Application of polymethoxyflavonol glycosides in the preparation of drugs for delaying intestinal aging

Abstract

The invention discloses the use of polymethoxy flavonol glycosides in the preparation of drugs for delaying intestinal aging, belonging to the field of medicine. Experiments show that the flavonol glycoside derivatives, namely CI and CA, can inhibit the excessive proliferation of intestinal stem cells of aging fruit flies, reduce intestinal barrier damage of aging fruit flies, reduce oxidative stress of aging fruit flies, reduce inflammatory response of aging fruit flies, prolong the life span of fruit flies, and increase the formation rate and budding rate of intestinal organoids of aging mice, indicating that CI and CA have excellent effects in delaying intestinal aging.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to an application of polymethoxy flavonol glycosides in the preparation of a medicine for delaying intestinal aging. Background Art

[0002] Aging is a complex and universal biological process that weakens the regenerative capacity of adult stem cells, especially in gastrointestinal tissues where cell renewal is rapid. As individuals age, the decline in intestinal stem cell (ISCs) function becomes a core cause of a series of age-related diseases, including inflammatory bowel disease and gastrointestinal cancer. Therefore, developing effective interventions to combat the aging process of ISCs is of vital importance for improving gastrointestinal health and prolonging life expectancy.

[0003] Although recent studies have highlighted the great potential of natural substances in anti-aging, especially those flavonoids, which are abundant in fruits and vegetables, have received extensive research and attention due to their low toxicity and ability to slow down aging and related diseases. However, despite the positive prospects of these natural substances for the treatment of aging, the resources of effective natural anti-aging drugs are still relatively limited. Therefore, it is particularly important to discover and develop new anti-aging drugs.

[0004] Drosophila melanogaster is a classic model for studying aging-related diseases. Its aging characteristics include abnormal proliferation of ISCs, which leads to imbalance of intestinal homeostasis and dysfunction. The diverse cell types and ISCs-driven regeneration ability of the intestine simulate the intestinal structure and function of mammals to a certain extent, providing an ideal biological system for studying the anti-aging effects of new natural compounds. At the same time, the mouse intestinal organoid model has become an important in vitro model for studying mammalian intestinal aging because it can more accurately simulate the function of stem cells in vivo. This model effectively reproduces the phenomenon of decreased intestinal regeneration ability and weakened stem cell proliferation and differentiation ability associated with aging, providing another ideal platform for evaluating the effects of new natural compounds on the aging process. Summary of the invention

[0005] In view of the above-mentioned prior art, the present invention discloses an application of polymethoxyflavonol glycosides in the preparation of a drug for delaying intestinal aging, which can provide a new drug research and development direction for the research and development of drugs for treating aging-related intestinal dysfunction.

[0006] In order to achieve the above-mentioned purpose, the technical solution adopted by the present invention is: polymethoxy flavonol glycosides are used to prepare drugs for delaying intestinal aging.

[0007] Furthermore, the drug is a drug that reduces intestinal oxidative stress.

[0008] Furthermore, this drug is a drug that reduces the expression level of intestinal inflammatory factors.

[0009] Furthermore, the drug is a drug for enhancing the intestinal barrier function.

[0010] Furthermore, this drug is a drug that enhances the activity of intestinal stem cells.

[0011] Furthermore, the polymethoxyflavonol glycoside is CI or CA, and their structural formulas are shown in Formula I and Formula II, respectively.

[0012]

[0013] Furthermore, polymethoxyflavonol glycosides are extracted from the Tibetan medicine Rhizoma Polygoni Multiflori, and the extraction steps include:

[0014] S1: extracting the whole herb of Long-stemmed Golden Waist with ethanol having a volume concentration of 60-80% at room temperature, and concentrating under reduced pressure to obtain an extract;

[0015] S2: adding a dispersant to the extract obtained in step S1 to disperse the extract, wherein the dispersant is 0% to 10% ethanol, and the ratio of extract to dispersant is 2 to 3:1 to 4, wherein the extract is measured in kilograms, and the dispersant is measured in liters, to obtain a dispersion;

[0016] S3: The dispersion obtained in step S2 is loaded onto a macroporous adsorption resin column, and gradient eluted with distilled water, 10% ethanol, 20% ethanol, 30% ethanol, 40% ethanol, 50% ethanol, 60% ethanol, 70% ethanol, 80% ethanol, and 95% ethanol in sequence, with each gradient elution lasting 3 to 6 column volumes, to obtain 10 fractions fr. H0 to H9;

[0017] S4: adding hot methanol to the 40% ethanol elution fraction fr.H4 obtained in step S3, kneading and filtering to obtain a methanol insoluble portion fr.H4-1 and a methanol soluble portion fr.H4-2;

[0018] S5: fr.H4-1 was subjected to silica gel column chromatography and eluted with a dichloromethane-ethanol solvent system of 16:1 to obtain 7 fractions fr.H4-1S1 to H4-1S7; among them, fr.H4-1S3 fraction was separated by preparative chromatography to obtain a compound with a structure represented by formula (II);

[0019] S6: The methanol-soluble portion fr.H4-2 obtained in step S4 is further applied to a Sephadex LH-20 column and eluted with 90% methanol to obtain eight fractions fr.H4-2L1 to H4-2L8; among them, the fr.H4 to 2L4 fractions are separated by preparative chromatography to obtain a compound having a structure represented by formula (I).

[0020] Furthermore, the drug also includes a pharmaceutically acceptable carrier or excipient.

[0021] The beneficial effects of the present invention are:

[0022] 1. CI and CA can significantly inhibit the accumulation of esg+ expressing cells (marking ISCs) in the midgut of aged Drosophila, and can also significantly reduce the number of Dl+ intestinal stem cells and pH3+ proliferating cells in the midgut of aged Drosophila, that is, CI and CA can effectively prevent the excessive proliferation of intestinal stem cells in aged Drosophila.

[0023] 2. CI and CA can enhance the tight junctions of the intestinal epithelium of aged fruit flies and protect the intestinal barrier function of aged fruit flies.

[0024] 3. CI and CA can significantly reduce the level of reactive oxygen species in aged fruit flies and effectively alleviate the oxidative stress in the intestine of aged fruit flies.

[0025] 4. CI and CA can significantly reduce the expression levels of upd2 and upd3, and can effectively inhibit the inflammatory response of aging fruit flies.

[0026] 5. CI and CA can extend the lifespan of fruit flies.

[0027] 6. CI and CA can significantly increase the formation rate and budding rate of intestinal organoids in aged mice, improve their stemness, and exert anti-aging effects.

[0028] 7. CI and CA can be used to prepare drugs for delaying intestinal aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of the process of feeding CI and CA to fruit flies;

[0030] Figure 2 Model for Drosophila midgut epithelial cell lineages and markers;

[0031] Figure 3 Representative immunofluorescence images of Drosophila (esg-GFP / CyO) midgut specifically labeled with Dl antibody under DMSO, 10 μM CI, or 10 μM CA treatment;

[0032] Figure 4 Figure 2 is the expression of esg-GFP in the intestine of Drosophila (esg-GFP / CyO) under the treatment of DMSO, 10 μM CI or 10 μM CA. + Statistical graph of the proportion of cells to the total cells in each target region (ROI);

[0033] Figure 5 Representative immunofluorescence images of the midgut of Drosophila (esg-GFP / CyO) under treatment with DMSO, 10 μM CI, or 10 μM CA;

[0034] Figure 6 After specific labeling with D1 antibody, D1 in Drosophila intestine + Statistical graph of the proportion of cells to the total cells in each target area;

[0035] Figure 7 For pH 3 + (phosphorylated histone H3) labeled cell number statistics;

[0036] Figure 8 Figure 4 shows the 40-day-old Drosophila melanogaster (W) treated with 10 μM DMSO, 10 μM CI and 10 μM CA. 1118 )'s representative images of smurf(-) and smurf(+);

[0037] Fig. 9 For 40-day-old Drosophila (W 1118 ) Quantitative results of the percentage of smurf(+) flies in the

[0038] Fig.10 Drosophila fed without CI and CA for 10 and 40 days (W 1118 ) and aged flies fed with 10 μM CI or 10 μM CA for 40 days (W 1118 ) Representative immunofluorescence images of the midgut after Arm immunofluorescence staining;

[0039] Fig.11 Representative immunofluorescence images of the midgut of 40-day-old Drosophila (esg-GFP / CyO) treated with DHE and supplemented with 10 μM DMSO, 10 μM CI, or 10 μM CA;

[0040] Fig.12 The quantitative results of DHE fluorescence intensity in each target area of ​​Drosophila (esg-GFP / CyO) midgut;

[0041] Fig.13 For fruit flies (W 1118 ) Statistical graph of relative expression levels of inflammatory factors upd2 and upd3 in the whole intestine;

[0042] Fig.14 For female fruit flies (W 1118 ) and male fruit flies (W 1118 )’s survival rate line graph;

[0043] Fig.15 Representative images of organoids from young and aged mice treated or not with CI and CA.

[0044] Fig.16Statistical graphs of the sprouting rate and organoid formation rate of young and aged mouse organoids treated or not with CI and CA;

[0045] Fig.17 Representative immunofluorescence images of organoids from young and aged mice treated or untreated with CI and CA after labeling with SOX9 and Ki67;

[0046] Fig.18 Statistical graph showing the proportion of SOX9+ cells and Ki67+SOX9+ cells in the total cells in each crypt. DETAILED DESCRIPTION

[0047] Implementation Materials and Methods

[0048] In this example, the fruit fly strains esg-GFP / CyO and w1118 (BDSC#3605) used were grown on standard cornmeal / agarose medium under the following conditions: 25°C, 65% humidity, and 12-hour light and 12-hour dark cycles. The flow chart of the fruit fly feeding CI and CA was shown in Figure 3 by feeding the 25-day-old middle-aged fruit flies esg-GFP / CyO and middle-aged fruit flies w1118 (BDSC#3605) with food containing CI and CA (three concentrations of 1, 10, and 100 μM, respectively) for 15 days. Figure 1 shown.

[0049] The specific implementation modes of the present invention are described in detail below with reference to the embodiments.

[0050] Example 1: Effects of CI and CA on the overproliferation of intestinal stem cells in aged Drosophila

[0051] like Figure 2 As shown in the midgut of aging fruit flies, intestinal stem cells (ISCs) regulate intestinal homeostasis through their proliferation and differentiation. As fruit flies age, ISCs show an abnormally increased proliferation rate, while their daughter enteroblasts (EBs) fail to effectively differentiate into mature intestinal cells. This leads to esg + Cells (including ISCs and EBs) accumulated in the midgut, while the number of mature enterocytes decreased, thus affecting intestinal function.

[0052] In order to monitor and quantify esg in real time + Cells, the present invention uses the esg-GFP / CyO reporter gene system. This system expresses green fluorescent protein (GFP) under the regulation of the escargot (esg) gene, such as Figure 3 As shown, DAPI (blue; nucleus) and esg-GFP (green; ISCs and progenitor cell markers), the aging-related ISCs overproliferation phenomenon can be intuitively observed in the figure.

[0053] By feeding 25-day-old middle-aged fruit flies (esg-GFP / CyO) with food containing CI and CA (three concentrations of 1, 10, and 100 μM, respectively) for 15 days, Figure 4 As shown, CI and CA, two natural small molecule compounds, can significantly reduce intestinal esg in aged Drosophila. + Cell accumulation, in addition Figure 5 As shown, DAPI (blue), esg-GFP (green), 10 μM concentration of CI and CA most effectively reduced the accumulation of intestinal esg+ cells in aged flies.

[0054] In addition, the Dl antibody was used to specifically label ISCs in aging flies, e.g. Figure 3 As shown, Dl (red), compared with the control group, Figure 6 As shown in Figure 2, the number of specifically labeled ISCs in aged flies treated with 10 μM CI and CA was significantly reduced. + The number of proliferating cells marked, e.g. Figure 7 As shown, it was shown that CI and CA significantly inhibited the overproliferation of ISCs in aged Drosophila, thus demonstrating the effectiveness of CI and CA in slowing down the overproliferation of intestinal stem cells in aged Drosophila.

[0055] Example 2: Protective effects of CI and CA on intestinal function of aged Drosophila

[0056] Aging is accompanied by a significant decline in intestinal function in Drosophila, including damage to the intestinal barrier. Armadillo (Arm) immunofluorescence staining and smurf assay were used to evaluate the effects of CI and CA on the intestinal barrier function of aged Drosophila. The smurf assay evaluates the integrity of the intestinal barrier by detecting the distribution of non-absorbable blue food dye in Drosophila, while Armadillo immunofluorescence staining is used to observe the tight junctions of intestinal epithelial cells. In the smurf assay, we first placed 20 aged Drosophila (W) in each group. 1118 ) for 2 hours, and then the aged fruit flies (W 1118 ) were transferred to a medium containing 2.5% w / v blue food dye and cultured for 12 hours. Figure 8 As shown in Figure 2, blue food dye leaked into the body cavity of aged flies, indicating impaired intestinal barrier function. These aged flies were labeled Smurf(+); in aged flies supplemented with CI and CA, as shown in Figure 2 Fig. 9 As shown in Figure 2, the number of Smurf(+) individuals was significantly reduced. Representative immunofluorescence images after Arm immunofluorescence staining, as shown in Figure 2 Fig.10As shown, DAPI (blue) and Armadillo (red; cell junction marker), it can be seen that aged fruit flies supplemented with CI and CA show stronger Arm signals in Arm immunofluorescence staining, indicating that the tight junctions between intestinal epithelial cells are strengthened. This indicates that CI and CA can effectively prevent or treat the dysfunction of the intestinal barrier in aged fruit flies, reduce intestinal barrier damage, and thus protect intestinal function from the effects of aging.

[0057] Example 3: CI and CA reduce oxidative stress in aged Drosophila

[0058] In addition to impaired intestinal barrier function, aged fruit flies also experience increased oxidative stress (ROS). The present invention uses DHE (dihydroethidium) fluorescent probe to detect ROS levels in the intestine to explore whether CI and CA can alleviate intestinal oxidative stress in aged fruit flies. First, the intestinal tissues of aged fruit flies (esg-GFP / CyO) cultured for 40 days in each group were dissected out, and then these intestinal samples were added to a solution containing 30μM DHE and incubated on a shaker at room temperature for 20 minutes, protected from light during the period. Subsequently, the intestinal samples were washed 3 times with PBS solution (phosphate buffer solution), each washing lasting 5 minutes. After that, the intestinal samples were fixed in a 4wt% paraformaldehyde solution at room temperature for 30 minutes, and then washed 3 times with PBS solution, and then immediately sealed and observed under a fluorescence microscope. All operations must be performed under light-proof conditions. The micrographs are shown in the following figure. Fig.11 As shown, DAPI (blue), esg-GFP (green), DHE (red; ROS marker).

[0059] Quantitative results of DHE fluorescence intensity in each target area of ​​the midgut of aged Drosophila (esg-GFP / CyO), such as Fig.12 As shown, the DHE level in aged flies (esg-GFP / CyO) supplemented with CI and CA was significantly reduced compared with that in aged flies (esg-GFP / CyO) not supplemented with CI and CA. This indicates that the supplementation of CI and CA can significantly reduce the level of ROS in aged flies, thereby alleviating oxidative stress.

[0060] Example 4: CI and CA reduce inflammatory responses in aged Drosophila

[0061] In aged Drosophila, increased inflammatory response is a typical feature of the aging process. In aged Drosophila, the expression of inflammatory factors upd2 and upd3 increases, and these two factors are conserved with IL-6 in mammals. To explore the effects of CI and CA on the intestinal immune response of aged Drosophila, 10-day-old Drosophila (W) treated with DMSO or 10 μM CI and 10 μM CA were added. 1118 ) and 40-day-old fruit flies (W1118 The relative expression levels of intestinal inflammatory factors upd2 and upd3 were measured in 15 flies per group (W 1118 ), the result is as follows Fig.13 As shown, the expression levels of these inflammatory factors were significantly reduced in aged fruit flies treated with CI and CA, indicating that CI and CA can effectively inhibit the inflammatory response of aged fruit flies.

[0062] Example 5: CI and CA extend the lifespan of Drosophila

[0063] Collect 120 fruit flies (W 1118 ), and evenly distributed them into 6 vials, and randomly divided into 3 groups, each group was fed with food containing standard medium, food containing standard medium supplemented with 10μM CI, and food containing standard medium supplemented with 10μM CA. Every two days, the number of dead fruit flies was counted, and fresh food was replaced for the surviving fruit flies. The results are shown in Fig.14 As shown, CI and CA can significantly extend the lifespan of male and female fruit flies.

[0064] Example 6: Effects of CI and CA on intestinal organoids in aged mice

[0065] Aging is a complex biological process that includes the decline of intestinal function. In mammals, this aging process can be manifested as a decrease in the number of intestinal crypts, a weakening of stem cell proliferation ability, and an increase in cell apoptosis. Correspondingly, a decrease in proliferation rate and budding rate was observed in intestinal organoids of aged mice cultured in vitro. In order to explore the potential anti-aging effects of CI and CA in the aging process of mammals, the present invention first measured the organoid formation rate of intestinal crypts of young and aged mice. These intestinal organoids were then treated with 10μM CI and 10μM CA, and after 7 days, it was observed that Fig.15 and 16 As shown in the figure, the budding rate and formation rate of aged intestinal organoids were significantly reduced, but treatment with CI and CA significantly increased the formation and budding rate of aged mouse organoids, almost restoring them to the levels observed in young intestinal organoid cultures. This suggests that the supplementation of CI and CA seems to be able to restore the function of aged organoids to a state similar to that of young organoids.

[0066] To further verify the anti-aging effects of CI and CA, SOX9 and Ki67 were used to mark mouse intestinal stem cells (ISCs) and proliferating cells, as shown in Fig.17 As shown, DAPI (blue), SOX9 (green; ISCs marker) and Ki67 (red; proliferating cell marker). Fig.18As shown in the figure, the number of SOX9-positive cells and Ki67 and SOX9 double-positive cells per crypt in CI- and CA-treated aged mouse intestinal organoids increased significantly compared with untreated cells. These results indicate that CI and CA can not only promote the formation rate and budding rate of aged intestinal organoids, but also promote the proliferation of stem cells, thereby exerting an anti-aging effect.

[0067] Although the specific implementation of the present invention is described in detail in conjunction with the embodiments, it should not be understood as limiting the scope of protection of this patent. Within the scope described in the claims, various modifications and variations that can be made by those skilled in the art without creative work still fall within the scope of protection of this patent.

Claims

1. The use of polymethoxyflavonol glycosides in the preparation of a drug for delaying intestinal aging, characterized in that: The polymethoxyflavonol glycoside is CI or CA, and their structural formulas are shown in Formula I and Formula II, respectively. ； The drug can reduce intestinal oxidative stress; or the drug can reduce the expression level of intestinal inflammatory factors; or the drug can enhance the intestinal functional barrier; or the drug can enhance the activity of intestinal stem cells.

2. The use according to claim 1, characterized in that: The polymethoxyflavonol glycosides are extracted from the Tibetan medicine Herba Lycopodii.

3. The use according to claim 1, characterized in that: The medicine also includes a pharmaceutically acceptable carrier or excipient.

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