MLM type structure triglyceride rich in DHA at sn-2 site as well as preparation method and application of MLM type structure triglyceride rich in DHA at sn-2 site

Abstract

The invention provides an MLM type structure triglyceride rich in DHA (docosahexaenoic acid) at sn-2 site as well as a preparation method and application of the MLM type structure triglyceride. The triglyceride has a glycerin skeleton, the sn-2 site of the glycerin skeleton is esterified with DHA, the sn-1 site and the sn-3 site of the glycerin skeleton are independently esterified with one of C6-C12 saturated medium-chain fatty acids, and at least one of the sn-1 site and the sn-3 site is esterified with lauric acid. The DHA is fixed at the sn-2 position, and lauric acid is introduced at the sn-1 and sn-3 positions, so that the effects of improving the DHA enrichment degree of the colon and cooperatively inhibiting the colorectal cancer are achieved. The triglyceride with the structure belongs to a typical lipid with a middle-long-middle (MLM) type structure. According to the molecular design, DHA is reserved in a monoacylglycerol form in the digestion process and acts on the colon in a positioned manner by utilizing the characteristic that digestive enzyme is selective to a triglyceride part, and meanwhile, lauric acid is rapidly absorbed to play an anti-tumor immunoregulation role, so that complementary advantages of two functional fatty acids are realized on the molecular level.

Description

Technical Field

[0001] This invention relates to the fields of functional lipids and gut health nutrition, specifically to a DHA-rich MLM-type triglyceride at the sn-2 position, its preparation method, and its application. Background Technology

[0002] Colorectal cancer is one of the most common malignant tumors worldwide. Despite advancements in surgery, radiotherapy, chemotherapy, and immunotherapy, survival rates for patients with advanced colorectal cancer remain poorly improved. Therefore, there is an urgent need to explore new adjuvant therapy strategies, including utilizing nutritional interventions and the anti-tumor effects of natural products.

[0003] DHA, a polyunsaturated fatty acid in Omega-3, has attracted attention due to its anti-inflammatory and anti-tumor properties. Studies have shown that DHA can trigger lipid peroxidation, enhance oxidative stress leading to DNA damage, inhibit COX-2 expression and pro-survival signals such as AKT, downregulate nuclear β-catenin levels, thereby activating the caspase-3-mediated apoptosis pathway, inducing autophagic apoptosis in colon cancer cells (such as HCT-116), and inhibiting tumor growth. In mouse experiments, a diet rich in DHA can reduce the activity of pro-inflammatory transcription factors such as NF-κB, inhibiting inflammatory responses and tumor-promoting signals in the tumor microenvironment. Another important natural fatty acid—lauric acid (dodecanoic acid, C12:0)—has also shown inhibitory effects on colorectal cancer. Recent studies report that lauric acid can significantly inhibit the proliferation of colorectal cancer cells, induce tumor cells to produce reactive oxygen species and undergo apoptosis. Simultaneously, high intake of lauric acid can significantly reduce the risk of colorectal cancer, thereby synergistically inhibiting tumor growth. Therefore, DHA and lauric acid, as n-3 long-chain polyunsaturated fatty acids and medium-chain saturated fatty acids respectively, have unique biological activities in anti-tumor and anti-inflammatory aspects.

[0004] However, direct intake of DHA and lauric acid still has some limitations. DHA is usually ingested as free fatty acids or randomly distributed triglycerides, and is largely absorbed and distributed throughout the body in the upper small intestine, resulting in limited DHA actually reaching the colon. This insufficient DHA in the colon may limit its direct effects on the colorectal mucosa and tumors. Furthermore, free DHA is easily oxidized and degraded, limiting its bioavailability. Medium-chain fatty acids such as lauric acid are mainly rapidly absorbed into the liver via the portal vein for energy, and their direct effects on the distal intestine are also limited. Therefore, how to effectively deliver and enrich DHA in the colon, while combining it with the effects of lauric acid to achieve targeted intervention for colorectal cancer, remains an unsolved problem in current technology.

[0005] Structured lipids offer a potential solution to these problems. They are typically designed and modified to improve the digestive and absorptive pathways and physiological functions of triglycerides by targeting their fatty acid composition and location. For example, in breast milk fat, more than half of the DHA is naturally located at the sn-2 position of triglycerides. Studies suggest that esterifying DHA to the sn-2 position of triglycerides can mimic the structure of breast milk fat, improving DHA stability and absorption while reducing its effective dosage. Because pancreatic lipases preferentially hydrolyze fatty acids at the sn-1 and sn-3 positions of triglycerides, DHA at the sn-2 position can be retained as monoacylglycerols during digestion and can be absorbed and utilized through the small intestinal mucosa without complete hydrolysis. Existing research has indicated that sn-2DHA structured triglycerides can improve the bioavailability and tissue uptake of DHA compared to ordinary fish oil: in animal experiments, groups fed sn-2DHA structured lipids had healthier blood lipid levels and significantly increased DHA / EPA content in the brain, liver, and erythrocytes. This indicates that structural modification to implant DHA at the sn-2 position of triglycerides can improve its function.

[0006] The combination of medium-chain fatty acids and long-chain fatty acids to form "medium- and long-chain triglycerides" (MLCTs) has become a hot topic in functional lipid research in recent years. However, existing products are not yet ideal in terms of improving the targeted absorption of active ingredients and anti-tumor effects, and the purity and function of the target products of structural lipids need to be improved. In particular, there are currently no reports on combining DHA and lauric acid, two of the best fatty acids for anti-colorectal cancer, into the same triglyceride molecule to achieve targeted enrichment of DHA in the colon and exert a synergistic anti-tumor effect. This invention addresses these gaps by providing a novel DHA / C12 structured triglyceride, its preparation, and its applications, overcoming the shortcomings of existing technologies. Summary of the Invention

[0007] Technical Problem to be Solved: To address the aforementioned technical problems, the present invention aims to provide a DHA-rich MLM-type triglyceride at the sn-2 position, its preparation method, and its applications. By fixing DHA at the sn-2 position and introducing lauric acid at the sn-1 and sn-3 positions, the invention achieves increased DHA enrichment in the colon and synergistic inhibition of colorectal cancer. The triglyceride structure of the present invention can be briefly described as "1,3-dilauroyl-2-docosahexaenoylglycerol," belonging to a typical "medium-long-medium" (MLM) type lipid structure. This molecular design aims to utilize the selectivity of digestive enzymes for triglyceride sites, allowing DHA to be retained in the form of monoacylglycerol during digestion and act locally on the colon, while lauric acid is rapidly absorbed to exert anti-tumor immunomodulatory effects, thereby achieving complementary advantages of the two functional fatty acids at the molecular level.

[0008] Technical solution: A DHA-rich MLM-type triglyceride at the sn-2 position, wherein the triglyceride has a glycerol backbone, wherein DHA is esterified at the sn-2 position of the glycerol backbone, and one of the saturated medium-chain fatty acids with C6 to C12 carbon atoms is independently esterified at the sn-1 and sn-3 positions, and at least one of the sn-1 and sn-3 positions is esterified with lauric acid (C12:0).

[0009] Furthermore, the triglyceride is a DHA-enriched triglyceride at the sn-2 position, meaning that the molar percentage of DHA at the sn-2 position is higher than the sum of the molar percentages of DHA at the sn-1 and sn-3 positions.

[0010] Furthermore, the molar percentage of DHA at the sn-2 position is not less than 70% of the total molar number of DHA in the triglyceride.

[0011] Furthermore, the molar percentage of DHA at the sn-2 position is not less than 90% of the total molar number of DHA in the triglyceride.

[0012] Furthermore, both the sn-1 and sn-3 positions are esterified with lauric acid (C12:0).

[0013] The method for preparing the above-mentioned DHA-rich MLM-type triglyceride at the sn-2 position includes the following steps: (1) Preparation of 2-mono-DHA glycerol intermediate: Using DHA-containing triglycerides as substrates, selective hydrolysis or alcoholysis at the 1,3 position is carried out in the presence of immobilized 1,3-specific lipase to obtain intermediate containing 2-mono-docosahexaenoic acid monoglyceride (2-MAG-DHA). (2) Introducing medium-chain fatty acids to form MLM structure: Using intermediate products as substrates, in the presence of immobilized 1,3-specific lipase, esterification or transesterification reactions are carried out with lauric acid and / or saturated medium-chain fatty acids with C6 to C12 carbons and their esters, so that the sn-1 and sn-3 positions of the glycerol skeleton independently esterify saturated medium-chain fatty acids with C6 to C12 carbons, and at least one of the sn-1 and sn-3 positions esterifies lauric acid (C12:0), to obtain a DHA-rich MLM-type triglyceride at the sn-2 position.

[0014] Furthermore, in step (1), the hydrolysis or alcoholysis reaction temperature is 40-60°C, the amount of immobilized 1,3-specific lipase added is 2-10 wt% of the substrate, and the reaction time is 4-24 hours.

[0015] Furthermore, in step (1), the immobilized 1,3-specific lipase is Lipozyme RM IM lipase (Trichoderma reesei lipase).

[0016] Furthermore, in step (2), the esterification or transesterification reaction temperature is 45-65°C, the molar ratio of medium-chain fatty acids to 2-MAG-DHA in the reaction system is 1.5:1-3.0:1, the amount of immobilized 1,3-specific lipase added is 5-15 wt% of the substrate mass, and the water generated in the reaction is continuously removed under reduced pressure or with the addition of a desiccant.

[0017] Furthermore, in step (2), the immobilized 1,3-specific lipase is Lipozyme TL IM lipase (Thermospora lancifolia lipase).

[0018] Furthermore, after step (2), a purification step of the reaction product is included, which includes molecular distillation or column chromatography separation to improve the purity of the MLM-type triglyceride and the enrichment of DHA at the sn-2 position.

[0019] Furthermore, the present invention also proposes an alternative preparation method for obtaining sn-2 DHA-rich MLM-type triglycerides through a one-step acid hydrolysis / ester exchange. This method can also involve: using DHA-containing triglycerides (such as triacyl-DHA glycerol or fish oil with high DHA content) and lauric acid and / or saturated medium-chain fatty acids with C6 to C12 carbon atoms as substrates, and performing a one-step ester exchange or acid hydrolysis reaction at 50–60°C in the presence of immobilized 1,3-specific lipase, thereby obtaining a triglyceride mixture mainly containing the aforementioned MLM-type triglycerides.

[0020] A composition containing an MLM-type triglyceride, the composition comprising any one of the MLM-type triglycerides described in any one of claims 1 to 3, wherein the MLM-type triglyceride accounts for 10 to 100 wt% of the total triglycerides in the composition.

[0021] The use of the above-mentioned sn-2 position DHA-rich MLM-type triglycerides or compositions containing MLM-type triglycerides in functional foods, health foods or nutritional compositions, wherein the MLM-type triglycerides account for 1 to 50 wt% of the total fat content in the functional foods or nutritional compositions.

[0022] Furthermore, the functional food or nutritional composition includes edible oil products, fortified foods, solid beverages, nutritional supplements, and pet food.

[0023] The above-mentioned sn-2 position DHA-rich MLM-type triglycerides or compositions containing MLM-type triglycerides are used in functional foods or nutritional compositions that nutritionally improve the integrity of the colonic epithelial barrier and / or regulate nutritional indicators related to intestinal inflammation. Beneficial effects

[0024] 1. The structured triglyceride of the present invention, through innovative molecular design and enzymatic preparation, achieves the organic combination of DHA and lauric acid in the same triglyceride molecule, overcomes the shortcomings of single-component application, and provides synergistic anti-cancer and health care effects. 2. The MLM-type structured triglycerides of this invention exhibit significant anti-colorectal cancer efficacy and physiological advantages both in vitro and in vivo. In vitro cell experiments showed that the digested product of this structured lipid effectively inhibited the proliferation and survival of colorectal cancer cells (HCT-116) and induced apoptosis and oxidative stress. Compared with the control group supplemented with DHA and lauric acid respectively, the structured lipids of this invention showed a stronger inhibitory effect, presumably because the 2-MAG-DHA released from the structured lipids can be more effectively taken up and utilized by intestinal cancer cells, while lauric acid also plays a synergistic role. In animal experiments, in colon cancer model mice (e.g., chemically induced colon tumor models), the tumor burden in the treatment group ingesting the structured lipids of this invention was significantly reduced, with both the average number and volume of tumors being less than the control group; simultaneously, the intestinal mucosal barrier function of the mice in the structured lipid group was improved, manifested by upregulated expression of tight junction proteins and reduced intestinal permeability. Furthermore, the structured lipids of this invention can reduce the levels of pro-inflammatory cytokines in the peripheral blood and intestines of colon cancer model mice, with a significant decrease in interleukin (IL) and tumor necrosis factor-α (TNF-α), indicating that they have anti-inflammatory effects. 3. The structured triglycerides of the present invention can not only serve as a nutritional preparation to increase the enrichment of DHA in the colon, but also have potential application value in the prevention and adjuvant treatment of colorectal cancer. 4. The product of this invention has high safety (all components are common edible fatty acids), and can be used in functional foods or nutritional supplements to help colorectal cancer patients improve prognosis and reduce inflammatory response. It can also be used for intestinal health management in the general population (e.g., to prevent malignant transformation of colon polyps). 5. This invention has significant novelty and innovation in the field of structured lipids. Its unique sn-2 DHA targeted enrichment and anti-tumor application expand the functional boundaries of structured lipids in food nutrition. Attached Figure Description

[0025] Figure 1 This is a diagram illustrating the inhibitory effects of Omega-3 long-chain fatty acids and medium-chain fatty acids on the proliferation of colorectal cancer cells in Example 1. Figure 2 The image shows the TLC diagram of 2-MAG-DHA obtained in step 1 of Example 2. Figure 3 The image shows the TLC diagram of the triglycerides obtained in step 2 of Example 2; Figure 4This study aims to validate the in vitro digestion model and cell experiments, as well as HCT-116 cell experiments. Specifically, A represents the inhibition rate of HCT-116 cells by the digestion products of the control group, Example 2, and Example 3 after 48 hours; B represents the cell state after treatment of the control group; C represents the cell state after treatment of Example 2; and D represents the cell state after treatment of Example 3. Figure 5 This is an evaluation of nutritional intervention in an animal model of colorectal cancer; where A is a trend graph of mouse body weight change; and B is a trend graph of colorectal tumor number change in mice. Figure 6 Comparison of mouse intestinal histological sections from Example 5; Figure 7 These are intestinal epithelial barrier-related indicators, including the ZO-1 gene and the Occludin gene; Figure 8 This represents the level of inflammatory factors in the serum. Detailed Implementation

[0026] This invention proposes a DHA-rich MLM-type triglyceride at the sn-2 position, its preparation method, and its applications. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the following examples will be used to further describe the invention in detail. It should be understood that the specific examples described herein are only for explaining the invention and are not intended to limit the invention.

[0027] Example 1: Screening of antitumor active fatty acids In vitro cellular screening experiments were conducted to determine the optimal fatty acid composition of the structural lipids of this invention. Using the human colorectal cancer cell line HCT-116 as a model, the inhibitory effects of Omega-3 long-chain fatty acids and medium-chain fatty acids on cancer cell proliferation were investigated. The selected Omega-3 fatty acids were docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and alpha-linolenic acid (ALA); the selected medium-chain fatty acids were C6 (caproic acid), C8 (octanoic acid), C10 (decanoic acid), and C12 (lauric acid). These fatty acids (in free form or in soluble forms that can be taken up by cells, bound to bovine serum albumin) were added to cultured cancer cells, and treated with the same molar concentration gradient for 48 hours. The cell viability inhibition rate was determined using the CCK-8 assay.

[0028] The screening results are as follows Figure 1As shown, among Omega-3 fatty acids, DHA exhibited the most significant inhibitory effect on the proliferation of two types of colorectal cancer cells, significantly stronger than EPA and ALA. Among medium-chain fatty acids, lauric acid (C12) showed the best inhibitory effect, superior to C6, C8, and C10 fatty acids. Taking HCT-116 cells as an example, after 48 h of treatment, at a treatment concentration of 200 μM, the cell viability of the DHA-treated group decreased significantly, while the EPA and ALA groups showed only mild inhibition. Similarly, lauric acid at a concentration of 2 mM inhibited cell viability by approximately 50%, while the cell inhibition rates of hexanoic acid, caprylic acid, and capric acid groups were relatively low. Therefore, the results of this embodiment indicate that among the tested Omega-3 fatty acids and medium-chain fatty acids, DHA and C12 showed the highest inhibitory activity on HCT-116 cells, and DHA and C12 are preferably used as the fatty acid composition of the triglycerides in this invention.

[0029] Example 2: Two-step preparation of sn-2 DHA-MLM structured triglycerides In this embodiment, a two-step enzymatic method was used to prepare MLM-type triglycerides with DHA at the sn-2 position and medium-chain fatty acids at the sn-1 and sn-3 positions, with lauric acid being the preferred medium-chain fatty acid.

[0030] Step 1: Preparation of sn-2 DHA-monoglyceride (2-MAG-DHA): DHA-rich triglycerides and purified docosahexaenoic acid glycerol (DHA-TAG) were used as substrates and mixed with immobilized 1,3-position specific lipase Lipozyme RM IM. The mixture was stirred at 50°C. The amount of enzyme used was 5% of the weight of the substrate oil. A small amount of water (0.2%) was added to the reaction system to promote enzyme hydrolysis. Under the action of the 1,3-specific enzyme, the fatty acids (mainly DHA) at the sn-1 and sn-3 positions of the triglyceride were preferentially hydrolyzed and released, while the DHA at the sn-2 position was retained due to the selective action of the enzyme. After 8 hours of reaction, the reaction progress was monitored by thin-layer chromatography (TLC). A large number of monoacylglycerol spots were found in the product, indicating that 2-MAG-DHA was generated. The reaction mixture was filtered to remove the enzyme, and the filtrate was collected. The crude product contained 2-MAG-DHA, free fatty acids (mainly free DHA), and small amounts of unreacted triglycerides and diacylglycerols. To improve purity, molecular distillation was performed at low temperature under high vacuum (<1 mbar) to separate the free fatty acid components, thereby enriching 2-MAG-DHA in the residue. After one short-path distillation, the purity of the obtained 2-MAG-DHA product was 85.29% (the remaining components were mainly diacylglycerols). The final 2-MAG-DHA glyceride was a pale yellow oily substance (e.g., ...). Figure 2 As shown), it exists stably at room temperature and is ready for use in the next esterification reaction; Step 2: Esterification to introduce lauric acid: 2-MAG-DHA obtained in step S1 was mixed with lauric acid at a molar ratio of 1:2.1. Immobilized 1,3-position specific lipase Lipozyme TL IM (8% of the total weight of the mixture) was added. Esterification was carried out under solvent-free conditions at 55°C with stirring. The reaction equilibrium was shifted towards the formation of triglycerides by reducing the system pressure (applying a 100 Pa vacuum). After 24 hours of reaction, samples were taken and the glyceride composition was analyzed by TLC. Numerous triglyceride spots (such as...) were found in the product. Figure 3 (As shown). Further HPLC-RID results showed that triglycerides (TAG) accounted for 87.25% of the total glycerides in the product, while monoacylglycerols (MAG) accounted for less than 1%, indicating that most of the 2-MAG-DHA had been esterified into triglycerides. Analysis of the fatty acid composition of TAG revealed that the substitution rate of C12 at the sn-1,3 position was 71.58%.

[0031]

[0032] After the reaction, the mixture was filtered to remove the enzyme preparation. The filtrate was the crude product oil, which may contain small amounts of unreacted lauric acid and intermediate glycerides. To improve the purity of the final product, appropriate post-treatment can be performed. In this example, the crude product oil was allowed to stand at 4°C to allow excess free lauric acid to crystallize out, which was then removed by alkaline washing and filtration. Molecular distillation was performed first, followed by silica gel column chromatography with a gradient elution using a hexane / ethyl acetate mixed solvent to further remove residual diacylglycerols. The purified final product was a high-purity structural triglyceride—1,3-dilauroyl-2-docosahexaenoylglycerol (triglyceride form)—with a recovery rate (based on DHA) of 68.72%. Therefore, the two-step enzymatic targeted synthesis of MLM deconstructed lipids has high synthetic purity.

[0033] Table 1. Comparison of lipid composition (%) after the two-step synthesis of S1 and purification. Oil components Raw material TAG Before S1 finishes purification S1 molecules were purified by distillation. After purification by S1 column chromatography TAG 98.98±0.93 10.78±0.53 2.25±0.08 0.53±0.04 FFA 0.25±0.03 13.73±0.87 1.16±0.08 1.03±0.06 DAG 0.77±0.08 21.32±0.97 11.30±0.77 1.57±0.12 MAG ND 54.17±1.23 85.29±1.39 96.87±1.12 Table 2 Comparison of lipid composition (%) after the two-step synthesis of S2 and purification. Oil components Before S2 finishes purification After S2 molecular distillation purification After purification by S2 column chromatography TAG 80.82±1.34 87.25±1.87 96.22±0.98 FFA 10.39±0.95 2.61±0.64 0.62±0.11 DAG 7.47±0.80 9.56±0.78 2.76±0.64 MAG 1.32±0.14 0.58±0.08 0.40±0.03 Example 3: One-step enzymatic transesterification preparation of MLM-structured triglycerides Using 100 g of DHA triglyceride and 40 g of lauric acid as substrates, the mixtures were thoroughly mixed in a reactor, and immobilized 1,3-specific lipase Lipozyme RM IM (5 wt% of the total substrate mass) was added. The transesterification reaction was carried out at 50–60 °C under reduced pressure for approximately 20 hours.

[0034] During this process, Lipozyme RM IM exhibits some selectivity for the sn-1 and sn-3 positions. However, because the substrate, product, and intermediate are in the same system, various partial transesterification and partial hydrolysis products are easily generated. After the reaction, molecular distillation yields a mixture containing triglycerides with the target MLM structure, but the proportion of the target MLM structure triglyceride in the triglyceride component is lower than that of the two-step method product. The proportion of triglycerides before purification is only 65.83%, far lower than that of the two-step method.

[0035] The product was separated by molecular distillation, and the triglyceride was finally separated with a purity of 78.63%. The fatty acid composition of TAG was analyzed and it was found that the substitution rate of C12 at the sn-1,3 position was 42.14%. Compared with the two-step product of Example 2, the structured lipid obtained by the one-step method had a significantly lower purity.

[0036] Therefore, its effect was relatively inferior in subsequent biological function evaluations (see Example 4 below). However, the one-step process is simple and involves fewer reaction steps, making it an alternative to the preparation method of this invention. The one-step method has certain practical value in applications where purity requirements are not high, such as general nutritional supplements. However, if a high sn-2 positioning rate and high purity of the structured triglyceride are required to exert specific biological activity, a two-step preparation method is still preferred. Furthermore, by measuring the content of fatty acids at the sn-2 position, it was found that the two lipases have good site specificity, ensuring a high retention of DHA at the sn-2 position.

[0037] Table 3. Comparison of lipid composition (%) before and after purification in the S2 step of the one-step synthesis and two-step synthesis methods. Oil components Before S2 finishes purification Before one-step purification After S2 molecular distillation purification After one-step molecular distillation TAG 80.82±1.34 65.83±2.75 87.25±1.87 78.63±2.90 FFA 10.39±0.95 15.35±1.23 2.61±0.64 8.42±1.32 DAG 7.47±0.80 14.82±1.97 9.56±0.78 10.61±2.17 MAG 1.32±0.14 4.00±0.62 0.58±0.08 2.34±0.47 Table 4. Molar content (mol%) of fatty acids at the sn-1 and sn-3 positions in triglycerides before and after structural lipid synthesis. fatty acid Abbreviation Before synthesis One-step method Two-step method butyric acid C4:0 ND ND ND hexanoic acid C6:0 ND ND ND bitter C8:0 0.017888337 ND ND Decanoic acid C10:0 0.004683702 0.091039598 0.146934997 Undecanoic acid C11:0 0.003742982 ND ND Dodecanoic acid C12:0 0.006414937 42.14127569 71.58118295 Tridecanoic acid C13:0 ND 0.039177084 0.067696751 Tetradecanoic acid C14:0 0.017862891 0.179771647 0.29354376 cis-9-tetradecenoic acid C14:1 ND ND ND Pentadecanoic acid C15:0 0.013870467 ND ND cis-10-pentadecaenoic acid C15:1 ND ND ND Hexadecanoic acid C16:0 0.617717414 0.206201143 0.215229409 cis-9-hexadecenoic acid C16:1 0.005618894 ND ND Heptadecanol C17:0 0.030389452 ND ND cis-10-heptadecenoic acid C17:1 ND ND ND Octadecyl carbonate C18:0 0.352459483 0.137435813 0.138285448 trans-9-octadecenoic acid C18:1n9t ND ND ND cis-9-octadecenoic acid C18:1n9c 0.35545153 0.116052512 0.098107337 trans, trans-9,12-octadecadienoic acid C18:2n6t 0.052925007 ND ND cis,cis-9,12-octadecadienoic acid C18:2n6c 0.790323089 0.291503561 0.24927678 diatocarbonate C20:0 0.333115148 0.111040271 ND cis,cis,cis-6,9,12-octadecadienoic acid C18:3n6 0.368489498 0.139300771 0.227768772 cis-11-eicosodenoic acid C20:1 0.026949473 ND ND cis,cis,cis-9,12,15-octadecadienoic acid C18:3n3 0.132191135 0.04643359 ND Twenty-one carbonic acid C21:0 0.062083973 ND ND cis,cis-11,14-eicosadienoic acid C20:2 ND ND ND Dodecanoic acid C22:0 0.300410793 0.176916607 ND cis,cis,cis-8,11,14-eicostrienoic acid C20:3n6 0.330441422 0.169056378 0.275289026 cis-13-docosahexaenoic acid C22:1n9 ND ND ND cis-11,14,17-eicoseriate C20:3n3 ND ND ND cis-5,8,11,14-eicosatetraenoic acid C20:4n6 0.180978019 0.095845041 0 Trichocarbonate C23:0 0.886702733 0.498572839 0.415647457 cis-13,16-docosadienoic acid C22:2 1.177540032 0.559426587 0.210325023 Twenty-four carbonic acid C24:0 0 0 0 cis-5,8,11,14,17-eicosapride C20:5n3 0 0 0.267551521 cis-15-tetracosanoic acid C24:1 0.041056248 0 0 cis-4,7,10,13,16,19-docosahexaenoic acid C22:6n3 93.89069334 55.00095086 25.81316077 Table 5. Molar content (mol%) of fatty acids at the sn-2 position in triglycerides before and after structural lipid synthesis fatty acid Abbreviation Before synthesis One-step method Two-step method butyric acid C4:0 ND ND ND hexanoic acid C6:0 ND ND ND bitter C8:0 ND 0.011809 0.011623 Decanoic acid C10:0 ND 0.003092 0.003072 Undecanoic acid C11:0 ND 0.002471 0.002453 Dodecanoic acid C12:0 ND 0.004235 0.004151 Tridecanoic acid C13:0 ND ND ND Tetradecanoic acid C14:0 ND 0.011792 0.011616 cis-9-tetradecenoic acid C14:1 ND ND ND Pentadecanoic acid C15:0 ND 0.009156 0.009018 cis-10-pentadecaenoic acid C15:1 ND ND ND Hexadecanoic acid C16:0 0.388971 0.407775 0.405093 cis-9-hexadecenoic acid C16:1 ND 0.003709 0.003644 Heptadecanol C17:0 ND 0.020061 0.020055 cis-10-heptadecenoic acid C17:1 ND ND ND Octadecyl carbonate C18:0 0.236387 0.23267 0.232602 trans-9-octadecenoic acid C18:1n9t ND ND ND cis-9-octadecenoic acid C18:1n9c 0.209447 0.234645 0.234577 trans, trans-9,12-octadecadienoic acid C18:2n6t ND 0.034937 0.034927 cis,cis-9,12-octadecadienoic acid C18:2n6c 0.511732 0.521717 0.519222 diatocarbonate C20:0 ND 0.2199 0.218784 cis,cis,cis-6,9,12-octadecadienoic acid C18:3n6 0.464582 0.243252 0.244362 cis-11-eicosodenoic acid C20:1 ND 0.01779 0.017679 cis,cis,cis-9,12,15-octadecadienoic acid C18:3n3 0.076603 0.087264 0.086766 Twenty-one carbonic acid C21:0 ND 0.040984 0.040166 cis,cis-11,14-eicosadienoic acid C20:2 ND ND ND Dodecanoic acid C22:0 ND 0.198311 0.200178 cis,cis,cis-8,11,14-eicostrienoic acid C20:3n6 ND 0.218135 0.210589 cis-13-docosahexaenoic acid C22:1n9 ND ND ND cis-11,14,17-eicoseriate C20:3n3 ND ND ND cis-5,8,11,14-eicosatetraenoic acid C20:4n6 0.133609 0.119469 0.119435 Trichocarbonate C23:0 0.672082 0.585341 0.5787 cis-13,16-docosadienoic acid C22:2 0.350089 0.777332 0.775159 Twenty-four carbonic acid C24:0 ND ND ND cis-5,8,11,14,17-eicosapride C20:5n3 0.533512 ND ND cis-15-tetracosanoic acid C24:1 ND 0.027103 0.027005 cis-4,7,10,13,16,19-docosahexaenoic acid C22:6n3 96.42299 95.96705 95.98912 Example 4: In vitro digestion model and cell experiment verification and HCT-116 cell experiment This embodiment obtains the digestion products of Embodiments 2 and 3 by simulating an in vitro digestion system, and evaluates their effect on cell proliferation in HCT-116 cells to verify the potential of structural design to improve colon-related indicators nutritionally.

[0038] Take 1 g of the product prepared in Example 2 and 1 g of the product prepared in Example 3 of this invention, add them to simulated gastric juice (containing an appropriate amount of pepsin, pH 2.0, 37°C) and shake to digest for 1 hour. Then add simulated small intestinal juice (containing bile salts and pancreatic enzymes, pH adjusted to 7.5) and continue to digest at 37°C with shaking for 2 hours. After digestion, extract the lipid products in the digestion system with ether, evaporate the ether to obtain the lipid extract of the simulated digestion product. For the control group, use an equal amount of physically mixed oils for the same digestion procedure: the control mixture is composed of DHA and lauric acid mixed in a molar ratio of 1:2 (simulating the simultaneous intake of free DHA and lauric acid). The control digestion product is also obtained after simulated gastrointestinal digestion.

[0039] Subsequently, the products obtained from the three simulated digestions were used in cell experiments. The extracted digestion products were dissolved in culture medium, and the concentration was adjusted so that the concentration of DHA and lauric acid contained therein was similar to that under physiological conditions in vivo. Then, they were added to HCT-116 cell culture and incubated for 48 hours to observe cell proliferation and apoptosis.

[0040] Experimental results are as follows Figure 4 As shown, compared with the untreated control, the proliferation of colorectal cancer cells in the treatment group of Example 2 was significantly inhibited, with a cell inhibition rate exceeding 80%, and apoptosis-related morphological changes such as cell shrinkage were observed. However, after treatment at the same concentration for 48 hours, the inhibitory effect of Example 3 on cancer cells was slightly weaker, reaching approximately 70%; the inhibitory effect of the digestion product of the control mixture on cancer cell proliferation was significantly weaker, with a survival rate decrease of approximately 50%. These effects were more significant compared to treatment with pure DHA or lauric acid alone. Furthermore, further observation of cell morphology revealed that the treated cells underwent varying degrees of apoptosis, with the number of apoptotic cells treated in Example 2 being significantly higher than that in Example 3. In summary, the combination of 2-MAG-DHA produced from the digestion of the structural triglycerides of this invention and lauric acid has a synergistic lethal effect on colorectal cancer cells. On the one hand, the 2-MAG form of DHA may be more easily incorporated into the cell membrane or taken up and utilized, triggering apoptosis signals; on the other hand, free lauric acid can induce oxidative stress and enhance the activity of immune cells. These results validate the rationality and superiority of the structural lipid design of this invention at the cellular level.

[0041] Example 5: Evaluation of nutritional intervention in an animal model of colon cancer The effects of the triglyceride structure of this invention on the development of colon tumors and intestinal health indicators were evaluated at the animal level. Healthy BALB / c mice (6 weeks old, both male and female) were randomly divided into groups of 10. A colon cancer model was established using the classic AOM / DSS induction method to induce neoplastic lesions in the colonic epithelium of the mice. Different dietary interventions were administered at the beginning of the modeling process.

[0042] Control group: fed normal feed, and subsequently given sterile water via injection and drinking water in equal volume; Model group: Colorectal cancer model was established by feeding normal diet and using AOM / DSS induction method; Experimental Group 1: The structural lipid intervention group was fed with 1% of Example 2, and a colon cancer model was established using the AOM / DSS induction method; Experimental group 2: 1% mixed lipids (DHA:C12=1:2) were added as a control, and a colon cancer model was established using the AOM / DSS induction method.

[0043] Mice had free access to food and water. The intervention lasted for 10 weeks, during which time the mice's weight, food intake, and other general conditions were recorded. Results are as follows: Figure 5 As shown, no abnormal behavior or significant side effects were observed in the structured lipid group mice during the experiment, indicating that the product of this invention has good safety.

[0044] Mice were sacrificed one week after the last DSS administration, and colon tissue was collected for analysis. Figure 6 As shown. Macroscopic observation revealed multiple tumor nodules on the colon surface of mice in the control group, with an average of 7.30 ± 2.10 tumors per mouse; while the number of colon tumors in the intervention group of Example 1 was significantly reduced, averaging only 3.80 ± 1.40, and the tumors were generally smaller. The average number of tumors in the intervention group of Example 2 was 5.90 ± 1.80, falling between the two groups. Statistical analysis showed that the tumor burden in the structural lipid group was reduced by approximately 48% compared to the model control group (…). p <0.01), which is also significantly different from the physical mixture group (reduced by approximately 36%). p <0.05).

[0045] Histological examination further confirmed the macroscopic observations: the tumors in the control group mice were mostly invasive adenocarcinomas, accompanied by severe dysplasia of the surrounding mucosa; the tumors in the structural lipid group were less severe, some being only benign adenomas or moderate dysplasia, with no obvious muscle layer infiltration. Furthermore, in the normal mucosal tissue of the structural lipid group mice, goblet cell density and mucus secretion were both superior to those in the control group, suggesting that the intestinal mucosal barrier function was protected.

[0046] To assess intestinal barrier function and inflammatory status, relevant biomarkers were detected in mice from each group. Colonic mucosal tissue was collected, total RNA was extracted, and cDNA was obtained through reverse transcription. Real-time quantitative PCR (qPCR) was used to detect the relative mRNA expression levels of tightly linked genes (ZO-1, Occludin) to reflect the expression status of intestinal epithelial barrier-related genes. Figure 7As shown, the number and average volume of colonic tumor-like lesions in mice in the structured lipid intervention group were significantly reduced, the intestinal mucosal structure was more intact, and the mRNA expression level of tight junction protein-related genes was significantly higher than that in the model group, indicating that colonic health-related indicators were improved under the intervention of structured triglycerides as dietary lipid components in this invention.

[0047] Finally, the levels of inflammatory factors in peripheral blood were measured. Figure 8 As shown, enzyme-linked immunosorbent assay (ELISA) results indicated that the serum levels of pro-inflammatory cytokines TNF-α and IL-6 were significantly elevated in the model group mice, while structural lipid intervention significantly reduced the levels of these inflammatory factors (TNF-α was reduced by 40% compared to the control group). p <0.05; IL-6 decreased by 30%, p <0.01). In comparison, while the physically mixed oil group also showed some reduction, it was not as significant as the structural lipid group. This is consistent with the histological results observed in the structural lipid group mice, which showed reduced inflammatory infiltration and fewer mucosal ulcers. It is speculated that the mechanism may be related to the synergistic anti-inflammatory effect of DHA and lauric acid: on the one hand, DHA can inhibit the release of inflammatory factors from macrophages through pathways such as GPR120 receptors; on the other hand, lauric acid may improve the gut microbiota or provide rapid energy to reduce the inflammatory response.

[0048] In summary, in a mouse model of colorectal cancer, the structured triglycerides of this invention significantly reduced tumor formation, improved intestinal barrier function, and lowered blood levels of inflammatory mediators, demonstrating superior intestinal protection and anti-tumor effects. Compared to administration of equal amounts of free DHA and lauric acid, the structured lipid form showed better results, proving the effectiveness and unique advantages of this invention. This result further confirms the value of increasing colonic DHA enrichment for colorectal tumor intervention: through molecular structural modification, this invention allows more DHA to act on the local colonic environment (whether directly acting on mucosal cells in the form of 2-MAG or enriching in intestinal tissue after absorption into the bloodstream), thereby maximizing its anti-cancer potential; simultaneously, the introduction of lauric acid provides additional anti-tumor and anti-inflammatory benefits.

[0049] The triglyceride structure of this invention holds promise for development into a novel nutritional supplement or functional food for the adjuvant treatment of colorectal cancer. It can be used in conjunction with conventional treatment to improve efficacy and reduce the risk of recurrence; it can also be used in daily health maintenance for individuals with intestinal polyps or a high-risk family history to prevent the occurrence of colorectal cancer.

[0050] In summary, the structural lipids provided by this invention are significantly innovative in improving the targeted utilization of DHA and synergistically inhibiting colon tumors, filling a gap in the prior art.

[0051] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A DHA-rich MLM-type triglyceride at the sn-2 position, characterized in that, The triglyceride has a glycerol backbone, wherein DHA is esterified at the sn-2 position, and one of the saturated medium-chain fatty acids with C6 to C12 carbon atoms is independently esterified at the sn-1 and sn-3 positions, and at least one of the sn-1 and sn-3 positions is esterified with lauric acid.

2. The sn-2 position DHA-rich MLM-type triglyceride according to claim 1, characterized in that, The triglyceride is a DHA-enriched triglyceride at the sn-2 position, meaning that the molar percentage of DHA at the sn-2 position is higher than the sum of the molar percentages of DHA at the sn-1 and sn-3 positions.

3. The sn-2 position DHA-rich MLM-type triglyceride according to claim 1, characterized in that, The molar percentage of DHA at the sn-2 position is not less than 70% of the total molar number of DHA in the triglyceride.

4. A method for preparing a DHA-rich MLM-type triglyceride at the sn-2 position according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Preparation of 2-mono-DHA glycerol intermediate: Using DHA-containing triglycerides as substrates, selective hydrolysis or alcoholysis at the 1,3 position is carried out in the presence of immobilized 1,3-specific lipase to obtain intermediate containing 2-mono-docosahexaenoic acid monoglyceride. (2) Introducing medium-chain fatty acids to form MLM structure: Using intermediate products as substrates, in the presence of immobilized 1,3-specific lipase, esterification or transesterification reactions are carried out with lauric acid and / or saturated medium-chain fatty acids with C6 to C12 carbons and their esters, so that the sn-1 and sn-3 positions of the glycerol skeleton independently esterify saturated medium-chain fatty acids with C6 to C12 carbons, and at least one of the sn-1 and sn-3 positions esterifies lauric acid (C12:0), to obtain a DHA-rich MLM-type triglyceride at the sn-2 position.

5. The preparation method according to claim 4, characterized in that: In step (1), the hydrolysis or alcoholysis reaction temperature is 40-60℃, the amount of immobilized 1,3-specific lipase added is 2-10 wt% of the substrate mass, and the reaction time is 4-24 hours.

6. The preparation method according to claim 4, characterized in that: In step (2), the esterification or transesterification reaction temperature is 45-65℃, the molar ratio of medium-chain fatty acids to 2-MAG-DHA in the reaction system is 1.5:1-3.0:1, the amount of immobilized 1,3-specific lipase added is 5-15 wt% of the substrate mass, and the water generated in the reaction is continuously removed under reduced pressure or with the addition of a desiccant.

7. The preparation method according to claim 4, characterized in that: The step (2) is followed by a purification step of the reaction product, which includes molecular distillation or column chromatography to improve the purity of the MLM-type triglyceride and the enrichment of DHA at the sn-2 position.

8. A composition containing an MLM-type triglyceride, characterized in that: The composition comprises any one of the MLM-type triglycerides described in any one of the following three, and the MLM-type triglycerides account for 10 to 100 wt% of the total triglycerides in the composition.

9. The use of a sn-2 position DHA-rich MLM-type triglyceride according to any one of claims 1-3 or a composition containing an MLM-type triglyceride according to claim 8 in a functional food, health food, or nutritional composition, characterized in that, The MLM-type triglycerides account for 1 to 50 wt% of the total fat content in the functional food or nutritional composition.

10. Use in a functional food or nutritional composition of a sn-2 position DHA-rich MLM-type triglyceride according to any one of claims 1-3 or a composition containing an MLM-type triglyceride according to claim 8, for nutritionally improving colonic epithelial barrier integrity and / or regulating nutritional indicators related to intestinal inflammation.

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