Application of 4-hydroxyphenyllactic acid in the preparation of intestinal development-promoting agents

Abstract

This invention belongs to the field of biopharmaceutical and functional food technology, and relates to the application of 4-hydroxyphenyllactic acid (4-HPHLA) in the preparation of formulations that promote intestinal development. This invention demonstrates that supplementation with 4-HPHLA can significantly promote intestinal development in early life. In vivo gavage experiments in suckling mice showed that it can promote body growth, increase small intestinal length and weight, and significantly lengthen duodenal and ileal villi, and increase ileal crypt density. In vitro intestinal organoid experiments further confirmed that 4-HPHLA can directly promote the proliferation, differentiation, and maturation of intestinal epithelial cells. The results indicate that 4-HPHLA can promote the maturation of intestinal structure and function in early life by regulating intestinal epithelial development and regeneration. This invention provides new application pathways and theoretical basis for the development of drugs, nutritional fortification agents, or dietary supplements for improving or promoting intestinal development in infants and young children.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical and functional food technology, specifically relating to the application of 4-hydroxyphenyllactic acid in the preparation of intestinal development-promoting agents. Background Technology

[0002] The gut is one of the most important organs in the human body, responsible for the digestion and absorption of nutrients and acting as a barrier against harmful substances, thus playing a crucial role in overall health. Early life is the most critical period for gut development and maturity. Gut maturation is essential for the healthy growth of infants and young children; conversely, immature gut development increases susceptibility to various diseases and hinders normal growth and development. Therefore, researching pharmaceutical preparations or fortified foods that can promote early gut development or alleviate intestinal problems is of great significance.

[0003] Breast milk is considered the gold standard for infant feeding, providing crucial nutritional support and infection protection for infants and playing a vital role in driving gut development in early life. Gut microbiota colonization is a critical event in gut development, and some of the many benefits of breast milk are mediated by the gut microbiota. Breastfeeding is also the strongest determinant of the composition and function of the gut microbiota in infancy. Human milk oligosaccharides (HMOs) are an important class of prebiotics abundant in breast milk, serving as substrates for specific gut microbes, such as Bifidobacteria. Bifidobacteria dominate the gut of breastfed infants and produce powerful prebiotic effects, effectively promoting gut development and immune maturation in early life.

[0004] The gut microbiota can influence the host's physiological or pathological state in various ways, such as through the production of metabolites. In recent years, many studies have focused on the contributions and mechanisms of Bifidobacteria and their metabolites to intestinal development and functional maturation in infancy, primarily concentrating on short-chain fatty acids (SCFAs). However, recent research indicates that breastfed Bifidobacteria convert aromatic amino acids into their respective metabolites, aromatic lactic acids, via aromatic lactate dehydrogenase (ALDH), including tryptophan-derived indolelacic acid (ILA), phenylalanine-derived phenyllactic acid (PLA), and tyrosine-derived 4-hydroxyphenyllactic acid (HPLA). Population cohort studies have found a significant increase in aromatic lactic acid levels in the feces of breastfed infants compared to formula-fed infants, and a significant positive correlation with typical Bifidobacterium species in infancy (such as *Bifidobacterium longum*, *Bifidobacterium breve*, and *Bifidobacterium bifidum*). In vitro experiments have validated the high aromatic lactic acid production capacity of these Bifidobacteria.

[10] Studies have shown that these aromatic lactic acids (HLAs) play a series of important functional roles in the body's health. For example, ILA prevents necrotizing enterocolitis (NEC) by activating aryl hydrocarbon receptors (AhRs) and inhibits tumorigenesis by guiding the differentiation of immature colonic macrophages; PLA extends healthy lifespan by enhancing mitochondrial function and stress recovery in *C. elegans* through SKN-1 / ATFS-1, and prevents obesity by regulating intestinal lipid metabolism. However, there is currently limited research on the potential physiological functions of HPLAs. Furthermore, we noted that in another study, the HPLA content in the feces of breastfed infants was increased by 2.56 times compared to formula-fed infants. In conclusion, we hypothesize that HPLAs, as a breast milk-dependent microbial metabolite, mediate important host-microbiota interactions in early life and may play a crucial role in intestinal development. Summary of the Invention

[0005] The technical problem to be solved by this patent application is to provide the application of 4-hydroxyphenyllactic acid in drugs or foods that promote intestinal development in early life, to explore the effects and mechanisms of 4-hydroxyphenyllactic acid on intestinal development in early life, and then to develop formulations or use them as dietary supplements by combining 4-hydroxyphenyllactic acid with pharmaceutically acceptable excipients or auxiliary ingredients.

[0006] Specifically, the present invention provides the following technical solution: This invention provides the application of 4-hydroxyphenyllactic acid in the preparation of intestinal development-promoting agents.

[0007] Furthermore, the formulation is used to promote gut development in early life, including gut development in infants and young children.

[0008] Furthermore, the preparation is a pharmaceutical preparation, a fortified food, or a dietary supplement.

[0009] Furthermore, the formulation contains 4-hydroxyphenyllactic acid as the active ingredient, and is formulated with pharmaceutically acceptable excipients or auxiliary ingredients.

[0010] Furthermore, the dosage form of the preparation is an oral preparation, including tablets, capsules, granules, oral liquids, or powders.

[0011] Furthermore, the administration method of the preparation is oral administration or gavage administration.

[0012] Further, the dosage of the preparation is 1-10 mg / kg / day; preferably, it is 2-5 mg / kg / day; most preferably, it is 3 mg / kg / day.

[0013] Furthermore, the promotion of intestinal development includes promoting intestinal morphological development, enhancing intestinal barrier integrity, promoting intestinal epithelial cell proliferation and differentiation, or regulating intestinal villus length and crypt density.

[0014] Furthermore, the formulation is also used to alleviate intestinal dysplasia, promote physical growth and development, or prevent intestinal-related diseases such as necrotizing enterocolitis (NEC).

[0015] Furthermore, the formulation is used to supplement breast milk or formula-fed infants to mimic the effects of breast milk-dependent microbial metabolites and promote gut microbiota-mediated development.

[0016] The beneficial effects of this invention include: demonstrating that 4-hydroxyphenyllactic acid supplementation can promote physical growth and development in suckling mice, regulate intestinal morphological development and maturation, and thus enhance the integrity of the intestinal barrier. Furthermore, 4-hydroxyphenyllactic acid supplementation can directly promote intestinal epithelial differentiation, indicating that 4-hydroxyphenyllactic acid supplementation may regulate intestinal development by influencing the differentiation of intestinal epithelial cells in early life, thereby promoting the maturation of intestinal morphology and function. This provides a theoretical basis for its application in the preparation of drugs or foods (dietary supplements) that promote intestinal development in infants and young children. Attached Figure Description

[0017] Figure 1 The promoting effect of 4-hydroxyphenyllactic acid on physical growth and intestinal development in suckling rats: A. Schematic diagram of the experimental process; B. Body weight; C. Body length; DE. Statistical analysis of small intestine length and weight; FG. Large intestine length and weight.

[0018] Figure 2 Effects of 4-hydroxyphenyllactic acid on the morphology of the duodenum, ileum, and colon in suckling rats.

[0019] Figure 3 Immunostaining was used to detect the expression of proliferating cell nuclear antigen (PCNA) in the intestinal epithelial crypts of suckling rats.

[0020] Figure 4 The changes in the number of two types of secretory cells in the intestinal epithelium of suckling rats were detected by PAS staining and immunostaining: A. goblet cells; B. Paneth cells.

[0021] Figure 5 The expression levels of Alpi (A) and Fapp1 (B) in the intestinal epithelium of suckling rats were detected using immunofluorescence staining.

[0022] Figure 6 HPLA promotes the differentiation of small intestinal epithelium in both normal and malnourished culture media. Detailed Implementation

[0023] The following detailed embodiments further illustrate the concept and technical effects of the present invention to fully understand its purpose, features, and effects. Unless otherwise specified, all methods described are conventional methods. Unless otherwise specified, all materials are available from publicly available commercial sources. The illustrative embodiments and descriptions of the present invention are used to explain the invention and do not constitute an undue limitation thereof. It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0024] Example 1: The effect of 4-hydroxyphenyllactic acid on the physical growth and intestinal development of suckling rats In this study, 4-hydroxyphenyllactic acid was administered by gavage to suckling mice starting on the sixth day after birth. The regulatory effect of 4-hydroxyphenyllactic acid on intestinal development in suckling mice was analyzed by assessing their physical growth and development, as well as the maturation of their small and large intestines.

[0025] 1. Animal experiments Several 8-week-old male and female C57BL / 6N mice were randomly divided into groups for mating. Pregnant females were separated into individual cages at day 16 (E16) until the end of the experiment. Two groups with births within two days of each other were selected as the control and treatment groups, respectively. The number of pups was evenly distributed between the two female cages to ensure that the baseline weight of newborn mice in both groups was consistent before the start of the gavage intervention. Oral gavage of 4-hydroxyphenyllactic acid (3 mg / kg / day) was administered to the mice starting on day 6 postpartum until day 16. The blank control group received the same volume of 1% DMSO (dissolved in PBS) once daily. The mice were weighed before gavage.

[0026] 2. Sampling After the intervention period ended (16 days after birth), the suckling mice were euthanized by cervical dislocation. After disinfection with medical alcohol, the intact intestines and spleen were removed with scissors. After measuring the baseline indicators, the intestinal contents were rinsed with ice-cold PBS. An appropriate amount was collected for protein and RNA extraction. The remaining intestinal segments were fixed with 4% paraformaldehyde at 4°C for 24-48 h, washed three times with PBS for 10 min each time, and then dehydrated and sectioned in paraffin.

[0027] 3. Analysis of body weight, body length, and the weight and length of the small and large intestines of suckling mice. The initial weight was taken as the weight on day 6 after birth (the start of the intervention period), and the weight was recorded daily thereafter until the end of the intervention period (day 16 after birth). Changes in weight can macroscopically indicate the growth rate of the suckling mice. As the suckling mice grow, their weight and body length will also change accordingly.

[0028] After the intervention period ended, the suckling mice were euthanized, and their body length was measured. After dissection, the weight and length of the small and large intestines were measured and the data were recorded for subsequent analysis.

[0029] 4. Experimental Results like Figure 1 As shown in Figure A, in this study, 6-day-old suckling rats were treated with 4-hydroxyphenyllactic acid (HPLA) via gavage at a dose of 3 mg / kg once daily until they reached 16 days of age. The results showed that, compared with the control group, HPLA intervention significantly promoted weight gain in the suckling rats. Figure 1 BC), and significantly increased the length and weight of the small intestine ( Figure 1 DE); however, no significant changes were observed in the length and weight of the large intestine (DE); Figure 1 (FG). In summary, HPLA has a significant promoting effect on the physical growth and intestinal development of suckling mice.

[0030] Example 2: Effects of 4-hydroxyphenyllactic acid on intestinal morphology in suckling rats This study analyzed the morphological changes of villi and crypt structures by performing HE staining on tissue sections of the duodenum, ileum, and colon of suckling mice, thereby assessing the effect of 4-hydroxyphenyllactic acid on the morphological maturation of various intestinal segments in suckling mice.

[0031] 1. Preparation of paraffin-embedded sections 1.1 Tissue dehydration The dehydration program is as follows: 50% ethanol, 30 min; 70% ethanol, 4-7.5 h; 80% ethanol, 1 h; 95% ethanol, 40 min; 100% ethanol I, 15 min; 100% ethanol II, 15 min; xylene: ethanol (volume ratio 1:1), 5 min; xylene I, 10 min; xylene II, 5 min; xylene: soft wax (volume ratio 1:1), 30 min; soft wax, 30 min; hard wax, 3 h; the temperature of the last 3 steps is set at 60℃ to dissolve the wax, and the remaining steps are carried out at room temperature.

[0032] 1.2 Paraffin Embedding The tissue was routinely embedded in paraffin, then cut into 5μm thick sections, and spread in a 42℃ water bath. The sections were observed under a stereomicroscope to see if they were fully spread. The spread sections were then retrieved with an adhesive slide, air-dried, and used for subsequent histopathological staining and immunostaining.

[0033] 2. Phenotypic analysis of mouse tissues (HE staining) Hematoxylin-eosin staining (HE staining) is the most common method used in cytology, histology, embryology, and pathology. It allows for direct observation of abnormal changes in tissue cells. The specific steps are as follows: Baking: 65℃, 1h; Dewaxing, hydration; Xylene I treatment for 15min; Xylene II treatment for 15min; Gradient ethanol treatment: 100% ethanol I and II each for 5min; 95% ethanol I and II each for 5min; 80% ethanol treatment for 5min; 70% ethanol treatment for 5min; Distilled water treatment for 5min; Hematoxylin treatment for 10min; Tap water treatment for 5min; 95% ethanol I and II treatments for 3min; Eosin treatment for 10s; 95% ethanol I and II each for 3min; 100% ethanol I and II each for 3min; Xylene I treatment for 10min; Xylene II treatment for 10min; Mounting, photographing.

[0034] 3. Experimental Results like Figure 2 As shown, this study analyzed different intestinal segments (duodenum, ileum, and colon) of suckling mice using hematoxylin-eosin (HE) staining. The results showed that HPLA gavage treatment significantly increased the villus length in the duodenum and ileum, and significantly increased the ileal crypt density. These results indicate that HPLA can effectively regulate the development and regeneration of the small intestinal epithelium in early-life suckling mice, promoting the maturation of intestinal epithelial morphology.

[0035] Example 3: Effects of 4-hydroxyphenyllactic acid on the proliferation and regeneration capacity of intestinal epithelium in suckling rats Proliferating cell nuclear antigen (PCNA) is a marker molecule for cell proliferation, with expression levels increasing primarily during the G1 / S phase and decreasing during the M phase. PCNA staining can locate proliferating cells and reflect the proliferative status of intestinal epithelium. This study assessed the effect of 4-hydroxyphenyllactic acid (4-PHALA) on the proliferation and regeneration capacity of intestinal epithelium in suckling mice by detecting PCNA expression through immunofluorescence staining of small intestinal tissue sections.

[0036] 1. Immunofluorescence staining Tissue sections were placed in sodium citrate antigen retrieval solution (pH 6.0) and boiled for retrieval. After cooling, 3% hydrogen peroxide solution was added to remove endogenous peroxidase. After blocking at room temperature for 1 h, primary antibody was added, and the mixture was incubated overnight at 4°C. After primary antibody incubation, the tissue sections were incubated with immunofluorescence secondary antibody at room temperature in the dark for 1 h. Subsequently, DAPI staining solution was added to counterstain the cell nuclei. Finally, the sections were mounted with antifluorescence quenching mounting medium and photographed using a laser confocal microscope.

[0037] 2. Experimental Results like Figure 3As shown, this study detected the expression characteristics of proliferating cell nuclear antigen (PCNA) in the crypts of the intestinal epithelium of suckling rats using immunostaining. The results showed that, compared with the control group, the number of PCNA-positive cells in the crypts of the HPLA intervention group was significantly increased, indicating that HPLA gavage can effectively enhance the proliferative activity of the intestinal epithelium of suckling rats and improve the regenerative potential of their intestinal tissue.

[0038] Example 4: Effect of 4-hydroxyphenyllactic acid on the composition of secretory cells in the intestinal epithelium of suckling rats Periodic acid-Schiff staining (PAS staining) primarily stains intracellular glycogen, extracellular matrix mucopolysaccharides, and mucus secreted by goblet cells in the intestinal mucosa. Goblet cells in the small intestine secrete mucus that participates in the formation of the intestinal barrier. PAS can specifically stain mucus granules within goblet cells, thus indicating the number of goblet cells. Paneth cells are specialized cells at the base of the small intestinal crypts, and Lyz (lysozyme) is a specific marker for them. This study used PAS staining and immunohistochemical staining on small intestinal tissue sections from suckling rats to detect changes in the number of goblet cells and Paneth cells, thereby assessing the effect of 4-hydroxyphenyllactic acid on the composition of secretory cells in the intestinal epithelium of suckling rats.

[0039] 1. PAS staining After dewaxing, periodic acid solution was added to the tissue sections and incubated at room temperature for 5 minutes. After soaking in tap water and distilled water, red sulfurous acid solution was added to the tissue sections and incubated at room temperature for 10 minutes. The sections were then immersed in tap water for 5-10 minutes to develop color, and finally, the reaction was terminated by immersion in pure water. Finally, hematoxylin staining and dehydration were performed followed by mounting.

[0040] 2. Immunohistochemical staining The preceding steps are the same as those in Example 3 for immunofluorescence staining. After primary antibody incubation, incubate secondary antibody at room temperature for 3 min. Then add horseradish enzyme-labeled streptavidin working solution and incubate at room temperature for 30 min. After membrane permeation and washing, add DAB chromogenic solution, and after about 1-2 min, immerse in pure water to stop the chromogenic process. Finally, perform hematoxylin staining and dehydration mounting.

[0041] 3. Experimental Results like Figure 4 As shown, this study detected changes in the number of two types of secretory cells (goblet cells and Paneth cells) in the intestinal epithelium of suckling rats using PAS staining and immunostaining. The results indicated that, compared to the control group, the number of PAS-positive goblet cells (…) in the intestinal epithelium of suckling rats after HPLA gavage… Figure 4 A) and Lyz-positive Paneth cells ( Figure 4 The number of cells in B) increased significantly, indicating that HPLA can significantly promote the differentiation of secretory cells in the intestinal epithelium of suckling rats, thereby accelerating the maturation of the intestinal mucus barrier and innate immune defense functions.

[0042] Example 5: Effects of 4-hydroxyphenyllactic acid on the differentiation, maturation, and absorption function of intestinal epithelium in suckling rats Alpi (intestinal alkaline phosphatase) is a marker protein of intestinal epithelial differentiation and maturation, mainly expressed in the brush border of mature absorptive cells in the small intestinal villi, and participates in nutrient absorption and maintenance of intestinal barrier function. Fabp1 (fatty acid-binding protein 1) is mainly expressed in differentiated mature intestinal epithelial cells and is responsible for fatty acid uptake, transport, and metabolism, making it a key protein for nutrient absorption in the intestinal epithelium. This study used immunofluorescence staining of small intestinal tissue sections from suckling rats to detect the expression of Alpi and Fabp1, thereby evaluating the effects of 4-hydroxyphenyllactic acid on intestinal epithelial differentiation and maturation and nutrient absorption function in suckling rats.

[0043] 1. Immunofluorescence staining The content is the same as in Example 3.

[0044] 2. Experimental Results As shown in Figure 5, this study used immunofluorescence staining to detect the expression levels of Alpi and Fapp1 in the intestinal epithelium of suckling rats. The results showed that, compared with the control group, the expression levels of Alpi (…) in the HPLA intervention group were significantly higher. Figure 5 A) and Fap1 ( Figure 5 B) The average fluorescence intensity was significantly increased. These results suggest that HPLA can effectively accelerate the differentiation and maturation of the intestinal epithelium in suckling rats and enhance intestinal nutrient absorption.

[0045] Example 6: 4-Hydroxyphenyllactic acid promotes intestinal epithelial differentiation under normal and malnourished conditions. In this study, small intestinal crypts of suckling mice were isolated in vitro and cultured into small intestinal organoids in normal culture medium (ENR) and malnutrition culture medium (50% ENR). 100 μM HPLA was added for intervention to explore the direct regulatory effect of HPLA on intestinal epithelial differentiation and maturation, as well as the effect of HPLA on improving intestinal development under malnutrition conditions.

[0046] 1. Isolation and culture of intestinal organoids from suckling mice Five-day-old suckling mice were euthanized, dissected, and 6-7 cm of small intestine was removed. After rinsing thoroughly, the intestine was cut longitudinally. The intestine was placed in a 50 mL centrifuge tube containing 20 mL of ice-cold PBS, and the washing was repeated 10 times. After washing, a new 20 mL of ice-cold PBS was added, along with 200 μL of EDTA solution, and the mixture was digested on an ice bath for 15 min on a shaker. The digestive fluid was washed away, and the mixture was placed in 20 mL of pre-chilled PBS, shaken vigorously 100 times, and transferred through a 70 μm cell sieve to a new 50 mL centrifuge tube. After centrifugation at 200 g for 5 min, the supernatant was discarded, and the pellet was resuspended in 10 mL of ice-cold DMEM / F12 medium. The pellet was transferred to a 15 mL centrifuge tube, centrifuged at 200 g for 5 min, and the supernatant was discarded. The pellet was resuspended in an appropriate amount of ENR medium, mixed with a 3:7 matrix gel, and 30 μL of the mixture was added to the center of each well of a 48-well plate to form a hemispherical shape. Invert the plate at 37 ℃ for 15 min, and add 270 μL of ENR or 50% ENR medium to each well. 50% ENR medium is prepared by replacing 50% of the Advanced DMEM / F12 medium in the ENR formulation with PBS while maintaining the same growth factor concentration. The experimental group medium was supplemented with 100 μM HPLA, and the control group medium was supplemented with an equal volume of blank solvent. Fresh medium and drugs were added on the third and fifth days of culture, and microscopic images were taken on the seventh day for subsequent statistical analysis.

[0047] 2. Experimental Results As shown in Figure 6, this study used 100 μM HPLA to intervene in primary small intestinal organoids from suckling rats. The results showed that under normal culture conditions (ENR), compared with the control group, HPLA treatment significantly promoted an increase in organoid cross-sectional area and the number of buds, indicating that HPLA can directly regulate the differentiation and maturation process of intestinal epithelium in early-life suckling rats. However, under malnourished culture conditions (50% ENR), HPLA intervention effectively reversed the reduction in organoid area and the decrease in the number of buds caused by malnutrition, indicating that HPLA can significantly improve the phenotype of intestinal developmental delay under malnutrition conditions.

Claims

1. The application of 4-hydroxyphenyllactic acid in the preparation of preparations that promote intestinal development in infants and young children.

2. The application according to claim 1, characterized in that, The formulation is intended to promote gut development in early life, including gut development in infants and young children.

3. The application according to claim 1, characterized in that, The preparation is a pharmaceutical preparation, a fortified food, or a dietary supplement.

4. The application according to claim 1, characterized in that, The formulation contains 4-hydroxyphenyllactic acid as the active ingredient, and is formulated with pharmaceutically acceptable excipients or auxiliary ingredients.

5. The application according to claim 1, characterized in that, The dosage form of the preparation is an oral preparation, including tablets, capsules, granules, oral liquids or powders.

6. The application according to claim 1, characterized in that, The preparation can be administered orally or by gavage.

7. The application according to claim 1, characterized in that, The dosage of the preparation is 1-10 mg / kg / day.

8. The application according to claim 1, characterized in that, The promotion of intestinal development includes promoting intestinal morphological development, enhancing intestinal barrier integrity, promoting intestinal epithelial cell proliferation and differentiation, or regulating intestinal villus length and crypt density.

9. The application according to claim 1, characterized in that, The formulation is also used to alleviate intestinal dysplasia, promote physical growth and development, or prevent intestinal-related diseases.

10. The application according to claim 1, characterized in that, The formulation is intended for infants who are breastfed or formula-fed to mimic the effects of breast milk-dependent microbial metabolites and promote gut microbiota-mediated development.

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