Conjugated fatty acids to promote visual and neural development and uses thereof

Abstract

The application discloses conjugated fatty acids for promoting visual development and neural development and application thereof, and belongs to the technical field of functional lipids. It is found by the application that conjugated polyunsaturated fatty acid c9,t11,c15-CLNA has the effect of promoting visual development and neural development in early life, specifically embodied in the following aspects: shortening the eye opening age in a safe range; increasing the number of retinal and choroidal cells; promoting the development of retinal and choroidal structures; improving the normal rate of visual reflex behavior experiments; improving the relative level of retinal epidermal growth factor EGFR, thereby promoting the development of retinal and visual development in early life; improving various indexes in open field and new object recognition behavior experiments, thereby promoting neural development and cognitive function in early life; and improving the concentration of excellent PUFAs in the liver and prefrontal cortex of the brain in early life.

Description

Technical Field

[0001] This invention relates to the technical field of functional lipids, specifically to a conjugated fatty acid that promotes visual and neural development and its applications. Background Technology

[0002] Polyunsaturated fatty acids (PUFAs) are essential nutrients for early brain, neural, and visual development, with alpha-linolenic acid (ALA) and docosahexaenoic acid (DHA) from the Omega-3 series playing particularly prominent roles. In the brain, DHA, as a core component of phospholipids in the cerebral cortex, synapses, and neuronal cell membranes, is closely related to superior cognitive function and learning / memory abilities when accumulated at high levels. ALA, as a precursor to DHA biosynthesis, can be converted into DHA in vivo through the action of desaturases and elongases, playing a vital role in maintaining normal nervous system function. In the visual system, DHA accounts for more than 50% of the total fatty acids in the retinal photoreceptor cell membranes, directly affecting the efficiency of visual signal transmission. Sufficient DHA intake during infancy has been proven to significantly promote visual function development. However, the extraction and preparation of high-purity DHA and ALA have long faced technical bottlenecks: due to the limited content of PUFAs in natural sources and their easy oxidation, when using processes such as supercritical fluid extraction and molecular distillation to obtain high-purity products, there are often problems such as large equipment investment, high operating costs, and low yields, resulting in high prices for end products and limiting their widespread application in the fields of fortified foods and medicines.

[0003] Existing research indicates that PUFAs promote brain, neural, and visual development through a multidimensional mechanism, primarily acting through a three-pronged approach involving structural construction, signal transduction, and protein regulation. At the structural level, PUFAs, led by DHA, are major components of cell membrane phospholipids. By embedding themselves in the lipid bilayer, they influence membrane fluidity, thickness, and curvature, thereby regulating the spatial conformation and functional activity of membrane proteins (such as ion channels, receptors, and transporters). At the signal transduction level, DHA can be metabolized by lipoxygenases and cyclooxygenases to generate a series of highly bioactive signaling molecules. For example, neuroprotectin D1, derived from DHA, has been shown to directly protect nerve cells from damage in the retina and brain by inhibiting oxidative stress and inflammatory responses. At the protein regulation level, PUFAs can activate specific nuclear receptors, such as peroxisome proliferator-activated receptor γ (PPARγ), and through transcriptional regulation, influence the expression levels of proteins related to neuronal development, synaptic plasticity, and lipid metabolism (such as retinal epidermal growth factor receptor EGFR), thus synergistically promoting the development and functional improvement of the nervous system through multiple pathways.

[0004] Because existing substances that promote brain, nerve, and visual development are expensive to prepare, there is an urgent need to find other readily available, inexpensive substances that also have good effects on promoting visual and nerve development. Summary of the Invention

[0005] This invention aims to overcome the shortcomings of existing technologies and provide a novel polyunsaturated fatty acid that promotes neural and visual development in early life and its applications. Through microbial fermentation and animal experiments, this invention yields a uniquely structured conjugated linolenic acid—C9,T11,C15-conjugated linolenic acid—which promotes visual development in newborn mammals, promotes neural development, improves behavioral indicators related to retinal function, lipid metabolism and distribution pathways, and enhances the expression of proteins and signaling pathways related to neuronal development in the prefrontal cortex and hippocampus.

[0006] The c9,t11,c15-conjugated linolenic acid (c9,t11,c15-CLNA) of this invention is obtained by fermenting ALA with Bifidobacterium breve CCFM683 to induce double bond conjugation, followed by liquid-liquid extraction and liquid-phase enrichment to obtain a high-purity substance.

[0007] Because there are many types of conjugated linolenic acid (CLNA), the double bonds at different positions result in significant differences in morphology, physiology, metabolism, and physiological functions. To date, no studies have found the role of c9,t11c15-CLNA in visual and neurodevelopment through animal experiments; only a few CLNAs with different double bond positions have shown effects in anti-cancer and anti-inflammatory aspects. Furthermore, no research has yet determined the reasons or mechanisms leading to these differences.

[0008] This invention provides the application of c9,t11,c15-conjugated linolenic acid (c9,t11,c15-CLNA) in the preparation of products that improve optic nerve development disorders.

[0009] In one embodiment of the invention, the product promotes visual and / or neural development in newborn mammals.

[0010] In one embodiment of the present invention, the product comprises a drug, food, food for special medical purposes, health product, nutritional supplement or feed additive.

[0011] In one embodiment of the present invention, the health product is used to relieve visual fatigue.

[0012] In one embodiment of the present invention, the pharmaceutical product contains the above-mentioned C9,T11,C15-CLNA, a drug carrier, and / or pharmaceutical excipients.

[0013] In one embodiment of the present invention, the drug carrier includes one or more of the following commonly used in medicine: fillers, adhesives, wetting agents, disintegrants, lubricants, and flavoring agents.

[0014] In one embodiment of the present invention, the pharmaceutical excipient includes excipients and / or additives.

[0015] In one embodiment of the present invention, the dosage form of the drug includes a liquid preparation, a solid preparation, or a semi-solid preparation.

[0016] In one embodiment of the present invention, the solid dosage form includes tablets, granules, powders, or blocks.

[0017] In one embodiment of the present invention, the liquid formulation includes an aqueous solution, a suspension, or an emulsion.

[0018] This invention provides the application of C9,T11,C15-conjugated linolenic acid in the preparation of health products that relieve visual fatigue.

[0019] In one embodiment of the present invention, the dosage of the c9,t11,c15-conjugated linolenic acid is at least 10 mg / kg.

[0020] Beneficial effects: This invention confirms that c9,t11,c15-CLNA can effectively improve optic nerve hypoplasia caused by malnutrition and promote visual and neural development in newborn mice, specifically in the following ways: 1) Within a safe range, promote the age of eye opening; 2) Improves visual development indicators in newborn mice with malnutrition during pregnancy; 3) Improves retinal pathological symptoms in newborn mice with maternal malnutrition; 4) Increase the relative expression level of EGFR in the retinal tissue of newborn mice with maternal malnutrition; 5) In the open field experiment, newborn mice with maternal malnutrition showed more autonomous movement trajectories and longer dwell time in the central area in unfamiliar environments, which improved their spatial exploration ability and environmental adaptability. The overall development level of the nervous system and the spatial cognitive ability under visual guidance were effectively promoted. 6) In the new object experiment, newborn mice with maternal malnutrition showed a significant increase in their discrimination index for novel objects, enhanced learning and memory abilities, and improved functional development of brain regions such as the hippocampus and visual recognition abilities. 7) Promotes the increase of omega-3 PUFAs content in the liver of newborn mice; 8) Promotes the increase of Ω-3 PUFAs content in the prefrontal cortex of newborn rat brains. Attached image description: Figure 1 This indicates c9, t11, c15-CLNA. 1 H NMR spectrum.

[0021] Figure 2 This indicates c9, t11, c15-CLNA. 13 C NMR spectrum.

[0022] Figure 3 This diagram illustrates the mass spectrometry fragmentation methods of c9, t11, and c15-CLNA.

[0023] Figure 4 The mass spectrometry fragment ion relative abundance diagrams for c9, t11, and c15-CLNA are shown.

[0024] Figure 5 This represents the chemical structural formula of c9, t11, c15-CLNA.

[0025] Figure 6 This section compares the effects of eye-opening age in different groups of mice. Eye-opening age is defined as the day the mouse is born (day 1) until its eyes are fully open.

[0026] Figure 7 This shows a comparison of the wet weight of a single eyeball in each group of baby mice.

[0027] Figure 8 This image shows a comparison of the retinal and choroidal structures of the eyes of different groups of mice after HE staining.

[0028] Figure 9 This represents a comparison of the total distance traveled by each group of pups in an open field behavioral experiment.

[0029] Figure 10 This represents a comparison of the movement speed of different groups of young mice in an open field behavioral experiment.

[0030] Figure 11 This shows a comparison of the movement trajectories of different groups of pups in an open field behavioral experiment.

[0031] Figure 12 This represents a comparison of the total time spent exploring new objects in the new object recognition behavior experiment among different groups of mice.

[0032] Figure 13 This represents a comparison of the percentage of time spent exploring new objects in the new object recognition behavior experiment among different groups of mice.

[0033] Figure 14 This shows a comparison of the effects of EGFR fluorescence staining on the eyes of different groups of baby mice.

[0034] Figure 15 This study compares the effects of different groups of mice on the level of retinal epidermal growth factor (EGFR) in their eyes.

[0035] Figure 16 This study compares the effects of different groups of mice on the concentration of arachidonic acid (ARA) in the liver.

[0036] Figure 17 This study compares the effects of different groups of mice on the concentration of docosahexaenoic acid (DHA) in the liver.

[0037] Figure 18 This study compares the effects of different groups of mice on the concentration of omega-3 series eicosapentaenoic acid (DPA) in the liver.

[0038] Figure 19 This study compares the effects of different groups of mice on the concentration of arachidonic acid (ARA) in the prefrontal cortex of their brains.

[0039] Figure 20 This study compares the effects of different groups of mice on the concentration of docosahexaenoic acid (DHA) in the prefrontal cortex of their brains.

[0040] Figure 21 This study compares the effects of different groups of mice on the concentration of omega-3 series eicosapentaenoic acid (DPA) in the prefrontal cortex. Detailed Implementation

[0041] The invention will be better understood through the following examples.

[0042] In this invention, unless otherwise specified, "%" or percentage used to describe concentration or proportion refers to weight percentage.

[0043] The C57BL / 6J mice used in the following examples were purchased from Spiford Biotechnology Co., Ltd.; the original strain of Bifidobacterium breve CCFM683 used in the following examples was isolated by the Biotechnology Center of the School of Food Science and Technology, Jiangnan University, and has been disclosed in patent CN105925514B with accession number CGMCC No.11828.

[0044] This invention relates to the following culture media: MRS liquid culture medium: 10 g tryptone, 10 g beef extract, 5 g yeast powder, 20 g glucose, 2 g diammonium citrate, 5 g sodium acetate, 2 g dipotassium hydrogen phosphate, 0.5 g magnesium sulfate heptahydrate, 0.25 g manganese sulfate monohydrate, 1 mL Tween 80, add water to 1000 mL.

[0045] MRS solid medium is obtained by adding 1.5% agar based on the total weight of the liquid medium to the above.

[0046] Example 1: Preparation and Verification of c9,t11,c15-CLNA The preparation methods of c9, t11, c15-CLNA in Examples 1-7 are as follows: A primary culture of *Bifidobacterium breve* CCFM683 was streaked onto MRS solid medium. Once single colonies emerged, they were inoculated into MRS liquid medium and cultured. When the bacteria reached the logarithmic growth phase, a 1% inoculum was added to MRS liquid medium containing 0.5 mg / mL ALA for fermentation. The culture was terminated after 24 hours. All culture conditions were performed in an anaerobic incubator. After collecting all fermentation broth, the supernatant was collected by centrifugation. The supernatant was then mixed with isopropanol in a 3:2:3 ratio in a separatory funnel, and the oil was extracted by inverting the funnel for 10 minutes. The supernatant was collected and the hexane was evaporated to dryness using a rotary evaporator. The obtained oil was then stored at -80°C after purging with nitrogen.

[0047] A portion of the oil was dissolved in methanol and purified by preparative liquid chromatography (HPLC). The HPLC conditions were: column temperature 25℃, isocratic elution with 95% methanol and 5% pure water (1% formic acid), flow rate 10 mL / min, detection wavelength 233 nm, sample volume 500 μL, and sample amount 100 mg. The column used was a Shanghai Yuexu Technology Co., Ltd. C30 column (model: XB-C30, column length: 21.2 mm x 250 mm, particle size: 5 μm, pore size: 120 Å). The obtained methanol-water solution was concentrated in a rotary evaporator to obtain high-purity c9,t11,c15-CLNAs.

[0048] The compound described in this invention is c9, t11, c15-conjugated linolenic acid (c9, t11, c15-CLNA), and its chemical structure was confirmed by nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS).

[0049] ¹H NMR (600 MHz, CDCl3), Figure 1 The characteristic signals include: δ 6.00 (2H, m, H-9, H-10), δ 5.56 (2H, m, H-11, H-12), δ 5.36 (1H, m, H-16), δ 5.32 (1H, m, H-15), δ 2.35 (2H, t, H-2), and δ 0.95 (3H, t, H-18). The signals at δ 6.00 and δ 5.56 indicate the presence of a conjugated trans double bond system, while the signals at δ 5.32 and δ 5.36 indicate the presence of a single cis double bond.

[0050] Carbon nuclear magnetic resonance spectroscopy¹³C NMR (150 MHz, CDCl₃, Figure 2 The characteristic signals include: δ 130.64 (C9), δ 130.33 (C10), δ 132.49 (C12), δ 131.60 (C11), δ 132.04 (C16), and δ 128.32 (C15). The δ values ​​of the middle carbons (C11, C12) in the conjugated double bond are lower than those of the outer carbons (C9, C10), which is consistent with the characteristics of a conjugated trans double bond. The δ values ​​of C15-C16 indicate that it is a cis configuration.

[0051] Mass spectrometry (MS) Figure 3-4 A molecular ion peak of m / z 277.2211 [MH] was detected in negative ion mode. - With molecular formula C 18 H 30 O2 is consistent. The main fragment ions include m / z 58.0062, 59.0140, 71.0141, 113.0616, 127.0777, and 181.5833, and their cleavage mode conforms to the β-cleavage pattern of conjugated triene fatty acids.

[0052] Based on the above spectral data, the compound described in this invention was ultimately determined to be c9, t11, c15-conjugated linolenic acid (LAA). Figure 5 ).

[0053] Example 2: Effects of c9,t11,c15-CLNA on the age of eye opening in maternally malnourished newborn mice The animal experimental design is as follows (Table 1): Three-week-old female mice were used as pregnant mice, and three-week-old male mice were used as breeding mice (after one week of acclimatization, female mice were 4 weeks old for modeling, which lasted for 28 days, and breeding began at week 8). Specific pathogen-free (SPF) mice were used. All mice were housed in an SPF-grade barrier facility at the Jiangnan University Animal Experiment Center, with a temperature of 22-26℃, relative humidity of 40-70%, a 12-hour light-dark cycle, and random access to sterile water and feed. The experiment was divided into a blank control group, a model group, a pregnant mouse intervention group, and a pup intervention group. The blank control group was fed a normal mouse diet throughout the experiment. Pregnant mice in the model group, pregnant mouse intervention group, and pup intervention group were fed a normal diet only during the acclimatization period. From the intervention period until sacrifice, they were fed a customized diet (fatty acid-free diet + 6.5g olive oil / 100g dry weight + 6.5g palm oil / 100g dry weight + 0.015g β-sitosterol / 100g dry weight). Control group: fed standard feed throughout. Female mice were introduced into paired cages at 8 weeks of age for conception. After the first puncture, the control group received daily oral administration of 0.9% saline solution. After the pups were born, gavage was stopped, and the mothers were allowed to produce milk naturally for their pups. Three days after birth, the pups were given daily oral administration of 0.9% saline solution until 4 weeks of age. Pups' weight and age at eye opening were recorded, and visual reflex behavior tests were conducted 3 days after eye opening. At 4 weeks of age, the pups underwent further behavioral tests (open field and novel object recognition). After these tests, the pups were anesthetized with 1% isoflurane, and blood was drawn from the heart. Subsequent dissections were performed to obtain complete retinal and cerebral cortex tissues.

[0054] Model Group: After one week of adaptive feeding, all female mice underwent a prenatal dietary deficiency model of Ω-3 PUFAs, using a customized diet for 28 days to allow them to metabolize most of their existing Ω-3 PUFAs. After modeling, the customized diet was continued until the end of the experiment. Male and female mice were introduced for mating at 8 weeks of age. After patency, pregnant mice in the model group were given 0.9% saline orally daily. After the pups were born, gavage was stopped, and the mother mice naturally lactated to feed the pups. Three days after birth, the pups were given 0.9% saline orally daily until 4 weeks of age. After birth, the pups' weight and age at eye opening were recorded, and visual reflex behavior tests were conducted 3 days after eye opening. At 4 weeks of age, the pups underwent further behavioral tests (open field and novel object recognition). After these tests, the pups were anesthetized with 1% isoflurane, and blood was drawn from the heart. Subsequent dissections were performed to obtain intact retina, cerebral cortex, and other tissues.

[0055] Pregnant mouse intervention group: After one week of adaptive feeding, all female mice underwent a prenatal dietary deficiency model of Ω-3 PUFAs, using a customized diet for 28 days to allow them to metabolize most of their existing Ω-3 PUFAs. After modeling, the customized diet was continued until the end of the experiment, and female mice began to conceive in the same cage at 8 weeks of age. After thromboembolism, pregnant mice in the CLNA group were orally administered 10 mg / kg c9,t11,c15-CLNA daily, pregnant mice in the DHA group were orally administered 10 mg / kg DHA daily, and pregnant mice in the ALA group were orally administered 10 mg / kg ALA daily. Drug administration was stopped one day before delivery, and the pregnant mice were allowed to naturally lactate to feed their pups, while simultaneously being fed the customized diet throughout the process. After birth, the weight and age at eye opening of the pups were recorded, and visual reflex behavior experiments were conducted 3 days after eye opening. Four weeks after the pups were born, they were subjected to the remaining behavioral experiments (open field, new object recognition). After the behavioral experiments were completed, the pups were anesthetized with 1% isoflurane, and blood was taken from their hearts. Then, they were dissected to obtain tissues such as the complete eyeballs, retina, and various cortical layers of the brain.

[0056] Pups Intervention Group: After one week of adaptive feeding, all female mice underwent a prenatal dietary deficiency model of Ω-3 and Ω-6 PUFAs, using a customized diet for 28 days to allow them to metabolize most of their existing Ω-3 PUFAs. After modeling, female mice were placed in the same cage for conception at 8 weeks of age. Once the patency was observed, pregnant mice were separated into different cages. No further intervention was provided to pregnant mice from the time of patency until delivery, allowing them to eat naturally (customized diet) and drink water. Pups were normally fed with milk after birth. Three days after birth, they were given daily oral administration of 10 mg / kg c9,t11,c15-CLNA (pups CLNA group), 10 mg / kg DHA (pups DHA group), and 10 mg / kg ALA (pups ALA group) until 4 weeks of age, during which time the mother mice were continuously fed a customized diet. Pups' weight and age at eye opening were recorded, and visual reflex behavior experiments were conducted 3 days after eye opening. Four weeks after the pups were born, they were subjected to the remaining behavioral experiments (open field, new object recognition). After the behavioral experiments were completed, the pups were anesthetized with 1% isoflurane, and blood was taken from their hearts. Then, they were dissected to obtain tissues such as the complete eyeballs, retina, and various cortical layers of the brain.

[0057] Table 1 Experimental Group Settings

[0058] The age at which the eyes of the control group pups opened was 14.50 days, while the age at which the eyes of the model group pups opened was 15.67 days, which was significantly delayed by 8.07% compared to the control group. Figure 6 (p < 0.05).

[0059] In a pregnancy intervention experiment in pregnant mice, mice in the group that were orally administered c9, t11, and c15-CLNA had an eye-opening age of 13.20 days, which significantly improved the delayed eye-opening caused by maternal malnutrition, shortening the time by 15.76% compared to the model group. Figure 6 (p < 0.05), its recovery effect was even better than the blank group by 1.3 days. It was comparable to the pregnant mouse oral administration of DHA positive drug group (13.38 days) and better than the pregnant mouse oral administration of ALA positive drug group (15.07 days) by 12.41%.

[0060] In the direct supplementation experiment of pups, the age of eye opening in the c9, t11, and c15-CLNA gavage group was 15.00 days, which also improved the delayed eye opening caused by maternal malnutrition, shortening it by 4.82% compared with the model group. Figure 6 (p>0.05), its effect was 87.80% of the effect of the oral positive drug DHA group (13.17 days) in pups and 90.00% of the effect of the oral positive drug ALA group (13.50 days) in pups.

[0061] Comparing the effects of direct dietary intake of c9, t11, c15-CLNA in pups and prenatal supplementation of c9, t11, c15-CLNA in pregnant mice on eye-opening age, it was found that dietary intake of c9, t11, c15-CLNA in pregnant mice was more effective than dietary intake in offspring, with the effect of dietary intake in offspring being only 89.20% of that in pregnant mice. Figure 6 ).

[0062] Example 3: Effects of c9,t11,c15-CLNA on ocular structure in maternally malnourished newborn mice The preparation and animal experiments of c9,t11,c15-CLNA are described in Examples 1 and 2.

[0063] After euthanizing the mice, the wet weight of their eyeballs was measured (results are shown below). Figure 7 After a simple wash with pre-cooled PBS, the ocular tissue sections were stained with hematoxylin and eosin (results are shown below). Figure 8 ).

[0064] The average wet weight of the eyeballs of the control group mice was 27.04 mg, while that of the model group mice was 21.26 mg. The wet weight of the eyeballs of the model group mice was only 78.62% of that of the control group, which was statistically significant. Figure 7 (p < 0.05).

[0065] In a pregnancy intervention experiment in pregnant mice, the wet weight of the eyeballs of pups in the group of pregnant mice orally administered c9, t11, and c15-CLNA was 26.66 mg, which significantly improved the decrease in eyeball wet weight caused by maternal malnutrition, and was 25.40% higher than that in the model group. Figure 7 (p < 0.05), its recovery effect basically reached the level of the blank group, which was 98.59% of the effect of the blank group. At the same time, it was superior to the effect of the oral DHA group (25.30 mg) in pregnant mice by 5.38%, and superior to the effect of the oral ALA group (22.28 mg) in pregnant mice by 19.66%.

[0066] In the direct supplementation experiment in pups, the wet weight of the eyeballs in the c9, t11, and c15-CLNA gavage group was 27.22 mg, which also improved the delayed eye opening caused by maternal malnutrition, increasing it by 28.07% compared to the model group. Figure 7 (p < 0.05), its recovery effect was even better than the blank group by 0.01%. It was 3.50% better than the effect of the pups orally administered positive drug DHA group (26.30 mg) and 1.08% better than the effect of the pups orally administered positive drug ALA group (26.93 mg).

[0067] Comparing the effects of direct dietary intake of c9, t11, c15-CLNA in pups and supplementation of c9, t11, c15-CLNA during pregnancy in pregnant mice on the ocular wet weight of pups, it was found that the effects of direct dietary intake of c9, t11, c15-CLNA in pups and supplementation of c9, t11, c15-CLNA during pregnancy in pregnant mice were basically the same, with direct intake in pups being slightly more effective than supplementation in pregnant mice. The effect of supplementation in pregnant mice during pregnancy was 97.94% of the effect of direct intake in pups. Figure 7 ).

[0068] In the control group, the retina and choroid of the mice were normal in morphology, with each layer intact and cells arranged neatly; while in the model group, the retinal morphology and structure were severely disordered, and the thickness of the inner and outer nuclear layers of the retina was significantly thinner. Figure 8 ).

[0069] In a pregnant mouse intervention experiment, pups in the group that received oral administration of c9, t11, and c15-CLNA showed significant improvements in ocular structure compared to the model group. Their retina and choroid morphology were normal, with abundant and regularly arranged cells, and significantly increased thickness of the inner and outer nuclear layers, even surpassing the control group in cell count. Pups in the ALA group showed lower cell counts, thinner inner and outer nuclear layers with gaps; while pups in the DHA group had normal and intact retina and choroid morphology, and significantly increased thickness of the inner and outer nuclear layers compared to the model group, the effect was still inferior to the CLNA group. Figure 8 ).

[0070] In the direct supplementation experiment in pups, the pups directly orally administered c9, t11, and c15-CLNA showed significant improvement in ocular structure compared to the model group. Their retina and choroid morphology were normal, with abundant and regularly arranged cells, and significantly increased thickness of the inner and outer nuclear layers, even surpassing the control group in cell count. In contrast, the pups in the ALA group exhibited lower cell counts, thinner inner and outer nuclear layers with gaps, and choroidal fragmentation. While the pups in the DHA group showed normal and intact retinal and choroid morphology, and significantly increased thickness of the inner and outer nuclear layers compared to the model group, the effect was still inferior to the pups in the CLNA group. Figure 8 ).

[0071] Overall, dietary intake of c9, t11, and c15-CLNA during pregnancy in pregnant mice and direct dietary intake of c9, t11, and c15-CLNA in pups can effectively promote retinal structural development in juvenile pups, but the effect is better in pregnant mice. Figure 8 ).

[0072] Example 4: Effects of c9,t11,c15-CLNA on visual developmental behavior in maternally malnourished newborn rat pups. The preparation and animal experiments of c9,t11,c15-CLNA are described in Examples 1 and 2.

[0073] Three days after the pups opened their eyes, a visual development behavioral experiment was conducted. The visual development experiment was carried out on the third day after the pups opened their eyes. The pups were held by their tails, head down, with their faces and forelegs facing the edge of a plane. The distance between the pups and the edge was gradually shortened. If the pups grasped the edge with their forelegs before their whiskers touched it, it was considered that their visual development was normal; otherwise, it was considered that their visual development was abnormal. The number of pups with normal visual development in each group was recorded.

[0074] The rate of normal visual development in the control group of mice was 93.33%, while that in the model group was only 66.67%.

[0075] In a prenatal intervention experiment in pregnant mice, the offspring of mice that ingested c9, t11, and c15-CLNA during pregnancy had a normal visual development rate of 91.67%, which was basically consistent with the control group and better than the model group (25.00%). The effect was also better than the ALA group (71.43%) (20.24%), but the DHA group showed the best results, with a 100% normal visual development rate in its offspring.

[0076] In the direct supplementation experiment in pups, the rate of normal visual development in pups directly supplemented with c9, t11, and c15-CLNA was 90.90%, which was basically the same as the control group and better than the model group by 24.23%. Furthermore, the effect of c9, t11, and c15-CLNA was 7.57% better than the ALA group (83.33%) and also better than the DHA group (83.33%) by 7.57%.

[0077] Comparative studies revealed that both gestational intake of c9, t11, and c15-CLNA in pregnant mice and direct intake of c9, t11, and c15-CLNA in offspring directly improved visual developmental problems in offspring caused by maternal malnutrition. The effects were essentially the same and significantly better than those ingested with ALA.

[0078] Example 5: Effects of c9,t11,c15-CLNA on open field behavior in maternally malnourished newborn rats. The preparation and animal experiments of c9,t11,c15-CLNA are described in Examples 1 and 2.

[0079] After the pups reached 4 weeks of age, they underwent open field behavioral experiments. A 45 cm side cube made of white acrylic was used as the test area, with a 20 cm x 20 cm area at the center of the bottom surface designated as the central area. A camera was mounted above this central area to record the mice's movement within the box. During the test, the mice were first placed in the box for 5 minutes to acclimatize, and then their movement trajectory was recorded for the next 5 minutes. Adaptation software was used to visualize the trajectory, and the total distance traveled and the average speed of the mice were analyzed and calculated.

[0080] In a supplementary experiment during the gestation period of pregnant mice, the total distance traveled by the control group pups in the open field was 1188.58 cm, while the total distance traveled by the model group pups was 1124.68 cm, a decrease of 5.38% compared to the control group. Figure 9 (p>0.05). Regarding movement speed, the average movement speed of the control group mice was 3.96 cm / s, while that of the model group mice was only 3.74 cm / s, a decrease of 5.56% compared to the control group. Figure 10 (p > 0.05). And according to Figure 11 The trajectory of the mice clearly shows that the time spent in the central area of ​​the open field by the blank group mice is significantly longer than that of the model group.

[0081] In a pregnant mouse intervention experiment, the offspring of mice in the c9, t11, c15-CLNA dietary intake group achieved a total distance of 1393.05 cm in the open field, which was 23.86% better than the model group. Figure 9 The effect was 19.27% ​​better than the positive control group (1167.98 cm / s) with ALA intake during pregnancy (p > 0.05), although it was not as good as the positive control group (1658.58 cm / s) with DHA intake during pregnancy (p > 0.05). Regarding movement speed, the offspring of pregnant mice supplemented with c9,t11,c15-CLNA (4.64 cm / s) were 1.06 times faster than the model group and 1.19 times faster than the positive control group (3.89 cm / s) with ALA intake during pregnancy (p > 0.05). Although it was not as good as the positive control group (5.53 cm / s) with DHA intake during pregnancy (p > 0.05), it was still 83.91% faster. Figure 10 As can be seen from the open field trajectory, compared with the model group, supplementation with DHA and CLNA during pregnancy in pregnant mice can increase the time their offspring spend in the central region, while the ALA group, although showing an improvement, has a poor overall effect. Figure 11 ).

[0082] In the direct supplementation experiment in pups, the group that ingested c9, t11, c15-CLNA moved a total distance of 1819.54 cm in the open field, which was 61.78% better than the model group. Figure 9The effect of direct supplementation with c9,t11,c15-CLNA (6.06 cm / s) in mice was significantly different from that in the model group (p < 0.05). Furthermore, it was superior to the positive control ALA group (1056.23 cm) by 72.27%, although it was less effective than the positive control DHA group (2131.28 cm), but still achieved 85.37% of the effect. Regarding movement speed, direct supplementation with c9,t11,c15-CLNA in mice (6.06 cm / s) showed a significant difference compared to the model group. Figure 10 The effect was 1.62 times that of the model group (p < 0.05), and 1.72 times that of the positive control ALA group (3.52 cm / s). Although it was not as good as the positive control group that directly ingested DHA (7.10 cm / s), it was 85.35% of the effect. The open field trajectory showed that, compared with the model group, direct supplementation of DHA and CLNA in the pups increased their residence time in the central region, while the ALA group, although showing an improvement, did not have a significant overall effect. Figure 11 ).

[0083] Based on all open field experiment data, direct dietary intake of c9, t11, c15-CLNA in pups was significantly better than that in pregnant mice during pregnancy in terms of total movement distance and speed. Specifically, the movement distance in pups with direct dietary intake of c9, t11, c15-CLNA was 30.62% higher than that in pregnant mice with direct dietary intake during pregnancy. Figure 9 In terms of average speed, the pups with dietary intake of c9, t11, c15-CLNA increased by 30.60% compared to the pregnant mice with dietary intake during pregnancy. Figure 10 This indicates that they have stronger spatial exploration and environmental adaptability, and that their overall developmental level of the nervous system and visually guided spatial cognitive abilities are effectively promoted.

[0084] Example 6: Effects of c9,t11,c15-CLNA on neoobject recognition behavior in maternally malnourished newborn rats. The preparation and animal experiments of c9,t11,c15-CLNA are described in Examples 1 and 2.

[0085] After the pups reached 4 weeks of age, following the open field behavioral experiment, a new object recognition behavioral experiment was conducted. The mice were placed on two objects and allowed to explore freely for a period of time. Then, a new object was selected to replace one of the original objects. Normally, the mice would spend more time exploring the new object. If the mice had memory loss or neurodevelopmental problems, they would spend the same amount of time exploring both objects during the experiment.

[0086] The blank group of mice spent 180.04 seconds exploring new objects, accounting for 60.00% of the total time, while the model group mice spent significantly less time exploring new objects. Figure 12 (120.87s) and exploration time percentage ( Figure 13There were significant differences between the model group and the control group in terms of the rate of improvement (40.28%) (p < 0.05), and the exploration time of the model group pups was only 67.14% of that of the control group.

[0087] In a pregnant mouse intervention experiment, the pups in the c9,t11,c15-CLNA group had a longer exploration time for new objects (168.17 s, accounting for 56.04% of the total time), significantly better than the model group (39.13%). Figure 12 , Figure 13 (p < 0.05). It was also superior to the positive control group that ingested ALA during pregnancy (136.66s, 45.53%) by 23.06%; although it was not as good as the positive control group that ingested DHA (177.39s, 59.11%), the effect was basically the same, only 5.20% lower than the pregnant mouse DHA group.

[0088] In the direct supplementation experiment with pups, the c9, t11, c15-CLNA group (193.77s, 64.57%) showed the best results. The total exploration time of the pups for the new object was 193.77s, accounting for 64.57% of the total time, which was 1.60 times that of the model group. Figure 12 , Figure 13 (p < 0.05), and it was 166.03% of the effect of the positive control ALA group (115.01s, 38.39%) and 121.78% of the effect of the positive control DHA group (159.13s, 53.02%).

[0089] Comparing pregnant mice that received C9, T11, and C15-CLNA during pregnancy with pups that received direct supplementation with C9, T11, and C15-CLNA, it was found that direct supplementation was more effective in the new object recognition experiment, with the effect being 115.22% of that observed in pregnant mice. This indicates that direct supplementation of C9, T11, and C15-CLNA in pups can enhance their learning and memory abilities, promote the functional maturation of brain regions such as the hippocampus, and improve visual recognition abilities. Figure 12 , Figure 13 ).

[0090] Example 7: Effects of c9,t11,c15-CLNA on EGFR levels in the retina of maternally malnourished newborn mice. The preparation and animal experiments of c9,t11,c15-CLNA are described in Examples 1 and 2.

[0091] After euthanizing the mice, whole eyeballs were collected for immunofluorescence staining to label the retinal epidermal growth factor receptor protein EGFR.

[0092] The relative expression level of retinal epidermal growth factor receptor protein EGFR in the control group was 1.00, while that in the model group was 0.77, showing a significant difference. Figure 14 , Figure 15 (p < 0.05).

[0093] In a pregnant mouse intervention experiment, the pups in the group receiving c9, t11, c15-CLNA in their diet had a relative EGFR level of 1.17, which was 51.95% higher than that in the model group. Figure 14 , Figure 15 (p < 0.05), the recovery effect was better than that of the blank group. It was also better than the positive control pregnant mouse ALA group (0.83) by 40.96% and better than the positive control pregnant mouse DHA group (0.96) by 21.88%.

[0094] In the direct supplementation experiment in pups, the relative EGFR content in the c9, t11, c15-CLNA dietary intake group reached 1.07, which was 38.96% better than the model group. Figure 14 , Figure 15 (p < 0.05), basically reaching the same level as the blank group. At the same time, it was superior to the positive control mouse ALA group (0.79) by 35.44% and superior to the positive control mouse DHA group (0.92) by 16.30%.

[0095] Comparing pregnant mice with dietary intake of c9, t11, and c15-CLNA during pregnancy and offspring mice with direct supplementation of c9, t11, and c15-CLNA, it was found that the effect of prenatal intake in promoting the retinal epidermal growth factor receptor protein EGFR was better, with an effect 9.35% higher than that of direct intake in offspring, which is more beneficial to the visual and retinal development of offspring in early life. Figure 14 , Figure 15 ).

[0096] Example 8: Effect of c9,t11,c15-CLNA on the concentration of high-quality PUFAs in the liver of maternally malnourished newborn rats The preparation and animal experiments of c9,t11,c15-CLNA are described in Examples 1 and 2.

[0097] After euthanizing mice, their livers were harvested, internal standards were added, and total lipids were extracted using the chloroform-methanol method. The total lipids were then methylated using the sulfuric acid-methanol method. Fatty acid methyl esters were detected using gas chromatography-mass spectrometry (GC-MS). GC conditions: Column: Rtx-wax (30 m × 0.25 mm × 0.25 μm); Temperature program: Initial column temperature 40℃, held for 5 min; increased to 120℃ at 20℃ / min; increased to 190℃ at 5℃ / min, held for 5 min; then increased to 220℃ at 5℃ / min, held for 17 min until the experiment ended; Vaporization chamber: 240℃; Helium (carrier gas) flow rate: 0.94 mL / min; Flow control mode: constant linear velocity; Injection method: split; Split ratio: 10:1. MS conditions: Ion source temperature: 220℃; Interface temperature: 250℃; Solvent delay time: 2.5 min; Acquisition method: scan; Mass number range: 50-550. Fatty acids were quantified using the internal standard method, with the ratio of the peak area of ​​the target substance to that of pentadecanoic acid or icosanoic acid being equal to the ratio of their concentrations.

[0098] Target peak area (obtained from integration in the previous step) : 15 (21) Alkanoic acid peak area (obtained from integration in the previous step) = Target concentration (to be measured) : 15 (21) Alkanoic acid concentration (actual amount added).

[0099] The concentration of arachidonic acid (ARA) in the liver of mice in the blank control group was 5.27 mg / g, while the concentration of ARA in the liver of pups in the model control group was 5.32 mg / g. The two were essentially the same, with no significant difference. Figure 16 (p>0.05). The concentration of docosahexaenoic acid (DHA) in the liver of mice in the blank control group was 3.84 mg / g, while the concentration of DHA in the liver of pups in the model control group was 0.30 mg / g. The blank group was 12.8 times that of the model group, which was statistically significant. Figure 17 (p < 0.05). The concentration of omega-3 docosapentaenoic acid (DPA) in the liver of mice in the blank control group was 0.43 mg / g, while the concentration of DPA in the liver of pups in the model control group was 0.23 mg / g. The blank group was 1.87 times that of the model group. Figure 18 ).

[0100] In a pregnant mouse intervention experiment, the arachidonic acid (ARA) concentration in the livers of pups in the c9, t11, c15-CLNA group was 18.40 mg / g, which was 3.46 times that of the model group pups. Figure 16The concentration of docosahexaenoic acid (DHA) in the liver of pups in the c9, t11, and c15-CLNA group was 5.23 mg / g (p < 0.05), which was 3.49 times that of the control group (12.26 mg / g). It was also 50.08% superior to the positive control group (ALA, 12.26 mg / g) and 45.34% superior to the positive control group (DHA, 12.66 mg / g). The concentration of DHA in the liver of pups in the c9, t11, and c15-CLNA group was 17.43 times that of the model group (p < 0.05). Figure 17 The effect was 1.36 times that of the blank control group of pups (p < 0.05), 3.68 times that of the positive control ALA group (1.42 mg / g), and 44.88% better than the positive control DHA group (3.61 mg / g). The concentration of omega-3 docosapentaenoic acid (DPA) in the liver of pups in the c9, t11, c15-CLNA group was 3.18 mg / g, which was 13.83 times that of the model group pups (p < 0.05). Figure 18 (p < 0.05), which is 7.40 times more effective than the blank control group of pups, 2.45 times more effective than the positive control group of pregnant mice with ALA (1.30 mg / g), and 85.96% more effective than the positive control group of pregnant mice with DHA (1.71 mg / g).

[0101] In the direct supplementation experiment in pups, the arachidonic acid (ARA) concentration in the liver of pups fed the c9, t11, and c15-CLNA diets was 14.81 mg / g, which was 2.78 times that of the model group pups. Figure 16 The concentration of docosahexaenoic acid (DHA) in the liver of pups in the c9, t11, c15-CLNA group was 4.39 mg / g, which was 14.63 times that of the model group (p < 0.05). This was 2.81 times higher than the control group (ALA, 7.57 mg / g) and 95.64% higher than the positive control group (DHA, 12.98 mg / g). Figure 17 The concentration of DPA (1.55 mg / g) in the liver of mice fed the c9,t11,c15-CLNA group was 2.43 mg / g, which was 1.14 times that of the control group (p < 0.05) and 2.83 times that of the positive control group (ALA, 1.55 mg / g). The concentration was second only to the positive control group (DHA, 4.72 mg / g), representing 93.01% of its effect. The concentration of DPA (europeanoic acid) in the liver of mice fed the c9,t11,c15-CLNA group was 2.43 mg / g, which was 10.57 times that of the model group (p < 0.05). Figure 18 (p < 0.05), which is 5.65 times the effect of the blank control group of mice, 2.45 times the effect of the positive control group of mice with ALA (0.99 mg / g), and 71.13% better than the positive control group of mice with DHA (1.42 mg / g).

[0102] Comparing pregnant mice with dietary intake of c9, t11, and c15-CLNA during pregnancy and pups directly supplemented with c9, t11, and c15-CLNA, it was found that the effect of supplementation during pregnancy was better in terms of the concentration of major PUFAs in the liver, with ARA concentration being 1.24 times higher in pregnant mice than in pups. Figure 16 The DHA concentration was 1.19 times that of the pups supplemented with it. Figure 17 The concentration of Ω-3DPA was 1.31 times that of the supplemented pups. Figure 18 This indicates that dietary intake of c9,t11,c15-CLNA during pregnancy in pregnant mice can increase the concentration of high-quality PUFAs in the liver of their offspring, providing a material basis for their transport to the brain.

[0103] Example 9: Effect of c9,t11,c15-CLNA on the concentration of high-quality PUFAs in the prefrontal cortex of maternally malnourished newborn rats The preparation and animal experiments of c9,t11,c15-CLNA are described in Examples 1 and 2.

[0104] After euthanizing mice, the prefrontal cortex of the brain was harvested, internal standard was added, and total lipids were extracted using the chloroform-methanol method. The total lipids were then methylated using the sulfuric acid-methanol method. Fatty acid methyl esters were detected by gas chromatography-mass spectrometry (GC-MS). GC conditions: Column: Rtx-wax (30 m × 0.25 mm × 0.25 μm); Temperature program: Initial column temperature 40℃ held for 5 min, increased to 120℃ at 20℃ / min, then increased to 190℃ at 5℃ / min, held for 5 min, then increased to 220℃ at 5℃ / min and held for 17 min until the end of the experiment; Vaporization chamber: 240℃; Helium (carrier gas) flow rate: 0.94 mL / min; Flow control mode: constant linear velocity; Injection method: split; Split ratio: 10:1. MS conditions: Ion source temperature: 220℃; Interface temperature: 250℃; Solvent delay time: 2.5 min; Acquisition method: scan; Mass number range: 50-550. Fatty acids were quantified using the internal standard method, with the ratio of the peak area of ​​the target substance to that of pentadecanoic acid or icosanoic acid being equal to the ratio of their concentrations.

[0105] Target peak area (obtained from integration in the previous step) : 15 (21) Alkanoic acid peak area (obtained from integration in the previous step) = Target concentration (to be measured) : 15 (21) Alkanoic acid concentration (actual amount added).

[0106] The concentration of arachidonic acid (ARA) in the prefrontal cortex of mice in the blank control group was 7.30 mg / g, while the concentration of ARA in the prefrontal cortex of pups in the model control group was 3.92 mg / g. The blank group showed a 1.86-fold increase in ARA concentration compared to the model group, indicating a statistically significant difference. Figure 19p < 0.05. The concentration of docosahexaenoic acid (DHA) in the prefrontal cortex of mice in the blank control group was 5.06 mg / g, while the concentration of DHA in the prefrontal cortex of pups in the model control group was 0.98 mg / g. The blank group was 5.16 times that of the model group, which was statistically significant. Figure 20 (p < 0.05). The concentration of omega-3 docosapentaenoic acid (DPA) in the prefrontal cortex of mice in the blank control group was 2.68 mg / g, while the concentration of DPA in the prefrontal cortex of pups in the model control group was 0.43 mg / g, which was 6.23 times that of the model group. Figure 21 ).

[0107] In a pregnant mouse intervention experiment, the concentration of arachidonic acid (ARA) in the prefrontal cortex of pups in the c9, t11, c15-CLNA group was 7.49 mg / g, which was 1.91 times that of the model group pups. Figure 19 The effect was 2.60% better than the control group (p < 0.05), and 40.53% better than the positive control group (ALA, 5.33 mg / g). It was also 86.69% of the effect of the positive control group (DHA, 8.64 mg / g). The concentration of docosahexaenoic acid (DHA) in the prefrontal cortex of pups in the c9,t11,c15-CLNA group was 5.13 mg / g, which was 5.34 times that of the model group pups. Figure 20 The effect was 1.01 times that of the control group, 2.93 times that of the positive control ALA group (1.75 mg / g), and 68.95% that of the positive control DHA group (7.44 mg / g). The concentration of omega-3 docosapentaenoic acid (DPA) in the prefrontal cortex of pups in the c9,t11,c15-CLNA group was 2.69 mg / g, which was 6.93 times that of the model group, 1.11 times that of the control group, 2.53 times that of the positive control ALA group (1.18 mg / g), and 63.29% that of the positive control DHA group (4.25 mg / g). (p < 0.05) Figure 21 ).

[0108] In the direct supplementation experiment in pups, the concentration of arachidonic acid (ARA) in the prefrontal cortex of the brain of pups fed the c9, t11, c15-CLNA diet was 7.02 mg / g, which was 1.79 times that of the model group. Figure 19The effect was consistent with the blank control group (p < 0.05), representing 96.16% of the effect in the blank control group of pups, and 31.21% better than the positive control group ALA (5.35 mg / g). It was also consistent with the positive control group DHA (7.01 mg / g). The concentration of docosahexaenoic acid (DHA) in the prefrontal cortex of pups in the c9,t11,c15-CLNA group was 7.03 mg / g, which was 7.17 times that of the model group (p < 0.05). Figure 20 The concentration of DPA (e.g., p < 0.05) in the prefrontal cortex of mice in the c9,t11,c15-CLNA group was 1.39 times that of the control group (ALA group, 2.94 mg / g) and 2.39 times that of the positive control group (ALA group, 2.94 mg / g). This concentration was second only to the positive control group (DHA group, 8.49 mg / g), representing 82.80% of its effect. The concentration of omega-3 docosapentaenoic acid (DPA) in the prefrontal cortex of mice in the c9,t11,c15-CLNA group was 2.10 mg / g, which was 4.88 times that of the model group, essentially consistent with the control group (78.36% of the effect of the control group), 1.15 times that of the positive control group (ALA group, 1.82 mg / g), and 70.47% that of the positive control group (DHA group, 2.98 mg / g). Figure 21 ).

[0109] Comparing pregnant mice with dietary intake of c9, t11, and c15-CLNA during pregnancy and pups with direct supplementation of c9, t11, and c15-CLNA, it was found that the effects of supplementation on the concentration of major PUFAs in the prefrontal cortex were basically the same. Regarding amino acids (AA), supplementation in pregnant mice was slightly better than direct supplementation in pups, exceeding the pup supplementation group by 6.70%. Figure 19 Regarding DHA, direct supplementation in pups was 37.04% more effective than supplementation during pregnancy in pregnant mice. Figure 20 Regarding Ω-3DPA, supplementation during pregnancy in pregnant mice was 30.58% more effective than direct supplementation in pups. Figure 21 This indicates that whether pregnant mice ingest c9, t11, c15-CLNA during pregnancy or pups ingest it directly, both can increase the concentration of high-quality PUFAs in the prefrontal cortex of the pups' brains, providing a prerequisite for their neural development and synaptic plasticity formation.

[0110] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. The application of a c9,t11,c15-conjugated linolenic acid in the preparation of products that improve optic nerve developmental disorders.

2. The application according to claim 1, characterized in that, The improvement of optic nerve development includes at least one of the following: promoting retinal functional development, increasing photoreceptor cell activity, improving visual signal transmission efficiency, promoting the development of cortical neurons, promoting hippocampal neuron development, promoting myelin formation, enhancing spatial learning and memory abilities, and reducing anxiety.

3. The application according to claim 2, characterized in that, The products mentioned are pharmaceuticals, food, special medical purpose formula food, health products, nutritional supplements, or feed additives.

4. The application according to claim 3, characterized in that, The drug also contains a drug carrier and / or pharmaceutical excipients.

5. The application according to claim 4, characterized in that, The drug carrier includes one or more of the following commonly used in medicine: fillers, adhesives, wetting agents, disintegrants, lubricants, and flavoring agents.

6. The application according to claim 5, characterized in that, The pharmaceutical excipients include excipients and / or additives.

7. The application according to claim 6, characterized in that, The dosage forms of the medicine include liquid, solid, or semi-solid preparations.

8. The application according to claim 7, characterized in that, The solid dosage forms include tablets, granules, powders, or blocks; the liquid dosage forms include aqueous solutions, suspensions, and emulsions.

9. Application of C9,T11,C15-conjugated linolenic acid in the preparation of health products to relieve visual fatigue.

10. The application according to claim 9, characterized in that, The dosage of the c9,t11,c15-conjugated linolenic acid is at least 10 mg / kg.

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