Application of pinoresinol diglucoside in increasing short-chain fatty acid in postmenopausal intestinal tract and relieving postmenopausal intestinal inflammation

Abstract

The invention discloses application of pinoresinol diglucoside in increasing short-chain fatty acids in postmenopausal intestinal tracts and relieving postmenopausal intestinal inflammation, and belongs to the field of medicines. The pinoresinol diglucoside can regulate the abundance of bacteria such as Akecmann bacteria, other mycobacteria, parabacteroides and ruminococcus in intestinal tracts and promote the expression of short-chain fatty acids in the postmenopausal intestinal tracts, so that the postmenopausal intestinal inflammation and osteoporosis are relieved. The invention opens up a new clinical approach for treating diseases related to intestinal flora disorder of postmenopausal women.

Description

Technical Field

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

[0002] After menopause, the sudden drop in estrogen levels in women leads to intestinal flora disorder, increased intestinal barrier permeability, flora imbalance and intestinal mucosal damage, which can cause lipopolysaccharide (LPS) to accumulate in the intestine and "leak" into the blood circulation, causing endocrine disorders, osteoporosis and systemic inflammatory changes. Short chain fatty acids (SCFAs) are metabolites produced by the fermentation of undigested carbohydrates by intestinal microorganisms. They can promote the secretion of anti-inflammatory factors, inhibit LPS-induced inflammatory responses, and promote the recovery of intestinal diseases. Pinoresinoldiglucoside (PDG) is a major component of the traditional Chinese medicine Eucommia ulmoides, which can be metabolized by intestinal flora into enterolactone or enterodiol, and has the effects of lowering blood pressure and anti-oxidation. The present invention aims to provide the use of pinoresinoldiglucoside in increasing short-chain fatty acids in the intestine after menopause and alleviating postmenopausal intestinal inflammation. Summary of the invention

[0003] In view of the above problems, the first object of the present invention is to provide the use of pinoresinol diglucoside in increasing short-chain fatty acids in the intestine after menopause, and the second object of the present invention is to provide the use of pinoresinol diglucoside in regulating intestinal flora.

[0004] The first object of the present invention is achieved by using pinoresinol diglucoside in increasing short-chain fatty acids in the intestinal tract after menopause, wherein the short-chain fatty acid is at least one of acetic acid, isobutyric acid, isovaleric acid or valeric acid.

[0005] The second object of the present invention is achieved by using pinoresinol diglucoside in regulating intestinal flora, and pinoresinol diglucoside regulates the intestinal flora after menopause by regulating the structure and abundance of Eckermanella, Alternaria, Lactobacillus, and Ruminococcus in the intestine. And pinoresinol diglucoside regulates the intestinal flora after menopause by increasing the Chao1 index, ACE index, and Shannon index in the β diversity of the intestinal flora.

[0006] The beneficial effects of the present invention are as follows: the present invention discloses the application of pinoresinol diglucoside in regulating postmenopausal intestinal bacteria metabolism and alleviating intestinal inflammation, opening up a new clinical approach for the treatment of diseases related to intestinal flora disorders in postmenopausal women. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0009] Figure 3 It is a three-dimensional image of the lower end of the distal femoral trabeculae of a rat;

[0010] Figure 4 This is a 2D image of the coronal longitudinal section of the femoral trabeculae of the rat;

[0011] Figure 5 The effect of PDG on the bone microenvironment of mice. DETAILED DESCRIPTION

[0012] In order to make those skilled in the art better understand the technical scheme of the present invention, the present invention is described in detail below in conjunction with specific embodiments. The experimental methods for which specific conditions are not indicated in the following examples are usually carried out under normal conditions or according to the conditions recommended by the manufacturer. The test materials used in the following examples, unless otherwise specified, are purchased from conventional biochemical reagent stores. Unless otherwise specified, percentages and parts are calculated by weight. Unless otherwise defined, all professional and scientific terms used in the text have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar to or equal to the recorded content can all be applied to the present invention. The preferred implementation methods and materials described in the text are only for demonstration purposes.

[0013] The endpoints and any values ​​of the ranges disclosed in this article 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 each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

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

[0015] The invention provides application of pinoresinol diglucoside in preparing medicine or food for increasing the content of short-chain fatty acids in the intestinal tract after menopause.

[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 an application of pinoresinol diglucoside in the preparation of a drug or food for regulating postmenopausal intestinal flora. In this application, pinoresinol diglucoside regulates the abundance of Eckermannella, Alternaria, Parabacteroides, and Ruminococcus in the intestine, thereby alleviating postmenopausal systemic inflammation, such as postmenopausal intestinal inflammation and osteoporosis.

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

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

[0020] The present invention further provides the use of pinoresinol diglucoside in preparing a drug for preventing or treating intestinal inflammation.

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

[0022] The auxiliary materials include one or more of 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, pills, suspensions, powders, injections, sustained-release preparations, controlled-release preparations or targeted preparations.

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

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

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

[0027] The sham group was a group in which the ovaries were only explored after the skin was cut and the abdominal cavity was exposed. The ovaries were not removed, and only the fat tissue of the same size as the ovarian tissue was removed to eliminate the interference of the surgical technical operation on the experimental results.

[0028] The OVX group was divided into two groups: the skin was cut open, the abdominal cavity was exposed, the bilateral fallopian tubes were ligated, and the bilateral ovaries of the mice were removed;

[0029] The PDG-L group: after removing both ovaries of rats, PDG was administered orally by gavage at 5 mg / kg / d, 0.2 ml / time, once / day, for 8 consecutive weeks;

[0030] The PDG-H group: after removing both ovaries of rats, PDG was administered orally by gavage at a dose of 10 mg / kg / d, 0.2 ml / time, once / day, for 8 consecutive weeks;

[0031] The other two groups were gavaged with equal volume of sterile distilled water as a control.

[0032] Experimental Example 1 Effect of pinoresinol diglucoside (PDG) on intestinal flora of OVX mice

[0033] Fecal and colon samples were collected from each group of mice, and genomic DNA of the samples was extracted by CTAB / SDS method. Beta diversity analysis was used to evaluate the species complexity differences of the samples, and QIIME software (Version 1.9.1) was used to calculate the Beta diversity on weighted and unweighted unifrac. Systematic sequencing analysis of the 16S rRNA gene in mouse fecal samples was performed to evaluate the changes in intestinal flora. The results are shown in Table 1-2.

[0034] Table 1 Effect of PDG on the diversity of intestinal microbial communities in OVX mice

[0035]

[0036] Note: (1) Compared with the Sham group, * P<0.05;⑵Compared with OVX group, Δ P<0.05.

[0037] Results: As shown in Table 1, compared with the Sham group, the Chao1 index of the OVX group decreased significantly, while it showed an overall upward trend after PDG intervention, and the increase was most obvious in the PDG-L group, indicating that the total number of species detected in the intestinal microbial community samples after castration showed a decreasing trend, and the total number of species had a recovery and increasing trend after PDG intervention. The Shannon index showed that compared with other groups, the PDG-L group had the most obvious upward trend, indicating that the community diversity of the PDG-L group was higher and the species distribution was more uniform.

[0038] Table 2 Effect of PDG on the relative abundance of the top 30 bacterial genera in the intestinal flora

[0039]

[0040] As can be seen from Table 2, PDG can regulate the abundance of intestinal flora in OVX mice, mainly composed of Eckermannia, Alternaria, Lactobacillus, Pseudomonas Prevotella, Parabacteroides, Ruminococcus and other genera. Compared with the Sham group, the Bacteroides, Lactobacillus, and Ruminococcus genera in the feces of OVX mice increased, while Eckermannia, Pseudomonas Prevotella, Parabacteroides Dischidnii, and Alternaria decreased; compared with the OVX group, the Eckermannia, Lachnospira_NK4A136, and Alternaria genera in the PDG low-dose group increased, while Lactobacillus, Pseudomonas Prevotella, and Parabacteroides Dischidnii decreased; the Eckermannia, Lachnospira_NK4A136, Pseudomonas Prevotella, Parabacteroides Dischidnii, and Alternaria genera in the PDG high-dose group increased, while Lactobacillus decreased. Among them, Ekmansia, Parabacteroides, Alternaria and Ruminococcus as differential bacterial genera can promote the synthesis of SCFAs, indicating that PDG can regulate these four differential bacterial genera to affect the structure and abundance of intestinal flora and may affect the synthesis of SCFAs.

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

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

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

[0044]

[0045] Note: ⑴Compared with the sham operation group, * P<0.05; ⑵Compared with the ovariectomy group, Δ P<0.05.

[0046] Results: As can be seen from 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 the feces was significantly increased, among which acetic acid, isobutyric acid, isovaleric acid and valeric acid showed 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 is widely colonized in the intestinal mucus layer of humans and animals and plays an important role in relieving intestinal inflammation, preventing aging, and improving metabolic diseases. Studies have found that g_Akkermansia can degrade mucins and ferment to produce SCFAs by producing a variety of biological enzymes. SCFAs, as a metabolite of intestinal microorganisms, 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 intestinal microbiota and can synthesize succinic acid and short-chain fatty acids such as acetate and propionate. It has an important regulatory effect on the host mucosal immune system. Its content is 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. Its metabolic end products are succinic acid, acetate, and propionate. In a Mendelian randomization study on bone density and intestinal microorganisms in different age groups, g_Alistipes was found to be a protective factor for bone density in people over 60 years old. Ruminococcus is a Gram-positive anaerobic bacterium of the Firmicutes phylum that can ferment complex carbohydrates and produce SCFAs. It has the dual potential of pathogenic bacteria and probiotics. The expression abundance of g_Ruminococcus is positively correlated with bone loss and intestinal barrier damage. In summary, g_Akkermansiag_, Alistipes, g_Parabacteroides, and g_Ruminococcus may be the target bacterial genera for PDG to regulate SCFAs.

[0048] Experimental Example 3 Effect of pinoresinol diglucoside (PDG) on intestinal inflammation in OVX mice

[0049] Colon tissues of mice in each group were obtained, and part of the colon tissues were cut and fixed with 4% paraformaldehyde fixative, and paraffin tissue sections were made for HE staining and immunofluorescence double labeling of Claudin-1 and ZO-1. The HE staining results are shown in Figure 5 In immunofluorescence double-labeling staining, green fluorescence represents the protein expression intensity of Claudin-1, red fluorescence represents the protein expression intensity of ZO-1, blue represents the cell nucleus stained with DAPI, and Merge is the picture after the three colors are merged. The detection results are shown in Table 4 and Figure 1 As shown;

[0050] Table 4 Effects of PDG on tight junction proteins in colon tissue 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;⑵Compared with OVX group, Δ P<0.05.

[0053] Tissue homogenate prepared from fresh colon tissue was tested for inflammatory factors such as interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor α (TNF-α), lipopolysaccharide (LPS), secretory immunoglobulin A (SIgA) and LPS in serum by enzyme-linked immunosorbent assay. The experimental results are shown in Table 5.

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

[0055]

[0056]

[0057] Note: (1) Compared with the Sham group, * P<0.05;⑵Compared with OVX group, Δ P<0.05.

[0058] Figure 2 The results showed that compared with the Sham group, the surface epithelium of the colon mucosa in the OVX group was damaged and shed, the goblet cells were reduced, the intestinal crypts were arranged irregularly, and the basal layer was thinned. After PDG intervention, the mucosal layer damage was alleviated, 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). After PDG intervention, the expression levels of intestinal tight junction proteins showed an upward trend, and the low-dose effect was most obvious (P<0.05), indicating that PDG can repair intestinal barrier damage in OVX mice and relieve intestinal inflammation.

[0059] As can be seen from Table 5, compared with the Sham group, the expression levels of IL-6, IL-1β, and TNF-α in the colon of the OVX group were significantly increased (P<0.001), the secretion level of SIgA was significantly decreased, and the LPS in serum and colon tissue was significantly increased (P<0.001), indicating that the mucosal immunity of colon tissue was reduced after ovariectomy, the expression level of inflammatory factors was increased, and LPS entered the blood through the damaged intestinal barrier, causing systemic inflammation. Compared with the OVX group, the secretion level of SIgA in the colon tissue of OVX mice was significantly increased and the expression level of inflammatory factors was decreased after PDG intervention (P<0.05), and the LPS level in the colon and serum was decreased (P<0.001), indicating that PDG can improve the mucosal immunity level of the colon of OVX mice, relieve intestinal inflammation, and reduce LPS entering the blood.

[0060] Experimental Example 4 Effect of PDG on Bone Density and Bone Transformation Index Factors in Experimental Animals

[0061] The femur of mice fixed on the micro CT stage was scanned by NEMO Micro CT system (model NMC-100) of PINGSENG Healthcare (Kunshan) Inc. to detect bone density or bone mineral density BMD (Bone Mineral Density, BMD), relative bone volume or bone volume fraction BV / TV, trabecular number Tb.N (Trabecular Number), trabecular separation Tb.Sp (trabecular Separation / apacing), and trabecular thickness Tb.Th (Trabecular Thickness). The test results are shown in Table 6 and Figure 3-4 shown.

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

[0063]

[0064]

[0065] Note: (1) Compared with the Sham group, * P<0.05;⑵Compared with OVX group, Δ P<0.05.

[0066] Enzyme-linked immunosorbent assay was used to detect the bone formation factors PINP and ALP and the bone resorption factors CTX-1 and TRACP-5b in mouse serum. The test results are shown in Table 7.

[0067] Table 7 Effects of PDG on bone formation factors and bone resorption factors 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] The bone density data of OVX mice showed that after PDG intervention, the number of trabeculae in the distal femur of ovariectomized mice increased significantly (see Figure 3-4 ); and from Table 1, it can be seen that the trabecular bone density (Tb.BMD) and trabecular bone volume fraction (Tb.BV / TV) of the low-dose group were significantly increased, the number of trabeculae (Tb.N) in both groups increased to varying degrees, and the trabecular separation (Tb.Sp) in both groups decreased significantly. It can be seen that pinoresinol diglucoside can increase the bone density of cancellous bone in ovariectomized mice.

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

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

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

[0074] Finally, it should be noted that the above description is only a preferred embodiment of the present invention. Under the guidance of the present invention, those skilled in the art can make a variety of similar expressions without violating the purpose and claims of the present invention, and such changes all fall within the protection scope of the present invention.

Claims

1. Application of pinoresinol diglucoside in the preparation of medicine or food for increasing the content of short-chain fatty acids in the intestinal tract after menopause.

2. The application according to claim 1, characterized in that: The short-chain fatty acid is at least one of acetic acid, isobutyric acid, isovaleric acid or valeric acid.

3. The use of pinoresinol diglucoside in the preparation of drugs or foods for regulating postmenopausal intestinal flora, characterized in that: Pinoresinol diglucoside can regulate the abundance of Eckermannella, Alternaria, Parabacteroides, and Ruminococcus in the intestine, thereby alleviating systemic inflammation after menopause.

4. The use according to claim 3, characterized in that: Pinoresinol diglucoside regulates postmenopausal intestinal flora by increasing Chao1 index, ACE index and Shannon index in the β-diversity of intestinal flora.

5. Application of pinoresinol diglucoside in the preparation of drugs for preventing or treating intestinal inflammation.

6. The use according to claim 1, 2 or 5, characterized in that: The drug uses pinoresinol diglucoside as an active ingredient and is prepared into a pharmaceutically acceptable dosage form using pharmaceutically acceptable excipients.

7. The use according to claim 6, characterized in that: The auxiliary materials include one or more of solvents, disintegrants, flavoring agents, preservatives, colorants, binders, lubricants, diluents and drug carriers.

8. The use according to claim 6, characterized in that: The dosage form includes any one of tablets, capsules, pills, powders, ointments, pills, suspensions, powders, injections, sustained-release preparations, controlled-release preparations or targeted preparations.

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