Extraction method and application of polygonum viviparum total flavonoids

Abstract

The invention relates to the technical field of biology, in particular to a polygonum viviparum general flavone extraction method and application, in the extraction method, multiple factors are screened, the method with the highest extraction efficiency is obtained, and the method has the advantages of being high in extraction rate, low in cost, easy and convenient to operate, safe, environmentally friendly and the like, and is high in comprehensive cost performance and suitable for large-scale production; the polygonum viviparum general flavone can restore the reduction of the thickness of an intestinal mucosa layer and enhance the intestinal barrier function by adjusting the level of inflammatory cytokines so as to protect the intestinal tract of an LPS-induced enteritis mouse; the effect of the DSS on the abundance of the microflora is reversed, the effects of regulating the intestinal flora and repairing the intestinal epithelial barrier injury are achieved, and the potential effects of treating the intestinal epithelial barrier injury, the intestinal flora disorder and the uric acid level abnormity in digestive tract diseases are achieved; the compound is expected to be further developed into a pure natural plant flavonoid medicinal preparation for treating digestive tract diseases.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for extracting total flavonoids from Polygonum vitripennis and an application thereof. Background Art

[0002] Piglet diarrhea is a common disease in veterinary clinics. It is one of the important digestive tract diseases that causes piglet deaths and causes huge economic losses to the pig industry. Studies have shown that the invasion of exogenous pathological factors such as Escherichia coli, the main pathogenic bacteria in piglet yellow and white diarrhea, leads to damage to intestinal epithelial cells, which is an important cause of diarrhea, and damage to the intestinal mechanical barrier is an important cause of diarrhea. In addition, the invasion of exogenous bacteria can also lead to intestinal flora disorders. Antibiotic therapy is currently the conventional means of treating piglet diarrhea, but due to the high incidence of drug resistance of the main pathogenic bacteria Escherichia coli, the clinical treatment effect is unsatisfactory, which is one of the important problems that has long plagued the healthy development of the breeding industry. At the same time, due to the long-term and widespread use of antibiotics, drug residues and environmental pollution problems are extremely prominent, seriously endangering the health of humans and animals. There is an urgent need for new alternative means to be used in the prevention and treatment of piglet diarrhea.

[0003] Currently, traditional Chinese veterinary medicine has become an important component of the prevention and treatment of livestock and poultry diseases in my country, and is one of the preferred antibiotic replacement strategies under the national policy of "reducing antibiotics and replacing them." Polygonum viviparum L. is a traditional Chinese medicinal material included in the pharmacopoeia. It has the functions of clearing heat and detoxifying, dispersing blood stasis and stopping bleeding. It is mainly used to treat intestinal jaundice, sore throat, etc. Its main components include flavonoids, organic acids, polysaccharides and other compounds with a wide range of pharmacological activities. However, its research on intestinal epithelial barrier damage, intestinal flora disorders and abnormal uric acid levels in gastrointestinal diseases is still lacking.

[0004] The present invention provides a preparation process of total flavonoids from Polygonum vitripennis by optimization, and systematically studies its pharmacological functions through in vivo and in vitro test models. It is expected to develop a pure natural plant flavonoid pharmaceutical preparation for treating digestive tract diseases, which has good application development prospects. Summary of the Invention

[0005] The primary purpose of the present invention is to provide a method for extracting total flavonoids from Polygonum villosa, comprising the following steps:

[0006] (1) Selecting Polygonum villosa medicinal material, placing it in a 50%-90% ethanol solution according to a solid-liquid ratio of 1:6-14, extracting for 0.5-2.5 hours, and extracting 1-5 times to obtain a crude extract of total flavonoids from Polygonum villosa;

[0007] (2) The total flavonoids from the Polygonum villosa obtained in step (1) are concentrated under vacuum at a concentration temperature of 60-80° C., and the relative density of the concentrated solution is 1.02-1.15, thereby obtaining a concentrated solution of total flavonoids from the Polygonum villosa;

[0008] (3) Weigh the pretreated resins separately, place them in centrifuge tubes, add the concentrated solution of total flavonoids from Polygonum villosa obtained in step (2), and elute at a flow rate of 1.0 mL·min-4.0 mL·min. Control the elution flow rate to be 1.0 mL·min-3.0 mL·min (3 BV / h) to obtain the total flavonoids from Polygonum villosa.

[0009] Preferably, the Polygonum villosa medicinal material in step (1) is coarse powder or flakes; the concentration of ethanol is 70%-80%, the material-liquid ratio is 1:10-14, the extraction time is 2.0-2.5h, and the number of extractions is 3-5 times.

[0010] Preferably, the Polygonum villosa medicinal material in step (1) is a thin slice; the concentration of ethanol is 70%, the material-liquid ratio is 1:10, the extraction time is 2.0h, and the number of extractions is 3.

[0011] Preferably, the relative density of the concentrated solution in step (2) is 1.06.

[0012] Preferably, the resin described in step (3) includes one or more of HP-20, HPD-100, HPD-600, D-101, and AB-8; the loading amount of the concentrated solution of total flavonoids from Polygonum vicifolium is 30 mL; and the eluent used for elution is water, 30% ethanol, 50% ethanol, 70% ethanol, and 95% ethanol.

[0013] Preferably, the eluent in step (3) is 50% ethanol-70% ethanol.

[0014] Preferably, the loading flow rate in step (3) is 2.0 mL·min, and the elution flow rate is 2.0 mL / min.

[0015] The second object of the present invention is to provide total flavonoids from Polygonum vitripennis extracted by the extraction method.

[0016] The third object of the present invention is to provide the total flavonoids of Polygonum villosa extracted by the extraction method or the use of the total flavonoids of Polygonum villosa in the preparation of medicines or health products for regulating intestinal flora disorders.

[0017] The fourth object of the present invention is to provide the total flavonoids of Polygonum villosa extracted by the extraction method or the use of the total flavonoids of Polygonum villosa in the preparation of medicines or health products for repairing intestinal epithelial barrier damage.

[0018] The beneficial effects of the present invention are as follows: the present invention provides a method for extracting total flavonoids from Polygonum villosa, in which multiple factors are screened to obtain a method with the highest extraction efficiency, which has the advantages of high extraction rate, low cost, simple operation, safety and environmental protection, high comprehensive cost performance, and is suitable for large-scale production; the total flavonoids from Polygonum villosa can protect the intestines of LPS-induced enteritis mice by regulating the levels of inflammatory cytokines, restoring the reduced thickness of the intestinal mucosal layer and enhancing the intestinal barrier function; and reverse the effect of DSS on the abundance of microbial communities, and is effective in regulating intestinal flora and repairing intestinal epithelial barrier damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Standard curve

[0020] Figure 2 Pathological sections of the jejunum of mice in each treatment group

[0021] Figure 3 IHC detection of Claudin-1, Occludin and ZO-1 protein expression levels

[0022] Figure 4 Western blotting to detect the expression levels of Claudin-1, Occludin and ZO-1 proteins

[0023] Figure 5 IHC detection of Claudin-1, Occludin and ZO-1 protein expression levels

[0024] Figure 6 Western blotting to detect the expression levels of Claudin-1, Occludin and ZO-1 proteins

[0025] Figure 7 Venn diagram (n=8)

[0026] Figure 8 α diversity: Chao 1 index, Shannon index, ACE index (n=8)

[0027] Figure 9 β diversity: PCA analysis, PCoA analysis, OPLS-DA analysis, NMDS analysis (n=8)

[0028] Figure 10 Microbial community structure at the phylum, genus, and species levels (n=8)

[0029] Figure 11 LEfSe multi-level species difference discriminant analysis (n=8)

[0030] Figure 12 PCA plot of metabolites (n=8)

[0031] Figure 13 OPLS-DA score diagram between group C and group M, and between group M and total flavonoids from Polygonum vitripennis group (n=8)

[0032] Figure 14 Volcano plot of differential metabolites (n=8)

[0033] Figure 15 Histogram of the fold differences of the top 10 up-regulated and down-regulated differential metabolites between group C and group M, and between group M and total flavonoids from Polygonum vitripennis group (n=8)

[0034] Figure 16 Bubble diagram of metabolic pathways enriched with differential metabolites between group C and group M, and between group M and total flavonoids from Polygonum vitripennis group (n=8)

[0035] Figure 17 Relative abundance of inosine, dGMP, ADP, and uric acid in colonic tissues of mice in different groups (n=8) DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they shall be carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially. Example 1. Study on the Optimization of the Extraction Process of Total Flavonoids from Polygonum vicifolium

[0037] 1. Medicinal materials

[0038] Polygonum villosa (production batch number: Y2206020) was purchased from Gansu Fuxinghou Biopharmaceutical Technology Co., Ltd. Anhydrous ethanol (production batch number: 10009218) was purchased from Sinopharm Chemical Reagent Co., Ltd.;

[0039] 2. Research Methods

[0040] 2.1 Single factor extraction test

[0041] The flavonoid content was determined by aluminum nitrate colorimetry using rutin as the reference. A standard curve was constructed with concentration x (mg / mL) as the horizontal axis and absorbance y as the vertical axis. Regression analysis was performed, and the regression equation was y = 6.5256x + 0.0032. 2 =0.9999, good linear relationship.

[0042] 2.1.1 Particle size

[0043] The test results are shown in Table 1. The yield and total flavonoid content of the coarsest powder and thin slices are not much different. Considering that in the actual production process, crushing will produce some fine powder, which is easy to clog the filter and affect the extraction and filtration process, thin slices were selected.

[0044] Table 1 Particle size of decoction pieces

[0045]

[0046] 2.1.2 Extraction solvent concentration

[0047] The test results are shown in Table 2. When extracted with 50% and 60% ethanol, the dry paste yield was higher, but the total flavonoids content was lower, indicating that more impurities were extracted. When extracted with 80% and 90% ethanol, the total flavonoids content was also lower. When extracted with 70% ethanol, the total flavonoids content was the highest and the dry paste yield was moderate, so 70% ethanol was used as the extraction solvent.

[0048] Table 2 Extraction solvent concentration

[0049]

[0050] 2.1.3 Material-liquid ratio

[0051] The test results are shown in Table 3. As the material-liquid ratio increases, the total flavonoid content gradually increases. When the material-liquid ratio exceeds 10 times, the increase in total flavonoid content is very small. Considering the extraction cost and number of extractions, the extraction solvent dosage is selected to be 10 times the material-liquid ratio.

[0052] Table 3 Material-liquid ratio

[0053]

[0054] 2.1.4 Extraction time

[0055] The test results are shown in Table 4. As the extraction time increases, the total flavonoid content gradually increases. When the extraction time exceeds 2 h, the increase in the total flavonoid content is very small. Considering the extraction cost and number of extractions, the extraction time is selected as 2 h.

[0056] Table 4 Extraction time

[0057]

[0058] 2.1.5 Number of extractions

[0059] The test results are shown in Table 5. When the extraction was performed three times, the total flavonoid content was the highest. Increasing the number of extractions resulted in a smaller increase in the total flavonoid content, while the production costs such as solvent input and energy consumption increased significantly. Therefore, three extractions were selected.

[0060] Table 5 Extraction times

[0061]

[0062] 2.2 Orthogonal Optimization

[0063] From the orthogonal test results and the variance analysis table (Table 6, 7), it can be seen that the F value of factor C is greater than the critical value of 19.00 (α=0.05), indicating that it has a significant effect on the comprehensive score. The F values ​​of factors A and B are both less than the critical value, and the effect is not significant. That is, the number of extractions (factor C) is the main influencing factor and has the greatest impact on the extraction of total flavonoids content. The range analysis results show that the optimal number of extractions is 3 times (C3). The solid-liquid ratio (factor A) and the extraction time (factor B) are secondary influencing factors. According to the range analysis results, the solvent dosage of 10 times (A3) and the extraction time of 2h (B3) are the best. Therefore, the optimal extraction parameters are A3B3C3, that is, using 10 times the amount of 70% ethanol, extracting 3 times, each extraction for 2h. The experimental results are consistent with the results of the single factor test.

[0064] Table 6 Orthogonal test arrangement and experimental results

[0065]

[0066] Table 7 Variance analysis table

[0067]

[0068] 2.3 Extraction process validation

[0069] The experimental results, shown in Table 8, indicate that the extraction process is stable, the parameters are reasonable, and the total flavonoid content of Polygonum villosa is high. Therefore, based on the results of single-factor experiments, orthogonal experiments, and process validation, the extraction process for total flavonoids from Polygonum villosa was determined to be three extractions using a 10-fold amount of 70% ethanol, each extraction lasting 2 hours.

[0070] Table 8 Extraction process verification

[0071]

[0072] Example 2: Study on the Concentration Process of Total Flavonoids from Polygonum villosa

[0073] Vacuum decompression is the most commonly used concentration method in modern pharmaceutical production, characterized by low concentration temperatures, high active ingredient retention, high production efficiency, and low energy consumption. In the ethanol reflux extraction of Polygonum villosa, the liquid contains a large amount of ethanol. To facilitate subsequent separation and purification operations, vacuum decompression concentration is used to recover the ethanol. The main factors influencing vacuum decompression concentration are concentration temperature, vacuum level, concentration time, and the relative density of the concentrate. However, in actual production and testing, the vacuum level of the equipment cannot be adjusted arbitrarily. In actual operation, the maximum continuous operating range of the vacuum pump is -0.07 to -0.08 MPa. Therefore, in concentration research, the vacuum level is fixed at -0.07 to -0.08 MPa. Therefore, the concentration temperature, concentration density, and concentration are mainly examined.

[0074] 1. Research Methods

[0075] 1.1 Concentration temperature investigation

[0076] The vacuum concentration temperature is generally controlled at 60-80℃. The concentration is very slow below 60℃, and the polysaccharides and tannin components are easily oxidized and discolored above 80℃, causing the liquid to darken in color and affecting the quality of the preparation. Therefore, the vacuum concentration to dryness was set at 60℃, 70℃, and 80℃ to investigate the effect of different concentration temperatures on the total flavonoids content of Polygonum villosa.

[0077] 1.2 Investigation of relative density of concentrated liquid

[0078] The total flavonoids from Polygonum villosa were purified using a macroporous resin. Excessive residual ethanol in the solution would produce a strong solvent effect, affecting the separation and purification results. Therefore, the relative density of the concentrate was investigated. Weigh 100 g of Polygonum villosa slices and add 10 times the volume of 70% ethanol. Reflux extraction was performed three times, each for 2 hours. The three extracts were combined and filtered. The filtrate was concentrated under reduced pressure at 60°C to 100 mL, 50 mL, and 25 mL, respectively. The filtrate was observed for the presence of ethanol and the relative density of the concentrate was measured. The total flavonoid content of Polygonum villosa was also determined in triplicate.

[0079] 2. Results

[0080] 2.1 Concentration temperature

[0081] The test results are shown in Table 9. The concentration temperature of 60-80°C has no significant effect on the total flavonoids content. Therefore, considering the fluctuation range of production operating temperature, it is appropriate to control the concentration temperature at 60-80°C.

[0082] Table 9 Concentration Temperature

[0083]

[0084] 2.2 Investigation of relative density of concentrated liquid

[0085] The test results are shown in Table 10. When the relative density of the concentrated solution is 1.06, there is no ethanol taste. Different relative densities have no significant effect on the total flavonoids content. Therefore, in order to ensure the purification effect of the macroporous resin, the Polygonum aviculare oral liquid is concentrated to a relative density of 1.06 and no ethanol taste.

[0086] Table 10 Relative density

[0087]

[0088] Example 3: Study on the purification process of total flavonoids from Polygonum villosa

[0089] Methods for separating and purifying the active ingredients of traditional Chinese medicines include the systemic solvent method and the macroporous resin method. The systemic solvent method uses organic solvents of varying polarity for extraction to initially separate the extract components. However, due to the large amount of organic solvents used in the extraction process, the production process is highly hazardous, poses occupational health risks to operators, pollutes the environment, and carries organic solvent residues, making it unsuitable for large-scale production. In recent years, macroporous resin purification technology has been widely used in the traditional Chinese medicine industry. Its low cost, good selectivity, large adsorption capacity, convenient regeneration, fast adsorption rate, and easy elution make it a separation and purification technology suitable for large-scale industrial production.

[0090] 1. Research Methods

[0091] 1.1 Resin screening

[0092] Choose a resin with a matching polarity based on the polarity of the target component. Non-polar resins (such as D101) are suitable for weakly polar components (such as fat-soluble terpenes); weakly polar resins (such as AB-8) are suitable for moderately polar components (such as flavonoids); and polar resins (such as NKA-9) are suitable for strongly polar components (such as polysaccharides and amino acids).

[0093] The pretreated macroporous resins were weighed separately and placed in 50 mL centrifuge tubes. The concentrated extract of Polygonum villosa was added and shaken on a shaker for 24 h. The concentration of the absorption solution was measured and the adsorption and desorption rates were calculated.

[0094] 1.2 Maximum sample loading capacity

[0095] Take the treated AB-8 macroporous resin column (2.0cm×20cm, BV=62.8mL, resin weight 60mg), take the concentrated solution of Polygonum vitripennis extract, and adjust the volume of the concentrated solution to an appropriate concentration according to the fluidity of the loaded drug solution and the preliminary test results. Take 0mL of the concentrated solution as 1 unit, and continuously load the sample for adsorption (flow rate of 1.0mL·min, about 1BV / h). Collect 1 portion every 20mL, collect continuously, and detect each portion of the effluent to determine the maximum drug loading capacity.

[0096] 1.3 Elution solvent selection

[0097] According to the instructions for use of the AB-8 macroporous resin, desorption solvents include ethanol and acetone. Ethanol is a commonly used solvent for traditional Chinese medicine extraction, but acetone has a low boiling point and is easily volatile, making production hazardous and potentially polluting the environment. Therefore, ethanol was chosen as the elution solvent. Solvents of varying polarity are typically used sequentially for elution, collecting the target component and discarding impurities to achieve effective separation and purification. Elution is performed in stages using water, 30% ethanol, 50% ethanol, 70% ethanol, and 95% ethanol. The target component content in each stage is measured to determine the primary elution interval. This primary elution interval is then carefully optimized using a 5% gradient increment to balance elution efficiency and cost.

[0098] 1.4 Sample loading flow rate investigation

[0099] According to the instructions for use of AB-8 macroporous resin, the sample adsorption flow rate is 1-4 BV / h. Therefore, the sample flow rate was examined within this flow rate range. Four pre-treated AB-8 macroporous resin columns (2.0 cm × 20 cm, BV = 62.8 mL, resin weight 60 mg) were taken and loaded with 30 mL of the concentrated extract of Polygonum vicifolium at flow rates of 1.0 mL·min' (1 BV / h), 2.0 mL·min (2 BV / h), 3.0 mL·min (3 BV / h), and 4.0 mL·min (4 BV / h), respectively. The effluent was collected and tested for the presence of total flavonoids from Polygonum vicifolium to determine the sample flow rate.

[0100] 1.5 Elution flow rate investigation

[0101] According to the instructions for use of AB-8 macroporous resin, the desorption flow rate is 0.5 to 3 BV / h. Therefore, the elution flow rate was investigated within this flow rate range. Three treated AB-8 macroporous resin columns (2.0 cm × 20 cm, BV = 62.8 mL, resin weight 60 mg) were taken, and 30 mL of the concentrated extract of Polygonum villosa was taken respectively. The sample was loaded at a flow rate of 2.0 mL min (2 BV / h), and eluted with ultrapure water 2 BV, 30% ethanol 2 BV, 50% ethanol 2 BV, 70% ethanol 3 BV, and 95% ethanol 1 BV in sequence. The elution flow rates were controlled at 1.0 mL min (1 BV / h), 2.0 mL min (2 BV / h), and 3.0 mL min (3 BV / h), respectively. The eluate was collected and the effluent was tested for total flavonoids of Polygonum villosa to determine the elution flow rate.

[0102] 2. Research Results

[0103] 2.1 Resin screening

[0104] The test results are shown in Table 11, which show that AB-8 macroporous resin has the highest adsorption rate for total flavonoids from Polygonum vitripennis, so AB-8 macroporous resin was selected for subsequent research.

[0105] Table 11 Resin screening

[0106]

[0107] 2.2 Maximum sample loading capacity

[0108] The test results are shown in Table 12. When the sample volume reached 30 mL, the total flavonoids of Polygonum vitripennis began to leak. Therefore, the maximum drug loading capacity of the AB-8 macroporous resin column is 30 mL. Taking into account the errors in the actual operation process, the sample volume was determined to be 30 mL to ensure that the target components were not leaked.

[0109] Table 12 Investigation of sample loading capacity of AB-8 macroporous adsorption resin

[0110]

[0111] 2.3 Elution solvent selection

[0112] The experimental results, shown in Table 13, indicate that the primary elution range is 50% to 70% ethanol. The water elution range primarily contains polar impurities such as polysaccharides and proteins. The 30% ethanol range contains some low-polarity impurities and a small amount of flavonoids. The 95% ethanol range (6.6%) likely represents fat-soluble pigments or resin residues. Using 70% ethanol as the primary eluent, shortening the elution volume in the remaining fractions, and increasing the column elution volume improves purification efficiency.

[0113] Table 13 Distribution of total flavonoid content in segmented elution

[0114]

[0115] 2.4 Sample loading flow rate

[0116] The test results are shown in Table 14. It was found that when the sample flow rate reached 3.0 mL / min (3 BV / h), the total flavonoids of Polygonum vitripennis began to leak. Therefore, considering the production time and production efficiency, the sample flow rate was determined to be 2.0 mL / min (3 BV / h).

[0117] Table 14 Sample loading flow rate

[0118]

[0119] 2.5 Elution flow rate

[0120] The test results are shown in Table 15. It was found that when the elution flow rate was 3.0 mL / min (3 BV / h), the total flavonoids content of Polygonum vitripennis was lower between 50% ethanol and 70% ethanol, and there was no significant difference between the other two elution flow rates. Therefore, considering the recovery rate and production efficiency, the elution flow rate was determined to be 2.0 mL / min (2 BV / h).

[0121] Table 15 Elution flow rate

[0122]

[0123] Example 4: Study on the efficacy of total flavonoids from Polygonum villosa

[0124] Effects of total flavonoids from Polygonum villosa on LPS-induced intestinal damage in mice

[0125] 1.1 Establishment of LPS-induced intestinal injury model in mice and grouping

[0126] After a week of acclimation, male C57BL / 6 mice were randomly divided into six groups of eight: control (CON), lipopolysaccharide (LPS), positive drug (DEX), low-dose PV (L), medium-dose PV (M), and high-dose PV (H). The DEX group received daily oral administration of dexamethasone (1 mg / kg bw), while the L, M, and H groups received daily oral administration of PV at 5, 10, and 20 mg / kg bw, respectively. After seven consecutive days of administration, the control group received an intraperitoneal injection of 0.2 mL of saline, while the other groups received an intraperitoneal injection of LPS (10 mg / kg bw, 0.2 mL) to establish an intestinal inflammation model. The control group received an intraperitoneal injection of 0.2 mL of saline. Eighteen hours after injection, all mice were weighed, anesthetized, sacrificed, and dissected, and organ tissues and serum were collected.

[0127] 1.2 Effects on LPS-induced intestinal damage in mice

[0128] Jejunum pathological sections of mice in each treatment group were as follows Figure 2 As shown in Figure 3 , a small amount of mucosal epithelial cell degeneration and necrosis, nucleolar lysis, and inflammatory cell infiltration were observed in the jejunum tissue of mice in the LPS group. On the other hand, no obvious pathological changes were observed in the jejunum tissue of mice in the blank control group and the Polygonum vitripennis total flavonoids groups at all doses.

[0129] 1.3 Effects of total flavonoids from Polygonum villosa on tight junction proteins in LPS-induced intestinal injury in mice

[0130] The expressions of tight junction proteins Claudin 1, Occludin and ZO-1 in mouse colon tissue were detected by IHC and WB, respectively. Figure 3 As shown. Compared with the blank control group, LPS significantly downregulated the positive expression and protein expression levels of Claudin 1, Occludin and ZO-1 in the colon tissue of mice (p<0.05). However, the three doses of total flavonoids from Polygonum vicifolium could reverse the effects of LPS on the expression of Claudin 1, Occludin and ZO-1 to varying degrees ( Figure 4 ), with high-dose total flavonoids from Polygonum villosa having the most significant effect, significantly upregulating the positive expression and protein expression levels of three tight junction proteins (p<0.05). These results suggest that total flavonoids from Polygonum villosa can protect the intestines of mice with LPS-induced enteritis by regulating the levels of inflammatory cytokines, restoring the reduced thickness of the intestinal mucosal layer, and enhancing intestinal barrier function.

[0131] 2. Effects of total flavonoids from Polygonum villosa on intestinal flora in DSS-induced intestinal damage

[0132] 2.1 Establishment of DSS-induced intestinal injury model in mice and grouping

[0133] Forty male C57BL / 6J mice (20 ± 2 g, 6-8 weeks old) were randomly divided into six groups: control (C), model (M), positive control (P), and Polygonum villosa flavonoids (PV) groups, with 10 mice in each group. The positive control (P) and PV groups were treated with SASP and PV, respectively, simultaneously with DSS stimulation. Within 3 days after modeling, the blank control and M groups were untreated except for free food and water, while the positive control and PV groups continued to receive SASP and PV, respectively. At the end of treatment, mice were anesthetized and sacrificed, and the colons were dissected and measured. The colon tissues were washed with PBS, then snap-frozen in liquid nitrogen and stored at -80°C until use.

[0134] 2.2 Effects on mouse tight junction proteins

[0135] Tight junction proteins between colonic epithelial cells are an important component of the intestinal barrier, maintaining the regulation of substances between cells. The expression of tight junction proteins was detected by IHC, such as Figure 5 As shown in the results, DSS induction significantly downregulated the positive expression of Claudin1, Occludin and ZO-1 proteins in the colon tissue of mice compared with the blank control group (p < 0.01), while PV significantly improved the inhibition of DSS on the expression of these three tight junction proteins in the colon tissue of mice. The expression levels of Claudin1, Occludin and ZO-1 proteins were further detected by WB assay. Figure 6 The results were consistent with those of IHC.

[0136] 2.3 Effects on intestinal flora in DSS-induced intestinal damage

[0137] To investigate the effects of total flavonoids from Polygonum villosa on the intestinal microbiota of mice in DSS-induced intestinal damage, cecal contents of mice in each treatment group were collected for 16S rRNA gene sequencing analysis. First, a Venn diagram can be used to display the number of common and unique features between samples, from which the overlap of features between samples can be visualized. Figure 7 As shown, a total of 21,140 OTUs were detected in samples from the four groups, of which 1,483 OTUs were shared.

[0138] Secondly, the Chao 1 index, Shannon index, and Ace index were used to evaluate the α diversity of the samples in each group. All indicators showed similar trends, that is, DSS treatment reduced the number and species diversity of microbial species in the cecal contents of mice to a certain extent, while the total flavonoids treatment of Polygonum villosa reversed the effect of DSS on the abundance of microbial communities, but the difference was not significant ( Figure 8 , p>0.05).

[0139] Subsequently, beta diversity analysis was used to assess the differences in the microbiome structure of each group of samples. Principal component analysis (PCA) and principal coordinate analysis (PCoA) were used to determine the overall differences in the microbial communities between groups, while non-metric multidimensional scaling (NMDS) and orthogonal projection discriminant analysis of latent structures (OPLSA-DA) score maps directly reflected the differences between and within groups. Figure 9 As shown in the figure, there were significant differences in the microbial communities of the cecal content samples of mice in the four treatment groups, indicating that different treatments had a significant impact on the structure of the mouse intestinal microbial community.

[0140] The results of microbial community structure analysis at the phylum, family, and genus levels were displayed using histograms ( Figure 10 ), where each histogram represents a sample, different colors are used to distinguish taxa, and the vertical axis represents the relative abundance of each taxon. The relative abundance of Firmicutes decreased in samples from the DSS group, while the relative abundance of Bacteroidetes increased. However, after treatment with total flavonoids from Polygonum vicifolium, these two phyla showed opposite trends.

[0141] Linear discriminant analysis-effect size (LEfSe) analysis was performed to identify species with significant differences between groups (LDA score > 3.5). Figure 11 As shown. At the phylum and genus levels, a total of 59 taxa were obtained from the four groups, including 18 taxa in group C, 7 in group M, 24 in group P, and 10 in the Polygonum villosa total flavonoid group. The most diverse taxa in group C were Erysipelothorax, Erysipelotrichaceae, and Duboisia. The most differentially abundant taxa in group M were Cyclosporum ileum, Cyclosporum jinshanensis, and Chrysobacterium. The most differentially abundant taxa in group P were Enterobacter, Enterobacter, and Enterococcus. The most differentially abundant taxa in the Polygonum villosa total flavonoid group were Shigella Escherichiae, Erysipelothrix, and Bacteroides vulgaris.

[0142] 2.4 Effects of total flavonoids from Polygonum villosa on metabolites in the cecum of mice with DSS-induced intestinal injury

[0143] To investigate the effects of total flavonoids from Polygonum villosa on metabolites and metabolic pathways in mice with DSS-induced intestinal damage, this study performed non-targeted metabolomics analysis of colon tissue samples from mice in four treatment groups based on the LC-QTOF platform. A total of 3,741 metabolites were identified. PCA statistical analysis of the test samples showed that the metabolic profiles of the four groups were highly differentiated, and the reproducibility of each group of samples was good ( Figure 12 ).

[0144] It is reported that although PCA is effective in extracting key information, it is not sensitive to variables with low correlation. Orthogonal projection discriminant analysis of latent structure (OPLS-DA) can solve this problem. The OPLS-DA model scores between group C and group M and between group M and total flavonoids group of Polygonum vicifolium are shown in Figure 2. Figure 13 The R2Y values ​​of the two models were both greater than 0.9, and the Q2Y values ​​were both less than 0.5, indicating that the two established models were stable and reliable and could be used for subsequent screening of differential metabolites.

[0145] In addition, projected variable importance (VIP) ≥ 1 and fold change (FC) ≥ 1 were used as screening criteria for metabolite screening. The volcano plot of differential metabolites is shown in Figure 2. Figure 14 As shown in the figure, a total of 1,389 differential metabolites were screened between the C and M groups, of which 249 were upregulated and 1,140 were downregulated. A total of 55 differential metabolites were screened between the M group and the Polygonum vitripennis total flavonoids group, of which 27 were upregulated and 28 were downregulated. After qualitative and quantitative analysis of the detected metabolites, the fold difference of the differential metabolites was processed by logarithmic transformation.

[0146] The test results are as follows Figure 15 The top 10 differential metabolites upregulated and downregulated in group C compared with group M, as well as the top 10 differential metabolites downregulated in group M compared with the total flavonoids group from Polygonum vitripennis, are shown in the figure. Complex metabolic reactions and their regulation in organisms do not occur in isolation, but usually form complex pathways and networks composed of different genes and proteins.

[0147] In order to more systematically understand the effects of total flavonoids from Polygonum villosa on the metabolic pathways of mice in DSS-induced intestinal damage, the KEGG database was used to annotate the screened differential metabolites. The bubble diagram of the KEGG enrichment factors of the differential metabolites between group C and group M and between group M and total flavonoids from Polygonum villosa is shown in Figure 2. Figure 16 As shown in Figure 3, the differential metabolites in the purine metabolic pathway were most enriched between the M group and TFTV.

[0148] Analysis of the important intermediates and products of purine metabolism, inosine, 2'-deoxyguanosine 5'-monophosphate (dGMP), adenosine diphosphate (ADP) and uric acid, showed that ( Figure 17 ), compared with the blank control group, the relative abundance of inosine, ADP and uric acid in the model group increased significantly ( Figure 17 , p < 0.05), and the relative abundance of dGMP was significantly reduced (p < 0.05). On the other hand, the effect of DSS on the relative abundance of inosine, dGMP, and ADP was significantly reversed in the group treated with total flavonoids from Polygonum viridis (P < 0.05). These results suggest that purine metabolism pathways are altered during colitis, and total flavonoids from Polygonum viridis can normalize purine metabolism and reduce intestinal uric acid levels.

[0149] This study focused on the pharmacological contribution of flavonoids, but lacked a systematic analysis of the synergistic effects of other active ingredients in Polygonum villosa (such as organic acids and volatile oils) and the metabolic regulatory network among multiple components, which may underestimate the overall effect of the traditional Chinese medicine Polygonum villosa. Although the DSS-induced intestinal injury model in mice has been widely used, its pathological characteristics may vary, and the potential heterogeneity of the immune microenvironment and metabolic pathways between species has not been considered.

Claims

1. A method for extracting total flavonoids from Polygonum villosa, characterized in that: The steps include: (1) Selecting Polygonum villosa medicinal material, placing it in a 50%-90% ethanol solution according to a solid-liquid ratio of 1:6-14, extracting for 0.5-2.5 hours, and extracting 1-5 times to obtain a crude extract of total flavonoids from Polygonum villosa; (2) The total flavonoids from the Polygonum villosa obtained in step (1) are concentrated under vacuum at a concentration temperature of 60-80° C., and the relative density of the concentrated solution is 1.02-1.15, thereby obtaining a concentrated solution of total flavonoids from the Polygonum villosa; (3) Weigh the pretreated resins separately, place them in centrifuge tubes, add the concentrated solution of total flavonoids from Polygonum villosa obtained in step (2), and elute at a flow rate of 1.0 mL·min-4.0 mL·min. Control the elution flow rate to be 1.0 mL·min-3.0 mL·min (3 BV / h) to obtain the total flavonoids from Polygonum villosa.

2. The extraction method according to claim 1, wherein The Polygonum villosa medicinal material in step (1) is coarse powder or thin slices; the concentration of ethanol is 70%-80%, the material-liquid ratio is 1:10-14, the extraction time is 2.0-2.5h, and the number of extractions is 3-5 times.

3. The extraction method according to claim 1, wherein The Polygonum villosa medicinal material described in step (1) is a thin slice; the concentration of ethanol is 70%, the material-liquid ratio is 1:10, the extraction time is 2.0 hours, and the number of extractions is 3 times.

4. The extraction method according to claim 1, wherein The relative density of the concentrated solution in step (2) is 1.

06.

5. The extraction method according to claim 1, wherein The resin described in step (3) includes one or more of HP-20, HPD-100, HPD-600, D-101, and AB-8; the loading amount of the concentrated solution of total flavonoids from Polygonum vitripennis is 30 mL; and the eluents used for elution are water, 30% ethanol, 50% ethanol, 70% ethanol, and 95% ethanol.

6. The extraction method according to claim 5, wherein The eluent in step (3) is 50% ethanol-70% ethanol.

7. The extraction method according to claim 1, wherein The loading flow rate in step (3) is 2.0 mL·min, and the elution flow rate is 2.0 mL / min.

8. The total flavonoids from Polygonum vitripennis obtained by the extraction method according to any one of claims 1 to 7.

9. Use of the total flavonoids of Polygonum vitripennis obtained by the extraction method according to any one of claims 1 to 7 or the total flavonoids of Polygonum vitripennis according to claim 8 in the preparation of medicines or health products for regulating intestinal flora disorders.

10. Use of the total flavonoids of Polygonum vitripennis obtained by the extraction method according to any one of claims 1 to 7 or the total flavonoids of Polygonum vitripennis according to claim 8 in the preparation of a medicine or health product for repairing intestinal epithelial barrier damage.

Images