Use of diacylglycerol in preparation of a drug for improving nutritional composition of breast milk and promoting neural development of offspring

Abstract

The application discloses application of diacylglycerol in preparation of medicines for improving nutritional composition of breast milk and promoting neural development of offspring, and belongs to the technical field of medicines. The complete technical chain of "maternal intake of DAG, remolding of breast milk lipids (enrichment of long-chain polyunsaturated ether lipids), activation of 2-AG signal pathways of offspring, and promotion of neural cognitive development" is provided, which provides a scientific basis for development of new maternal nutrition products, and has extremely high clinical application value and industrialization prospect.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the use of diacylglycerol (DAG) in the preparation of drugs for improving the lipid composition of breast milk during lactation, particularly enriching long-chain polyunsaturated ether lipids, and promoting the neurocognitive development of infants and / or offspring. Background Technology

[0002] Breast milk is the only natural source of nutrition for infants in the early stages of life. Its lipid composition (accounting for 40-50% of total energy) plays a decisive role in the growth and development of infants, especially the development of the brain and nervous system. Breast milk lipids are mainly composed of triglycerides (TAG, 95-98%), and also contain trace amounts of phospholipids, cholesterol, and highly bioactive ether lipids (such as phosphatallipids). Although present in minute quantities, ether lipids are key structural components of neuronal membranes and myelin sheaths, playing an irreplaceable role in early neural development.

[0003] Although the basic composition of breast milk lipids is relatively fixed, its specific molecular structure is dynamically regulated by factors such as maternal diet and metabolic state. The source of fat in the maternal diet directly affects the fatty acid composition of breast milk, which in turn affects the infant's cognitive abilities and lipid profile. However, current research on the targeted regulation of trace but functionally critical lipid components (such as ether lipids) in breast milk through specific nutritional interventions remains very limited.

[0004] Diacylglycerols (DAGs) are a natural component of edible oils (typically comprising 2-10%) and are metabolic intermediates and signaling molecules in mammals. DAG exists in two isomers: 1,2-DAG and 1,3-DAG. The metabolic pathway of 1,3-DAG differs from that of regular TAGs during digestion and absorption; it is not easily resynthesized into TAGs for storage but tends to be oxidized for energy or participate in phospholipid synthesis. DAG oils rich in the 1,3-DAG isomer have been shown to have health benefits such as reducing body fat and improving insulin sensitivity. However, as a novel functional oil, the effects of DAG intake during lactation on the remodeling of breast milk lipid profiles and its influence on offspring development (especially nervous system development) have not yet been reported. Summary of the Invention

[0005] The purpose of this invention is to provide the use of diacylglycerol in the preparation of medicaments that improve the nutritional composition of breast milk and / or promote the neural development of offspring.

[0006] The diacylglycerol component contains two isomers: 1,3-diacylglycerol (1,3-DAG) and 1,2-diacylglycerol (1,2-DAG), with the 1,3-DAG isomer comprising more than 50% of the total diacylglycerol, preferably more than 70%. The fatty acid composition of the DAG can be derived from common vegetable oils such as soybean oil, rapeseed oil, sunflower oil, and corn oil.

[0007] Preferably, the improvement of breast milk nutritional composition involves enriching long-chain polyunsaturated ether lipids, increasing phospholipid content, and reducing medium- and short-chain fatty acid ether lipids in breast milk.

[0008] Further preferably, the long-chain polyunsaturated ether ester includes an ether ester with C20:4 linked at the sn-1 position.

[0009] More preferably, the phospholipid is phosphatidylcholine (PC) or phosphatidylethanolamine (PE).

[0010] Further preferably, the ether esters of the medium- and short-chain fatty acids are ether esters with C10:0 or C10:1.

[0011] Preferably, the promotion of offspring neural development refers to promoting the improvement of offspring cognitive function and enhancing offspring spatial learning and memory abilities.

[0012] Preferably, the drug further includes a pharmaceutically acceptable carrier; the carrier is a conventional pharmaceutical excipient or adjuvant.

[0013] Preferably, the dosage form of the drug is a liquid dosage form or a solid dosage form. The liquid dosage form is a solution, suspension, or emulsion, and the solid dosage form is a tablet, capsule, pill, powder for injection, sustained-release preparation, or microparticle delivery system.

[0014] The State Administration for Market Regulation issued and implemented the "Catalogue of Permitted Health Functions Claimed by Health Foods (Non-Nutritional Supplements (2020 Edition)" and related regulations for nutrient supplements in 2020. This catalogue adjusted, merged, and standardized the original 27 functional claims, ultimately establishing 24 permitted health functions. These functional claims must strictly use the standard terminology specified in the catalogue and may not be modified or exaggerated. The 24 currently permitted health functions are as follows: Enhancing immunity, assisting in lowering blood lipids, assisting in lowering blood sugar, anti-oxidation, assisting in improving memory, relieving eye fatigue, promoting lead excretion, clearing the throat, assisting in lowering blood pressure, improving sleep, promoting lactation, relieving physical fatigue, improving hypoxia tolerance, providing auxiliary protection against radiation hazards, weight loss, improving growth and development, increasing bone density, improving nutritional anemia, providing auxiliary protection against chemical liver damage, treating acne, treating melasma, improving skin moisture, improving skin oiliness, regulating intestinal flora, promoting digestion, relieving constipation, and providing auxiliary protection against gastric mucosal damage.

[0015] Offspring who drank breast milk containing the DAG group of this invention showed a significant improvement in spatial learning and memory abilities.

[0016] This invention also provides the application of diacylglycerol in the preparation of health products or nutrient supplements that help improve memory.

[0017] Beneficial effects Compared with the prior art, the present invention has the following beneficial effects: 1. Significantly remodels the breast milk lipid profile: Maternal intake of the DAG described in this invention during lactation can finely remodel breast milk lipids without altering total breast milk production and total fat content. Specifically, this manifests as follows: Specific enrichment of key ether lipids: significantly upregulated 43 long-chain polyunsaturated ether lipids in breast milk, among which the abundance of ether lipids with C20:4 linked at sn-1 position (such as TG(20:4e_18:2_20:4)) increased to 1.82 times that of the control group.

[0018] Increased phospholipid content: Significantly increased the content of key phospholipids for neurodevelopment, such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE), in breast milk, with an overall increase of 1.60 times in PC lipids.

[0019] Reduced non-essential lipids: Significantly downregulated 30 ether lipids containing medium- and short-chain fatty acids (C10:0, C10:1) in breast milk, optimizing the lipid composition of breast milk.

[0020] 2. Significantly promotes offspring neural development: Offspring who drank breast milk from the DAG group of this invention showed significant improvements in spatial learning and memory abilities. In the Morris water maze test, the DAG group offspring spent more than 30% longer in the target quadrant than the control group, and significantly increased the number of times they crossed the original platform positions, while there was no difference in motor ability, indicating that the improvement in cognitive function is specific.

[0021] 3. Activation of key neural signaling pathways: The serum level of 2-arachidonic acid glycerol (2-AG) in the DAG group was significantly higher than that in the control group, providing a clear molecular mechanism for improving cognitive function.

[0022] 4. Good safety: DAG intervention does not affect the reproductive performance and milk production of female mice, nor does it affect the basal growth (weight), metabolic indicators (blood glucose, blood lipids) and immune function (immunoglobulins, cytokines) of offspring, indicating that it has good safety as a nutritional intervention method during lactation.

[0023] In summary, this invention provides a scientific basis for developing novel maternal nutrition products through a complete technical chain of "maternal intake of DAG → remodeling of breast milk lipids (enrichment of long-chain polyunsaturated ether lipids) → activation of offspring 2-AG signaling pathway → promotion of neurocognitive development," and has extremely high clinical application value and industrialization prospects. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the experimental procedure and a graph of basic maternal indicators for the present invention. Figure A is the experimental design flowchart; Figure B is a statistical comparison of litter sizes between the two groups of mother mice; Figure C is a comparison of milk production between the two groups of mother mice under different litter sizes; and Figure D is a comparison of breast milk fat content between the two groups of mother mice.

[0025] Figure 2 This is a global analysis diagram of breast milk lipidomics according to the present invention. Among them, Figure A is a pie chart of the distribution of lipid categories in breast milk of the TAG control group; Figure B is a pie chart of the distribution of lipid categories in breast milk of the DAG experimental group; Figure C is a principal component analysis (PCA) diagram based on quality control samples; and Figure D is an orthogonal partial least squares discriminant analysis (OPLS-DA) diagram of the lipid profiles of the two groups of breast milk.

[0026] Figure 3 This is a graph of differential lipid analysis in breast milk according to the present invention. Graph A is a heatmap of differential lipid categories; Graph B is a volcano diagram of differential lipid molecules.

[0027] Figure 4 This diagram illustrates the structural characteristics of fatty acid chains in different lipid classes.

[0028] Figure 5 This is a specific enrichment analysis diagram of breast milk ether lipids according to the present invention. Figure A is a lipid ontology clustering analysis diagram of significantly upregulated lipids; Figure B is a lipid ontology clustering analysis diagram of significantly downregulated lipids; Figure C is a statistical diagram of the number of upregulated and downregulated ether lipids; Figure D is a fatty acid chain structural feature analysis diagram of upregulated and downregulated glycerol ether esters. In Figures A and B, the numbers next to the bars represent the number of lipids in the corresponding category.

[0029] Figure 6 This is a maternal gut microbiota abundance analysis diagram of the present invention. Figure A shows a comparison of the gut microbiota composition at the genus level between the two groups of female mice; Figure B shows the variation in gut microbiota composition at the genus level at the individual level; Figure C shows a significant difference analysis of the two groups of microbiota based on machine learning.

[0030] Figure 7 This is a diagram of maternal gut microbiota category analysis according to the present invention. Figure A is a principal component analysis (PCA) diagram of two groups of microbiota at the genus level; Figure B is a correlation analysis diagram between differentially expressed genera.

[0031] Figure 8 This is a correlation analysis diagram of maternal gut microbiota and breast milk ether lipids according to the present invention.

[0032] Figure 9 These are the behavioral test results of the Morris water maze offspring of this invention. Figure A shows a representative swimming trajectory; Figure B is a spatial exploration heatmap; Figure C shows the percentage of time spent in each quadrant; Figure D shows the average swimming speed; Figure E shows the total swimming distance; Figure F shows the time spent in the target quadrant; Figure G shows the swimming distance in the target quadrant; Figure H shows the time spent at the platform location; and Figure I shows the time taken to first find the platform location.

[0033] Figure 10 These are the results of the physiological and biochemical index detection of offspring in this invention. Figure A shows the climbing time statistics; Figure B shows the weight at weaning; Figure CH shows the metabolic indexes, including fasting blood glucose (Figure C), glutathione (Figure D), total cholesterol (Figure E), serum triglycerides (Figure F), high-density lipoprotein (Figure G), and low-density lipoprotein (Figure H); Figure IO shows the immune function indexes, including serum immunoglobulins IgG1 (Figure I), IgG2a (Figure J), ​​and IgG2b (Figure K), as well as interleukins IL1b (Figure L), IL4 (Figure M), IL6 (Figure N), and IL10 (Figure O); Figure PQ shows the cytokine profile, including transforming growth factor TGFb (Figure P) and tumor necrosis factor TNFa (Figure Q); Figure R shows the ATP content in brain tissue; Figure S shows the 5-hydroxytryptamine (5-HT) content in brain tissue; and Figure T shows the serum 2-arachidonic acid glycerol (2-AG) content.

[0034] Figure 11 These are the base peak chromatograms for lipidomics in milk. AC chromatogram: DAG group positive ion mode; DF chromatogram: TAG group positive ion mode; GI chromatogram: DAG group negative ion mode; JL chromatogram: TAG group negative ion mode.

[0035] Figure 12 This is a total ion chromatogram of milk lipidomics. AC plot: positive ion mode of DAG group; DF plot: positive ion mode of TAG group; GI plot: negative ion mode of DAG group; JL plot: negative ion mode of TAG group. Detailed Implementation

[0036] To more clearly illustrate the present invention, specific embodiments and comparative examples are provided below. It should be understood that these embodiments are for illustrative purposes only and do not constitute any limitation on the scope of protection of the present invention.

[0037] The definition of diacylglycerol (DAG) in this invention is as follows: The diacylglycerol (DAG) described in this invention refers to an edible oil composition rich in diacylglycerol, prepared through processes such as enzymatic transesterification and physical refining. The total diacylglycerol content in the DAG accounts for more than 40% of the total weight of the oil, preferably more than 80%. The diacylglycerol component contains two isomers: 1,3-diacylglycerol (1,3-DAG) and 1,2-diacylglycerol (1,2-DAG), with the 1,3-DAG isomer accounting for more than 50% of the total diacylglycerol, preferably more than 70%. The fatty acid composition of the DAG can be derived from common vegetable oils such as soybean oil, rapeseed oil, sunflower oil, and corn oil, but the fatty acid distribution pattern on its glycerol backbone is fundamentally different from that of ordinary triglyceride oils. The "control group" or "TAG group" mentioned in this invention refers to test subjects who ingested ordinary triglyceride oil, wherein the ordinary triglyceride oil contains >95% triglycerides and <5% diacylglycerols.

[0038] Example 1: Preparation of a lactating maternal dietary composition containing DAG 1.1 Experimental Feed Formulation The high-fat diet (HFD) used in this invention is based on the AIN-93G purified feed formula, with a total fat energy ratio of 45%. The control group (TAG group) feed sourced its fat from ordinary soybean oil (mainly triglycerides). The experimental group (DAG group) feed sourced its fat from DAG (DAG content >80%, of which 1,3-DAG isomers accounted for approximately 70%), replacing all the fat in the control group feed through an equal energy substitution method. Except for the different fat source, the two feeds maintained the same nutritional components (protein, carbohydrates, cellulose, vitamins, minerals, etc.) and energy density. The feed was processed and prepared by the Guangdong Provincial Medical Laboratory Animal Center and stored at -20℃ for later use.

[0039] 1.2 Subjects and Intervention Program ( Figure 1 A shows the timeline of uniform feeding of ordinary high-fat diet during pregnancy, group intervention during lactation (i.e., TAG control group and DAG experimental group) and subsequent detection and analysis. Eight-week-old SPF (Specific Pathogen Free) C57BL / 6J mice (60 females and 30 males) were purchased from the Guangdong Provincial Laboratory Animal Center. After one week of acclimatization, they were mated together at a female:male ratio of 2:1. The next morning, the female mice were examined for vaginal plugs; those with plugs were recorded as day 0.5 of gestation. The pregnant mice were randomly divided into two groups, both fed the same high-fat diet as the control group (TAG) during gestation to establish a consistent maternal metabolic background.

[0040] After giving birth, the mother mice (with the number of pups in each litter adjusted to 4-5 to ensure uniform nursing) were randomly divided into two groups: Control group (TAG group, n=10): continued to be fed ordinary high-fat diet (TAG-HFD).

[0041] Experimental group (DAG group, n=10): Starting from day 1 of breastfeeding, the diet was switched to a high-fat diet containing DAG (DAG-HFD).

[0042] The above dietary intervention was continued throughout the lactation period (21 days postpartum). All animals had free access to food and water, and the housing environment was maintained at a temperature of 22±2℃, humidity of 50-60%, and a 12-hour light / dark cycle.

[0043] Example 2: Effects of maternal DAG intake on breast milk production and basic components 2.1 Experimental Methods On day 14 of lactation, breast milk was collected using a milk collection method. The mother mice were briefly separated from their pups (4 hours) to promote milk accumulation, followed by an intraperitoneal injection of 0.5 IU / kg oxytocin. The mammary glands were gently massaged, and breast milk was collected using a self-made capillary pipette. A portion of the collected milk was used for milk fat percentage determination, and another portion was frozen at -80°C for lipidomics analysis.

[0044] Milk fat percentage determination (Creamatocrit method): Breast milk is drawn into a heparinized capillary glass tube, one end is sealed, and centrifuged at 12,000 rpm for 5 minutes. The percentage of the milk fat layer length to the total milk column length is measured and converted to absolute fat content (g / dL) according to the standard curve.

[0045] Milk production assessment: Isolate the mother mouse and the pups for 4 hours to allow the mother's milk to accumulate in the mammary glands. Weigh the pups. Put the pups back in the same cage with the mother mouse for 1 hour and weigh the pups again. Subtract the two weights. The difference is the amount of milk the pups ingested. The sum of the weight differences for each litter of pups is the milk production of the mother mouse during that period.

[0046] 2.2 Experimental Results The results show (reference) Figure 1(B, 1C, 1D) DAG intervention did not adversely affect the reproductive performance and baseline lactation parameters of the mothers. There were no significant differences in litter size between the two groups (DAG group 4.5±0.5 vs (Control, Versus) TAG group 4.6±0.6, p>0.05) or pup weaning survival rate (DAG group 94.74% vs TAG group 95.73%). There were also no statistically significant differences in average daily milk production between the DAG and TAG groups under different litter sizes (2-7 pups). In mothers with litter sizes standardized to 4-5 pups, the fat content of breast milk in the DAG group was 11.9±0.8%, which was not significantly different from the 12.0±0.7% in the TAG group (p>0.05). This indicates that maternal intake of DAG is safe and does not affect the baseline yield or macroscopic fat content of breast milk.

[0047] Example 3: Maternal DAG intake reshapes breast milk lipidomics characteristics (lipid composition detection) 3.1 Experimental Methods A breast milk sample frozen on day 14 of the lactation period in Example 2 was used for non-targeted lipidomics analysis.

[0048] Lipid extraction: A modified Folch method was used. 50 μL of breast milk was added to a pre-cooled chloroform:methanol (2:1, v / v) mixture, vortexed, and separated by aqueous phase after an ice bath. The organic phase was collected and the extraction was repeated once. The organic phases were combined and dried under vacuum. The residue was redissolved in isopropanol, filtered through a 0.22 μm filter membrane, and then analyzed.

[0049] Liquid chromatography-mass spectrometry (LC-MS / MS) analysis: An ultra-high performance liquid chromatography-tandem mass spectrometry system (such as Thermo Fisher Q Exactive Plus) was used. The chromatographic column was an ACQUITY UPLC BEH C18 column (1.7µm, 2.1×100mm), and the column temperature was 50℃. Mobile phase A was acetonitrile:water (60:40 v / v, containing 0.1% formic acid and 10mM ammonium formate), and mobile phase B was isopropanol:acetonitrile (90:10 v / v, containing 0.1% formic acid and 10mM ammonium formate), with gradient elution. Mass spectrometry was performed using an electrospray ionization (ESI) source, acquiring data separately in positive and negative ion modes.

[0050] Data processing: LipidSearch software was used for lipid identification and peak area quantification. Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to compare the differences in the lipid profiles of the two groups of breast milk.

[0051] 3.2 Experimental Results Figure 11 This is the baseline chromatogram of lipidomics in milk. Figure 12This is a total ion chromatogram of milk lipidomics.

[0052] Lipomics results show (reference) Figure 2 A, 2B, 2C, 2D Figure 3 A, 3B Figure 3 A shows the relative abundance changes of major lipids between the two groups. Figure 3 B shows significantly upregulated and downregulated lipid molecules; the lipid profiles of breast milk from the DAG group and the TAG group were significantly separated. Figure 2 As shown in pie charts A and B, compared with the TAG group, the DAG group did not show a significant difference in the number of lipid categories identified; however, based on the content of different lipids, the two groups were significantly separated. Figure 2 C, 2D Figure 2 The C-display detection system shows good stability. Figure 2 (D shows a significant separation between the two lipid profiles). Heatmaps revealed a significant enrichment of phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and lysophosphatidylcholine (LPC) in the DAG group breast milk, while the levels of diglycerides (DG) and some triglycerides (TG) were relatively reduced. Volcano plot analysis further revealed hundreds of significantly different lipid molecules. This demonstrates that maternal DAG intake can finely "reshape" breast milk lipids at the molecular level.

[0053] Example 4: Maternal DAG intake specifically enriches long-chain polyunsaturated ether lipids in breast milk 4.1 Experimental Methods Based on the lipidomics data from Example 3, in-depth analysis and structural analysis were conducted on the identified ether lipids. The changing trends of alkyl-diacylglycerols and phospholipid precursors were analyzed in detail, and they were classified and statistically analyzed according to the number of carbon atoms and degree of unsaturation of their fatty acid chains.

[0054] 4.2 Experimental Results The results showed (reference) Figure 4 and Figure 5(See Table 1). The effect of DAG on breast milk ether lipids exhibits precise structure selectivity. Among the 39 significantly upregulated ether lipids, the vast majority are triglyceride-type ether lipids (TG-e), characterized by a long-chain polyunsaturated alkyl chain (e.g., C18:1e, C18:2e, C20:3e, C20:4e) attached to the sn-1 position, and long-chain fatty acids (≥C18) esterified at the sn-2 and sn-3 positions. For example, the abundance of TG(20:3e_16:0_18:1) and TG(20:4e_18:2_20:4) in the DAG group was 1.66 times and 1.82 times that in the TAG group, respectively. Conversely, the 28 significantly downregulated ether lipids were mainly TG-e lipids containing medium- and short-chain fatty acids (such as C10:0, C10:1, C12:0), such as TG(14:0e_10:1_10:1) (fold change FC=0.29). This indicates that maternal DAG intake promotes the preferential assembly and secretion of long-chain polyunsaturated ether lipids with higher neurodevelopmental potential in the mammary gland.

[0055] Table 1. Significant changes in ether lipids in breast milk.

[0056]

[0057]

[0058] Example 5: Effects of maternal DAG intake on offspring development 5.1 Experimental Methods The pups were weaned on day 21 after birth. After weaning, their growth and development, neurocognitive function, and biochemical indicators were assessed.

[0059] Morris water maze test: Testing began in the first week after weaning. The pool was 120cm in diameter, with a hidden platform 1cm underwater. Orientation and navigation training was conducted for four consecutive days. On the fifth day, the platform was removed, and a spatial exploration experiment was performed. The time the mice spent in the target quadrant and the time it took to first reach the platform were recorded to evaluate their spatial memory ability.

[0060] Physiological and biochemical indicator detection: After behavioral testing, the pups were euthanized, and serum and brain tissue were collected. ELISA kits were used to detect serum levels of 2-arachidonic acid glycerol (2-AG), immune factors (IL1b, IL4, IL6, IL10, IgG1, IgG2a, IgG2b), and cytokines (TNFa, TGFb), as well as brain tissue levels of ATP and serotonin (5-HT). Fasting blood glucose and blood lipids (TC, TG, HDL, LDL) were also measured simultaneously.

[0061] 5.2 Experimental Results Neurocognitive Development: Morris Water Maze Test Results (Reference) Figure 9 In the spatial exploration experiment, the DAG group mice spent significantly more time in the target quadrant, swam more distance, and crossed the original platform position more times than the TAG group (p<0.05), while there was no difference in swimming speed between the two groups, indicating that the spatial memory ability of the DAG group mice was significantly enhanced.

[0062] Key molecular mechanism: Discovered by ELISA detection (reference) Figure 10 In the TAG group, the serum level of 2-arachidonic acid glycerol (2-AG) was significantly higher than that in the TAG group (p<0.01). There were no significant differences in brain ATP and 5-HT levels, or other immune and inflammatory markers in serum between the two groups (reference). Figure 10 This indicates that the improvement in cognitive ability in the DAG group pups was closely related to the increase in 2-AG levels, rather than being caused by nonspecific inflammation or changes in basal metabolism.

[0063] Basic growth and development: There were no significant differences between the two groups of pups in weaning weight, fasting blood glucose, blood lipid profile, muscle strength, and immune function indicators (reference). Figure 10 This further confirms the safety of DAG intervention for offspring.

[0064] Example 6: Preliminary Exploration of the Mechanism of Action of DAG 6.1 Experimental Methods Feces were collected from female mice on day 14 of lactation, genomic DNA was extracted, and high-throughput sequencing was performed on the V3-V4 region of the 16S rRNA gene to analyze the composition of the gut microbiota and perform correlation analysis with breast milk lipidomics data.

[0065] 6.2 Experimental Results The results show (reference) Figure 6 , Figure 7 , Figure 8 ; Figure 6 C is a graph showing the significant differences between the two groups of bacterial communities based on machine learning, reflecting the differences in genus diversity between the two groups. Under all four algorithms, the DAG group showed a higher trend in bacterial community diversity, and this difference was statistically significant under the Goods coverage algorithm. Figure 8A heatmap showing the correlation between differentially expressed bacterial genera and differentially expressed lipid molecules reveals a strong positive correlation between specific bacterial genera, such as *Turicibacter*, and specific lipids, such as those containing C20:4. DAG intake significantly altered the gut microbiota structure of maternal mice, with a significant increase in the relative abundance of genera such as *Turicibacter* and *Adlercreutzia*. Correlation analysis showed a very strong positive correlation between the abundance of *Turicibacter* and the level of arachidonic acid (C20:4)-rich lipids in breast milk. This suggests that DAG may indirectly affect lipid synthesis and metabolism in the mammary gland by regulating the maternal gut microbiota, providing deeper mechanistic evidence for the applications of this invention.

[0066] Result comparison: Compared with the DAG group in Example 1, the TAG group showed the following significant differences: The lipidomic characteristics of breast milk were different: no enrichment of long-chain polyunsaturated ether lipids was observed in the breast milk of the TAG group, but the proportion of ether lipids bound to medium and short-chain fatty acids was relatively high; the content of phospholipids (PC, PE, etc.) was significantly lower than that of the DAG group (p<0.05).

[0067] The maternal gut microbiota differed: the relative abundance of bacteria such as Turicibacter and Adlercreutzia in the gut of TAG group mothers was significantly lower than that in DAG group (p<0.01).

[0068] Different cognitive functions in offspring: Morris water maze test showed that the TAG group pups spent significantly less time in the target quadrant, swam less distance, and crossed the original platform position less often than the DAG group (p<0.05), indicating that their spatial learning and memory abilities were poorer.

[0069] The offspring had different 2-AG levels: the serum 2-AG level in the TAG group was significantly lower than that in the DAG group (p<0.01).

[0070] Conclusion: Compared with ordinary TAG oil, only the DAG of this invention can specifically reshape the lipid composition of breast milk, enrich long-chain polyunsaturated ether lipids, and ultimately significantly promote the neurocognitive development of offspring by regulating the maternal gut microbiota. This indicates that the technical solution of this invention has achieved unexpected technical effects and possesses outstanding substantive features and significant progress.

[0071] Industrial applicability The application of diacylglycerol provided by this invention in the preparation of compositions that improve the nutritional composition of breast milk and promote the neural development of offspring has clear prospects for industrialization. The DAG can be added as a functional oil component to health products such as maternal and infant formula, nutritional supplements, and special medical purpose formulas. The product preparation process is simple, can be scaled up using existing industrial production lines, has controllable costs, and has demonstrated good safety and efficacy in animal experiments, making it suitable for promotion and application in the clinical nutrition and mass consumer markets.

Claims

1. The use of diacylglycerol in the preparation of drugs that improve the nutritional composition of breast milk and / or promote the neural development of offspring.

2. The application according to claim 1, characterized in that, The diacylglycerol contained in the composition of two isomers, 1,3-diacylglycerol and 1,2-diacylglycerol, with the 1,3-diacylglycerol isomer accounting for more than 50% of the total diacylglycerol.

3. The application according to claim 2, characterized in that, The 1,3-diacylglycerol isomer accounts for more than 70% of the total diacylglycerol component.

4. The application according to claim 1, characterized in that, The improvement of breast milk nutritional composition involves enriching long-chain polyunsaturated ether lipids, increasing phospholipid content, and reducing medium- and short-chain fatty acid ether lipids in breast milk.

5. The application according to claim 4, characterized in that, The long-chain polyunsaturated ether esters include ether esters with C20:4 linked at the sn-1 position.

6. The application according to claim 4, characterized in that, The phospholipids mentioned are phosphatidylcholine and phosphatidylethanolamine.

7. The application according to claim 4, characterized in that, The ether esters of the medium- and short-chain fatty acids mentioned are ether esters with C10:0 and C10:

1.

8. The application according to claim 4, characterized in that, The aforementioned promotion of offspring neural development refers to promoting the improvement of offspring's cognitive function and enhancing their spatial learning and memory abilities.

9. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable carriers.

10. Application of diacylglycerol in the preparation of health products or nutrient supplements that help improve memory.

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