Application of plant polyphenol flemingia philippinensis A in relieving intestinal inflammation diseases

Abstract

The invention discloses an application of plant polyphenol flemingia philippinnin A. The flemingia philippinnin A is a flavonoid compound, the molecular formula of the flemingia philippinnin A is C30H32O6, and the optimal concentration of the flemingia philippinnin A compound in relieving the intestinal inflammation disease is 1 mu M. The invention further discloses a preparation method of the flemingia philippinnin A. The invention further discloses an application of the flemingia philippinnin A compound in relieving the intestinal inflammation disease. The invention finds that the Flemiphilippinin A has no toxic or side effect on normal cells and has a good anti-inflammatory effect. Compared with a control group, the inflammatory intestinal organ treated by the Flemiphilippinin A has the advantages that the cell apoptosis level is reduced in phenotype; according to the present invention, the inflammatory factor marker gene is subjected to up-regulation expression on the expression level and the gene level, the good promotion effect is provided in the proliferation, differentiation and function aspects during the normal intestinal organ treatment process, the pharmacodynamic research and the safety evaluation of the in vivo model are further performed, and the foundation can be laid for the preclinical research.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine cultivation, and specifically relates to an application of plant polyphenol trichomoniasis A in alleviating intestinal inflammatory diseases. Background Art

[0002] Intestinal inflammatory diseases, such as inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, are chronic, recurrent intestinal diseases characterized by persistent inflammation of the intestinal mucosa. These diseases are prevalent worldwide, and the incidence is increasing year by year. At present, conventional treatments for intestinal inflammation include drug therapy, surgical treatment, and nutritional support. Drug therapy mainly involves anti-inflammatory drugs such as corticosteroids, 5-aminosalicylic acid drugs, immunosuppressants, and biologics. In some refractory or severe cases, surgical treatment may be required. However, there are common problems such as tolerance, side effects, and treatment costs. In recent years, plant polyphenols, as a class of natural compounds widely present in nature, have rapidly emerged in the field of biomedicine. Plant polyphenols exhibit multiple pharmacodynamic effects such as antioxidant, anti-inflammatory, antiviral, antibacterial, and cardioprotective. They are abundant in nature and can be obtained sustainably. Summary of the invention

[0003] An object of the present invention is to solve at least the above problems and / or disadvantages and to provide at least the advantages which will be described hereinafter.

[0004] Another object of the present invention is to provide the use of plant polyphenol trichomoniasis A in alleviating intestinal inflammatory diseases.

[0005] To this end, the technical solution provided by the present invention is:

[0006] A use of a plant polyphenol, tricholoma A, in alleviating intestinal inflammatory diseases. Trichoderma A is a flavonoid compound derived from the plant Trichoderma.

[0007] Preferably, in the application of the plant polyphenol phytosporin A in alleviating intestinal inflammatory diseases, the molecular formula of the phytosporin A is C 30 H 32 O6, its chemical formula is as follows:

[0008]

[0009] Preferably, in the application of the plant polyphenol tricholoma A in alleviating intestinal inflammatory diseases, the plant polyphenol tricholoma A has the purpose of promoting the expression of proliferation and differentiation genes of intestinal organoids in the establishment of an intestinal organoid model.

[0010] Preferably, the plant polyphenol tricholoma A is used in alleviating intestinal inflammatory diseases, and the plant polyphenol tricholoma A is used in locally slowing down the expression of LPS-induced intestinal organoid inflammatory factors in the establishment of an intestinal organoid model.

[0011] Preferably, in the use of the plant polyphenol tricholoma A in alleviating intestinal inflammatory diseases, the effective concentration of the tricholoma A is 1 μM.

[0012] The present invention has at least the following beneficial effects:

[0013] The present invention verifies the application of Flemiphilippnin A in alleviating intestinal inflammatory diseases by using intestinal organoids. Flemiphilippnin A, a polyphenol compound extracted from plants, exhibits significant anti-inflammatory activity in studies using intestinal organoid models, and after careful evaluation and verification, it is confirmed that it is non-toxic to cells at therapeutic concentrations. This discovery provides a promising candidate molecule for the development of new natural anti-inflammatory drugs, and also brings innovative strategies for the treatment of intestinal-related diseases such as inflammatory bowel disease.

[0014] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 An image of inflammatory intestinal organoid culture in an embodiment of the present invention is shown.

[0016] Figure 2 The images show the growth rate and development status of normal organoids after Flemiphilippnin A treatment in the examples of the present invention.

[0017] Figure 3 The expression graph of genes related to proliferation and development of normal organoids after Flemiphilippnin A treatment in the examples of the present invention is shown.

[0018] Figure 4 The images show the growth rate and development status of inflammatory intestinal organoids after Flemiphilippnin A treatment in the examples of the present invention.

[0019] Figure 5 The inflammatory factor gene expression graph of inflammatory intestinal organoids after Flemiphilippnin A treatment in the examples of the present invention is shown. DETAILED DESCRIPTION

[0020] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0021] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0022] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0023] Naturally derived anti-inflammatory compounds have less side effects and toxicity. Flemiphilippnin A has been widely used in traditional medicine, but they may face challenges in terms of efficacy, stability and bioavailability, and their anti-inflammatory effects need to be further proven. The plant polyphenol Flemiphilippnin A has been shown to have strong free radical scavenging and anti-tumor effects in the human body, but its toxic side effects and anti-inflammatory effects have not been mentioned. Intestinal organoids can simulate the complexity of the intestine and provide an in vitro model for disease research and drug screening, while maintaining a high degree of genetic stability and the potential for personalized treatment. Intestinal organoids were then chosen to verify whether Flemiphilippnin A has toxic side effects on normal cells and relieves inflammation.

[0024] Application of a plant polyphenol Flemiphilippnin A in relieving intestinal inflammatory diseases, Flemiphilippnin A is a flavonoid compound derived from the plant Flemiphilippnin, and the molecular formula of Flemiphilippnin A is: C 30 H 32 O6, molecular weight: 488; chemical formula is as follows,

[0025]

[0026] In one embodiment of the present invention, preferably, the plant polyphenol tricholoma A is used in alleviating intestinal inflammatory diseases, and the tricholoma A is used to promote the expression of proliferation and differentiation genes of intestinal organoids in the establishment of intestinal organoid models.

[0027] In one embodiment of the present invention, preferably, the phycoerythrin A is used to locally slow down the expression of LPS-induced intestinal organoid inflammatory factors in the establishment of an intestinal organoid model.

[0028] In one embodiment of the present invention, preferably, the effective concentration of the phytohormone A is 1 μM. The optimum concentration of the phytohormone A compound for relieving intestinal inflammatory diseases is 1 μM.

[0029] The Flemiphilippnin A (Flemingiaphilippinensis) is widely used in traditional Chinese medicine to treat rheumatism, arthritis and osteoporosis, and the polyphenols contained in it are generally believed to have anti-inflammatory, anti-estrogen, anti-tumor, cytotoxic, immunosuppressive and antioxidant activities. It has been proven that Flemiphilippnin A has strong free radical scavenging and anti-tumor effects in the human body. The invention shows that Flemiphilippnin A has no toxic side effects and anti-inflammatory effects.

[0030] In order to enable those skilled in the art to better understand the technical solution of the present invention, the following embodiments are provided for further explanation:

[0031] Embodiment 1:

[0032] See also Figure 1-Figure 5 As shown, a plant polyphenol Flemiphilippnin A is used in relieving intestinal inflammatory diseases. The molecular formula of Flemiphilippnin A is: C 30 H 32 O6.

[0033] Raw material selection: First, determine the natural raw materials containing the target compound (such as "Vegetable A"), such as plants, microorganisms or marine organisms. This is usually based on previous research or literature reports.

[0034] Flemiphilippnin A is a natural plant polyphenol extracted from the plant Flemingia philippinensis (MCE catalog number HY-N4316), which is widely used in traditional Chinese medicine to treat rheumatism, arthritis and osteoporosis. The polyphenols contained in it are generally believed to have anti-inflammatory, anti-estrogen, anti-tumor, cytotoxic, immunosuppressive and antioxidant activities. Among them, Flemiphilippnin A has been proven to have strong free radical scavenging and anti-tumor effects.

[0035] The present invention provides an application of plant polyphenol Flemiphilippnin A in alleviating intestinal inflammatory diseases, and the steps thereof are mainly divided into three parts:

[0036] (1) Establishment of LPS-induced intestinal organoid validation model;

[0037] (2) Flemiphilippnin A promotes the proliferation and differentiation of normal cells;

[0038] (3) Flemiphilippnin A reduces the expression level of inflammatory factor marker genes.

[0039] Step (1) Establishment of LPS-induced intestinal organoid validation model, specifically:

[0040] (1) Intestinal organoid recovery: The frozen intestinal organoids (extracted from the jejunal tissue of the small intestine of newborn healthy pigs) were quickly thawed in warm water at 37°C, centrifuged with 4 times the volume of 90% Advanced DMEM / F12 (Gibco Product No.: 12634-010) + 10% FBS (4°C, 200rcf, 5 min), and embedded with 1:1 culture medium (Hunan Ograde Biotechnology Co., Ltd., customized product Crypt Boosting Medium) and matrix gel Matrigel, cultured in 24-well plates to form 20 μL droplets, incubated in an incubator for 20 min to solidify, added with 500 μL culture medium, and cultured for 3 days before maturation;

[0041] (2) Establish a stable intestinal organoid model: After the pig intestinal organoids are revived, they are passaged to P1. After the culture medium is aspirated, TE digestion solution is added for digestion. After 3 minutes, 3-5 times the volume of 90% Advanced DMEM / F12+10% FBS is added for centrifugation (4°C, 200rcf, 5 minutes). 1:1 culture medium and Matrigel are added for embedding. Incubate in the incubator for 20 minutes to wait for solidification, and 500μL culture medium is added. Intestinal organoids can grow stably, and organoid budding is stable and the number meets the experimental requirements;

[0042] (3) Passage again to establish an intestinal organoid inflammation model: After absorbing the culture medium, add TE digestion solution (TE digestion solution is Trypsin / EDTA Solution, belongs to life technologies, product number R001100) for digestion. After 3 minutes, add 3-5 times the volume of 90% Advanced DMEM / F12+10% FBS for centrifugation (4°C, 200rcf, 5min), add 1:1 culture medium and Matrigel for embedding, and wait for coagulation in the incubator for 20 minutes. The modeling is successful. The successfully modeled intestinal organoid inflammation model was divided into two groups, one of which was added with 500μL culture medium containing 200μg / mL LPS and cultured until maturity, and the other group was added with 500μL culture medium as the control group;

[0043] (4) Microscopic observation and phenotypic analysis: Phenotypic analysis showed that the intestinal organoids induced by LPS in step (3) showed a lower budding level and an increased cell death rate compared with the control group without LPS.

[0044] pass Figure 1 Bright field results showed that compared with the control group without LPS, the budding level was reduced and the cell death rate was increased.

[0045] (5) qPCR detection, gene level analysis: After maturation, intestinal organoid RNA was extracted for qPCR analysis: the culture medium was aspirated, washed with PBS, and 1 mL of RNAiso Plus lysis buffer was added. After sufficient lysis, 200 μL of chloroform was added, and the cells were placed on a vortexer for full oscillation for 20 seconds, stabilized at room temperature for 3 minutes, and separated by centrifugation (4°C, 12000 rpm, 15 minutes). 500 μL of supernatant was aspirated, 500 μL of isopropanol was added, mixed, and stored at -20°C overnight. After overnight, the supernatant was discarded by centrifugation, and the precipitate was washed with 75% ethanol solution prepared with DEPC water. After completion, it was placed on a clean bench to dry. The dried RNA precipitate was dissolved in DEPC water on ice for 30 minutes, the RNA concentration and purity were measured, and the corresponding dilution was performed, with a final concentration of 59 ng / μL. The mRNA was reverse transcribed into cDNA, and the system was 12.5 μL. Real-time PCR, according to the requirements of the kit, the ligand solution, the final system is 12.5μL, the annealing temperature Tm is uniformly 58℃, which can be used for qPCR detection and analysis. At the gene level, if there is inflammation, the expression of inflammatory factor-related marker genes such as IL-6 and TNF-ɑ is upregulated.

[0046] pass Figure 5 qPCR results showed that compared with the control group, LPS induced upregulation of the expression of inflammatory factor-related marker genes IL-6, IL-8, and TNF-ɑ in intestinal organoids.

[0047] Step (2) Flemiphilippnin A promotes the proliferation and differentiation of normal cells; specifically:

[0048] (1) Intestinal organoid recovery: The frozen intestinal organoids were quickly thawed in warm water at 37°C, centrifuged with 4 times the volume of 90% Advanced DMEM / F12+10% FBS (4°C, 200 rcf, 5 min), and embedded with 1:1 culture medium and Matrigel. The wells were cultured in a 24-well plate to form 20 μL gel droplets. The wells were incubated in an incubator for 20 min to solidify, and 500 μL culture medium was added. The wells were cultured for 3 days before maturation.

[0049] (2) Establish a stable intestinal organoid model: After the pig intestinal organoids are revived, they are passaged to P1. After the culture medium is aspirated, TE digestion solution is added for digestion. After 3 minutes, 3-5 times the volume of 90% Advanced DMEM / F12+10% FBS is added for centrifugation (4°C, 200rcf, 5 minutes). 1:1 culture medium and Matrigel are added for embedding. Incubate in the incubator for 20 minutes to wait for solidification, and 500μL culture medium is added. Intestinal organoids can grow stably, and organoid budding is stable and the number meets the experimental requirements;

[0050] (3) Flemiphilippnin A treatment of normal P2 intestinal organoids: After aspirating the culture medium, add TE digestion solution for digestion. After 3 minutes, add 3-5 times the volume of 90% Advanced DMEM / F12+10% FBS for centrifugation (4°C, 200rcf, 5 minutes). Add 1:1 culture medium and Matrigel for embedding. Incubate in an incubator for 20 minutes to wait for solidification. Add 500 μL culture medium containing 1 μM Flemiphilippnin A and culture until maturity.

[0051] (4) Microscopic observation and phenotypic analysis: Phenotypically, the budding rate of intestinal organoids treated with Flemiphilippnin A was increased compared with the control group;

[0052] pass Figure 2 Bright field results showed that the germination rate was increased compared with the control group.

[0053] (5) qPCR detection, gene level analysis: qPCR detection, gene level analysis: After maturation, extract intestinal organoid RNA for qPCR analysis: aspirate the culture medium, wash with PBS, and add 1mL RNAiso Plus lysis buffer. After sufficient lysis, add 200μL chloroform, place on a vortex and fully shake for 20s, stabilize at room temperature for 3min, and centrifuge after stratification (4℃, 12000rpm, 15min), aspirate 500μL supernatant, add 500μL isopropanol, mix well, and incubate at -20℃ overnight. After overnight, centrifuge and discard the supernatant, and wash the precipitate with 75% ethanol solution prepared with DEPC water. After completion, place it on a clean bench to dry. Dissolve the dried RNA precipitate with DEPC water on ice for 30min, measure the RNA concentration and purity, and dilute accordingly, with a final concentration of 59ng / μL. Reverse transcribe mRNA into cDNA, with a system of 12.5μL. Real-time PCR, according to the requirements of the kit, the ligand solution, the final system is 12.5μL, the annealing temperature Tm is uniformly 58℃, which can be used for qPCR detection and analysis. At the gene level, Flemiphilippnin A has no adverse effects on normal intestinal organoids and promotes the expression of proliferation and differentiation genes in intestinal organoids.

[0054] pass Figure 3 qPCR results showed that compared with the control group, the expression of proliferation and differentiation-related marker genes ChgA, MUC2, and PCNA were upregulated.

[0055] Step (3) Flemiphilippnin A reduces the expression level of inflammatory factor marker genes; specifically:

[0056] (1) Establishment of control and experimental groups: Culture to P1, aspirate the culture medium after subculture, add TE digestion solution for digestion, add 3-5 times the volume of 90% Advanced DMEM / F12 + 10% FBS after 3 minutes, centrifuge (4°C, 200rcf, 5 minutes), add 1:1 culture medium and Matrigel for embedding, wait for coagulation in the incubator for 20 minutes, add 500μL culture medium containing 200μg / mL LPS (control group) and 1μM Flemiphilippnin A + 200μg / mL LPS, and culture until maturity;

[0057] (2) Microscopic observation and phenotypic analysis: Phenotypically, the budding rate of the Flemiphilippnin A-treated experimental group increased and the cell death rate decreased compared with the control group;

[0058] pass Figure 4 Bright field results showed that compared with the LPS group, the budding rate increased and the cell death rate decreased.

[0059] (3) qPCR detection, gene level analysis: qPCR detection, gene level analysis: After maturation, intestinal organoid RNA was extracted for qPCR analysis: the culture medium was aspirated, washed with PBS, and 1 mL of RNAiso Plus lysis buffer was added. After sufficient lysis, 200 μL of chloroform was added, and the mixture was placed on a vortexer for full oscillation for 20 seconds, stabilized at room temperature for 3 minutes, and then separated by centrifugation (4°C, 12000 rpm, 15 minutes). 500 μL of supernatant was aspirated, 500 μL of isopropanol was added, mixed, and stored at -20°C overnight. After overnight, the supernatant was discarded by centrifugation, and the precipitate was washed with 75% ethanol solution prepared with DEPC water. After completion, it was placed on a clean bench to dry. The dried RNA precipitate was dissolved in DEPC water on ice for 30 minutes, the RNA concentration and purity were measured, and the corresponding dilution was performed, with a final concentration of 59 ng / μL. The mRNA was reverse transcribed into cDNA, and the system was 12.5 μL. Real-time PCR, according to the requirements of the kit, the ligand solution, the final system is 12.5μL, the annealing temperature Tm is uniformly 58℃, which can be used for qPCR detection and analysis. At the gene level, Flemiphilippnin A slows down the expression of LPS-induced intestinal organoid inflammatory factors.

[0060] pass Figure 5 qPCR results showed that compared with the LPS group, the expressions of inflammatory factor-related marker genes IL-6, IL-8, and TNF-ɑ were downregulated.

[0061] The plant polyphenol Flemiphilippnin A in the present invention can be used not only for intestinal organoid inflammation, but also for other inflammations; the present invention provides an application of plant polyphenol Flemiphilippnin A in alleviating intestinal inflammatory diseases. The plant polyphenol is an innovative anti-inflammatory drug option, which reduces the toxic side effects of existing drugs on normal cells, and the specific analysis of related mechanisms helps to target specific molecular pathways.

[0062] Although the embodiments of the present invention are disclosed above, they are not limited to the applications listed in the specification and the embodiments. It can be fully applied to various fields suitable for the embodiments of the present invention. For those familiar with the art, other modifications can be easily realized. Therefore, without departing from the general concept defined by the claims and equivalent scopes, the embodiments of the present invention are not limited to the specific details and the legends shown and described here.

Claims

1. A use of plant polyphenol ramie A in alleviating intestinal inflammatory diseases, characterized in that: Echinopsin A is a flavonoid compound derived from the plant Echinops truncatula.

2. The use of the plant polyphenol ramie A in alleviating intestinal inflammatory diseases as claimed in claim 1, characterized in that: The molecular formula of the vine-like quinoline A is C 30 H 32 O6, its chemical formula is as follows:

3. The use of the plant polyphenol ramie A in alleviating intestinal inflammatory diseases as claimed in claim 1, characterized in that: The described ramophilin A is used to promote the expression of proliferation and differentiation genes of intestinal organoids in the establishment of intestinal organoid models.

4. The use of the plant polyphenol ramie A in alleviating intestinal inflammatory diseases as claimed in claim 3, characterized in that: The use of the described ramophilin A in locally slowing down the expression of LPS-induced intestinal organoid inflammatory factors in the establishment of an intestinal organoid model.

5. The use of the plant polyphenol ramie A in alleviating intestinal inflammatory diseases as claimed in any one of claims 3 or 4, characterized in that: The effective concentration of the phytohormone A is 1 μM.

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