Comparative Example 1 22.92±1.38 197.74±10.13 7.33±0.56 Comparative Example 2 22.40±2.05 207.31±1.692 11.43±0.59 Comparative Example 3 27.50±1.68 212.26±6.17 12.24±1.36 Comparative Example 4 23.35±1.75 226.56±19.66 10.97±0.46 Comparative Example 5 24.77±2.42 205.36±20.71 10.16±0.45

[0125] Table 3 shows the effects of different combinations of branched-chain fatty acids on LPS-induced pro-inflammatory factors in BV2 microglia.

[0126] It can be seen from the table that after LPS induction, the content of pro-inflammatory factors NO, TNF-a and IL-1β in microglia increased significantly, indicating that LPS induction caused serious damage to microglia and produced a large number of pro-inflammatory factors. In the experimental group and the control group treated with different branched fatty acid compositions and LPS, it can be seen that the pro-inflammatory factors have been reduced to varying degrees, as shown in Table 3. It can be seen from the table that the amount of NO produced in the embodiments and comparative examples is lower than that in the model group, wherein the amount in embodiment 1 is 15.71 μM / L, reaching the level of the control group before LPS treatment. After one-way ANOVA difference significance analysis of each group, it was found that embodiments 1 to 5 were all significantly lower than the model group (p < 0.0001), and comparative examples 1 and 2 were also significantly lower than the model group (p < 0.0001), but comparative example 3 had a poor effect. Although the amount of NO produced was lower than that of the model group, after significant difference analysis, it was found that there was no significant difference with the model group (p > 0.05). Although comparative examples 4 and 5 were significantly lower than the model group (p < 0.01 and p < 0.05), the amount of NO in comparative examples 4 and 5 was still higher than that in embodiments 1 to 5, and after significant difference analysis, it was found that embodiments 1 to 3 were significantly lower than comparative example 5 (p < 0.01), and embodiment 1 was significantly lower than comparative example 4 (p < 0.05).

[0127] It can be seen from Table 3 that different branched fatty acid compositions have a stronger inhibitory effect on TNF-a. Examples 1 to 5 are all significantly reduced to the level of the control group, especially the production of TNF-a in Examples 1 to 4 is lower than that in the control group. Comparative Examples 1 to 5 also inhibit the production of TNF-a, but the effect is not as good as that of the examples, and the production of TNF-a is higher than that of Examples 1 to 5.

[0128] For the anti-inflammatory factor IL-1β, the branched fatty acid composition has a significant inhibitory effect. Compared with the model group, it is found that each group has a significant reduction compared with the model group (p < 0.0001), but the production of IL-1β in Examples 1 to 5 is lower than that in Comparative Examples 1 to 5, and after a significant difference analysis, it is found that Example 1 is significantly lower than Comparative Examples 1 to 5 (p < 0.01 and p < 0.0001), and Examples 2 to 5 are extremely significantly lower than Comparative Examples 2 to 5 (p < 0.0001). It can be seen that when anteiso-C15:0 accounts for 35%-53%, it has a significant effect of inhibiting the production of anti-inflammatory factors.

[0129] Table 4 Effects of different branched-chain fatty acid compositions on LPS-induced pro-inflammatory cytokines in BV2 microglia

[0130] 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 1 227.62±19.39 43.47±2.19 Example 2 220.78±10.91 39.92±2.24 Example 3 223.368±6.64 39.59±2.99 Example 4 218.37±11.41 37.26±1.78 Example 5 218.31±13.56 38.50±1.07 Comparative Example 1 215.89±18.93 33.92±0.90 Comparative Example 2 200.38±12.50 33.19±1.42 Comparative Example 3 199.98±14.21 32.17±1.41 Comparative Example 4 188.59±22.20 32.86±2.13 Comparative Example 5 159.75±12.99 29.85±0.85

[0131] Table 4 shows the effects of different combinations of branched fatty acids on LPS-induced anti-inflammatory factors in BV2 microglia. It can be seen from the table that different branched fatty acid compositions have different effects on the production of anti-inflammatory factors. For the anti-inflammatory factor IL-10, the present invention finds that both Examples 1 to 5 and Comparative Example 1 have the effect of enhancing the production of anti-inflammatory factor IL-10, while the production of anti-inflammatory factor IL-10 in Comparative Examples 2 to 5 is close to that in the model group, or lower than that in the model group, and has no effect on the production of anti-inflammatory factor IL-10. After significant difference analysis, it was found that Examples 1 to 5 were not significantly different from the control group (p>0.05), further verifying that the generation of anti-inflammatory factor IL-10 in the example can achieve the effect of the untreated control group; at the same time, Table 4 shows the effect of each group on the production of anti-inflammatory factor IL-4, and the results show that the overall branched fatty acid is not very effective in promoting the production of IL-4, but the production of IL-4 in Examples 1 to 5 is higher than that in the model group, while IL-4 in Comparative Examples 1 to 5 is lower than that in the model group, indicating that the branched fatty acid composition of the comparative example has no effect on promoting the production of IL-4. Combining the production of the above two anti-inflammatory factors, it is proved that when anteiso-C15:0 accounts for 35%-53%, it has a significant effect on promoting the production of anti-inflammatory factor IL-10.

[0132] 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 expression of mRNA of BV2 cell M1 phenotype marker iNOS and M2 phenotype marker CD206. The results are as follows: Figure 1 As shown. As can be seen from the figure, the iNOS marker characterizing M1 cell polarization in the five embodiments is significantly lower than that in the model group (p < 0.0001), especially the mRNA expression of iNOS in Example 1 and Example 2 is significantly lower than that in the control group. The mRNA expression of CD206 in the five embodiments is significantly higher than that in the model group (p < 0.0001), and the production of CD206 in Example 1 and Example 3 is close to that of the control group. The above results further prove that the branched fatty acid composition of the five embodiments has the effect of regulating LPS-induced BV2 cell neuroinflammation, can promote the differentiation of microglia to M2 type, and promote 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 and C17:0 are in the anti-isomer form; In terms of quality, The content ratio of the C15:0 to the C17:0 is 3:7 to 6:

4.

2. The composition according to claim 1, characterized in that The content ratio of the C15:0 to the C17:0 is 3.5:6.5 to 5.5:4.

5.

3. The composition according to claim 1 or 2, characterized in that Based on the total mass of the branched-chain fatty acid composition, The content of C15:0 is 30% to 60%, and the content of C17:0 is 40% to 70%.

4. Use of the 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. The use according to any one of claims 4 to 6, characterized in that: The food is candy, beverage, dairy product or bakery product.

8. The use according to claim 7, characterized in that The candies include at least one of hard candies, jelly candies, crisp candies, compressed candies and aerated candies; the beverages include at least one of carbonated beverages, tea beverages, coffee beverages, fruit and vegetable juice beverages and lactic acid bacteria beverages; the dairy products include at least one of fermented milk, cheese and milk powder; the baked goods include at least one of bread, cakes and biscuits.

9. 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.

10. The use according to claim 9, characterized in that The assistance in maintaining the health of the central nervous system includes alleviating neuroinflammation for non-therapeutic purposes.

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