Use of pinoresinol diglucoside in increasing short-chain fatty acids in the intestine of postmenopausal women and relieving inflammation in the intestine of postmenopausal women

Abstract

The application discloses application of pinoresinol diglucoside in increasing short-chain fatty acids in the postmenopausal intestinal tract and relieving postmenopausal intestinal inflammation, and belongs to the medical field. The pinoresinol diglucoside can regulate the abundance of Akkermansia, another branch bacteria, parabacteroides, rumenococcus and other genera in the intestinal tract, promote the expression of short-chain fatty acids in the postmenopausal intestinal tract, and then relieve postmenopausal intestinal inflammation and osteoporosis. The application opens up a new clinical approach for the treatment of postmenopausal women and diseases related to intestinal flora disorder.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to the application of pinoresinol diglucoside in increasing short-chain fatty acids in the postmenopausal intestine and alleviating postmenopausal intestinal inflammation. Background Technology

[0002] After menopause, the sharp drop in estrogen levels in women leads to gut microbiota dysbiosis and increased intestinal barrier permeability. This imbalance and damage to the intestinal mucosa cause lipopolysaccharide (LPS) to accumulate in the intestines and "leak" into the bloodstream, resulting in endocrine disorders, osteoporosis, and systemic inflammatory changes. Short-chain fatty acids (SCFAs) are metabolites produced by the fermentation of undigested carbohydrates by gut microbes. They can promote the secretion of anti-inflammatory factors, inhibit LPS-induced inflammatory responses, and promote the recovery of intestinal diseases. Pinoresinoldiglucoside (PDG), a major component of the traditional Chinese medicine Eucommia ulmoides, can be metabolized by gut microbiota into enterolactones or enterodiols, and has antihypertensive and antioxidant effects. This invention aims to provide the application of pinoresinoldiglucoside in increasing short-chain fatty acids in the postmenopausal gut and alleviating postmenopausal intestinal inflammation. Summary of the Invention

[0003] To address the aforementioned problems, the first objective of this invention is to provide the application of pinoresinol diglucoside in increasing short-chain fatty acids in the postmenopausal gut, and the second objective of this invention is to provide the application of pinoresinol diglucoside in regulating gut microbiota.

[0004] The first objective of this invention is achieved by the application of pinoresinol diglucoside in increasing short-chain fatty acids in the postmenopausal gut, wherein the short-chain fatty acids are at least one of acetic acid, isobutyric acid, isovaleric acid, or valeric acid.

[0005] The second objective of this invention is achieved through the application of pinoresinol diglucoside in regulating the gut microbiota. Pinoresinol diglucoside regulates the postmenopausal gut microbiota by influencing the structure and abundance of gut microbiota, particularly by regulating the presence of *Akermansia*, *Alternaria*, *Lactobacillus*, and *Ruminococcus*. Furthermore, pinoresinol diglucoside regulates the postmenopausal gut microbiota by increasing the Chao1 index, ACE index, and Shannon index in gut microbiota β-diversity.

[0006] The beneficial effects of this invention are as follows: This invention discloses the application of pinoresinol diglucoside in regulating postmenopausal gut microbiota metabolism and alleviating intestinal inflammation, opening up a new clinical approach for the treatment of postmenopausal women and diseases related to gut microbiota dysbiosis. Attached Figure Description

[0007] Figure 1The effect of PDG on intestinal tight junction proteins in OVX mice;

[0008] Figure 2 The effects of PDG on mouse colon tissue;

[0009] Figure 3 A three-dimensional image of the lower end of the trabecular bone in the distal femur of a rat;

[0010] Figure 4 A 2D longitudinal section of the trabecular bone of the rat femur in coronal view;

[0011] Figure 5 The effects of PDG on the bone microenvironment in mice. Detailed Implementation

[0012] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be described in detail below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise specified, the test materials used in the following embodiments were purchased from conventional biochemical reagent stores. Unless otherwise stated, percentages and parts are by weight. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar with the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0013] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0014] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0015] This invention provides the application of pinoresinol diglucoside in the preparation of drugs or foods that increase the content of short-chain fatty acids in the postmenopausal intestine.

[0016] The short-chain fatty acid is at least one of acetic acid, isobutyric acid, isovaleric acid, or valeric acid.

[0017] The present invention further provides the application of pinoresinol diglucoside in the preparation of drugs or foods for regulating postmenopausal intestinal flora. In this application, pinoresinol diglucoside alleviates postmenopausal systemic inflammation, such as postmenopausal intestinal inflammation and osteoporosis, by regulating the abundance of Ekkermansia, Alternaria, Parabacterium, and Ruminococcus in the intestine.

[0018] Pinoresinol diglucoside can promote the expression of short-chain fatty acids in the gut by regulating the structure and abundance of gut microbiota.

[0019] Pinoresinol diglucoside regulates the postmenopausal gut microbiota by increasing the Chao1 index, ACE index, and Shannon index in gut microbiota β-diversity.

[0020] The present invention further provides the application of pinoresinol diglucoside in the preparation of drugs for the prevention or treatment of intestinal inflammation.

[0021] The drug uses pinoresinol diglucoside as its active ingredient and is formulated into a pharmaceutically acceptable dosage form using pharmaceutically acceptable excipients.

[0022] The excipients include one or more of the following: solvents, disintegrants, flavoring agents, preservatives, colorants, binders, lubricants, diluents, and drug carriers.

[0023] The dosage form includes any one of tablets, capsules, pills, powders, ointments, elixirs, suspensions, powders, injections, sustained-release preparations, controlled-release preparations, or targeted preparations.

[0024] The following are the experimental animals used in the experimental examples and their grouping.

[0025] 1. Laboratory animals: SPF-grade C57BL / 6J female mice, purchased from Spiford (Beijing) Biotechnology Co., Ltd.;

[0026] 2. Experimental groups: including sham operation group (Sham), ovarian castration group (OVX), low-dose pinoresinol diglucoside group (PDG-L) and high-dose pinoresinol diglucoside group (PDG-H), with 5 animals in each group.

[0027] The Sham group consisted of individuals who, after incising the skin and exposing the abdominal cavity, only accessed the ovaries. The ovaries were not removed; instead, only adipose tissue of the same size as the ovarian tissue was excised to eliminate any interference from the surgical technique with the experimental results.

[0028] The OVX group consisted of mice whose skin was incised, abdominal cavity exposed, bilateral fallopian tubes were ligated, and bilateral ovaries were removed.

[0029] The PDG-L group consisted of rats whose bilateral ovaries were removed and who were given oral PDG at a dose of 5 mg / kg / d, 0.2 ml once a day for 8 consecutive weeks.

[0030] The PDG-H group consisted of rats whose bilateral ovaries were removed and who were then administered PDG orally by gavage at a dose of 10 mg / kg / d, 0.2 ml once a day for 8 consecutive weeks.

[0031] The other two groups were controlled by gavage with equal volume of sterile distilled water.

[0032] Experiment Example 1: Effects of Pinoresinol Diglucoside (PDG) on the Gut Microbiota of OVX Mice

[0033] Fecal and colonic samples were collected from mice in each group. Genomic DNA was extracted from the samples using the CTAB / SDS method. Beta diversity analysis was used to evaluate the species complexity differences in the samples. Weighted and unweighted beta diversity on unifrac was calculated using QIIME software (Version 1.9.1). A systematic sequencing analysis of the 16S rRNA gene in mouse fecal samples was performed to assess changes in the gut microbiota. The results are shown in Tables 1-2.

[0034] Table 1. Effects of PDG on gut microbial community diversity in OVX mice

[0035]

[0036] Note: (1) Compared with the Sham group * P < 0.05; (2) Compared with the OVX group, Δ P < 0.05.

[0037] Results: Table 1 shows that compared with the Sham group, the Chao1 index of the OVX group decreased significantly, while it generally increased after PDG intervention, with the PDG-L group showing the most significant increase. This indicates that the total number of species detected in the gut microbiota samples after castration tends to decrease, while the total number of species recovers and increases after PDG intervention. The Shannon index showed the most significant increase in the PDG-L group compared to other groups, indicating higher community diversity and more even species distribution in the PDG-L group.

[0038] Table 2 shows the percentage effect of PDG on the relative abundance of the top 30 genera of gut microbiota.

[0039]

[0040] As shown in Table 2, PDG can regulate the abundance of intestinal flora in OVX mice, mainly consisting of *Akkermansia*, *Alternaria*, *Lactobacillus*, *Prevotella*, *Parabacteroides*, and *Ruminococcus*. Compared with the Sham group, the OVX group showed an increase in *Bacteroides*, *Lactobacillus*, and *Ruminococcus* in feces, and a decrease in *Akkermansia*, *Prevotella*, *Parabacteroides*, and *Alternaria*. Compared with the OVX group, the low-dose PDG group showed an increase in *Akkermansia*, *Trichophyton* (NK4A136), and *Alternaria*, and a decrease in *Lactobacillus*, *Prevotella*, and *Parabacteroides*. The high-dose PDG group showed an increase in *Akkermansia*, *Trichophyton* (NK4A136), *Prevotella*, *Parabacteroides*, and *Alternaria*, and a decrease in *Lactobacillus*. Among them, *Akermansia*, *Pseudomonas*, *Alternaria*, and *Ruminococcus* are differentially expressed bacterial genera that can promote the synthesis of SCFAs, indicating that PDG can regulate the influence of these four differentially expressed bacterial genera on the structure and abundance of the intestinal flora and may affect the synthesis of SCFAs.

[0041] Example 2: Effect of pinoresinol diglucoside (PDG) on the secretion level of SCFAs in feces of OVX mice

[0042] Experimental methods: Feces from mice in each group were collected, and the content of short-chain fatty acids in the fecal samples was detected by GC-Q-MS target metabolomics. The results are shown in Table 3.

[0043] Table 3. Effects of PDG on the concentration of short-chain fatty acids in feces of OVX mice.

[0044]

[0045] Note: (1) Compared with the sham surgery group * P < 0.05; (2) Compared with the ovarian castration group, Δ P < 0.05.

[0046] Results: As shown in Table 3, compared with the Sham group, the content of short-chain fatty acids in the feces of mice in the OVX group was significantly reduced. After PDG intervention, the content of short-chain fatty acids in feces was significantly increased, with acetic acid, isobutyric acid, isovaleric acid, and valeric acid showing a significant increasing trend, indicating that PDG can promote the production of short-chain fatty acids.

[0047] Akkermansia is a Gram-negative bacterium belonging to the phylum Verrucomicrobia. It widely colonizes the intestinal mucus layer of humans and animals, playing an important role in alleviating intestinal inflammation, preventing aging, and improving metabolic diseases. Studies have found that *g_Akkermansia* can produce various enzymes to degrade mucin and ferment it to produce SCFAs (strain-soluble fatty acids). SCFAs, as metabolic products of gut microbiota, play an important role in regulating osteoclast metabolism and maintaining bone mass. *g_Parabacteroides* is a Gram-negative bacterium belonging to the phylum Bacteroidetes. It is a core component of the gut microbiota, synthesizing succinic acid and short-chain fatty acids such as acetic acid and propionic acid. It plays an important regulatory role in the host mucosal immune system, and its levels are significantly negatively correlated with disease states such as obesity, non-alcoholic fatty liver disease, and inflammatory bowel disease. *g_Alistipes* is a relatively new genus isolated from clinical samples. It is a Gram-negative bacterium of the phylum Bacteroidetes, and its metabolic end products are succinic acid, acetic acid, and propionic acid. A Mendelian randomized study on bone mineral density and gut microbiota across different age groups found that *g_Alistipes* was a protective factor against bone mineral density in individuals over 60 years of age. *Ruminococcus*, a Gram-positive anaerobic bacterium of the Firmicutes phylum, ferments complex sugars and produces SCFAs, possessing dual potential as both a pathogen and a probiotic. The expression abundance of *g_Ruminococcus* was positively correlated with both bone loss and intestinal barrier damage. In conclusion, *g_Akkermansiag_*, *Alistipes*, *g_Parabacteroides*, and *g_Ruminococcus* may be target bacterial genera for PDG regulation of SCFAs.

[0048] Experimental Example 3: Effects of Pinoresinol Diglucoside (PDG) on Intestinal Inflammation in OVX Mice

[0049] Colon tissue was collected from mice in each group. A portion of the colon tissue was cut and fixed with 4% paraformaldehyde fixative, and paraffin sections were prepared. HE staining and double immunofluorescence staining with Claudin-1 and ZO-1 were performed. The HE staining results are shown below. Figure 1 As shown in the image. In the immunofluorescence double staining, green fluorescence represents the protein expression intensity of Claudin-1, red fluorescence represents the protein expression intensity of ZO-1, and blue represents the cell nuclei stained with DAPI. Merge is the image after combining the three colors. The detection results are shown in Table 4 and... Figure 1 As shown;

[0050] Table 4. Effects of PDG on tight junction protein in the colon of OVX mice.

[0051] Group Only Claudin-1 (fluorescence intensity value) ZO-1 (fluorescence intensity value) Sham Group 3 <![CDATA[0.21±0.00 ΔΔΔ ]]> <![CDATA[0.20±0.01 ΔΔ ]]> OVX group 3 <![CDATA[0.18±0.00 *** ]]> <![CDATA[0.17±0.00 ** ]]> PDG-L group 3 <![CDATA[0.20±0.01 Δ ]]> 0.18±0.00 PDG-H group 3 <![CDATA[0.19±0.01 Δ ]]> 0.17±0.00

[0052] Note: (1) Compared with the Sham group * P < 0.05; (2) Compared with the OVX group, Δ P < 0.05.

[0053] Fresh colon tissue homogenate was prepared and subjected to enzyme-linked immunosorbent assay (ELISA) to detect inflammatory factors interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), lipopolysaccharide (LPS), secretory immunoglobulin A (SIgA), and serum LPS. The experimental results are shown in Table 5.

[0054] Table 5. Effects of PDG on inflammatory factors, LPS, SIgA, and serum LPS in colonic tissue of OVX mice.

[0055]

[0056]

[0057] Note: (1) Compared with the Sham group * P < 0.05; (2) Compared with the OVX group, Δ P < 0.05.

[0058] Figure 2 The results showed that, compared with the Sham group, the OVX group had damage and shedding of the colonic mucosal epithelium, reduced goblet cells, irregular arrangement of intestinal crypts, and thinning of the basal layer. After PDG intervention, the mucosal damage was relieved, the intestinal crypts were arranged neatly, and the thickness of the basal layer increased. Figure 1 The results showed that, compared with the Sham group, the expression levels of intestinal tight junction proteins Claudin-1 and ZO-1 in the OVX group were significantly reduced (P<0.05), while the expression levels of intestinal tight junction proteins increased after PDG intervention, with the low dose showing the most significant effect (P<0.05). This indicates that PDG can repair intestinal barrier damage in OVX mice and alleviate intestinal inflammation.

[0059] As shown in Table 5, compared with the Sham group, the OVX group showed significantly increased expression levels of IL-6, IL-1β, and TNF-α in the colon (P<0.001), significantly decreased SIgA secretion levels, and significantly increased LPS levels in serum and colonic tissue (P<0.001). This indicates that ovariectomy reduces the mucosal immunity of the colonic tissue, increases the expression levels of inflammatory factors, and allows LPS to enter the bloodstream through the damaged intestinal barrier, causing systemic inflammation. Compared with the OVX group, PDG intervention significantly increased SIgA secretion levels and decreased inflammatory factor expression levels in the colonic tissue of OVX mice (P<0.05), and decreased LPS levels in the colon and serum (P<0.001). This suggests that PDG can improve the mucosal immunity of the colon in OVX mice, alleviate intestinal inflammation, and reduce LPS entry into the bloodstream.

[0060] Example 4: Effects of pinoresinol diglucoside (PDG) on bone mineral density and bone turnover markers in experimental animals.

[0061] The femur of mice fixed on a microCT stage was scanned using a NEMO Micro CT (model NMC-100) system from PINGSENG Healthcare (Kunshan) Inc. Bone mineral density (BMD), relative bone volume (BV / TV), trabecular number (Tb.N), trabecular separation (Tb.Sp), and trabecular thickness (Tb.Th) were measured. The results are shown in Table 6. Figure 3-4 As shown.

[0062] Table 6. Effects of PDG on bone parameters in OVX mice.

[0063]

[0064]

[0065] Note: (1) Compared with the Sham group * P < 0.05; (2) Compared with the OVX group, Δ P < 0.05.

[0066] The levels of bone formation factors PINP and ALP, and bone resorption factors CTX-1 and TRACP-5b in mouse serum were detected by enzyme-linked immunosorbent assay (ELISA). The results are shown in Table 7.

[0067] Table 7. Effects of PDG on bone formation factor and bone resorption factor in OVX mice.

[0068]

[0069] Note: (1) Compared with the Sham group, *P<0.05; (2) Compared with the OVX group, P<0.05.

[0070] OVX mouse bone mineral density data showed that after intervention with pinoresinol diglucoside (PDG), the number of trabeculae in the distal femur of castrated mice was significantly increased (see...). Figure 3-4 Furthermore, as shown in Table 6, the low-dose group exhibited significantly increased trabecular bone mineral density (Tb.BMD) and trabecular bone volume fraction (Tb.BV / TV), and both groups showed varying degrees of increase in trabecular bone number (Tb.N), while both groups showed a significant decrease in trabecular bone separation (Tb.Sp). This demonstrates that pinoresinol diglucoside can increase the bone mineral density of cancellous bone in ovariectomized mice.

[0071] The serum ELISA data in Table 7 show that after PDG intervention, the bone formation factors PINP and ALP in OVX mice increased to varying degrees, while the bone resorption factors CTX-1 and TRACP-5b decreased to varying degrees, suggesting that PDG can promote bone formation and inhibit bone resorption.

[0072] like Figure 5 HE staining of femoral tissue revealed that PDG intervention increased the thickness and integrity of bone trabeculae and reduced bone marrow fat vacuoles, indicating that PDG can improve the bone microstructure of OVX mice.

[0073] Based on the above experimental results, it can be shown that PDG can effectively promote bone formation, inhibit bone resorption, increase bone density and improve bone microstructure, and can be used clinically to alleviate postmenopausal osteoporosis.

[0074] Finally, it should be noted that the above description is only a preferred embodiment of the present invention. Those skilled in the art, under the guidance of the present invention, can make various similar representations without departing from the spirit and claims of the present invention, and such modifications all fall within the protection scope of the present invention.

Claims

1. The application of pinoresinol diglucoside in the preparation of drugs for treating postmenopausal intestinal flora imbalance, characterized in that, Pinoresinol diglucoside can increase the abundance percentage of Ekkermansia, Alternaria, and Parabacterium in the postmenopausal gut, and decrease the abundance percentage of Ruminococcus in the postmenopausal gut.

2. The application according to claim 1, characterized in that, The drug uses pinoresinol diglucoside as its active ingredient and is formulated into a pharmaceutically acceptable dosage form using pharmaceutically acceptable excipients.

3. The application according to claim 2, characterized in that, The excipients include one or more of the following: solvents, disintegrants, flavoring agents, preservatives, colorants, binders, lubricants, diluents, and drug carriers.

4. The application according to claim 2, characterized in that, The dosage form includes any one of tablets, capsules, suspensions, sustained-release formulations, controlled-release formulations, or targeted formulations.

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