Application of compound saussurea involucrata composition in intestinal tract protection

Abstract

The invention discloses an application of a compound saussurea involucrata composition in protecting intestinal tracts, the application of the compound saussurea involucrata composition taking saussurea involucrata as a main raw material in protecting the intestinal tracts is found for the first time, deep research and development are carried out on the basis, and the compound saussurea involucrata composition is found to be capable of protecting the intestinal tracts from multiple target points; the compound saussurea involucrate composition can be used for protecting intestinal tracts by improving intestinal wall barriers and maintaining the steady state of the intestinal tracts, and experiments prove that the compound saussurea involucrate composition can be used for maintaining the steady state of the intestinal tracts by improving intestinal microbe disorder by up-regulating probiotics such as lactobacillus and butyric acid producing bacteria; the intestinal wall barrier is improved by increasing the height of intestinal villi and improving the expression of intestinal repair proteins ZO-1 and Occludin, and the multi-target effect is achieved, so that the intestinal tract is protected, and the application of the medicine is further expanded.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the application of compound snow lotus composition in protecting the intestines. Background Technology

[0002] The intestines are the longest digestive tract in the human body, comprising the duodenum, jejunum, ileum, cecum, colon, and rectum, with a total length of approximately 7 meters. The jejunum and ileum are coiled in the center of the abdominal cavity, surrounded by the colon. The inner wall of the small intestine is densely covered with villi, and it is the main site of digestion and absorption. Here, various digestive juices break down chyme into small-molecule nutrients such as glucose and amino acids, which are then absorbed into the body. The remaining residue enters the large intestine, where water is absorbed and gradually forms feces, which are temporarily stored in the left colon and rectum before being excreted. Abnormal intestinal function can easily lead to digestive and absorptive disorders, potentially causing a series of health problems such as abdominal pain, diarrhea, and malnutrition, significantly impacting overall bodily function.

[0003] Snow lotus is a rare and endangered traditional medicinal material commonly used in Chinese medicine and ethnic minority medicine. It encompasses a variety of species, including *Saussurea involucrata*, *Saussurea medusa*, and *Saussurea involucrata*, among which *Saussurea involucrata* is considered the best in quality and is the only snow lotus variety recorded in the *Chinese Pharmacopoeia*. *Saussurea involucrata* has a wide range of medicinal uses, including antioxidant, anticancer, analgesic, anti-inflammatory, and anti-brain-damage effects. In-depth research has revealed that *Saussurea involucrata* contains abundant flavonoids, lactones, lignans, alkaloids, volatile oils, and polysaccharides, with flavonoids being the most prominent and abundant component. The raw materials for Compound Snow Lotus Capsules, a traditional Chinese medicine, are composed of Tian Shan Snow Lotus, Notopterygium incisum, processed Aconitum carmichaelii, Angelica pubescens, processed Aconitum kusnezoffii, vinegar-processed Corydalis yanhusuo, Chaenomeles speciosa, and Periploca sepium. Typically, Tian Shan Snow Lotus is the principal ingredient, Notopterygium incisum, processed Aconitum carmichaelii, Angelica pubescens, and processed Aconitum kusnezoffii are the assistant ingredients, and vinegar-processed Corydalis yanhusuo, Chaenomeles speciosa, and Periploca sepium are the adjuvant ingredients. The proportions of these ingredients may vary between different manufacturers. Currently, Compound Snow Lotus Capsules are mainly used to treat rheumatoid arthritis, ankylosing spondylitis, and various degenerative osteoarthritis. It has the effects of warming the meridians and dispelling cold, dispelling wind and dampness, resolving blood stasis and reducing swelling, and relaxing muscles and tendons. However, there are currently no publicly available reports that Compound Snow Lotus Capsules can protect the intestines. Summary of the Invention

[0004] During their research on the treatment of rheumatic diseases, the inventors of this application discovered that Compound Snow Lotus Capsules can protect the intestines from multiple targets. Based on this, they conducted in-depth research and development, applying the Compound Snow Lotus composition to protect the intestines and further expanding the application of the drug.

[0005] The technical solution provided by this invention is as follows:

[0006] The use of the compound snow lotus composition in the preparation of a medicine having at least the following pharmacological effects, wherein the compound snow lotus composition is composed of the following raw materials in parts by weight:

[0007] Tian Shan Snow Lotus 60-120 parts, Corydalis Rhizome (vinegar-processed) 15-50 parts, Notopterygium Root 15-50 parts, Aconitum Carmichaelii (processed) 40-85 parts, Angelica Pubescens Root 15-50 parts, Aconitum Carmichaelii (processed) 30-80 parts, Chaenomeles Root 15-50 parts, Periploca Bark 18-60 parts.

[0008] The pharmacological effects include improving the intestinal barrier and maintaining intestinal homeostasis.

[0009] Preferably, the compound snow lotus composition is composed of the following raw materials in parts by weight: 72 parts of Tian Shan snow lotus, 18 parts of Corydalis yanhusuo (vinegar-processed), 18 parts of Notopterygium incisum, 60 parts of Aconitum carmichaelii (processed), 18 parts of Angelica pubescens, 60 parts of Aconitum kusnezoffii (processed), 18 parts of Chaenomeles speciosa, and 24 parts of Periploca sepium.

[0010] Preferably, the drug maintains intestinal homeostasis by improving intestinal microbial imbalance.

[0011] Preferably, the drug improves the intestinal barrier by increasing the height of the small intestinal villi and enhancing the expression of intestinal repair proteins ZO-1 and Occludin.

[0012] Preferably, the drug is an oral preparation.

[0013] Preferably, the drug is in the form of capsules.

[0014] Based on the same inventive concept, the present invention also provides the application of the aforementioned drug in the preparation of intestinal protection agents.

[0015] Based on the same inventive concept, the present invention also provides the use of the aforementioned drug in the preparation of a formulation for treating inflammatory bowel disease.

[0016] The beneficial effects achieved by this invention are as follows:

[0017] This invention is the first to discover that a compound snow lotus composition with snow lotus as the main raw material can be used for intestinal protection. Based on this, in-depth research and development were carried out, and it was found that the compound snow lotus composition can protect the intestine from multiple targets. It can protect the intestine by improving the intestinal wall barrier and maintaining intestinal homeostasis. Through experimental verification, the compound snow lotus composition can maintain intestinal homeostasis by improving intestinal microbial imbalance by upregulating probiotics such as Lactobacillus and Odoribacter, and improve the intestinal wall barrier by increasing the height of small intestinal villi and increasing the expression of intestinal repair proteins ZO-1 and Occludin. This multi-target action can protect the intestine and further expand the application of the drug.

[0018] Instruction manual illustrations

[0019] Appendix Figure 1 A comparison chart of species richness and evenness of the blank group, model group and compound snow lotus composition group in the embodiments of the present invention: Chao1 index, Simpson index and Shannon index;

[0020] Appendix Figure 2 This is a diagram showing the OTU composition of the blank group, model group, and compound snow lotus composition group in this embodiment of the invention;

[0021] Appendix Figure 3 These are species abundance diagrams for the blank group, model group, and compound snow lotus capsule group in embodiments of the present invention. a: Species abundance diagram at the phylum level; b: Species abundance diagram at the genus level;

[0022] Appendix Figure 4 HE staining of mice in the blank group, model group, and compound snow lotus capsule group of this invention embodiment;

[0023] Appendix Figure 5 These are the intestinal wall permeability results of the blank group, model group, and compound snow lotus capsule group in the embodiments of the present invention, as well as the immunofluorescence staining and quantitative analysis of ZO- and Occludin.

[0024] Appendix Figure 6 The intestinal villi are shown in the blank group, model group, and compound snow lotus capsule group of the present invention.

[0025] Appendix Figure 7 This describes the expression of FGL1 and downstream related inflammatory factors in this invention. a: TNF-α; b: IL-23; c: IL-1β. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Example:

[0028] 1) Grouping and Model Preparation

[0029] Thirty-six SPF-grade male ICR mice, 5 weeks old and weighing 20±2 g, were selected. All the mice were purchased from Beijing Sibefu Biotechnology Co., Ltd.

SCXK(Beijing) 2021-0006

[0030] The compound Xue Lian composition in this embodiment is composed of the following raw materials in parts by weight: 72 parts of Saussurea involucrata, 18 parts of Corydalis yanhusuo (processed with vinegar), 18 parts of Notopterygium incisum, 60 parts of Aconitum carmichaeli (processed), 18 parts of Angelica pubescens, 60 parts of Aconitum kusnezoffii (processed), 18 parts of Chaenomeles sinensis, and 24 parts of Periploca sepium.

[0031] The above is the Chinese medicine composition of the applicant's finished product drug, compound Xue Lian capsules.

[0032] Control (control group): Each mouse was intragastrically administered 1 ml of normal saline;

[0033] Model (model group): Each mouse was intragastrically administered 0.5 ml of peanut oil + 0.2 ml of 30% fructose solution;

[0034] FXLC (drug group): On the basis of intragastrically administering 0.5 ml of peanut oil plus 0.2 ml of 30% fructose solution, a suspension of the compound Xue Lian composition with a concentration of 0.16 g / kg was intragastrically administered.

[0035] Preparation of the suspension of the compound Xue Lian composition:

[0036] Each raw material was taken in proportion, crushed separately, passed through an 80-mesh sieve and then mixed. The mixed medicinal powder was added to water according to a solid-liquid ratio of 1:20 (g / ml), and after stirring evenly, it was ready. Before intragastric administration, the suspension of the compound Xue Lian composition was stirred to make the drug evenly dispersed in water.

[0037] Each group of mice above was intragastrically administered once a day for 14 consecutive days.

[0038] The diet, activities and death conditions of the mice in the FXLC group were counted, and no dead individuals were found in the FXLC group of mice.

[0039] 2) 16S flora sequencing

[0040] The initial feces of the mice 1 day after the last intragastric administration were collected, and the feces were used to detect the flora with a fecal DNA extraction kit.

[0041] (1) DNA extraction and 16S rRNA sequencing

[0042] Total DNA was extracted from feces using a fecal DNA extraction kit (Tiangen; DP328). DNA purity and concentration were detected and calculated using a NanoDrop 1000, and the quality of DNA extraction was determined using a 1% agarose gel. 12.5 ng of DNA was used to amplify the bacterial V3 / V4 region using a pair of specific primers 341F / 805R. The amplification system consisted of 25 μl of sterile double-distilled water, 5 μl of 5*FastPfu buffer, 2 μl of 2.5 mM dNTPs, 1 μl each of 16S F primer (5 μM) and 16S R primer (5 μM), and 12.5 ng of MicrobiaL DNA. The program was as follows: 98°C pre-denaturation for 1 min, 25 cycles (98°C denaturation for 15 s, 58°C annealing for 15 s, 72°C extension for 15 s), and 72°C extension for 5 min (PCR instrument: Veriti Dx 96 Well Thermal Cycler). PCR products were recovered using magnetic beads and detected by 2% agarose gel electrophoresis.

[0043] Analysis of the sequencing results revealed significant differences in the Chao1, Simpson, and Shannon indices among the blank group, model group, and compound snow lotus combination group. Figure 1 As shown in the figure. The results suggest that the species richness and evenness of the high-fat mouse community were reduced, and the biodiversity was low, while the compound snow lotus composition can improve this phenomenon.

[0044] like Figure 2 As shown, the blank group and the model group together had 289 OTU bacteria, the model group and the compound snow lotus capsule group together had 363 OTU bacteria, and the three groups together had 724 OTU bacteria.

[0045] like Figure 3As shown in the heatmap, the bacterial species differences between the CON and MOD groups are significant. Compared to the CON group, the MOD group shows a significant increase in the relative abundance of bacteria such as Prevotella, Bacteroidetes, Bifidobacterium, Porphyromonas, Mucispirillum, and Parabacteroidos, while the abundance of Desulfovibrio, Acinetobacter, and Staphylococcus is significantly decreased. The heatmap showing the bacterial species differences between the MOD and FXLC groups further demonstrates the significant differences in bacterial species composition between the two groups. Compared with the MOD group, the relative abundance of Prevotella, Bifidobacterium, and Parabacteroidos was significantly reduced in the FXLC group, while the relative abundance of Rhodococcus, Streptococcus, Turicibacter, Aggregatibacter, Candidatus Arthromitus, and Lactococcus was significantly increased. These bacteria are the unique dominant bacteria that emerged after the improvement by FXLC.

[0046] (3) Small intestinal barrier-related experiments

[0047] One day after the last gavage, mice from each group were randomly selected for dissection, and their ileum tissue was collected. The organs of the dissected mice were observed, and no abnormalities were found.

[0048] 1) HE staining analysis:

[0049] Ileal tissue was fixed in 4% paraformaldehyde, then embedded in paraffin for sectioning. The specific staining steps are as follows:

[0050] ① Dewaxing paraffin sections to water: Place the sections in xylene I for 20 min, II for 20 min, anhydrous ethanol I for 5 min, II for 5 min, and 75% ethanol for 5 min in sequence, and then rinse with tap water.

[0051] ② Staining: For intestinal HE staining, stain sections in hematoxylin staining solution for 3-5 min, wash in tap water, separate in differentiation solution, wash in tap water, stain again in blue reflux solution, and rinse in running water. Dehydrate sections in 85% and 95% alcohol for 5 min each, and stain in eosin staining solution for 5 min. For spinal safranin-fast green staining, stain sections in safranin staining solution for 1-2 h, wash in tap water, separate in differentiation solution, and fast green stain for 3-5 min.

[0052] ③ Dehydration and mounting: Place the slides in anhydrous ethanol I, II, and III for 5 minutes each, and in xylene I and II for 5 minutes each, and then place them in a transparent container sealed with neutral glue.

[0053] ④ Examine the samples under a microscope, collect images, and analyze them.

[0054] 2) Immunofluorescence analysis:

[0055] Ileal tissue was fixed in 4% paraformaldehyde, then embedded in paraffin for sectioning. The specific immunofluorescence single-labeling experimental procedure is as follows:

[0056] Dewaxing paraffin sections to water: The sections are placed in xylene I for 15 min, xylene II for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min, and then washed with distilled water.

[0057] Antigen retrieval: Tissue slides were placed in a retrieval chamber filled with EDTA antigen retrieval buffer (pH 9.0) and microwaved for antigen retrieval. Microwave on medium heat for 8 minutes, then turn off for 8 minutes, and then microwave on medium-low heat for 7 minutes. During this process, excessive evaporation of the buffer should be prevented, and the slides should not be allowed to dry. After natural cooling, the slides were placed in PBS (pH 7.4) and washed three times on a decolorizing shaker for 5 minutes each time.

[0058] Blocking: The slides were placed in a 3% hydrogen peroxide solution and incubated at room temperature in the dark for 25 minutes to block endogenous peroxidase. The slides were then placed in PBS (pH 7.4) and washed three times on a decolorizing shaker for 5 minutes each time.

[0059] Serum blocking: After slightly drying the section, draw a circle around the tissue with a histochemical pen (to prevent antibody from flowing away), and add 10% goat serum to the circle and incubate at room temperature for 30 minutes.

[0060] Add antibody: Gently shake off the blocking solution, add the prepared primary antibody (ZO-1 / Occludin) to the slide, and incubate the slide flat in a humidified chamber at 4°C overnight. (Add a small amount of water to the humidified chamber to prevent antibody evaporation). Place the slide in PBS (pH 7.4) and wash three times on a decolorizing shaker for 5 minutes each time. After slightly drying the slide, add a secondary antibody (HRP-labeled goat anti-rabbit) of the corresponding species to the primary antibody to cover the tissue in the circle, and incubate at room temperature in the dark for 50 minutes.

[0061] Add TSA reagent: Place the slide in PBS (pH 7.4) and wash three times on a decolorizing shaker for 5 min each time. Add tyramine fluorescein (prepare fresh before use with PBST containing 0.003% H2O2) to the tissue and incubate at room temperature for 20 min.

[0062] Diamidino-phenyl-indole (DAPI) counterstaining of cell nuclei: Slides were placed in PBS (pH 7.4) and washed three times on a destaining shaker for 5 minutes each time. After slightly drying the sections, DAPI staining solution was added to the inner circle and incubated at room temperature in the dark for 10 minutes.

[0063] Mounting and photography: Slides were placed in PBS (pH 7.4) and washed three times on a decolorizing shaker for 5 minutes each time. After drying, sections were mounted with anti-fluorescence quenching mounting medium. Sections were observed and images were acquired under a fluorescence microscope.

[0064] Data collection and analysis:

[0065] The above HE staining ( Figure 4 Images obtained from intestinal villus staining and immunofluorescence staining were imported into ImageJ for quantitative analysis to obtain the height of the small intestinal villi. Figure 6 The expression levels of ZO-1 protein were measured. Data are expressed as mean ± standard error of mean (SEM). One-way ANOVA was performed using Graphpad Prism 8.0 to evaluate statistical significance among multiple groups. Statistical significance was defined as p < 0.05.

[0066] ZO-1 and Occludin are tight junction proteins, which are essential for barrier function and effective mucosal repair. Figure 5 As shown, the protein expression levels of ZO-1 and Occludin in the model group were significantly lower than those in the blank group (*P<0.001), while the FXLC group effectively improved this phenomenon (#P<0.01).

[0067] Combination Figure 4 and Figure 6It is evident that the intestinal villi in the model group mice were shorter than those in the control group, suggesting damage to the morphology and structure of the small intestine. In contrast, the villi in the FXLC group mice were longer than those in the model group, indicating that the compound snow lotus composition can help increase the height (length) of the intestinal villi.

[0068] Following intestinal damage, the richness and evenness of the gut microbiota in mice are negatively affected, and beneficial bacteria such as Lactobacillus and Odoribacter are also reduced. Furthermore, the height of the small intestinal villi decreases, and the expression of the intestinal repair protein ZO-1 is reduced. As demonstrated in the above experiments, the compound snow lotus composition can significantly improve these phenomena. The richness and evenness of the gut microbiota, the height of the intestinal villi, and the expression of the intestinal repair protein ZO-1 essentially reflect the health of the gut. Therefore, the compound snow lotus composition can improve intestinal damage and protect the gut by regulating the intestinal microecology, increasing the expression of protein ZO-1, and increasing the height of intestinal villi.

[0069] Inflammatory bowel disease (IBD) is a group of diseases characterized by chronic, relapsing nonspecific inflammation of the intestines, mainly including two clinical subtypes: ulcerative colitis (UC) and Crohn's disease (CD). Typical clinical manifestations include chronic diarrhea, abdominal pain, and weight loss, severely impacting patients' quality of life. Recent studies have shown that the pathogenesis of IBD is closely related to significant abnormalities in the structure and function of the intestinal barrier, with a characteristic change being increased intestinal permeability, commonly referred to as the "leaky gut" phenomenon. Intestinal barrier dysfunction is not only seen during disease activity but also persists during clinical remission, suggesting it may be a key early event in the development of IBD and play a sustained driving role in the chronicity of the disease. Specifically, impaired barrier integrity allows intestinal symbiotic microorganisms and their metabolites to more easily translocate to the lamina propria of the mucosa, activating abnormal innate and adaptive immune responses, forming and maintaining a vicious cycle of "inflammation—barrier disruption—microbiota translocation—further inflammation," thereby promoting disease progression and recurrent episodes.

[0070] In this pathological context, the compound snow lotus composition can exert a synergistic effect on intestinal protection through dual intervention targeting the inflammatory response and intestinal barrier repair, thereby improving and treating inflammatory bowel disease.

[0071] (4) Anti-inflammatory related experiments

[0072] ELISA was used to detect the expression levels of serum TNF-α, IL-1β, and IL-23. The kit was equilibrated at room temperature for 30 minutes beforehand. Serum samples were allowed to coagulate naturally at room temperature for 10-20 minutes, then centrifuged at 3000 rpm for 20 minutes. The supernatant was used for detection, taking care to avoid precipitation. Serum was stored at -80℃ for enzyme-linked immunosorbent assay (ELISA). The assay was performed according to the manufacturer's instructions. Finally, the absorbance was measured using a microplate reader to obtain the levels of TNF-α, IL-1β, and IL-23. Results are shown below. Figure 7 .

[0073] Depend on Figure 7 The ELISA results of serum TNF-α, IL-1β and IL-23 levels showed that the expression levels of TNF-α, IL-23 and IL-1β were significantly increased in the MOD group, while the serum pro-inflammatory factor levels in the FXLC group treated with the compound snow lotus composition decreased to varying degrees. There were significant differences in the expression levels of TNF-α, IL-23 and IL-1β between the FXLC group and the MOD group, indicating that the compound snow lotus composition has a significant anti-inflammatory effect.

[0074] The present invention and its embodiments have been described above. This description is not restrictive, and the embodiments shown are only one of the embodiments of the present invention. The actual structure is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. Use of a compound saussurea composition in the preparation of a medicament having at least the following effects, characterized in that, The compound saussurea composition is composed of the following raw materials in parts by weight: Saussurea involucrata 60-120 parts, Corydalis tuber (vinegar) 15-50 parts, Notopterygium 15-50 parts, Radix Aconiti Kweichow 40-85 parts, Angelica pubescens 15-50 parts, Radix Aconiti 30-80 parts, Chaenomeles speciosa 15-50 parts, Cortex Meliae 18-60 parts; The effects include improving the intestinal wall barrier and maintaining intestinal homeostasis.

2. Use according to claim 1, characterized in that, The compound saussurea composition is composed of the following raw materials in parts by weight: Saussurea involucrata 72 parts, Corydalis tuber (vinegar) 18 parts, Notopterygium 18 parts, Radix Aconiti Kweichow 60 parts, Angelica pubescens 18 parts, Radix Aconiti 60 parts, Chaenomeles speciosa 18 parts, Cortex Meliae 24 parts.

3. Use according to claim 1 or 2, characterized in that, The medicament maintains intestinal homeostasis by improving intestinal microbial imbalance.

4. Use according to any one of claims 1 to 3, characterized in that, The medicament improves the intestinal wall barrier by increasing the height of the small intestinal villi and increasing the expression of intestinal repair proteins ZO-1 and Occludin.

5. Use according to any one of claims 1 to 4, characterized in that, The medicament is an oral preparation.

6. Use according to claim 5, characterized in that, The medicament is a capsule.

7. Use of the medicament of any one of claims 1 to 6 in the preparation of an intestinal protection preparation.

8. Use of the medicament of any one of claims 1 to 6 in the preparation of an inflammatory bowel disease treatment preparation.

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