Use of galangal polysaccharide in preparation of a preparation for improving alcohol-induced intestinal flora disorder

Abstract

The application belongs to the technical field of biological medicine, and discloses application of high galangal polysaccharide in preparation of a preparation for improving alcohol-induced intestinal flora disorder. The application prepares an oral preparation with high galangal polysaccharide as an active ingredient, and aims at alcohol-induced intestinal flora disorder. According to a dosing amount of 125-250 mg / kg body weight / day, the alcohol-induced intestinal barrier damage can be repaired, the expression of tight junction proteins can be up-regulated, the intestinal flora alpha diversity can be improved, the intestinal flora structure can be regulated, the relative abundance of beneficial bacteria can be up-regulated, the relative abundance of harmful bacteria can be down-regulated, the alcohol-induced flora structure deviation can be reversed, and the intestinal short-chain fatty acid content can be increased, so that the alcohol-induced intestinal flora disorder can be well regulated.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the application of galangal polysaccharide in the preparation of formulations for improving alcohol-induced intestinal flora imbalance. Background Technology

[0002] Gut microbiota homeostasis is a core foundation for maintaining human gut health and metabolic balance. Various external factors can trigger gut microbiota dysbiosis, with alcohol intake being a common risk factor. Long-term or excessive alcohol consumption can damage the intestinal epithelial barrier function, cause deviations in gut microbiota structure, and subsequently induce a series of related diseases such as alcoholic enteropathy and alcoholic fatty liver. With the expansion of the domestic alcohol-drinking population, the number of people suffering from these diseases continues to rise, creating an urgent need for safe and effective targeted interventions.

[0003] In existing technologies, natural plant polysaccharides have become a research hotspot in the field of intestinal microecological intervention due to their low toxicity and side effects and clear activity in regulating gut microbiota. Among them, galangal polysaccharide has been proven to have a certain effect on regulating gut microbiota. The disclosed related technology CN119925415A discloses that galangal polysaccharide can be used to improve gut microbiota imbalance induced by hyperuricemia. Through a dosage of 75~250mg / kg, it can achieve intestinal barrier repair and microbiota structure regulation under the influence of hyperuricemia.

[0004] However, existing research and technical solutions focus on intervening in gut microbiota dysbiosis induced by hyperuricemia, without exploring their application value in alcohol-induced gut microbiota imbalance. Studies have confirmed that alcohol-induced gut microbiota imbalance has unique pathological characteristics. Its impact on gut microbiota diversity, molecular characteristics of barrier damage, and changes in short-chain fatty acid metabolism are all significantly different from those of hyperuricemia-induced gut microbiota dysbiosis. Existing administration regimens of galangal polysaccharides for hyperuricemia cannot meet the intervention needs of alcohol-induced damage. Currently, there is still a lack of safe and effective targeted intervention programs for alcohol-induced gut microbiota imbalance. Summary of the Invention

[0005] The present invention aims to solve the technical problems that in the prior art, Alpinia officinarum polysaccharide can only be used for intervention scenarios of intestinal flora imbalance induced by high uric acid, and cannot cover the intervention needs of intestinal flora imbalance induced by alcohol. Furthermore, there is currently a lack of safe, efficient and targeted intervention methods for alcohol-induced intestinal flora imbalance.

[0006] To address the aforementioned issues, this invention provides the application of galangal polysaccharide in the preparation of formulations for improving alcohol-induced intestinal flora imbalance, wherein the dosage of galangal polysaccharide is 125-250 mg / kg body weight / day.

[0007] Furthermore, in the above applications, the formulation is used to repair alcohol-induced intestinal barrier damage and upregulate the expression of tight junction proteins ZO-1 and Occludin in colon tissue.

[0008] Furthermore, in the above applications, the formulation is used to regulate gut microbiota diversity, enhance gut microbiota alpha diversity, increase chao1 index, ACE index, Shannon index and Simpson index, while regulating gut microbiota beta diversity and reversing alcohol-induced gut microbiota structure deviation.

[0009] Furthermore, in the above applications, the formulation is used to regulate the abundance of intestinal flora at the phylum level, upregulating the relative abundance of Bacteroidetes and Dethiobacterium, and downregulating the relative abundance of Firmicutes.

[0010] Furthermore, in the above applications, the formulation is used to regulate the abundance of intestinal flora genera, upregulating the relative abundance of Bacteroides, Parabacterium, and Paratella, and downregulating the relative abundance of Clostridium, Corynebacterium, Enterobacter, Klebsiella, and Biliophilia.

[0011] Furthermore, in the above applications, the formulation is used to increase the content of short-chain fatty acids in the intestine.

[0012] Furthermore, in the above applications, the preparation is an oral preparation.

[0013] Compared with the prior art, the present invention has the following significant advantages:

[0014] This invention is the first to apply galangal polysaccharide to the intervention of alcohol-induced intestinal flora imbalance, expanding the application scenarios of this natural active ingredient and filling the application gap in the existing technology.

[0015] The technical solution of this invention can be specifically adapted to the unique pathological characteristics of alcohol-induced gut microbiota imbalance, and can achieve precise repair of alcohol-induced intestinal damage, providing a safe and efficient intervention method for alcohol-related intestinal and metabolic damage.

[0016] This invention can improve the intestinal tight junction protein ZO induced by alcohol. The relative expression levels of 1 and Occludin were downregulated, which helped repair the intestinal barrier function.

[0017] This invention demonstrates excellent efficacy in regulating alcohol-induced changes in alpha and beta diversity of gut microbiota.

[0018] This invention demonstrates excellent effects in regulating alcohol-induced changes in the composition of gut microbiota at both the phylum and genus levels.

[0019] This invention can effectively increase the content of short-chain fatty acids in the intestine, regulate intestinal immune function, and protect the intestine. Attached Figure Description

[0020] Figure 1 The results show the relative expression levels of tight junction proteins ZO-1 and Occludin in the colon tissue of mice in different experimental groups; where A is the electrophoretic band diagram of the relative expression levels of tight junction proteins ZO-1 and Occludin in the colon tissue of mice in different experimental groups, B is the statistical result of the relative expression level of ZO-1 protein, and C is the statistical result of the relative expression level of Occludin protein.

[0021] Figure 2 The results show the alpha and beta diversity of gut microbiota in mice from different experimental groups; where A is the result of the chao1 index, B is the result of the Shannon index, C is the result of the Simpson curve, and D is the result of the beta diversity.

[0022] Figure 3 The results show the composition of the major phyla of the gut microbiota in mice from different experimental groups. Among them, A is the abundance of phylum-level clustering of gut microbiota in mice from different experimental groups, B is the relative abundance of Firmicutes, C is the relative abundance of Bacteroidetes, D is the relative abundance of Dethiobacterium, and E is the relative abundance of Planktonicum.

[0023] Figure 4 The results show the composition of major genera in the gut microbiota of mice in different experimental groups. Among them, A is the abundance of genera-level clustering of gut microbiota in mice in different experimental groups, B is the relative abundance of Bacteroides, C is the relative abundance of Clostridium, D is the relative abundance of Corynebacterium, E is the relative abundance of Enterobacter, F is the relative abundance of Parabacterium, G is the relative abundance of Paradactylus, H is the relative abundance of Klebsiella, and I is the relative abundance of Bacillus.

[0024] Figure 5 The results show the determination of short-chain fatty acid composition in mice from different experimental groups; where A represents the acetic acid content in mouse feces, B represents the propionic acid content, C represents the isobutyric acid content, D represents the butyric acid content, E represents the isovaleric acid content, F represents the valeric acid content, and G represents the total short-chain fatty acid content. Detailed Implementation

[0025] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0026] Unless otherwise specified, the experimental and detection methods in the following embodiments are conventional methods; the reagents and materials mentioned are commercially available unless otherwise specified; and the index data are measured using conventional methods unless otherwise specified.

[0027] The main experimental materials used in the embodiments of this invention are as follows: 1. Laboratory animals SPF-grade male C57BL / 6J mice, 6 weeks old, weighing 18±2g, were purchased from a qualified laboratory animal supplier and housed in an SPF-grade animal laboratory environment with environmental parameters of 22±2℃, 50±10% relative humidity, and 12h light-dark cycle. The experiment was conducted after acclimatization feeding.

[0028] 2. Active ingredients Galangal polysaccharides were prepared by extraction and purification according to the process described in patent CN119925415A.

[0029] 3. Main reagents and consumables 1) Feeding materials: Lieber-DeCarli standard control diet and Lieber-DeCarli liquid alcohol diet, purchased from Xiaoshuyoutai Beijing Biotechnology Co., Ltd., were used for group feeding of mice and the construction of an alcohol-induced model. The Lieber-DeCarli standard control diet contains 1.0 kcal per milliliter, of which 16.7% comes from fat, 64% from carbohydrates, and 19.3% from protein. Preparation and usage: Take 221.78g of the control diet powder, add water to a final volume of 1L, mix thoroughly within 30 seconds, and then dispense. The prepared diet should be used within 3 days. The Lieber-DeCarli liquid alcohol diet contains 1.0 kcal, of which 35% comes from fat, 11% from carbohydrates, 18% from protein, and the remaining 36% from alcohol. Preparation and usage instructions: Take 132.18g of the alcohol-based feed powder, add 57.3mL of 95% ethanol, then add water to bring the volume to 1L. Mix thoroughly within 30 seconds and then dispense. The prepared feed should be used within 3 days if possible.

[0030] 2) Protein immunoblotting assay reagents: RIPA buffer containing protease and phosphatase inhibitors, BCA protein quantification kit (purchased from Shanghai Beyotime Biotechnology Co., Ltd.), SDS-PAGE gel, PVDF transfer membrane, skim milk powder, ZO-1 specific primary antibody for tight junction protein (purchased from Chengdu Zhengneng Biotechnology Co., Ltd.), Occludin specific primary antibody (purchased from Chengdu Zhengneng Biotechnology Co., Ltd.), HRP-labeled secondary antibody (purchased from Abimate (Shanghai) Biopharmaceutical Co., Ltd.), TBST washing buffer, and ECL chemiluminescence imaging substrate (purchased from Icoin (Wuhan) Biotechnology Co., Ltd.), for the detection of tight junction protein expression levels in colon tissue.

[0031] 3) Microbial community sequencing detection reagents: bacterial genomic DNA extraction kit, 16S rRNA gene V3-V4 region amplification primers, PCR product purification kit, used for 16S high-throughput sequencing analysis of intestinal microbiota in cecal contents.

[0032] 4) Short-chain fatty acid detection reagents: 15% phosphoric acid solution, ethyl acetate, and GC-MS detection kits are used to detect the short-chain fatty acid content in mouse fecal samples.

[0033] Example 1 This embodiment describes an animal experiment demonstrating the effect of galangal polysaccharide on improving alcohol-induced intestinal damage in mice.

[0034] 1. Experimental Methods After one week of acclimatization feeding, the mice were randomly divided into four groups of six mice each, based on their body weight: control group, alcohol model group, low-dose galangal polysaccharide group, and high-dose galangal polysaccharide group.

[0035] Grouping and treatment: The control group was fed a standard Lieber-DeCarli diet, while the other three groups were fed a Lieber-DeCarli liquid alcohol diet to establish an alcohol-induced intestinal flora imbalance model. Simultaneously, two groups were administered medication by gavage: the low-dose group received 125 mg / kg body weight of galangal polysaccharide solution daily, and the high-dose group received 250 mg / kg body weight of galangal polysaccharide solution daily. The control and model groups received an equal volume of physiological saline. All mice were administered the medication for 15 consecutive days. After the last administration, all mice were deprived of food and water for 24 hours. Blood was then collected by enucleation, serum was separated, and the mice were euthanized by cervical dislocation, yielding colon tissue and fecal samples for subsequent assays.

[0036] 2. Experimental Results 1) Mouse status During the administration period, the mice in the control group were in good spirits, had normal activity, and had glossy fur; the mice in the model group showed reduced activity, decreased food intake, and dull fur. The above abnormal conditions in the mice in the Alpinia officinarum polysaccharide administration group were significantly alleviated.

[0037] 2) Intestinal barrier To verify the repair effect of Alpinia galanga polysaccharide on the intestinal barrier, the expression of tight junction proteins ZO-1 and Occludin in mouse colon tissue was further detected: 20 mg of mouse colon tissue was weighed and homogenized in RIPA buffer containing protease and phosphatase inhibitors; after lysis for 30 min, the tissue was centrifuged at 10,000 rpm for 10 min at 4 °C, and the supernatant was collected. The protein concentration was determined using a BCA assay kit; the protein was then separated on a 10% SDS-PAGE gel and electrotransferred to a PVDF membrane; the membrane was blocked with 5% skim milk powder for 2 h at room temperature, and then incubated overnight with a specific primary antibody at 4 °C; after three 15 min TBST washes, the membrane was incubated with the corresponding HRP-labeled secondary antibody for 2 h at room temperature; after three more TBST washes, the protein bands were visualized with ECL substrate and captured using a gel imaging system; the band density was quantitatively analyzed using ImageJ software; the final data are presented as the ratio of target protein grayscale to internal control grayscale.

[0038] ZO, a tight junction protein in mouse colon tissue The results of the determination of the relative expression levels of 1 and Occludin are shown in the figure. Figure 1 Figure A shows the protein electrophoresis bands, Figure B shows the relative expression statistics of ZO-1 protein, and Figure C shows the relative expression statistics of Occludin protein. The results showed that compared with the control group, the expression of ZO-1 and Occludin in the model group mice was significantly downregulated, while administration of Alpinia galanga polysaccharide significantly upregulated the expression of these two proteins, repairing alcohol-induced intestinal epithelial barrier damage. Repair of the intestinal epithelial barrier can prevent endotoxin leakage and slow down the occurrence of intestinal flora imbalance.

[0039] 3) Gut microbiota The DNA genome was extracted from mouse cecal contents, and the V3-V4 region of bacterial 16S rRNA was amplified using forward and reverse primers. The PCR amplicon was purified and quantified. After quantification, gut microbiota diversity analysis, alpha diversity analysis, and beta diversity analysis were performed. The results are shown in Table 1. Figures 2-4 As shown.

[0040] Table 1: Results of Alpha diversity index measurement of gut microbiota in mice of different experimental groups

[0041] Figure 2The results show the measurement of gut microbiota diversity in mice from different experimental groups. A represents the chao1 index, B the Shannon index, C the Simpson curve, and D the Beta diversity. The results showed that compared with the control group, the chao1, ACE, Shannon, and Simpson indices were all decreased in the model group, indicating that alcohol intake led to a decrease in Alpha diversity and dysbiosis in the gut microbiota. Alpinia galanga polysaccharide administration significantly increased these indices, improving the reduction in Alpha diversity. Meanwhile, Beta diversity analysis showed that the microbiota structure in the model group deviated significantly from that in the control group, while the microbiota structure in the Alpinia galanga polysaccharide-treated group reverted towards the control group, suggesting that Alpinia galanga polysaccharide can effectively improve alcohol-induced microbiota structure deviation.

[0042] Figure 3 The results show the phylum-level composition of the gut microbiota in mice from different experimental groups. A represents the phylum-level cluster abundance diagram, and B-E represent the relative abundance statistics of Firmicutes, Bacteroidetes, Dethiobacteria, and Planktonicia, respectively. The results showed that compared with the control group, the model group exhibited a highly significant increase in the relative abundance of Firmicutes (p<0.001), a highly significant decrease in the relative abundance of Bacteroidetes (p<0.001), a significant decrease in the relative abundance of Dethiobacteria (p=0.01), and a decrease in the relative abundance of Planktonicia. Administration of Alpinia galanga polysaccharide reversed these abnormal changes in the abundance of the above-mentioned microbiota, helping to restore the phylum-level microbiota structure to normal levels. Furthermore, Alpinia galanga polysaccharide could regulate the F / B ratio (the relative abundance ratio of Firmicutes to Bacteroidetes), indicating that Alpinia galanga polysaccharide can alleviate obesity caused by alcohol-induced alcoholic fatty liver disease in mice.

[0043] Figure 4The figures show the genus-level composition of the gut microbiota in mice from different experimental groups. Figure A shows the genus-level cluster abundance diagram, and figures B through I show the relative abundance statistics of *Bacteroides*, *Clostridium*, *Anaerobes*, *Enterobacter*, *Parabacteria*, *Paratella*, *Klebsiella*, and *Biliophores*, respectively. The results showed that compared to the control group, the model group had significantly lower relative abundances of *Bacteroides* (Figure B, p<0.01), *Parabacteria* (Figure F, p=0.08), and *Paratella* (Figure G, p=0.08), while significantly higher relative abundances of *Clostridium* (Figure C, p<0.001), *Anaerobes* (Figure D, p<0.001), *Enterobacter* (Figure E, p<0.001), and *Klebsiella* (Figure H, p=0.001). The *Biliophores* (Figure B, p<0.001) cluster abundance was significantly higher. The relative abundance of bacteria (I, p=0.67) was increased, and the administration of Alpinia galanga polysaccharide could effectively reverse the abnormal abundance of the above-mentioned genus-level flora and improve the genus-level flora disorder caused by alcohol. Among them, Bacteroides has a unique polysaccharide site, which can efficiently decompose polysaccharides to produce short-chain fatty acids, which can regulate intestinal immune function and protect the intestine. Paradactylus can produce 3-phenyllactic acid and N-acetyl-L-leucine, thereby reducing oxidative stress and protecting the intestine and hematopoietic system.

[0044] 4) Short-chain fatty acids To detect the content of short-chain fatty acids in mouse feces: Weigh 20 mg of mouse feces, add 15% phosphoric acid and ethyl acetate, grind thoroughly, centrifuge at 4℃ and 10000 rpm for 10 min, take the supernatant and pass it through a membrane, and use GC-MS to detect short-chain fatty acids.

[0045] Figure 5 Figure 1 shows the results of short-chain fatty acid composition determination in mice of different experimental groups. Figures A through G show the content of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, and total short-chain fatty acids, respectively. The results showed that compared with the control group, the levels of acetic acid (Figure A, p<0.001), propionic acid (Figure B, p<0.001), butyric acid (Figure D, p<0.001), valeric acid (Figure F, p<0.001), and total short-chain fatty acids (Figure G, p<0.001) in the feces of mice in the model group were all significantly reduced; the isovaleric acid content was significantly reduced (Figure E, p=0.04), but there was no significant difference in isobutyric acid content among the groups. Administration of Alpinia officinarum polysaccharide can, to a certain extent, increase the content of short-chain fatty acids, improve alcohol-induced insufficient short-chain fatty acid synthesis, and thus improve the intestinal microenvironment.

[0046] The above embodiments can well illustrate the technical solution of the present invention, but they are only describing preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, all kinds of changes and improvements made by those skilled in the art to the technical solution of the present invention should fall within the protection scope defined by the present invention.

Claims

1. The application of galangal polysaccharide in the preparation of a formulation for improving alcohol-induced intestinal flora imbalance, wherein the dosage of galangal polysaccharide is 125~250 mg / kg body weight / day.

2. The application according to claim 1, characterized in that, The formulation is used to repair alcohol-induced intestinal barrier damage and upregulate the expression of tight junction proteins ZO-1 and Occludin in colon tissue.

3. The application according to claim 1, characterized in that, The formulation is used to regulate gut microbiota diversity, enhance gut microbiota alpha diversity, increase chao1 index, ACE index, Shannon index and Simpson index, while regulating gut microbiota beta diversity and reversing alcohol-induced gut microbiota structure deviation.

4. The application according to claim 1, characterized in that, The formulation is used to regulate the abundance of intestinal flora at the phylum level, upregulating the relative abundance of Bacteroidetes and Dethiobacterium, and downregulating the relative abundance of Firmicutes.

5. The application according to claim 1, characterized in that, The formulation is used to regulate the abundance of intestinal flora genera, upregulating the relative abundance of Bacteroides, Parabacterium, and Paratella, and downregulating the relative abundance of Clostridium, Corynebacterium, Enterobacter, Klebsiella, and Biliophilia.

6. The application according to claim 1, characterized in that, The formulation is used to increase the content of short-chain fatty acids in the intestine.

7. The application according to claim 1, characterized in that, The preparation is an oral preparation.

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