A composition for regulating the development of the HPA axis in infants and young children and its application

Abstract

This invention belongs to the field of composition technology, specifically relating to a composition for regulating the development of the HPA axis in infants and young children and its application. The composition for regulating the development of the HPA axis in infants and young children provided by this invention, based on manganese element, comprises the following components in parts by mass: 0.015 to 0.506 parts manganese source, 12 to 80 parts nucleotides, and 100 to 506 parts choline. The composition provided by this invention can increase the expression level of GR mRNA in the hypothalamus and pituitary gland, alleviate the increase in serum COR caused by stress stimulation, thereby regulating HPA axis development, reducing early stress-induced excessive activation of cortisol, and supporting the healthy development of infants' cognition, emotion, and neurobehavioral functions, showing good application prospects and market value.

Description

Technical Field

[0001] This invention belongs to the field of composition technology, specifically relating to a composition for regulating the development of the HPA axis in infants and young children and its application. Background Technology

[0002] The hypothalamus-pituitary-adrenal (HPA) axis is the core stress regulation system of the neuroendocrine system. When the body is stimulated, the hypothalamus secretes corticotropin-releasing hormone (CRH), which prompts the pituitary gland to release adrenocorticotropic hormone (ACTH), thereby stimulating the adrenal cortex to synthesize and secrete cortisol (COR). Under normal physiological conditions, COR binds to glucocorticoid receptors (GR) highly expressed in the hypothalamus and pituitary gland, initiating a negative feedback inhibitory pathway, allowing the HPA axis to automatically reset after the stress is eliminated, and COR levels subsequently decrease. In infants aged 0-3 years, the HPA axis regulatory capacity is not yet mature and they are highly sensitive to the care environment. Harmful stresses such as mother-infant separation or adverse environments can lead to overactivation of the HPA axis, impairing GR function and weakening negative feedback regulation, manifesting as elevated basal cortisol levels or circadian rhythm disorders. Long-term exposure can further damage hippocampal nerves, and in severe cases, can lead to cognitive and emotional developmental delays.

[0003] In current technologies, the treatment of HPA axis dysfunction in infants and young children mainly relies on tactile stimulation, environmental intervention, or later drug intervention. On the one hand, tactile stimulation and environmental intervention are difficult to standardize and have a slow onset of action, with minimal effect on existing GR functional impairment. On the other hand, drug intervention focuses on reducing COR levels, neglecting the core mechanism of GR expression downregulation leading to negative feedback failure, and lacks targeted regulation of neuroendocrine pathways. In addition, existing infant formula products at the neurodevelopmental level mostly focus on optimizing the composition and ratio of macronutrients, vitamins, and minerals, and there are no compositions specifically designed for programmed intervention of the HPA axis, resulting in inaccurate dosage and difficulty in stably supporting neurodevelopment. Summary of the Invention

[0004] In view of this, the present invention provides a composition for regulating the development of the HPA axis in infants and young children and its application. The composition for regulating the development of the HPA axis in infants and young children provided by the present invention includes a manganese source, nucleotides, and choline. This composition can reduce early stress-induced excessive activation of cortisol by regulating the development of the HPA axis in infants and young children, thereby supporting healthy cognitive, emotional, and neurobehavioral development in infants and young children.

[0005] To achieve the above-mentioned objectives, the embodiments of the present invention employ the following technical solutions: In a first aspect, the present invention provides a composition for regulating the development of the HPA axis in infants and young children, wherein the manganese source, calculated by the elemental manganese, comprises the following components in parts by mass: 0.015 to 0.506 parts of manganese source, 12 to 80 parts of nucleotides, and 100 to 506 parts of choline.

[0006] Compared to existing technologies, the composition for regulating the development of the HPA axis in infants and young children provided by this invention uses nucleotides to promote gastrointestinal development and immune system maturation, indirectly regulating the HPA axis through the gut-brain axis and providing a stable peripheral environment for the development of the HPA axis in infants and young children; manganese sources, as components or activators of various enzymes, directly participate in brain function development and neurotransmitter metabolism, enhancing the neuroendocrine system's ability to regulate the HPA axis by improving the neuronal metabolic environment; choline, as a precursor of acetylcholine, directly participates in neurotransmitter synthesis, improving neural regulation efficiency by participating in signal transduction in downstream processes, promoting the timely recovery of the HPA axis to homeostasis after stress stimulation, and reducing the adverse effects of excessive cortisol activation; the three components work together to maintain the homeostatic balance of the HPA axis in infants and young children's nervous, immune, and endocrine systems.

[0007] Preferably, the manganese source is calculated as manganese element, and the composition for regulating the development of the HPA axis in infants and young children includes the following components in parts by weight: 0.02 to 0.30 parts of manganese source, 20 to 58 parts of nucleotides, and 120 to 300 parts of choline.

[0008] More preferably, the manganese source includes at least one of manganese sulfate, manganese chloride, manganese carbonate, manganese citrate, or manganese gluconate.

[0009] More preferably, the nucleotide comprises the following components by mass percentage: 30%–40% disodium 5'-cytidine, 20%–28% disodium 5'-uridine, 18%–23% adenosine monophosphate, 10%–16% disodium 5'-inosine, and 5%–10% disodium 5'-guanylate.

[0010] More preferably, the choline includes at least one of choline tartrate or choline chloride.

[0011] More preferably, the composition for regulating the development of the HPA axis in infants further comprises the following components in parts by weight: 9,000 to 19,000 parts of protein, 18,000 to 30,000 parts of fat, and 40,000 to 72,000 parts of carbohydrates.

[0012] More preferably, the composition for regulating the development of the HPA axis in infants further comprises the following components in parts by weight: 12,000 to 18,000 parts of protein, 20,000 to 28,000 parts of fat, and 45,000 to 67,000 parts of carbohydrates.

[0013] More preferably, the protein includes at least one of skim milk powder, whey protein concentrate, casein, or whole milk powder.

[0014] More preferably, the fat includes at least one of soybean oil, corn oil, sunflower oil, coconut oil, or flaxseed oil.

[0015] More preferably, the carbohydrate includes at least one of lactose, maltodextrin, fructooligosaccharides, or galactooligosaccharides.

[0016] For example, the components in the composition for regulating the development of the HPA axis in infants provided by the present invention can be prepared simply by mixing them.

[0017] Secondly, the present invention provides a product for regulating the development of the HPA axis in infants and young children, comprising the above-mentioned composition for regulating the development of the HPA axis in infants and young children.

[0018] Preferably, the product includes, but is not limited to, infant formula or regular food. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0020] Unless otherwise specified, the raw materials and reagents used in this invention are all conventional commercially available products; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0021] 1. Laboratory animals SPF-grade SD rats, weaned pups (PND 21), half male and half female, provided by Spiford (Suzhou) Biotechnology Co., Ltd. (China), Certificate No.: SCXK (Su) 2022-0006.

[0022] 2. Main Reagents, Materials, and Test Kits (1) Main reagents and materials TRIzol, product number: 15596026, is from Thermo Fisher Scientific (China) Co., Ltd.

[0023] Chloroform, product number: 10006818, is sourced from Sinopharm Chemical Reagent Co., Ltd.

[0024] Isopropyl alcohol, batch number: WXBD5854V, sourced from Sigma-Aldrich, USA. Ethanol, product number: 100092680, is sourced from Sinopharm Chemical Reagent Co., Ltd.

[0025] Manganese sulfate (manganese provided) is sourced from DSM FineMeyers Nutrition (Shanghai) Co., Ltd.

[0026] Choline tartrate (provides choline) is sourced from DSM FineMeyers Nutrition (Shanghai) Co., Ltd.

[0027] The nucleotides (5'-cytidine disodium 35%, 5'-uridine disodium 24%, 5'-monophosphate adenosine 20.3%, 5'-inosine disodium 13.8% and 5'-guanylate disodium 6.9%) were sourced from Nanjing Tongkai Zhaoye Biotechnology Co., Ltd.

[0028] The skimmed milk powder is sourced from Junlebao Dairy Group Co., Ltd., and the concentrated whey protein is sourced from Daiweilin International Trade (Shanghai) Co., Ltd.

[0029] Soybean oil and corn oil are sourced from Langfang Development Zone Yijia Grain and Oil Food Supply Chain Management Co., Ltd.

[0030] Lactose is sourced from Daiweilin International Trading (Shanghai) Co., Ltd.

[0031] Fructooligosaccharides are sourced from Baolingbao Biotechnology Co., Ltd.

[0032] (2) Test kit Serum cortisol (COR) enzyme-linked immunosorbent assay kit, catalog number: COR31-K01, is from Beijing Huaying Biotechnology Research Institute.

[0033] Example 1 This embodiment provides a composition for regulating the development of the HPA axis in infants and young children, comprising the following components by weight: 41 μg manganese sulfate (15 μg manganese element), 12 mg nucleotides, 100 mg choline, 14 g protein (provided by equal weights of skim milk powder and whey protein concentrate), 27 g fat (provided by equal weights of soybean oil and corn oil), and 53 g carbohydrates (provided by equal weights of lactose and fructooligosaccharides).

[0034] Use a dry mixing process to mix until homogeneous.

[0035] Example 2 This embodiment provides a composition for regulating the development of the HPA axis in infants and young children, comprising the following components by weight: 110 μg manganese sulfate (40 μg manganese), 50 mg nucleotides, 120 mg choline, 14 g protein (provided by equal weights of skim milk powder and whey protein concentrate), 27 g fat (provided by equal weights of soybean oil and corn oil), and 53 g carbohydrates (provided by equal weights of lactose and fructooligosaccharides).

[0036] Use a dry mixing process to mix until homogeneous.

[0037] Example 3 This embodiment provides a composition for regulating the development of the HPA axis in infants and young children, comprising the following components by weight: 1389 μg manganese sulfate (506 μg manganese), 80 mg nucleotides, 506 mg choline, 14 g protein (provided by equal weights of skim milk powder and whey protein concentrate), 27 g fat (provided by equal weights of soybean oil and corn oil), and 53 g carbohydrates (provided by equal weights of lactose and fructooligosaccharides).

[0038] Use a dry mixing process to mix until homogeneous.

[0039] Comparative Example 1 This embodiment provides a composition that differs from Example 2 in that manganese sulfate is replaced with an equal mass of nucleotides; Specifically, it includes the following components by mass: 50.11 mg of nucleotides, 120 mg of choline, 14 g of protein (provided by equal masses of skim milk powder and whey protein concentrate), 27 g of fat (provided by equal masses of soybean oil and corn oil), and 53 g of carbohydrates (provided by equal masses of lactose and fructooligosaccharides).

[0040] Use a dry mixing process to mix until homogeneous.

[0041] Comparative Example 2 This embodiment provides a composition that differs from Example 2 in that the nucleotides are replaced with an equal amount of choline. Specifically, it includes the following components by mass: 110 μg manganese sulfate (40 μg manganese), 170 mg choline, 14 g protein (provided by equal masses of skim milk powder and whey protein concentrate), 27 g fat (provided by equal masses of soybean oil and corn oil), and 53 g carbohydrates (provided by equal masses of lactose and fructooligosaccharides).

[0042] Use a dry mixing process to mix until homogeneous.

[0043] Comparative Example 3 This embodiment provides a composition that differs from Example 2 in that choline is replaced with an equal amount of nucleotides. Specifically, it includes the following components by mass: 110 μg manganese sulfate (40 μg manganese), 170 mg nucleotides, 14 g protein (provided by equal masses of skim milk powder and whey protein concentrate), 27 g fat (provided by equal masses of soybean oil and corn oil), and 53 g carbohydrates (provided by equal masses of lactose and fructooligosaccharides).

[0044] Use a dry mixing process to mix until homogeneous.

[0045] Comparative Example 4 This embodiment provides a composition comprising the following components by weight: 14g of protein (provided by equal weights of skim milk powder and whey protein concentrate), 27g of fat (provided by equal weights of soybean oil and corn oil), and 53g of carbohydrates (provided by equal weights of lactose and fructooligosaccharides).

[0046] Use a dry mixing process to mix until homogeneous.

[0047] Application examples 1. Sample preparation The compositions provided in Examples 1-3 and Comparative Examples 1-4 were respectively mixed with vitamins and minerals to prepare products, which were designated as experimental groups 1-3 and control groups 1-4, respectively. In addition to the composition, each 100g of product also contains the following vitamin and mineral components in the following amounts: Vitamin A 400μgRE, Vitamin D3 18μg, Vitamin E 5.8mg α-TE, Vitamin K1 30μg, Vitamin B1 400μg, Vitamin B2 800μg, Vitamin B6 280μg, Vitamin B... 12 1μg, niacin 3mg, folic acid 85μg, pantothenic acid 2.6mg, vitamin C 63mg, biotin 10.3μg, inositol 35mg, sodium 150mg, potassium 380mg, copper 320μg, magnesium 32mg, iron 3.0mg, zinc 3.2mg, calcium 290mg, phosphorus 190mg, iodine 85μg, chlorine 260mg, selenium 17μg, taurine 25mg, L-carnitine 8.5mg.

[0048] 2. Animal grouping Weaned SD rat pups (PND 21) of SPF grade were selected for the experiment. They were provided by Spiford (Suzhou) Biotechnology Co., Ltd. (China), with certificate number SCXK (Su) 2022-0006. A total of 96 rats were selected, 12 rats per group, with half males and half females in each group. They were randomly divided into 8 groups, which were designated as blank control group, experimental groups 1-3 and control group 1-4.

[0049] The feeding environment conditions included: temperature of 20℃~24℃, relative humidity of 40%~60%, light-dark cycle of 12h / 12h, free access to water, and feed formulas for experimental groups 1~3 and control groups 1~4, which were the products of their respective groups. The feed formula for the blank control group was the same as that for control group 4. The animals were fed for 30 days.

[0050] 3. Establishment of animal models During the feeding period, after 7 days of acclimatization, all weaned rats (PND 21), except for the control group, were subjected to chronic unpredictable mild stress (CUMS) for 21 days. Specific stressors included: restraint (4 h), cage tilt (45° for 24 h), damp bedding (24 h), fasting / water deprivation (24 h), tail clamping (1 cm from the tail tip, 3 min), swimming in cold water (4°C, 3 min), and day-night reversal (24 h). During the experiment, rats were housed individually in single cages and randomly subjected to one stressor daily. For the last two days, they were fed only, with free access to food and water, without any other treatment.

[0051] 4. Detection of serum COR levels in rats After the establishment of the rat model and the completion of 30 days of feeding, the rats were anesthetized by intraperitoneal injection of 10% chloral hydrate at a dose of 350 μL / kg. Blood was collected from the abdominal aorta into EP tubes, incubated on ice for 1 hour, and then centrifuged at 3000 rpm for 15 minutes at 4°C. The supernatant was carefully transferred to new EP tubes and stored at -80°C until use. Serum COR levels in each group of rats were measured using an enzyme-linked immunosorbent assay (ELISA). The ELISA kit was obtained from Beijing Huaying Biotechnology Research Institute, and the assay was performed according to the instructions. The results are shown in Table 1.

[0052] Experimental data are expressed as mean ± standard deviation. One-way ANOVA was used to compare the means among multiple groups, and P < 0.05 was considered statistically significant.

[0053] Table 1 Results of serum COR level detection in rats In Table 1, the same letter indicates no significant difference between groups, while different letters indicate significant differences between groups (P < 0.05).

[0054] As shown in Table 1, the COR level in the blank control group was significantly lower than that in control group 4, indicating that stress stimulation led to an increase in blood COR levels. The COR levels in experimental groups 1-3 were significantly lower than those in the control group, indicating that the intake of the composition alleviated the increase in COR caused by chronic stress. The COR level in experimental group 2 was lower than that in experimental groups 1 and 3, but there was no significant difference among the three experimental groups, indicating that the composition can alleviate the increase in serum COR caused by stress stimulation within the range of the examples.

[0055] 5. Detection of mRNA expression levels of GR in rat hypothalamus and pituitary gland (1) Tissue sampling and total RNA extraction Tissue sampling: After blood collection from rats in each group, the hypothalamus and pituitary gland were quickly separated on ice, the capsule was removed, and the tissue was transferred to a liquid nitrogen tank for fixation and preservation.

[0056] Total RNA extraction: Hypothalamic and pituitary tissue blocks preserved in liquid nitrogen were quickly removed. Approximately 50 mg of the tissue block was added to 1 mL of pre-chilled TRIzol, and the mixture was repeatedly homogenized on ice using a handheld high-speed homogenizer to form a homogenate sample. 0.2 mL of chloroform was added to the homogenate, and the mixture was vigorously shaken for 15 s. After incubation at room temperature for 5 min, the mixture was centrifuged at 12000 rpm for 20 min at 4°C. The supernatant was carefully transferred to a new tube, and an equal volume of isopropanol was added. The mixture was inverted and mixed, incubated at room temperature for 10 min, and then centrifuged at 3000 rpm for 15 min at 4°C. The supernatant was discarded, and the RNA precipitate was collected. 1 mL of 75% ethanol was added to the precipitate for rinsing, and the precipitate was rinsed twice. The precipitate was dried under vacuum and dissolved in 50 μL of RNase-free ultrapure water to obtain the RNA sample for testing.

[0057] Quality testing: Take 2 μL of RNA sample to be tested, dilute it to 80 μL with RNase-free ultrapure water, and determine the concentration of nucleic acid using a UV spectrophotometer. After passing the test, take 2 μL of sample and perform 0.8% agarose gel electrophoresis to determine the purity of nucleic acid.

[0058] The remaining RNA samples after extraction were frozen and stored at -70℃ for later use.

[0059] (2) Reverse transcription (RT) reaction to synthesize cDNA RT reaction system: 5×Buffer 6μL; dNTP 1.5μL; OligoT 2μL; RNasin 1.5μL; MLV 1.5μL; RNA sample to be tested 3μL, add RNase-free ultrapure water to 30μL.

[0060] RT reaction conditions: The RT reaction system was incubated in a water bath at 37°C for 5 min, at 42°C for 60 min, and at 70°C for 10 min in sequence to obtain cDNA. The cDNA was then stored at -20°C.

[0061] (3) Real-time quantitative PCR (qPCR) reaction Primers: synthesized by Beijing Aoke Biotechnology Co., Ltd., with an expected amplified fragment length of 271 bp. Primer sequences are shown in Table 2.

[0062] Table 2 Primer Sequences The target gene qPCR reaction system is as follows: ddH2O 8.5μL; PCR mix 12.5μL; upstream primer of the target gene 0.5μL; downstream primer of the target gene 0.5μL; cDNA 3.0μL, with a total reaction volume of 25μL.

[0063] The internal reference gene qPCR reaction system consisted of: ddH2O 8.5 μL; PCR mix 12.5 μL; upstream primer of the internal reference gene 0.5 μL; downstream primer of the internal reference gene 0.5 μL; cDNA 3.0 μL, with a total reaction volume of 25 μL.

[0064] qPCR reaction conditions: The target gene and internal reference gene qPCR reaction system are loaded onto the instrument, and the amplification cycle program is set as shown in Table 3. The qPCR reaction is performed, and the qPCR amplification product is obtained after the reaction is completed.

[0065] Table 3 qPCR reaction conditions Relative quantitative analysis: 10 μL of qPCR amplification product was subjected to 1.2% agarose gel electrophoresis for 1 h at a voltage of 100V. 3 μL of a 100bp marker was loaded. Images were acquired using the ImageMaster VDS gel imaging system, and the optical density of the electrophoretic bands was analyzed using the accompanying image recording and analysis system. The total absorbance (A) value was expressed as the average absorbance multiplied by the band area. The ratio of the A value of the sample amplification product to the A value of the band in the β-actin amplification product was used as a parameter for mRNA expression level. Semi-quantitative analysis of GR mRNA expression was performed, and the results are shown in Table 4.

[0066] Experimental data are expressed as mean ± standard deviation. One-way ANOVA was used to compare the means among multiple groups, and P < 0.05 was considered statistically significant.

[0067] Table 4. Results of GR mRNA expression level detection in hypothalamus and pituitary gland In Table 4, the same letter indicates no significant difference between groups, while different letters indicate significant differences between groups (P < 0.05). Table 4 shows that the GR mRNA expression levels in the hypothalamus and pituitary gland of the blank control group were higher than those in control group 4, indicating that chronic stress significantly reduced the GR mRNA expression levels in the hypothalamus and pituitary gland, leading to overactivation of the HPA axis in rats and forming a vicious cycle of hypercortisol-receptor downregulation, manifested as persistently elevated COR and persistently hyperactive HPA axis function. The GR mRNA expression levels in the hypothalamus and pituitary gland of experimental groups 1–3 were significantly higher than those in control groups 1–3, indicating that the intake of specific components and proportions in this regimen can increase the GR mRNA expression levels in the hypothalamus and pituitary gland, thereby regulating HPA axis development and reducing early stress-induced cortisol overactivation.

[0068] Based on the above effects, the composition of this solution can be used in products that regulate the development of the HPA axis in infants and young children.

[0069] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A composition for modulating the development of the HPA axis in an infant, characterized in that, The manganese source, calculated by elemental manganese, comprises the following components in parts by mass: 0.015 to 0.506 parts manganese source, 12 to 80 parts nucleotides, and 100 to 506 parts choline.

2. The composition for regulating development of HPA axis in an infant or young child according to claim 1, wherein The manganese source, calculated as elemental manganese, comprises the following components in parts by weight: It includes the following components in parts by weight: 0.02 to 0.30 parts manganese source, 20 to 58 parts nucleotides, and 120 to 300 parts choline.

3. The composition for regulating development of HPA axis in an infant or young child according to claim 1 or 2, characterized in that, The manganese source includes at least one of manganese sulfate, manganese chloride, manganese carbonate, manganese citrate, or manganese gluconate.

4. The composition for regulating development of HPA axis in an infant or young child according to claim 1 or 2, characterized in that, The nucleotide comprises the following components by weight percentage: 30%–40% disodium 5'-cytidine, 20%–28% disodium 5'-uridine, 18%–23% adenosine monophosphate, 10%–16% disodium 5'-inosine, and 5%–10% disodium 5'-guanylate.

5. The composition for regulating development of HPA axis in an infant or young child according to claim 1 or 2, characterized in that, The choline includes at least one of choline tartrate or choline chloride.

6. The composition for regulating development of HPA axis in an infant or young child according to claim 1 or 2, characterized in that, The composition for regulating the development of the HPA axis in infants and young children also includes the following components in parts by weight: 9,000 to 19,000 parts of protein, 18,000 to 30,000 parts of fat, and 40,000 to 72,000 parts of carbohydrates.

7. The composition for regulating development of HPA axis in an infant or young child according to claim 6, wherein The protein includes at least one of skim milk powder, whey protein concentrate, casein, or whole milk powder.

8. The composition of claim 6, wherein the composition is formulated to be administered to the infant in an amount of 0.1 to 10 mg / kg of the infant’s body weight per day. The fat includes at least one of soybean oil, corn oil, sunflower oil, coconut oil, or flaxseed oil.

9. The composition for regulating the development of the HPA axis in infants and young children as described in claim 6, characterized in that, The carbohydrate includes at least one of lactose, maltodextrin, fructooligosaccharides, or galactooligosaccharides.

10. A product for regulating the development of the HPA axis in infants and young children, characterized in that, The composition comprising any one of claims 1 to 9 for regulating the development of the HPA axis in infants and young children.

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