Application of composite active component in preparation of medicine for treating ulcerative colitis

Abstract

The invention belongs to the technical field of application of active components, and relates to application of a composite active component in preparation of a medicine for treating ulcerative colitis, the composite active component is prepared by mixing indigo blue, indirubin and isatin according to a mass ratio of (13.84-13.87): (0.15-0.17): (0.11-0.13); the dosage of the composite active component per day is 80 mu g / ml. The compound active component is prepared by mixing indigo blue, indirubin and isatin, the components are simple, and the research on the therapeutic action mechanism of the compound active component on ulcerative colitis shows that the compound active component has the effects of treating and improving ulcerative colitis, and can be used for preparing the ulcerative colitis therapeutic medicine.

Description

Technical Field

[0001] The invention belongs to the technical field of active component application, and relates to the application of a composite active component in preparing a medicine for treating ulcerative colitis. Background Art

[0002] Ulcerative colitis (UC) is a chronic, nonspecific intestinal inflammatory disease of unknown etiology. The disease site is the rectum and distal colon, with sporadic ulcers. The clinical manifestations are abdominal pain, multiple diarrhea and mucus, pus and blood in the stool. The exact pathogenesis of UC is still not fully understood, but some factors, including immune response disorders, changes in intestinal microbiota, genetic susceptibility and environmental factors, have been shown to be closely related to ulcerative colitis. The early goal of UC treatment is mainly to induce remission and reduce intestinal inflammatory response and improve quality of life; the late goal is mainly to reduce surgical risks and colorectal cancer risks. At present, clinical drugs for UC mainly include glucocorticoids, 5-aminosalicylic acid, sulfasalazine, mesalazine, etc., but these drug treatments cannot effectively prevent the development of UC, and long-term use is prone to a large number of side effects and drug resistance. Therefore, it is necessary to find safe and effective anti-UC drugs.

[0003] Traditional Chinese medicine has been increasingly valued for its advantages such as good efficacy, multiple targets, strong tolerance, few side effects, and low toxicity. Modern Chinese medicine scholars have classified UC into the categories of "diarrhea", "dysentery", "chronic dysentery", "intestinal problems", etc. In the existing studies on the treatment of UC with traditional Chinese medicine, compound Guchang Zhixie pills or tryptanthrin are used. For example, compound Guchang Zhixie pills (Guchang Zhixie pills), the ingredients include black plum, coptis root, dried ginger, poppy shell and Corydalis, the function is to harmonize the liver and spleen, astringe the intestines and relieve pain; it is used for liver and spleen disharmony, diarrhea and abdominal pain, chronic nonspecific ulcerative colitis; for example, tryptanthrin is an important secondary metabolite with a wide range of biological activities: it has good anti-tumor, cancer prevention, antibacterial and anti-inflammatory, antimalarial, anti-leishmania, anti-trypanosomal and other activities; it can also be used to treat ulcerative colitis.

[0004] Although there are drugs available to treat ulcerative colitis, their ingredients are complex and the mechanism by which they exert their therapeutic effects is not very clear. Summary of the invention

[0005] In view of the technical problem that the existing drugs for treating ulcerative colitis have complex ingredients and the mechanism of their therapeutic effects is not very clear, the present invention provides an application of a composite active component in the preparation of a drug for treating ulcerative colitis.

[0006] In the present invention, indigo, indirubin and isatin are mixed in a specific mass ratio to obtain a composite active component; the composition is simple, and the therapeutic action mechanism of the composite active component on ulcerative colitis is studied, and it is found that the composite active component has a therapeutic and improving effect on ulcerative colitis, and can be used for the preparation of a drug for treating ulcerative colitis.

[0007] The invention discloses an application of a composite active component in preparing a medicine for treating ulcerative colitis.

[0008] The invention discloses an application of a composite active component in preparing a medicine for treating ulcerative colitis by inhibiting the secretion of NO in cells induced by LPS.

[0009] A composite active component is used in the preparation of a drug for treating ulcerative colitis by inhibiting the expression of iNOS protein, p-STAT3 protein and p-P38 protein in a MAPK signaling pathway in cells induced by LPS.

[0010] A composite active component is used in the preparation of a drug for treating ulcerative colitis by inhibiting the expression of pro-inflammatory factors TNF-α and IL-6 and increasing the expression of anti-inflammatory factor IL-10.

[0011] A composite active component is used in preparing a drug for treating ulcerative colitis by reducing the MPO activity in mouse colon tissue.

[0012] A composite active component is used in the preparation of a drug for treating ulcerative colitis by restoring colon structure and crypt structure and reducing inflammatory cell infiltration.

[0013] It is further defined that the composite active component is a mixture of indigo, indirubin and isatin in a mass ratio of (13.84-13.87): (0.15-0.17): (0.11-0.13).

[0014] It is further defined that the composite active component is a mixture of indigo, indirubin and isatin in a mass ratio of 13.87:0.15:0.11.

[0015] It is further defined that the daily dosage of the composite active ingredient is 80 μg / ml.

[0016] The beneficial effects of the present invention are:

[0017] 1. In the present invention, the composite active component is a mixture of indigo, indirubin and isatin in a mass ratio of (13.84-13.87): (0.15-0.17): (0.11-0.13). The three active ingredients are simple, and the therapeutic mechanism of the composite active component on ulcerative colitis is studied. It is found that the composite active component has a therapeutic and improving effect on ulcerative colitis and can be used for the preparation of a drug for the treatment of ulcerative colitis.

[0018] 2. The present invention found through model experiments that the composite active component can inhibit the secretion of NO in RAW264.7 cells induced by LPS, and inhibit the expression of iNOS, p-STAT3 and p-P38 proteins in the MAPK signaling pathway of RAW264.7 cells induced by LPS, indicating that the production of NO through the oxidation of L-arginine to L-citrulline is reduced, and the occurrence of inflammatory response is reduced; thereby inhibiting the continued aggravation of inflammation.

[0019] 3. The present invention found through model experiments that the composite active components can inhibit the expression of pro-inflammatory factors TNF-α and IL-6, increase the expression of anti-inflammatory factor IL-10, thereby regulating the release of multiple inflammatory factors and exerting an anti-inflammatory effect.

[0020] 4. The present invention found through model experiments that the composite active component can significantly reduce the MPO activity in the colon tissue of mice, indicating that MIX can reduce the degree of inflammation in the colon tissue of mice with ulcerative colitis.

[0021] 5. The present invention found through model experiments that the composite active components can restore the colon structure and crypt structure and reduce inflammatory cell infiltration. MIX improves ulcerative colitis in mice.

[0022] 6. The active ingredients of the present invention, indigo, indirubin and indigo carmine, can be extracted from plant species, have no toxic side effects and are safe. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the effect of the mixing ratio on the survival rate of RAW264.7 cells (n=3);

[0024] Figure 2 The effects of indigo, indirubin, and indigo carmine on NO production in RAW264.7 cells (n=3);

[0025] Figure 3 The effect of the mixed ratio on NO production in RAW264.7 cells (n=3);

[0026] Figure 4 is the change of NF-κB protein expression inside and outside the cell nucleus after MIX administration (n=3);

[0027] Figure 5is the changes in STAT3 and iNOS protein expression after MIX administration (n=3);

[0028] Figure 6 is the change of MAPK protein expression after MIX administration (n=3);

[0029] Figure 7 is the effect of MIX on DAI scores of DSS-induced UC mice (n=6);

[0030] Figure 8 is the effect of MIX on pathological changes of colon tissue in UC mice (×200);

[0031] Fig. 9 MIX was used to score the colon histopathology of UC mice (n=3);

[0032] Fig.10 is the effect of MIX on serum inflammatory cytokines in UC mice (n=6);

[0033] Fig.11 The effect of MIX on MPO activity in colon tissue of UC mice (n=6). DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0035] The composite active component provided by the invention is prepared by mixing indigo, indirubin and indigo carmine in a mass ratio of (13.84-13.87): (0.15-0.17): (0.11-0.13).

[0036] Preferably, indigo, indirubin and isatin are mixed in a mass ratio of 13.84:0.15:0.11.

[0037] Preferably, indigo, indirubin and isatin are mixed in a mass ratio of 13.87:0.17:0.13.

[0038] Preferably, indigo, indirubin and isatin are mixed in a mass ratio of 13.85:0.16:0.12.

[0039] The composite active component provided by the invention can be used in the preparation of medicine for treating ulcerative colitis. In the application, the daily dosage of the composite active component is 80 μg / ml.

[0040] In the present invention, the three active ingredients of indigo, indirubin and isatin can be extracted from plants.

[0041] Preferably, since Indigo Naturalis contains indigo, indigotin and indigo carmine at the same time, these three active ingredients can be extracted from Indigo Naturalis; the above active ingredients can also be extracted from other plants using conventional methods.

[0042] Indigo Naturalis (IN) is a dry powder, agglomerate or granule made from the leaves or stems of Baphicacanthus eusia Bremek, Polygonum tinctofium Ait or Isatis indigotica Fort. It has a long history of medicinal use and can relieve infantile malnutrition, weight loss and kill insects. The Compendium of Materia Medica records that it can "relieve irritability and heat, vomit blood, hemoptysis, acne, genital acne and kill evil insects". Modern pharmacological studies have shown that Indigo Naturalis has anti-tumor, antibacterial, analgesic and anti-inflammatory effects.

[0043] The therapeutic effect of the composite active component on ulcerative colitis is illustrated below by means of a model example.

[0044] It should be noted that, unless otherwise specified, the chemicals, reagents, etc. used in the following model examples are all conventional products purchased from the market.

[0045] Example 1: In vitro cell level

[0046] In this example, LPS was used to stimulate RAW264.7 cells to construct an inflammation model, and the anti-inflammatory mechanism of the composite active component was demonstrated through in vitro cell tests.

[0047] 1.1 Model Construction

[0048] Forty SPF-grade C57BL / 6 male mice, 6-8 weeks old, with a body weight of (20±2 g) were selected.

[0049] It was divided into five groups: control group (CON), model group (Model), positive drug group (sulfasalazine, SASP), Indigo naturalis administration group (IN), and Indigo naturalis mixed ratio administration group (MIX).

[0050] The mice in the CON group drank pure water, and the mice in the other groups drank 2.5% DSS to induce ulcerative colitis models.

[0051] The mice in the CON and Model groups were gavaged with distilled water, the SASP group was gavaged with 125 mg / kg, the IN group was gavaged with 200 mg / kg, and the MIX group was gavaged with 27.74 mg / kg indigo + 0.3 mg / kg indirubin + 0.22 mg / kg indigo suspension. The gavage volume was 10 mL / kg, and the gavage was continued for 12 days.

[0052] 1.2 Test methods

[0053] The MTT method was used to investigate the effects of IN, IN, INDI, INDIRUPIN, IStain, and INDI mixed ratio group (MIX) on cell viability.

[0054] The NO level was determined by Griess method, and the administration concentration with anti-inflammatory activity was selected.

[0055] ELISA was used to determine the contents of inflammatory factors such as TNF-α, IL-6, IL-1β and IL-10; Western Blot was used to detect the expression levels of iNOS, NF-κB, IκBα, p-IκBα, STAT3, p-STAT3, p-ERK, ERK, p-P38, P38, p-JNK and JNK proteins.

[0056] 1.3 Test results

[0057] 1.3.1 Effects on the survival rate of RAW264.7 cells in each group

[0058] A: Indigo group (80 μg mL -1 )B: Indigo group (11.096 μg mL -1 ); C: indirubin group (0.12 μg·mL -1 ); D: indigo carmine group (0.088 μg mL -1 ); E: indigo + indigotin + indigo carmine group (11.096 μg mL -1 +0.12μg·mL -1 +0.088μg·mL -1 ); F: indigo + indirubin group (11.096 μg mL -1 +0.12μg·mL -1 ); G: indigo + indigo group (11.096μg·mL-1+0.088μg·mL- 1 ); H: indigo carmine + indigo carmine group (0.12μg·mL-1+0.088μg·mL -1 ), the result is as follows Figure 1 shown.

[0059] See also Figure 1According to the mass ratio of indigo, indirubin and indigo carmine (13.87%:0.15%:0.11%), 80 μg / mL of indigo naturalis, 11.096 μg / mL of indigo, 0.12 μg / mL' of indigo carmine and 0.088 μg / mL of indigo carmine with a survival rate within a reasonable range were selected for pairwise and triple ratios. It was found that the above concentration mixed ratios had basically no effect on the growth of LPS-induced RAW264.7 cells. Therefore, the above mixed ratio groups can be selected for subsequent experiments.

[0060] 1.3.2 Effects of each group on NO secretion in LPS-stimulated RAW264.7 cells

[0061] A: CON group, B: LPS group; C: Indigo group (80 μg mL -1 ); D: indigo + indigotin + indigo carmine group (11.096 μg mL -1 +0.12μg·mL -1 +0.088μg·mL -1 ); E: indigo group (11.096 μg·mL -1 ); F: Indirubin group (0.12 μg·mL -1 ); G: indigo carmine group (0.088 μg mL -1 ); H: indigo + indirubin group (11.096 μg mL -1 +0.12μg·mL -1 ); I: indigo + indigo group (11.096 μg mL -1 +0.088μg·mL -1 ); J: indigo carmine + indigo carmine group (0.12 μg mL -1 +0.088μg·mL -1 ); compared with the CON group, ## P<0.01; compared with the model group, * P<0.05, ** P<0.01.

[0062] Effects of indigo, indirubin, and indigo carmine on NO production in RAW264.7 cells (n=3) Figure 2 ; Effect of mixing ratio on NO production in RAW264.7 cells Figure 3 .

[0063] See also Figure 2 Compared with the LPS group, the same molar concentrations of indigo, indirubin and indigo carmine had a significant inhibitory effect on the NO production induced by LPS in RAW264.7 cells (P<0.01), and indigo carmine had a better inhibitory effect on NO than indigo, and indigo was better than indirubin.

[0064] See also Figure 3 Compared with the LPS group, none of the two mixed ratio groups had a significant inhibitory effect on the NO secretion of RAW264.7 cells, but the indigo, indirubin and indirubin mixed ratio group (MIX) could significantly reduce the secretion of NO (P<0.05). Therefore, the MIX group (11.096μg / mL indigo + 0.12μg / mL indirubin + 0.088μg / mL indirubin) was finally selected for further synergistic anti-inflammatory mechanism research.

[0065] 1.3.3 Effect of MIX on protein expression in LPS-stimulated RAW264.7 cells

[0066] The IN group was administered at a concentration of 80 μg mL -1 ; The MIX group's drug concentration was 11.096 μg·mL -1 Indigo + 0.12 μg·mL -1 Indirubin + 0.088 μg·mL -1 Indigo carmine; compared with the CON group, # P<0.05, ## P<0.01; compared with the model group, * P<0.05, ** P<0.01. Figure 4 As shown, Figure 4 A is the expression diagram of different histones; Figure 4 B are the expressions of NF-κB in the nucleus, NF-κB in the cytoplasm and p-IκBa protein.

[0067] See also Figure 4 Compared with the control group, the expression of nuclear NF-κB protein in RAW264.7 cells stimulated by LPS was significantly increased (P<0.05); the expression of NF-κB protein in the cytoplasm was significantly decreased (P<0.01); and the relative expression level of p-IκBa was significantly increased (P<0.05). Compared with the LPS stimulation group, the expression of nuclear NF-κB and p-IkBa protein in the Qingdai group was decreased, and the expression of NF-κB protein in the cytoplasm was increased with significant difference (P<0.05). The MIX group had no effect on the expression of nuclear NF-κB, cytoplasmic NF-κB and p-IκBa protein after administration. The above results indicate that MIX administration failed to inhibit the activation of NF-κB signaling pathway.

[0068] 1.3.4. Expression of STAT3 and iNOS proteins after MIX administration

[0069] The IN group was administered at a concentration of 80 μg mL -1 ; The MIX group's drug concentration was 11.096 μg·mL -1 Indigo + 0.12 μg·mL -1Indirubin + 0.088 μg·mL -1 Indigo carmine; compared with the CON group, # P<0.05, ## P<0.01; compared with the model group, * P<0.05, ** P<0.01. Figure 5 As shown, Figure 5 A is the expression diagram of different histones; Figure 5 B shows the changes of p-STAT3 protein expression and iNOS protein expression.

[0070] See also Figure 5 , the expression of p-STAT3 in the LPS group was significantly upregulated compared with the control group (P<0.01). After administration of MIX, the expression of p-STAT3 was significantly reduced compared with the model group, which was statistically significant (P<0.05). This indicates that the activation of the STAT3 signaling pathway in LPS-induced RAW264.7 cells is inhibited by MIX, thereby reducing related inflammatory cytokines and exerting an anti-inflammatory effect. The expression of iNOS protein in the LPS group was significantly increased compared with the control group (P<0.01), and the protein expression was reduced after MIX intervention, which was statistically significant (P<0.05). This indicates that the MIX group can exert an anti-inflammatory effect by inhibiting the expression of iNOS protein and reducing the release of NO.

[0071] 1.3.5 Changes in MAPK protein expression after MIX administration

[0072] The IN group was administered at a concentration of 80 μg mL -1 ; The MIX group's drug concentration was 11.096 μg·mL -1 Indigo + 0.12 μg·mL -1 Indirubin + 0.088 μg·mL -1 Indigo carmine; compared with CON group, P<0.05, ## P<0.01; compared with the model group, * P<0.05, ** P<0.01. Figure 6 As shown, Figure 6 A is the expression diagram of different histones; Figure 6 B shows the expression changes of p-ERK, p-P38 and p-JNK proteins respectively.

[0073] See also Figure 6Compared with the control group, the expression of p-ERK, p-P38, and p-JNK proteins in the LPS group increased significantly (P<0.05). Compared with the LPS group, the expression of p-P38 in the MIX group was significantly decreased (P<0.05), but the MIX group had no significant effect on the expression of p-ERK and p-JNK proteins in the cells. This indicates that MIX can exert an anti-inflammatory effect by inhibiting the protein expression of p-P38 in the MAPK signaling pathway of RAW264.7 cells induced by LPS.

[0074] In summary, the administration of indigo, indirubin and indigo carmine in a mixed ratio can significantly reduce the secretion of NO. This is because when the concentrations of indigo, indirubin and indigo carmine are 11.096 μg·mL -1 , 0.12 μg·mL -1 , 0.088 μg·mL -1 The results showed that MIX could significantly reduce the expression of p-P38 protein after administration, but had no significant effect on the expression of p-ERK and p-JNK proteins in cells. This showed that MIX could regulate the release of multiple inflammatory factors and exert anti-inflammatory effects by inhibiting the expression of p-P38 protein in the MAPK signaling pathway of RAW264.7 cells induced by LPS.

[0075] Further Western Blot experiments showed that after LPS stimulation of RAW264.7 cells, iNOS protein levels increased significantly (P<0.05), while MIX administration significantly inhibited the increase of iNOS, indicating that MIX can inhibit the expression of iNOS protein, thereby reducing the production of NO through the oxidation of L-arginine to L-citrulline and reducing the occurrence of inflammatory response. After MIX intervention, p-STAT3 protein expression decreased with significant difference (P<0.05), indicating that MIX can inhibit abnormal STAT3 phosphorylation, thereby inhibiting the continued aggravation of inflammation.

[0076] After being stimulated by external factors, the cascade reaction of MAPK kinase causes the phosphorylation of P38, ERK1 / 2 and JNK1 / 2 in its family. The phosphorylated proteins are transferred to the cell nucleus and dephosphorylated, which can activate the expression of corresponding downstream proteins, thereby regulating the release of various inflammatory factors.

[0077] Compared with the group treated with indigo, MIX had less cytotoxicity and produced better anti-inflammatory activity, which significantly inhibited the release of NO and cellular proinflammatory factors TNF-α and IL-6. At the same time, MIX exerted anti-inflammatory effects by inhibiting the expression of iNOS, p-STAT3, and p-P38 proteins.

[0078] Example 2: In vivo animal level evaluation

[0079] Since one of the main characteristics of UC affecting the mucosa is the infiltration of inflammatory cells (such as neutrophils and T lymphocytes) into the crypts through the gaps between damaged epithelial cells to form crypt abscesses, free drinking of DSS has direct toxicity to the intestinal epithelial cells of the basal crypts, affecting the integrity of the mucosal barrier and leading to the occurrence of intestinal inflammation, which is characterized by weight loss, diarrhea, blood in the stool, epithelial ulcers and neutrophil infiltration.

[0080] This example mainly uses 2.5% dextran sulfate sodium salt (DSS) to induce C57BL / 6 mice to establish an ulcerative colitis model, and illustrates the anti-inflammatory mechanism of the composite active ingredients through in vivo analysis.

[0081] 2.1 Model construction and grouping

[0082] Forty SPF-grade C57BL / 6 male mice, 6-8 weeks old, with a body weight of (20±2 g) were selected.

[0083] It was divided into five groups: control group (CON), model group (Model), positive drug group (sulfasalazine, SASP), Indigo naturalis administration group (IN), and Indigo naturalis mixed ratio administration group (MIX).

[0084] The mice in the CON group drank pure water, and the mice in the other groups drank 2.5% DSS to induce ulcerative colitis models.

[0085] The mice in the CON and Model groups were gavaged with distilled water, the SASP group was gavaged with 125 mg / kg, the IN group was gavaged with 200 mg / kg, and the MIX group was gavaged with 27.74 mg / kg indigo + 0.3 mg / kg indirubin + 0.22 mg / kg indigo suspension. The gavage volume was 10 mL / kg, and the gavage was continued for 12 days.

[0086] 2.2 Test results

[0087] 2.2.1. MIX score for DAI in DSS-induced UC mice

[0088] The changes in the body weight and blood in stool of mice before and after administration were recorded to score the disease activity index. Figure 7 shown.

[0089] See also Figure 7 The DAI scores of mice in the model group increased, which was significantly different from that in the control group (P<0.01), indicating that the UC model of mice was successfully established. Compared with the model group, the DAI scores of each drug-treated group gradually increased, but the increase was slower than that of the model group (P<0.05), indicating that Indigo and MIX have a certain protective effect on UC mice.

[0090] 2.2.2. Observation of colon tissue morphology by H&E staining

[0091] The SASP group was given a dose of 125 mg kg -1 : The dose of IN group was 200 mg kg -1 The MIX group was given a dose of 27.74 mg kg -1 Indigo + 0.3 mg kg -1 Indigo carmine + 0.22 mg kg -1 Indigo. The results are as follows Figure 8 shown.

[0092] See also Figure 8 The colon tissue structure of mice in the control group was intact, the crypt structure was normal, and there was no inflammatory infiltration. In the model group, increased inflammatory cell infiltration was observed in the colon, the crypts were atrophied and moved upward and irregular in shape, and goblet cells were greatly reduced. After the administration of Indigo and MIX, the colon structure and crypt structure were restored to a certain extent, and the inflammatory cell infiltration was reduced. This shows that the medication can improve DSS-induced colon inflammation in UC mice to a certain extent and reduce colon tissue damage in UC mice.

[0093] 2.2.3. MIX scoring of colonic histopathology in UC mice

[0094] The SASP group was given a dose of 125 mg kg -1 : The dose of IN group was 200 mg kg -1 The MIX group was given a dose of 27.74 mg kg -1 Indigo + 0.3 mg kg -1 Indigo carmine + 0.22 mg kg -1 Indigo carmine; compared with the CON group, ## P<0.01; compared with the Model group, * P<0.05, ** P<0.01. Fig. 9 shown.

[0095] See also Fig. 9 Compared with the control group, the scores of mice in the model group were significantly increased (P<0.01), while the scores of the Indigo group and MIX group were significantly decreased compared with the model group (P<0.01), and the effects of the two groups were comparable.

[0096] 2.2.4 Effect of MIX on inflammatory cytokines in serum of DSS-induced UC mice

[0097] The ASP group was given a dose of 125 mg kg -1 : The dose of IN group was 200 mg kg -1The MIX group was given a dose of 27.74 mg kg -1 Indigo + 0.3 mg kg -1 Indigo carmine + 0.22 mg kg -1 Indigo carmine; compared with the CON group, ## P<0.01; compared with the Model group, ** P<0.01. Fig.10 shown.

[0098] See also Fig.10 The levels of pro-inflammatory factors IL-6 and TNF-α in the serum of mice in the model group were significantly higher than those in the control group (P<0.01); the level of anti-inflammatory factor IL-10 was significantly lower than that in the control group (P<0.01). The levels of pro-inflammatory factors in the serum of mice in the Qingdai and MIX groups were significantly lower than those in the model group (P<0.01); the levels of anti-inflammatory factors were significantly higher (P<0.01).

[0099] 2.2.5 Effect of MIX on MPO activity in colon tissue of DSS-induced UC mice

[0100] MPO is an inflammatory mediator in tissue damage and various inflammatory diseases, which can reflect the severity of inflammation. The higher the MPO level, the more severe the inflammation.

[0101] The SASP group was given a dose of 125 mg kg -1 The IN group was given 200 mg kg -1 The MIX group was given a dose of 27.74 mg kg -1 Indigo + 0.3 mg kg -1 Indigo carmine + 0.22 mg kg -1 Indigo carmine; compared with the CON group, ## P<0.01; compared with the Model group, * P<0.05. Fig.11 shown.

[0102] See also Fig.11 The MPO activity in the colon tissue of mice in the DSS group was significantly increased (P<0.01). After administration of MIX, the MPO activity in the colon tissue of mice was significantly decreased (P<0.05), and the degree of decrease was comparable to that in the colon tissue of mice in the Indigo group, indicating that MIX can reduce the degree of inflammation in the colon tissue of mice with ulcerative colitis.

[0103] In this embodiment, a DSS-induced UC mouse model was selected. After the mice drank DSS freely, the DAI score of the DSS group increased, which was significantly higher than that of the blank control group (P<0.01), and the mice lost weight, moved slowly, clustered, and had symptoms of diarrhea and bloody stools. There was no significant increase in the DAI scores of the mice in the drug-treated group. Histopathological results showed that the colon crypts of the mice in the DSS group were severely deformed, the crypts moved upward and had irregular edges, and neutrophils accumulated in large numbers in the crypts. After the administration of MIX, the colon structure and crypt structure were restored to a certain extent, and the infiltration of inflammatory cells was reduced; This shows that MIX has an improving effect on ulcerative colitis in mice.

[0104] In summary, the levels of pro-inflammatory factors TNF-α and IL-6 in the serum of the model group mice of the present invention were significantly increased (P<0.01), and the levels of inflammatory factors were significantly decreased after MIX administration (P<0.05). The anti-inflammatory factor IL-10 was significantly increased after MIX administration. This shows that MIX can reduce intestinal inflammatory response by inhibiting the expression of pro-inflammatory factors TNF-α and IL-6 and increasing the expression of anti-inflammatory factor IL-10.

[0105] Obviously, 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 from these are still within the scope of protection of the invention.

Claims

1. Use of a composite active component in the preparation of a drug for treating ulcerative colitis.

2. Use of a composite active component in the preparation of a drug for treating ulcerative colitis by inhibiting the secretion of NO in cells induced by LPS.

3. Use of a composite active component in the preparation of a drug for treating ulcerative colitis by inhibiting the iNOS protein expression, p-STAT3 protein expression and p-P38 protein expression of the MAPK signaling pathway in cells induced by LPS.

4. A composite active component is used in the preparation of a drug for treating ulcerative colitis by inhibiting the expression of pro-inflammatory factors TNF-α and IL-6 and increasing the expression of anti-inflammatory factor IL-10.

5. Use of a composite active component in the preparation of a drug for treating ulcerative colitis by reducing the MPO activity in mouse colon tissue.

6. Use of a composite active component in the preparation of a drug for treating ulcerative colitis by restoring colon structure and crypt structure and reducing inflammatory cell infiltration.

7. The use according to any one of claims 1 to 6, characterized in that: The composite active component is prepared by mixing indigo, indirubin and isatin in a mass ratio of (13.84-13.87): (0.15-0.17): (0.11-0.13).

8. The use according to claim 7, characterized in that: The composite active component is prepared by mixing indigo, indirubin and indigo carmine in a mass ratio of 13.87:0.15:0.

11.

9. The use according to claim 7, characterized in that: The daily dosage of the combined active component is 80 μg / ml.

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