A natural active composition, extract for alleviating inflammatory bowel disease and its preparation method and application

Abstract

The application discloses a natural active composition for relieving inflammatory bowel disease, an extract and a preparation method and application thereof, and belongs to the technical field of biological medicine. The application provides a composition containing myricitrin, ellagic acid and quercetin. By using the composition in an extremely low dose, the composition can effectively improve the status of hematochezia, restore the colon length and repair the mucosal barrier, and the effect is even significantly better than that of pure myricitrin monomer in a high dose of 100 mg / kg. The composition of the application simultaneously intervenes in the inflammation network and the barrier network, and increases the content of allocholic acid, thereby promoting the repair of the intestinal tract. The composition provided by the application is derived from nature, the extraction process is simple, is suitable for preparing functional food or medicine for preventing or relieving inflammatory bowel disease, and has extremely high practical application value and development prospect.

Description

Technical Field

[0001] This invention relates to a natural active composition, extract, preparation method and application for alleviating inflammatory bowel disease, belonging to the field of biomedical technology. Background Technology

[0002] Inflammatory bowel disease (IBD), primarily including ulcerative colitis (UC) and Crohn's disease, is a chronic, relapsing inflammatory bowel disease. Clinical treatment often employs aminosalicylic acids, glucocorticoids, and biologics; however, long-term use frequently results in serious adverse reactions such as target tolerance and weakened immunity. In recent years, the extraction of monomeric compounds (such as myricetin and myricetin) from natural plants for anti-inflammatory purposes has become a research hotspot. However, the separation and purification of high-purity monomers is extremely costly, and their bioavailability in vivo is low. Furthermore, single-target intervention often struggles to counteract the complex systemic inflammatory network of IBD, leading to a bottleneck in efficacy even with high-dose administration. Therefore, exploring natural compound formulations or precise extract combinations with multi-target network regulatory capabilities and the ability to exert potent anti-inflammatory and mucosal repair effects at low doses is a pressing technical challenge in this field. Summary of the Invention

[0003] [Technical Issues] To overcome the shortcomings of existing high-dose monomeric drugs, such as single target, high cost, and limited efficacy, this invention provides a natural active composition and myrica extract based on a specific mass ratio. Surprisingly, this invention has discovered that when myricetin, ellagic acid, and quercetin are combined in a specific ratio, a strong synergistic effect is produced. Even at extremely low myricetin equivalents, it can exert colonic barrier repair and anti-inflammatory effects significantly superior to 10 times the effect of high-dose pure myricetin monomer.

[0004] [Technical Solution] The first object of the present invention is to provide a natural active composition for the prevention, relief or treatment of inflammatory bowel disease, the composition comprising myricetin, ellagic acid and quercetin; wherein the mass ratio of myricetin, ellagic acid and quercetin is (4~6):(2~4):(1~3).

[0005] Preferably, the mass ratio of myricetin, ellagic acid and quercetin is 5:3:2.

[0006] A second object of the present invention is to provide a bayberry extract, wherein the extract contains the natural active composition described herein.

[0007] Preferably, the natural active composition accounts for 4.0% to 6.0% of the total dry weight of the bayberry extract.

[0008] In one embodiment of the present invention, the method for preparing the bayberry extract includes the following steps: after removing the pits from the bayberries, juicing them, and centrifuging them to obtain a supernatant, the supernatant is purified of impurities and then extracted with alcohol to obtain the bayberry extract.

[0009] In one embodiment of the present invention, the method for preparing the bayberry extract includes the following steps: (1) taking fresh bayberry fruit, removing the pit, juicing and centrifuging, and collecting the supernatant to obtain a crude extract; (2) passing the crude extract from step (1) through a macroporous adsorption resin column, first washing with water to remove water-soluble impurities, and then eluting with an ethanol aqueous solution of 60%~80% volume concentration, and collecting the eluent; (3) concentrating the eluent from step (2) under reduced pressure and freeze-drying under vacuum to obtain the extract.

[0010] A third object of the present invention is to provide a product for the prevention, relief or treatment of inflammatory bowel disease, comprising the aforementioned natural active composition.

[0011] In one embodiment of the present invention, the content of the natural active composition is 1% to 10%.

[0012] In one embodiment of the present invention, the product includes a drug or a functional food.

[0013] The present invention also provides the use of the aforementioned natural active composition, or the aforementioned bayberry extract, in the preparation of drugs or functional foods for the prevention, relief or treatment of inflammatory bowel disease; In one embodiment of the present invention, the preparation method of the bayberry extract is as follows: after removing the pits from the bayberries, juicing, and centrifuging to obtain the supernatant, the supernatant is purified and extracted with alcohol to obtain the bayberry extract.

[0014] In one embodiment of the present invention, the inflammatory bowel disease is ulcerative colitis.

[0015] In one embodiment of the present invention, the application is as follows: (1) Repair the physical barrier of the intestinal mucosa and upregulate the expression of Occludin, ZO-1 and MUC2 in intestinal tissue; (2) Downregulates the expression of pro-inflammatory factors IL-6 and TNF-α; (3) Activate the Nrf2 / HO-1 antioxidant pathway.

[0016] [Beneficial Effects] 1. Synergistic Effect: This invention combines myricetin, ellagic acid, and quercetin in a very low dose (10 mg / kg equivalent of myricetin) of the composition to significantly improve problems such as weight loss, shortened colon length, and impaired intestinal mucosal barrier in colitis, restoring these indicators to essentially the same level as in normal control mice. This is significantly superior to the high dose of pure myricetin monomer (100 mg / kg). The three natural active ingredients in the composition of this invention produce a synergistic effect, enhancing bioavailability, overcoming the barrier of action of single components, and more effectively overcoming the risk of toxic side effects that may arise from relying on high doses of a single component in existing technologies.

[0017] 2. The composition of this invention simultaneously intervenes in both the inflammatory network and the barrier network. On the one hand, it significantly inhibits pro-inflammatory factors (IL-6, TNF-α), and on the other hand, it activates the body's Nrf2 / HO-1 antioxidant defense system and strongly upregulates tight junction proteins such as Occludin. Further investigation will be conducted to explore the repair function of the composition in increasing allocholic acid levels in intestinal inflammation.

[0018] 3. The bayberry extract of the present invention specifically enriches the core polyphenols and flavonoids through macroporous resin, eliminating the need for expensive monomer chromatography purification steps, greatly reducing production costs, and has broad prospects for pharmaceutical or functional food transformation. Detailed Implementation

[0019] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0020] Drugs and reagents Dextran sulfate sodium (DSS, molecular weight 36,000–50,000) was purchased from MPBiochemicals, Inc. (Santa Ana, California, USA). Myricitrin (MYRR): CAS No. #17912-87-7; Ellagic acid: CAS No. 476-66-2; Quercetin: CAS No. #117-39-5.

[0021] Statistical analysis In this study, experimental data were processed using Microsoft Excel 2022, and graphs were created using GraphPad. All experimental data were obtained from three parallel experiments, and all results are expressed as mean ± standard deviation (SD). A p-value < 0.05 was considered statistically significant using Duncan's multiple range test. To predict functional changes in the microbiome, researchers obtained KO metabolic enzyme genes showing significant differences between groups (p < 0.0001, FDR < 0.05). Gene function was predicted using PICRUSt2 software and analyzed via the KEGG database website (http: / / huttenhower.sph.harvard.edu / galaxy / , https: / / www.kegg.jp / kegg / ).

[0022] Example 1: Preparation of Composition A The natural active composition A of the present invention was prepared by mixing commercially available standard products with a purity >98% according to the mass ratio of myricetin: ellagic acid: quercetin = 5:3:2.

[0023] Example 2: Preparation of Composition B The natural active composition B of this invention was prepared by mixing commercially available standard products with a purity >98% according to the mass ratio of myricetin: ellagic acid: quercetin = 6:4:3.

[0024] Example 3: Preparation of Composition C The natural active composition C of this invention was prepared by mixing commercially available standard products with a purity >98% according to a mass ratio of myricetin: ellagic acid: quercetin = 5:2:1.

[0025] Example 4: Preparation of Composition D The natural active composition D of this invention was prepared by mixing commercially available standard products with a purity >98% according to the mass ratio of myricetin: ellagic acid: quercetin = 6:3:3.

[0026] Example 5: The effect of the extracts and compositions of the present invention on the phenotype of ulcerative colitis mice. 1. Preparation of bayberry juice extract: Fresh bayberries were pitted and juiced, and the supernatant was collected by centrifugation at 4000 r / min for 15 min. The crude extract was passed through an AB-8 macroporous adsorption resin column at a flow rate of 2 BV / h, first eluted with water to remove sugars, and then eluted with 70% ethanol. The ethanol eluent was collected, concentrated under reduced pressure, and freeze-dried under vacuum to obtain bayberry extract (BJ freeze-dried powder).

[0027] 2. Laboratory animals: The animals used in the experiment were healthy male SPF-grade C57BL / 6 mice (8 weeks old, weighing 20±2 grams), all provided by Shanghai Silex Laboratory Animal Co., Ltd. All mice were housed in a constant environment (temperature 23±1℃, humidity 60%), with free access to standard feed and water. This research protocol was approved by the Animal Ethics Committee of Jiangnan University.

[0028] 3. Establishment and treatment of ulcerative colitis in mice Forty-eight mice were randomly divided into six groups (n=8). After acclimatization for seven days, except for the normal control group (Con), the other groups were given 2.5% DSS solution (sodium dextran sulfate solution) in drinking water to induce a colitis model (Mod). Simultaneously, each intervention group received continuous gavage administration of the following medications: Low-dose monomer group (MYRR-Low): pure myricetin 10 mg / kg; High-dose monomeric group (MYRR-High): pure myricetin 100 mg / kg; Myrica extract group (BJ): Myrica juice extract 400 mg / kg (calculated, its myricetin equivalent is approximately 10 mg / kg). Combo: Composition A of Example 1, 20 mg / kg (of which the myricetin equivalent is 10 mg / kg).

[0029] Mice were monitored for body weight and clinical symptoms daily. On day 7 of the experiment, mice were euthanized humanely and then analyzed. Table 1: Evaluation results of macroscopic pathological phenotypes in mice of each group

[0030] (Note: ##P<0.01vsCon group;) P<0.05, P<0.01 (vsMod group) The results are shown in Table 1. The BJ group and Combo group, which contained extremely low myricetin equivalent (10 mg / kg), achieved extremely significant efficacy in terms of weight recovery, reduction of DAI, and improvement of colon length. Moreover, this efficacy was statistically significantly better than that of 10 times higher dose of pure myricetin monomer (MYRR-High), and significantly higher than that of the same dose of pure myricetin monomer (MYRR-Low).

[0031] 4. Combination therapy and adjunctive therapy for ulcerative colitis Total RNA was extracted from mouse colon tissue using the TRIzol method. After NanoDrop assay, 1 μg of RNA was reverse transcribed into cDNA. Amplification was performed using SYBR Green qPCR premix on a real-time quantitative PCR instrument. GAPDH was used as an internal reference gene, and the relative expression levels of target genes (Occludin, ZO-1, MUC2, IL-6, TNF-α, Nrf2, HO-1) were calculated using the 2^(-ΔΔCt) method.

[0032] Table 2: Detection results of colonic tight junctions and mucus barrier related indicators

[0033] (Note: ##P<0.01vsCon group;) P<0.05, P<0.01 (vsMod group) Table 3: Relative expression levels of pro-inflammatory factors and antioxidant pathway genes in colon tissue

[0034] (Note: ##P<0.01vsCon group;) P<0.05, P<0.01 (vsMod group) The results are shown in Tables 2 and 3. Compared with the model group (Mod), although the high-dose pure myricetin monomer group (MYRR-High) could significantly upregulate the expression of Occludin, ZO-1 and MUC2 in colon tissue (P<0.05 or P<0.01), the myricetin extract group (BJ) and the combination group (Combo) showed more outstanding repair effects: the expression levels of Occludin and ZO-1 in the Combo group were close to the levels of the normal control group (95% and 92%, respectively), and the expression level of MUC2 reached 85% of the normal group, which was significantly better than the MYRR-High group (P<0.01). This fully demonstrates that the composition and extract of the present invention can repair the physical barrier of the intestinal mucosa more efficiently.

[0035] Regarding the regulation of pro-inflammatory factors and activation of antioxidant pathways, the expression levels of IL-6 and TNF-α in the model group (Mod) were significantly increased (1.98 times and 1.51 times that of the Con group, respectively), while the expression levels of Nrf2 and HO-1 were significantly decreased (only 45% and 52% of the normal group, respectively). Although the MYRR-High group could inhibit pro-inflammatory factors and activate antioxidant pathways, the effect was limited. The BJ group reduced IL-6 to 1.08 times that of the normal group, TNF-α to 0.85 times, and Nrf2 and HO-1 to 98% and 105% of the normal group, respectively. The Combo group showed the most significant effect, with IL-6 and TNF-α expression levels close to normal levels (0.95 times and 0.78 times, respectively), and Nrf2 and HO-1 expression levels even slightly higher than those of the normal group (1.02 times and 1.12 times, respectively). All indicators were significantly better than those of the MYRR-High group (P<0.01).

[0036] Example 6: The regulatory effect of the composition of the present invention on the gut microbiota-metabolite-host axis. 1. Gut microbiota 16S rRNA sequencing analysis: Fecal DNA was extracted from mice in each group and sequenced. Results showed that the model group (Mod) mice exhibited abnormally high gut microbiota diversity (Shannon and Simpson indices) and disordered microbiota structure. After intervention with the composition of this invention, the microbiota structure significantly approached that of the normal control group. LEfSe analysis and correlation network analysis indicated that the composition of this invention specifically enriches probiotics with mucosal repair and anti-inflammatory potential, particularly *Akermansia myxophilus* (…). Ackermansia muciniphila ), Sylvesica schlegelii ( Mucispirillum schaedleri ) and Bacteroides multiforme ( Bacteroides thetaiotaomicron Its relative abundance was significantly higher than that of the Mod group (P<0.05).

[0037] 2. Serum and fecal metabolomics analysis: Untargeted metabolomics analysis of mouse serum and feces was performed using LC-MS / MS. Orthogonal partial least squares discriminant analysis (OPLS-DA) showed clear differentiation of metabolic profiles among groups. Enrichment of differentially expressed metabolite pathways indicated that the composition of this invention significantly modulates the primary bile acid biosynthesis pathway. In both serum and feces, the composition of this invention significantly upregulated the levels of allocholic acid (ACA) and cholic acid (CA).

[0038] Multi-omics analysis confirmed that the composition of the present invention achieves systemic intervention for inflammatory bowel disease through the microecological-metabolic axis of "reshaping the gut microbiota structure - upregulating key metabolites (such as ACA)".

[0039] Example 7: Validation of Key Metabolic Barrier Repair Function Based on Colonic Organoid Model To further verify in vitro the direct repair effect of the core metabolites (such as ACA) generated by the composition of the present invention on the physical barrier of the intestinal mucosa, a mouse 3D colon organoid model was constructed.

[0040] (1) Modeling and intervention: Crypts were extracted from the mid-colon of normal mice and cultured in Matrigel. A TNF-α-induced intestinal barrier injury model (simulating in vitro inflammation, Mod group) was added to the differentiation medium. In the intervention group, in addition to the TNF-α-induced intestinal barrier injury model, the differentiation medium also contained allocholic acid (ACA, 20% v / v), the core metabolite upregulated by the natural active composition of Example 1 of this invention, and the intervention lasted for 24 h.

[0041] Differentiation medium: ENR medium: basal medium, 50 ng / mL EGF, 100 ng / mL Noggin, 500 ng / mL R-spondin 1.

[0042] The basal medium was Advanced DMEM / F12, supplemented with 1×B27, 1×N2, 1 mM N-acetylcysteine, 10 mM HEPES, 1×GlutaMAX, and 1×penicillin-streptomycin.

[0043] (2) Immunofluorescence detection: 3D fluorescence imaging of the core mucin MUC2 in organoids was performed using laser confocal microscopy.

[0044] The results showed that the organoids in the normal control group exhibited strong and continuous MUC2 green fluorescence; the organoids in the Mod group damaged by TNF-α showed edge destruction, weak and discontinuous MUC2 fluorescence signal, indicating severe damage to the mucosal barrier; after ACA intervention, the MUC2 fluorescence intensity and continuity of the organoids were significantly restored (quantitative analysis P<0.01), even better than the level of the normal control group.

[0045] As can be seen from the in vitro organoid model, the composition of the present invention can directly stimulate colonic epithelial goblet cells to synthesize and secrete MUC2 by targeting and upregulating specific bile acid metabolites (ACA), thereby fundamentally achieving the repair of the physical barrier of the intestinal mucosa.

[0046] In summary, the natural active composition and bayberry extract of the present invention, through synergistic effects, form a systemic regulation from physical barrier repair and inflammation inhibition to antioxidant defense. This not only improves the symptoms of ulcerative colitis at the phenotypic level, but also achieves multi-target precision intervention at the molecular level, providing a safe and efficient natural solution for the prevention, relief and treatment of inflammatory bowel disease.

[0047] Comparative Example 1 Compared to Example 1, ellagic acid was omitted from the natural active composition, and myricetin:quercetin was administered in a 5:5 ratio (total dose 20 mg / kg).

[0048] Comparative Example 2 Compared to Example 1, quercetin was omitted from the natural active composition, and myricetin: ellagic acid was administered in a 5:5 ratio (total dose 20 mg / kg).

[0049] Comparative Example 3 Compared to Example 1, ellagic acid in the natural active composition was replaced with gallic acid, and the dosage was administered according to the ratio of myricetin:gallic acid:quercetin = 5:3:2 (total dose 20 mg / kg). Comparative Example 4 Compared with Example 1, quercetin in the natural active composition was replaced with kaempferol, and the dosage was administered according to the ratio of myricetin: ellagic acid: kaempferol = 5:3:2 (total dose 20 mg / kg).

[0050] The results showed that mice in Comparative Examples 1 to 4 all exhibited improvements in weight loss rate, colon length shortening, and DAI score compared to the Mod group, but all indicators were still significantly inferior to the composition of Example 1 (Combo group, P<0.05).

[0051] The colon length in Comparative Examples 1 and 2 only recovered to about 6.8 cm (7.85 cm in the Combo group of Example 1), indicating that the lack of ellagic acid or quercetin would lead to a significant reduction in the anti-inflammatory effect.

[0052] The experimental results of Comparative Examples 3 and 4 show that simple substitution with other polyphenols or flavonoid monomers with similar structures (such as gallic acid and kaempferol) cannot reproduce the target synergistic effect induced by the specific combination of the three components of the present invention.

[0053] Comparative Example 5 Compared to Example 1, the natural active compositions contained only quercetin, and the total dosage was adjusted to 10 mg / kg, 20 mg / kg, and 30 mg / kg.

[0054] Comparative Example 6 Compared to Example 1, the natural active composition contained only ellagic acid, and the total dosage was adjusted to 10 mg / kg, 20 mg / kg, and 30 mg / kg.

[0055] Table 4: Symptoms, DAI scores, and relative expression levels of intestinal mucosal barrier proteins

[0056] (Note: ##P<0.01vsCon group;) P<0.05, P<0.01 (vsMod group) Table 5: Relative expression levels of inflammatory factors and antioxidant pathway genes in colon tissue

[0057] Tables 4 and 5 show that, based on the in vivo pharmacodynamic evaluation results of Comparative Example 5 (quercetin monotherapy group) and Comparative Example 6 (ellagic acid monotherapy group), compared with the model group, the quercetin monotherapy groups (10 mg / kg, 20 mg / kg, 30 mg / kg) showed a reduced rate of weight loss, a lower DAI score, and upregulated expression levels of Occludin, ZO-1, and MUC2 proteins in colon tissue, while downregulated expression levels of IL-6 and TNF-α genes and increased expression levels of Nrf2 and HO-1 genes. The ellagic acid monotherapy groups also showed a similar improvement trend, which was dose-dependent. However, comparing the two groups reveals that a single component is unlikely to achieve the therapeutic effect of a combination, and high-dose administration may bring a higher risk of toxic side effects.

[0058] However, a comparative analysis of the high-dose group of the above-mentioned comparative example with that of Example 1 of the present invention (Composition A group, total dose only 20 mg / kg) reveals that the natural active composition of the present invention with a specific mass ratio (myricetin: ellagic acid: quercetin = 5:3:2), under the premise of significantly reduced total dosage load and extremely low absolute exposure of each monomer (ellagic acid only 6 mg / kg and quercetin only 4 mg / kg), is significantly superior to the high-dose group (30 mg / kg) of quercetin or ellagic acid alone in terms of inhibition of mouse weight loss, improvement of DAI score, repair of intestinal mucosal barrier proteins Occludin, ZO-1 and MUC2, and inhibition of IL-6 and TNF-α inflammatory factors in colonic tissue and activation of Nrf2 / HO-1 antioxidant pathway. All key indicators show statistically significant differences compared with the high-dose group of monomers.

[0059] The present invention has been disclosed above with reference to preferred embodiments, but it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be defined by the claims.

Claims

1. A natural active composition for the prevention, relief, or treatment of inflammatory bowel disease, characterized in that, The composition comprises myricetin, ellagic acid and quercetin; wherein the mass ratio of myricetin, ellagic acid and quercetin is (4~6):(2~4):(1~3).

2. The natural active composition according to claim 1, characterized in that, The mass ratio of myricetin, ellagic acid and quercetin is 5:3:

2.

3. A bayberry extract, characterized in that, The extract contains the natural active composition according to claim 1 or 2; preferably, the natural active composition accounts for 4.0% to 6.0% of the total dry weight of the bayberry extract.

4. The method for preparing the bayberry extract according to claim 3, characterized in that, The process includes the following steps: after removing the pits from the bayberries, juicing them, and centrifuging them to obtain the supernatant, the supernatant is then purified of impurities and extracted with alcohol to obtain the bayberry extract; Preferably, the steps are as follows: (1) Take fresh bayberry fruit, remove the pit, squeeze the juice and centrifuge, collect the supernatant to obtain crude extract; (2) Pass the crude extract of step (1) through a macroporous adsorption resin column, first wash with water to remove water-soluble impurities, and then use 60%~80% volume concentration of ethanol aqueous solution for elution, and collect the eluent; (3) Concentrate the eluent of step (2) under reduced pressure and freeze dry under vacuum to obtain the final product.

5. A product for the prevention, relief, or treatment of inflammatory bowel disease, characterized in that, Contains the natural active composition as described in claim 1 or 2.

6. The product according to claim 5, characterized in that, The content of the natural active composition is 1% to 10%.

7. The product according to claim 5, characterized in that, The products include pharmaceuticals or functional foods.

8. The use of the natural active composition according to claim 1 or 2, or the bayberry extract according to claim 3 or 4, in the preparation of drugs or functional foods for the prevention, relief or treatment of inflammatory bowel disease; The preparation method of the bayberry extract is as follows: After removing the pits from the bayberries, juicing, and centrifuging to obtain the supernatant, the supernatant is purified and extracted with alcohol to obtain the bayberry extract.

9. The application according to claim 8, characterized in that, The inflammatory bowel disease mentioned is ulcerative colitis.

10. The application according to claim 8, characterized in that, The specific applications are as follows: (1) Repair the physical barrier of the intestinal mucosa and upregulate the expression of Occludin, ZO-1 and MUC2 in intestinal tissue; (2) Downregulates the expression of pro-inflammatory factors IL-6 and TNF-α; (3) Activate the Nrf2 / HO-1 antioxidant pathway.

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