Branched chain fatty acid composition and use thereof in regulating brain immune balance

Abstract

A branched chain fatty acid composition, and a use thereof in regulating brain immune balance. The branched chain fatty acid composition comprises iso-C15:0, anteiso-C15:0, anteiso-C17:0 and iso-C16:0. The branched chain fatty acid composition inhibits the polarization of microglia into M1-phenotype, promotes the polarization of microglia into M2-phenotype, and also promotes the secretion of anti-inflammatory factors such as IL-4 and IL-10, inhibits the secretion of pro-inflammatory factors such as NO, TNF-a, and IL-1β, can regulate brain immune balance, and maintains the health of the central nervous system.

Description

Branched-chain fatty acid compositions and their uses in regulating brain immune balance Technical Field

[0001] This invention relates to branched-chain fatty acid compositions and their use in regulating brain immune balance, and more specifically to branched-chain fatty acid compositions containing C15:0, C17:0 and C16:0 and their use in regulating brain immune balance and nervous system health, belonging to the field of nutritional research. Background Technology

[0002] Branched-chain fatty acids (BCFAs) are fatty acids with one or more branched alkyl groups (mainly methyl groups) on their carbon skeleton. When the branched alkyl group is located on the second carbon atom from the alkyl end, it is called an isomer or iso-fatty acid; when it is located on the third carbon atom from the alkyl end, it is called an anteiso-fatty acid. Due to their unique branched structure, BCFAs possess excellent physicochemical properties, including: low freezing and pour points; good thermal and oxidative stability; good solubility; low foaming properties; and good air and water permeability of the membranes they form. BCFAs are widely distributed in nature, but in low amounts. It has been reported that BCFAs with different structures are present in the milk and internal tissues of ruminants (such as cow and sheep milk and meat products), and in human milk. BCFAs are more abundant in vernix caseosa and the neonatal intestine; the BCFA profile of meconium shows that BCFAs are the main components of the gastrointestinal tract of normal, healthy, full-term newborns. Premature infants have significantly fewer BCFAs in their intestines than normally healthy infants, and are more likely to develop necrotizing enterocolitis.

[0003] The enteric nervous system is crucial for coordinating processes such as absorption, digestion, motility, secretion, regulation of the gut microbiota, and maintenance of intact barrier function. It possesses a high degree of autonomy and forms an external connection with the central nervous system through the autonomic nervous system, creating the gut-brain axis. Glial cells are immune cells of the central nervous system, and their participation in neuroinflammatory processes and inducing nerve damage repair are key mechanisms for their immune function. For a long time, controlling neuroinflammatory diseases has been a focus of treatment for degenerative diseases and traumatic injuries of the central nervous system. Currently, there are no effective medical treatments for many neurodegenerative diseases, such as Alzheimer's disease and stroke. The gut-brain axis has become a breakthrough point in medical treatment. By regulating the gut microbiota through drugs or fecal microbiota transplantation, and altering the gut microenvironment, the gut microbiota can be continuously restored from multiple dimensions of the microenvironment, such as microbiota structure and short-chain fatty acids, thereby improving the occurrence and development of neurodegenerative diseases. Reference 1 discloses the interaction between the gut microbiome and oligodendrocytes, as well as the role of gut microbiome-derived metabolites in neurodegenerative diseases, indicating that the gut microbiome can regulate oligodendrocyte maturation and myelin production, and that changes in the gut microbiome are related to the pathogenesis of neurodegenerative diseases. However, many mechanisms remain unclear.

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

[0005] For example, cited reference 2 discloses a composition containing branched-chain fatty acids such as 14-methylpentadecanoic acid, 12-methyltetrazoic acid, and 13-methyltetradecanoic acid. This composition can inhibit the generation of ROS in a LPS-induced calf small intestinal epithelial cell inflammation model; and can regulate the levels of inflammatory factors in the intestines of DSS-induced colitis mice and improve the gastrointestinal health of mice.

[0006] Reference 3 discloses the addition of branched-chain fatty acids (BCFAs) to a CaCo2 cell culture system and the gavage administration of BCFAs to mice with DSS-induced enteritis. The results showed that BCFAs could induce hyper-SUMOylation of intestinal proteins in CaCo2 cells, thereby reducing the expression of pro-inflammatory cytokines. In addition, BCFAs also inhibited DSS-induced intestinal inflammation, reduced the activity of intestinal cell deaminases, and promoted intestinal epithelial integrity.

[0007] Similarly, reference 4 also discloses that branched-chain fatty acids can inhibit intestinal inflammation in mice with DSS-induced enteritis and enhance intestinal resistance. Reference 5 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.

[0008] References

[0009] Cited literature 1: Loh, JS, Mak, WQ, Tan, LKS et al. Microbiota–gut–brain axis and its therapeutic applications in neurodegenerative diseases. Sig Transduct Target Ther 9, 37 (2024).

[0010] Reference 2: CN118416112B;

[0011] Reference 3: WO2023031226A1;

[0012] Reference 4: CN115300530A;

[0013] Reference 5: Ran-Ressler RR, et al. Branched chain fatty acids reduce the incidence of necrotizing enterocolitis and alter gastrointestinal microbial ecology in a neonatal rat model. PLoS One. 2011; 6(12): e29032. Summary of the Invention

[0014] The problem the invention aims to solve

[0015] Currently, the study of neurodegenerative diseases focuses more on the gut microbiota as a target, aiming to improve the occurrence and development of neurological diseases by regulating the gut microecological environment.

[0016] In the course of researching branched-chain fatty acids (BCFAs), this invention unexpectedly discovered that BCFAs also have certain effects in regulating brain immune balance and alleviating and improving neuroinflammation. Therefore, this invention screened a BCFA composition that can maintain the health of the nervous system by regulating microglial cell phenotypic polarization.

[0017] It should be noted that the neuroinflammation mentioned in this invention refers to a non-pathological state in which the nerves are not fully healthy due to the presence of inflammatory factors.

[0018] Solution for solving the problem

[0019] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0020] [1]. A branched-chain fatty acid composition, wherein the branched-chain fatty acid comprises iso-C15:0, anteiso-C15:0, anteiso-C17:0 and iso-C16:0;

[0021] Based on the total mass of the four branched-chain fatty acids, iso-C15:0, anteiso-C15:0, anteiso-C17:0, and iso-C16:0,

[0022] The content of iso-C15:0 is not less than 10%, the content of anteiso-C15:0 is 10% to 34%, the content of anteiso-C17:0 is 15% to 35%, and the content of iso-C16:0 is not less than 20%.

[0023] [2]. According to the composition described in [1], wherein, based on the total mass of the four branched fatty acids, iso-C15:0, anteiso-C15:0, anteiso-C17:0 and iso-C16:0,

[0024] The content of iso-C15:0 is 10% to 25%, the content of anteiso-C15:0 is 12% to 34%, the content of anteiso-C17:0 is 17% to 35%, and the content of iso-C16:0 is 20% to 35%.

[0025] [3]. The composition according to [1] or [2], wherein, based on the total mass of the four branched fatty acids iso-C15:0, anteiso-C15:0, anteiso-C17:0 and iso-C16:0,

[0026] The content of iso-C15:0 is 15% to 25%, 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-C16:0 is 25% to 35%.

[0027] [4]. Use of any of the branched-chain fatty acid compositions described in [1] to [3] in the preparation of foods that help regulate the immune balance of the brain.

[0028] [5]. According to the use described in [4], the aid in regulating brain immune balance includes regulating microglia polarization.

[0029] [6]. According to the use described in [5], wherein the regulation of microglia polarization includes at least one of inhibiting microglia polarization to the M1 phenotype, promoting microglia polarization to the 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] Use of the branched-chain fatty acid composition described in any one of [1] to [3] in the preparation of food for the purpose of assisting in the maintenance of the health of the central nervous system.

[0031] [8]. According to the use described in [7], wherein the aid in maintaining the health of the central nervous system includes the relief of neuroinflammation for non-therapeutic purposes.

[0032] [9]. The use according to any one of [4] to [8], wherein the food is a confectionery, beverage, dairy product or baked food.

[0033]

[0010] . According to the use described in [9], the food is an oral preparation, which includes any one or more of tablets, pills, granules, powders, capsules and oral liquids.

[0034] The effects of the invention

[0035] The branched-chain fatty acid composition provided by this invention comprises C15:0, C17:0, and C16:0, and more specifically, iso-C15:0, anteiso-C15:0, anteiso-C17:0, and iso-C16:0. Results from the application of this branched-chain fatty acid composition to LPS-induced microglia show that it promotes the differentiation of microglia into M2 phenotype microglia more than it promotes their 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-α, and IL-1β. The iso-C15:0, anteiso-C15:0, anteiso-C17:0, and iso-C16:0 in the branched-chain fatty acid composition have a synergistic effect, which can regulate brain immune balance and maintain the health of the central nervous system. Detailed Implementation

[0036] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0037] Furthermore, to better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the present invention.

[0038] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0039] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0040] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0041] In this specification, "optional" and "optionally" mean that the events or circumstances described below may or may not occur, and the description includes both cases where the events or circumstances occur and cases where the events or circumstances do not occur.

[0042] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0043] In this invention, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0044] In this invention, the terms “a”, “an”, or “the” can mean “one”, “one or more”, “at least one”, or “one or more”.

[0045] In this invention, the terms "comprising," "having," "including," or "containing" can mean included or open-ended, and do not exclude additional, uncited elements or method steps. At the same time, "comprising," "having," "including," or "containing" can also mean closed-ended, excluding additional, uncited elements or method steps.

[0046] In this invention, the term "about" is used to define that the numerical ranges and parameters of this invention are approximate values, while specific related values ​​have been presented as precisely as possible. Unless otherwise explicitly stated, it should be understood that all ranges, quantities, values, and percentages used in this invention are modified by "about". Here, "about" generally means that the actual value is within ±5%, ±3%, ±1%, or ±0.5% of a specific value or range.

[0047] Unless otherwise defined, other technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0048] This invention is mainly based on the following insights:

[0049] Branched-chain fatty acids (BCFAs) have been widely reported for improving gut health, but their other functions remain to be explored and studied. This invention uses LPS-induced microglia as an in vitro neuroinflammation model. First, BCFAs with low cytotoxicity and appropriate intervention concentrations are selected. Then, different concentrations of BCFAs are blended to obtain BCFA compositions, exploring how these compositions can improve neuroinflammation and thus regulate brain immune balance.

[0050] I. Branched-chain fatty acid composition

[0051] The branched-chain fatty acid composition of the present invention comprises C15:0, C17:0 and C16:0, specifically comprising isomer iso-C15:0, trans isomer anteiso-C15:0, trans isomer anteiso-C17:0 and isomer iso-C16:0.

[0052] In this invention, "iso-branched fatty acid" or "iso-branched fatty acid (iso-BCFA)" refers to a fatty acid with a methyl branch on the penultimate carbon atom of the fatty acid molecule's carbon chain backbone. "Anti-iso-branched fatty acid" or "antiso-BCFA" refers to a fatty acid with a methyl branch on the penultimate carbon atom of the fatty acid molecule's carbon chain backbone.

[0053] In addition to the four branched-chain fatty acids iso-C15:0, anteiso-C15:0, anteiso-C17:0, and iso-C16:0 described above, the compositions of the present invention may optionally contain other types of branched-chain or straight-chain fatty acids. The content of these other fatty acids in the composition may be less than 50% by mass, less than 30% by mass, or less than 10% by mass.

[0054] Further, in the composition, based on the total mass of the four branched-chain fatty acids iso-C15:0, anteiso-C15:0, anteiso-C17:0, and iso-C16:0, the content of iso-C15:0 is not less than 10%, preferably 10% to 25%, more preferably 15% to 25%, 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%, 13.8%, 13%... 0.9%, 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%, 1 7%, 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%, 1 9%, 20%, 21%, 22%, 23%, 24%, 25%; the content of anteiso-C15:0 is 10%–34%, preferably 12%–34%, more preferably 15%–34%, 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%, 2 7%, 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 15%–35%, preferably 17%–35%, more preferably 20%–35%, for example 15%, 16%, 17%, 18%, 19%, 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%, 23.7%, 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%, 34.5%, 35%; the content of iso-C16:0 is not less than 20%, preferably 20% to 35%, more preferably... The percentages range from 25% to 35%, for example: 20%, 21%, 22%, 23%, 24%, 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%.

[0055] This invention does not particularly limit the source of branched-chain fatty acids. For example, they can be isolated and extracted from natural substances containing branched-chain fatty acids, such as bacterial biofilms, animal sebum, mammary tissue, and plants. They can also be prepared through biological or chemical synthesis, such as using microorganisms or *C. elegans* for synthesis. Exemplarily, branched-chain fatty acids are synthesized in microorganisms or *C. elegans* using branched-chain amino acids as substrates through enzymatic reactions. Currently, the commonly used method in this field is to isolate and extract branched-chain fatty acids from lanolin. Lanolin undergoes a saponification reaction to prepare free lanolin alcohol and free lanolin acid soap. Branched-chain fatty acids from lanolin are then obtained through multiple steps, including alcohol-soap separation and fatty acid preparation and extraction.

[0056] II. Food

[0057] The food products of this invention contain or use the above-mentioned branched-chain fatty acid compounds, thereby containing C15:0, C17:0 and C16:0, specifically iso-C15:0, anteiso-C15:0, anteiso-C17:0 and iso-C16:0.

[0058] This invention does not impose any particular limitation on the absolute content of C15:0, C16:0, and C17:0 in food, as long as it complies with relevant laws and regulations, such as the quantitative limits for fatty acids in food as specified 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 0.0033 g / 100g in solid foods, for example, 0.0001–0.0033 g / 100g; and not higher than 0.0013 g / 100g in liquid foods, for example, 0.0001–0.0013 g / 100g. In some embodiments, the content of C16:0 is no higher than 0.0066 g / 100g in solid foods, for example, 0.0001–0.0066 g / 100g; and no higher than 0.0026 g / 100g in liquid foods, for example, 0.0001–0.0066 g / 100g. In some embodiments, the content of C17:0 is no higher than 0.0066 g / 100g in solid foods, for example, 0.0001–0.0066 g / 100g; and no higher than 0.0026 g / 100g in liquid foods, for example, 0.0001–0.0026 g / 100g.

[0059] This invention does not specifically limit the types of food products, but may include candies, beverages, dairy products, or baked goods. Examples of candies include hard candies, shortbread candies, gel candies, compressed candies, and aerated candies. Examples of beverages include carbonated drinks, tea drinks, coffee drinks, fruit and vegetable juices, and lactic acid bacteria drinks. Examples of dairy products include fermented milk, cheese, and milk powder. Examples of baked goods include bread, cakes, and biscuits. Furthermore, the food products described in this invention can also be health foods, such as various types of oral preparations, including tablets, pills, granules, powders, capsules, and oral liquids.

[0060] In addition to C15:0, C17:0, and C16:0 in the branched-chain fatty acid composition, in some embodiments, depending on the type of food and the final needs of the target audience, the food of the present invention may also contain any one or more of the following ingredients: plant-based ingredients, animal dairy ingredients, animal meat ingredients, functional additives, and any acceptable excipients.

[0061] Examples of plant-based ingredients include fruits such as fig, pomegranate, kiwi, orange, tangerine, pineapple, strawberry, apple, banana, grape, pear, cherry, blueberry, blackberry, blackcurrant, cranberry, raspberry, melon, amla, and bilberry, or their extracts; vegetables such as onion, cucumber, tomato, cauliflower, carrot, spinach, kale, Brussels sprouts, garlic, basil, and oregano, or their extracts; and rice (indica rice). Grains or their extracts, including japonica rice, glutinous rice, cereals (wheat, barley, oats, rye), corn, sorghum, millet, sorghum, yellow millet, buckwheat, soybeans, broad beans, peas, mung beans, red beans, kidney beans, etc.; nuts or their extracts, including walnuts, pistachios, cashews, hazelnuts, almonds, apricot kernels, pine nuts, peanuts, sunflower seeds, chestnuts, macadamia nuts, ginkgo nuts, etc.; coffee or its extracts; and some medicinal and edible plant-based Chinese medicinal materials or their extracts.

[0062] Animal dairy product ingredients can 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, whey protein concentrate, desalted whey powder, whey protein powder, and hydrolyzed whey protein powder.

[0063] Examples of animal meat product ingredients include those from pork, beef, mutton, seafood, or poultry.

[0064] Examples of functional additives include vitamin supplements, mineral supplements, nucleotide supplements, dietary fiber, and functional polyunsaturated fatty acid supplements.

[0065] Any acceptable excipients may include solvents, antioxidants, antibacterial agents, thickeners, diluents, cosolvents, stabilizers, emulsifiers, fillers, disintegrants, lubricants, coating materials, anti-caking agents, flavoring agents, sweeteners, food flavorings, and food colorings.

[0066] III. Uses that help regulate the brain's immune balance

[0067] This invention unexpectedly discovered that branched-chain fatty acid compositions containing specific C15:0, C17:0, and C16:0, specifically those containing iso-C15:0, anteiso-C15:0, anteiso-C17:0, and iso-C16:0, have a regulatory effect on brain immune balance. Furthermore, when iso-C15:0, anteiso-C15:0, anteiso-C17:0, and iso-C16:0 are combined, they exhibit a synergistic effect, further enhancing the efficacy of regulating brain immune balance. Therefore, the branched-chain fatty acid compositions containing iso-C15:0, anteiso-C15:0, anteiso-C17:0, and iso-C16:0 described in this invention, as well as foods containing or using said branched-chain fatty acid compositions, all have a regulatory effect on brain immune balance. The regulation of brain immune balance described in this invention is not intended for the prevention or treatment of diseases.

[0068] Furthermore, the present invention provides the use of branched-chain fatty acid compositions containing iso-C15:0, anteiso-C15:0, anteiso-C17:0, and iso-C16:0 in the preparation of foods that help regulate the brain's immune balance.

[0069] In some embodiments, the effect of assisting in regulating brain immune homeostasis includes regulating microglia polarization. In some specific embodiments, regulating microglia polarization includes at least one of inhibiting microglia polarization towards the M1 phenotype, promoting microglia polarization towards the M2 phenotype, increasing the amount of IL-4 and IL-10 released by microglia, and decreasing the amount of TNF-α, IL-1β, and / or NO released by microglia. In some specific embodiments, regulating microglia polarization includes inhibiting the tendency of microglia to polarize towards the M1 phenotype, promoting the tendency of microglia to polarize towards the M2 phenotype, increasing the amount of IL-4 and IL-10 released by microglia, and decreasing the amount of TNF-α, IL-1β, and / or NO released by microglia. In some specific implementations, the regulation of microglia polarization includes inhibiting the tendency of microglia to polarize towards the M1 phenotype, promoting the tendency of microglia to polarize towards the M2 phenotype, increasing the amount of IL-4 and IL-10 released by microglia, and reducing the amount of TNF-α, IL-1β, and NO released by microglia.

[0070] Furthermore, the aforementioned role in regulating the brain's immune balance includes promoting the development of brain nerves and the nervous system.

[0071] In some implementations, the nervous system is the central nervous system.

[0072] IV. Uses to assist in improving or maintaining the health of the central nervous system

[0073] This invention unexpectedly discovered that combining iso-C15:0, anteiso-C15:0, anteiso-C17:0, and iso-C16:0 can help improve or maintain the health of the central nervous system, especially by alleviating discomfort or lack of well-being caused by neuroinflammation in a non-pathological state. Furthermore, when combined, the four components exhibit a synergistic effect, further enhancing the improvement of neuroinflammation. Therefore, the branched-chain fatty acid composition containing iso-C15:0, anteiso-C15:0, anteiso-C17:0, and iso-C16:0 described in this invention also has the effect of helping to improve or maintain the health of the central nervous system and alleviating discomfort or lack of well-being caused by neuroinflammation in a non-pathological state.

[0074] Furthermore, the present invention provides the use of branched-chain fatty acid compositions containing iso-C15:0, anteiso-C15:0, anteiso-C17:0, and iso-C16:0 in the preparation of foods for the purpose of assisting in the maintenance of the health of the central nervous system.

[0075] In some implementations, the assistance in maintaining central nervous system health includes non-therapeutic relief of neuroinflammation that does not reach a medically pathological level.

[0076] Example

[0077] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0078] 1. Instruments and reagents

[0079] Mouse microglia BV-2 cell line (Shanghai Enzyme Research Institute, catalog number CC-Y2022), fetal bovine serum (Vicente, catalog number 085-150), high glucose DMEM medium (Ciscotech, catalog number CA0004-500ML), antibiotics, culture flasks, well plates, centrifuge tubes, trypsin, dimethyl sulfoxide, lipopolysaccharide, kits, etc.

[0080] 2. Experimental Methods

[0081] 2.1 Cell Culture

[0082] Mouse microglia (BV2) cell line was cultured in high-glucose DMEM medium (containing 10% fetal bovine serum) and placed in a 5% CO2 incubator (37°C, saturated humidity).

[0083] 2.2 Cytotoxicity assay

[0084] BV2 microglia viability was detected using the CCK8 assay (CCK8 kit purchased from White Shark, catalog number BS350A).

[0085] Methods for evaluating the cytotoxicity of nutrients: BV2 cells in the logarithmic growth phase were selected and cultured at a density of 5 × 10⁶ cells per well. 4 Cells were seeded at a rate of [number] cells / mL in 96-well plates and allowed to adhere overnight. Then, different concentrations of branched-chain fatty acids were added for 12 hours. After treatment, 10 μL of CCK8 solution was added to each well, and the plates were incubated at 37°C for 4 hours. The absorbance at 450 nm was then measured using a microplate reader.

[0086] According to the formula: Cell viability = [(Absorbance of experimental wells - Absorbance of blank wells) / (Absorbance of control wells - Absorbance of blank wells)] × 100% (where experimental wells contain cells, culture medium, nutrients of different concentrations and CCK-8 solution; blank wells contain culture medium and CCK-8 solution, but not cells and nutrients; control wells contain cells, culture medium and CCK-8 solution, but not nutrients), the cell viability is finally calculated.

[0087] 2.3 Evaluation of anti-inflammatory efficacy

[0088] 1) Establishment of a microglial cell inflammation model and nutritional intervention methods

[0089] Select BV2 cells in the logarithmic growth phase, using 5 × 10⁶ cells per well. 4 Cells were seeded at a rate of 100 cells / mL in 96-well plates and allowed to adhere overnight before being divided into groups for intervention. (1) Control group: cells were cultured using conventional methods; (2) LPS group (model group): BV2 cells were treated with LPS (1 μg / mL) for 24 h to establish a cell neuroinflammation model; (3) Experimental group (example and comparative example): BV2 cells were stimulated with LPS for 24 h, and then 6 different combinations were added for 12 h of intervention.

[0090] 2) NO level measurement

[0091] The nitrate reductase method was used to detect the NO (nitric oxide) level in BV2 microglia culture medium. Experimental grouping and nutrient intervention methods were as shown in 1) above. Cell culture medium was collected from each group. The NO detection kit was used according to the instructions, and the absorbance of each tube was measured at a wavelength of 550 nm and a light path of 0.5 cm. The NO content was then calculated.

[0092] 3) Measurement of inflammatory factor levels

[0093] The levels of IL-1β, TNF-α, IL-10, and IL-4 in the supernatant of BV2 cells were detected using ELISA. The experimental grouping and nutrient intervention methods were the same as described in 1) above. Cell supernatant was collected from each group, and the ELISA kit was operated according to the instructions. The absorbance values ​​of each group were measured sequentially at a wavelength of 405 nm using a microplate reader to detect the levels of IL-1β, TNF-α, IL-4, and IL-10.

[0094] 2.4 Detection of iNOS and CD206, phenotypic markers of microglia M1 and M2 phenotypes

[0095] The mRNA expression levels of iNOS and CD206-related genes were detected using qRT-PCR, and the changes in the content of M1 and M2 cell populations were then determined. Third-generation BV2 microglia suspension was seeded into 24-well culture plates at a cell density of 4 × 10⁶ cells / well. 5Cells were processed in groups according to the experimental design, one cell per well. After treatment, cells were washed twice with PBS (200 μL per well). Trypsin digestion was performed for 2.5 min, followed by termination with 400 μL of culture medium. Cells were collected by pipetting into enzyme-free centrifuge tubes. Total RNA was extracted from each group of cells. cDNA was obtained by reverse transcription using a reverse transcription kit following the prescribed procedure. The amplified cDNA was mixed with primers, fluorescent probes, and qPCR Master Mix. The appropriate PCR cycle program and parameters were set according to qPCR requirements, and the corresponding mRNA expression levels were analyzed using a real-time PCR detection system. Specific primer information is as follows:

[0096] iNOS:

[0097] Upstream: 5'-GGCTTGCCCCTGGAAGTTT-3' (SEQ ID NO.1),

[0098] Downstream: 5'-TGCAAGTGAAATCCGATGTGG-3' (SEQ ID NO.2);

[0099] CD206:

[0100] Upstream: 5'-TTCAGCTATTGGACGCGAGG-3' (SEQ ID NO.3),

[0101] Downstream: 5'-GAATCTGACACCCAGCGGAA-3' (SEQ ID NO.4);

[0102] GAPDH:

[0103] Upstream: 5'-GGTTGTCTCCTGCGACTTCA-3' (SEQ ID NO.5),

[0104] Downstream: 5'-TGGTCCAGGGTTTCTTACTCC-3' (SEQ ID NO.6).

[0105] 3. Detection of cytotoxicity of various branched-chain fatty acids

[0106] This study selected six branched-chain fatty acids (iso-C14:0, iso-C15:0, anteiso-C15:0, iso-C16:0, iso-C17:0, and anteiso-C17:0) to investigate the regulatory effects of different combinations on neuroinflammation. First, the cytotoxic effects of the six branched-chain fatty acids on microglia were examined. The results are shown in Table 1. The table shows that the cytotoxic effect of iso-C17:0 decreased to 90.79% at a concentration of 25 μmol / L, while the cell viability of the other four branched-chain fatty acids remained above 100% at a concentration of 25%. Therefore, the concentration of branched-chain fatty acids was uniformly set at 25 μmol / L in this study.

[0107] Table 1. Effects of different concentrations of branched-chain fatty acids on microglial cell viability (%)

[0108] 4. The inhibitory effect of branched-chain fatty acids on neuroinflammation

[0109] Microglia, as resident immune cells of the central nervous system, are key participants in neuroinflammation. LPS-induced treatment of microglia accelerates the transcription of inflammatory genes by activating the Toll-like receptor (TLR4) NF-κB signaling pathway, further leading to the differentiation of microglia into M1 and M2 phenotypes.

[0110] M1 phenotype microglia secrete pro-inflammatory cytokines such as NO, IL-1β, and TNF-α, exacerbating the inflammatory response. NO free radicals are synthesized by enzymes such as NOS (eNOS) and iNOS via the L-arginine pathway. Under normal physiological conditions, iNOS remains dormant in dormant cells. However, under pathological conditions, it produces large amounts of NO and plays a dual role in chronic infection and inflammation. Reducing NO production may be an effective strategy to improve inflammation. IL-1β has strong pro-inflammatory activity and can induce various pro-inflammatory mediators, such as cytokines and chemokines. Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine produced by various cells in response to inflammatory responses and immune regulation, and can induce apoptosis.

[0111] M2 macrophages secrete anti-inflammatory cytokines such as IL-4 and IL-10, reducing neuroinflammatory responses. IL-4, a typical type II immune cytokine, plays a crucial immunomodulatory role in type II immune diseases such as asthma, allergic dermatitis, and urticaria. Studies have shown that IL-4 can promote macrophage differentiation into M2 macrophages, thereby exerting an anti-inflammatory effect. Interleukin-10, a multicellular, multifunctional cytokine, regulates cell growth and differentiation, participates in inflammatory and immune responses, and is a recognized inflammatory and immunosuppressive factor.

[0112] Therefore, regulating the conversion of microglia to the M2 type is a potential strategy for suppressing neuroinflammation. This invention investigated the effects of different proportions of branched-chain fatty acids mixed with LPS on LPS-induced inflammation in BV2 microglia.

[0113] Examples 1-3

[0114] A microglial cell inflammation model was established following step 1) in section 2.3 above, and nutritional intervention was implemented. The branched-chain fatty acid composition is shown in Table 2, and the concentration of branched-chain fatty acids in the microglial cell inflammation model was 25 μmol / L. After the intervention, NO levels, inflammatory factor levels, and the contents of iNOS and CD206 were measured.

[0115] Comparative Examples 1-6

[0116] A microglial cell inflammation model was established following step 1) in section 2.3 above, and nutritional intervention was implemented. The branched-chain fatty acid composition is shown in Table 2, and the concentration of branched-chain fatty acids in the microglial cell inflammation model was 25 μmol / L. After the intervention, NO levels, inflammatory factor levels, and the contents of iNOS and CD206 were measured.

[0117] Table 2. Design of different combinations of branched-chain fatty acids (%)

[0118] By applying different branched-chain fatty acid compositions to a microglial inflammation model, the inventors unexpectedly discovered the regulatory effect of a composition containing four branched-chain fatty acids, consisting of three different carbon chains and different α and I structures, on neuroinflammation. The specific results are as follows:

[0119] Table 3. Effects of different branched-chain fatty acid compositions on LPS-induced pro-inflammatory cytokines in BV2 microglia.

[0120] Table 3 shows the effects of branched-chain fatty acid compositions of different proportions on the production of pro-inflammatory factors by LPS-induced BV2 microglia. The table shows that the levels of pro-inflammatory factors NO, TNF-α, and IL-1β produced by microglia significantly increased after LPS induction, indicating that LPS induction severely damaged microglia and led to the production of large amounts of pro-inflammatory factors.

[0121] In the examples and comparative examples after different branched-chain fatty acid compositions were co-treated with LPS, it can be seen that pro-inflammatory factors were reduced to varying degrees.

[0122] Among them, all branched-chain fatty acid compositions, including the comparative and example compositions, showed good reduction effects on the pro-inflammatory factor IL-1β. IL-1β was significantly lower than that of the control group, and the difference was found to be extremely significant after one-way ANOVA analysis (p < 0.0001). Further comparison revealed that the IL-1β production in Examples 1-3 was below 8 ng / L, while the production in the comparative examples ranged from 8.23 ​​to 9.45 ng / L. Clearly, the production of the pro-inflammatory factor IL-1β in the examples was lower than that in the comparative examples. Regarding the pro-inflammatory factor NO, the table shows that the NO levels were reduced in both the examples and comparative examples compared to the model group. Except for Comparative Example 6, which showed no significant difference from the model group (p > 0.05), all other groups significantly reduced NO production. However, further comparison revealed that the NO production in Examples 1-3 was lower than that in the comparative examples. Regarding the pro-inflammatory factor TNF-α, the inventors found that all compositions inhibited TNF-α production to some extent after treatment. However, after difference analysis, only Examples 1-3 and Comparative Example 1 (p < 0.0001) showed significant differences from the model group (p = 0.041), while Comparative Examples 2-6 showed no significant differences from the model group (p > 0.05).

[0123] The above results indicate that the four branched-chain fatty acids iso-C15:0, anteiso-C15:0, anteiso-C17:0 and iso-C16:0 in the fatty acid composition have a good effect on reducing the production of pro-inflammatory factors induced by LPS treatment at 15.5%-21%, 15%-34%, 20.00%-34.5% and 25%-35%, respectively.

[0124] Table 4. Effects of different branched-chain fatty acid compositions on LPS-induced pro-inflammatory cytokines in BV2 microglia.

[0125] Table 4 shows the effects of different combinations of branched-chain fatty acid treatments on LPS-induced anti-inflammatory factors in BV2 microglia. The table shows that the IL-4 production in Examples 1-3 was higher than that in the model group (35.59 ng / L), while the IL-4 production in Comparative Examples 1-6 was comparable to or lower than that in the model group. Regarding the anti-inflammatory factor IL-10, Examples 1-3 promoted its production, and after significant difference analysis, Examples 1-3 reached the level of the control group, with no significant difference (p > 0.05). In contrast, the IL-4 production in Comparative Examples 1-6 was at or lower than that in the model group. Therefore, these conclusions indicate that Examples 1-3 have a regulatory effect on the production of anti-inflammatory factors.

[0126] To determine whether the inhibition of inflammatory response in BV2 cells by the branched-chain fatty acid composition is related to phenotypic polarization, this invention used RT-qPCR to detect the expression of mRNA of the M1 phenotypic marker iNOS and the M2 phenotypic marker CD206 in BV2 cells. The results are shown in Table 5.

[0127] Table 5. RT-PCR detection of iNOS and CD206 mRNA expression levels in BV2 cells of each group.

[0128] As shown in the table, the mRNA expression level of iNOS, a marker of M1 cell polarization, was significantly lower in Examples 1-3 than in the model group (p < 0.0001). The mRNA expression level of CD206 was significantly higher in Examples 1-3 than in the model group (p < 0.0001). However, the iNOS content was significantly higher in the six comparative examples than in Examples 1-3, while the CD206 content was significantly lower (p < 0.05). These results further demonstrate that the four branched-chain fatty acids iso-C15:0, anteiso-C15:0, anteiso-C17:0, and iso-C16:0 effectively reduce the production of pro-inflammatory factors induced by LPS treatment at concentrations of 15.5%-21%, 15%-34%, 20%-34.5%, and 25%-35%, respectively.

Claims

1. A branched-chain fatty acid composition, characterized in that, The branched-chain fatty acids include iso-C15:0, anteiso-C15:0, anteiso-C17:0, and iso-C16:0; Based on the total mass of the four branched-chain fatty acids, iso-C15:0, anteiso-C15:0, anteiso-C17:0, and iso-C16:0, The content of iso-C15:0 is not less than 10%, the content of anteiso-C15:0 is 10% to 34%, the content of anteiso-C17:0 is 15% to 35%, and the content of iso-C16:0 is not less than 20%.

2. The composition according to claim 1, characterized in that, Based on the total mass of the four branched-chain fatty acids, iso-C15:0, anteiso-C15:0, anteiso-C17:0, and iso-C16:0, The content of iso-C15:0 is 10% to 25%, the content of anteiso-C15:0 is 12% to 34%, the content of anteiso-C17:0 is 17% to 35%, and the content of iso-C16:0 is 20% to 35%.

3. The composition according to claim 1 or 2, characterized in that, Based on the total mass of the four branched-chain fatty acids, iso-C15:0, anteiso-C15:0, anteiso-C17:0, and iso-C16:0, The content of iso-C15:0 is 15% to 25%, 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-C16:0 is 25% to 35%.

4. Use of the branched-chain fatty acid composition according to any one of claims 1 to 3 in the preparation of foods that help regulate the immune balance of the brain.

5. The use according to claim 4, characterized in that, The benefits of regulating the brain's immune balance include modulating microglia polarization.

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

7. Use of the branched-chain fatty acid composition according to any one of claims 1 to 3 in the preparation of a food for the purpose of assisting in the maintenance of 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 the relief of neuroinflammation for non-therapeutic purposes.

9. The use according to any one of claims 4 to 8, characterized in that, The food products mentioned are candies, beverages, dairy products, or baked goods.

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

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