Preparation method of amomum tsao-ko flavone and application of amomum tsao-ko flavone in preparing medicine for treating colitis

Abstract

The invention discloses a preparation method of amomum tsao-ko flavone and application of the amomum tsao-ko flavone in preparation of a medicine for treating colitis. The preparation method of the amomum tsao-ko flavone comprises the following steps: drying and crushing amomum tsao-ko, soaking the amomum tsao-ko with an ethanol solution, and performing ultrasonic extraction to obtain a flavone crude extract; separating and purifying by adopting an HPD300 macroporous resin chromatographic column, eluting by adopting 15%-35% ethanol to obtain an amomum tsao-ko flavone extracting solution, and freeze-drying to obtain amomum tsao-ko flavone powder. According to the present invention, the high purity amomum tsao-ko flavone elution component is prepared by optimizing the amomum tsao-ko flavone extraction and purification process, the component has strong in vitro oxidation resistance, the DSS induced mouse UC symptom is relieved by regulating the intestinal flora-LPS / TLR4 / NF-kappa B / NLRP3 inflammation signal pathway, and the amomum tsao-ko flavone elution component can be used for treating or improving colitis.

Description

Technical Field

[0001] The invention relates to the field of natural product medicines, and in particular to a method for preparing tsaoko flavonoids and an application thereof in preparing medicines for treating colitis. Background Art

[0002] Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by periodic recurrences, with lesions mainly occurring in the rectum, colon mucosa, and submucosa. UC is characterized by recurrent abdominal pain and diarrhea, thick mucus and blood in the stool, and severe weight loss. The severity of the disease varies, and is often accompanied by skin, liver, gallbladder, and upper gastrointestinal inflammation. Long-term intestinal inflammation increases the risk of colon cancer. Clinically, UC disease activity is often assessed by colonoscopy, imaging manifestations, and inflammatory index measurements. Treatment plans are determined based on the extent and severity of the disease. Patients with mild disease do not require immunosuppression or surgical treatment, while patients with severe disease require surgical resection of the colon, and subsequent recurrences are still possible, requiring patients to bear higher time costs and economic losses.

[0003] Tsaoko (Amomum tsaoko Crevost et Lemarie) is a perennial evergreen herbaceous plant of the genus Cardamom in the ginger family. It is one of the major varieties of Chinese medicinal materials for both medicinal and edible purposes. Tsaoko has a long history of application. It was first recorded in the Ming Dynasty's "Compendium of Materia Medica", which listed tsaoko as a top-grade herb. It has the effects of warming the spleen and stomach, stopping vomiting, eliminating food stagnation, and expelling cold pain in the abdomen. It can nourish the stomach and clear the spleen when decocted with other medicinal materials. In addition to medicinal uses, tsaoko can also be used as a spice. Its special aroma is often used to remove fishy smells and stimulate appetite. Among the large amount of research data on the efficacy of tsaoko, there are no reports on its improvement of inflammatory bowel disease. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a method for preparing tsaoko flavonoids and its application in preparing drugs for treating or improving colitis. The present invention obtains tsaoko flavonoids by extraction and purification, and establishes a DSS-induced UC mouse model to reveal the improvement effect of tsaoko flavonoids on ulcerative colitis.

[0005] In order to solve the above technical problems, the technical solution of the present invention is as follows: a method for preparing tsaoko flavonoids, the preparation method comprising the following steps:

[0006] Step 1: Dry and crush the Amomum villosum, soak it in an ethanol solution, and then perform ultrasonic extraction to obtain a crude flavonoid extract;

[0007] Step 2: Use HPD300 macroporous resin chromatography column to separate and purify the crude flavonoid extract, and gradient elute with 0-50% ethanol solution (elution starts with pure water, and the gradient increases with 8-15% ethanol concentration), to obtain 15-35% ethanol elution of amomum villosum flavonoids eluate, and freeze-dry to prepare amomum villosum flavonoids powder.

[0008] Preferably, in step one, the concentration of the ethanol solution is 55-65%, the solid-liquid ratio of the tsaoko powder and the ethanol solution is 1:35-1:40 (w / v), the soaking time is 10-15h, the ultrasonic power is 160-180W, the ultrasonic time is 40-50min, and the ultrasonic temperature is 65°C-70°C.

[0009] Preferably, in step 2, when HPD300 macroporous resin chromatography column is used for separation and purification, the adsorption column-to-material ratio is 1:15-25; the loading concentration is 2.5-3.0 mg / mL; the loading solution pH is 1.8-2.2; the adsorption temperature is 20-25°C; and the elution flow rate is 1.4-1.6 BV / h.

[0010] The invention also provides the amomum villosum flavonoids obtained according to the preparation method.

[0011] The invention also provides application of the amomum villosum flavonoids in preparing medicine for treating colitis.

[0012] As a further description of the above scheme: the colitis is acute or chronic ulcerative colitis.

[0013] As a further description of the above scheme: the Amomum villosum flavonoids can significantly inhibit the significant weight loss caused by colitis, reduce the DAI score, inhibit the shortening of the colon and the destruction of the colon tissue structure.

[0014] As a further description of the above scheme: at the molecular level, the amomum villosum flavonoids significantly inhibit the abnormal increase of LPS, TNF-α and IL-1β content, significantly downregulate the mRNA expression levels of proinflammatory factors TNF-α and IL-1β, and effectively improve the immune disorder caused by overexpression of F4 / 80, CD68, CD11b, and CD14, while significantly upregulating the mRNA expression of intestinal barrier factors ZO-1, Occludin and Claudin4; the amomum villosum flavonoids can significantly downregulate the gene expression of TLR4, NF-κB, MyD88, TRAF6, NLRP3, and Caspase-1, thereby inhibiting the activation of the TLR4 / NF-κB / NLRP3 inflammatory signaling pathway.

[0015] As a further description of the above scheme: the amomum villosum flavonoids can significantly affect the structure and composition of intestinal microbial communities, promote the recovery of intestinal flora, increase dominant bacterial genera and reduce harmful bacterial genera.

[0016] As a further description of the above scheme: the drug is prepared into a pharmaceutically acceptable dosage form with the amomum villosum flavonoids as the active ingredient, alone or in combination with other drugs, and with the addition of pharmaceutically acceptable excipients, auxiliary ingredients or carriers.

[0017] The present invention obtains tsaoko flavonoids through extraction, separation and purification processes, evaluates the in vitro antioxidant activity of different elution fractions, and selects elution fractions with higher flavonoid content to carry out anti-colitis activity research. The main research results are as follows:

[0018] (1) Based on the single factor experiment to study the effect of ethanol concentration, solid-liquid ratio, ultrasonic power, ultrasonic time and ultrasonic temperature on the yield of tsaoko flavonoids, the response surface Box-Behnken experimental design was further used to obtain the optimal extraction process conditions. When the ethanol concentration was 60%, the solid-liquid ratio was 1:39 (w / v), the ultrasonic power was 160W, the ultrasonic time was 47min, and the ultrasonic temperature was 69℃, the yield of tsaoko flavonoids reached 3.33%.

[0019] (2) Obtaining the purification process conditions of Amomum villosum flavonoids. First, the adsorption / desorption capacity of 10 different macroporous resins for adsorbing Amomum villosum flavonoids was compared, and X-5, HPD100, HPD300 and HP-20 macroporous resins were selected for pseudo-first-order kinetic model, pseudo-second-order kinetic model and intra-particle diffusion kinetic model fitting. The Langmuir and Freundlich equations were used to explore the adsorption process of these four resins, and HPD300 was determined to be the best resin. Subsequently, the effect of HPD300 on the content of Amomum villosum flavonoids under different loading and elution conditions was investigated. Under the conditions of adsorption time of 8h, adsorption temperature of 25℃, loading concentration of 2.79mg / mL, pH value of 2, column-to-material ratio of 1:20, elution flow rate of 1.5BV / h, and elution of 10% to 50% ethanol, the elution fraction with higher flavonoid content was obtained; among them, the Amomum villosum flavonoids content obtained by 20% and 30% ethanol elution exceeded 90%. Finally, the mixture of 20% and 30% elution fractions (1:1 mixture) was detected using plant broad targeted metabolomics technology, and it was found that the relative abundance of flavonoids was over 73%, mainly containing epicatechin (30.88%), isoquercetin (17.33%), astragaloside (9.52%), kaempferol-3-O-rutinoside (8.46%), proanthocyanidin B2 (6.82%) and rutin (5.65%).

[0020] (3) Evaluation of the in vitro antioxidant capacity of the flavonoid extract of Amomum villosum. The DPPH, ABTS and OH levels of the fractions eluted with 10%, 20%, 30%, 40% and 50% ethanol were determined. －The free radical scavenging rate and FRAP value showed that the comprehensive antioxidant capacity of the 20% and 30% ethanol elution components was stronger, while the antioxidant capacity of the crude extract was the weakest, that is, the antioxidant capacity order was 30%>20%>40%>10%>50%>crude extract.

[0021] (4) Evaluation of the anti-colitis activity and mechanism of Amomum villosum flavonoids. The anti-colitis activity of Amomum villosum flavonoids (a 1:1 mixture of Amomum villosum flavonoids eluted with 20% and 30% ethanol) was systematically investigated in a DSS-induced ulcerative colitis mouse model (C57BL / 6J). It was found that high-dose Amomum villosum flavonoids (100 mg / kg) intervention could significantly inhibit the significant decrease in body weight, increase in DAI scores, shortening of colon length, and destruction of colon tissue structure in DSS-induced UC mice. At the molecular level, Amomum villosum flavonoids significantly inhibited the abnormal increase of serum LPS, TNF-α and IL-1β levels in UC mice induced by DSS (P < 0.05), significantly downregulated the mRNA expression levels of colon proinflammatory factors TNF-α and IL-1β in UC mice (P < 0.05), and effectively improved the immune disorder caused by overexpression of F4 / 80, CD68, CD11b, and CD14 in the colon of UC mice (P < 0.05), while significantly upregulated the mRNA expression of intestinal barrier factors ZO-1, Occludin and Claudin4 in UC mice (P < 0.05). It was also found that Amomum villosum flavonoids intervention could significantly downregulate the gene expression of TLR4, NF-κB, MyD88, TRAF6, NLRP3, and Caspase-1 (P < 0.05), thereby inhibiting the activation of the TLR4 / NF-κB / NLRP3 inflammatory signaling pathway.

[0022] (5) The effect of Amomum villosum flavonoids on the intestinal microbial diversity of UC mice was investigated based on the 16S rRNA gene high-throughput detection technology. It was found that Amomum villosum flavonoids could significantly affect the structure and composition of the intestinal microbial community of UC mice. Amomum villosum flavonoids intervention caused the intestinal flora of UC mice to converge towards the normal control group in terms of structure and composition, as manifested by the ability to inhibit the increase of Firmicutes / Bacteroidota (F / B) in the intestinal flora of UC mice induced by DSS and to reverse the abnormal relative abundance of microorganisms at the family and genus levels of Proteobacteria, Desulfobacterota, Verrucomicrobiota and Actinobacteriota. The results of differential species analysis showed that amomum tsaoko flavonoids could significantly reduce the relative abundance of Escherichia-Shigella, Colidextribacter, Oscillospiraceae_UCG-005, Oscillibacter and other bacterial genera (P < 0.05), and increase the relative abundance of UBA1819, Citrobacter, Bifidobacterium, Clostridium_innocuum_group, Flavonifractor, Ruminococcus_gauvreauii_group, unclassified_f__Atopobiaceae and other bacterial genera (P < 0.05).

[0023] (6) Intestinal flora may mediate the improvement of DSS-induced colon inflammation in mice by tsaoko flavonoids. Bivariate correlation analysis was used to explore the association between specific differential bacterial genera and conventional pathological phenotype indicators, intestinal permeability indicators, inflammatory indicators, cell markers and related inflammatory pathways of UC mice. Among them, Escherichia-Shigella, Oscillospiraceae_UCG-005, unclassified_f__Anaerovoracaceae, Colidextribacter, Defluviitaleaceae_UCG-011, Ruminiclostridium, Oscillibacter, Clostridium_innocuum_group and unclassified_f__Atopobiaceae were strongly correlated with multiple indicators. PICRUSt2 functional prediction analysis also found that the functional characteristics of the intestinal flora of mice in the tsaoko flavonoids intervention group were related to pathways such as cell growth and apoptosis, circulatory system, material metabolism and human diseases. These results indicate that Amomum villosum flavonoids can effectively improve the intestinal flora and its functional properties, thereby inhibiting intestinal inflammation, maintaining intestinal barrier integrity, and slowing down the development of UC disease.

[0024] Compared with the prior art, the present invention has the following beneficial effects: the present invention obtains a high-purity tsaoko flavonoid elution fraction by optimizing the extraction and purification process of tsaoko flavonoids, which has a strong in vitro antioxidant capacity and may alleviate the symptoms of DSS-induced UC in mice by regulating the intestinal flora-LPS / TLR4 / NF-κB / NLRP3 inflammatory signaling pathway. The research results lay a data foundation for in-depth research on the material basis and mechanism of action of tsaoko against colitis, and also provide a scientific basis for the application of tsaoko in inflammatory diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The single factor experiment was used to optimize the extraction conditions of flavonoids from Amomum villosum; Note: (A) soaking time, (B) ethanol concentration, (C) solid-liquid ratio, (D) ultrasonic power, (E) ultrasonic time, (F) ultrasonic temperature. All data are expressed as mean ± standard error (mean ± SEM), and different letters indicate significant differences (P

[0026] <0.05), the same letters had no significant difference (P>0.05).

[0027] Figure 2 To compare the adsorption / desorption performance of different macroporous adsorption resins on tsaoko flavonoids;

[0028] Figure 3 This is the adsorption kinetic model curve of tsaoko flavonoids; Note: (A) adsorption kinetic curve, (B) pseudo-first-order kinetic model, (C) pseudo-second-order kinetic model, (D) intra-particle diffusion kinetic model.

[0029] Figure 4 This is the adsorption isotherm curve of amomum villosum flavonoids by HPD300 macroporous resin;

[0030] Figure 5 This is the fitting curve of the adsorption isotherm equation of amomum tsaoko flavonoids by HPD300 macroporous resin; Note: (A) Langmuir model, (B) Freundlich model.

[0031] Figure 6 The effect of sample concentration on the adsorption rate and adsorption amount of HPD300 macroporous resin;

[0032] Figure 7 The effect of pH on the adsorption rate and adsorption amount of HPD300 macroporous resin;

[0033] Figure 8 The effect of column-to-material ratio on the adsorption rate and adsorption amount of HPD300 macroporous resin;

[0034] Fig. 9The effect of different elution conditions on the content of flavonoids in Amomum villosum; Note: (A) The change of ethanol volume (BV) and flavonoid content (mg) required under different flow rates of 0% to 100% ethanol elution conditions, where each gradient of ethanol was eluted to colorless; (B) The flavonoid content (%) of the flavonoid elution fractions obtained by 0% to 50% ethanol elution. All data are expressed in the form of mean ± standard error (mean ± SEM).

[0035] Fig.10 Comparison and component analysis of Amomum villosum flavonoids before and after purification; Note: (A) Purity comparison of Amomum villosum flavonoids before and after purification; (B) TIC graph of Amomum villosum flavonoids eluted with 20% and 30% ethanol; (C) Component analysis of Amomum villosum flavonoids; (D) Analysis of the top 15 flavonoid compounds in relative abundance in Amomum villosum flavonoids. All data are expressed as means ± SEM.

[0036] Fig.11 This is the experimental procedure of the effect of Amomum villosum flavonoids on DSS-induced UC mice;

[0037] Fig.12 The improvement of basic physiological indicators of DSS-induced colitis by tsaoko flavonoids; Note: (A) Survival curve; (B) Changes in mouse body weight; (C) DAI score; (D) Colon image; (E) Colon length; (F) Liver index; (G) Spleen index; (H) Kidney index. All data are expressed as mean ± standard error (means ± SEM). n = 10-12. # indicates compared with the CON group, * indicates compared with the DSS group, using unpaired two-tailed T test, *P < 0.05, **P < 0.01, ***P < 0.001; #P < 0.05, ##P < 0.01, ###P < 0.001.

[0038] Fig.13 The improvement of colon tissue morphology by Amomum villosum flavonoids;

[0039] Fig.14 The regulation of pro-inflammatory and anti-inflammatory factors by tsaoko flavonoids; Note: (A) Expression of TNF-α in colon; (B) Expression of IL-1β in colon; (C) Expression of IL-10 in colon; (D) Expression of IL-18 in colon; (E) Expression of IL-6 in colon; (F) Expression of INF-γ in colon. All data are expressed as mean ± SEM. n = 8-10. # indicates compared with the CON group, * indicates compared with the DSS group, using unpaired two-tailed T test, *P < 0.05, **P < 0.01, ***P < 0.001; #P < 0.05, ##P < 0.01, ###P < 0.001.

[0040] Fig.15The inhibition of tsaoko flavonoids on inflammatory cell infiltration; Note: (A) Colon F4 / 80 expression; (B) Colon CD68 expression; (C) Colon CD11b expression; (D) Colon CD14 expression; (E) Colon Ly-6G (Gr-1) expression; (F) Colon CD115 expression; (G) Representative immunostaining images of colon sections F4 / 80 (200 μm). All data are expressed as mean ± standard error (means ± SEM). n = 8-10. # indicates compared with the CON group, * indicates compared with the DSS group, using unpaired two-tailed T test, *P < 0.05, **P < 0.01, ***P < 0.001; #P < 0.05, ##P < 0.01, ###P < 0.001.

[0041] Fig.16 The inhibition of LPS and inflammatory factors in mice by Amomum villosum flavonoids and the protection of intestinal barrier integrity; Note: (A) Serum LPS expression; (B) Serum TNF-α expression; (C) Serum IL-1β expression; (D) Colon ZO-1 expression; (E) Colon Occludin expression; (F) Colon Claudin4 expression; (G) Colon Muc2 expression. All data are expressed as mean ± standard error (means ± SEM). n = 8-10. # indicates compared with the CON group, * indicates compared with the DSS group, using unpaired two-tailed T test, *P < 0.05, **P < 0.01, ***P < 0.001; #P < 0.05, ##P < 0.01, ###P < 0.001.

[0042] Fig.17 Amomum villosum flavonoids significantly inhibited the upregulation of mRNA expression of genes related to the TLRs / NF-κB / NLRP3 signaling pathway; Note: (A) Colon TLR2 expression; (B) Colon TLR4 expression; (C) Colon TLR5 expression; (D) Colon NF-κB expression; (E) Colon MyD88 expression; (F) Colon TRAF6 expression; (G) Colon NLRP3 expression; (H) Colon Caspase-1 expression; (I) Colon ASC expression; (J) Representative immunostaining images of TLR4 and NLRP3 in colon sections (50 μm). All data are expressed as mean ± standard error (mean ± SEM). n = 8-10. # indicates compared with the CON group, * indicates compared with the DSS group, using unpaired two-tailed T test, *P<0.05, **P<0.01, ***P<0.001; #P<0.05, ##P<0.01, ###P<0.001.

[0043] Fig.18 For the dilution curve and Rank_abundance curve.

[0044] Fig.19 This is the PCoA principal coordinate analysis based on different distance algorithms; Note: (A) Bray-Curtis distance algorithm; (B) unweighted UniFrac distance algorithm; (C) Hellinger distance algorithm.

[0045] Fig. 20 Comparison of relative abundance of different species in each group at the genus level. DETAILED DESCRIPTION

[0046] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments, but the present invention is not limited to the following technical solution.

[0047] Example 1 Extraction, separation and purification of tsaoko flavonoids

[0048] Extraction of Flavonoids from Amomum villosum

[0049] Weigh 4.00g of cardamom powder for use, soak the cardamom powder in ethanol solution and then perform ultrasonic extraction. When evaluating the effect of ethanol concentration on the yield of cardamom flavonoids, the cardamom powder was soaked in 40%, 50%, 60%, 70%, and 80% ethanol at a solid-liquid ratio of 1:25 (w / v) for 12 hours, and then the ultrasonic power was set to 200W, the ultrasonic time was 40min, and the ultrasonic temperature was 25°C. When optimizing the solid-liquid ratio conditions, the cardamom powder was soaked in 60% ethanol at a solid-liquid ratio of 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, and 1:50 (w / v) for 12 hours, and then extracted under the conditions of ultrasonic power 200W, ultrasonic time 40min, and ultrasonic temperature 25°C. When investigating the ultrasonic power, ultrasonic time and ultrasonic temperature, a 60% ethanol solution with a solid-liquid ratio of 1:35 (w / v) was prepared for soaking the cardamom fruit for 12 hours, and the extraction was carried out under the conditions of ultrasonic power of 120, 140, 160, 180, and 200 W (ultrasonic time of 40 min, temperature of 25°C), ultrasonic time of 10, 20, 30, 40, 50, and 60 min (power of 180 W, ultrasonic temperature of 25°C), and ultrasonic temperature of 25, 35, 45, 55, 65, and 75°C (power of 180 W, time of 50 min).

[0050] The results of optimization of extraction conditions of flavonoids from Amomum villosum are as follows Figure 1 As the soaking time increases, the flavonoids in the grass fruit continue to precipitate. After the soaking time reaches 12 hours, the flavonoid content in the soaking solution tends to be stable ( Figure 1 A). The immersion time of 12h was determined, and the effects of different ethanol concentrations, solid-liquid ratios, ultrasonic power, ultrasonic time, and ultrasonic temperature on the extraction of flavonoids from Amomum villosum were investigated. Figure 1As shown in Figures B and 1C, with the increase of ethanol concentration and the decrease of solid-liquid ratio, the yield of flavonoids from Amomum villosum showed a trend of first rising and then falling. When the ethanol concentration was 60% and the solid-liquid ratio was 1:35 (w / v), the flavonoid content was the highest, which was 2.91% and 3.11%, respectively. Then, different ultrasonic parameters were set. When the ultrasonic power, ultrasonic time and ultrasonic temperature reached certain conditions, the flavonoid yield increased from rising to flat ( Figure 1 D to 1F). Considering time and energy saving, the optimal extraction conditions were finally selected: 60% ethanol concentration, 1:35 (w / v) solid-liquid ratio, 180 W ultrasonic power, 50 min ultrasonic time, and 65 °C ultrasonic temperature.

[0051] In order to further optimize the extraction process parameters of flavonoids from Amomum villosum, the single factor extraction conditions were optimized by Design-Expert 8.0.6 software. The Box-Behnken test combination design was used to study the effects of solid-liquid ratio (A), ultrasonic power (B), ultrasonic time (C) and ultrasonic temperature (D) on flavonoids in Amomum villosum (Table 1).

[0052] Table 1 Response surface experimental design factors and levels

[0053]

[0054]

[0055] Design-Expert 8.0 software was used to optimize the combination of process parameters, and the prediction results of relevant extraction conditions are shown in Table 2. Considering the feasibility of actual operation, three verification tests were carried out under the process conditions of solid-liquid ratio 1:39 (w / v), ultrasonic power 160W, ultrasonic time 47min, and ultrasonic temperature 69℃. The maximum yield of tsaoko flavonoids was 3.33%, which was close to the predicted value, indicating that the process conditions optimized by the response model were accurate and reliable, as shown in Table 2.

[0056] Table 2 Prediction model validation results

[0057]

[0058] Separation and Purification of Amomum villosum Flavonoids by Macroporous Resin

[0059] This example investigates the separation and purification of tsaoko flavonoids by various macroporous resins, and the physical property parameters of the resins are shown in Table 3. The pretreatment of the macroporous resin is as follows: soaking in 95% ethanol for 12 hours, washing with UP water (ultrapure water) until tasteless; soaking in 5% sodium hydroxide for 4 hours, washing with UP water until neutral; soaking in 5% hydrochloric acid for 4 hours, washing with UP water until neutral, and finally soaking in UP water for standby use.

[0060] Table 3 Physical properties of macroporous resin

[0061]

[0062]

[0063] Different resins have different adsorption and desorption capabilities for tsaoko flavonoids. Figure 2 As shown, different resins have different adsorption and desorption abilities for amomum villosum flavonoids, and resins with lower polarity show good adsorption abilities. Among them, the adsorption rates and adsorption amounts of non-polar resins X-5, HPD100, HPD300 and HP-20 for amomum villosum flavonoids are higher than those of other resins. Comparing the physical properties of the resins in Table 3, it is found that the HPD300 resin has the smallest average pore size and a large specific surface area, and exhibits better adsorption and desorption abilities than other resins. The specific surface areas of X-5 and HPD100 resins are similar, but the average pore size of X-5 is larger, and it has a stronger affinity with the adsorbate molecules, and its desorption ability for amomum villosum flavonoids is relatively weaker than that of HPD100, HPD300 and HP-20 macroporous resins. Therefore, the HPD300 macroporous resin has the strongest comprehensive adsorption / desorption ability and is suitable for the separation and purification of amomum villosum flavonoids. Kinetic fitting of the adsorption of amomum villosum flavonoids by macroporous resins

[0064] In order to better highlight that HPD300 resin is more suitable for the separation and purification of amomum villosum flavonoids, the adsorption rates of four resins with stronger adsorption performance (X-5, HPD100, HPD300 and HP-20) and the possible rate-limiting steps of adsorption were studied and compared.

[0065] Comparison of the adsorption kinetics of tsaoko flavonoids by different macroporous resins

[0066] The kinetic curves of tsaoko flavonoids on X-5, HPD100, HPD300 and HP-20 resins are shown in Figure 2. Figure 3 A. When different resins adsorb tsaoko flavonoids, their adsorption amount Q t Both increased with the extension of adsorption time. In the first 120 minutes of the adsorption process, the adsorption amount of tsaoko flavonoids on the resin increased rapidly, and the adsorption curve gradually stabilized after 360 minutes. When the adsorption equilibrium was reached, the HPD300 resin had the highest adsorption rate for tsaoko flavonoids, followed by X-5, HPD100 and HP-20.

[0067] Study on the adsorption process of flavonoids from Amomum villosum based on pseudo-first-order or pseudo-second-order kinetic model

[0068] The pseudo-first-order kinetic model is usually suitable for describing the initial stage of the adsorption process, and simply associates the adsorption rate with the number of unadsorbed sites on the resin. The pseudo-second-order kinetic model is often used to predict the behavior of the entire adsorption process, mainly combining the adsorbed sites and the unadsorbed sites to more comprehensively explore the factors affecting the adsorption rate. The experimental data were fitted with the pseudo-first-order model and the pseudo-second-order model, and the adsorption kinetic equations and related parameters of the amomum villosum flavonoids on X-5, HPD100, HPD300 and HP-20 macroporous resins were calculated. The results show that the linear fit of the experimental points of the pseudo-second-order kinetic model is more accurate than that of the pseudo-first-order model ( Figure 3 B and 3C), by the regression coefficient R 2 It can be seen that the pseudo-second-order model (0.9996, 0.999, 0.9993, 0.9997) has a higher fit than the pseudo-first-order model (0.7480, 0.8242, 0.7880, 0.7918) (Table 4). In addition, the theoretical maximum adsorption capacity Q of the pseudo-second-order model is e(cal) (40.24, 39.11, 42.32, 38.77 mg / g) and experimental value Q e(exp) (38.46, 37.54, 39.58, 36.76 mg / g) are relatively close, while the theoretical maximum adsorption capacity Q of the pseudo-first-order model is e(cal) (14.70, 16.05, 19.85, 16.74 mg / g) are much smaller than the experimental values. Therefore, the pseudo-second-order kinetic model is more suitable for describing the adsorption process of amomum villosum flavonoids by X-5, HPD100, HPD300 and HP-20 macroporous resins.

[0069] Study on the intra-particle diffusion behavior of amomum villosum flavonoids adsorbed by macroporous resin

[0070] The intraparticle diffusion kinetic model can determine whether intraparticle diffusion is the limiting step in adsorption rate. Figure 3 D shows that the adsorption amount Q t With t 1 / 2 The fitting curve shows three linear relationships, indicating that the adsorption process of amomum villosum flavonoids by X-5, HPD100, HPD300 and HP-20 macroporous resins is controlled by three consecutive steps. The sharp rise in the first part represents the film diffusion stage, that is, amomum villosum flavonoids diffuses from the solution to the outer surface of the resin; the linear gradual inclination of the second part indicates that internal diffusion and pore diffusion are dominant at this time, that is, amomum villosum flavonoids enter the interior of the resin; the final stable stage indicates that amomum villosum flavonoids are adsorbed on the inner surface of the resin and reach adsorption equilibrium. In addition, the M value is the intercept of the intraparticle diffusion kinetic model diagram, which indicates the thickness of the boundary layer. When M = 0, the boundary layer thickness is 0, indicating that intraparticle diffusion is the only rate-limiting step, and the intraparticle diffusion kinetic curves established for amomum villosum flavonoids by X-5, HPD100, HPD300 and HP-20 macroporous resins do not pass through the origin ( Figure 3 D), indicating that the rate-limiting step is not only intraparticle diffusion, but also boundary layer diffusion during adsorption. As shown in Table 4, the rate constants k3 of the intraparticle diffusion model of X-5, HPD100, HPD300 and HP-20 macroporous resins are 1.2571, 1.1944, 1.4131 and 1.2472, respectively. Among them, the rate constant value of HPD300 resin is the largest, indicating that Amomum villosum flavonoids are more easily diffused in HPD300 than other resins, making its adsorption speed of Amomum villosum flavonoids faster and the adsorption amount is larger, reflecting the adsorption advantage of macroporous resin HPD300. Therefore, HPD300 is more suitable for the adsorption of Amomum villosum flavonoids than X-5, HPD100 and HP-20 macroporous resins.

[0071] Table 4 Fitting parameters of adsorption kinetic model of macroporous resin for tsaoko flavonoids

[0072]

[0073]

[0074] Adsorption isotherm fitting of HPD300 macroporous resin for tsaoko flavonoids

[0075] Comparison of isotherms of adsorption of amomum villosum flavonoids by macroporous resin at different temperatures

[0076] In order to further describe the adsorption process of amomum villosum flavonoids by HPD300 macroporous resin, the equilibrium adsorption concentration C e is the horizontal axis, and the equilibrium adsorption amount Q e As the ordinate, draw the adsorption isotherms of HPD300 resin for amomum villosum flavonoids under different temperature conditions. Figure 4 As shown in the figure, with the increase of adsorption concentration, the adsorption amount shows an upward trend. At the same time, under the adsorption conditions of 25℃, 35℃, and 45℃, the adsorption capacity of the same concentration of Amomum villosum flavonoids on HPD300 macroporous resin weakens successively. The higher the temperature, the worse the adsorption capacity of HPD300 resin, indicating that the increase of temperature is not conducive to the adsorption of Amomum villosum flavonoids on HPD300 resin.

[0077] Study on the adsorption process of flavonoids from Amomum villosum based on Langmuir model and Freundlich model

[0078] At present, the Langmuir model and the Freundlich model are usually used to describe the adsorption isotherm curve, thereby revealing the interaction between the adsorbent and the adsorbate. The Langmuir model is usually suitable for describing the adsorption process on a monolayer, based on the fact that the adsorption sites of the resin are evenly distributed and have the same energy, and there is no interaction between the adsorbed molecules. The Freundlich model is based on the uneven distribution of the adsorption sites and energy of the resin, and can be used for multi-molecular layer adsorption. Figure 5 As shown in Figure 2, both models can well describe the adsorption process of HPD300 macroporous resin on tsaoko flavonoids, but the linear fitting of the Freundlich model is better than that of the Langmuir model. 2 (0.9786, 0.9979, 0.9946) are also higher than the Langmuir model (0.8218, 0.9851, 0.9619) (Table 5), indicating that the Freundlich model is more suitable for describing the adsorption behavior of amomum villosum flavonoids by HPD300 macroporous resin. L and constant k F are important parameters of the Langmuir model and the Freundlich model, respectively, and are related to the adsorption energy of the adsorbent. In this application, when the adsorption temperature increases from 25°C to 45°C, k L The value decreased from 0.2528 mL / mg to 0.2389 mL / mg, k F The value dropped from 2.3579 mL / mg to 2.1891 mL / mg, indicating that high temperature is not conducive to the adsorption of HPD300 macroporous resin (Table 5). The index n is an indicator of the affinity of the adsorbent to the adsorbate. When 0.1≤1 / n<0.5, the adsorbate is easily adsorbed, while when 1≤1 / n, adsorption is difficult to occur. As shown in Table 5, the 1 / n at 25℃, 35℃, and 45℃ are 0.4241, 0.4570, and 0.4568, respectively, all between 0.1 and 0.5, indicating that the adsorption process of amomum villosum flavonoids by HPD300 macroporous resin is spontaneous within this temperature. When the temperature increases from 25℃ to 45℃, the corresponding 1 / n value increases, indicating that the adsorption process gradually becomes difficult. In summary, 25℃ as the adsorption temperature is more suitable for further experiments on amomum villosum flavonoids by HPD300 macroporous resin.

[0079] Table 5 Adsorption isotherm parameters of HPD300 macroporous resin for tsaoko flavonoids at different temperatures

[0080]

[0081] Optimization of the process conditions for separation and purification of tsaoko flavonoids using macroporous resin

[0082] (1) Optimization of resin adsorption conditions

[0083] The effects of loading concentration, pH, and column-to-material ratio on macroporous resin adsorption were investigated. A crude extract of tsaoko flavonoids with a certain flavonoid concentration was prepared, and after adjusting the pH with HCl solution (1 mol / L) or NaOH solution (1 mol / L), it was mixed with 5 g of pretreated macroporous resin and shaken on a shaker for 8 h (25 ° C, 140 r / min). After reaching adsorption equilibrium, the macroporous resin was retained and thoroughly washed with UP water, and then 100 mL of 80% ethanol (v / v) was added and shaken in a shaker for desorption for 24 h (25 ° C, 140 r / min). The flavonoid concentration in the solution was measured, and the adsorption rate and adsorption amount were calculated.

[0084] Determination of loading concentration

[0085] The concentration of the amomum villosum flavonoids loading solution has an important influence on the adsorption capacity of the HPD300 macroporous resin. Figure 6 As shown in the figure, with the increase of sample concentration, the adsorption amount of HPD300 macroporous resin also gradually increased. This may be because more and more flavonoid molecules bind to the various adsorption sites of the resin, resulting in a continuous increase in adsorption. When the flavonoid concentration reaches 2.79 mg / mL, the unadsorbed sites on the HPD300 macroporous resin gradually decrease, and the rate of increase in the resin adsorption amount gradually slows down. In addition, the adsorption rate of cardamom flavonoids on the HPD300 macroporous resin decreases with the increase of sample concentration, which may be related to the impurities in the sample solution competing with flavonoid molecules for the active sites of the resin. Therefore, the optimal sample concentration was selected as 2.79 mg / mL.

[0086] Determination of pH

[0087] The pH of the sample solution can affect the surface charge characteristics of the macroporous resin and the degree of ionization of the adsorbed molecules, thereby directly or indirectly determining the strength of the adsorption capacity of the macroporous resin. Figure 7 As shown in the figure, the pH value of the sample has a significant effect on the HPD300 macroporous resin. Compared with the higher pH (6.0-8.0), the lower pH (1.0-3.0) showed better adsorption capacity, which may be related to the fact that the higher pH value weakened the interaction between the HPD300 macroporous resin and the flavonoids of Amomum villosum. When the pH value was 2.0, the adsorption rate and adsorption amount of the HPD300 macroporous resin were the largest, which were 83.64% and 49.24 mg / g, respectively. Therefore, the pH of the optimal loading solution was determined to be 2.0.

[0088] Determination of column-to-material ratio

[0089] The effect of the ratio of the amount of macroporous resin to the volume of the sample on the adsorption capacity is as follows: Figure 8As shown. When the amount of macroporous resin is fixed, as the volume of the sample solution increases, the adsorption amount of amomum villosum flavonoids by HPD300 macroporous resin gradually increases, and gradually tends to be flat after the column material ratio is 1:20. The adsorption rate change trend is the opposite. After the column material ratio is 1:20, the adsorption rate is less than 80%. When the column material ratio is relatively large, the macroporous resin can basically completely adsorb the flavonoid molecules in the sample solution, resulting in a higher adsorption rate. The adsorption amount is related to the quality of the resin. When the amount of macroporous resin is dominant in the column material ratio, the adsorption amount is relatively low. The appropriate column material ratio is conducive to the adsorption of the resin. Therefore, after comprehensive consideration, 1:20 is selected as the optimal adsorption column material ratio.

[0090] (2) Optimization of resin elution conditions

[0091] In order to improve the purity of flavonoids in the amomum villosum extract, a dynamic desorption test was conducted on the adsorption saturated HPD300 macroporous resin using the gradient elution method. Since flavonoids with different structures and existence states have different chemical properties, 0% to 100% ethanol solution (v / v) was set for elution, and each ethanol concentration was eluted until colorless. Fig. 9 As shown in A, the faster the elution flow rate, the more asymmetric the flavonoid content curve obtained by elution, and the more ethanol is consumed. Among them, 3BV / h consumes about 2 to 3BV of ethanol more than 0.75BV / h and 1.5BV / h. Although 0.75BV / h consumes the least ethanol, it takes the longest time, about 2 to 4 times that of 1.5BV / h and 3BV / h. In addition, 0% to 50% ethanol can basically elute the flavonoid molecules in the resin completely. Therefore, the flavonoid elution components obtained by eluting with 0% to 50% ethanol are vacuum freeze-dried and their flavonoid content is determined. Fig. 9 B shows that the contents of each flavonoid elution component of 1.5 BV / h are all above 50%, among which the flavonoid contents obtained by 20% and 30% ethanol elution are relatively high, 91.42% and 90.68% respectively. In summary, 1.5 BV / h is determined to be the optimal elution flow rate, and the required volumes of 0% to 50% ethanol (elution starts from pure water, and the ethanol concentration gradually increases by 10%) are 2.3 BV, 6.7 BV, 16.7 BV, 14.3 BV, 8.5 BV, and 3.3 BV.

[0092] Analysis of Amomum villosum flavonoids

[0093] According to the optimal process conditions for separation and purification of amomum villosum flavonoids by HPD300, i.e., loading concentration 2.7 mg / mL, pH=2, column-to-material ratio 1:20, adsorption time 8 h, and elution flow rate 1.5 BV / h, the freeze-dried powders of amomum villosum flavonoids obtained by elution with 20% and 30% ethanol were mixed, and the content was determined by NaNO3-Al(NO3)3-NaOH colorimetry to be 90.24%, which was 4.16 times higher than that obtained by response surface optimization of ultrasound-assisted extraction ( Fig.10 A). At the same time, the amomum villosum flavonoids obtained by elution with 20% and 30% ethanol were subjected to plant-wide targeted metabolomics analysis. According to the retention time and peak area of ​​the TIC graph, a total of 1257 compounds were detected, which can be roughly divided into flavonoids, polyphenols, alkaloids, plant hormones, terpenes, aromatics, keto acids, fatty acyl, vitamins, nucleotides and others ( Fig.10 B and 10C). Among them, the relative abundance of flavonoids was the highest (73.83%), mainly including epicatechin (30.88%), isoquercetin (17.33%), astragaloside (9.52%), kaempferol-3-O-rutinoside (8.46%), proanthocyanidin B2 (6.82%), rutin (5.65%) and other substances ( Fig.10 D).

[0094] The effects of flavonoid components separated and purified by macroporous resin on DPPH, ABTS and OH － The influence of free radical scavenging rate and FRAP value. With the increase of the concentration of each component, the scavenging ability of different free radicals and the iron ion reducing ability also increase continuously. Among them, the antioxidant capacity of the tsaoko flavonoids component (30% tsaoko flavonoids) obtained by eluting with 30% ethanol is relatively high, which is close to the control sample Vc, followed by the tsaoko flavonoids (20% tsaoko flavonoids) obtained by eluting with 20% ethanol, while the antioxidant capacity of the crude extract and the 50% obtained component is relatively weak. In addition, it was found that the 20% and 30% tsaoko flavonoids components have relatively high free radical scavenging ability, and the crude extract is the weakest.

[0095] Example 2 Application of Amomum villosum flavonoids in improving colitis in mice

[0096] DSS-induced modeling is widely used in animal experiments of ulcerative colitis. Compared with other chemical modeling methods, it has lower intestinal corrosiveness and is closer to animal physiological lesions. DSS can directly stimulate intestinal epithelial cells to destroy the intestinal barrier, prompt the body to secrete inflammatory factors, disrupt intestinal homeostasis, and thus induce the occurrence of acute enteritis, manifested as diarrhea, intestinal bleeding, neutrophil infiltration, etc., which are basically consistent with the clinical UC manifestations. Different DSS concentrations can establish acute or chronic UC models, and 2% to 5% DSS solution is a commonly used dose for acute UC. In the early stage of the present invention, a preliminary experiment of DSS-induced UC mouse colitis was conducted. 3% DSS solution can cause UC mice to show obvious weight loss, bloody stools, and soft stools within 7 days, with a low mortality rate, and the colon length of UC mice is significantly shortened, showing typical UC clinical characteristics. Therefore, 3% DSS solution can meet the experimental needs. On this basis, the present application established a 3% DSS-induced UC mouse acute colitis model.

[0097] Design and grouping of animal experiments

[0098] 60 C57 / 6J male mice, weighing 20-23 g, were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. (SPF grade), animal license number SCXK (Beijing) 2021-0011. They were kept in the animal laboratory of the Yunnan Provincial Key Laboratory of Precision Nutrition and Personalized Food Manufacturing and approved by the Animal Ethics Committee of Yunnan Agricultural University. The animals were kept in a constant temperature environment (24±1℃, 12h light and dark alternation), with free diet (basic feed). After one week of adaptation, the mice were divided into groups for experiments, and there was no significant difference in the average weight of the mice in each group.

[0099] Set up 5 experimental groups ( Fig.11 ): (a) CON group (ultrapure sterilized water, n = 12); (b) DSS group (3% DSS, n = 12); (c) DSS + LATF group (25 mg / (kg · bw) Amomum villosum flavonoids + 3% DSS, n = 12); (d) DSS + MATF group (50 mg / (kg · bw) Amomum villosum flavonoids + 3% DSS, n = 12); (e) DSS + HATF group (100 mg / kg · bw) Amomum villosum flavonoids + 3% DSS, n = 12). The gavage volume was 0.2 mL / 10 g body weight. The experimental process is as follows Figure 2 As shown, before DSS intervention, mice in the CON and DSS groups were gavaged with ultrapure water, and mice in the ATF dose group were gavaged with 25, 50, and 100 mg / kg·bw of Amomum villosum flavonoids, respectively, for 14 consecutive days, and mice were allowed to drink water freely for the rest of the time. From the 15th day, mice in the CON and DSS groups were gavaged with ultrapure water, and mice in the ATF dose group were gavaged with 25, 50, and 100 mg / kg·bw of Amomum villosum flavonoids, respectively, and mice were allowed to drink 3% DSS aqueous solution freely for the rest of the time except for the CON group, and mice were sacrificed on the 22nd day. The Amomum villosum flavonoids used in the experimental group were a mixture of freeze-dried powder of Amomum villosum flavonoids obtained by eluting with 20% and 30% ethanol.

[0100] Mouse dissection and tissue collection methods

[0101] Serum: At the end of the experiment, mice were killed by CO2 asphyxiation, and the abdominal cavity of the mice was immediately opened. Blood was collected from the vein with a 1 mL syringe and stored in a sterile EP tube at 4°C for later use. The blood samples were incubated at 37°C for 30 min, centrifuged (4000 rpm, 4°C) for 10 min, and serum was obtained and stored at -20°C for later use.

[0102] Liver, kidney and other major organs: Disassemble the mouse liver, spleen, kidney and other organs and record their weight.

[0103] Colon tissue: After the mice were killed, the entire intestine was removed, the mesentery and other tissues were removed, the proximal colon and cecum were taken, and the length of the colon segment of each mouse was recorded. The colon was flushed with PBS at 4°C pH 7.4, and the colon contents were collected. The colon tissue was cut into 3 parts, one of which was fixed in 10% neutral buffered formalin fixative for later use; the other 2 parts were wrapped in sterile tin foil, stacked and frozen in liquid nitrogen, and stored in a -80°C refrigerator for later use.

[0104] Cecal contents: Record the weight of the cecum before and after removing the contents. Collect the cecal contents by cutting the cecum open and rinsing it in 2 mL EP containing 1.0 mL cold sterile water. After the collected cecal contents are frozen in liquid nitrogen, they are stored in a -80°C refrigerator for later use.

[0105] DAI Scoring Method

[0106] During the DSS induction period, the weight change, stool characteristics and blood in the stool of the mice were recorded every day, and the disease activity index score (DAI) was calculated as the average of the sum of the three scores. Among them, weight loss was calculated as the ratio of daily weight change to initial weight (day 0), the stool condition was slightly modified with reference to the Bristol stool typing, and blood in the stool was determined according to the instructions of the fecal occult blood kit. The detailed DAI scoring criteria are shown in Table 6.

[0107] Table 6 Mouse DAI scoring criteria

[0108]

[0109] Colon Histomorphological Analysis (H&E Staining) Method

[0110] Morphological analysis of colon and other tissues fixed in 4% formaldehyde solution was performed, including paraffin embedding, sectioning and H&E staining. The thickness of tissue sections was controlled at 3 μm. Two clear and complete H&E-stained sections were obtained from each mouse for subsequent histological observation and analysis. High-definition images of H&E-stained sections were taken using a biological microscope.

[0111] Data analysis

[0112] The data were analyzed by one-way ANOVA test in SPSS26.0 software. Different letters indicate significant differences between the two (P < 0.05). GraphPad Prism 8.0 software was used for drawing and data, and the results were expressed as mean ± standard error (mean ± SEM). * indicates that compared with the CON group, # indicates that compared with the DSS group, using unpaired two-tailed T test, *P < 0.05, **P < 0.01, ***P < 0.001; #P < 0.05, ##P < 0.01, ###P < 0.001. Spearman's r coefficient was used to calculate bivariate correlation; multivariate analysis was performed using the cloud platform of Meiji Biopharmaceutical Technology Co., Ltd.; heat maps were constructed using OmicStudio (https: / / www.omicstudio.cn / tool); unless otherwise stated in the legend, the results were considered statistically significant when P < 0.05.

[0113] The results of the experiment on the application of tsaoko flavonoids in the treatment of colitis in mice

[0114] To evaluate the protective effect of tsaoko flavonoids on colitis, the body weight and DAI of mice were monitored and recorded daily during the DSS intervention. No abnormal symptoms were observed in the CON group during the entire experiment. After DSS intake, 2, 2, 1, and 0 mice died in the DSS group, DSS+LATF group, DSS+MATF group, and DSS+HATF group, respectively ( Fig.12 A). Compared with CON, the body weight of mice in the DSS group decreased significantly from the 5th day after DSS intervention (P<0.01, Fig.12 B), accompanied by diarrhea, bloody stools, etc., which led to a significant decrease in the DAI score of mice (P<0.01, Fig.12 C). At the same time, the colon in the DSS group was significantly shortened (P<0.01, Fig.12 D and 12E), indicating that the 3% DSS-induced colitis mouse model was successfully established. After intervention with low, medium and high doses of Amomum villosum flavonoids, it was found that the high-dose treatment of mice could significantly reverse the weight loss of mice, reduce the DAI score and inhibit the shortening of the colon (P<0.01, Fig.12 B to 12E), indicating that high doses of Amomum villosum flavonoids can effectively improve the symptoms of DSS-induced colitis in mice. In addition, it was observed that after DSS intake, the liver, spleen, and kidneys of mice showed a certain degree of swelling ( Fig.12 F to 12H), suggesting that the colitis mice had immune disorders and inflammatory imbalance.

[0115] Amomum villosum flavonoids significantly improve colon histological damage in colitis mice

[0116] DSS has toxic effects on colonic epithelium, easily causing erosion, damaging barrier integrity, and increasing colonic epithelial permeability, further allowing pro-inflammatory factors to spread to the primary tissues and disrupting the balance between the immune system and intestinal flora. Fig.13 As shown in the figure, compared with CON, the colon mucosal tissue cells of mice in the DSS group were severely damaged, the number of crypt cells and goblet cells was significantly reduced, and a large number of inflammatory cells infiltrated. In contrast, the inflammatory cell infiltration in the DSS+HATF group was significantly lower than that in the model group, the number of goblet cells was larger, and the mucosal structure was relatively more complete. Therefore, Amomum villosum flavonoids can significantly improve DSS-induced colon damage, which may be related to the ability of Amomum villosum flavonoids to improve the intestinal barrier and reduce colon inflammation.

[0117] Example 3 Study on the mechanism of amomum villosum flavonoids improving colitis in mice

[0118] Regulation of Amomum villosum flavonoids on the gene expression of inflammatory factors in mice with colitis

[0119] Abnormal expression of inflammation-related factors is often found in UC. To explore the anti-inflammatory effect of Amomum villosum flavonoids, the mRNA levels of cytokines in the colon tissue of UC mice were measured. Fig.14 Compared with the CON group, the DSS group significantly upregulated the expression of TNF-α and IL-1β in the colon tissue of mice (P<0.05, Fig.14 A to 14B), inhibited the mRNA expression of anti-inflammatory factor IL-10 in the colon (P < 0.05, Fig.14 C). After intervention with Amomum villosum flavonoids, the levels of TNF-α and IL-1β in the colon of mice were significantly reduced. The expressions of IL-18, IL-6 and INF-γ mRNA showed a downward trend (P>0.05, Fig.14 G to 14I), indicating that tsaoko flavonoids can inhibit the inflammatory response caused by DSS in colitis mice.

[0120] Effect of Amomum villosum flavonoids on the alleviation of inflammatory cell infiltration in mice with colitis

[0121] Effects of Amomum villosum flavonoids on cell-specific markers in colon tissue of mice with DSS-induced colitis Fig.15 Compared with the CON group, the DSS group showed higher immune activity and could significantly promote the mRNA expression levels of F4 / 80, CD68, CD11b, CD14 and Ly-6G (P < 0.05, Fig.15 A to 15E), but had no significant effect on CD115 (P>0.05, Fig.15F). The intervention of tsaoko flavonoids can significantly reverse the upregulation of F4 / 80, CD68, CD11b, CD14 and Ly-6G mRNA expression in the mouse colon (P < 0.05). In addition, immunohistochemistry was used to analyze the expression of mouse macrophage marker F4 / 80, and it was found that the expression of F4 / 80 in the colon increased with the occurrence of DSS-induced UC and decreased with the treatment of tsaoko flavonoids ( Fig.15 G), which was consistent with the RT-qPCR results, indicating that Amomum villosum flavonoids could inhibit the infiltration of inflammatory cells and protect colon tissue.

[0122] Amomum villosum flavonoids can significantly inhibit the secretion of LPS and inflammatory factors in UC mice and improve intestinal barrier damage

[0123] The release of toxins and inflammatory factors is closely related to the damage of the intestinal barrier. Compared with the CON group, the levels of serum LPS, TNF-α and IL-1β in the DSS group were significantly increased (P < 0.05, Fig.16 A to 16C). At the same time, the mRNA expressions of ZO-1, Occludin, Claudin4 and Muc2 in the colon tissues of mice with DSS-induced colitis were significantly decreased (P < 0.05, Fig.16 D to 16G), indicating that UC mice have high toxin content, damaged intestinal barrier, and released inflammatory factors. Compared with the DSS group, the DSS+HATF group can significantly reduce the levels of serum LPS, TNF-α, and IL-1β, and significantly upregulate the mRNA expressions of ZO-1, Occludin, and Claudin4 (P < 0.05, Fig.16 A to 16F). Although tsaoko flavonoids had no significant effect on the expression level of Muc2 mRNA (P>0.05, Fig.16 G), but Amomum villosum flavonoids can significantly reduce the content of LPS, inhibit the occurrence of intestinal inflammation and promote the upregulation of tight junction proteins, thereby improving intestinal homeostasis.

[0124] Amomum villosum flavonoids inhibit the activation of the inflammatory pathway TLR4 / NF-κB / NLRP3

[0125] To further study the effect of tsaoko flavonoids intake on intestinal tissue inflammation in DSS-induced colitis mice, the mRNA expression of TLRs / NF-κB / NLRP3 signaling pathway-related factors in the colon tissue of mice was detected. Fig.17 Compared with the CON group, the DSS group had no significant effect on the mRNA expression of TLR2 (P>0.05, Fig.17 A), but it could significantly up-regulate the mRNA expression of TLR4 and TLR5 (P<0.05, Fig.17B and 17C). Compared with the DSS group, the DSS+HATF group was able to significantly inhibit the upregulation of TLR4 mRNA expression, but had no significant regulatory effect on TLR2 and TLR5. In addition, the mRNA expressions of NF-κB pathway-related factors NF-κB, MyD88, TRAF6, and NLRP3 pathway-related factors NLRP3, Caspase-1, and ASC were significantly upregulated in the colon tissues of DSS-induced colitis mice (P < 0.05, Fig.17 E to 17I), while Amomum villosum flavonoids could significantly reverse this phenomenon. In summary, Amomum villosum flavonoids can inhibit the activation of TLR4 / NF-κB / NLRP3 signaling pathway, reduce the overexpression of colonic inflammatory mediators, and thus improve DSS-induced colonic inflammation.

[0126] Amomum villosum flavonoids improve DSS-induced ulcerative colitis by regulating intestinal flora in UC mice

[0127] ASV analysis

[0128] The ASVs sequenced in the V3-V4 region of the 16S rRNA gene were annotated and classified, and the composition and abundance information of the microbial community was obtained in the taxonomic analysis. The abundance and distribution uniformity of species in the samples were evaluated by the Rank-abundance curve. Fig.18 As shown in A, as the species richness in the sample increases, the curve gradually flattens, indicating that the uniformity of species composition gradually increases. Compared with the CON group, the curve of the DSS group spans a large range on the horizontal axis, and its species richness is higher, which may be related to the large number of harmful bacteria produced by DSS intake, while the curve of the DSS+HATF group is close to that of the CON group, indicating that Amomum villosum flavonoids can improve the richness and uniformity of intestinal microorganisms in UC mice. In addition, a certain number of sequences were randomly selected from ASV, and their corresponding α-diversity index was counted and a curve was drawn. The results are shown in Fig.18 As shown in B. The Sobs index rarefaction curves of each sample platform currently sequenced tend to be flat, indicating that the sequencing depth is sufficient to reflect the diversity of the samples and the sequencing results are credible.

[0129] Analysis of changes in bacterial flora structure after intervention with tsaoko flavonoids based on β-diversity

[0130] The PCoA principal coordinate analysis method is a commonly used β-diversity analysis method. It can use a two-dimensional visual scatter plot to show the similarities and differences between the control group and the treatment group, and use the distance between samples to reflect the degree of aggregation of the sample community. Fig.19The PCoA graphs of different distance algorithms are shown. It was observed that the PCoA analysis based on the Bray-Curtis distance algorithm was 38.35% representative of the overall microbiota, while the unweighted UniFrac distance algorithm and the hellinger distance algorithm were relatively low, at 20.73% and 36.26%, respectively. In addition, by comparing the R values ​​of different distance algorithms, it was found that the R value (0.7285) based on the hellinger distance algorithm was closer to 1, indicating that the difference between the groups under this algorithm was larger, followed by the Bray-Curtis distance algorithm (R = 0.6678), and finally the unweighted UniFrac distance algorithm (R = 0.4793). In summary, the Bray-Curtis distance algorithm is more suitable for PCoA analysis of the intestinal microbiota of UC mice treated with tsaoko flavonoids. In the PCoA diagram based on the Bray-curtis distance algorithm, the CON group, DSS group and DSS+HATF group were clearly separated, and the DSS+HATF group was closer to the CON group, indicating that amomum villosum flavonoids changed the β-diversity of the intestinal flora of UC mice (P=0.001) and had a certain restorative effect on the DSS-mediated intestinal flora.

[0131] Intervention with tsaoko flavonoids can significantly reverse the relative abundance of specific species

[0132] LEfSe analysis can usually be used to analyze the differences in species between different groups, and the LDA value is combined to measure the degree of influence of species on the difference effect to obtain the dominant species of different groups at multiple levels. According to the nodes of different colors, the enrichment of the microbial group in the group can be judged. There are 88 different groups in the three different groups, including 4 phyla, 6 classes, 10 orders, 20 families, and 48 genera. Among them, the CON group, DSS group, and DSS+HATF group had 31, 32, and 25 dominant bacterial groups at different levels, respectively, indicating that DSS intake and intervention with tsaoko flavonoids have a significant effect on the intestinal flora of mice.

[0133] To further determine which bacterial communities Amomum villosum flavonoids may play a key role in the intervention process of UC, statistical analysis of the differential species at the genus level was performed. Fig. 20Compared with the CON group, the DSS group increased the relative abundance of many genera, including norank_f__Desulfovibrionaceae, Escherichia-Shigella, unclassified_f__Anaerovoracaceae, Colidextribacter, Oscillospiraceae_UCG-005, Defluviitaleaceae_UCG-011, Enterococcus, and Oscillibacter, and Amomum villosum flavonoids could significantly reverse the abnormal increase in their relative abundance (P < 0.05, Fig. 20 Although Faecalibaculum, norank_f__Clostridium_methylpentosum_group, Ruminiclostridium, and Lachnospiraceae_UCG-006 were less affected by DSS, their relative abundance in UC mice was significantly reduced after intervention with tsaoko flavonoids (P < 0.05, Fig. 20 B). In addition, compared with the DSS group, tsaoko flavonoids could also significantly increase the relative abundance of Citrobacter, unclassified_f__Enterobacteriaceae, Parasutterella, Erysipelatoclostridium, Bifidobacterium, Clostridium_innocuum_group, Flavonifractor, UBA1819, unclassified_f__Erysipelatoclostridiaceae, Ruminococcus_gauvreauii_group, Klebsiellaunclassified_f__Atopobiaceae, and Clostridioides, which were decreased by DSS intervention (P<0.05, Fig. 20 C and 20D).

[0134] It is worth noting that Escherichia-Shigella is a common pathogenic bacteria, which is closely related to the development of UC. The relative abundance of Escherichia-Shigella was the highest in the DSS group, and Amomum villosum significantly reduced the relative abundance of Escherichia-Shigella in UC mice. It is speculated that the decrease in the relative abundance of Escherichia-Shigella may be one of the important signals of Amomum villosum flavonoids in the intervention of UC. After the intervention of Amomum villosum flavonoids, the strains with higher relative abundance included Akkermansia, Colidextribacter, and Bifidobacterium, among which Akkermansia had the highest relative abundance.

[0135] The above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the present invention.

Claims

1. A method for preparing tsaoko flavonoids, characterized in that: The preparation method comprises the following steps: Step 1: Dry and crush the Amomum villosum, soak it in an ethanol solution, and then perform ultrasonic extraction to obtain a crude flavonoid extract; Step 2: Separate and purify the crude flavonoid extract using a HPD300 macroporous resin chromatography column, gradiently elute with a 0-50% ethanol solution to obtain an amomum villosum flavonoid eluate eluted with 15-35% ethanol, and freeze-dry to prepare amomum villosum flavonoid powder.

2. The method for preparing tsaoko flavonoids according to claim 1, characterized in that: In step 1, the concentration of the ethanol solution is 55-65%, the solid-liquid ratio of the tsaoko powder and the ethanol solution is 1:35-1:40 (w / v), the soaking time is 10-15h, the ultrasonic power is 160-180W, the ultrasonic time is 40-50min, and the ultrasonic temperature is 65°C-70°C.

3. The method for preparing tsaoko flavonoids according to claim 1, characterized in that: In step 2, when HPD300 macroporous resin chromatography column is used for separation and purification: the adsorption column-to-material ratio is 1:15-25; the loading concentration is 2.5-3.0 mg / mL; the loading solution pH is 1.8-2.2; the adsorption temperature is 20-25°C; and the elution flow rate is 1.4-1.6 BV / h.

4. The amomum villosum flavonoids obtained according to the preparation method described in any one of claims 1 to 3.

5. Use of the amomum villosum flavonoids according to claim 4 in the preparation of drugs for treating colitis.

6. The use of Amomum villosum flavonoids according to claim 5 in preparing a drug for treating colitis, characterized in that: The colitis is acute or chronic ulcerative colitis.

7. The use of Amomum villosum flavonoids according to claim 5 in preparing a drug for treating colitis, characterized in that: The amomum villosum flavonoids can significantly inhibit the significant weight loss caused by colitis, reduce the DAI score, and inhibit the shortening of the colon and the destruction of the colon tissue structure.

8. The use of Amomum villosum flavonoids according to claim 5 in preparing a drug for treating colitis, characterized in that: At the molecular level, the amomum villosum flavonoids significantly inhibit the abnormal increase of LPS, TNF-α and IL-1β content, significantly downregulate the mRNA expression levels of pro-inflammatory factors TNF-α and IL-1β, and effectively improve the immune disorder caused by overexpression of F4 / 80, CD68, CD11b, and CD14, while significantly upregulating the mRNA expression of intestinal barrier factors ZO-1, Occludin and Claudin4; the amomum villosum flavonoids can significantly downregulate the gene expression of TLR4, NF-κB, MyD88, TRAF6, NLRP3, and Caspase-1, thereby inhibiting the activation of the TLR4 / NF-κB / NLRP3 inflammatory signaling pathway.

9. The use of Amomum villosum flavonoids according to claim 5 in preparing a drug for treating colitis, characterized in that: The amomum villosum flavonoids can significantly affect the structure and composition of intestinal microbial communities, promote the recovery of intestinal flora, increase dominant bacterial genera and reduce harmful bacterial genera.

10. The use of Amomum villosum flavonoids according to claim 5 in the preparation of a drug for treating colitis, characterized in that: The medicine uses the amomum villosum flavonoids as an active ingredient, which is prepared into a pharmaceutically acceptable dosage form by adding pharmaceutically acceptable excipients, auxiliary ingredients or carriers alone or in combination with other medicines.

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